Oxygen Radicals in Chemistry and Biology: Proceedings, 3. Internat. Conference, Neuherberg, Federal Republic of Germany, July 10-15, 1983 [Reprint 2011 ed.] 9783110866537, 9783110097047

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Oxygen Radicals in Chemistry and Biology

Oxygen Radicals in Chemistry and Biology Proceedings Third International Conference Neuherberg, Federal Republic of Germany July 10-15,1983 Editors Wolf Bors · Manfred Saran · David Tait

W DE G Walter de Gruyter · Berlin · New York 1984

Editors Wolf Bors, Dr. rer. nat. Manfred Saran, Dipl. Phys. David Tait, Ph. D. Gesellschaft für Strahlenund Umweltforschung mbH Institut für Biologie Abteilung für Strahlenbiologie Ingolstädter Landstraße 1 D - 8 0 4 2 Neuherberg Federal Republic of Germany

CIP-Kurztitelaufnahme der Deutschen Bibliothek Oxygen radicals in chemistry and biology: proceedings, 3. internat, conference, Neuherberg, Fed. Republic of Germany, July 10-15,1983 / ed. Wolf Bors ... - Berlin; New York: de Gruyter, 1984. ISBN 3-11-009704-4 NE: Bors, Wolf [Hrsg.]

Library of Congress Cataloging in Publication Data Oxygen radicals in chemistry and biology. Bibliography: p. Includes index. 1. Active oxygen—Congresses. 2. Radicals (Chemistry) —Congresses. I. Bors, Wolf, 1940II. Saran, Manfred, 1936III. Tait, David, 1953 QP535.010935 1984 546'.721424 84-1691 ISBN 3-11-009704-4

Copyright © 1984 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany.

PREFACE It is now more than a decade since Joe McCord and Irwin Fridovich discovered that erythrocuprein catalyses the dismutation of the superoxide radical, a discovery which called the attention of the scientific community to the participation of radical reactions in biological processes. Since that time the spectrum of radicals of biological interest has broadened: hydroxyl, peroxyl, and alkoxyl radicals have appeared

on

the

scene,

oxygen activation

by metal-oxygen

complexes

and

metal-catalyzed radical generating processes have evoked great interest, and our knowledge about the implications of radical reactions for life processes has grown tremendously. In response to the growing importance of this topic, Ajit Singh and Abram Petkau organized in 1977 a multi-disciplinary meeting a t Pinawa (Canada) on 'Singlet Oxygen and Related Species in Chemistry and Biology', and three years later Mike Rodgers and Larry Powers hosted a conference on 'Oxygen and Oxy-Radicals' in Austin (Texas). The Munich conference was the third in the series but contained more medical and

clinical

aspects

thus

following the

example

of

related

conferences on Superoxide Dismutase. We hope that the meeting succeeded in its goal of promoting communication and understanding among researchers of many different disciplines and of stimulating them to collaborate in solving the riddles of this rapidly developing field. The proceedings book has been produced by the camera-ready technique for the sake of early publication. This has meant some sacrifices to the appearance of the book. The poster sessions are now combined with the oral contributions and the papers have been arranged in an order different to that in which they were delivered a t the conference. The organization of a congress involves a tremendous amount of work 'behind the scenes'. We were therefore very lucky that Hildegard Buchhart was there with her team (Marie-Claude Lapointe, Christiane Lumpe, Sigrid Moeltner and Hannelore

VI

Tait) to cope so effectively with the host of logistic problems and to ensure a smoothly running conference. Our thanks go to Christa Michel for organizing the poster exhibition and to Beate Kreileder, Evelyn Rumpf, Ulf Faltermeier and Alf Kruse for dealing with the projection and other technical equipment.

Transcribing the discussions was one of the most demanding jobs. This would not have been possible without the help of Ursula Welscher, Roselie Werneyer and especially Beate Kreileder and Michael Erben-Russ, who deciphered the tape recordings and brought them into ' p r i n t e t ^ ' form. Nevertheless, we think the effort was worthwile. As well as providing the reader with some interesting scientific details, the discussions might convey a little bit of the convivial atmosphere of the meeting that we personally enjoyed very much.

We hope finally that this book brings an account of the latest developments in this fascinating area of research to both experienced workers and newcomers in the field as well as to an interested greater public. Wolf Bors, Manfred Saran, David Tait.

ACKNOWLEDGEMENTS

On behalf of all congress participants we would like to thank the GSF for the financial and administrative support which made this conference possible. The following organisations are also gratefully acknowledged for their financial assistance: Boehringer-Mannheim (Tutzing), Chemie Grünenthal (Stolberg), Hoffmann-LaRoche (Basel), A. Nattermann & Cie (Bochum) and Paul-Martini-Stiftung (Mainz).

VII ICOR III

SCIENTIFIC COMMITTEE

Steven D. Aust

(USA)

Wolf Bors

(W. Germany)

Gidon Czapski

(Israel)

Erich F. Elstner

(W. Germany)

Leopold FLohé

(W. Germany)

Klaus Gollnick

(W. Germany)

A. Michael Michelson

(France)

Giuseppe Rotilio

(Italy)

George Scholes

(England)

Volker Ullrich

(W. Germany)

CONTENTS

A - PHYSICO-CHEMICAL AND CHEMICAL PROPERTIES OF OXYGEN RADICALS (Chair: G. Scholes) Spectral and kinetic properties of the products resulting from the reactions of HO2/O2 with Mn(Il) complexes. B.H.J. Bielski, R.L. Arudi and D.E. Cabelli

1

New mechanism of the reaction of O5 with H2O2: significance for biology. I.B. Afanas'ev, N.S. Kuprianova and A.V. Letuchaia

17

Generation of activated oxygen species by electron t r a n s f e r reduction of dioxygen in the presence of protons, chlorinated hydrocarbons, methyl viologen and transition metal ions. D.T. Sawyer, XL. Roberts, T. Tsuchiya and G.S. Srivatsa 25 On reaction of KO2 with sterol hydroperoxides. N.M. Made Gowda and L.L. Smith

35

The O5 radical reactions in neutral and alkaline solutions. J. Holcman, K. Sehested, E. Bjergbakke and E.J. Hart

Ψ3

Reactivities of tert-butoxy radicals in aqueous solution. W. Bors, D. Tait, M. Erben-Russ, C. Michel and M. Saran

Ψ9

Formation and reactions of halothane peroxy f r e e radicals, CF3CHCIO2. 3. Mönig and K.-D. Asmus

57

Electronic and geometrical structures of ethylene peroxy cation and anion radicals: mechanisms of oxygenation reactions via electron transfers. K. Yamaguchi

65

ESR and Ε Ν DOR studies of f r e e radicals having unpaired spin density localized primarily on oxygen. H.C. Box and E.E. Budzinski

77

Corona discharge as a source of superoxide. H.C. Sutton

87

Simultaneous multi-wavelength kinetic spectroscopy: A new set-up for pulse radiolysis studies of oxygen radicals. M. Saran, G. Vetter, M. Erben-Russ and W. Bors

91

R a t e constants of sparingly water-soluble phenolic antioxidants with hydroxyl radicals. W. Bors, C. Michel, M. Erben-Russ, B. Kreileder, M. Saran and D. Tait

95

χ Α pulse radiolysis study of the addition of hydroxyl f r e e radicals to nitrone spin traps. R. Sridhar, P.C. Beaumont and E.L. Powers General discussion

101 105

Β - GENERATION AND REACTIONS OF INORGANIC AND ORGANIC RADICALS (Chair: E.F. Elstner) The reactivities of organic oxygen (oxy) radicals. M.G. Simic and E.P.L. Hunter

109

Autoxidative cytotoxicity: is there metal-independent formation of hydroxyl radicals? Are there "crypto-hydroxyl" radicals? D.C. Borg, K.M. Schaich and A. Forman

123

Role of oxygen activation in adriamycin-mediated DNA strand scission and the e f f e c t of binding on the redox properties of the drug. R.J. Youngman, F. Götz and E.F. Elstner 131 A comparative kinetic study of the initiation of lipid peroxidation with ·ΟΗ radicals and ferrous ion. L.H. Piette, L.H. Baxley, T.A. Grover and P.J. Harwood

137

Evidence for the initiation of lipid peroxidation by a ferrous-dioxygen-ferric chelate complex. S.D. Aust, J.R. Bucher and M. Tien

1Ψ7

The biochemical role of ubiquinone and ubiquinone derivatives in the generation of hydroxyl radicals from hydrogen peroxide. H. Nohl and W. Jordan

155

Oxidative metabolism of phenols: conversion of bromocatechol to protein binding species by O3. T. Wolff and R.J. Youngman

165

Generation of superoxide in the autoxidation of ascorbate and glutathione. A. Rigo, M. Scarpa, E. Argese, P. Ugo and P. Viglino

171

Role of metal chelating agents as catalysts in an -OH-forming process - a comparison with the Haber-Weiss reaction. H.C. Sutton and C.C. Winterbourn

177

Reaction of paraquat radicals with H2O2: Effect of O2 and comparison of radicals generated enzymatically and by irradiation. C.C. Winterbourn and H.C. Sutton

185

The chemistry and biochemistry of the radicals in cigarette smoke: ESR evidence for the binding of the tar radical to DNA and polynucleotides. W.A. Pryor, K. Uehara and D.F. Church

193

XI

Oxygen as an inhibitor in the radical-induced chain isomerisation of the (5-nitrofuryl)acrylamide, AF-2. I. Wilson, E.D. Clarke and P. Wardman

203

Generation of the superoxide radical in the red blood cell. M. Scarpa, A. Rigo, E.F. Orsega and P. Viglino

207

Superoxide degrading activity of desferrioxamine. J. Sinaceur, C. Ribière, J. Nordmann and R. Nordmann

211

Free radical production from the reaction of t-butyl hydroperoxide with iron complexes. P.J. Thornalley, R.J. Trotta and A. Stern

215

The e f f e c t of cell-bound copper on the toxicity of superoxide and vitamin C. 3. Aronovitch, D. Godinger, A. Samuni and G. Czapski

219

On the mechanisms of cytotoxicity induced by superoxide. G. Czapski, J. Aronovitch, D. Godinger, A. Samuni and M. Chevion 225 Microsomal denitrosation of N-nitrosodimethylamine. H. Kuthan, H.-J. Haussmann and J. Werringloer

231

C - ACTIVATED OXYGEN SPECIES IN FATTY ACID AND LIPID PEROXIDATION (Chair: S.D. Aust) Oxidation mechanisms of poly-unsaturated f a t t y acids. N.A. Porter, L.S. Lehman and D.G. Wujek

235

Preparation, isomerisation and breakdown of hydroperoxides from methyl oleate. W. Grosch and J. Megele

249

Photolysis of unsaturated f a t t y acid hydroperoxides. P. Schieberle, W. Grosch and J. Firl

257

Isomers of linoleic acid hydroperoxides from peroxidation of microsomal lipids. H. Frank, M. Wiegand, D. Thiel and H. Remmer

267

Oxidation of phosphatidylcholine and its inhibition by vitamin E and vitamin C. E. Niki, Y. Yamamoto and Y. Kamiya 273 Product distribution of unsaturated phospholipid oxidation in organic solvent and aqueous emulsion. L.S. Lehman and N.A. Porter

281

Involvement of activated oxygen species in membrane peroxidation: possible mechanisms and biological consequences. R.A. Floyd and M.M. Zaleska

285

XII

Hydroperoxides, free radicals and prostaglandin synthesis. P.J. Marshall, R.J. Kulmacz and W.E.M. Lands

299

Spin-trapping agents protect against microsomal lipid peroxidation. R. Sridhar, C.A. Crossley and W.W. Wise

309

Low-level chemiluminescence, alkane production and gluthathione depletion in isolated hepatocytes caused by a diffusible product of lipid peroxidation, 2 with NADH if you "stretched it out", as you said. But not so much when it reacted with HO2. What do you mean by "stretched"? BIELSKI: Free NADH in solution exists in two helical configurations which are in equilibrium with a co-planar form (Sarma and Kaplan, ( 1 9 7 0 ) , Biochem. 9, S39), while when bound to lactate dehydrogenase it is attached in an uncoiled configuration or in a stretched-out form (Chandrasekhar e t . a l .

( 1 9 7 3 ) , J. M o l . B i o l . 7 6 ,

503) .

NEW MECHANISM OP THE REACTION OP 0* WITH HgOgj SIGNIFICANCE POR BIOLOGY

I.B.Afanas'ev, N.S.Kuprianova, A.V.Letuchaia All-Union Research Vitamin Institute, 117246, Moscow, USSR

Introduction Recently, much attention has been given by biochemists to the mechanism of the interaction of superoxide ion with hydrogen peroxide. It is due to the fact that this reaction may represent a mechanism by which 0| results in lipid peroxidation. In 1970 Beauchamp and Fridovich (1) proposed that 0 2 production by the action of xanthine oxidase on xanthine leads to the formation of hydroxyl radicals by the HaberWeiss reaction 0* + H 2 0 2 0 2 + HO· + HO" (1) Later it was confirmed (2) that is indeed a precursor of hydroxyl radicals in biological systems, in spite of the fact that the rate of reaction (1) is small (3)· Therefore, the catalytic mechanism of the Haber-Weiss reaction with the participation of ferric ions (or their complexes) became generally accepted (2,4): 0£ + Pe 3+ »- 0 2 + Pe 2+ (2) 2+ 3+ Pe + H202 • Pe + HO· + H0~ (3) However, in spite of the previous extensive discussion (2,4), the mechanism of the interaction of 0 2 with H 2 0 2 remains obscure. Recently, it has been shown (5,6) that 0 2 reacts in aprotic media with even weaker acids than HgOg, such as water and alcohols, by splitting off a proton with a rate constant of (O.5-3.5)·10~ 3 M -1 s" 1 . Therefore other routes of interaction of Ol with H o 0 o must be considered. In this work we

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

18

1 »2,3.4.5,6,7 - Absorption spectrum 1.3.6,9,40,43* and 46 min after mixing the reactants in AH. [Oil = 5.44 ΊΟ - - 3 Μ,ΓΗ„0 ο 1.= 0.0302 Μ, ΓΗ„01 = 0.147 M. 8 - AbskrjttSon spectrum of L ¿ ¿ J ° 0* in AH. ι ¿ -i0 investigated the reaction of electrochemically generated Og with HgOg in aprotic media (acetonitrile, AH, and dimethylformamide, DMF) using a procedure described earlier (7)·

Results The reaction of superoxide ion with hydrogen peroxide was carried out at room temperature in the cell of a Gary 219 spectrophotometer. Mixing the OT, and ^ O g >p2]o

^isappearence

solutions at

[H2O2J o

superoxide ion band and to

a sharp increase in the optical density at 200-270 nm (Fig.1). Then, in the AH solution the optical density in this region decreased, and two maxima at 212-215 nm and 249 nm appeared (in DMF these maximη cannot be observed owing to the strong solvent absorption at

λ < 255 nm).

After the reaction was completed, the optical density at 220250 nm became negative. This indicated a sharp reduction of the HgOg concentration, because the spectra were recorded against an original solution of HgOg· Indeed, the titration of the final solution by 0.1 Ν solution of KMnO^ confirmed a more than twofold decrease in the H 2 0 2 concentration. When the reaction was carried out at

Γ°2] 0 > ΓίΙ2°2ΐο'

°2

19

D

0.8

1

2 0.7

3 4

0.6 0.5

200 220 240 260 280 nm Figure 2 1,2,3,4 - Absorption spectrum 1,5,10, and 15 min after mixing the reactants in AN. ΓΟΓΙ = 3.18·10-3Μ, ΓΗο0ο1 = 1.82·10~3Μ, JH201 = 8.82·10-3 Μ . ι 2jo [ 2 2jo 5 - Absorption spectrum of H00~ in AN. ma-ri m im at 249 nm slowly decreased, and the maximum at 210220 nm appeared (Pig.2). A maximum at 249 nm which is seen on Fig.1 corresponds to the 0g maximum, i.e. superoxide ion is formed during subsequent stages of the reaction. A maximum at 212 nm seems to belong to hydroperoxide ion, H00~, as the same spectrum was obtained by mixing the AN solutions of HgOg and NBu^OH (Pig.2). The spectrum of the reaction mixture changed drastically when catalytic quantities of Fe"^+ ions were added. As is seen from Pig.3, the spectrum recorded immediately after mixing the reactants corresponded to the final stage of the reaction in the absence of F e r i o n s where the maximum at 212-215 nm belonging to hydroperoxide ion already became apparent. Moreover, in contrast to the small increase in superoxide concentration during the reaction in the absence of Fe^ + ions (Curves 2 and 3 in Fig 1), the OZ concentration increased 3+

considerably when Fe^

was present (Curves 1 and 2 in Fig.3)·

Discussion Thus, our experiments prove that superoxide ion does react with hydrogen peroxide in aprotic media. Recently, an analogous conclusion was made by Ozawa and Hanaki (8), but they believe that the reaction proceeds via the Haber-Weiss

20

Figure 3 1,2,3 - Absorption spectrum 1,3, and 6 min after mixing the reactants in AN in the presence of Fe^+ ions. ^OgJ0=4.17*1 M, |^H202Jo=0.0445 M, p 2 0j Q =0.293 M, [Fe2(SC>4)3] Q=1.2-10"4 M. mechanism. However, the appearance of a strong absorption at 200-270 nm immediately after the reactant mixing owing to the H00~ formation seems to indicate that the interaction of 0 2 with HgOg occurs via deprotonation and that reaction (1) must be unimportant. We found that H 2 0 2 was consumed in considerably greater quantities than 0 2 . This fact and the increase in the 0 2 concentration during the reaction point out a chain mechanism which can be represented by reactions (4)-(6) 0* + H 2 0 2 — H00· + H00~ (4) H00· + 0 2 • H00" + 0 2 (5) H00" + H 2 0 2 0 2 + HO· + H 2 0 (6) The possibility of reaction (6) proposed by Roberts et al.(9) was confirmed by the results obtained in the gas phase (10) where a fast reaction was observed H00~ + H 2 0 2 — H 0 ~ + H 2 0 + 0 2 (7) Reaction (7) appears to include stages (6) and (8) 0 2 + HO0 2 + HO" (8) The experiments which were carried out in the presence of Fe^+ ions confirmed that Fe^+ can catalyse the interaction between 0 2 and H 2 0 2 · Indeed, as is seen from Fig.3, rates of the H00" decomposition and the 01 formation essentially

21

increase in the presence of

ions. Therefore we propose

that reactions (9) and. (10) take place Pe3+ (Fe00H)

2+

+

H

2

+ 0

2

(Fe00H) 2 +

H00~ — - 0* +

HO· +

Pe

3+

+

(9) HgO

(10)

Thus, the interaction of 0 2 with HgOg may lead to a chain reaction with the formation of HO· both in the presence and. in the absence of F e ^ + ions. As the rate of this process can be much greater than that of the disappearance of 0 2 , the interaction of 0^ with HgOg m a y initiate lipid peroxidation. In addition to Η0· , this reaction leads to the formation of H00~ and (FeOOH) 2 + . In view of recent work (11,12), both of theee species may be considered as reactive intermediates in lipid peroxidation. The possibility of generating hydroxyl radicals in the reaction of H 2 0 2 with other reductants should be examined Red

+

H202

Ox

+

HO'

+

HO"

(11)

Ascorbate (AH 2 ), NADH, NADPH, etc. have been discussed recently (13) as possible participants in reaction (11). It has been suggested that these compounds can be more important reductants than 0 2 , as they are present in the cells in considerably greater quantities. But it is difficult to agree with that, as 0 2 has the lowest redox potential among these compounds E}(0 / o p

= (-0.11) -

(-0.15)V (14), Β|(ΑΗ·/ΑΗ 2 ) = 0.30V (15), Εγ(NAD·/NADH)= O.3OV (16), and therefore the rate of reaction (11) for A H 2 and NADH must be smaller even than the rate of reaction (1) which, as is known (3), is very small. Therefore, the direct electron transfer from such reductants to HgOg via reaction (11) seems to be very improbable.

22 References 1. Beauchamp, C.O., Fridovieh, I.: J.Biol.Chem. 245» 46414646 (1970). 2. Kellogg, E.W., Fridovich, I.: J.Biol.Chem. 252, 6721-6728 (1977); Diguiseppi, J., Fridovich, I.: Arch.Biochem. Biophys. 20¿, 323-33O (1980); Gutteridge, J.M.C., Rowley D.A., Halliwell, B. : Biochem.J. 206, 605-609 (1982); Rosen, Η., Klebanoff, S.J.: Arch.Biochem.Biophys. 208, 512-519 (1982). 3. Weinstein, J., Bielski, B.H.J.: J.Am.Chem.Soc. 101, 5862 (1979). 4· Richmond, R., Halliwell, B., Chauhan, J., Darbe, Α.: Anal.Biochem. _1_18, 328-330 (1981); Floyd, R.A. : Biochem. Biophys.Res.Commun. 99, 1209-1216 (1981). 5· Nanni, B.J., Stalling, M.D., Sawyer, D.T.: J.Am.Chem.Soc. 102, 4481-4485 (1980). 6. Afanas'ev, I.B., Kuprianova, U.S.: Int.J.Chem.Kinet. 15, 000 (1983). 7· Afanas'ev, I.B., Polozova, N.I., Samokhvalov, G.I.: Bioorgan.Chem. 434-439 (1980). 8. Ozawa, T., Hanaki, Α.: Chem.Pharm.Bull. 29, 926-929(1981). 9· Roberts, J.L., Morrison, M.M., Sawyer, D.T.: J.Am.Chem. Soc. 100, 323-330 (1978). 10. Mead, R.D., Schulz, F.Α., Lineberger, W.C.: J.Am.Chem.Soc. 103, 6262-6264 (1981). 11. Adams, P.A., Berman, M.C.: J.Inorg.Biochem.17, 1-14 (1982) 12. Tien, Μ., Svingen, B.A., Aust, S.D.: Federation Proc. 4 0 , 179-183 (1981). 13. Halliwell, B.: Biochem.J. 205, 461-462 (1982); Winterbourn, C.C.: BiochemTJT 205, 463 (1982). 14. Afanas'ev, I.B.: Usp.Khim. 48, 977-1014 (1979). 15. Steenken, S., Neta, P.: J.Phys.Chem. 86, 3661-3667 (1982). 16. Farrington, J.Α., Land, E.J., Swallow, A.J.: Biochim. Biophys.Acta 590, 273-277 (1980); Anderson, R.F.: Biochim.Biophys.Acta 590, 272-273 (1980).

23 DISCUSSION

SCHOLES: The Haber-Weiss reaction has been talked about for years, 50 years now, I guess. To what extent do you think the anion of the hydrogen peroxide will react with hydrogen peroxide in a normal aqueous environment. You are looking at this in an aprotic solvent - in acetonitrile. What happens when you have water around? SAWYER: Possibly, I can answer that. The anion of hydrogen peroxide does react with hydrogen peroxide, but very slowly. The rate constant is, I believe, 10 - 8 M - 1 s - 1 . The data also is fairly old, which may mean that metals were present. One can only speculate whether it goes by the same mechanism or not because the rate constants are so small. All we know is that base causes hydrogen peroxide to decompose slowly. JONES: If I could just add to that, I think the comments referred to the old DUKE and HAAS data (J. Phys. Chem. (1961) 65, 304). In fact EDWARDS and coworkers (J. Am. Chem. Soc. (1963) 85, 2263) have shown, that if you clean up the reagents and add BOTA to the system, the reaction disappears entirely, so that there is no evidence, I think, in aqueous solution for direct reaction of HO2 with H2O2. On the other hand, there is evidence for the reaction of the anions of peroxy acids with peroxy acid molecules - this is also mainly work of EDWARDS. But in that case there is no evidence of radical formation. The products, oxygen and carboxylic acid, seeme to arise entirely from a bimolecular reaction. AFANAS'EV: I would like to say that I think it is not very important what the rate of this reaction is in biological systems, because in the cell the superoxide ion always exists. These reactions can be very slow, but they can be pathological. CZAPSKI: You showed that in the earlier reaction iron may play a role in catalyzing the effect. In your last reaction, reaction /II/, ascorbate together with hydrogen peroxide produces OH radicals. I think there is good evidence in many biological systems, whole cells and also in DNA damage, that ascorbate causes this deleterious effect, apparently through OH radical. Yet this is catalyzed by metal ions and can be blocked in the biological systems adding chelating agents. I think that also in acetonitrile there is a very fair chance that reaction /II/ may be catalyzed by metal ions. AFANAS'EV: I don't believe that the direct electron transfer from ascorbate to hydrogen peroxide is possible. It can be seen from comparison of one electron reduction potentials of the reactants. WABNER: Could you give some information about your electrochemical method of producing 05? AFANAS'EV: It is an electro-chemical cell with two electrodes, platinum and mercury. We obtained very stable solutions of superoxide ion using tetrabutylammonium Perchlorate as a supporting electrolyte.

GENERATION OF ACTIVATED OXYGEN SPECIES BY ELECTRON-TRANSFER REDUCTION OF DIOXYGEN IN THE PRESENCE OF PROTONS, CHLORINATED HYDROCARBONS, METHYL VIOLOGEN, AND TRANSITION METAL IONS. Donald T. Sawyer, Julian L. Roberts, Jr., Tohru Tsuchiya, G. Susan Srivatsa Department of Chemistry, University of California Riverside, California 92521, U.S.A.

The reaction of ground-state dioxygen (^02) with spin-paired diamagnetic species is limited because triplet-singlet processes are spin-forbidden.

Activation of dioxygen, which in

the present context means to make more reactive, requires a change in the spin multiplicity of

O2-

The most dd.rect

means to accomplish this is by the one-electron reduction of dioxygen to 0- (or HO,· in the presence of proton sources).''"' 3 4 Recent reviews ' confirm that superoxide ion is a versatile reactant in aprotic solvents, which can act as (a) an effective BrgSnsted base (able to abstract protons from weak oxygen and nitrogen acids (pK& 2 is unit molarity). These data indicate that + 1. 20 +0.70

02

-0.16, ~ w - x o ) 02~

+0

·89

Ί

> H 2 O 2 +°- 5 1 > H 2 0 + -OH i I

+0.36

+2

· 1 8 > 2H 2 0

(1)

+1.35

+ 0.41

+0. 86 the most limiting step is the first electron-transfer to 0 2 , and that an electron source adequate for the reduction of 0 2 will produce all of the other reduced forms of dioxygen via

27

reduction, hydrolysis, and disproportionation steps. -0.16 V, °2

+

6

HO,

CO.

H2O

H

HO.

2°2'

(2)

•OH, OH )

When 0 2 is electrochemically reduced in aprotic media (e.g.., dimethylformamide

(DMF)), the process yields a stable solu-

tion of superoxide ion by reversible electron transfer 1).

(Figure

The presence of proton sources such as E^O in DMF causes the 0 2

to decompose via a

proton-induced disproporg tionation. Superoxide is reduced by a second electron

4:1 PHOH-.O, I: 1 HCHVO* 4.8 mM Ot 7\ \ f Aj

at -1.7 V vs. NHE (eq. 3). Figure 1 also illustrates

: t /

/V yι

Ί

the effect on the 0 2 reduc-

•'// if ι

tion process of a 1:1 mole

τ ΜμΑ 1 1 1V I 1 0 -0.5 -1.0 E,V «. SCE

1

ratio of (H 3 0)C10 4 relative to 0 2 , and of a 4:1 mole ratio of Ph0H:0 2 ·

The

latter system drives the

Figure 1• Cyclic voltaramqgrams in DMF (0.1 M TEAP) of 0 2 at a Pt electrode (0.033 cm 2 ); scan rate, 0.1 V s - 1 .

reaction of eq. 3 to completion by a post electrontransfer proton-induced disproportionation to give

an overall two-electron reduction of 0 2 to H 2 0 2 ·

However, in

the presence of strong acids the primary process is shifted to more positive potentials to give H0 2 · as the primary product (eq. 4). °2

-0.60

+

-1.7 V h2O

V

HO,

+ OH (3)

h2o 1/2 H 2 0 2 + 1/2 0 2 + OH °2

+

h3o

+ e

+0.12

V.

HO,

h2o (4)

k >10 7 M

1

s

1

> 1/2 H 2 0 2 + 1/2 0 2

28 A previous study® has shown that addition of base to aprotic solvents promotes a rapid disproportionation reaction _ 2H 2 0 2 + OH

1, 11U η

_ 02

+ 2H 2 0 + "OH

(5)

Thus, as in the case of aqueous media, the one-electron reduction of 0 2 yields significant fluxes of several reactive oxygen species via facile hydrolysis and disproportionation reactions (see eq. 2). Reductive Activation of 0 2 in the Presence of Halogenated Hydrocarbons and Carbonyl Compounds.

In aprotic media 0~ 2 10

rapidly reacts with chlorinated hydrocarbons ' compounds

and carbonyl

via nucleophilic addition and displacement reac-

tions to form peroxo radical intermediates.

Subsequent reduc-

tion, disproportionation, and auto-catalytic steps yield reactive peroxides and oxygenated products. of C>2 to 0 2

Thus,'reduction

in the presence of excess quantities of these

co-factors yields reactive intermediates.

Reaction schemes

for O- with butyl chloride (BuCl), carbon tetrachloride (CC14), phenyl benzoate [PhC(0)0Ph], and benzil [PhC(O)C(O)Ph] are outlined by the following sets of equations. (a)

BuCl + 0 2 + e~

BuOO· + Cl -

(6)

BuOO · + e~

-

(7)

BuOO

BuOOBu + CI -

BuOO~ + BuCl (b)

CC1 4 + 0 2 + e

-

Cl3COO· + e~

(8)

-+• Cl3COO· + Cl

-

(9)

Cl 3 COO -

(10)

Cl,COO~ + CCI. + Cl,COOCCl, + cl 3 4 3 3

-

(11)

Cl 3 COOCCl 3 + 2e~ + 2[C1 3 C0 _ ] + 2C1 2 C=0 + 2C1(c)

PhC(0)OPh + 0 2 + e PhC (0)00· + e"

(d)

-

+ PhC (0)00· + PhO

-

(13)

PhC (0) 00 -

PhC (O) C (0) Ph + ¿0_ + e

ι PhCC(0)Ph ι 0-0·

(12)

(14) > 2PhC(0)0

(15)

29 Reductive Activation of

in the Presence of Methyl Viologen,

Hydrazines, Ascorbic Acid, and Catechols in Aprotic Media. The electron-transfer reduction of 0 2 in the presence of excess quantitites of these co-factors results in coupling reactions with 0_

to give reactive intermediates. Reaction 2+12 schemes for methyl viologen (MV ), Phenylhydrazine (PhNHNH 2 ), 2 1,2-diphenylhydrazine (PhNHNHPh),2 ascorbic acid (H 2 Asc), 13 and 3,5-di-tert-butylcatechol lined:

(DTBCH2)6 are out-

Scheme (a) represents a rare example of a radical-

radical coupling reaction (0„

+ MV*) that results from the 2+ . Schemes (b)

concerted one-electron reductions of 0 2 and MV

(c), and (d) are examples of hydrogen atom abstraction by superoxide ion, while Scheme (e) illustrates the ability of 02

to act as a base and effective one-electron oxidizing

agent via the use of substrate protons to stabilize (a) M V 2 + + 0 2 + 2e~ ->- M V + — 0 0 ~

(b)

M V + — 0 0 ~ + Me2SO •+ [MVO] + Me2SC>2

(17)

PhNHNH 2 + C>2 + e~ •+ [PhN=NH] + 0H~ + -OH

(18)

[PhN=NH] + 0 ~

(19)

Ph· + N. + HO-~ PhH

1/2 PhPh

(c)

PhNHNHPh + 0 2 + e~ ->• PhN=NPh + 0H~ + · OH

(d)

H 2 Asc + 0 2 + e~ 2 [Asc~ ] + H 2 0 dehydroAsc

(e)

(16)

[Asc7] + H 2 °2 HAsc" + dehydroAsc

(21) + 0H~

+ 0 2 + 2e~ ->- threonate + HC 2 0 4 ~

DTBCH 2 + 0 2 + e~

(20)

DTBSQ~ + H 2 0 2

2DTBSQ~ + H O î DTBQ + DTBCH~ + OH~

(22) (23) (24) (25)

Reductive Activation of 0 2 in the Presence of Metal Ions in Aprotic Media. Reduction of 0_ in the presence of excess zinc II 2+ cation [Zn (bipy)2 ], tetraphenylporphinatoiron(III) ion , and cuprous ion (Cu1) results in formation of

30

reactive metal-superoxo and -peroxo species.

+ 1.0

0

-1.0

-2.0

E , V vs. SCE

Figure 2. Cyclic v o l t a m m o g r a m s in DMF (0.1 M TEAP) of (a) 0 , (b) F e X I I T P P C l , (c) C>2 + and (d) O + FeIIlTPPCl, FelllTPP(N-Melm)+. Measurem e n t s were m a d e with a glassy carbon electrode (area, 0.11 cm ) at a scan rate of 0.1 V s temperature, 25°C.

+2.0

+1.0

Figures 2 and 3

0.0

-1.0

-2.0

E, V vs SCE Figure 3. Cyclc v o l t a m m o g r a m s in M e C N (0.1 M TEAP) of (a) Ο , (b) C u x ( M e C N ) 4 C I O 4 , (c) 0 2 + CuI(MeCN)4C104Measurements were made with a platinum electrode (area, 0.23 cm^) at a scan rate of 0.1 V temperature, 25°C.

illustrate the cyclic voltammograms for (Fe 111 !??)CI and Cu1(MeCN)4CIO4,

respectively, in the absence and in the

presence of 0,.

Reaction schemes for the three metal-0~

31

systems are outlined. (a)

Zn 1 1 (bipy)

+

+ C>2 + e~ "°·5 V > [Zn11 (bipy) 3 00-] +

(26)

[ZnII(bipy)300-]+ + e" - Zn 11 (bipy) 3 (0 2 ) (b)

XI

Fe TPP + 0 2 + e" Ρ β Ι:[Ι τΡΡ +

+

°^Fe

ο, + 2e~ 2

ν „ DMF, Me- Im

Il:C

(27)

TPP"

(28)

o^FeinTpp^y

^

1/2 TPPFe III -0-Fe III TPP + H 2 0 2 + OH~ (c)

CuI(MeCN)4 C10 4 + 0 2 + e" l:C

Cu

1

(0-) + e" " · Δ

35 V

~°' 35

V

a CuI:C(02)

I

> Cu (0.)" £.

(29)

2

Cu^^O + 2 OH

(30) -

(31)

Cul:E0 + e" "¿'β ν » cu 1 (OH)-2

(32)

Cul:C(02)

(33)

+2,1 V

> Cu 1 1 + 0 2 + 2e~

2CU1 + 0 2 + 2e °· 25 V > Cu I OOCu I 1 c^oocu [CuII00CuII]2+ + 0 . 05 V

2Cu^O 2e-

(34) (35)

Discussion and Conclusions 7 Recent discussions suggest that the oxygenase enzymes bind oxygen only in the presence of substrate to form the ternary complex, ESC>2, with concommitant activation of the dioxygen for oxygenation of the substrate, either by an ionic mechanism (interaction of metal-ion unpaired spins with bound dioxygen to change the O 2 spin state) or by a radical mechanism that involves the reduction of by the reduced form of some 14 co-factor. Both of these mechanisms are consistent with the observation that every oxygenase has one or two characteristics: (a) The active site involves a transition metal ion (usually iron or copper), and (b) the enzyme has a cofactor or substrate whose reduced state loses an electron or the equivalent of a hydrogen atom to give a resonance stabilized free radical.

32

The present results illustrate that the reduction by electron transfer of 0 2 in the presence of protons, organic co-factors and transition-metal ions yields reactive radical species [H02·, -OH, ROO·, RC(0)00·, and (Ζη ΙΙ 00·) + 1, organo-peroxides (ROO-, RC (0) 00~, and MV + -00~), and metal-peroxides [FeIl;CTPP(02) , Cu I I (0 2 ), and Zn^fC^)].

Hence, activation of oxygen

is achieved by its reduction in the presence of co-factors that are reactive with superoxide ion.

In the case of

transition-metal co-factors the resultant metal-peroxides are 14 analogous to the reaction intermediates of oxygenases, and the reductive mechanism for activation appears to be similar to that for the oxygenases.

The investigations have focused

on the reduction of dioxygen in the presence of several cofactors that are especially reactive with O^ -

If the co-

factor is also electroactive, then an effective two-electron activation is accomplished by two concerted one-electron < ) reductions (e.£. , Fe l:CI TPP + + 0 2 + 2e~ j ^.FeIIITPP~) , which is similar to the mechanism for cytochome P-450. rich variety of oxy- and peroxy-radicals

The

and other active

forms of oxygen that are produced by reductive activation of 0 2 also provides insight to the mechanism of oxidases and for the auto-oxidation of organic substrates.

Acknowledgment.

This work was supported by the National

Science Foundation under Grant No. CHE-8212299.

33 References 1.

Sawyer, D.T., Nanni, E.J., Jr.: "Oxygen and Oxy-Radicals in Chemistry and Biology," Rodgers, M.A.J., Powers, E.L., eds.; Academic Press (1981); pp. 15-40.

2.

Sawyer, D.T., Nanni, E.J., Jr., Roberts, J.L., Jr.: in Adv. in Chem. Series, No. 201, Kadish, K.M., ed. (1982); pp. 585-600.

3.

Sawyer, D.T., Valentine, J.S.: Acc. Chem. Res., 14, 393 (1981).

4.

Sawyer, D.T., Roberts, J.L., Jr., Calderwood, T.S., Tsuchiya, T., Stamp, J.J.: in "Oxy Radicals and Their Scavenger Systems, Vol. 1, Molecular Aspects," Cohen, G., Greenwald, R.A., eds; Elsevier Biomedical, New York; 1983; pp. 8-19.

5.

Borg, D.C., Schaich, K.M., Elmore, J.J., Jr.: in "Oxygen and Oxy-Radicals in Chemistry and Biology," Rodgers, M.A.J., Powers, E.L., eds.; Academic Press (1981); pp. 177-195.

6.

Nanni, E.J., Jr., Stallings, M.D., Sawyer, D.T.: J. Am. Chem. Soc., 102, 4481 (1980).

7.

Nozaki, Μ., Yamamoto, S., Ishimura, Y., Coon, M.J., Ernster, L., Estabrook, R.W., eds.: in "Oxygenases and Oxygen Metabolism," Academic Press, New York (1982).

8.

Chin, D.-H., Chiericato, G., Jr., Nanni, E.J., Jr., Sawyer, D.T.: J. Am. Chem. Soc., 104, 1296 (1982).

9.

Roberts, J.L., Jr., Morrison, M.M., Sawyer, D.T.: J. Am. Chem. Soc., 10£, 329 (1978).

10.

Roberts, J.L., Jr., Sawyer, D.T.: J. Am. Chem. Soc., 10 3, 712 (1981).

11.

Gibian, M.J., Sawyer, D.T., Ungermann, T., Tangpoonpholvivat, Morrison, M.M.: J. Am. Chem. Soc., 101, 640 (1979).

12.

Nanni, E.J., Jr., Angelis, C.T., Dickson, J., Sawyer, D.T.: J. Am. Chem. Soc., 103, 4268 (1981).

13.

Sawyer, D.T., Chiericato, G., Jr., Tsuchiya, T.: J. Am. Chem. Soc., 1£4, 6273 (1982).

14.

Hamilton, G.A., Reddy, C.C., Swan, J.S., Moskala, R.L., Mulliez, Ε., Naber, N.: in "Oxygenases and Oxygen Metabolism," Nozaki, Μ., et al., eds., Academic Press, New York (1982); pp. 111-123.

15.

Ullrich, V., Castle, L., Haurand, M.: in "Oxygenases and Oxygen Metabolism," Nozaki, Μ., et al., eds., Academic Press, New York (1982); pp. 497-509.

34 DISCUSSION SINGH: Two brief questions: (i) how sure are you that OH is formed from 05 + 1.2-diphenylhydrazine, and (ii) can some of the reactions which you have attributed to 05 be due to direct electrolysis of substrates? SAWYER: 1.2-Diphenylhydrazine plus superoxide in an 0 2 -saturated solution gives by all chemical criteria azobenzene, there is no question about that. The stoichiometry has been worked out by doing a titration. Although I showed electrochemical experiments, we can synthesize, either electrochemically or chemically, stable superoxide solutions. So there is no electrochemistry and therefore no chance for so-called side-electrochemistry to go on. To the second question: our most recent results do not support the idea that we produce any free OH radical. When oxygen is excluded and superoxide and 1.2-diphenylhydrazine are combined, an anion radical is formed. The latter produces azobenzene upon exposure to O2. AFANAS 1 EV: You give the extinction coefficient for superoxide ion as about 6000 in dimethylformamide. It is very strange, because it is 2500 in water, and we found the same value in dimethylformamide. What do you think about it? And what about the strong absorption of dimethylformamide at 253 nm? SAWYER: I think the molar absorptivity data was not for dimethylformamide but for acetonitrile, because we are aware of the problem that most aprotic solvents absorb where superoxide ion does. We cannot explain why we get such a large value and all of the previous values are lower. With chemically synthesized tetramethylammonium superoxide, which we have carefully purified and assayed, the molar absorbtivity in acetonitrile approaches 10.000, which is four times BIELSKI's value in water. When we dilute this material in 90% water and 10% acetonitrile it is still about 8.000. I don't know the explanation. The problem of trace levels of metals is formidable and it is conceivable that the other studies may not have been as successful in getting rid of the last vestiges of trace metals.

ON REACTION OF K O , WITH STEROL

N.M. Made Gowda and Leland L.

HYDROPEROXIDES

Smith

Division of B i o c h e m i s t r y , University G a l v e s t o n , TX 77550 U.S.A.

Reduction of

of Texas Medical

organic h y d r o p e r o x i d e s

model for the

(ROOH) by

related reaction b e t w e e n H2O2 and 0 2 ~

now viewed

as proceeding by the process of Eq. 1.

reactions

(Eq. 2-4) are not supported ROOH ROOH

+

ROOH

+

ROOH ROO" + Product

o2-

+

+

in

posed

as

(1,2)

is

Alternative

experimentally experimentally

+ HOO ·

*· RO · + H O

°2~ o2-

°2_ (CH3)2S0

alcohols

ROO

Branch,

-

+

(3-6) .

Eq. 1 °2 02

Eq. 2

>-

R0- + HO · ROO • + HOO-

Eq. 4

>"

RO~ +

Eq. 5

•

the p o p u l a r

+

(CH3)2S02

system

Eq. 3

of K O j - c r o w n

ether-

(CH ),S0 are then derived by subsequent reaction of ROO 3 with solvent (Eq. 5) and protonation of RO~, with observed 0 induced O ^ -

liberation a matter of proton

2

disproportionation

(3,4) . In attempt cies

to use cholesterol

(7) implicated

examined

the KO^

hydroperoxide the

reduction of

absence of

cholesterol

(IIb) were p r o d u c t s , being

1:1.

from IIa

oxygen

and

detected;

and

spe-

system

we

stoichiometry

(III)

a minor one from la. thus, cholesterol

(IIa) .

stenediols

In 5a-

cholest-5-ene-3 β, 7α-diol determined

With added cholesterol

3ß-hydroxycholest-5-en-7-one

were

7a-hydroperoxide

only corresponding

(lb)

reaction

ether-(CH^)^SO

3ß-hydroxy-5a-cholest-6-ene-5-

(la) and cholesterol

cholest-6-ene-3β,5-diol

metrically

to intercept active oxygen

in the KO^-crown

(1-10 equii/ . ),

was also a major

product

No other oxidized did

not intercept

species in these cases. The same results were

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

iodo-

sterols active

obtained

36

ΗΟ^^οΓ^

OR la.

R =

Ib.

R = H

OH

Il a . R =

H O - ^ ^ ? "

OH

III.

Il b . R = H

"(O), «»η-

using that

IV a. η = 2

ν a. η =

IV b. η = 1

V b. η = 1

other bases strong b a s e

Other

sterols

instead

of KC>2

in air yield

also a f f e c t e d

Cholesterol

homologues

sitosterol)

caused

(Table

2

1),

0 ~ in these

it being systems

the c o u r s e of the

stigmasterol

III f o r m a t i o n ,

recognized

(3,5,8) .

IIa

reductions.

and b r a s s i c a s t e r o 1

cho 1e stan-3 β - o 1 , 5 β - C h o l e s t a n - 3 β - ο 1 , and c h o l e s t e r o l T r a c e s of c h o l e s t a - 3 , 5 - d i e n - 7 - o n e tered tate

in some

c a s e s , and

occurred.

4-en-3ß-ol

re

trast,

Reductions

in which

in

duct a l c o h o l s tem with slower was

A d d i t i o n a l l y , both

duction

cholesterol,

added

times without

from III were

only

c h o l e s t e r o l , but by added

la and

small

regard

by all

amounts

reactions

of

sterols

of III w e r e

gave

the

in the in

C,H. 6

6

ot a b s e n c e

IIa gave

same

(CH^J-^SO were

Reduction

III was p r e s e n t

to the p r e s e n c e

Reaction

3ß-ace-

IIa o x i d i z e d c h o l e s t -

cholesterol.

after 6 d a y s , b u t

(no lib d e t e c t e d ) .

encoun-

as a s e p a r a t e m a t t e r . By

of la w a s u n a f f e c t e d case

not

3ß-acetate.

of c h o l e s t e r o l

of la and IIa by KO2 6 6 and 7 - k e t o n e III as o b t a i n e d

and u n a f f e c t e d

incomplete

derived

some h y d r o l y s i s

to cho1e s t - 4 - e n - 3 - o n e

(but

as did chole s t - 4 - e n - 3 β - o 1 , S a -

IIb and

of

from

consave had. prosysmuch

of

la

early

cholesterol

III with

or

37 Table

1. R e a c t i o n s

No. Reaction

of Sterol

System

Hydroperoxides

Products, No cholesterol-

Products j With cholesterol-

From

From

la From

IIa

la

From

d lb ,I I I -

IIa

1 .

κ ο 2 , (CH 3 ;, 2 S 0

lb-

2 .

κο2.

6 6 (CH 3 ) 2 s o

lb,Ill- IIb,Ill-

lb. I I I ^

IIb,Ill-

No rx .

No rx .

NO rx .

No rx .

C H 6 6 KOH , (CH 3 ) 2 S 0

No rx . C,h Ib-'-

No rx . C,h IIb— —

NO rx .

No rx .

5. 6.

KCl , (CH 3 ) 2 S 0

No rx .

7 .

H 2 0 , (CH 3 ) 2 S 0

No rx .

3. 4.

C

H

Ilb^

IIb,III

-

-

No rx .

-

-

No rx .

-

-

ji R e a c t i o n c o n d i t i o n s : ImM R O O H , I m M KO^ (or other c o m p o n e n t ) i n 0.3M c r o w n e t h e r , r o o m t e m p e r a t u r e , 4 5 m i n . TLC a n a l y s e s with C g H g - e t h y l a c e t a t e (17:8) and ( 7 : 1 ) , d e t e c t i o n by UV l i g h t , Ν , N - d i m e t h y 1 - p - p h e n y l e n e d i a m i n e for R O O H , S 50% Η 2 3 0 ^ . All i d e n t i t i e s c o n f i r m e d by i s o l a t i o n and m . ρ . , i n f r a r e d s p e c t r a , and a d d i t i o n a l c h r o m a t o g r a p h y , b Same c o n d i t i o n s as in a b u t held for 22 h . c^ Same p r o d u c t s w i t h o u t crown ether but at slower rate. ^ Found at low l e v e l . ^ R e a c t i o n i n c o m p l e t e at 22 h . f_ R e a c t i o n i n c o m p l e t e at 6 d a y s , g R e a c t i o n c o m p l e t e at 3 d a y s . h Same p r o d u c t s with N a O H , K O t B u , o r Na O instead of KO .

without The

cholesterol,

7 - k e t o n e III

sterols in these ments

II

thermal

experiments,

after

decomposition

(9,10), but t h e r m a l p r o c e s s e s

the

as no III was

same w a y . M o r e o v e r ,

experiments

from

cholesterol

added

with K 0 2

of

is a m a j o r

complete

found

III was not

IIb as s u g g e s t e d as

interceptor.

3 days. product

do not a c c o u n t

in other

formed cases

Cholesterol

of

for

in c o n t r o l

(IIa w i t h o u t K O 2 or IIa with KO., but w i t h o u t

treated

The

reaction being

III

experi-

cholesterol) in

control

(11)

or

does not

from

react

(12) .

7 - k e t o n e III is also a m i n o r t h e r m a l sterols

I,but as m o r e p r o m i n e n t

ducts c h o l e s t a - 4 , 6 - d i e n - 3 - o n e 4,6-triene

and

lib

from

lb

and

decomposition

thermal lib

(9,10) were

from

product

decomposition la and

pro-

cholesta-2,

not d e t e c t e d ,

thermal

38 processes do not account for III from la.

Thus,

III does not

arise directly from substrates la and Ila or products lb lib but must derive from undetected

intermediates

and

implicated.

On general chemical principles, III is viewed a s a

product of

termination reactions of radicals such as 3 β-hydroxycho1 est-5 ene-7a-peroxy1

(IVa)

or 3ß-hydroxycholest-5-en-7a-oxy1

radicals derived from the anion ROO (Eq.l).

Oxygen radicals not HOO-

of IIa formed

(IVb)

initially

have been demonstrated

related systems involving ROOH reductions by O^

in

(1,4,5,11,13,

14) . Termination reactions of IVa involving tetroxide formation

intermédiate

(Eq.6) may account for III

disterol

(and lib) from

IIa. Decomposition of the tetroxide and subsequent oxyl cal disproportionation within the solvent cage

(Eqs.

radi-

7,8) or

decomposition via the Russell mechanism involving a cyclic termediate and hydrogen transfer 2 ROO · ROOOOR [RO-O

ROOOOR =»- [RO-O

-OR]

ROOOOR [RO·0 ·0 R] 2

ROO· + (CH 3 ) 2 S0

(Eq.9) yield III and Eq.6 -OR]

Eq.7

Ì— ROH + R'R"C = 0 + 0 2

Eq.8

ROH + R'R"C = 0 + 0 2

Eq.9

2RO · + 0Δ9

Eq.10

RO· + ( C H 3 ) 2 S 0 2

Eq.ll

Yet other possibilities involve generation of 7 α -oxyl IVb from IVa by decomposition of or solvent participation

(Eq.ll)

in-

IIb.

-the (3,4).

tetroxide

radical

(Eqs.

7,10)

Subsequent Β-scission

of IVb then yield III. Any lib derived from competing abstraction by IVb or tetroxide decomposition would

hydrogen be indis-

tinguishable from lib derived by processes of Eq. 1 and Eq. 5. Although these processes account for III of III from la requires additionally of a

6

A -5a-sterol.

from IIa, derivation

the allylic

rearrangement

As lib is not among products from la in

any experiment, rearrangement of la to Ila or of lb to lib not supported.

is

Accordingly, III must arise from la by a rear-

39 rangement of 3ß-hydroxy-5a-cholest-6-ene-5-peroxyl

(Va) or 3g-

hydroxy-5 α-cho1est-6-en-5-oxy1

conditions

resulting

in regiospecificity

luding lib

(Vb) radicals under

favoring III formation and exc-

formation.

In trying to devise means of choice between peroxyl and

oxyl

radicals as precursor of III, we also oxidized la and IIa in a biphasic system with Ce (NH ) (NO ) , a system in which peroxyl 4 2 3 6 radicals are implicated (15). Oxidation of la gave lb and III; oxidation of IIa gave lib and III.

The common pattern of cor4+ oxidation,

responding alcohol and ketone suggests that the Ce KO z-, reduction in C H , and KO reduction in 6 6 2 cholesterol all involve peroxyl radicals.

(CH ) SO containing 3 2

However, our experiments do not address means by which IV or V arise in the O^

systems from parent ROOH or

anions, but metal-catalyzed or thermal peroxide bond is not indicated.

homolysis

Rather, cholesterol appears to provoke

molecule-ass isted homolysis radicals

radicals derived

(Eq.12) that may generate

a

peroxyl

(Eq.13). ROOH

cholesterol B- R0- + -OH

RO· + ROOH

»»

ROH + ROO"

,„ Eq.12 Eq.13

We conclude that there are at least two processes in these systems, participation

an ionic one in ( C H ^ ^ S O

implicated

involving

solvent

in which lb form from la and lib from IIa

(Eqs.

1,5), the other in

(CH.,) „SO containing cholesterol or in C,H, 3 2 6 6 involving peroxyl and/or oxyl radicals yielding III and lb from la. III and lib from IIa

(Eqs.6-11).

On balance our results demonstrate further the sensitivity these O^

systems to influences of solvent and extraneous

ponents and confirm the complexity previously recognized others

(3-5,8,13-16).

These several items clearly

against reliance on such 0 s y s t e m s

as models for

of comby

mitigate any biolo-

gical process, but in fancy the influence of cholesterol may be viewed as a solution counterpart of the recognized

effects

40 of c h o l e s t e r o l on m o r e c o m p l e x s y s t e m s l i p o s o m e s , and b i o l o g i c a l m e m b r a n e s . Acknowledgement. Foundation, ES-02394)

Financial

Houston,TX

is g r a t e f u l l y

support

involving

micelles,

of the R o b e r t A .

and the U . S . P u b l i c

Health

Welch

Service

(grant

acknowledged.

References 1. P e t e r s , J . W . ,

F o o t e , C . S . : J . A m . C h e m . Soc . 9 8 , 8 7 3 - 8 7 5 ( 1 9 76) •

2. T h o m a s , M . J . , M e h 1 , Κ . S . , P r y o r , W . A . : C o m m u n . 83 ,927-932 (1978) .

Βiochem.Βiophys.Res.

3. G i b i a n , M . J . , U n g e r m a n n , Τ . : J . O r g . C h e m . 4. G i b i a n , M . J . , (1979) .

Ungermann,Τ.:

41,25 0 0 - 2 5 0 2 ( 1 9 76) •

J.Am.Chem.Soc.

101,1291-1293

5. S t a n l e y , J . Ρ . : J . O r g . C h e m .

45,1413-1418(1980) •

6. S a w y e r , D . T . , G i b i a n , M . J . :

Tetrahedron

7. G u m u l k a , J . , P y r e k , J . S . ,

Smith,L.L.:

35 , 1 4 7 1 - 1 4 8 1 (1979) •

Lipids

8. H y l a n d , K . , A u c l a i r , C . : 5 3 1 - 5 3 7 (1981) .

Β iochem.Βiophys.

9. T e n g , J . I . , K u l i g , M . J . , (1973) .

Smith,L.L.:

1 7 , 1 9 7 - 2 0 3 (1982)

Res.

J.Chromatog.

10. S m i t h , L . L . , K u l i g , M . J . ,Teng , J.I - : S t e r o i d s 11. Le Β er re , A . , B e r g u e r , Y . : (1966) .

Teng,J.I.:

13. T h o m a s , M . J . , M e h l , K . S . , 8347 (1982 ) .

Pryor,W.A.:

75,108-113

22,627-635(1973) .

Bu11.Soc.Chim.France

12. S m i t h , L . L . , K u l i g , M . J . , 2 1 1 - 2 1 5 (1977) .

2363-2368

Chem.Phys.Lipids J.Biol.Chem.

14. M e r r i t t , M . V . , J o h n s o n , R . A . : J . A m . C h e m . S o c . (1977) . 15. H o w a r d , J . Α . , (1968) .

Commun.102,

20,

257,8343-

99 , 3 7 1 3 - 3 7 1 9

Ingo1d,Κ.U.:

J.Am.Chem.Soc.

90,1056-1058

16. J e f f o r d , C . W . , C a d b y , P . A . : (1979) .

He1ν.Chim.Acta

62,1866-1871

17. A r u d i , R . L . , A l l e n , Α . Ο . , 265-267(1981) .

Β i e 1 s k i , Β . Η . J . : FEBS

Letters

13 5,

41 DISCUSSION

PRYOR: Did I understand that y o u have cholesterol hydroperoxide to w h i c h y o u add m o r e c h o l e s t e r o l and it c h a n g e s the products? Have you done that e x p e r i m e n t w i t h a d i f f e r e n t hydroperoxide, for instance t-butyl hydroperoxide? SMITH: No, we have not. W e have the two sterol hydroperoxides, and because we feel very c o n f i d e n t that we c a n detect every product that forms, our work has b e e n limited to these. PRYOR: O l e f i n s accelerate the d e c o m p o s i t i o n of hydroperoxides. Do y o u know that superoxide is involved? Or c o u l d it be just a n accelerated d e c o m p o s i t i o n of the hydroperoxide c a u s e d by the double bond in c h o l e s t e r o l ? SMITH: W e d o not know that superoxide is directly involved, but the a d ded cholesterol in the system tends to slow the reaction down. The reaction would be faster w i t h o u t cholesterol. PRYOR:

I d o n ' t know if that answers the question.

SMITH:

No, I would say not.

PRYOR: A p e r o x y l radical could react w i t h cholesterol to give the o x y l radical and c h o l e s t e r o l epoxide. Could superoxide oxidize the o x y l radical to the ketone?

alkalk-

SMITH: W e did not see e p o x i d e s formed. W e have carefully looked at these products, because I had preconceived notions about w h a t we m i g h t see in the system. W e did not see anything other than what I reported. PRYOR: Just one more just for cholesterol?

question.

Do y o u observe

this

for

all

olefins

SMITH:

No, b o t h 5 - - c h o l e s t a n o l and 5-ß-cholestanol do the same thing.

PRYOR:

W h a t about o l e f i n s that aren't sterols?

or

SMITH: W e have not added anything but sterols. Again, because we were c o n f i d e n t of finding any products derived from either the hydroperoxide or from the added sterol.

THE O, RADICAL REACTIONS IN NEUTRAL AND ALKALINE SOLUTIONS

Jerzy Holcman, Knud Sehested, Erling Bjergbakke Accelelerator Department Rise National Laboratory DK 4000 Roskilde, Denmark Edwin J. Hart 2115 Hart Road Port Angeles, WA 98362, USA

Introduction Although the ozonide radical ion has been studied extensively by means of pulse radiolysis and flash photolysis

(1,2), not

much is known about the reactivity of O^ itself. The only well established reaction of the 0^ is the equilibrium with oxygen and 0~

(eq. 1).

la)

0¡

0 2 + O"

lb)

0_ + 2

0~

Ol 3

k l a = 3.3 χ 10 3 s - 1

(ref. 3)

k,, = 3 χ 10 9 dm 3 lb

(ref. 4)

Equilibrium 1) poses some experimental difficulties in studying the reactivity of 0^. As reaction lb) is used to form 0^ the concentration of any solute present is limited by its k (S + 0~) [0~] reactivity with 0 /OH and if ^ (g + 0 ~ ) - 0~ + 0 2

8 6 = 1. 1 X 10 9 k 7 = 1. 6 χ 10 k

•1 -1 s

•1 -1 dm 3 mol~ s dm 3 mol

Although the reaction of Η atoms with ozone is fast

•1 -1 s

(ref. 9),

k(H + 0 3 ) = 3.65 χ 1 0 1 0 dm 3 mol~ 1 s" 1 , G(0~) initially formed equals

G

( e a g ) ' when the Η atoms are scavenged by 0 3 . When the

Η atoms are scavenged by oxygen, G(0~) = G ( e a g ) + G(H) independent of pH. Moreover, when carbonate is used to scavenge the OH radicals, G(CO") = G(OH) + G(H) in the former experiment and G(C0 3 ) = G(OH) in the latter. In acid solutions of ozone (7), it was found that the product of the reaction, H + 0 3 , is analogous to an OH radical with respect to both spectra and

47 and kinetics. On the basis of these findings it is concluded that the HO^ species, if formed at all, is either extremely unstable 10) HO^ -*• OH + 0^ or it is kinetically indistinguishable from the OH radical. In conclusion, a determination of the pK of the O^ does not seem to be experimentally attainable as it would require measurements of either equilibrium concentrations according to reactions 5-6) or of the rate constant for the reaction: H0 3

H + + 0~.

References 1.

Bielski, B.H.J, Gebicki, J.M.: "Advances in Radiation Chemistry", Vol. 2. Eds. M. Burton and J.L. Magee, WileyInterscience, 1970, p. 248.

2.

Czapski, G.: Annu. Rev. Phys. Chem. 22,

3.

Gall, B., Dorfman, L.M.: J. Am. Chem. Soc. 91, 2199

4.

Farhataziz, Ross, Alberta B.: Natl. Stand. Ref. Data Ser. (U.S. Natl. Bur. Stand.) No. 59 (1977).

5.

Holcman, J., Sehested, Κ., Bjergbakke, Ε., Hart, E.J., J. Phys. Chem. 86_, 2069 (1982).

6.

171

(1971). (1969).

Sehested, K., Holcman, J., Bjergbakke, Ε., Hart, E.J., J. Phys. Chem. 86.. 2066

(1982).

7.

Sehested, Κ., Holcman, J., Bjergbakke, E., Hart, E.J., submitted for publication in J. Phys. Chem.

8.

Christensen, H., Sehested, Κ., Radiat. Phys. Chem. 1.6, 183 (1979).

9.

Sehested, Κ., Holcman, J., Hart, E.J., to appear in May issue of J. Phys. Chem.

10. Gear, C.W., Commun. ACM 14, 185-90

(1971).

11. Buxton, G.V., Trans. Farad. Soc. 65., 2150 12. Jonah, C., Hart, E.J., Unpublished result.

(1969).

48 DISCUSSION

BIELSKI: I would like to make a comment on a similar study. We have studied at pH 2 the reaction HO2 + OH H2O3. When one uses long pulses (> 100 msec) the system runs out of oxygen and the H and OH radicals attack the H203 yielding most likely HO3 O3 + H+. This transient disappears subsequently by second and first order kinetics giving as one of the end products ozone. HOLCMAN: I wonder if you could positively identify the HO3 so that you can measure the deprotonation rate constant. CZAPSKI: The negative pK-value of HO3 has never been measured, we found that the pK's of all the HnOra compounds - I mean H2O, OH, H 2 0 2 , HO2 and H 2 0 3 - all lie on a straight line when you plot the ratio O/H versus the pK (H.A. SCHWARZ, Ann. Rev. Phys. Chem. (1965) 347; CZAPSKI in "Radiation Chemistry of Aqueous Systems", G. Stein, ed.? Weizmann Sci. Press, Jerusalem (1968), pp. 211). If this applies for HO3 also, then the pK is negative. SARAN: You stated that the neutralization reaction is extremely fast, almost lO^l. If you go to neutral or acid pH where you have an appreciable amount of H + , this neutralization might become dominant over the other O3 reaction that gives you 0 2 . You told us that 70% of your 0 3 disappears via the OJ pathway. In . your computer simulation did you take into consideration the competition by the neutralization reaction? HOLCMAN: Well, the reaction of OH plus O3 was studied down to pH 10 and the neutralization of O3 accounted for in the computer simulation. There are some difficulties in studying it quantitatively in the ozone system, although we made a qualitative check. You can't isolate this reaction in pulse radiolysis, because it is a matter of finding the right conditions where this reaction is enhanced. Even then it is only a certain percentage of all the reactions, because if you are to produce O3 in alkaline solution, you have to convert your OH into O". So you have to calculate how many OH radicals are left for the reaction with O3. This kind of system can only be tackled by computer simulation.

REACTIVITIES OF tert-BUTOXY RADICAIS IN AQUEOUS SOLUTIONS

W. Bors, D. Tait, M. Erben-Russ, C. Michel arid M. Saran Institut für Biologie, Abteilung für Strahlenbiologie, GSF ftirschungszentrum, 8042 Neuherberg, F.R.G.

Introduction We have determined the reactivities in aqueous solutions of numerous substances with photolytically produced alkoxy radicals fron tert-butyl hydroperoxide and hydroperoxides of polyunsaturated fatty acids (PUFA; ref. 1-3). In these studies we used the indirect assay of inhibiting the radical-induced bleaching of the water-soluble carotenoid crocin by adding various amounts of the substances to be measured (1,2). What little is known about alkoxy radicals, however, indicates that they are very unstable in aqueous solutions (4,5). The aim of our kinetic studies was always intended to be the determination of absolute reaction rate constants. Ihis requires the unequivocal verification of the participation of alkoxy radicals in these reactions. Previous studies have established that crocin itself is unsuitable for pulse-radiolytic experiments, since it scavenges the hydrated electrons which are necessary to reduce (hydro)peroxides to alkoxy radicals (6). Encouraging results came from a study of the structure-activity relationship for phenolic compounds. In spite of the rather wide scatter of the data the correlation was sufficient to indicate electrophilic interactions by both "OH and _t-BuO' radicals (3). the present study is a continuation of our efforts to confirm the radiolytic generation of alkoxy radicals in aqueous solution and to determine their absolute reaction rate constants.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

50 Materials and Methods Peroxide precursors of alkoxy radicals were either tert-butyl hydroperoxide [t-Bu00H; 80% in di-tert-butyl peroxide, Merck, Darmstadt]; ditert-butyl peroxide [(t-BuO>2; technically pure, Merck] and 13-hydroperoxy-linoleic acid [13-IÛOH, produced enzymatically with soybean lipoxygenase-1 (7), gift of W. Grosch, Garching]. ifordihydroguaiaretic acid [NDGA], caffeic acid (3.4-dihydroxy-cinnamic acid), 4-methoxyacetophenone and 4-vinylbenzenesulfonic acid were from Aldrich-Europe, and Trolox c a gift of Hoffman La-Roche, Basel - all were used as supplied. GC experiments were performed in a Perkin & Elmer F20 Fraktometer, the column Carbowax 1500 (15%, 80-100 mesh) accepting direct injection of aqueous solutions without need of derivatization. 4-Methoxyacetophenone (0.1 mM) was added to (t-BuO)2 solutions in these experiments to accelerate the photolytic cleavage of the peroxide (8). HPLC experiments to isolate trapped alkoxy adducts used a Waters MPG with M440 fixed wavelength detector with Extended Wavelength Module for scanning at 214 and 229 nm. Some pulse-radiolytic experiments were done at the Elbena facility of the Hahn-Meitner-Institute in Berlin, with the help of Dr. K.-D. Asmus and Dr. M. Bonifacio. These included determination of absolute reaction rates of hydrated electrons with various precursors and evaluation of build-up kinetics. Our own facility (details see Saran et al., these proceedings) was employed for determining transient spectra and competition kinetics. Results and Discussion The most obvious method of identifying alkoxy radicals is by chemical or spin trapping, i.e forming a more or less stable adduct (5). In organic solvents alkoxy radicals have been shown to add only to activated double bonds apart from abstracting hydrogen atoms (9-12). Suitable water-soluble derivatives of such traps, however, are hard to come by. Crocin itself as a highly conjugated substance is bleached very effectively by alkoxy radicals, but attempts to isolate and identify adducts by HPLC were unsuccessful. We are presently investigating 4-vinyl benzenesulfonic acid and preliminary data suggest the formation of several HPIC-separable products. Indirect approaches, in which not an adduct of alkoxy radicals but a specific reaction product is determined, have recently been tried. Et>r example, QC-analysis of the ratio of the production of acetone vs. tert-butanol has occasionally been used to arrive at reaction rates of t-BuO" radicals (13,14). Ihe assay is based on the competition of ß-fragmentation

51

to yield acetone and reaction of t-BuO* via Η-abstraction to yield t-BuOH,as shown in R. /I/ and /2/: ß-fragm.| (CH3) 3C-O·

PhOH

(CH3)2C=O + *CH3

/l/

(CH3)3C-Π+ PhO'

/2/

Using GC-analysis, we have been unable thus far to detect t-BuOH in photolyzed aqueous (t-BuO)2"Solutions. Although ß-fragmentation is very rapid in polar solvents, especially water (5,13,15), we thought that highly reactive antioxidants might react fast enough with t-BuO" to produce t-BuOH. Pulse-radiolytic experiments were therefore carried out using the catechol antioxidant nordihydroguaiaretic acid (NEGA) as radical scavenger. Figure 1 and löble I depict the NDGA transient absorption maxima and peak ratios observed under alkoxy radical-generating conditions. They differ from those produced by "OH,

or "CH3 attack.

FIGURE 1 - TRANSIENT SPECTRA OF NORDIHYDROGUAIARETIC ACID (0.1 mM) WI1H VARIOUS TYPES OF RADICALS IN NEUTRAL AQUEOUS SOLUTION (a) oxidizing radicals: (•) "CH radicals (N O-saturated solutions); (A) t-BuO" raflicals (N_-saturated solutions containing 10 mM t-butanol and 0.13 M t-BuOOH); (γ) 13-LO" radicals (^-saturated solution containing 10 mM t-butanol and 50 ^uM 13-IßOH). (b) different concentrations of t-BuOOH (in N_-saturated solutions containing 10 mMisopropanol) : (•) 0.13 M t-BuOOH (mixture of t-BuO" and _t-BuOO" ) ; (A) 1 mM t-BuOOH (mostly t-BuO") ; (ψ) 0.01 mM t-BuOOH (mixture of t-BuO" and e ). aq (c) reducing radicals: (A) e (N.-saturated solutions containing 10 ml isopropanol); (ψ) "CH, radicals (deoxygenated solution containing 10 mM t-butanol and saturated with CH CI).

52 TABLE I - SPECTROSCOPIC DATA OF TRANSIENTS OF NORDIHYDROGUAIARETIC ACID WITH SEVERAL RADICAL SPECIES IN AQUEOUS SOLUTION.

Radical

Scavenger/ Convertant

Concentration (mM)

Wavelength region (nm)

Ratio

•CH

N2O

12

255, 315

0.5

e"ag

t-BuOH

10

250, 300

1.7 2.2 2.4

II

II

IPOH t-BuOH

•CH3

t-BuO· II

II

t-BuO· + e η

iq

13-LO· + e

äq

II

—

II

_

II

__

II

_

__

II

_

CH3CI

10 sat.

t-BuOH t-BuOOH

10 133

IPOH t-BuOOH

10 133

t-BuOH t-BuOOH

100 1.6

IPOH t-BuOOH

10 1.0

IPOH t-BuOOH

10

t-BuOH 13-DXH

10 0.05

a) very strong signal (120 mAU b) slower build-up kinetics at c) different build-up kinetics over 50 msec; d) initial decay of both peaks

270, 300 __

II

_

250, 300 __

II

_

a

)

2.4 b)

0.75 c> 0.71 1.35 1.5 d> 1.9 d)

0 . 0 1

265, 320, 360

(1.7)

at 315 nm); 300 nm shift ratio to smaller values; for 270, 300 nm-peaks, both rather stable far more rapid than at higher [t-BuOOH].

The spectra at 80 ^usec after the pulse have been plotted in arbitrary absorbance units for better comparison of the absorption maxima and the ordinate scale is not identical for the individual transients, the strong transient absorption after "CH attack (120 mAU at 315 nm) is unique and is presumably due to the semiquinone. A weaker absorbing species with similar peak ratio, but with a 15-20 nm shift of both wavelength peaks is observed at the highest t-BuOCH concentration employed. A gradual shift of 7\ m a x and ratio occurs with decreasing hydroperoxide concentration until we reach the transient absorption after e~ attack - which is aq nearly identical to the transient formed with "CH.. radicals.

53 TO account for these differences, we propose the following reactions: PhOH

+ "OH

»

PhO* + H 2 0

/3/

PhOH + e" aq

* ")

PhOH + *CH3

• J

t-BuOOH + e" aq

•

t-BuO' + 0H~

/6/

t-BuO" + PhOH

»·

PhO" + t-BuOH

/2/

t-BuO" + t-BuOOH

»

t-BuOO" + t-BuOH

/7/

t-BuOO' + PhOH

•

PhO" + t-BuOOH

/8/

unknown transient

/4/ /5/

Only R. /3/ is straight-forward, since it has been demonstrated for numerous catechol compounds (16-18). While absolute absorption maxima and peak ratios can only be given for transients corrected for solute depletion, we assume that absorption maxima at 270 nm and 300-310 nm and a peak ratio of 0.5 - 0.75 represent the semiquinone PhO". We could thus compare effective and calculated yields at 300 nm to arrive at a reasonable estimate for the reaction rate of R. /2/. Et>r this calculation in addition to R. /7/ and /8/, ß-fragmentation (f), self-reaction of two t-BuO" radicals (s), reaction with the alcohol solvent (t; which scavenges the "OH radicals) and disproportionation of the semiquinone (d), must be considered: _ d[t^BuOJ_

=

kj[t-BuO"] [PhOH] + k?[t-BuO"] [t-BuOOH] + k f [t-BuO"] + ks[t-BuO"]2+ kt[t-BuO"][RCH2OH]

1

=

k2[t-BuO"] [PhOH] + kg [t-BuOO*] [PhOH] -k d [PhO*] 2

Except for ß-fragmentation ( ) IO 6 s" 1 ; réf. 5), none of the reaction rates are actually known. Nevertheless, making a reasonable assumption of k_ = IO 6 , k /

S

= IO 8 , k. = 103 and k 0 = 106 (all in M - 1 s - 1 ) and taking into L·

Ö

account the various concentrations of t-BuOOH - only at the highest, 0.13 M, would R. /7/ be of any significance - we arrive at approximately 5 χ 109 M" 1 s" 1 for R. /2/.

54 This hypothetical value is somewhat lower than an average value of (2.5+0.6) χ 10

M s

which we obtained from evaluating the build-up

kinetics at 300 nm. Both these absolute reaction rate constants of t-BuO" with phenolic antioxidants in aqueous solutions are very high. This implies that alkoxy radicals have indeed to be considered as highly reactive species in aqueous solutions.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Bors, W., Michel, C., Saran, M. : Bull. Eur. Physiopath. Resp., Suppl·, 17' 1 3 - 1 8 (1981) Bors, W., Michel, C., Saran, M.: in Oxygen and Oxy-Radicals in Chemistry and Biology (M.A.J. Rodgers, E.L. Powers, Etís.), Academic Press, New York, 75-81 (1981) Bors, W., Michel, C., Saran M. : in Oxy Radicals and their Scavenger Systems, Vol. I. Molecular Aspects (G. Cohen, R.A. Greenwald, EÜs.), Elsevier, New York, 38-43 (1983) Gilbert, B.C., Holmes, R.G.G., Laue, H.A.H., Norman, R.O.C.: JCS, Perkin II, 1047-52 (1976) Gilbert, B.C., Marshall, P.D.R., Norman, R.O.C., Pineda, Ν., Williams, P.S.: JCS, Perkin II, 1392-1400 (1981) Bors, W., Michel, C., Saran, M.: Int. J. Radiat. Biol. 41, 493-501 (1982) Funk, M.O., Isaac, R., Porter, N.A.: Lipids U , 113-117 (1976) Griller, D., Ingold, K.U., Scaiano, J.C.: J. Magnet. Reson. 38, 169-171 (1980) Surzur, J.M., Bertrand, M.P.: Bull. Soc. Chim. France, 1861-67 (1973) Elson, I.H., Mao, S.W., Kochi, J.K.: J. An. Chem. Soc. 97, 335-339 (1975) Wong, P.C., Griller, D., Scaiano, J.C.: J. Am. Chem. Soc. 104, 5106-08 (1982) I&illez, B., Bertrand, M.P., Surzur, J.-M.: JCS, Perkin II, 547-553 (1983) Kochi, J.K.: J. Am. Chem. Soc. 84, 1193-97 (1962) Oleina, M.V., Lissi, E.A.: Int. J. Chem. Kinet. 10, 657-667 (1978) Walling, C., Padwa, Α.: J. Am. Chem. Soc. 85, 1593-97 (1963) Adams, G.E., Michael, B.D.: Trans. Faraday Soc. 63, 1171-80 (1967) Neta, P., Ftessenden, R.W.: J. Phys. Chem. 78, 523-529 (1974) Bors, W., Saran, Μ., Michel, C.: Biochim. Biophys. Acta 582, 537-542 (1979)

55 DISCUSSION

von SONNTAG: From your data, you might now get a better idea about the rate constant of decomposition o.f the tert-butoxy radical into acetone and methyl radical than those reported by GILBERT et. al. (JCS, Perkin Trans. II (1981), 1392). BORS: Well, we expect to be able to recalculate the probable limit of this ß-fragmentation rate when we get more accurate values of the intermolecular reaction rates of the t-BuO· radical. SIMIC: Dr. NETA and myself have measured cumyl alkoxy radicals and we got a fragmentation rate constant of l o ' sec-^·, so they are very fast. I don't know if tert-butoxy radicals are going to be similarly fast or a little bit slower. But if you put a phenyl on it, it is certainly going to be faster. BORS: I agree with you that the cumyl alkoxy radical is definitely stable than the tert-butoxy radical.

less

FORMATION AND REACTIONS OF HALOTHANE PEROXY FREE RADICALS, CF3CHCIO2'

Jörg Mönig and Klaus-Dieter Asmus Hahn-Meitner Institut für Kernforschung Berlin GmbH, Bereich Strahlenchemie, D-1000 Berlin 39, Postbox, Fed.Rep.of Germany

Introduction Halothane (2-Bromo-2-chloro-1,1,1-trifluoroethane), which is widely used as general anaesthetic, undergoes considerable biotransformation((1). Products of this process have been identified. Bromide ions (Br ) and trifluoroacetate (TFA) were shown to be the major metabolites in oxic environments (2). In addition, F -ions and the volatile compounds CF^CH^Cl and CF2CHCI are produced under hypoxic conditions (3,4). Therefore distinction was made between two completely different metabolic pathways, i.e. reductive metabolism in the absence of oxygen and oxidative metabolism (5). Recently it was shown that free radicals are intermediates in the biotransformation of halothane. These free radicals could be identified by several means (6,7) under hypoxic conditions. It has been suggested that these free radicals initiate the liver damage, which occasionally occurs after halothane-anaesthesia (8). Radiation-chemical methods offer the possibility to produce the radical of interest and allow the study of its specific reactions.

Methods Solutions were prepared according to the radiation-chemical standards. The pulse radiolysis set-up is described elsewhere (9). Steady-state radiations were carried out with a ^Co-γ-

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

58 source with dose-rates of about 320 Gy/h. Products were identified by means of ion chromatography

(10) with a DIONEX 201Oi

equipped with a separation column AS4 and a conductivity cell.

Results One-electron reduction of halothane in aqueous solution e~ g

+

CF^CHBrCl

• Br"

+

CFjCHCl

(1)

leads almost quantitatively to the formation of bromide ions and 2-chloro-1,1,1-trifluoroethyl radicals. (The "OH radicals produced upon radiolysis of water were quantitatively removed by added t-butanol). The CF^CHCl radical reacts by various mechanisms to the end products which include F , CF^CI^Cl and CF_CHCl (11). All these processes appear to be quite slow with 5-1-1 rate constants k>

b

>

0

ST0

" 3 G (kcal/mol).

positions of propene or vinylalcohol as i l l u s t r a t e d i n 2 5 a n d 2 6 ·

The

relative s t a b i l i t y between the intermediates i s shown in Table 5.

From

Table 5, the 3-attack (26) i s preferred to the 2-attack (25), showing the electrophilic nature of l·^· in contrast to the nucleophilicity of 0£. Ab-initio geometry-optimization calculations have also performed for 1 2 addition products by molecular oxygen ( 0 2 , 0 2 ) . hydroxy radical (·0Η), and oxygen atom ( 3 0 ) .

Table 5 summarizes the relative energies obtained.

From Table 5, a b - i n i t i o calculations confirm the electrophilic nature of these oxyradicals (13).

Then the regioselectivity i s opposite between

oxyradical (X·) addition to electron-rich olefins and oxyanion (ΧΘ) addition to cation radicals of the species as i l l u s t r a t e d in Fig. 4. Conclusions A b - i n i t i o MO calculations have provided the following results: CI: The charge-transfer (CT) interaction between π* of 0¿ and the LUMO of a substrate radical-cation (S*) i s of particular importance in the recombination reactions between S* and 0¿

as shown in 1 .

Therefore

the (2+2) cycloaddition between S* and 0| i s symmetry-allowed (see3 , 4 ) . Both CT and Coulombic interactions indicate the predominant attack of 0¿

Fig. 4 Schematic i l l u s t r a t i o n s of potential curves for addition reactions of oxyanions and oxyradicals to electron-rich olefins.

3-attack

2-attack

74 to the positive center of radical cations of olefins with unsymmetrical electron-donating groups ( 9 , 1 1 ). * C2: The CT (i.e., LUMO-H^) and Coulombic interactions indicate that nucleophilic additions of

to olefins with electron withdrawing groups (21~

2 3 ) and to carbonyl groups of esters and related species are likely. The net charge populations of addition intermediates confirm the nucleophilicity of 0¿ as well as oxyanions such as θΟΗ, ©(^H and 0* .which could be generated from C3: The nucleophilicity of action in protic media.

in

a

P P r o P r i a t e conditions.

is suppressed by the hydrogen bonding interAlternatively, a protonated form of 0£, i.e.,

hydroperoxyradical (HO,,·), could be an initiator of radical chain reactions.

H0„·

undergoes electrophilic additions to electron-rich double 13 3 bonds (26,28 ) a s i n the cases of oxyradicals such as ·0Η, ' 0 ¿ and 0, which could be generated from

or Η,,Ο,, in chemical and biological

systems. References 1. Sawyer, D. T., Valentine, J. S.: Accounts Chem. Res. 14_, 393-400 (1981 ). 2. Eriksen, J.,Foote, C. S.: J. Am. Chem. Soc. 102, 6083-6088 (1980). 3. Yamagucgi, K.: Singlet Oxygen, Chap. 5, Frimer, A. A. Ed., CRC Press, Boca Raton, FL., 1984. 4. Mattes, S. L., Farid, S.: J. Am. Chem. Soc. 104, 1454-1456 (1982). 5. Nami, E. J. Jr., Angelis, C. T., Dickson, J., Sawyer, D. T.: J. Am. Chem. Soc. 103, 4268-4270 (1981). 6. Yamaguchi, K.: Chem. Phys., 25, 215-235 (1977). 7. Clennan, E. L., Simmons, W., Almgren, C. W.: J. Am. Chem. Soc. 103, 20982099 (1981 ). 8. Barton, D. H. R., Haynes, R. K., L e d e r e , G., Magnus, P. D., Menzies, I. D.: J. Chem. Soc. Perkin I, 2055-2065 (1975). 9. Frimer, Α. Α., Rosenthal, I., Hoz, S.: Tetrahedron Letters,4631-4634(1977). 10. Ashadi, M., Kebarle, P.: J. Phys. Chem. 74. 1483-1485 (1970). 11. Böhme, D. K., Mackay, G. I.: J. Am. Chem. Soc. 103, 978-979 (1981). 12. Gebicki, J. M., Bielski, B. H. J.: J. Am. Chem. Soc. 103, 7020-7022 (1981). 13. Yamaguchi, K., Yabushita, S., Fueno, T., Houk, K. N.: J. Am. Chem. Soc. 103 , 5043-5046(1981).

75 DISCUSSION

SINGH:

Would 0;j react with olefins in the gas phase?

YAMAGUCHI: Ab-initio MO calculations have shown that the radical adduct (29) between 0 2 and ethylene (X=H) is more stable than the nucleophilic adduct (30) by 15.3 kcal/raol, whereas 29 is less stable than 30 by 15.2 kcal/mol in the case of the OJ plus acrylonitrile (X=CN) system.

•z Theroretical studies indicate that, in gas phase, the radical additions to ethylene-type compounds are preferred to the nucleophilic additions. (We discussed only the nucleophilic attack for comparisons with reported experimental results in section 3). SINGH: It would be interesting to look for this reaction in the gas phase; both in aqueous and aprotic media, O2 does not react with olefins. YAMAGUCHI: Certainly, ab-initio calculations clearly confirm that 05 exhibits nucleophilicity towards acrylonitrile (and other olefins with electron-accepting groups) and carbonyl compounds in both gas phase and aprotic media (see section 3 of my paper). The magnitude of radical reactivity of O2 to olefins in gas and solution phase remains a problem. Although the activation energy for the radical addition of 05 to ethylene is not yet calculated, it is probably over 40 kcal/ mol as in the case of the radical addition of 'o2 to ethylene. Therefore the radical additions of OJ to olefinic compounds are probably unfavourable at ordinary temperature even in the gas phase. Actually, there are no reports in the literature of such reactions. However, radical additions of OJ may be expected for hindered olefins and compounds with unstable double bonds (note that both 3 0 2 and ^-02 react with these compounds, and that abstracts a hydrogen atom from flavin analogs; NANNI and SAWYER (J. Am. Chem. Soc. (1980) 102, 7591) . It is known that 0~ as well as 3 0 2 , like the active oxygen radicals OH and H0 2 , exhibit high radical reactivity with olefins and hydrogen donors. Interestingly, ab-initio calculations indicate significant localization of the unpaired electron on one of the oxygen atoms and the hydrogen-bonding stabilization of the 0~-site by cluster formation of 02 with water: O — 0 - - (Η2θ)η. This implies an increase of radical reactivity of 0 2 and a decrease of its nucleophilicity by cluster formation with protic compounds in the gas phase. So, I have great interest in future gas-phase experimental studies of the reactions between olefins and O2 clustered with water and other protic compounds (HA) (for example, FAHEY et al., (1982), J. Chem. Phys. 2Í» 1799). These experiments enable us to compare the reactivities of OJ, O2 (HA)n and H0 2 with olefins in the gas phase.

ESR AND ENDOR STUDIES OF FREE RADICALS HAVING UNPAIRED SPIN DENSITY LOCALIZED PRIMARILY ON OXYGEN

Harold C. Box and Edwin E. Budzinski Biophysics Department, Roswell Park Memorial

Institute

Buffalo, New York 14263, USA

Introduction The focus of attention in t h i s conference i s on oxygen radicals in chemi s t r y and biology.

Many interesting oxygen

free radical

species can be

produced by the effects of ionizing radiation on organic c r y s t a l s . relevance of single crystal

The

studies to t h i s conference i s that ESR and

ENDOR measurements on oriented oxygen radicals have provided the clearest insights

into the electronic

structure of these species.

have revealed a surprising variety of oxygen free radical

Such

studies

types.

A few preliminary remarks need to be made concerning the nature of these studies.

The two principal features that characterize the ESR absorptions

of oxygen free radicals are the g value and the hyperfine pattern.

The

former i s a measure of the effective magnetic moment of the unpaired electron; the l a t t e r indicates the strength of the magnetic interaction between the unpaired electron and each magnetic nucleus in i t s Both characteristics

depend

upon the orientation

molecule with respect to an applied magnetic f i e l d .

of the

vicinity.

free

radical

The anisotropic be-

havior of the g value and of each hyperfine s p l i t t i n g can be described completely by means of a tensor. defined with respect to i t s

A tensor description i s simplest when

principal

axes.

These are coordinate axes

fixed with respect to the free radical molecule; frequently the orientation of the principal the molecules.

axes coincides with obvious structural

features of

Then the tensor i t s e l f can be specified by just

three

quantities, called principal values, which include the maximun observable

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

78 value of the q value (or hyperfine s p l i t t i n g ) , i t s minimun value and some intermediate value.

The g tensor i s deduced from ESR measurements of the

q value at various orientations of the free radical molecule with respect to an applied magnetic f i e l d . ally

Hyperfine coupling tensors are not gener-

deducible with sufficient accuracy directly

patterns.

from the ESR hyperfine

Preferably, hyperfine tensors are deduced from ENDOR (electron-

nuclear double resonance) frequencies measured at various orientations of the radical with respect to the f i e l d (1). Our intention i s

to present insights

into the electronic

structure ob-

tained from ESR and ENDOR studies on oriented oxygen free radical

species.

All experimental data i s reported in terms of principal values.

Di scussi on An oxygen free radical can be defined as one in which the main concentration of spin density is on one or more oxygen atoms. free radical

The earliest oxygen

to be characterized from ESR and ENDOR studies was the hy-

droxyl radical, which i s , of course, of fundamental importance in biochemi s t r y and radiation biology.

The principal values of the g tensor and of

the proton hyperfine coupling tensor are given in Table 1 for the OH radicals created by oxidation of water molecules in X-irradiated single crystals of ice.

The observations are consistent with the expectation that

the unpaired spin resides mainly in an oxygen 2JD o r b i t a l .

The hyperfine

coupling arises from dipole-dipole interaction between the proton and the electron spin density on the oxygen atom, but also from a contact interaction due to a small negative density at the proton i t s e l f . mim hyperfine

splitting

occurs with the f i e l d

parallel

Thus, mini-

to the OH bond,

maximun with the f i e l d perpendicular to both the OH bond and the axis of the 2p o r b i t a l .

Minimum g value occurs with the f i e l d

parallel

to the

axis of the oxygen 2p o r b i t a l , and maximun with the f i e l d parallel to the OH bond (2).

HO I h2 C I c

HO I HJC

Yí

I

O O-CH.-CH-C *»0

l\H

Ho

H O /

I HO ΠΓ»

HO CHJO I c H /I ι/η

HZC o \H \l V V ι Ηi/i I/ Ο — ρ : C I OH

l\OH HO o \\L l C I Η

C H /I 1/ H

HO I HZC

Vi Λ C

l\HO HO HOΟ Ν» C

Η

I H

I HO

S»

C

V IS b

O'

NHz ΗΝ

CH

HC I HC V

.CH CH \H C Η

r

Η / — •C I o" IS α

CH-CH,-Ο — P = 0 * I O H

CH II CH I CHJ

Cl

•

ι

NHJ — CH — C ,

1 0 1 CH 2 HCOH I HOCH I HCOH I CHJOH Hb Figure

1.

Various oxygen free radicals w h o s e characteristics are described in the text and in Table 1.

80 Table 1.

The principal values of the g tensor and of the proton hyperfine coupling tensors which characterize various oxygen free radicals. Except for species I (hydroxyl radicals) the various free radicals are identified in Fig. 1. Principal values of the hyperfine tensors are given in MHz. Ia

g A

2.0597 9.2

g A

2.0571 19.4

g A

2.0581 15.6

g Αι A2

2.079 334 67.6

g Ai A3

2.077 187.11 266.50

g A! A2 A3

2.074 28.30 30.46 16.66 12.38

2.0089 -124.4

2.0028 -80.0

2.0089 -124.4

2.0031 -81.2

2.0088 -126.2

2.0027 -78.2

2.005 313 50.1

1.998 311 48.5

2.056 171.38 247.94

1.969 164.91 243.14

2.004 12.34 20.40 9.36 9.68

1.969 11.02 15.40 -4.04 8.84

2.0063 128.66 11.80 12.80

1.9999 125.56 11.42 -5.46

2.0087 25.46 14.80 9.36 11.89

2.0037 24.18 13.51 -1.26 9.54

-19.8 -18.9

-7.1 -7.3

lb

Ic

IIa

IIb

Illa

A4

I IIb g Αχ A2 A3

2.0627 145.44 26.62 18.10

g Ax A2 A3 A4

2.0270 28.36 16.96 20.67 23.96

g Ai A2

2.008 -25.1 -26.1

IV

V

81 The crystal

structure of ice i s such that hydroxyl

r a d i c a l s are created in

three d i s t i n c t environments, which accounts for the three e n t r i e s in Table 1.

for OH

This circonstance provides an opportunity to observe how much

the structure of a trapped free radical

is

perturbed by i t s

environment.

In t h i s instance, the effect i s minimal. The next free radical

species to be discussed i s the alkoxy r a d i c a l .

representative alkoxy r a d i c a l s are shown in F i g .

1.

are

serine

produced

by r a d i a t i on-induced

phosphate (4) r e s p e c t i v e l y . included in Table 1.

oxidation

of

(3),

and glucose

The tensor d e s c r i p t i o n s of these r a d i c a l s are

The outstanding feature of the hyperfine pattern ex-

exhibited by these species i s the occurrence of extremely large couplings.

Some

Species I I a and l i b

β proton

In oxidized serine one of the proton couplings has a maximin

value of 319 MHz. present several show preferential

Polyhydroxy compounds, such as glucose phosphate, which

sites

for possible r a d i a t i on-induced oxidation, generally

oxidation at a p a r t i c u l a r

site.

However, three of the

four hydroxy groups in glucose phosphate are oxidized to some degree. oxidation s i t e g i v i n g r i s e to each of the absorptions i s r e a d i l y

The

identi-

fied since the d i r e c t i o n of maximum g value coincides with the d i r e c t i o n of C-0 bond as known from the c r y s t a l

structure.

Another v a r i e t y of oxygen free radical the alkoxy r a d i c a l s j u s t described.

i s an i n t e r e s t i n g counterpoint In t h i s v a r i a t i o n the

to

β protons as

well as the γ protons contribute to the hyperfine pattern of the ESR spectrum although t h e i r

individual

However, the individual surements.

contributions

coupling

usually cannot be resolved.

tensors can be deduced from ENDOR mea-

The s i g n i f i c a n t involvement of γ protons in the hyperfine i n -

teraction i s the d i s t i n g u i s h i n g feature of t h i s alkoxy v a r i a n t .

Two exam-

ples of these species are I l i a and 11 lb ( F i g . I ) derived by oxidation of deoxycytidine monophosphate and x y l i t o l

respectively

descriptions

Species

are included in Table 1.

(5, 6 ) .

I I and I I I

The tensor give r i s e

to

such d i f f e r e n t hyperfine patterns because of a subtle difference in t h e i r electronic structure. the orbital

The extreme cases are i l l u s t r a t e d in F i g . 2.

bearing the unpaired electron takes the o r i e n t a t i o n

When

depicted

by the dashed o u t l i n e , the ß proton hyperfine coupling i s minimum.

More-

82

over

proton couplings become important in the latter case presimably be-

cause of spin polarization introduced with carbon bonding systems. ever the character of the g tensor in radical substantially the same.

types I I and I I I

How-

remains

Some del ocal i zation of the electron away from the

oxygen atom, perhaps 10-20%, occurs in these radicals (7).

Nunerous types

I I and I I I radicals have been identified in irradiated single crystals of polyhydroxy and carbohydrate compounds (8, 9, 10, 11) H

Figure 2.

Electronic structure of radicals where s o l i d and dashed outlines represent orbital of unpaired electron in type I I and type I I I radical respectively.

S t i l l other oxygen radicals exhibit somewhat atypical g tensors and an unusual set of proton couplings.

An example is the free radical IV (Fig. 1)

produced by radiation-induced oxidation of sucrose (10). maximum principal

The minimim and

values of the g tensor are not as extreme as those ob-

served in types ( I I ) and ( I I I ) . The nunber of proton couplings i s larger than can be accounted for in terms of β and γ couplings. Fortunately ENDOR spectroscopy

is

sufficiently

sensitive

extramolecular protons can be detected.

so that even weakly

interacting

From these almost pure dipole-

dipole couplings, together with the g tensor, i t can be demonstrated that the main concentration of spin density i s localized as shown in IVa (Fig. 1).

The atypical character!sties of the absorption are due to s i g n i f i c a n t

del ocal ization of the spin as represented by the structure IVb.

This de-

localization away from the oxygen diminishes the g value extrema and generates two additional proton couplings (Table 1).

83

As electron spin density on oxygen i s reduced through del ocal i za t i on, the hallmark of the oxygen free radical, i t s g s h i f t , becomes l e s s prominent. Spin density i s , of course, highly delocalized in aromatic molecules.

An

example of an aromatic oxygen free radical i s V which i s produced by radiation-induced oxidation of tyrosine (12).

The spin density at the carbon

atom positions ortho to the carbonyl group can be deduced from the coupl i n g tensors for the protons at these positions (Table 1). sity on each of these carbons i s 0.31. about 0.38.

The spin den-

The spin density on the oxygen i s

Maximim g value for this species ( f i e l d parallel to the C-0

bond) i s only 2.008. The recent surge of interest in oxygen radicals

i s due in part to the

l i k l i h o o d that in vivo autoxidation originates a free radical process that could account for indirect i n i t i a t i o n as well as promotional carcinogenesis.

effects

in

The free radicals generated in the autoxidation step are

often oxygen free radicals.

I t would be of interest therefore to study

these radicals in single crystals using ionizing radiation to produce the oxidized products and ESR and ENDOR spectroscopies to characterize them. In this laboratory efforts toward t h i s end are in progress.

The molecules

of interest are often relatively small planar conjugated species.

Since

planar conjugated molecules tend to interact strongly in the c r y s t a l l i n e state, a d i f f i c u l t y i s encountered, namely that ionization electrons are not trapped but tend to return to the parent oxidized molecules.

A possi-

ble way out of t h i s problem i s suggested by the tyrosine study mentioned above where the phenol rings are isolated from each other in the crystal by intervening non-conjugated components.

Acknowl edgement This work was supported by grant CA 25027 from the National Cancer I n s t i tute.

84 References

1.

Box, H.C.: Radiation Effects: ESR and ENDOR Analysis, Academic Press. New York 1977.

2.

Box, H.C., Budzinski, E.E., Lilga, K.T., Freund, H.G.: J. Chem. Phys. 53, 1059-1065 (1970).

3.

Lee, J.Y., Box, H.C.: J. Chem. Phys. 59, 2509-2512 (1973).

4.

Locher, S.E., Box, H.C.: J. Chem. Phys. 72, 828-832 (1980).

5.

Box, H.C., Budzinski, E.E., Potienko, G.: J. Chem. Phys. 73, 20522056 (1980). Budzinski, E.E., Potter, W.R., Box, H.C.: J. Chem. Phys. 72, 972975 (1980).

6. 7.

Budzinski, E.E., Freund, H.G., Potienko, G., Box, H.C.: J. Chem. Phys. 77, 3910 (1982).

8.

Kim, H-s., Alexander, C.: J. Chem. Phys. 77, 4879-4883 (1982).

9.

Bernhard, W.A., Close, D.M., Huttermann, J., Zehner, H.: J. Chem. Phys. 67, 1211 (1977).

10.

Samskog, P.O., Kispert, L.D., Lund, Α.: J. Chem. Phys. 77, 23302335 (1982).

11.

Madden, K.P., Bernhard, W.A.: J. Chem. Phys. 70, 2341 (1979).

12.

Pryor, W.A.: New York Acad. Sci. 393, 1-22 (1982).

13.

Slaga, T.J., Klein-Szanto, A.J.P., T r i p l e « , L.L., Yotti, L.P., Trosko, J.E.: Science , 1023-1025 (1981).

DISCUSSION BORG: A small experimental point. I noticed the value of the free proton ferquency in the ENDOR spectrum you showed. If I can trust the results from my pocket calculator, I conclude that you must have carried out your ESR at 70 GHz. Is that right? BOX:

Yes, 70 GHz.

BORG: That is going to make small shifts in g-value very prominent and will also spread out your ENDOR spectral lines. So is it important, in order to do what you did, to operate at that high microwave frequency? BOX: Yes, I think it is. In fact, I think that a lot of the oxygen radicals have not been reported in the literature, just because they haven't been shifted out from other radicals. SARAN: If I understood it correctly, the outstanding feature of the alkoxy radicals is that they have an extremely high degree of Q-proton coupling. What would that mean at room temperature in terms of chemical behaviour of the alkoxy radical? Would it be stabilized by this?

85 BOX:

No, they are not stable at high temperature.

SCHOLES:

Could you give any indication as to what they decay to.

BOX: No, I can't. This is certainly one thing we want to look into, but I really can't make a statement now. von SONNTAG: You reported the interesting observation that the g value of your oxyl radical at 0-3' was very different from what you usually observe with oxyl radicals and you also told us that there is considerable spin at the neighbouring carbon. This oxyl radical should be especially prone to the fragmentation reaction that you have shown. I wonder if the g value of oxyl radicals can give us some information on the ease of fragmentation of these species in a similar way as it has been shown for some coupling constants by BEHRENS et al. (Int. J. Radia t. Biol. in the r (1978) 33.» i work on the release of phosphate from phosphatesubstituted alkyl radicals. Did you ever try such a correlation? BOX: No, I never have, but I am interested and happy to have a suggestion now. I suppose we could look at the relative stability of the various alkoxy radicals which have exhibited some variation in g tensor and see whether any kind of correlation can be established. I am very anxious that some of the stuff we do is relevant to this audience.

CORONA DISCHARGE AS A SOURCE OF SUPEROXIDE

Harry C. Sutton

Institute of Nuclear Sciences, DSIR, Lower Hütt, New Zealand

Introduction Corona discharge from a gold or platinum needle point in air at negative voltages high enough to give appreciable current emits energetic electrons leading to the production of ozone by reactions such as e~ + 0 2 5»0 + 0 + e~ 0

+ o2

o3

in yields which exceed 10 to 20 molecules per negative ion. Bastien and Lecuiller (1) showed that one can reduce these ozone yields dramatically by carrying out the discharge in nitrogen (to thermalise the electrons) and flowing the products into air, thus producing superoxide by the reaction e" + 0 2

0~

This superoxide can be collected in an earthed aqueous solution below the discharge needle. I have modified their apparatus so that the ions (mainly 0 2 ) which carry the current impinge on a stirred aqueous solution but are discharged to earth in an anode cell separated from this by a capillary tube. Consequently superoxide chemistry occurring in the aqueous solution may be studied without interference from anodic oxidation. Details of this and other modifications will be published, the following brief account illustrates some of the properties of this source.

Results It was shown firstly that ozone yield is reduced appreciable by Bastien's

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

88

technique to values of typically 1 or 2 molecules per negative ion which are nearly independent of the voltage on the corona needle and hence of its current. The fraction of this which reacts with reagents in the solution depends on their reactivity with ozone and may exceed 0.3 for potassium iodide, but can be reduced by appropriate design of the apparatus so that it is very small for dilute ferrous sulphate, which reacts much more slowly that iodide ions with ozone.

2 Minutes exposure at

4 15^uA

Fig. 1: Reduction of cytochrome c solutions by the products of corona discharge, as described in text. Nitrogen was flowed over a platinum needle tip at 300 ml/min.; oxygen was flowed over the exposed solution at 60 ml/min. O-12/uM cyt. c solution in phosphate buffer at pH 7.2 + EDTA (50/uM) + catalase (10/ug/ml) .D-same solutions + sodium formate (lmM).A- same solutions + SOD (0.03 mg/ml, = 500nM) The behaviour of the system is illustrated in Fig. 1 which shows the reduction of cytochrome c solutions by

produced in the corona source,

running at 15^ua with -lOkV on the needle tip. If all the negative ions in this current produced

in the 5 ml of solution, reacting quantitatively

with cyt. C, then the yield of reduced cyt. c would be expressed by the interrupted line in this figure. In fact, the yield is only 60% of this and this percentage is only slightly increased by increasing the initial cyt. c

89 concentration or decreasing the corona current. Nevertheless, the reduction m y be attributed to

since it is suppressed by superoxide dismutase

(SOD) in the manner calculated from its reactivity with 0^· The "missing" 40% may therefore be reasonably attributed to dismutation of 0~ at the solution surface and this view is supported by studies on the reduction of eerie sulphate in acid solution, in which (contrary to earlier reports with a similar corona source in air (2)), the yield approaches 100% of theoretical. Both HO- and Η,Ο, resulting from its dismutation reduce Ce 4 + with 2 + 2 +

equal efficiency. Studies on ferrous sulphate and on Fe

-Cu

mixtures

indicate comparable yields of scavengeable superoxide but are complicated by a small interference from ozone. Nitrogen oxides might be expected in the discharge but only traces of nitrite were found in treated solutions, equivalent to less than 5% of the yield of superoxide. This technique has disadvantages of which ozone production is one, as noted above. Another is evaporation of water or volatile solutes caused by the carrier gas and the "electron wind" associated with the corona. There is evidence that tetranitromethane solutions react partially in the vapour above the solution, thus leading to reduction yields near 100% which are not reduced in the expected manner by other volatile superoxide scavengers such as bromine. Nevertheless, the source has useful applications, particularly since it produces 0~ free of OH, as evidenced by the absence of any effect of added formate on reaction yields as illustrated in Fig. 1 for cyt. c. It has been used to confirm that superoxide will effectively reduce methaemoglobin to oxyhaemoglobin in the presence of menadione though not in its absence, owing to conversion of menadione to a semiquinone which is more reactive (3). In related studies it has also been used to show that paraquat has a similar effect even in the presence of oxygen, as noted by Winterbourn (4), presumably owing to the extreme reactivity of the very low concentration of paraquat radicals which exist in equilibrium with oxygen: 0~ + PQ++

5-

0 2 + PQ +

90

References

1. Bastien, F., Lecuiller, M.: J. Chim. Phys. 70, 1692-69 (1973) 2. Rippe, Β., Lecuiller, M., Koulkes-Pujo, A.-M.: J. Chim. Phys. 1185-1190 (1974) 3. Sutton, H.C., Sangster, D.F.: J. Chem. Soc., Faraday Trans. 1, 695-711 (1982) 4. Winterbourn, C.C.: Biochem. Int. (in press)

SIMULTANEOUS MULTI-WAVELENGTH KINETIC SPECTROSCOPY: A NEW SET-UP EOR HJISE RADIOLYS IS STUDIES OF OXYGEN RADICAIS

Manfred Saran, Georg Vetter, Michael Erben-Russ and Wolf Bors. Institut für Biologie, Abt. Strahlenbiologie, GSF Forschungszentrum, 8042 Neuherberg, W. Germany

Introduction Pulse radiolysis techniques have in the past proven their extreme usefulness for investigating the chemical properties of oxygen radicals. The hydroxyl radical

(ΌΗ) can be obtained by fragmenting water molecules

with ionizing radiation H20

>

'OH, e¡ g , Η"

(ίψ 2 , H 2 , H 3 0 + )

/l/

Especially in ^O-saturated solutions where the primary yield of hydrated electrons (e~ ) is converted to Ό Η , aq N o 0 + e~ ¿

+ H„0

aq

> "OH + N. + OH"

¿

/2/

¿

the reactions of this important oxygen radical can easily be followed by spectrophotometry kinetic techniques. The superoxide ion

(Oj), the other oxygen radical of particular bio-

chemical importance, is amenable to investigation in oxygen-saturated solution, where hydrated electrons are scavenged by oxygen to give 0~ e

¡q+02

or in formate-containing

>

°2~

/V

solution, where also

"OH radicals are even-

tually converted to O^ by a series of conversion reactions "CH + HCDO"

>

œ

?

2

+

°2

H 2 0 + CD~ °2

+ 00

2

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

/4/ /5//

92

Of

the

carbon

containing

oxygen

radicals,

CD2

can

be

produced

by

reaction /4/ under anaerobic conditions, and the carbonate ion radical "00~ by reaction of "OH with dissolved bi-carbonate "OH + HCD~

>

"CD"

+ H20 .

/6/

When the radical under investigation has an absorption in the spectral range between 200 and 700 nm its kinetic parameters can be determined directly by mathematical evaluation of the absorbance build-up or decay curves. If other transient species hide or distort the spectral features of the radical under study, competition techniques using added radical scavengers can help elucidate the original reaction. It is quite clear that

the efficiency of pulse-radiolysis

laboratory equipment

largely

depends on the ease with which spectral and kinetic information can be collected at wavelengths between 200 and 700 nm and it was therefore decided to design a multi-wavelength simultaneous detection set-up that would provide maximal spectro-kinetic information from a single accelerator shot. Time-dependent spectral sc eins can be obtained by several different techniques e.g.: use of diode arrays, CCD's

(charge coupled devices) and

vidicon cameras. Hiere are commercially available optical multichannel analysers (OMA's) which have the advantage of extremely high spectral resolution (see SJMIC and HUNTER, these proceedings) but spectra can only be taken with a limited time resolution. Consideration of the inherent features of all these devices showed that only an array of photomultipliers, each connected to a separate channel of a fast transient digitizer, would meet the requirements of sensitivity in the far LA/ as well as of high time resolution. The following characteristics are essential for satisfactory performance of the system: (i) coverage of the spectral range of 200-700 nm with a wavelength resolution of at least 5 nm, (ii) time resolution less than 1 microsecond, (iii) detection limit under the least favourable conditions of less than 1% absorbance change.

93

Realization of the concept System

Overview

The specifications of the different components of the block diagram are: I)

Optical system:

Oonmercially available 1.5 m spectrograph

(RSV,

Hechendorf/Germany; Ebert-design). Two turn-table mounted gratings allow the following spectral ranges to be projected on the 15 multipliers at the 250 nm long exit slit: a) grating 1200 grooves/mm: spectral coverage 130 nm, 9 nm FM to EM distance b) grating 300 grooves/mm: spectral coverage 500 nm, 35 nm FM to EM distance II)

Photomultipliers and associated electronics: -

EMI side window tubes type 9783R, extended S5 spectral response

-

ZENER diode stabilized voltage divider chain for maximum sensiti-

(wavelength range 165-750 nm) vity and gain linearity

94 -

current to voltage conversion by high speed FET operational amplifier (LH 0062CH)

-

self regulating circuitry for high voltage supply

III) Btperiment control:

CñMAC compatible mainframe with circuitry for

operating light shutter, cuvette support, solvent delivery valves, light beam adjustment, high voltage regulation etc. For solution handling a step-motor operated syringe drive (HAMILTON) was interfaced to the computer allowing two different solutions to be mixed directly into the cuvette with mixing ratios from 100:1 to 1:100. IV) Data acquisition and evaluation: -

16 channel transient digitizer (VUKO-VKMC), computer controllable via IEEC-interface, vertical resolution 8 bits

(= 0.4%), time

resolution 500 ns/cycle -

WANG VP computer with periphery: graphic display, plotter, diskette drive

As a typical example of system performance the absorption spectra of the superoxide anion radical obtained from a single accelerator

shot are

given in the figure. As can be seen the spectral as well as time resolution are sufficient to cover and evaluate all relevant spectro-kinetic parameters of 0~ decay.

mAU ¿8

24

0

225

250

275

300[nm]

Spectra were taken at a) 100 us b) 9 ms c) 53 ms d) 210 ms after the pulse

RATE CONSTANTS OF SPARINGLY WATER-SOLUBLE PHENOLIC ANTIOXIDANTS WITH HYDROXYL RADICAIS.

W. Bors, C. Michel, M. Erben-Russ, B. Kreileder, D. Tait, M. Saran Institut für Biologie, Abteilung für Strahlenbiologie, GSF Forschungszentrum, 8042 Neuherberg, F.R.G.

In the course of our studies of the radical scavenging capabilities of phenolic antioxidants, based on the inhibition of the radical-induced bleaching of the water-soluble carotenoid crocin, we were interested in the rate constants of these compounds with both "CH and organic radicals (1). Furthermore, the contribution of a wide variety of non-functional substituents to the reactivities of phenols, e.g. their antioxidative efficiency, make quantitative structure activity correlation (QSAR) studies feasible (2-5). For a number of phenolic antioxidants or phenolic plant components (6-9) neither rate constants with "CH radicals nor transient spectra of the respective phenoxy or semiquionone radicals are known - assuming that these are ultimately formed after oxidative attack by "OH. Since kinetic studies of "OH radicals can only be performed in aqueous solution, problems arise fron the poor solubility in water of sane of the antioxidants. IVo different approaches will be described to overcome this problem. Hie phenolic compounds isoeugenol (4-hydroxy-3-methoxy-l-propenyl benzene) and sinapinic acid (3.5-dimethoxy-4-hydroxy-cinnamic acid) were fron Fluka, the others (ferulic acid: 4-hydroxy-3-methoxy-cinnamic acid; caffeic acid: 3.4-dihydroxy-cinnamic acid; the dihydroxy-acetophenones and nordihydroguaiaretic acid) except for Trolox c (6-hydroxy-2.5.7.8-tetramethylchromane-2-carboxylic acid; a gift of Hoffmann-LaRoche, Basel) were from Aldrich-Europe. tert-Butanol and acetonitrile were from Fluka and 2.6-dichlorophenol indophenol (DCIP) from Κ & Κ. All reaction rates were determined by competition kinetics (10), using the multi-wavelength array of our pulse radiolysis facility (for details see Saran et al., these proceedings) .

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

96 One obvious approach to cope with the poor hydrophilicity of these substances is to lower the required concentrations by using very sensitive competitors. In previous pulse-radiolytic studies we had established that the electron acceptor DCIP is a highly effective scavenger of *0H radicals (4.0xl010 M - 1 s~ 1 ; ref. 11), with the added advantage that the 610 ran absorption is far beyond any absorption by phenoxy radical species. We now determined that not only the initial bleaching yield but a transient absorption at 440-460 run both allowed the evaluation of competition kinetics, provided there was no superimposed transient absorption of the phenol. The drawback of DCIP is the fact that the bleaching rate is very sensitive towards interference by secondary radicals (formed from the competitors). Another approach to avoid the solubility problem would be to add a fully water-miscible solvent promoter. Pcetonitrile (ACN) proved to be a good candidate, because it is both a widely used solvent and has a rather low reaction rate with 'CH radicals (2.5xl07 M - 1 s - 1 ; ref. 12). In the case of ACN experiments, DCIP cannot be used as "OH-scavenger because the ACN Q radical readily reacts with this compound. Tert-butanol = 5.2x10 -1

M

-1

s

; réf. 13) was used instead, in addition allowing the direct obser-

vation of the phenoxy radical transient spectra. Determination of k . œ of the vitamin E model compound Trolox C (14) at three concentrations of ACN showed that only at the highest concentration used (100 mM), an increase in k . ^ was observed - probably due to adventitious scavenging of the ACN radical itself (Table I). TABLE I -

[Trolox c] /UM 100 II tl

5-50 1.5 - 15

REACTION RATES OF TROIjOX c WITH -OH RADICAIS AT VARIOUS ACETONITRILE CONCENTRATIONS. [ACN] mM

[t-BuOH] mM

[DCIP] /uM

k -0H xlO" 10 M " 1 s _ 1

100 20 5

1.0 - 10 1.0 - 7.5 1.0 - 10

-

-

7.1240.65 3.38+0.79 3.44+0.53

-

-

-

-

35 10

4.42+0.30 3.54+0.55

-

r q (%) (see text)

22.5 - 42.4 6.4 - 12.8 1.4 - 3.5 -

r q : kinetic probability of formation of ftCN radicals by direct scavenging of 'CH radicals.

97 The Tq-values (reaction probability, obtained from the respective reaction rates and concentrations) show that below 15% of ACN radicals being formed by direct scavenging of "CH there is no interference in the competition kinetics. Thus, 20 itM solutions of £CN can act to promote the water solubility of marginally soluble compounds without disturbing the determination of the rate constants with hydroxyl radicals. Por the evaluation of the competition kinetics, the transient absorption bands of the radical intermediates (hydroxycyclohexadienyl as primary and phenoxy/semiquinone radicals as secondary species) cannot easily be assigned (11,15). The selection of suitable absorption bands for evaluation of competition kinetics requires that these absorptions represent primary radical species and that there be no overlap of solute depletion and product formation. Ctoing to the multi-wavelength arrangement, we were able to evaluate several absorption and transmission bands simultaneously. Figure 1 depicts the transient spectrum of Trolox c after "OH attack. Of the three absorption peaks, only the one at 255 nm gave a reasonable competition plot - it also did not decay during the observation period.

280

360

440

520

L

Figure 1 - Transient spectrum of Trolox c with ·ΟΗ radicals. Concentration of Trolox c 0.1 mM, of acetonitrile 20 mM, ^O-saturated solutions at {4! 8.2; dose per pulse - 2 krad; Observation periods: 40 /usee ( • ), 160 /usee ( A ), 1.18 msec ( Τ ); ordinate scale 0.1 AUFS. Table II lists all the reaction rates with *CH radicals which we obtained in this study, together with the wavelength regions which gave the most accurate values of the competition kinetics.

98 TABLE II - RAIE CONSTANTS OF PHENOLIC ANTIOXIDANTS WITH -CH RADICAIS. compound

[ACN]

competitor

mM isoeugenol _

II

-

_ -

ferulic acid

observed wavelength (region)

xlO

-IO 0 0 -! -1 M s

t-BuOH DC IP

286 610

2.82+0.10 2.69+0.23

DCIP

455,610

0.61+0.05

-

t-BuOH DCIP

360 610

1.38+0.14 1.64+0.10

-

t-BuOH

265,325,442

2.41+0.60

-

DCIP

460,610

1.78+0.30

2' ,5'-dihydroxyacetophenone

-

DCIP

610

0.46+0.01

nord ihydroguaiaretic acid

-

t-BuOH

314

1.3340.17

5 20

t-BuOH

255

-

DCIP

3.44+0.53 3.38+0.79 4.42+0.30 3.54+0.55

sinapinic acid _

II

_

caffeic acid 2 '. 4 1 -dihydroxyacetophenone

Trolox c _

II

_

_

II

_

_

II

_

-

20

—

II

II

II

610 455

In summary we have demonstrated (a) two competition methods which are mutually complementary for phenolic compounds, (b) that rate constants with *0H of water-insoluble compounds can be obtained by using up to 20 mM solution of ACN to promote (enhance) solubility, (c) that the transient spectra of phenolic antioxidants after "CH attack are too diverse to unequivocally identify primary and secondary radicals. Detailed studies of the pulse-radiolytic oxidation of such compounds by various types of radicals should help clarify the individual mechanisms. The fact that practically all k . ^ are in the diffusion-limited range demonstrates the high efficiency of these substances to scavenge radicals, an important aspect of their antioxidant efficacy (16-18).

References : 1. Bors, W., Michel, C., Saran, M.: Oxy Radicals and Their Scavenger Systems, Vol. I. Molecular Aspects (G. Cohen, R.A. Greenwald, Etìs.), Elsevier, New York, 38-43 (1983) 2. Job, D., Dunford, H.B.: Eur. J. Biochem. 66, 607-614 (1976) 3. Slabbert, N.P.: Itetrahedron 33, 821-824 (1977) 4. Dewhirst, F.E.: Prostaglandins 20, 209-222 (1980)

99 5. Lee, T.T., Starratt, A.N., Jevnikar, J.J.: Phytochem. 21, 517-523 (1982) 6. Pratt, D.E.: J. Rxx3 Sci. 30, 737-741 (1965) 7. Daniels, D.G.H., Martin, H.F.: J. Sei. Fbod Agrie. 18, 589-593 (1967) 8. Cort, W.M.: Food Technol. 28, 60-66 (1974) 9. Dugan, L.R.: in Autoxidation in Food and Biological Systems (M.G. Simic, M. Karel, 0äs.), Plenum Press, New York, 261-282 (1980) 10. Aîams, G.E., Boag, J.W., Currant, J., Michael, B.D.: in Pulse Radiolysis (M. Ebert, J.P. Keene, A.J. Swallow, J.H. Baxendale, EÜs.), Akademie Press, New York, 131-143 (1965) 11. Bors, W., Saran, Μ., Michel, C.: J. Phys. Chem. 83, 2447-52 (1979) 12. Neta, P., Schuler, R.H.: J. Phys. Chan. 79, 1-6 (1975) 13. Willson, R.L., Greenstock, C.L., Aîams, G.E., Wageman, R., Dorfman, L.M. : Int. J. Radiat. Phys. Chan. 2' 211-220 (1971) 14. Cort, W.M., Scott, J.W., Araujo, Μ., Mergens, W.J., Cannalonga, M.A., Osadca, M., Harley, H., Parrish, D.R., Pool, W.R.: J. An. Oil Chem. Soc. 52, 174-178 (1975) 15. Steenken, S., Neta, P.: J. Phys. Chan. 83, 1134-37 (1979) 16. Slater, T.F.: in Oxygen Free Radicals and Tissue Damage (CIBA Pound Symp.), 65, 143-76 (1979) 17. Wills, E.D.: Int. J. Radiat. Biol. 37, 403-414 (1980) 18. logani, M.K., Davies, R.E.: Lipids 15, 485-495 (1980)

A PULSE R A D I O L Y S I S

STUDY OF THE A D D I T I O N OF H Y D R O X Y L

R A D I C A L S TO N I T R O N E

R.

SPIN

FREE

TRAPS

Sridhar

Biomembrane Research Foundation, Oklahoma

P. C. Beaumont

Laboratory, Oklahoma Medical C i t y , O k l a h o m a 7 3 1 0 4 , USA

and E. L.

Research

Powers

L a b o r a t o r y of R a d i a t i o n B i o l o g y , D e p a r t m e n t of U n i v e r s i t y of T e x a s , A u s t i n , T e x a s 78712, USA

Zoology,

Introduction

Hydroxyl normal

radicals

(HO") m a y be

and p a t h o l o g i c a l

involved

processes

in a v a r i e t y

(1).

by r a d i o l y s i s of w a t e r has b e e n a s s o c i a t e d tained

by cells exposed

to

ionizing

with damage

radiation

o b s e r v a t i o n of h i g h l y r e a c t i v e

ephemeral

HO" by e l e c t r o n

spectroscopy

cult.

spin r e s o n a n c e

Such r a d i c a l s

with

compound

the s h o r t - l i v e d

(3).

free r a d i c a l

produce a more stable radical more amenable

to i n v e s t i g a t i o n

have been synthesized ability

Direct such

as

indirect

technique,

is allowed

('the s p i n ' )

to

a

suitable react

in o r d e r

to

('the spin a d d u c t ' ) which

is

by E S R .

A v a r i e t y of

for use as spin traps based

to p a r t i c i p a t e

HO"

sus-

(ESR) is d i f f i -

by the

In this

('the spin t r a p ' )

(2).

radicals

are u s u a l l y d e t e c t e d

t e c h n i q u e of spin trapping diamagnetic

of

The f o r m a t i o n of

in a d d i t i o n

on

nitrones their

r e a c t i o n s of the type

shown

below.

+ Ar-C=IjJ-C(CH,) 3 3 H 0_ spin t r a p

+

HO' short-lived

> radical

HO Ar-C-N-C(CH,), HO. spin a d d u c t

where Ar- is PBN

;2-PyBN

;3-PyBN

;4-PyBN

; 3-PyOBN

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

; 4-PyOBN

102

Before spin trapping can emerge as a reliable technique for the quantitative analysis of reactive species such as HO", it is necessary to know the kinetics and the pattern of addition of the radical to the spin trap, as well as the kinetics of other competing reactions that can deplete the spin adduct

Results and Discussion Pulse radiolysis and time-resolved spectrophotometric techniques were applied to study the kinetics of addition of HO* to a series of nitrone spin traps.

The rate constants for the

reaction of HO* with the spin traps were determined by measuring the rate of growth of the transient species in nitrous oxide staturated aqueous solutions (Table 1 ; Figure 1).

Figure 1. Transient absorption spectra obtained after the reaction of PBN (panel a) and 3-PyOBN (panel b) with HO* radicals generated by pulse radiolysis (absorbed dose of about 40 Gy) of N 2 0 saturated aqueous solutions. In each case the top spectrum (A) was obtained 8 ys after the pulse and the bottom spectrum (J) was obtained 269 MS after the pulse. The other spectra were obtained at intermediate times as shown.

103

Table 1.

Bimolecular rate constants for the reaction of spin

traps with both the hydroxyl radical and the hydrated electron Bimolecular rate 10 9 L m o l - 1 Spin trap

s-1

HO' radical

PBN

6.1

e

aq 18

(lit. (4) value 8.5)

3-PyOBN

4.5 (lit. (5) value 4.5)

25

4-PyOBN

4.0 (lit. (5) value 3.5)

31

2-PyBN

9.6

25

3-PyBN

4.6

24

4-PyBN 8.3 29 Errors in the measurement of rate constants in our experiments are estimated to be ± 10%.

The transient absorption spectrum resulting from the addition of HO* to PBN showed two peaks decaying at different rates, possibly suggesting formation of more than one product by the addition of HO' to the spin trap.

While HO* can add to

the nitrone group of the spin trap, (α-phenyl N-tert-butylnitrone

(PBN)), to yield the spin adduct with a nitroxyl

func-

tion, the possibility of HO* addition to the aromatic ring of PBN cannot be discounted.

Since e

formed by radiolysis of water, can react with spin aq _ traps, the rates of ea(^ addition to the various spin traps were also determined butanol

(Table 1) in experiments in which tert-

(0.1 M) was added as a scavenger for HO*

radicals.

Nitrous oxide saturated solutions of 4-PyOBN (1 to 3 χ 10-1* M) containing isopropanol

(0.1 M) were also studied.

The

principal species produced in this system is ( C H 3 ) 2 C - O H , the carbon centered radical produced by the action of HO* on isopropanol.

It was possible to monitor the rate of addition

of this carbon centered radical to 4-PyOBN spectrophotometrically at 270 nm and a bimolecular rate constant of 1 χ

104 IO 8 M - 1 s - 1

w a s found for this r e a c t i o n .

For s p i n

trapping

s t u d i e s w i t h 4 - P y O B N , HO* r a d i c a l s w e r e g e n e r a t e d by Fenton system

(2.5 χ ΙΟ'Ή F e S O H ,

(50 m M , pH 7 . 2 ) ) . was

included

radical.

1% H 2 0 2

In p a r a l l e l e x p e r i m e n t s ,

in o r d e r to p r o d u c e

the

in p h o s p h a t e

buffer

isopropanol

( C H 3 ) 2 C - O H as the

(1 M)

secondary

The E S R s i g n a l d u e to the HO* s p i n a d d u c t of

4-

P y O B N w a s a b o u t 20 t i m e s less i n t e n s e t h a n the s i g n a l d u e the s p i n a d d u c t from

( C H 3 ) 2 C - O H and 4 - P y O B N .

a c c u m u l a t i o n of E S R - d e t e c t a b l e s p i n a d d u c t s w a s m u c h c o m p a r e d to the b i m o l e c u l a r r a t e c o n s t a n t radiolysis)

for the a d d i t i o n of H O ' or

T h e s e r e s u l t s s u g g e s t that

(CH3)2C-OH,

to

T h e r a t e of lower

(found by p u l s e

( C H 3 ) 2 C - O H to

4-PyOBN.

in c o n t r a s t to

HO",

a d d s m o r e s e l e c t i v e l y to the n i t r o n e m o i e t y of 4 - P y O B N and s p i n a d d u c t from

( C H 3 ) 2 C - O H m a y be s t a b l e

a d d u c t d e r i v e d from HO* and

t h a n the

the

spin

4-PyOBN.

References 1.

Willson, R.L.: O x y g e n F r e e R a d i c a l s and T i s s u e D a m a g e (Ciba F o u n d a t i o n S y m p o s i u m 6 5 ) , p p 19-42. Excerpta M e d i c a , A m s t e r d a m , 1979.

2.

Powers, E.L.:

I s r a e l J. C h e m .

10, 1199-121

(1972).

3.

Janzen, E.G.:

A c c o u n t s C h e m . R e s . _4, 31-40

(1971).

4.

Greenstock, C.L., Wiebe, R.H.: 1563 (1982).

5.

N e t a , P., S t e e n k e n , S., J a n z e n , E . G . , S h e t t y , R . V . : P h y s . C h e m . 84, 532-534 (1980).

C a n . J. C h e m . j>0,

1560J.

Acknowledgements T h e e x p e r i m e n t s and a n a l y s e s of the d a t a p r o d u c e d w e r e p e r f o r m e d at the C e n t e r for Fast K i n e t i c s R e s e a r c h at the U n i v e r s i t y of T e x a s at A u s t i n . T h e C F K R is s u p p o r t e d j o i n t l y by the B i o t e c h n o l o g y B r a n c h of the D i v i s i o n of R e s e a r c h R e s o u r c e s of N I H (RR00886) a n d by the U n i v e r s i t y of T e x a s . This research was partially supported by grant #CA-3022 from NCI, DHHS. We t h a n k M s . A n i t a H i l l for a s s i s t i n g in the p r e p a r a t i o n of t h i s manuscript.

105

GENERAL DISCUSSION

BORG: I want to make a comment about both Don SAWYER'S & Leland SMITH'S presentations. Each of them raised the question of whether chemistry in aprotic solvents had biological significance. This issue has been discussed at previous conferences by Don SAWYER and me, as well as by others. In the hydrophobic centers of membranes composed of phospholipid bilayers might we find such chemistry? At hydrophobic sites on enzyme surfaces might we find such chemistry? I want to report a very preliminary result from Aryeh FRIMER of Bar Ilan University (who is spending a sabbatical year with us) which is consistent with the idea that chemistry within membranes may be similar to that in aprotic solvents. To start with, we are studying what is only a weak surrogate for membranes: namely, we are using bulk lipids. Our first example was oleic acid, and as a test for the nucleophilic behaviour of superoxide FRIMER utilized K0 2 added to stilbene dibromide. If 0 2 acts as a strong nucleophile, as it does in strongly aprotic solvents, then one will observe formation of a blue colour over some minutes as dehydro-dehalogenation of the stilbene dibromide occurs. By that criterion, oleic acid is a weakly protic environment, because the blue colour did not materialize, and the yellow K0 2 slowly disappeared, presumably by disproportionation. However, a different result was obtained by using the ester, methyl oleate, rather than the somewhat more polar acid. Even after the ester was allowed to hydrate as much as possible by being shaken with water and separated, it appeared to be an aprotic environment by the K0 2 /stilbene dibromide test. Of course this preliminary result doesn't tell us what the centers of lipid bilayers in liposomes and cellular membranes are like, but FRIMER may be able to cast more light on that soon. Nonetheless, these first findings do suggest that aprotic chemistry may, indeed, be of direct interest to life scientists. SMITH: I hope you did not misunderstand what I said about K0 2 in crown ether and DMSO. This is a useful system for many things, but it is so complicated that I would not recommend it as model of any biological system. SINGH: Prof. AFANAS'EV, did you look for any products that may have been formed inadvertently or unexpectedly from your solvent acetonitrile? AFANAS'EV: in.

No, acetonitrile is very inert under the conditions we worked

FOOTE: I want to make one brief comment on Prof. SMITH'S and also Don SAWYER'S paper. Long before anybody suspected that metal ions might be that important, we did some work on reactions of tetramethylammonium superoxide with tert-butyl and tert-amyl hydroperoxide. The important finding was that, whatever the mechanism, a very large yield of alkoxy radicals was produced. The mechanism is probably not the (organic) Fenton reaction, but there definitely was a very large yield of tertiary alkoxy radicals. BORS:

I want to come back to the comment of Don BORG. I don't want to

106 discredit his argument, but going from an aqueous solution via a micellar solution to an aprotic solvent, the kinetic parameters change considerably. Thus, the background of our studies of alkoxy radicals in aqueous solution was the question: would it be possible for hydroperoxides derived from membrane phospholipids to be released into the aqueous bulk of the cell and then be reduced to alkoxy radicals. From our pulse-radiolysis experiments it seems that such alkoxy radicals are reacting extremely rapidly and that they are more reactive in aqueous solution than they are in an organic solvent. BORG: There is no question about the reactivity of alkoxyl radicals, but there is also biological significant reactivity of some peroxy radicals, especially certain smaller ones such as the secondary peroxyl radical from dimethyl sulfoxide (DMSO) following hydroxyl radical attack. We also heard discussion today of a reactive secondary peroxyl radical from halothane. The point is that a peroxyl 1 radical need not to be the termination of a sequence of biologically important reactions, because some are quite reactive in a biochemical milieu. Jim RALEIGH will have something to say about the relevance of the peroxy radical in terms of biological damage form DMSO. Many biologists and biochemists use DMSO as a scavenger for OH radicals, because of the high rate constant for the reaction and then appear to assume that's the end of the chain of oxidative damage. Jim, can I induce you to say something about that? RALEIGH: I would like to ask Dr. SMITH whether he knows if the oxygen from DMSO is incorporated into cholesterol and whether dimethyl sulfide is formed in this system. SMITH: We did not look at any of the liquid products. Any of the other possible oxidized products might be formed. A few other things about this system. DMSO may potentially complex with hydroperoxide so that one may have other reactions which could possibly explain ketone formation. One might try to find out whether cholesterol frees the putative R00H-DMS0 complex from whatever forces might be involved, thereby getting reactions further than would be seen in the absence of cholesterol. POWERS: Ulis is to follow-up Dr. BORG's remarks and to comment also on interpretation of experiments with hydroxyl radical scavengers in cell systems. There is much evidence that free radicals are formed in irradiated dry bacterial spores and that oxygen must combine with these before they become part of the oxygen effect. However, while the formation of the radical-oxygen complex is very rapid (— "diffusion—controlled") and is irreversible, the peroxy radical does not result in biological injury unless an undescribed set of time- and temperature-dependent reactions occur. After exposure to oxygen, the spores must be raised to 60°C for 30 minutes for the full oxygen effect to develop. This biological system requires the peroxy radical to do energy-requiring chemistry before they are damaging. Second point: the fact that an alcohol reduces radiation sensitivity of a cell is no proof that OH radicals are involved in radiation damage. Some alcohols (e.g. ethyl) that form -carbon alcohol radicals after OH reaction do protect if the concentration is right; but others (e.g. t-butyl) that form fl-carbon alcohol radicals do not protect. It is the alcohol radical that is the protector, removal of OH per se is

107 riot important. Third point: the use of DMSO can give no information at all on OH action, whatever the result (POWERS and TALLENTIRE, in "Physical and Chemical Actions of Radiations", M. Haissinsky, ed., Masson, Paris (1968), Vbl. 12, pp. 3; EWING and POWERS, in "Radiation Biology in Cancer Research", Meyn and Withers, eds., Raven Press, New York (1980), pp. 143). So DMSO we reject completely as a hydroxyl radical scavenger in bringing about its effects. As a matter of fact that compound is so mysterious that we don't allow it in our lab at all! PRYOR: In reference to Smith's comment about DMSO and cholesterol hydroperoxide forming a complex: we really should study the decomposition of a variety of hydroperoxides. All hydroperoxides undergo 0-0 bond horaolysis with the same rate constant. That is, rate of radical production by unimolecular homolysis is the same for all ROOH, regardless of the nature of R. The thermodynamic data is very clear on that. Yet one bit of old data that I know on linoleic acid hydroperoxide gives an activation energy of something like 20 kcal/mol for its homolysis. That may be a chain process and an induced decomposition or a molecule-assisted decomposition (W.A. PRYOR in "Free Radicals in Biology", Academic Press (1976) , Vol. I, pp. 9) . Nevertheless, it is true that peroxides that we are most interested in (such as cholesterol hydroperoxide and linoleic acid hydroperoxide) are the very hydroperoxides for which there are not good data on the rates of homolysis. If we had good data on linoleic acid hydroperoxide or cholesterol hydroperoxide, then we could ask the question: does DMSO influence the rate of decomposition? BIELSKI : I would like to make a very short comment on the use of DMSO. Some time ago we attempted to use CH3SOCH3-KO2 systems for various studies of 05 but had to give it up because commercial K0 2 contains a significant amount of KOH. The latter reacts with DMSO to yield the "dimsyl anion", CH3SOCH3 + KOH — CH3SOCH2 + K+ + H 2 0. Tests with various superoxide indicators proved that the dimsyl anion is much more reactive toward these compounds than is 0 2 . Since the amount of dimsyl anion formed in this system is dependent upon the quantities of KOH and H 2 0 present, controls are not very reproducible. NIKI: I am interested in the interactions between 0 2 and hydroperoxide. Prof. FOOTE has shown that t-butoxy radical is formed, but I've been wondering whether it comes directly from the hydroperoxide or it comes from the tertiary peroxy radical. We know that the bimolecular interactions of tertiary peroxy radicals produce alkoxy radicals. I've been wondering if someone has worked with secondary hydroperoxides and 0 2 , because secondary peroxy radicals will not give alkoxy radicals. So the question is, whether or not secondary alkoxy radicals are formed by the interaction between 0 2 and secondary hydroperoxide. FOOTE: Our only evidence on the tertiary system was that we did not see the cage recombination product, di-t-butyl peroxide, which we would have expected to see if the alkylperoxy radicals had been formed.

THE REACTIVITIES OF ORGANIC OXYGEN (OXY) RADICALS

Michael G. Simic, Edward P. L. Hunter Center for Radiation Research, and Center for Chemical Physics National Bureau of Standards Washington, DC 20234, USA

Introduction The organic oxy radicals also referred to as oxyl radicals have commanded a substantial share of interest in fields as diverse as combustion (1), degradation of polymers (2) and food chemistry (3).

Lately, the role of oxy radicals in radi-

ation biology, stress physiology, pathology, ageing and metabolism is becoming increasingly a topic of various general and specialized conferences (4).

In addition, participation of

oxy radicals in drug metabolism, a variety of physiological disorders and perhaps even in neuro and brain chemistry should be expected to become a pertinent topic in the coming years. What are the species we are talking about —

the species we

are increasingly becoming aware of and concerned about?

The

oxy radicals can be conveniently subdivided into three major classes : HROO· or HArOO·

peroxy radicals

HRO· HArO·

alkoxy radicals aroxy radicals

where HR- represents an alkyl radical and HAr- an aryl (aromatic ring) radical (not an aromatic substituted alkyl radical). Purely for convenience of writing chemical reactions involving these radicals, the aliphatic compounds are represented as H 2 R since in a majority of cases they behave as two electron redox

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

110

systems.

This allows us to indicate abstraction of a hydrogen

atom from the parent compound and oxidation of the resulting radical.

For example when ·0Η is used as a free radical gener-

ator (5) : H 2 R + -OH ->• HR· + BJC HR· +

Ox + R

(1)

+ Red (H+)

(2)

where Ox represents an oxidizing agent and Red its reduced form. Abstraction reactions from aromatic rings are much less favorable because of their high bond energies, e.g. C-Η in benzene is 112 kcal/mol (6).

Consequently the same reactant used in

reaction (1) would add to the ring rather than abstract: HAr + *0H + HÀr-OH

(3)

HÂr-OH + Ox + Ar-OH + Red (H+)

(4)

For substituted aromatic compounds the reactant may attack both the aliphatic substituents and the aromatic ring, resulting in a mixture of alkyl and cyclohexadieny1 free radicals. *-H,R-ÂrH-0H

(5a)

•HR-ArH + H 2 0

(5b)

H,R-ArH + ·OH The advantages of this type of notation are obvious from these three sets of reactions, and using this presentation more complex structures could be constructed when necessitated. This notation is to be compared to e.g. ROO·, RO·, φ0· etc., frequently found in literature. A great deal of information concerning oxy radicals has been derived from the measurements of products in various autoxidation processes (2, 7), via indirect deductions and reconstruction of the associated transient free radical steps.

Inspite

of intrinsic difficulties some remarkable conclusions regarding oxy radicals were reached. More recently, the trend has been towards rapid in situ generation (pulse radiolysis (5), pulsed laser photolysis (8),

111

and direct monitoring (ESR, kinetic spectrophotometry, kinetic conductivity, etc.) of oxy radicals.

These techniques have

contributed greatly towards characterization of the oxy transient species, the description of their properties and kinetic parameters and have lead to the rebirth of autoxidation and antioxidant research as witnessed in particular by this III. International Conference on Oxygen Radicals. There are many classes of aryloxy (9) or simply aroxy radicals as we prefer to call them which are derived from aromatic hydroxyl derivatives.

Very often they are erroneously referred

to as phenoxy type radicals and are designated as φ0·.

We are

also suggesting here that proper distinction should be made between various classes of aroxy radicals and we shall distinguish here a few. Phenoxy radicals, ΗφΟ·. They are derived from the hydroxy and polyhydroxy derivatives of benzene and substituted benzenes and are the intermediates in the reactions of man-made (BHA, BHT, etc.) and natural (sesamol, caffeic acid, eugenol, capsaicin, etc.) antioxidants. Naphthoxy radicals, HNO·. These are derived from the hydroxy derivatives of naphthalene, which are not found among natural products. Chromanoxy radicals, HChO·. They are derived from hydroxy chroman (10) whose derivatives are known as natural (Vitamin E) and man-made (Trolox (11)) antioxidants. In this presentation we shall briefly address some basic modes of generation of aroxy radicals and also highlight a simple organic oxy radical COj, known as the carbonate radical, which can be conveniently exploited for the generation of these particularly interesting and relevant oxy radicals.

112

Experimental Pulse radiolysis and detection systems Primary water radicals (e , ·0Η, Η) were generated by a Febeaq tron 705, which can deliver a variable dose in a 50 ns pulse of 2 Mev electrons (12) .

Secondary radicals and further reaction

intermediates were monitored by a kinetic spectrophotometry system consisting of a Xe lamp (Varian, PS 300-1), monochromator (Bausch & Lomb, 1350 grooves/mm) and a photomultiplier (RCA 4840).

The signals were processed by a transient digitizer

(Tektronix 7612 AD) capable of sampling up to 200 MHz with 8bit resolution, and which was interfaced to a computer (DEC 11/32) where digitized data can be stored and analyzed.

Doses

of 200-800 rad were used for the formation and 5-10 krad for the decay kinetics measurements. Transient absorption spectra Broad band absorption spectra of transients were obtained by dividing the analyzing light beam with an Inconel beam splitter, with the transmitted beam focused onto the monochromator and the reflected beam onto a spectrograph (Jarrell Ash, Mark X, No 82-484) coupled to an optical multichannel analyzer (EG & G Princeton Applied Research) referred to as OMA system (12, 13).

This system consists of a silicon photodiode array (Model

1420) and a controller and a processor unit (Model 1218 and 1215) .

This system was modified to be used with an external

pulsed power supply so that gated spectra could be recorded at varying times after a single radiation pulse, with observation times ranging from 1-50 us depending upon the competing needs of temporal resolution and signal integration. Transient absorption spectra were derived from the transmission spectra before and after the pulse, Fig. 1.

113

λ, nm Fig.l.

IQ and I transmission spectra before and after radiation pulse, respectively; DC, dark count (signal without analyzing light). Aqueous solution of 1 mM 1-naphthol, 0.1 M Na 2 C0 3 , pH = 11.2, 20 °C.

The processor unit allows arithmetic operations such as dark count subtraction, conversion to logarithmic ratios for a whole spectrum, thereby converting raw signal data shown in Fig. 1 into transient absorption spectra presented in Figs. 4 and 5 (12, 14).

At present, spectra could be recorded in the 300-650

nm region, which could be extended down to 200 nm with a better optical system.

In Fig. 1, the signal cutoff at 360 nm is a

consequence of the solute absorption. Arrangements were also made to have a simultaneous recording of the OMA and the photomultiplier signals.

Thus, whole transient

spectra at a given time interval and absorbance at a given wavelength could be recorded with a single radiation pulse*. *

Certain commercial equipment, instruments, or materials are identified in this paper in order to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified are necessarily the best available for the purpose.

114

Results and

Discussion

The hydroxy radical,

·0Η, w a s g e n e r a t e d r a d i o l y t i c a l l y

in

aque-

ous solutions saturated w i t h N 2 0 , w h i c h w a s u s e d to convert h y d r a t e d e l e c t r o n to

-OH (5).

Consequently

tions were almost exclusively a m o u n t of Η a t o m

(8%).

the deprotonated form

OH radical

A t h i g h p H the ·0~, p K

a hence

a strong oxidizing agent, COf-

= 11.9

+

the reaction

(92%) w i t h a

small

»0H r a d i c a l e x i s t s (5).

The

k = 4.5 χ 10

1

M" s"

1

in

·0Η r a d i c a l

·0Η ·+ C 0 7 + 0 H ~ 8

the

condi-

is

(6)

(15, 16)

T h e c a r b o n a t e r a d i c a l is a s i m p l e o r g a n i c o x y r a d i c a l , a n d can b e r e p r e s e n t e d by a r e s o n a n c e s t r u c t u r e .

It d i s a p p e a r s

in a

second order process to give products that have not y e t been satisfactorily

characterized 2CCf^ ->· p r o d u c t s k = 1.0 χ 1 0 7 M ^ s " 1

(7) IO6

A s i m i l a r r a t e c o n s t a n t w a s o b s e r v e d p r e v i o u s l y k = 7.5 χ M s "

1

(15, 16).

The

long l i f e t i m e of the C O j r a d i c a l is m o s t

l i k e l y d u e to its r e s o n a n t s t r u c t u r e o r an u n s t a b l e Both reactions

(6) a n d

dimer.

(7) w e r e f o u n d to p r o c e e d w i t h

negligi-

ble activation energy, E constants

~ 0 , s i n c e the c o r r e s p o n d i n g r a t e a s h o w e d n o t e m p e r a t u r e d e p e n d e n c e in the 10-70 °C t e m -

perature region. reaction

It h a s b e e n s u g g e s t e d t h a t the p r o d u c t s

(7) c o u l d r e s u l t f r o m t h e t r a n s f e r of

·0

(16), b u t s u c h a p r o c e s s s h o u l d r e q u i r e a c o n s i d e r a b l e

activa-

t i o n e n e r g y , e v e n t h o u g h the b o n d b r e a k i n g a n d f o r m a t i o n occur corcertedly.

A process more consistent with

b e d i m e r i z a t i o n to p e r o x y d i c a r b o n a t e . reaction

is

~ 0 would

(17, 18).

The

(17)

4COj + 02

+ 2CO§- + 2 C 0 2

(8)

B e i n g a s t r o n g o x i d a n t , the CO3 r a d i c a l is e x p e c t e d to s t r a c t e a s i l y an e l e c t r o n

would

Whatever the product

(7) the u l t i m a t e p r o d u c t is o x y g e n

overall stoichiometry

in

to f o r m C O * -

f r o m t h e c o n j u g a t e b a s e s of

naphthols and trolox c carbinol, whose structures

are

abphenol,

of

115

OH

OH

phenol

1-naphthol

2-naphthol

The general reaction transfer

trolox

for t h e i r o x i d a t i o n

c-carbinol

is a d i r e c t

electron

process HArO

+ CO3

HArO·

+ C0|"

(9)

a n d t h e a s s o c i a t e d r a t e c o n s t a n t s a r e l i s t e d in T a b l e

1.

T A B L E 1. K i n e t i c a n d s p e c t r o s c o p i c p a r a m e t e r s for a r o x y r a d i c a l s in a q u e o u s s o l u t i o n s of 0.1 M N a 2 C 0 3 , 1 a t m N 2 0 , p H = l 1 . 2 , a t 20 °C. Solute, S

Phenol 1-Naphthol 2-Naphthol Trolox C carbinol

Radical,

k(co;+s) , 1

1

•R

M~ s ~ (a)

Ηφ0· 1-HNO· 2-HNO·

4.7x10® 3.lxlO9 1.3x10 9

HChO

2.2x10 9

V

• "MX ,

kcal/ mol ~0 1.9 ~0

ran

400 393 Fe 3 + 0 2 > Fe 2 + 0 2 > Fe 3 + 0 2 H- + L·

(6) (7) (8)

F i n a l l y , the modified Fenton mechanism acting on trace l i p i d hydroperoxide i s generally accepted as being the most important i n i t i a t i o n reaction in the continued peroxidation of l i p i d . The reaction merely involves the metal catalyzed breakdown of l i p i d hydroperoxide. Fe2+ + LOOH

> Fe 3 + + LO· + .OH

(9)

As mentioned above, c r i t i c a l to all of these schemes i s the presence of reduced iron Fe 2 + either free or complexed. The source of reducing equivalents to keep the iron in the reduced state is either from .O2- radicals or by direct enzymatic reduction by appropriate reductase. * This work supported in part by the National Cancer Institute Research Grant CA15655 and CA10977

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

138 In an attempt to shed some l i g h t on the r e l a t i v e importance of these v a r i o u s mechanisms, we have c a r r i e d out k i n e t i c s t u d i e s i n which we f o l l o w e d p e r o x i d a t i o n by diene formation monitored by i t s absorption at 232 nm and by e l e c t r o n s p i n resonance (ESR) s p i n t r a p p i n g . In the l a t e r case, one takes advantage of the f a c t that c e r t a i n n i t r o n e d e r i v a t i v e s are capable of t r a p p i n g l a b i l e r a d i c a l s , such as .OH and .O2", forming r e l a t i v e l y s t a b l e r a d i c a l s t h a t can be monitored i n k i n e t i c r e a c t i o n s d i r e c t l y by ESR. There has been concern expressed by several i n v e s t i g a t o r s (1-3) t h a t s p i n t r a p p i n g may not be d e t e c t i n g l a b i l e .OH r a d i c a l s and t h a t the n i t r o n e adduct used f o r i t s i d e n t i f i c a t i o n may have r e s u l t e d from rearrangement or r e a c t i o n s of n i t r o n e adducts other than the .OH adduct. It has been suggested t h a t the popular n i t r o n e DMPO which i s used t o t r a p .OH r a d i c a l s t o y i e l d DMPO-OH may y i e l d t h i s same adduct by a l t e r n a t e r o u t e s , such as: DMPO + .HO2 DMPO-OOH + Fe 2 + 2 DMP0-00H

> > >

DMP0-00H (10) DMP0-0H + OH + Fe + 3 (ID DMP0-0H + non-paramagnetic species (12)

thus r a i s i n g some question as to whether or not the DMPO-OH adducts reported by a number of i n v e s t i g a t o r s i n l i p i d p e r o x i d a t i o n s t u d i e s a c t u a l l y r e s u l t e d from the r e a c t i o n of DMPO + .OH > DMPO-OH. Experimental M a t e r i a l s . L i n o l e n i c acid and a r a c h i d o n i c acid were obtained from NuChek Prep, E l y s i a n , Minnesota. These f a t t y acids were approximately 99% pure. Further p u r i f i c a t i o n was c a r r i e d out by the method of Gardner (4) or by high pressure l i q u i d chromatography. EDTA, DETAPAC, ADP, i r o n s a l t s , mann i t o l and hydrogen peroxide were a l l reagent grade and used as i s . L i p o x i d a s e and c a t a l a s e were obtained from Sigma. Twice c r y s t a l l i z e d c a t a l a s e was p u r i f i e d by column chromatography before use. Instruments. A Vari an E-104 i n t e r f a c e d with a V71 computer was used f o r a l l s p i n - t r a p p i n g s t u d i e s . A H i t a c h i model 110-A u . v . - v i s i b l e double beam spectrophotometer was used f o r diene measurements. Procedures. Diene formation was f o l l o w e d at 232 nm. A l l reagents used i n k i n e t i c runs i n v o l v i n g i r o n (Fe2 + ) or the Fenton reagent were prepared d a i l y and c a r e f u l l y degassed with Argon. F a t t y acids used f o r o p t i c a l s t u d i e s were s o l u b i l i z e d as d i s p e r s i o n s with 0.04% l u b r o l . The pH of the s o l u t i o n s was adjusted by a d d i t i o n of the appropriate amount of NaOH or HCl. A t y p i c a l k i n e t i c r e a c t i o n monitoring diene production would c o n t a i n , 0.55 mM f a t t y a c i d ( l i n o l e n i c or a r a c h i d o n i c acid) dispersed with 0.04% w/v l u b r o l , 0.1 mM Fe 2 + as the s u l f a t e and equal molar H2O2 when using the Fenton reagent. The blank would c o n t a i n a l l reagents except f a t t y a c i d . In the ESR s t u d i e s , f a t t y a c i d (0.55 mM) was prepared without l u b r o l . The opaque d i s p e r s i o n s obtained by vigorous v o r t e x i n g were used d i r e c t l y . The Fenton reagent used was 0.5 mM, the s p i n t r a p DMPO was 100 mM. In the l i p o x i d a s e s t u d i e s 4500 u n i t s of l i p o x i d a s e was added to 0.3 ml of 2.75 mM f a t t y a c i d . Catalase was used at concentrations from 4.9 to 9.8 χ ΙΟ" 9 M. Results To answer the question of whether or not the r e a c t i o n of DMP0-00H with Fe 2 +

139

will yield DMPO-OH (reaction 11), we carried out the experiments i l l u s t r a t e d in Figure ι . in these experiment ·02" radicals were generated external to the ESR cavity using the photocatalyzed reduction of oxygen by r i b o f l a v i n in the presence of an electron donor, such as EDTA or DETAPAC.

FIG. 1. E f f e c t of Fe 2 " 1 and Fe3» o n T H I . JHPO-OOH s o i n adduct. Reaction mixtures contained 0.3 im r i b o v l a v i n , S n« EDTA, and 50 ιτΜ OHPO. S o l u t i o n s «ere i r r a d i a t e d 15 seconds with a hi9h pressure Hg u . » . lanp and then placed i n the ESR c a v i t y , a.) control s o l u t i o n b.) 5 nfl Fe3* added a f t e r i r r a d i a t i o n c . ) 5 r * F e 2 + added a f t e r i r r a d i a t i o n d . ) d i f f e r e n c e s p e c t r i n produced by s u b t r a c t i n g s p e c t r i n lc and l a .

When the l i g h t i s extinguished .O2" production ceases and the sample i s placed in the ESR cavity. As can be seen from Figure l a , both DMPO-OH and DMP0-00H adducts are formed. The former originating possibly from the photochemical generation of oxazarine from DMPO which then hydrolyses to yield DMPO-OH. It could also possibly be formed from the reduction of trace Fe3 + , which i s always present in EDTA solutions, by .O2" and then reaction of t h i s Fe 2 + with H2O2 formed in the dismutation of .O2" to yield .OH. In Figure l b , Fe 3 + i s added with no change in the spectrum. However, when Fe2 + i s added as in Figure l c , the DMP0-00H signal i s decomposed to nonparamagnetic species leaving only the original DMPO-OH. Figure Id i s a difference spectrum produced by subtracting lc from la which leaves only the DMP0-00H adduct. This clearly demonstrates that DMPO-OH i s not formed from iron catalysed decomposition of DMP0-00H. I f the same reaction i s carried out using DETAPAC as the electron donor, spectra identical to la and lb are obtained when Fe2 + i s added either after the l i g h t i s extinguished or before. In other words, no change in the

140

spectra are observed, thus i l l u s t r a t i n g that Fe^ + chelated with DETAPAC does not react with the DMPO-OOH adduct. Experiments in which Fe^* i s added during photolysis of riboflavin result in no formation of the DMPO-OOH adduct because of the rapid reduction of Fe3+ by .0?" to Fe2+ and subsequent reaction with the DMPO-OOH adduct. The same results are obtained i f other sources of O2" are used, such as the NADPH dependent cytochrome P-450 reductase reduction of oxygen or the xanthine-xanthine oxidase reaction. In these later cases, however, one cannot stop product i o n of .O2" as easily as in the photolysis of r i b o f l a v i n . Comparing the i n i t i a l rates of diene production in the direct peroxidation of linolenic acid by Fe2+ alone with that of a typical Fenton reagent cons i s t i n g of Fe2 + and H2O2 one can see (Table I) that the i n i t i a l rates of formation with the Fenton are as high as nine times that of iron alone. In the later case, however, the reaction continues for a longer period of time albeit much slower. This i s i l l u s t r a t e d in Figure 2 where the change in absorption at 232 nm i s followed after i n i t i a t i o n with the Fenton reagent or iron alone. Table 1 Rate of diene formation as a function of Fe2+ concentration with and without hydrogen peroxide a Fe2+ ( μ Μ)

a

Η202 (0.1 mM)

no H2O2

No.A 232/min 0.5 0.066 0.018 0.093 0.023 1.0 0.156 2.5 0.028 5.0 0.240 0.035 0.342 10.0 0.039 Reaction mixtures contained 0.55 mM linolenic acid, 3 mM NaCI, 0.04% lubrol and the indicated amount of Fe2+ and H2O2, pH 5. Rates given are i n i t i a l rates.

In Figure 3 we see that the subsequent addition of aliquots of Fe2+ to a reaction o r i g i n a l l y i n i t i a t e d by the Fenton reagent, produces short rapid bursts of diene formation which quickly level o f f , suggesting that the .OH radical i s less efficient in sustaining l i n o l e n i c acid peroxidation than FE2+ alone. In the later case, continued i n i t i a t i o n must be due to the iron catalysed breakdown of linolenic acid hydroperoxides. The importance of these hydroperoxides in t h i s reaction is i l l u s t r a t e d in Figure 4 where we see an increase in diene formation as a function of the level of hydroperoxide present. V e r i f i c a t i o n by means of spin trapping that fatty acid hydroperoxides are formed in these reactions and that their breakdown i s iron catalyzed i s i l l u s t r a t e d in Figure 5. If one i n i t i a t e s peroxidation with a Fenton reagent, lets the reaction run for ten minutes, and then adds the spin trap DMPO, no detectable adducts are observed with the exception of a weak carbon centered adduct (Figure 5a). I f , however, after addition of DMPO, one adds catalase followed by Fe2+ the spectrum in Figure 5b i s obtained.

141

1

2

3

M/NUTE5

4

F I G . 2. D i e n e f o r m a t i o n 1» l i n o l e n i c a c i d i n i t i a t e d by the Fenton reaqent o r F e z + . R e a c t i o n m i x t u r e s c o n t a i n e d 0 . 5 5 e« l i n o l e n i c a c i d , 3 nrt N a C I , 0 . 1 irti H2O2 and 0 - 0 4 * « / * l u b r o l , pH 5. a.) Fenton r e a c t i o n s t a r t e d by adding 0 . 1 irM F e 2 * t o samóle and b l a n k , b . ) r e a c t i o n i n i t i a t e d by adding 0 . 1 nM F e 2 4 t o sample and b l a n k . No H ^ was p r e s e n t . Blanks contained e v e r y t h i n g except f a t t y a c i d .

1

2

3

4

5

MINUTES F I G . 3. E f f e c t of a d d i t i o n a l F e * + on Fenton induced d i e n e f o r m a t i o n . R e a c t i o n m i x t u r e sane as 2a except a f t e r i n i t i a l r e a c t i o n r a t e l e v e l e d a d d i t i o n a l a l i q u o t s of 0 . 0 5 irti F e ' * were added a s i n d i c a t e d .

off,

0.90-

.5

0.75

>

0.60

5

0.45

ε

«-OJO

0.02

0.04

0.06 A232

0.08

030

nm

F I G . 4 . E f f e c t o f h y d r o p e r o x i d e s on diene f o n d a t i o n r a t e s . L i n o l e n i c acid ( 0 . 5 5 trH) was a u t o x i d i z e d f o r v a r y i n g t i m e s u n t i l a p a r t i c u l a r A232 * * * reached ( x - a x i s ) . F e ' * (60 μΗ) was then added t o t h e s e s o l u t i o n s and the r a t e s of d i e n e f o r m a t i o n measured at 232 nm, pH S .

This spectrum i s c l e a r l y that of the DMPO-OH adduct superimposed on a weak carbon centered radical adduct which i s derived from the c a t a l y t i c decomposition of LOOH into LO.+.OH. The addition of catalase i s necessary to remove traces of H2O2 formed during the Fenton reaction with l i n o l e n i c

142 acid, wherein the OH radicals generated not only abstract hydrogen from the fatty acid, but also recombine to yield H2O2· A quantitative comparison of the amount of DMPO-OH adduct formed, as determined by integration of the ESR spectrum, and the amount of diene generated, as measured by 232 nm absorption, indicates that approximately 15% of the .OH generated in the decompositon of LOOH is actually trapped. Similar comparisons can be made in which LOOH is generated not by the Fenton reaction but by the action of lipoxidase (Figure 6). Figure 6a is the trace taken after allowing the reaction to proceed 10 minutes followed by addition of the spin trap. Figure 6b shows the result of adding catalase followed by Fe2+ as in Figure 5. Evidence that these adducts are in fact derived from the trapping of .OH in the iron catalysed breakdown of LOOH is given in Figure 7 where increasing amounts of ethanol are added prior to Fe2 + addition. As can be seen in these spectra, two distinct adducts are formed; one identified as DMPO-OH and the other as the carbon centered ethanol-DMP0 adduct. The later, formed as a result of hydrogen abstraction by .OH, increases with ethanol concentrations. Similarly, if one adds mannitol in sufficient concentration, i.e., equal molar to DMPO, no DMPO-OH adduct is observed in the Fe2+ catalysed breakdown of LOOH.

FIG. 5. ESR spin t r a p studies of the reaction of F e * + with linolenic acid hydroperoxide, a.) after 10 minutes of Fenton induced peroxidation of linolenic acid, DMPO (100 nfi) was added, b.) t o solution 5a was added catalase (4.9 χ I O " 9 H) followed by F e 2 * (0.25 m ) . pH • 3.65

FIG. 6. ESR spin trao studies of the reaction of F e ¿ + with linolenic acid hydroperoxides generated by lipoxidase. DMPO (100 nti) added after 10 minut e s of reaction of 4500 units of lipoxidase w i t h 2.75 irti linolenic acid, b.) addition of 4.9 χ ΙΟ" 9 M catalase followed by f e 2 + 0.5 n « , pH 6 . 9 5 .

There has been considerable discussion in the literature as to the requirement for chelation of iV-on in the stimulation of lipid peroxidation (3,5,6). It is well known that the chelation of iron can alter its redox potential, however chelation may in fact decrease the availability of the iron depending upon the ratio of chelator to iron employed. In the case of

143

EDTA, i t has been demonstrated that chelation with Fe 2 + rapidly oxidizes the iron to Fe3 + (5). For example, we have shown that in a one-to-one complex, one minute after mixing less than 10% of the iron in the Fe2+ state i s available for reaction with either bathophenanthroline sulfonate, which gives a strong colored complex only with Fe2 + , or with HgOg to give a typical Fenton reaction producing .OH radicals as evidenced by spintrapping. It has been reported however that EDTA does enhance the Fe2+ dependent peroxidation of l i p i d that i s dispersed (5) and that i t inhibits peroxidation when used in a Fenton reaction with l i p i d (7). It has also been suggested that Fe3+/EDTA with ADP stimulates superoxide dependent l i p i d peroxidation in liposomes (5).

FIG. 7. Effect of ethanol on the spin trapped signal from F e * + breakdown of LOOH. a.) 5 ul ethanol added t o reaction s o l u t i o n as In F i g . 6b p r i o r t o addition of F e 2 * b.) 10 ul ethanol c . ) 15 pi ethanol d.) 50 ul ethanol. Arrow indicates carbon centered radical from ethanol. Reaction volime was 0.5 ml.

We report here s p e c i f i c a l l y the effects of chelation of Fe 2 + by EDTA, ADP and DETAPAC on l i p i d hydroperoxide dependent peroxidation. As mentioned e a r l i e r , i t i s generally accepted that metal catalyzed decomposition of l i p i d hydroperoxide accounts for most of the l i p i d peroxidation observed. In experiments similar to those described in Figures 5 and 6 fatty acid hydroperoxide was generated either by a Fenton reaction or lipoxidase for approximately ten minutes. The hydroperoxide, LOOH, was then reacted with Fe2+ and EDTA, ADP or DETAPAC to give a 1:1 complex. In Figure 8b only, a small DMPO-OH signal and weak carbon centered adduct results from the react i o n of Fe2+/EDTA with the LOOH formed by lipoxidase reaction with the fatty acid. This small signal is probably due to the reaction of the small amount of free Fe2+ not oxidized upon chelation which i s in equilibrium with the complex. In Figure 8c, however, no DMPO-OH adduct i s observed when LOOH formed either in a Fenton reaction or from lipoxidase i s reacted

144 with Fe2+/DETAPAC (1:1). There is instead, only a carbon centered adduct formed. This could be due to the complex acting not on LOOH to yield .OH and LO·, but its reaction with other peroxide derivatives formed during lipid peroxidation. Similarly, no DMPO-OH adduct is formed with Fe 2 + /ADP as shown in Figure 8d; however, again a weak carbon centered radical is detectable. Similar results were obtained when diene formation was monitored during the peroxidation of linolenic acid containing trace amounts of hydroperoxide by these various Fe2+ complexes. Table 2 tabulates the diene results. Table II Effect of chelators on rates of diene formation in linolenic acid micelles containing trace hydroperoxides a No.A 232/min

Fe2+

a

0.250

Fe2+/ADP 0.040 Fe 2 + /DETAPAC 0.038 Fe2+/EDTA 0.003 Reaction nixtures contained 0.55 mM linolenic acid, 3mM NaCl, 0.04% lubrol, pH5. Reaction was started by addition of 0.05 mM p e 2 + or F¿2+/ C hel ator.

-Λ-VM/V-V^ FIG. 8. Effect of c h e l a t e s on Fe^ + catalysed breakdown of L00H. Linoienic acid h y o r o o e r o m o e s *ere generated Dy reaction witn 4500 units of lioojudase, pH 7.0 for 10 p m u t e s . a.) addition of 0.5 nfl Fe2*" b.) addition of 0.22 rtt F e 2 V £ : T A , pH 7.0 c.) addition of 0.44 n « F e 2 + / D £ ' A P A C d.) addition of 0.4 rti F e ' V A O P , pH 7.0.

It would appear that only free Fe2 + is effective in the metal catalysed decompositon of lipid hydroperoxide to yield .OH radicals, the others could be acting on other peroxides but not LOOH. All of these complexes are effective, however, in the decomposition of hydrogen peroxide to yield OH radicals as evidenced by the spin-trapping results shown in Figure 9. Here we see that F e 2 + yields trapped OH as DMPO-OH which is unstable. Fe z + /EDTA yields a smaller signal due most likely to the oxidation of F e 2 + to Fe·3"1". Fe 2 + /DETAPAC and Fe2+/ADP are very effective in producing DMPO-OH which is quite stable at these pH's of approximately 7.0.

145 In conclusion the data presented suggests that under certain conditions where a Fenton reaction can take place, the subsequent production of OH radicals is very effective in initiating lipid peroxidation. Continued peroxidation, however, occurs primarily by free F e 2 + decomposition of the lipid hydroperoxide formed in the early initiation reaction.

FIG. 9. Effect of chelators on the Fenton reaction. All reaction mixtures contained 0.5 nrt H 2 0 2 and 100 nM ÇHP0 a.) addition of 0.5 nrt Fe 2 *, pH 3.65 b.) addition of O.Z2 «M F e 2 V E 0 T A 1 minute prior to mixing witn H 2 0 2 and DMPO, pH 7.0 c.) addition of 0.44 ml F e 2 V D E T A P A C as 1n 9b, pH 7.0 d.) addition of 0.4 «M Fe 2 + /ADP as 1n 9b, pH 7.0.

References 1.

Finkelstein, E., Cohen, F.J., and Raukman, E.L.: Arch. Biochem. Biophys. 200,1 (1980).

2.

Eisworth, J.F., Lamchen, M.: J.S. Afr. Chem. Inst. 24,196 (1974).

3.

Aust, S.D., Svingen, B.A.: Free Radicals in Biology, Vol. 5, editor U.A. Pryor, Academic Press, N.Y. (1982), p. 1.

4.

Gardner, H.W.:

5.

Tien, M., Morehouse, L.A., Bucher, J.R., Aust, S.D.: Arch. Biochem. Biophys. 218,450 (1982).

6.

Poyer, J.L., McCay, P.B.: J. Biol. Chem. 246, 263 (1971).

7.

Tien, M. Svingen, B.A., and Aust, S.D.: Arch. Biochem. Biophys. 216,142 (1982).

J. Lipid Res. 11,311

(1970).

EVIDENCE FOR THE INITIATION OF LIPID PEROXIDATION BY A FERROUSDIOXYGEN-FERRIC CHELATE COMPLEX

*

Steven D. Aust, John R. Bucher, Ming Tien DepartmentState of Biochemistry Michigan University, East Lansing, MI

48824

Introduction Chelated

ferrous

added

to

iron

will

aerobic

initiate

incubations

polyunsaturated fatty acids. anism of initiation chelator (1,2). rapid

lipid of

peroxidation lipids

when

containing

The efficiency and apparent mech-

is dependent upon the nature of the iron

For example, ferrous chelates of EDTA undergo

autoxidation,

and

generate

the

·0Η

(3).

This

can

initiate peroxidation in lipids which are dispersed with detergent,

EDTA-Fe^ +

however,

promote

*0H

formation

and

during

other

ferrous-chelates

autoxidation

do

not

which

initiate

peroxidation of lipids which are in a liposomal configuration (2). anism

Several lines of evidence indicate that a different mechis

required

microsomes

(4,5).

to

initiate

peroxidation

Nucleotide-ferrous

in

liposomes

chelates

or

apparently

initiate liposomal peroxidation through the formation of ironoxygen

complexes

during

their

autoxidation

(1,5).

Recent

results have indicated that the actual initiator is a ferrousdioxygen-ferric examines

these

implications

of

nucleotide

chelate

findings these

data

in in

complex

detail, terms

(6).

and of

This

report

discusses

possible

the

cellular

strategies for preventing lipid peroxidation. * Present Address-Forest Products Laboratories, Forest Service, USDA, Madison, WI 53705

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

148

Materials and Methods Superoxide

dismutase

diphosphate,

(EC

1.15.1.1),

thiobarbituric

2,900

u/mg,

adenosine

acid, and mannitol were

5'

purchased

from Sigma Chemical Co.

Catalase

was from Millipore Corp.

Enzymes were passed over Sephadex G -

25

and

buffers

ferrous

over

chelates

Chelex

were

(EC 1.11.1.6), 44,000

resin

prepared

prior

by

argon-purged solutions of ADP, pH 7.0. rat liver (5).

phospholipids

to

adding

were prepared

use.

FeC^

u/mg,

Ferric or

FeCl^

and to

Liposomes of extracted as per Pederson et al.

Lipid peroxidation reaction mixtures were constituted in

a shaking water bath at 37° under an air atmosphere.

Rates of

lipid peroxidation were assessed by determining malondialdehyde (MDA) formation

(6).

Results Lipid

peroxidation

nucleotide

occurs

chelates,

in

incubations

lipid,

and

a

containing

variety

of

ferriccellular

reductants, including ascorbate, GSH, cysteine and superoxide. Lipid

peroxidation

products

are

observed

immediately

reactions progress at constant rates (2,5).

and

the

The reactions are

not inhibited by catalase or ·0Η scavengers, and this suggests that

the

simple

reduction

of

necessary to form an initiator.

the

chelate

is

all

that

is

Consequently, the direct addi-

tion of ferrous chelates to lipid should initiate lipid peroxidation.

However, when A D P - F e ^ + was added to lipid

polyunsaturated immediately, periods

of

fatty

rather from

5

these experiments. simple

acids

there to

10

a

minutes

short are

did

lag

not

(Fig.

frequently

commence 1).

observed

Lag in

This suggests that something other than a

ADP-Fe^+-dioxygen

initiation of lipid

peroxidation

was

containing

complex

peroxidation.

is

responsible

for

the

149

IO •

2 and analyzes the nature of the ultimate binding species.

*Hie amount of irreversibly protein bound metabolites is termed "binding"

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

166

Materials and Methods ^C-p-Bromophenol (5 mCi/mnol) was obtained from Amersham, England; ~^C-bromocatechol (1 mCi/mmol) was prepared by incubation of the phenols with rat liver microsomes and NADPH and was purified by TLC. Liver microsomes were prepared from male Wistar rats pretreated with phénobarbital. Diaphorase [NADPH-cytochrome c-(ferredoxin)-oxido reductase] was prepared from Euglena gracilis, according to (4,5). Other substances were obtained from commercial sources. 14 Binding of

C-labelled compounds was determined on paper filter disks

after precipitation of the protein and extensive washing procedures with organic solvents as described in (6). Incubations were carried out in two enzymatic systems: The "diaphorase-system" contained 50^ug diaphorase, 100 nmol paraquat, Q.5yumol NADPH and 1 mg bovine serum albumin in 1 ml 70 mM potassium phosphate, pH 7.5. The mixture was incubated at 25°C for 30 min. The "microsomal system" contained 1 mg/ml microsomal protein, glucose-6-phosphate dependent NADPH-regenerating system in 70 mM phosphate, pH 7.5, and 14 was incubated for 15 min at 37°C. The concentration of C-bromocatechol in both systems was 200 ,uM.

Results C>2 as activating species. The conversion of

14 C-bromocatechol to protein bound adducts by defined

oxygen species was first studied in the diaphorase system. In the presence of NADPH, bromocatechol formed protein adducts at a rate of about 3 nmol/ 30 min. This protein binding was lowered to about 30% in the absence of NADPH and was 20% of the binding in the complete system when the binding protein was added after termination of the reaction (Fig. 1). 250 U SOD decreased the binding to control values observed in the absence of NADPH. 250 U of catalase in contrast had no effect on the binding.

167

loo * 100-

50-

τ -NADPH

!

ÍL

albumin added after termination

SOD

50-

A

+ catalase

14_ Fig. 1: Binding of """""C-bromocatechol to albumin in the diaphorase

1

- NADPH

+ SOD

Fig. 2: Binding of

system

* diaphorasesystem

14 C-bro-

mocatechol in the microsomal system

A 2-fold level of binding was noted when intact rat liver microsomes were 14 used as the activation system for C-bromocatechol. When the diaphorase system was additionally present the binding to microsomes was further increased by 50% (Fig. 2). The role of OH"-radicals in the activation process formed possibly from Oj and H„0_ in the microsomal system was investigated by studying 14 the binding of C-bromophenol in the presence of the radical scavengers mannitol (5 mM) and t-butanol (5 mM). Neither reagent decreased the binding, but showed a small stimulatory effect. Similarly, these reagents 14 did not affect the binding of C-bromocatechol in the microsomal activation system. Is bromo-o-benzoquinone the binding species? The preceding results strongly indicate that C>2 directly converts bromocatechol to the binding species which could be either the ortho-semiquinone radical or the ortho-quinone by disproportionation of the semiquinone. In order to determine the role of the quinone in the binding pro14 14 cess, we have synthesized C-l-bromo-3,4-benzoquinone from C-bromocatechol by oxidation with 1,2,3,4-tetrachloro-o-benzoquinone according to (7). The reaction yielded 75% of a yellow coloured product with an absorption maximum of 382 nm (in acetonitrile/H90 4:1).

168

When this compound was incubated with albumin in the absence of cofactors and the activating diaphorase enzyme about 10% became bound to the protein (Tab. 1). About 50% more quinone became protein bound when albumin was replaced by α-casein, a cysteine free protein. Bromocatechol in contrast, did not bind to

α-casein during incubation in the diaphorase

system. The binding of bromoquinone and bromocatechol was differently affected by the quinone scavenger polylysine. Polylysine (2mM) slightly decreased the binding of bromocatechol in the microsomal system by 29% and in the diaphorase system by 16%. The binding of 40 and 80^uM bromoquinone to albumin was markedly lowered by 75% by the same concentration of the inhibitor. Table 1: Binding of l4C-bromocatechol and l4C-bromo-benzoquinone to different target proteins.

Compound

activating system

cofactor

bromocatechol (200/uM)

diaphorase It

-NADPH +NADPH

0.5 1.5

0.6 0.4

none It tl

none It II

2.3 5.4 7.2

3.9 7.4 12.7

bromoquinone 25/uM 50/uM 75/uM

binding to albumin α-casein (nmol/mg prot.)

The binding of bromocatechol was further studied in the presence of sulfhydryl reagents. Cysteine (lmM) or glutathione both decreased the binding by two third in both enzymatic activation systems. A covalent binding between the sulfhydryl group of glutathione and the electrophilic position of the binding intermediate is indicated by the isolation of two glutathione conjugates by HPLC (8). Methionine, carrying a methyl substituted sulfhydryl group, did not show an inhibitory effect.

169 Discussion The present paper provides direct evidence for the essential role of C>2 in the formation of reactive intermediates from phenols during microsomal metabolism. Our model compound, bromocatechol, formed protein bound adducts in an enzymatic system, selectively generating C^. This system, consisting of NADPH, and NADPH-dependent diaphorase and paraquat, has been shown to transfer electrons from NADPH to O^ via the paraquat radical (5). In the presence of SOD or catalase, this system selectively produces, respectively, 1^0^ or 0 2 · We conclude from the inhibitory effect of SOD and the ineffectiveness of catalase that only 0 2 but not or

OH" "fricáis, eventually formed from R.£>2· activates bromo-

catechol in the diaphorase system. This also holds true for the microsomal system, as indicated by the increase of the bromocatechol binding when the diaphorase system was additionally present and by the negative effects of te OH*-radical scavengers. Attempts to determine whether the semiquinone radical or the quinone formed from bromocatechol by reaction with 0~ is more likely to be 14 the binding species relevated that C-bromo-o-benzoquinone, although it spontaneously bound to protein in a high yield, seems not to be the major binding species of bromocatechol. This is demonstrated by the finding that the reactive intermediate formed from bromocatechol in the diaphorase system did not bind to α-casein, which, on the other hand, was an excellent target protein for bromoquinone. Furthermore, the poor effectiveness of the quinone scavenger, polylysine, to inhibit the binding of bromocatechol also suggests a minor role of the quinone as binding species. Probably, the bromoquinone is only present at a very low steady state concentration under the reducing conditions of our incubation mixtures indicating the semiquinone radical to be the ultimate binding species. The activation mechanism presented here has also been discussed for a number of other catechols or hydroquinones of biological relevance (1,2,9). The diaphorase system containing appropriate target molecules offers a suitable method to study the role of oxygen species and the nature of binding intermediates.

170

References

1. Dybing, E., Nelson, S.D., Mitchell, J.R., Sasaroe, H.A., Gillette, J.R.: Molec. Pharmacol. 12, 911-920 (1976). 2. Nelson, S.D., Mitchell, J.R., Dybing, Ε., Sasame, H.A.: Biochem. Biophys. Res. Comm. 70, 1157-1165 (1976). 3. Wolff, T.: "Oxy Radicals and Their Scavenger System", Vol. Is Molecular Aspects, Eds. G. Cohen and R.A. Greenwald, Elsevier, New York, pp. 304307 (1983). 4. Lengfelder, E., Elstner, E.F.: Ζ. Naturforschung 34c, 374-380 (1979). 5. Youngman, R.I., Osswald, W., Elstner, E.F. : "Oxy Radicals and Their Scavenger System", Vol. II: Cellular and Medical Aspects, Eds. R.A. Greenwald and G. Cohen, Elsevier, New York, pp. 212-217 (1983). 6. Wallin, H., Schelin, C., Tunek, A., Jergil, B.: Chem. Biol. Interact. 38, 109-118 (1981). 7. Harner, L., Dürckheimer, W.: Z. Naturforschung 14b, 741-752 (1959). 8. Cumpelik, 0., Oliv, K.H.: Naunyn-Schmiedeb. Arch. Pharmacol. 322, Suppl., 423 (1983). 9. Marks, F., Hecker, E. : Biochem. Biophys. Acta 187, 250-265 (1969).

DISCUSSION

KUTHAN: We have determined that about 10 pM/rag protein of superoxide dismutase and about 5 pM/mg protein of catalase are firmly associated with the microsomal fraction, prepared according to standard procedures, and these amounts could not be lowered significantly by extensive washings. Did you check the superoxide dismutase, catalase and diaphorase content of your preparations and what were the concentrations of your added enzymes in the experiments? WOLFF: We have found that catalase is present in our microsomal preparations but we did not look for SOD and diaphorase. The SOD concentration used in our microsomal incubation system was 250 U = 500 pMol/mg protein. Catalase was present in the diaphorase system at a concentration of 16 pMol/ml containing 1 mg of binding protein. The diaphorase concentration was 0.1 mg protein/ml.

GENERATION OF SUPEROXIDE IN THE AUTOXIDATION OF ASCORBATE AND GLUTATHIONE

Adelio Rigo, Marina Scarpa I n s t i t u t e of General Pathology, U n i v e r s i t y of Padova Padova, I t a l y Emanuele Argese, Paolo Ugo, Paolo V i g l i n o I n s t i t u t e of Physical Chemistry, U n i v e r s i t y of Venice Venice, I t a l y

Introduction In connection with the debate on superoxide ion, 0^, as an agent of oxygen t o x i c i t y ( 1 , 2 ) , i t appears i n t e r e s t i n g to i n v e s t i g a t e on the generation of t h i s radical in the autoxidation of ascorbate (AH^) and of reduced g l u t a thione (GSH), which are ubiquitous compounds in b i o l o g i c a l

systems.In s p i t e

of the vast number of papers dealing with the k i n e t i c s and the mechanisms of the autoxidation of these compounds, there i s weak or contradictory e v i dence on the generation of superoxide (3,5). In t h i s paper we report on the production of 0^ during the autoxidation of AH^ and of GSH either in h i g h l y p u r i f i e d systems or in the presence of c a t a l y t i c amounts of Fe and Cu i o n s .

Results and D i s c u s s i o n Evidence for Superoxide Generation in the Autoxidation of AH^ and of GSH. The generation of superoxide in aqueous s o l u t i o n s can be measured u t i l i s i n g the oxidized form of Cu,Zn superoxide dismutase (Cu + + = 100%), which in the presence of a flow of superoxide ion reaches the steady-state condition ( E„ ++ Cu

?

E„ + = 50% ) via a f i r s t - o r d e r process ( k i n e t i c rate constant r Cu

proportional to the f l u x of 0,,). The reduction of E C u ++ during t h i s process can be e a s i l y followed adding ^ F

to the s o l u t i o n and measuring the

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

172 F i g u r e 1.

-

Behaviour of SOD in the presence of ascorbate.

Experimental c o n d i t i o n s :

10

-4

M A H ^ 10

-6

6

M Fe( I I I

)-EDTA,

10

-7

2X10~ M SOD, 0.5 M F~ in 0.02 M phosphate b u f f e r at pH 7.4. O ) s t r i c t l y anaerobic c o n d i t i o n s ;

M catalase, -

• ) i n the presence of 2.5x10

nuclear magnetic r e l a x a t i o n time, Τ , of

-4

M 0.

19 ++ F . In f a c t the Cu present 19 -

the o x i d i z e d form of Cu,Zn superoxide dismutase (SOD) r e l a x e s the

F

in ve-

ry e f f i c i e n t l y . By t h i s method c l e a r evidence of 0^ production d u r i n g the a u t o x i d a t i o n of AH„ and GSH was obtained. The r e s u l t s are summarized i n

173 F i g . 1 for AH . From the f i g u r e i t r e s u l t s that, in the presence of a i r , -4 the addition of 10 M AH^ to a s o l u t i o n containing oxidized SOD determines a r e l a t i v e l y f a s t reduction { ' t , = 5 minutes) of E. ++ from 100% to J î Cu 50% level (steady-state c o n d i t i o n ) . On the contrary in s t r i c t l y anaerobic conditions E„ ++ was reduced from 100% to a f i n a l value of E„ ++ = Cu Cu 10% with a rate constant of orders of magnitude lower than that measured in the presence of 0^. Furthermore the i n j e c t i o n of a i r in the s y s tem containing the reduced SOD b r i n g s again the E(~ u ++ level to about 50%, being the oxidation rate comparable with that observed in the reduction of Eç u ++

in the presence of a i r . This experiment c l e a r l y shows that

lar-

ge f l u x e s of superoxide ion are generated in the oxidation of ascorbate -3 by molecular oxygen. An analogous experiment c a r r i e d out with 5x10

M

GSH gave s i m i l a r r e s u l t s . In t h i s case the f i n a l content of E^ u ++,when the reduction was c a r r i e d out in anaerobic c o n d i t i o n s , was about 5% and the r a t i o between the k i n e t i c rate constants of E„Cu ++ reduction in the presence and in the absence of 0,, was about 20. In these experiments no change in SOD a c t i v i t y was observed. Oxidation of AH„ and of GSH by Molecular Oxygen: Reaction Order and I n fluence of MetaT Ions. The oxidation of GSH and AH^ was followed in the UV region in a c o n t r o l led atmosphere using reagents with the highest p u r i t y . The solutions,when p o s s i b l e , were p u r i f i e d from traces of heavy metals by Chelex 100, because we found that the oxidation rate of AH^ and GSH increases in the presence of c a t a l y t i c amounts of metal ions such as copper or i r o n .

In

p a r t i c u l a r the oxidation of AH^ appears s e n s i t i v e to the presence of these ions which,at micromolar concentrations, enhance the oxidation rate of orders of magnitude. In the case of GSH the oxidation rate i n creases at the maximum of a factor 3. All

measurements have been c a r r i e d out at 25°C,in the presence of 10 ^M

c a t a l a s e , SOD f r e e , to minimize the p o s s i b l e reactions of H O . genera-

174 ted in the initial stages of the oxidation process. In these conditions we found that the reaction order with respect to the reductant is one for the GSH both in the presence and in absence of C u + + , while for AH^ a progressive saturation of the initial reaction rate was observed increasing the AH^ concentration in the presence of Cu{11)-hi sti di ne or Fe(111)-EDTA complexes. Since Cu or Fe ions (Me) appear to act as catalysts of the AH^

and

GSH oxidation by molecular oxygen we found quite unexpectedly that in both cases the oxidation rates do not increase linearly with the concentration of metal ion and clearly show a saturation effect. Accordingly the plots of the reciprocal of the initial oxidation rates against [Me] ^ are straight lines. Evaluation of Superoxide Flow; Effect of Superoxide Dismutase. Addition of SOD in the range 10

10

5

M halves the AH^ oxidation rate

both in the presence of Cu or Fe complexes while in the case of GSH no change of the oxidation rate was observed. This signifies that 0^ is directly involved in the AH^

oxidation while it appears to be only

a pro-

duct of the autoxidation of GSH. These hypotheses have been further confirmed by measurements of the rate of production of superoxide ion carried out in the presence of F . The addition of F

determines only a small

variation of the oxidation rates due to the increase of the ionic strength. -5 -4 Under all experimental conditions (AH , 1.0x10 -6

6

6

- 1.5x10 6

l.OxlO - 5.0X10~ M; Cu(II)-histidine,0.8xl0" -6.0xl0" M; of AH^ oxidation halves in the presence of SOD

M; Fe(111)-EDTA, pH 7.4) the rate

10 ^M and the rate of su-

peroxide production appears to be, within the experimental

errors,equal

to the rate of AH^ oxidation in the absence of SOD. These results indicate that one molecule of superoxide ion is produced for each molecule of ascorbate oxidized by 0„. In the GSH oxidation the rate of generation of -9 -1 -9 -1 superoxide resulted 5x10 M séc at pH 9.2 and 1x10 M sec at pH 7.4. Furthermore the production of 0. appears independent of the thiol concen-4 -2 tration in the GSH range 4x10 - 1.6x10 M and increases slightly with

175

the concentration of Cu i o n s . As a consequence the f r a c t i o n of

conver-

ted into 0. increases when the GSH concentration i s lowered and r e s u l t s -4 -7 about 1 at 2x10 M GSH and 8.7x10 M C u ( I I ) . From the a n a l y s i s of k i n e t i c data i t appears that the mechanisrrEof the oxidation of GSH and of AH,, by 0^ share some c h a r a c t e r i s t i c s . In fact the experimental data can be e a s i l y explained with the f o l l o w i n g reaction pattern:

RH + 0„

+Me,k MRH-Og)^. » -Me,k.

k (RH-0 2 -Me)

R" + 0~

RH

ρ

+

H2O2

2R*

being k c · [ R H ] « k , for AH and k c . [RH! ~ k^ for GSH b b ¿ b b where RH i s AH2 or GSH, R' and Ρ t h e i r r a d i c a l s and reactions products, r e s p e c t i v e l y . The reaction products (dehydroascorbic acid and GSSG) were detected spectrophotometrically and/or p o l a r o g r a p h i c a l l y . According to the above reaction scheme, the complex RH-O^-Me i s fundamental to explain the saturation effect of oxidation rate with respect to the metal ion,and apart from undergoing a monomolecular decomposition, i t can react, in the case of the t h i o l , with the GSH i t s e l f . The superoxide generated in step 6 reacts q u a n t i t a t i v e l y with AH2 or i t s radical doubling the oxidation rate. On the contrary GSH and GS", under our experimental

conditions,

seem not to react with 0 2 and consequently the oxidation rate appears independent of the presence of the SOD. In conclusion superoxide ion i s

176 generated in the autoxidation of GSH and AH ~9 -8 -1 range 10

- 10

M sec

at physiological

with rates which are in the conditions. Therefore we must

consider these compounds as potential sources of superoxide in biological systems. References 1.

Fee,J.: Trends Biochem. Sci. 84-86

(1982).

2.

Halliwell,B. : Trends Biochem. Sci.

3.

Puget,K., Michel son,M.: Biochimie 56, 1255-1267

4.

Halliwell,Β., Foyer,C.: Biochem. J. J_55, 697-700

5.

Misra.H.: J. Biol. Chem. 249, 2151-2155

6.

Rigo,A r , Ugo,P., Viglino,P., Rotilio,G.: FEBS Letters ]_32, 78-80(1981).

270-272

(1982). (1974). (1976).

(1974).

DISCUSSION

AUST: W e find that the autoxidation of substances like glutathione, c y s teine, etc., does not occur in the absence of metal and that the metal chelator greatly affects the rate and mechanism of autoxidation. Do you see the same results, i.e. the same inhibition by SOD, with each chelator? RIGO: This is exactly what we obtained with each kind of chelator we used: ADP, EDTA, histidine, it is a general result. Yes, we have measured the rate of oxidation of ascorbate by NMR and we found that in the presence of SOD the rate is altered. BIELSKI: W e recently published a study on the reaction of H 0 2 / 0 2 with ascorbic acid as a function of pH (0.3 to about 11) and found that although OJ reacts with AH-, the reaction product is not A·(CABELLI and BIELSKI, J. Phys. Chem. (1983), 87, 1809-1812). RIGO: We found that the EPR spectrum of the ascorbate radical is independent of the experimental conditions. W e varied all experimental conditions, and the concentration was almost the same. W e have done some c a l culations and we found that the rate of the reaction between O J and the ascorbate radical under our conditions is about, or a bit less than, the reaction rate between superoxide ion and ascorbic acid. These experiments were all done at pH 7. BIELSKI: I am puzzled by your ESR observations since our search for a n absorbance due to A · - formation at pH 8 in the range between roughly 300 and 650 nm gave negative results. RIGO:

This is our result.

ROLE OF METAL CHELATING AGENTS AS CATALYSTS IN AN Ό Η FORMING PROCESS A COMPARISON WITH THE HABER-WEISS REACTION

Harry C. Sutton Institute of Nuclear Sciences, DSIR, Lower Hütt, New Zealand Christine C. Winterbourn Clinical Biochemistry Department, Christchurch Hospital, Christchurch, New Zealand

Introduction In recent work we have studied a number of oxidation reactions which occur when paraquat radicals (PQ+') react with Ü2°2 in the presence of substrates such as sodium formate. We have evidence that these reactions are mediated by "OH radicals generated in the reaction PQ + · + H 2 0 2

"OH + 0H~ + PQ + +

(1)

but only in the presence of metal chelate compounds such as Fe-EDTA and iron chelated with diethylenetriaminepentaacetic acid, which we denote as Fe-DTPA. We have also reached some conclusions about its mechanism, which we shall try to summarise insofar as they relate to the corresponding Haber-Weiss reaction: °2

+ H

2°2

*

'0H +

0H

~

+

°2

t2)

Method Paraquat radicals are strongly reducing but do not dismutate. They are therefore stable in oxygen free solutions and we prepared them either by irradiating paraquat dichloride in air-free solutions of sodium formate, or by enzymatic methods using xanthine oxidase or glutathione reductase. We then mixed them with air-free solutions of H,0_ in the presence of

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

178

various reagents and looked for oxidation products. As reagents for 'OH we chose formate which is ultimately oxidised to CC>2 by a known simple mechanism, and methanol which in aqueous solution is oxidised to formaldehyde. We also studied the decomposition of deoxyribose to aldehyde-type products which can be assayed with considerable sensitivity. This can also be mediated by "OH though by a more complex mechanism (1).

Results In the absence of chelators we could not detect CO- or formaldehyde, al¿ though PQ+ ' was fairly rapidly decomposed by H 2 0 2 with the stoichiometry 2 PQ+· + H 2 0 2

>· 2 PQ + + + 2 OH~

(3)

This confirms earlier reports of other authors (2), although our kinetics were very different for reasons which we attribute to trace metal ions in our buffer solutions. In the presence of chelators a different reaction occurred, producing C0 2 from formate and formaldehyde from methanol in yields of typically 4 molecules per paraquat radical at pH 7. Deoxyribose was decomposed to aldehydetype products. H 2 0 2 was consumed in these reactions in similar yields to those of product formation. Product yields were suppressed by adding 'OH scavengers (isopropanol for formate, benzoate & formate for deoxyribose) approximately in accordance with their known rates of reaction with "OH. All this indicates that the reaction occurred by a small chain sequence in which one of the carriers is the "OH radical. The length of the chain sequence increased under some conditions, particularly at pH 6. Similar results were obtained using DTPA but the product yields were smaller, generally about half those for EDTA. The most striking feature of the reaction is its kinetics, illustrated in Fig. (1) for 30 mM formate solutions at pH 6. Under these conditions the chain sequence is rather long, giving 15 molecules of product per paraquat radical, and the reaction is slow enough to follow the rate of consumption of both H 9 0, and paraquat radicals. The figure shows that these two rates

179

2.4

ε 1.8

(M O (M Χ

Ο—01.2 ο

10

20

MINUTES

Fig. (1). Decomposition of paraquat radicals (PQ+·) and of H2O2 after mixing oxygen-free solutions of PQ+· [made by irradiating PQCI2 (2 mM) in sodium formate (30 mM) at pH 6] with oxygen free H2O2, in the presence of EDTA (60 /uM), at 22°C. are proportional to each other (with a slight discrepancy due to experimental conditions), and that both rates are nearly constant until all the PQ+' radicals are consumed. In most of our work the reaction rates were too rapid to follow HjOj consumption or CC>2 formation, but we could monitor the decay rate of [PQ+']. The results showed that this rate is independent of chelator concentration from 20 to 100 ^uM, and is nearly independent of [PQ+*] from 2 to 100 ^uM, but is proportional to [f^C^]. This accounts for most of the slight curvature in Fig. (1) since [HjC^] decreased two-fold during the reaction. This implies that the rate determining step involves reactions of Η,Ο, with sites + at low concentrations which are either in equilibrium with PQ ' or are rapidly restored by it. The rate at constant [HjOj], and therefore the concentration of these sites, increases with the concentration of buffer salts (which are known to contain iron), is decreased by passing the buffer through a Chelex column which partially removes metals, and is increased by adding Fe3+-EDTA or Fe3+-DTPA in proportion to their concentration. These data support a mechanism analogous to that commonly assumed for the Haber-Weiss reaction. For paraquat-hydrogen peroxide in EDTA the rate determining step is the initiation reaction (4), followed (in the case of formate) by the three rapid propagation reactions (5), (6) and (7), all of which would be expected to be quantitative under our conditions:

180

Initiation:

H2C>2 + Fe2+-EDTA

Propagation:

*0H + HC0~

> H 2 0 + CO~"

CO"' + PQ ++

> C02 + PQ+"

+

> Fe3+-EDTA + Ό Η + OH

3+

2+

PQ · + Fe -EDTA H 2 O 2 + HCO2

H 2 O + OH

(5) (6)

) Fe -EDTA + PQ >

(4)

++

(7)

+ CO2

This accounts for the observed chain reaction and its kinetics but not for the consumption of PQ+" which ultimately stops the chain. Two features of this chain termination should be noted. The first is that it follows the same kinetics as reaction (4), and therefore probably competes with 2+

this in a reaction between H 2 0 2 and Fe

-EDTA which does not form "CH.

The second is that its overall stoichiometry corresponds with 2 PQ+· + H 2 0 2

2 PQ ++ + 2 OH~

(3)

which was also observed with similar (though faster) kinetics in the absence of chelators. This can be considered as the sum of two reactions in which the first is probably a two electron oxidation by H.09 of Fe 2+

Fe

or +

-EDTA to a tetravalent compound which is then rapidly reduced by PQ ' 2+

back to Fe -EDTA. The details are not as yet established but they do not affect the major requirement of this mechanism, which is that the rate of product formation should equal that of reaction (4), i.e. d[C0,]/dt = 2+ k 4 [Fe -EDTA][H202]. We have also been able to verify this (at least approximately) by methods based on the decay rate of [PQ+*] in solutions containing known added (micromolar) concentrations of Fe-EDTA, and we find values of k 4 ( 104 M ^ s - 1 at pH 7) which agree with those reported in the literature for reaction (3).

Conclusions

These studies show that suitable catalysts enable paraquat radicals, like superoxide, to react with H 2 0 2 to produce 'OH radicals, and by a mechanism which is very similar to that commonly assumed for the Haber—Weiss reaction·

181

The difference is that PQ+ is both more stable (non-dismutating) and more reducing than 0~ (Ee = -0.45V in comparison with -0.15V) and, in our experience (based partly on flash photolysis studies which indicate k?

108 M _ 1 s _ 1 , both for EDTA and DTPA) can rapidly reduce both Fe3+-EDTA 3+

and Fe

2+

-DTPA to their corresponding Fe

-

compounds, whilst O^ can only

reduce the former at appreciable rate (4,5). Thus, both are effective in the paraquat system, though with different efficiency, which we attribute to their behaviour in the chain terminating reaction noted above. Another and probably related difference is that the paraquat reaction produced substantial yields of 'CXI at extremely low Fe-chelator concentrations, at which the Haber-Weiss reaction is scarcely detectable. Perhaps a more inçortant conclusion relates to the catalytic requirements for this reaction. We have seen that no 'CH is produced unless the reaction is catalysed by metal chelating compounds like Fe-EDTA. We now have evidence that this has nothing to do with the electron donating properties of EDTA, as we once thought, but instead is almost certainly related to the thermodynamic properties of Fe-EDTA, which Koppenol has pointed out (6). On this view, the essential feature of EDTA and DTPA is that they bind ferric ion much more effectively than ferrous ion (about l O ^ 3+

2+

times more). This lowers the redox potential of Fe /Fe

by 0.65V, thus

making "OH production thermodynamically possible in reaction (4) at neu trai pH, whereas the reaction does not occur using uncomplexed iron. Our studies fully support this argument, and if it is taken to its logical conclusion then it imposes considerable requirements on any metal catalysts for the Haber-Weiss reaction which act by this pathway. If these involve iron, acting by a Fenton type reaction such as (4),3+ then2+ the thermodynamic argument requires that they must lower the Fe /Fe

redox

potential by at least 0.3V to less than 0.46V at pH 7. However, the active product may not necessarily be "OH to have biological significance as discussed in the following paper (7).

182 References

1. Halliwell, Β., Gutteridge, J.M.C.: FEBS Lett. 128, 347-352 (1981) 2. Levey, G., Rieger, A.L., Edwards, J.O.: J. Org. Chem. 46, 1255-1260 (1981) 3. Borgaard, O.K., Farrer, D., Anderse, V.S.: Acta Chem. Scand. 25, 3541-3543 (1971) 4. Cohen, G., Sinet, P.M.: FEBS Lett. 138, 258-260 (1982) 5. Butler, J., Halliwell, B.: Arch. Biochem. Biophys. 218, 174-178 (1982) 6. Koppenol, W.H.: Proc. 3rd Int. Conf. on Superoxide and Superoxide Dismutase, G. Cohen and R. Greenwald, eds., Elsevier, Amsterdam (1982) 7. Winterbourn, C.C.: these proceedings

DISCUSSION ELSTNER: Is the reaction thermodynamically favoured, where a formate radical (CO2) is supposed to reduce paraquat to the paraquat radical with a midpoint potential of lower than -450 mV? SUTTON: CX>2 reduces paraquat very rapidly. Its rate constant, as we measured it, is nearly diffusion-controlled. FORMAN: According to FARRINGTON et al. (Biochim. Biophys. Acta (1973) 314, 372-281), the rate constant for the reaction of oxygen with paraquat is about diffusion-limited and one wonders, how you can make a hydrogen peroxide solution that is absolutely free of oxygen. So the question is, did you add SOD to your reaction to eliminate the possibility that some of this was due to superoxide acting as an intermediate. SUTTON: Oh, you don't have to worry about oxygen, because paraquat radicals react with oxygen tremendously fast and straight away remove it. Also, everything was done in air-free solution. FORMAN: What was the exact amount of how much PQ radical was present?

in your R2P2 solution and

SUTTON: Well, again, it would not have mattered, because the paraquat radicals would have consumed it rapidly. There could conceivably be up to 1 /uM oxygen in our solution and this would indeed react with paraquat radicals to form superoxide. However, such concentrations are negligible in comparison with those of the paraquat radicals which we used - which are typically 100 /uM. They are even smaller in comparison with the

183 p r o d u c t (C0 2 ) y i e l d s w h i c h typically exceed 400 /uM. So the e f f e c t is negligible. Further, superoxide could not produce our o b s e r v e d p r o d u c t s in the presence of DTPA, it is effective only w i t h EDTA. AUST: I w o u l d like to point o u t that we g e t very similar results w i t h NADPH-cytochrome P-450 reductase. The enzyme w i l l not reduce A D P - F e 3 + but w i l l readily reduce E D T A - F e 3 + or D T P A - F e 3 + , w h i c h c a n then reduce other iron chelators if they are present. It should be p o i n t e d o u t that EDTA is p r e s e n t in m a n y things, and iron c o n t a m i n a t i o n is d i f f i c u l t to avoid, so it is important to check for artifactual E D T A - F e 3 + or D T P A F e 3 + redox reactions.

REACTION OF PARAQUAT RADICALS WITH H ^ : EFFECT OF 0 2 AND COMPARISON OF RADICALS GENERATED ENZYMATICALLY AND BY IRRADIATION

Christine C. Winterbourn Department of Clinical Biochemistry, Christchurch Hospital, Christchurch, New Zealand Harry C. Sutton Institute of Nuclear Sciences, D.S.I.R., Lower Hütt, New Zealand

Introduction In the preceding paper we showed how paraquat radicals (PQ+) react with H2O2 in the presence of certain iron chelates to produce OH" radicals. The mechanism proposed for the reaction is: PQ+

+

Fe ^(chelate ) 2+

>

Fe2+(chelate) 3+

H202

+

Fe (chelate )

>

Fe (chelate)

PQ+

+

H202

>

PQ 2+

+

OH~

+

+

PQ 2 +

+

0H~

(1) +

OH'

(2)

OH-

The characteristics of the reaction were studied under anaerobic conditions and with ΕΙΧΓΑ or DTPA present, but its significance as a pathological mechanism depends on its being able to occur in a biological environment. This would normally not be strictly anaerobic, iron if present would not be complexed to EDTA or DTPA, and paraquat reduction would predominantly occur enzymatically. We have therefore compared the reactions of PQ+ produced by 2- irradiation and enzymatically ( from xanthine oxidase, ferredoxin reductase or glutathione reductase), and determined whether OH ' production could occur in the presence of 0,.

Methods

Solutions of paraquat chloride in air-free Na formate were irradiated to give approx. 100 μΜ PQ+ which was then mixed with H 2 0 2 and either Na formate or deoxyribose. Alternatively PQ+ was produced continuously

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

186

in the presence of H^O^ from paraquat chloride and either xanthine and xanthine oxidase (1), or NADPH and glutathione reductase (2) or ferredoxin reductase (3). Enzyme reactions were carried out in stoppered glass tubes either with a ^

atmosphere or air/^ mixtures prepared by bubbling with

N^ and replacing the requisite volume of N^ with air. Tubes containing air mixtures were mixed by rotation. OH" was detected by its ability to oxidize formate to CO^, or deoxyribose to thiobarbituric acid - reactive products (4) as in the preceding paper. Glassware was washed in 30% nitric acid and deionized distilled water, and reactions were carried out in chelex- treated phosphate buffers (pH 7.4) containing ~·Η -C 4J O C C o o Ε Ή 4-1 Ι4- co O -rt "D C CO O ti •H h •4-1 -H CO i-i CD CO .C CL-U ω ω t LOOH + L· (3) k

2 LOO·

t

> nonradical products

(4)

The rate of chain initiation was determined by the inhibitor method (5).

In the presence of phenolic antioxidant, IH,

the chains are terminated not by reaction 4 but by reactions 5 and 6.

As shown in Fig. 1, the addition of antioxidant

suppresses the oxidation and the length of time during which the oxidation is suppressed is called induction period, t^ The rate of oxidation during the induction period, R ^ ^ '

275

given by Eq. 8, where η and k ^ ^ are the stoichiometric factor for the antioxidant and the rate constant for reaction 5 respectively (6). k. inh LOO· + IH LOOH + I · (n-1) LOO· + I· -> stable products

(6)

t.

(7)

=

n h

n[IH]/R.

(5)

(8)

R. , = k L[LH] R. /nk. , [IH] inh p ι inh

The oxidizabilities of bisallylic hydrogens were similarly independent of parent substrate (PUFA or PC) and medium (homogeneous solution, micelle, and bilayer liposome in aqueous emulsion). α-Tocopherol (vitamin E) is known to act as an antioxidant in vivo and in vitro (1).

As shown in Fig. 1, the addition of

vitamin E suppresses the oxidation and produces an induction period.

When vitamin E is exhausted, the induction period is

over and the oxidation proceeds at the same rate as that in the absence of an antioxidant.

In the oxidation of methyl

linoleate in t-butyl alcohol at 37°C, the induction period was

Time F i g . 1 Rate of o x y g e n uptake with and without antioxidant

276 proportional to the amount of vitamin E added, as sugaested by Eq. 7.

Eqs. 7 and 8 give Eq. 9.

The rate constant k

was

k· v. = k [ L H l / R ^ t ^ inh

(9)

estimated to be 100 M "'"s ^ at 37°C from the literature values (2,7). Since [LH] is known and and can be obtained experimentally from Fig. 1, the inhibition rate constant can be calculated from Eq. 9. The k. , was obtained as inh „„-I -1 for vitamin E, which is in fair agreement 5.1 χ 10"^ M ""' ss with those reported previously (1,6). It was found that vitamin C also acted as a chain-breaking antioxidant and produced a distinct induction period.

The rate

of oxygen uptake after the induction period was equal to that in the absence of an antioxidant.

The induction period was

proportional to [vitamin C]/R.. ... . The rate constant k. , for 1 inh 4-1-1 from the oxidation vitamin C was calculated as 7.5 χ 10 M s of methyl linoleate at 37°C. 100

U ! >. X

40

60

Time/min

Fig. 1

Inhibition of oxidation of methyl linoleate in t-butyl alcohol/ methanol

(3/1) by vitamin C and vitamin E, 37°C, [AMVNl = 0.01 M. 4 2 3 1 54.8 54 . 8 0 0 [vit C] /UM 30.4 30.4 0 0 [ vi L Ej /uM

Curve

t. ,_.,_.,_· /min inhibition

0

28 . 5

40 . 3

65.6

277 It has been suggested that a-chromanoxy radical

(vitamin

E radical) interacts with vitamin C and glutathione to regenerate vitamin E (8-10).

Galvinoxyl, a stable phenoxy radical

used as a model for vitamin E radical, incorporated into micelle or liposome reacted with vitamin C, glutathione, and cysteine in aqueous phase, although with slower rate than in homogeneous solution. Fig. 2 shows the oxygen uptake in the oxidation of methyl linoleate.

The addition of vitamin E and vitamin C suppressed

the oxidation and gave a clear induction period.

Vitamin E

gave a slower rate of oxidation during the induction period than vitamin C constant

apparently because of a higher inhibition rate ( and possibly higher n) than for vitamin C.

When both vitamin E and vitamin C were added, the induction period was lengthened to the sum of the induction period observed when either vitamin E or vitamin C was used alone. What is interesting is that the rate of oxidation was the same throughout the induction period as the inhibited rate when vitamin E was used alone.

0

50

It is noteworthy that the rate

100 150 Time/min Fig. 3 Disappearance of vitamin E a n d C in the oxidation of methyl linoleate at 37°C in t - b u t y l alcohol/methyl alcohol (3/1 b y v / v ) . [ L H ] = 0.60 M , [ A M V N ] = 0.010 M, [ v i t E] = 0.595 mM, [ v i t C] = 0.620 mM

278

5 constant obtained in this system vms

= 4.0 χ 10

-1-1 M s ,

which is larqer than that for vitamin C and close to that for vitamin E. The rate of consumption of vitamin E and vitamin C durinq the oxidation of methyl linoleate was measured. of the results is shown in Fig. 3.

The examole

When either vitamin E or

vitamin C was added alone, they disappeared linearly with time. However, when both vitamin E and vitamin C were used, vitamin C was consumed first but vitamin E remained almost constant and then it was consumed after vitamin C disappeared. These results suggest that vitamin E scavenges the chain carrying peroxy radical more quickly than vitamin C but the vitamin E radical reacts with vitamin C to regenerate vitamin E.

This sequence must contribute to the synergistic inhibition

of oxidation by vitamin E and vitamin C and to the maintenance of vitamin E level in tissue.

References 1.

Niki, E., Tanimura, R., Kamiya, Y.: Bull. Chem. Soc. Jpn. 55, 1551-1555 (1982), and references cited therein.

2.

Yamamoto, Y., Niki, E., Kamiya, Y.:Lipids _17' 870-877 (198?)

3.

Porter,Ν. Α., Weber, Β. Α., Weenen,H., Khan, J. Α.: J. Am. Chem. Soc. 102, 5597-5601 (1980).

4.

Barclay, L.R.C., Ingold, K.U.: J. Am. Chem. Soc. 103, 6478 -6485 (1981).

5.

Boozer, C.E., Hammond, G.S., Hamilton, C.E., Sen, J.N.: J. Am. Chem. Soc. 77, 3233-3237 (1955).

6.

Burton, G.W., Ingold, K.U.: J. Am. Chem. Soc. 103, 64726477 (1981). Howard, J.A., Ingold, K.U.: Can. J. Chem. £5, 793-802 (1967)

7. 8.

Packer, J.E., Slater, T.F., Willson, R.L.: Nature 278, 737 -738 (1979).

9.

Niki, E., Tsuchiya, J., Tanimura, R., Kamiya, Y.: Chem. Lett. 789-792 (1982).

10. Tsuchiya, J., Niki, E., Kamiya, Y.: Bull. Chem. Soc. Jpn.. 56, 229-232 (1983).

279 DISCUSSION

PRYOR: Did you show that the thiol protects vit. C which protects vit. E? Was there a cascade, or did you just show that C protected E? NIKI: Well, in the slides I just showed that vit. C can react with a vit. E radical. But in a different experiment we could show that glutathione reacted with a vit. E radical and regenerated vit. E. And we can trap the thiyl radical from cysteine or glutathione. PRYOR: It would be interesting to know what the ultimate repository of reducing power is, ascorbate or glutathione. NIKI: If we look at the rate constants, vit. C is the most reactive, followed by cysteine and glutathione. Glutathione is the least reactive toward the vit. E radical. That's semi-quantitative though. RALEIGH: Dr. NIKI, you are following the oxygen consumption as a function of time in the presence of inhibitors. Once the inhibitor was consumed, oxygen consumption proceeded at an increased rate. It is often observed that once vit. E is oxidized, it becomes a pro-oxidant and so the rate of oxygen consumption might be expected to increase. Did you ever see any pro-oxidant activity of vit. E in your system? NIKI: Not much, that's the short answer. But we are studying how vit. E changes and what the product is when it reacts with the peroxy radical. Some people say it's a tocopheryl-quinone and we are trying to find out whether that is true. But as for the rate of oxidation, we don't observe much enhanced rate after the induction period. BORG: If I followed correctly, the refurbishing of vit. E by vit. C that you showed was in homogenous alcohol solution. NIKI: That's right. BORG: In the cell there is not much vit. C within membranes, it is mostly outside. Yet vit. E is probably deep within the membrane bilayer. How do you propose that the redox exchange between them takes place, and how efficiently do you think it occurs in comparison with what you showed in homogeneous solution? NIKI: We have asked ourselves that for a long time, and we are still working on it; but there is some interesting data. If we use oil-soluble initiators in liposomes, i.e. generate radicals in the oil phase, vit. C is not effective. But if we have vit. E and vit. C and generate radicals in the oil phase, then vit. C disappears and is effective as antioxidant. So, I don't know how it goes, but I'm sure if we have a vit. E radical in liposomes, it can react with vit. C. Whether it comes into liposomes or vit. E goes out, I don't know, but they can react, even though the rate is slower than in the homogeneous system. FORMAN: As far as the ultimate repository of reducing power in mammalian cells is concerned, there are two enzymes that exist for the oxidation of

280 vit. C. One is an NADH-dependent serai-dehydroascorbate reductase and the other is a glutathione-dependent dehydroascorbate reductase. Their distribution is variable from cell to cell. SIMIC: For the sake of accuracy I want to make a correction. Al TAPPEL in 1962 (in "Vitamins & Hormones", 20, pp. 493) was probably the first to suggest a reaction of the vit. E radical with vit. C, not PACKER, SLATER and WILLSON in 1979 (ref. 8). NIKI:

I should have mentioned that, yes.

SIES: You just mentioned you could give us a semi-quantitative figure for the difference in rate constant of reaction between vit. C and vit. E radical compared to cysteine and vit. E radical. Is it orders of magnitude or is it similar? NIKI: In micelle systems the reaction with glutathione and cysteine is an order of magnitude smaller than with vit. C. In liposomes we still don't have a very accurate number yet. SIES: So, if one considers concentrations, all the reactions could have similar importance, because glutathione is present at about 10 mM, and vit. C is certainly somewhat less.

PRODUCT DISTRIBUTION OF UNSATURATED PHOSPHOLIPID OXIDATION IN ORGANIC SOLVENT AND AQUEOUS EMULSION

Laura S. Lehman, Schering-Plough Corporation, Bloomfield, NJ Ned A. Porter, Duke U n i v e r s i t y , P. M. Gross Chemical Durham, N.C.

07960

Laboratories,

27706

Introduction We have investigated the oxidation of l i n o l e i c 1-palmitoyl,2-1inoleoyl

phosphatidylcholines

glycerophosphatidylcholine (1-P,2-LGPC) and

1,2-Dilinoleoylglycerophosphatidylcholine

(D-LGPC) membrane systems.

To

examine the possible effect that l i p i d peroxidation might have on the structural aggregation of phospholipids in multilamellar v e s i c l e s (MLVs), the autoxidation of 1-P,2-LGPC was monitored by 31p

nmr

spectroscopy.

Selected 31p nmr spectra are shown in Figure 1, which were taken during the autoxidation of 30 mM 1-P,2-LGPC in D 2 0 at 37°C. Phospholipids in MLVs give r i s e to broad asymmetric

nmr spectra with

low f i e l d shoulders, whereas phospholipids in other structural

aggregates

such as unilamellar v e s i c l e s , micelles, and reverse micelles e x h i b i t much narrower symmetrical ^lp

nmr

spectra.(1)

During the f i r s t 1.5 days,

1-P,2-LGPC retained i t s multilamellar structure and oxidized over 20%.(2) After 5 days, the asymmetrical narrow s i g n a l .

31

P nmr signal collapsed to a symmetrical

These experiments therefore suggest that l e c i t h i n s

retain

their multilamellar structure i f the oxidation of the system i s maintained to less than 20% conversion.

Note:

1-Palmitoyl, 2-linoleoylglycerophosphaticycholine (1-P, 2-LGPC) is denoted as PLinPC in Figures 1 and 2

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

282

•2 1

Figure I.

30

Ρ nmr

Spectra

of

PLinPC

0

-30

Observed

Multilamellar

30

0

During

Autoxidation

Vesicles

-30

30

0

ppm (Relative to H3PO4 = 0 ppm) When studying products formed during model membrane o x i d a t i o n , i t i s important to know the kind of environment in which the products are being formed.

The four hydroperoxide products of l i n o l e a t e oxidation have been

p r e v i o u s l y i d e n t i f i e d as the 9- and 13-substituted t r a n s , c i s ( t , c ) and t r a n s , t r a n s ( t , t ) isomers.(3)

To i n v e s t i g a t e the e f f e c t of aggregation on

product d i s t r i b u t i o n , 1-P,2-LGPC was autoxidized in MLVs and reverse micelles.

Autoxidation of 1-P,2-LGPC i n multilamellar v e s i c l e s was

i n i t i a t e d by xanthine oxidase system.(4)

Autoxidation of 1-P,2-LGPC i n

reverse micelles was studied as a function of concentration of l e c i t h i n i n o-dichlorobenzene and in Figure 2 i s presented a plot of the t , c / t , t vs. 1-P,2-LGPC molarity.

ratio

283

Figure 2. [ t . c l / C t . t ] Oxidation

Products from

PLinPC

in o-Dichlorobenzene.

0.05

0.10 Molarity of

0.15 PLinPC

0.20

Unlike autoxidation in reverse micelles, the product distribution resulting from l e c i t h i n autoxidation in vesicles does not vary with molarity of the l e c i t h i n .

The size of the aggregate is not determined by

the total molarity of the l e c i t h i n in aqueous emulsion as i t i s in organic solvent.(5)

At similar concentrations, (0.3 rtiM) the product ratio

resulting from 1-P.2-LGPC autoxidation is approximatey twice as high in vesicles ( t , c / t , t = 0.63 ±0.04) than i t is in reverse micelles ( t , c / t , t = 0.30 ±0.01).

This result most l i k e l y r e f l e c t s the higher kinetic

s t a b i l i t y of vesicles.

A lower t , c / t , t ratio would be anticipated from

lecithins in more dynamic structures.

284 References 1.

Cull i s , P.R., Hope, M.J.: Nature 271, 672 (1978).

2.

Percent oxidation was determined by reverse phase HPLC as described in the following references:

(a) Crawford, C.G., P l a t t n e r , R.D.,

Sessa, D . J . , Ruckis, J . J . : L i p i d s 15, 91 (1980); (b) Porter, N.A., Wolf, R.A., Weenen, H. i b i d . 15, 163 (1980). 3.

Chan, H.W.S., Levett, G. i b i d . 12, 99 (1977).

4.

F r i d o v i c h , S . E . , Porter, N.A. J. B i o l . Chem. 256, 260 (1981).

5.

Fendler, J.H. J. Phys. Chem. 84, 1485 (1980).

INVOLVEMENT OF ACTIVATED OXYGEN SPECIES IN MEMBRANE PEROXIDATION:

POSSIBLE MECHANISMS AND BIOLOGICAL CONSEQUENCES

Robert A. Floyd and Malgorzata M. Zaleska Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73204 USA

Introduction It is difficult to quantitate a particular molecular species if it exists at nanomolar or less and is formed and decays at about the same rate and then leaves very few if any tangible byproducts.

The above set of circumstances may very well

describe the endogenous occurrence of oxygen free radicals and membrane lipid peroxidation.

The metabolism of drugs such as

CCl^ (1), paraquat (2), plumbagen (3), certain aromatic nitro compounds (4), some carcinostatic agents (5), or some neurotoxins (6,7) cause an enhanced production of oxygen free radicals and in most cases membrane lipid peroxidation. During aging lipofuscin, a nondescript collection of products which form by the condensation of lipid aldehydes, produced during peroxidation, with primary amines of proteins or lipids, accumulates in tissue, notably brain.

Neurons are post

mitotic cells and thus the loss of these cells may have drastic consequences.

For this reason, we have studied peroxida-

tion processes in brain to understand the role of oxygen free radicals and iron in this process.

Model systems where the

oxidation state of iron, its ligation to biologically meaningful compounds, and exploitation of the spin-trapping technique (8) were used to quantify the oxygen free radicals.

We found

that only when certain chelators were present was it possible to obtain substantial yields of OH from H 2 0 2 following the

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

286 a d d i t i o n of F e ( I I )

(9).

In p h o s p h a t e b u f f e r , as w e l l

o t h e r s , Fe(II) r a p i d l y b e c a m e b r e a k d o w n to y i e l d O H . l a t o r s such as D E T A P A C considered this metal

to e x a m i n e s o m e b i o l o g i c a l

if t h e y w o u l d l í g a t e Fe(II) s u c h

+ ÔH + O H - + F e ( I I I ) .

(5,5-dimethylpyrroline-1-oxide),

w i t h ÒH

(~3 χ 1 0 9 M - 1

H202 che-

(diethylene triamine pentaacetate),

ion w o u l d c a r r y o u t the f o l l o w i n g

Fe(II) + H 2 0 2 DMPO

i n c a p a b l e of c a t a l y z i n g

R a t h e r t h a n using s y n t h e t i c iron

it m o r e m e a n i n g f u l

p o u n d s and d e t e r m i n e

as

sec-1)

we

com-

that

reaction:

Utilizing

the

which reacts

and y i e l d s a u n i q u e

spin-trap rapidly

1:2:2:1,

A

= A . = 14.9 g a u s s E P R s p e c t r u m in a q u e o u s s o l u t i o n , we w e r e JN ρ a b l e to m a k e p e r t i n e n t o b s e r v a t i o n s (10,11).

Results Figure

1 presents results

important points.

(10) w h i c h

DMPO

is a d d e d to a s o l u t i o n

the s p i n - t r a p D M P O as w e l l as A D P

has been added Fe(II)Cl2 in e x c e s s .

several

T h e top s c a n s h o w s the E P R s p e c t r u m of

s p i n - t r a p p e d ÒH o b t a i n e d w h e n H 2 0 2 containing

illustrate

(2 m M ) to

which

(100 μΜ) and t h e n w i t h i n 30 s e c

If any o n e of the c o m p o n e n t s

is o m i t t e d ,

ADP

(-)AOP (-)F.(I) (-)Η,Ο,

F i g u r e 1. E l e c t r o n s p i n r e s o n a n c e s p e c t r a of D M P O t r a p p e d ÔH in a n A D P - F e ( I I ) - H 2 0 2 s y s t e m (10).

spin-

H202

287

(second spectrum), Fe(II) (third spectrum) or H 2 0 2 spectrum), then no OH is spin-trapped.

(bottom

We have tested many

biologically important compounds and have found that only a very few will act as a ligander of Fe(II) in the same manner as ADP such as to allow this metal to catalyze ÔH formation in high yield from H 2 0 2 .

The yield of spin-trapped OH is about

20 μΜ or one-fifth the amount of Fe(II) added.

The yield of

OH in fact is directly proportional to the amount of Fe(II) added when H 2 0 2 is in excess (10). Figure 2 demonstrates that the amount of ÒH spin-trapped from Fe(II) mediated breakdown of H 2 0 2 increases in a sigmoidal manner up to 0.6 mM ADP but then increases in a more gradual but linear fashion at higher concentrations (10).

A somewhat

similar pattern was observed for ATP, only more ÔH was spintrapped at all concentrations as compared to ADP.

Interest-

ingly, AMP at all concentrations tested did not cause an enhancement of ÒH formed over that observed in the bicarbonate buffer only.

I have investigated the guanosine, cytosine and

thymidine tri-, di- and monophosphate nucleotides in a similar fashion and have found that in all cases the di- and triphosphate nucleotides were very effective in allowing Fe(II) to

NudeoMa (mM)

Figure 2. The amount of ÔH spin-trapped as a function of nucleotide concentration (10).

288 catalyze OH formation from H 2 0 2 , but the monophosphate nucleotides were not (11).

In all cases the triphosphate nucleo-

tides were more effective than the diphosphate nucleotides. Somewhat similar curves were obtained as a function of nucleotide concentration as was obtained with the adenosine nucleotides . The nucleotides are usually considered as being complexed with M g + + and therefore we have determined if Fe(II) binding to the di- and triphosphate nucleotides is modified by M g + + such that OH formation from H 2 0 2 is altered

(11).

Figure 3 shows that

M g + + does not effectively compete with Fe(II) in nucleotide binding.

That is, the initial Fe(II) level was 50 μΜ whereas

the ADP concentration was 2 mM.

Adding M g + + before the addi-

tion of Fe(II) to a level of 100 times that of the iron caused only about a 20% reduction in the amount of ÒH spin-trapped. This demonstrates that M g + + , even if present at 100 times the Fe(II) level and 2.5 times the nucleotide level, does not effectively compete in a manner such as to prevent formation of the Fe(II)-nucleotide complex.

The ferrous-nucleotide complex is much more effective in catalyzing OH formation from H 2 0 2 than is the ferric-nucleotide

50

σ>

? _

eg σ> 05

'

25 _

Λ -

_

·

1.0

2.0

3.0

4.0

ADP 2mM, Fe (II) 50>jM, Mg + + (mM) Figure 3. The effect of M g + + concentration on the amount of ÔH spin-trapped in the ADP—Fe(II)—H 2 0 2 system.

5.0

289 complex.

Kinetic studies

the other

nucleotides

have shown that ADP, and presumably

as w e l l , p r e s e r v e s

c a p a b l e of c a t a l y z i n g

ÒH formation

in t h e p r e s e n c e o f b i c a r b o n a t e time

much longer

buffer only.

In fact,

than

the half

f o r t h e l o s s o f a c t i v i t y w a s 160 s e c i n t h e p r e s e n c e o f

ADP but was about (11).

7.5 s e c in the a b s e n c e of this

Thus, we think

o x i d a t i o n of ferrous cally

active

esting the

the iron in a state

from H 2 0 2

that the nucleotides to ferric w h i c h

in ÒH formation

and most

to the ferrous

OH formation along with

t h e n is l e s s

from added H 2 0 2 . reduce

state which

from H 2 0 2 .

the

the rapid catalyti-

Another

likely biologically meaningful

fact that ascorbate will

complex

nucleotide

prevent

the results of the various

is

ferric-nucleotide

then effectively

This result

inter-

observation

is s h o w n

catalyzes

in F i g u r e 4

necessary

control

exper-

iments .

investigated

lipid peroxidation

the role of o x y g e n

m e n t of iron in the p r o c e s s . isolated, homogenized

c. 100 o>

σι in CD >

« «

to p e r o x i d i z e

60

of p u r e

I

Γ • α

-

80

1

I -

40 20 Conditions

er

and then allowed

- η -

0 1 υ »

o 20* E φ I 10


. aO

Pantane

in 0.8 .c «£! 0.6 C x O 01 O A

x

τΐοο

Ethane

AO

< 1 c 0.2

20 0

0 0

10 2 0 7.

30

Methemoglobin

E Φ I

Fig.II: E f f e c t of different globin

methaemo-

concentration

o n the f o r m a t i o n of alkanes a n d the h a e molysis

(n=6). For

i n c u b a t i o n see Methods. Mean +

SD.

344

The measurement of pentane and ethane produced in red cells is a useful technique regarding peroxidising effects of foreign compounds and effects of antioxidants. Free radicals capable of abstracting hydrogen atoms from unsaturated fatty acids, such as hydroxyl radicals, induce lipid peroxidation, whereas transition metal ions catalyse propagation reactions. In all instances, hydroxyl radical participation in initiation of lipid peroxidation has been proposed for the superoxidedependent lipid peroxidation. The basic mechanism proposed for the formation of hydroxyl radicals in superoxide-dependent lipid peroxidation is based on a combination of the reactions that constitute Fenton's reagent and the Haber-Weiss reaction. In agreement, our results suggest that after formation of methaemoglobin by nitrite, hydrogen peroxide-induced lipid peroxidation is diminished, since methaemoglobin is not able to release a superoxide radical like haemoglobin and thus, haemiron cannot act as a catalyst promoting red cell lipid peroxidation .

References 1. Riely, C.A., Cohen, G., Lieberman, M.: Science 183, 208 210 (1974) . 2. Frank, H., Hintze, T., Bimboes, D., Remmer, H.: Toxicol Appi Pharmacol 56, 337 - 344 (1980). 3. Tappel, A.L.: Free Radicals in Biology, Vol. IV, Ed.: W.A. Pryor, Academic Press, New York (1980). 4. Clemens, M.R., Remmer, H.: Blut 45, 329 - 335 (1982). 5. Lieberman, M., Kunishi, A.T., Mapson, L.W., Wardale, D.A.: Biochem J 97, 449 - 459 (1965).

IN V I T R O

ASSESSMENT

OF H E P A T I C

MALONDIALDEHYDE

Albrecht

Wendel

PEROXIDATION

BY

DETERMINATION

and Rudolf

Physiologisch-Chemisches D-7400

LIPID

OR E T H A N E

Institut

Reiter der

Universität

Tübingen,Hoppe-Seyler-Str.1

Introduction

Xenobiotic

- i n d u c e d LPO

primary

event

carbon

exhalation

in h e p a t o c e l l u l a r

invasive

assay

assessment

of L P O

Another rial

material (MDA).

which

mination

adapted tation an

the

estimate and

pentane

the

poor

correlations

gas evolution of

to c h e c k in-vitro

technique LPO

under

the

organs

-mortem

mate-

reactive

malondia1dehyde

quantitatively metabolic

(2),the

comeli-

mitochondrial

during

between

LPO in m i c e

the validity

non-

for

acid

to as

as a r t i f a c t s

upon drug-induced

in o r d e r

venience

in p o s t

o f LPO a r e

and

hydro-

perfused

dye b e t w e e n MDA and t h i o b a r b i t u r i c

for

ters observed

in

ari s e . D i f f e r ent

as w e l l

the

is a v a i l a b l e

as

used

possible

n o n - d e s t r u e t i ve

referred

indexes

of e t h a n e

of MDA,

the t r i m e t h i n the reason

two

a

of t h i o b a r b i t u r i c

differences

rates

metabolism

well

of L P O , m a i n l y

as a

destructi on.With

(1)

as

is g e n e r a l l y

large

discussed

high sensitivity

i η - ν ivo

determination

If t h e s e

pared,

technique

of

estimate

is the

is c u r r e n t l y

processing acid may

these

parame-

(3 ) . T h e r e fore

to t h e of b o t h aspects

sensitivity.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

in-vitro both

we

situ-

techniques of

be

as

con-

346 When

mouse

liver mierosomes,peroxidized to

different

ex-

tents,were assayed for ethane evolution as well as for thiobarbituric acid reactive

material,a very

close

correlation

of both parameters was found.As shown in Fig.2, one molecule of ethane

is evolved per 2200 molecules

oxidative

disintegration

trast,a

large

of

scattering

MDA upon

per-

microsomal membranes.In

con-

of

the

of

parameter

pair pentane

evolution / MDA formation was observed. This technique can be of

various

chemical

pro-

practically applied for the screening

or

interference

considerations

are

anti-

oxidative

in

system

a

compounds

were

without

pharmacokinetic

distribution phenomena play no role.The

results of such experiments where different drugs were added to mouse liver

homoqenate are shown in the following

table.

Table I : E f feet of various compounds on lipid peroxidation in mouse liver homogenate as monitored by ethane evolution compounds added

at

finally

ethane/hour per mg of protein

inert

1

mM

,rates

(all

given in pmoles

,blank = 4.6 ).

inhibitory

stimulatory

benzphetamine,

4.5

octylamine,

2.4

C Cl4

25.4

ethylmorphine ,

A. 8

phenetidine,

2.8

paraquat,

15.2

campher,

4.5

12.2

p-nitroanisol

1.8

daunomycin

ethoxycoumarine,4.5

phenacetine,

1.6

DEF

9.8

phorone,

4.6

paracetamol,

1.5

DEM

9.2

a-methyl-dopa ,

4.7

DEF=diethyl

fumarate, DEM=diethyl

maleate

adriamycin

7.8

furosemide

7.4

menadione

5. 3

347 Results

Figure

1

illustrates

that a c o n v e n t i o n a l

be m o d i f i e d

in such

microsomal

suspensions

glass syringe

a way that gas e v o l u t i o n can be m e a s u r e d

rates

(l)from

at d i f f e r e n t

times

under defined atmospheric conditions without dilution fects in the g a s p h a s e . T h i s sampling without and a

simple

the l i m i t a t i o n s

time-course

resolution

device

allows

of the h e a d - s p a c e

of LPO

in

the

can

ef -

isobaric technique

very same

in-

cubation.

FIG.2

FIG. 1

M A L O N D I A L D E H Y D E FORMATION EVOLUTION FROM M I C R O S O M A L

AND ETHANE SUSPENSIONS

η moles M D A oiygen supply

(ampNng

poM tor

0

In the p r e s e n c e of an N A D P H r e g e n e r a t i n g

20

40

60 ρ moles ethane

system,liver

micro-

s o m e s from m a l e m i c e e v o l v e d 0 . 9

p m o l e s of e t h a n e per mg

p r o t e i n per m i n

phosphate pH=7.4,

protein,

(O.lmM p o t a s s i u m

3.3 mM Mg C l 2 , l

mM N A D P ,

isocitrate dehydrogenase,

of F e / A D P this rate w a s s t i m u l a t e d e f f e c t s d e c r e a s e d by 8b% 525 A was a d d e d in 0 . 2 5 mM

10 mM i s o c i t r a t e ,

1 ^jg/ml r o t e n o n e ,

22

of

2 mg/ml 0.5

U/ml

).By

addition

to 1.3 p m o l e / m g /

min.Both

w h e n the m o n o o x y g e n a s e concentration.

blocker

SKF

348 It

seems

produce

remarkable

hepatic

in the

the c o m p o u n d s

injury,e.g.paracetamol,

e.g. ο ι - m e t h y 1 - d o p a , dants

that some of

show either

in-vitro

or

no effect

known

to

neurodegeneration or act as

antioxi-

system.

Di scuss ion

The

close

correlation

microsomal

LPO

essentially

the same

were

around

line

in F i g . 2

only

for

orders

suggests

of

magnitude

material.In

other

sensitivity.Thus conditions be e q u a l l y LPO,in with

this

remarkable

intact

detection about

that h y d r o c a r b o n

less

limits

than

the

study ethane

shows

of

and c o m p a r a b l y contrast

to

monitor ethane

of MDA

can

the

straight

evolution

accounts

which

is a b o u t

yield

in

-

like for

and MDA

sensitive

MDA

three

compensates

that u n d e r

formation

following

for

one nmol

,the s l o p e

of LPO p r o d u c t s ,

MDA

parameters

words,stoichiometry

in vitro useful

and

both

range,while

measured.However

a small part

ethane

that

event.The

the ρ mol

be a c c u r a t e l y

between

indicate

appropriate formation

parameters

the s i t u a t i o n

when

may for

working

animals.

Re ferences

1.Wendel.A.,Dume1 i η,E.E.: Meth.Enzymo 1.77,10-16

(19B1).

2. F i l s e r , J . G . , B o l t , H . M . , M u l i a w a n , H . , K a p p u s , H . :

Arch.Toxicol.

52,135-147. 3.Wendel,A.Feuerstein.S.,Konz,Κ.H.: 2051-2055

(1979).

Biochem.Pharmac.28,

349 DISCUSSION

LANDS: You have emphasized that you can measure nanomolar levels of MDA. I had the impression from the report of ASAKAWA that about 10 to 50 nM peroxide were needed to get about 1 nM of MDA. What amount of lipid hydroperoxide do you have to have in order to get 1 nMol of MDA assayed? HENDEL: Well, I can just calculate back from the oxygen consumption which we can measure in this system, and that would yield a figure of about 5% of malondialdehyde, a ratio of 1:20. SRIDHAR: There is a paper by JORDAN & SCHENKMANN (Biochem. Pharm. (1982) 31, 1393-1400) which describes the correlation between malonaldehyde production and arachidonic acid loss during microsomal lipid peroxidation. WENDEL: Well, we looked also at the fatty acids which are affected, and at least in these ¿n vivo experiments using that massive drug challenge, we find that the loss of 22:6, docosahexaenoic acid, and ethane evolution correlates, while we don't see a correlation with other fatty acids.

DETECTION OF MALONDIALDEHYDE BY HPLC

Johanna Lang, Peter Heckenast, Hermann Esterbauer Institut für Biochemie, Universität Graz A 8010 Graz Trevor F. Slater Department of Biochemistry, Brunei University Uxbridge, Middlesex, U.K.

Introduction Malondialdehyde (MA), a product of lipid peroxidation is generally measured by the TBA assay. This method however is not specific, as so called "malonaldehyde-like material" is also reactive with TBA. Poyer and McCay^ have used Sephadex G 10 chromatography to demonstrate the presence of free MA in peroxidizing liver microsomal suspensions. High performance 2

liquid chromatography or an ODS column

(HPLC), using a size exclusion column

was also tried to assay free MA. These

methods however require extensive sample pretreatment (ultrafiltration, steam destination) to avoid peak interference, moreover with the ODS column, the MA peak is eluted near or with the void volume and its retention time cannot be increased by altering the mobile phase. We 4have developed an HPLC method for the determination of free MA , using an aminophase column.

Results and Discussion Samples, such as incubation mixtures of peroxidizing microsomes, mitochondria and autoxidized polyunsaturated fatty acids are directly injected (20 μΐ loop) into the HPLC instrument and

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

352

separateci with an aminophase column (Spherisorb S 5 NI^, Waters carbohydrate analysis column) with Tris buffer 0,03 M pH 7,4/ acetonitrile 9:1 (v/v) as eluent, flow = 1,0 ml/min. We recommend the use of an ODS precolumn to protect the analytical column. The effluent is monitored at 270 nm, which is the absorption maximum of free MA in the enolate anionic form. Peak identification and calibration is done by running a reference 'chromatogram with a solution of freshly prepared free MA. For protein rich samples, such as total homogenates, crude nuclear fraction and erythrocytes, samples are pretreated by addition of an equal volume of acetonitrile, which will remove most of the protein by precipitation. The sample is centrifuged and the clear supernatant is used for HPLC analysis. For samples, where lipid peroxidation was stimulated by ascorbate/iron, the eluent was modified, since ascorbate elutes very near to the MA peak and therefore interferes with the MA detection. Addition of 0,1 mM I^C^ to the eluent leads to a rapid and complete oxidation of the ascorbate to dehydroascorbate, which shows no interference with malonaldehyde.

no

e

ffect

on malonaldehyde retention time and malonaldehyde concentration. Fig. 1 shows HPLC chromatograms of a MA standard and suspensions of peroxidizing liver microsomes, either directly injected or treated with an equal volume of acetonitrile and centrifuged prior to the injection. Free MA was estimated by the HPLC method described above in suspensions of liver microsomes, mitochondria and crude nuclear fraction as well as total homogenates of liver,kidney, lung, brain, spleen, skeleton muscle and heart muscle, in which lipid peroxidation had been stimulatee by ADP/Fe, Fe or ascorbate/Fe. In all of these systems MA was also assayed simultaneously by the TBA method"'. In none of the experiments could a noteworthy difference between TBA and HPLC values be found, which indicates, that the TBA-reactive material in these peroxidized biological samples is in fact free MA. The close agreement

353 b)

Fig. 1

HPLC chromatograms of

0,016 AU a) microsomes, incubated for 45 min. in the presence of ADP/Fe and HA

MA

precipitated with an equal volume of acetonitrile.

vJU

^

b) as a), exept that the complete microsomal suspension was injected into the HPLC.

d)

c)

c) as a), immediately after addition MA

MA

of ADP/Fe. d) reference malonaldehyde (20 μΜ)

ι ι ι ι ι I

4

8

I I I I · I

12

4

8

in ο,Ι M Tris buffer pH 7,4.

12

min. ret. time

between the two methods is shown in tab.1 for liver microsomes and mitochondria. Tab. 1 Malonaldehyde (nmoles/mg protein) in suspensions of rat liver microsomes and mitochondria, stimulated by ADP/Fe 3

5

2+

4

2+

5

(1,6.10~ M/1,8.10~ M) or ascorbate/Fe (5.10~ M/2.10" M) after 30 min. of incubation. The protein concentration was approx. 1 mg/ml. Microsomes ADP/Fe2+ Microsomes asc/Fe2+ Mitochondria ADP/Fe 2+ TBA

HPLC

TBA

HPLC

TBA

HPLC

50,0

48,9

46,6

46,9

26,3

24,8

The HPLC method was also used to follow the kinetic of free MA formation during autoxidation of arachidonic acid (0,1mg/ml in Tris buffer/KCl pH 7,4) in the presence of ascorbate/Fe -4 -5 (5.10 M/2.10 M). Interestingly enough, a remarkable difference was found in this experiment between free MA as estimated

354 Fig.

δ io

LA' + H"

Free-radical initiation

(2)

LA"

>

LA'-

Double bond rearrangement

Sequence I protein absent

(DC)

S e q u e n c e II protein present

(3) L A 1 ' + 0 2 > LA'00' l e a d i n g to c l a s s i c a l peroxidation pathway

LA'' + Prot

»

LA' +

Prof

W h e n LA is e x p o s e d to f r e e - r a d i c a l a c t i v i t y i n vitro i n

the

p r e s e n c e o f a l b u m i n a p r o p o r t i o n of the l i p i d is s t a b i l i s e d the p r o t e i n

(Sequencell)

ation pathway

(Sequence

: some f o l l o w s

the c l a s s i c a l

by

peroxid-

I). H o w e v e r , v i r t u a l l y a l l DC

measur-

a b l e i n b i o l o g i c a l m a t e r i a l is L A ' - i . e . the s t a b i l i s e d

prod-

uct of S e q u e n c e II. It is p r e s e n t e i t h e r as a free fatty

acid

(eg. i n d u o d e n a l j u i c e ) or e s t e r i f i e d i n p h o s p h o l i p i d s , glycerides and cholesteryl

esters. Current mass

s t u d i e s s u g g e s t that it is a n i s o m e r or m i x t u r e LA'

of i s o m e r s

(6). The c h a n g e s w h i c h o c c u r in the p r o t e i n as the

of the s t a b i l i s i n g r e a c t i o n m a y be s i m i l a r by W i c k e n s

et al

(8), a n d are b e i n g f u r t h e r

tri-

spectroscopic

to t h o s e

of

result

described

investigated.

References 1. Di L u z i o , N . R . : J. A g r . F o o d C h e m . , 20, ¿ 8 6 - 4 9 0

(1972).

2. B r a g a n z a , J . M . , W i c k e n s , D . G . , C a w o o d , P . , D o r m a n d y , Lancet, in press. 3. L u n e c , J . , H a l l o r a n , S . P . , W h i t e , A . G . , D o r m a n d y , J. of R h e u m a t o l . 8, 2 3 3 - 2 4 5 (1981).

T.L.:

T.L.:

k. W i c k e n s , D . G . , W i l k i n s , M . H . , L u n e c , J . , B a l l , G . , D o r m a n d y , T . L . : A n n a l s of C l i n . B i o c h e m . 18, 1 5 8 - 1 6 2 (1981). 5. B a r b e r , A . A . , B e r n h e i m , F . : A d v . G e r o n t o l . R e s . 2, (1967). 6. C a w o o d , P., I v e r s e n , S . A . , D o r m a n d y ,

T . L . : to be

355-Λ03

published.

7. A u s t , S . D . , S v i n g e n , B . A . : F r e e R a d i c a l s in B i o l o g y v o l . V, E d . P r y o r , W . A . , A c a d e m i c P r e s s , N e w Y o r k , (1982). 8. W i c k e n s , D . G . , N o r d e n , A . G . , L u n e c , J., D o r m a n d y , B i o c h i m . B i o p h y s . A eta 7J¿, 6 0 7 - 6 1 6 (1983).

T.L.:

THE ROLE OF IRON IN L I P I D PEROXIDATION INDUCED BY ADRIAMYCIN DURING REDOX CYCLING IN LIVER MICROSOMES*

H. Kappus, H. Muliawan, and M.E. Scheulen Department of Dermatology (FB 3, WE 15), Free U n i v e r s i t y of B e r l i n , Augustenburger Platz 1, D-1000 B e r l i n 65, F.R.G., and Department of Internal Medicine (Tumor Research), U n i v e r s i t y of Essen, Hufelandstr. 55, D-4300 Essen 1, F.R.G.

Introduction Adriamycin ( d o x o r u b i c i n ) , an anthracycline a n t i b i o t i c , i s used in tumor therapy. The cytotoxic a c t i v i t y of adriamycin has been related to the increased formation of oxygen r a d i c a l s occurring during redox c y c l i n g of adriamycin which i s catalyzed by reductases ( 1 , 2). I t has a l s o been suggested that l i p i d peroxidation induced by these oxygen r a d i c a l s

is

responsible f o r the t o x i c side e f f e c t s observed during tumor therapy, e.g. c a r d i o t o x i c i t y (1 - 3). However, contradictory r e s u l t s on the a b i l i t y of adriamycin to provoke l i p i d peroxidation are reported (4 - 6). Theref o r e , as a new measure for l i p i d peroxidation we determined ethane and n-pentane. As enzymic scource we selected rat l i v e r microsomes which are well known to catalyze an NADPH-dependent redox cycle of adriamycin.

Methods The methods used are described in detail elsewhere ( 7 ) . In general, rat l i v e r microsomes without contaminations of iron ions were incubated a e r o b i c a l l y i n T r i s buffer with an NADPH-regenerating system ( N a - i s o c i t r a t e , i s o c i t r a t e dehydrogenase and NADP + ). Adriamycin was added to microsomes 2 min before the s t a r t of the reaction with FeCl 2 and NADP+. Ethane and n-pentane were determined as already described ( 7 ) . *) Supported by the Deutsche Forschungsgemeinschaft

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

360 Results The Table demonstrates that our microsomal system was f a i r l y free of i r o n . This i s shown by the fact that in the presence of NADPH ethane and n-pentane were not formed (Table). Only a f t e r a d d i t i o n of FeCl 2 and NADPH ethane and n-pentane were observed. Furthermore, i t i s important to note that in the presence of NADPH neither 1 χ 10~ 5 M nor 1 χ 10"4 M adriamycin stimulate ethane and n-pentane formation (Table). Under these conditions a redox cycle of adriamycin was measurable s i m i l a r l y as demonstrated recently (8). But in the presence of NADPH, FeCl 2 and adriamycin r e l a t i v e l y high amounts of ethane and n-pentane were formed which exceeded the amounts observed with FeCl 2 and NADPH 2 - 3

f o l d (Table). With FeCl 2 and

adriamycin but in the absence of NADPH only very low amounts of ethane and n-pentane were formed.

Discussion We demonstrate here that the adriamycin-induced l i v e r microsomal

lipid

peroxidation depends on the presence of i r o n i o n s . Probably the authors who p r e v i o u s l y measured adriamycin-induced l i p i d peroxidation used microsomes or buffer systems which contained traces of iron i o n s . This i s mainl y shown by the lack of l i p i d peroxidation in the presence of adriamycin and NADPH where high amounts of oxygen are consumed (8) and superoxide anions are formed (1 - 3). This indicates that redox c y c l i n g of adriamycin r e s u l t s in a c t i v e oxygen species which are unable to react with unsaturated f a t t y a c i d s . We conclude that during redox c y c l i n g of adriamycin l i p i d peroxidation i s not occurring unless unbound iron ions are present. Our r e s u l t s can be interpreted by the following scheme ( F i g u r e ) : Superoxide anions are formed during the enzyme-catalyzed redox c y c l i n g of adriamycin.

Independently

ferrous ions induce l i p i d peroxidation. One of the key events in i r o n induced l i p i d peroxidation i s presumably the reduction of the f e r r i c to the ferrous state of i r o n (9, 12). The s t i m u l a t i o n of iron-induced l i p i d peroxidation observed with adriamycin i s probably the r e s u l t of an

361 TABLE Alkane formation in rat liver microsomes during aerobic incubation with or without an NADPH-regenerating system (n.d. = not detectable) nmol Alkane / mg Microsomal Protein / 15 min (η = 4) Ethane

n-Pentane

NADPH

n.d.

n.d.

+ NADPH

n.d.

n.d.

+ FeCl 2 (2.4 X 10-5 M) NADPH

0.,006 ± 0. 002

0..016 ± 0. 004

+ FeCl 2 (2.4 X 10-5 M) + NADPH

0..126 ± 0. 030

0..199 ± 0. 045

-

+ Adriamycin NADPH

(1

+ Adriamycin +

(1 χ 10-5 M)

χ 10-5 M)

n.d.

n.d.

n.d.

n.d.

(1 χ 10-5 M) FeCl;, (2.4 X 10-5 M) NADPH

0..006 ± 0. 002

0,.015 ± 0. 004

+ Adriamycin (1 χ 10-5 m) + FeCl, (2.4 X 10-5 M) + NADPH

0..316 ± 0. 001

0..450 ± 0. 044

NADPH

+ Adriamycin +

+ Adriamycin (1 χ 10-4 M) NADPH

n.d.

n.d.

+ Adriamycin (1 χ 10-4 M) + NADPH

n.d.

n.d.

+ Adriamycin (1 χ 10-4 M) + FeCl 2 (2.4 X 10-5 M) NADPH

0..004 ± 0. 002

0,.011 ± 0. 003

+ Adriamycin (1 χ 10-4 M) + FeCl (2.4 X 10-5 M) 2 + NADPH

0..266 ± 0.024

0,.627 ± 0. 011

increased reduction of ferric ions by superoxide anions formed during redox cycling of adriamycin. In both cases the involvement of cytochrome P-450 reductase is very likely (Figure). Furthermore, it is also possible that an adriamycin-iron-complex activates oxygen to radical species which elicit lipid peroxidation (10, 11).

362 Redox Cycle: Adriamycin . 1 NADPH . Η*

p^o"Adriamycin-Semiquinone

Adriamycin-Semiquinone • 02 Lipid [LH) Peroxidation

LH . 02 . Fe2* . Η* L· L-O-O· Fe3'

Stimulation :

•

.

-

Adriamycin

L"

.

Fe3*

•

L-O-O'

LH \ NADPH. H*

•

H202

O,

. \ NADP*

L-O-OH NADPH-Cytochrome P-450

Reductase

Fe*·

Fe3*

.

L'

^NADP*

Fe'*

FIGURE: Proposed scheme of stimulation by adriamycin of iron-catalyzed microsomal l i p i d peroxidation

References 1. Kappus, H., Sies, H.: Experientia 37, 1233-1241 (1981). 2. Trush, M.A., Mimnaugh, E.G., Gram, T.E.: Biochem. Pharmacol. 3 U 3335-3346 (1982). 3. Lown, J.W., Joshua, A.V., Chen, H.-H.: In: Free Radicals, L i p i d Peroxidation and Cancer, (D.C.H. McBrien, T.F. S l a t e r , e d s . ) , pp. 305-328, Academic Press, New York 1982 4. Muliawan, H., Scheulen, M.E., Kappus, H.: Res. Commun. Chem. Pathol. Pharmacol. 30, 509-519 (1980). 5. Mimnaugh, E.G., Trush, M.A., Gram, T.E.: Biochem. Pharmacol. 30, 2797-2804 (1981). ~ 6. Babson, J . R . , Abel!, N.S., Reed, D.J.: Biochem. Pharmacol. 30, 2299-2304 (1981). 7. Muliawan, Η., Scheulen, M.E., Kappus, H.: Biochem. Pharmacol. 31, 3147-3150 (1982). 8. Scheulen, M.E., Kappus, H., Nienhaus, Α . , Schmidt, C.G.: J. Cancer Res. Clin. Oncol. JJ)3, 39-48 (1982) 9. Kornbrust, D . J . , Mavis, R.D.: Mol. Pharmacol. V7. 400-407 (1980). 10. Myers, C.E., Gianni, L . , Simone, C.B., Klecker, R., Greene, R. : Biochemistry 2i_, 1707-1713 (1982). 11. Sugioka, K . , Nakano, M.: Biochim. Biophys. Acta 7J2, 333-343 (1982). 12. Gutteridge, J.M.C.: FEBS L e t t . ^50. 454-458 (1982).

COMPARATIVE EVALUATION OF SUBSTRATE EFFECT ON CUMENE HYDROPEROXIDE-DEPENDENT LIPID PEROXIDATION IN MITOCHONDRIA AND MICROSOMES

Alberto Bindoli, Marina Valente, Lucia Cavallini Centro Studio Fisiologia Mitocondriale CNR, and Istituto di Chimica Biologica, Via Marzolo 3, 35131 Padova, Italy.

Introduction Lipid peroxidation is a degenerative process of unsaturated fatty acids, that is involved in the mechanism of ageing, oxygen toxicity, membrane alteration and other pathological events (1). Natural or synthetic hydroperoxides may be the initiators of lipid peroxidation "in vivo" since it was shown that they are able to induce this phenomenon "in vitro" either in liver microsomes (2,3) or in mitochondria (4). In the present paper we have compared the extent of peroxidation on both mitochondria and microsomes and the preventing effect elicited by substrates of the mitochondrial and microsomal electron transport systems. The present work is based on the contention that a cytochrome P-450 is present in inner mitochondrial membrane (5,6) and is different from the microsomal one. Results and Discussion In Fig.1 the peroxidation initiated by eumene hydroperoxide (CHP) in either microsomes or submitochondrial particles (SMP) of rat liver is reported. Microsomes appear to be more susceptible than mitochondria to lipid peroxidation measured both as malondialdehyde (MDA) and lipid hydroperoxides (LOOH)formation. This lipid peroxidation is an enzymatic reaction mediated by the hemoprotein cytochrome P-450. In fact: a) it does not occur in boiled membranes nor in liposomes; b) it is not mediated by free iron ions activation of CHP since our experiments were performed in the presence of millimolar concentrations of EDTA; c) well known inhibitors of cytochrome P-450 (SKF 525A, metyrapone and aniline) prevent this type of lipid peroxidation (Table I); d) the larger extent of lipid peroxidation in microsomes

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

364 compared to that in mitochondria is related to the different concentration of cytochrome P-4S0, which is about 0.15 nmoles/mg protein in rat liver mitochondria (7) and about 1.0 nmoles/mg protein in rat liver microsomes (8).

CHP

tmM)

Fig.1: CHP-induced formation of MDA and LOOH in mitochondrial and microsomal membranes. Rat liver microsomes (1 mg/ml) and SMP (2 mg/ml) were incubated in 0.125 M KCl buffered with 15 irM HEPES and 6.5 mM Tris at pH 7.4 and containing 1 mM EDTA, for 10 minutes at 30°C with the indicated concentrations of CHP. Aliquots of 1 ml were withdrawn, MDA was determined according to (9) and LOOH with the thiocyanate method (10) modified as in (3). Values are expressed as nmoles/mg protein. Microsomes: LOCH (Δ—Δ), MDA (O—o) ; SMP: LOOH (A—•), MDA (·-·).

TABLE I : EFFECT OF P-450 INHIBITORS ON MDA-FORMATION IN MICROSOMES AND SUBMITOCHONDRIAL PARTICLES. Additions MDA None SKF 525A (0.6mM) Metyrapone (2mM) Aniline (1mM)

13.50 2.05 8.24 4.69

Microsomes "êlnhibition -

85 40 65

MDA 2.83 0.16 1.10 0.70

SMP %Inhibition -

95 61 73

Microsomes and SMP were incubated as described in the legend of Fig. 1 in the presence of 0.5 mM CHP. MDA was determined according to (9) and expressed as nanomoles/mg protein.

365

The active forms of oxygen (superoxide, hydrogen peroxide, hydroxy radical and singlet oxygen) are scarcely involved in this type of lipid peroxidation since a small effect of the specific scavengers (superoxide dismutase, catalase, mannitol and histidine, respectively) was seen either in microsomes or mitochondria, while the antioxidants vitamin E and butylated hydroxyanisole were active (data not shown). In mitochondria incubated with 0.5 mM CHP, in the presence of 1 mM KCN to prevent respiratory oxygen depletion, all the substrates tested (succinate, glutamate, (3-hydroxybutyrate, malate, isocitrate) exhibit a protective effect against lipid peroxidation which is more evident when LOOH are measured (Table II). Succinate, p,-hydroxybutyrate + NAD+ and reduced pyridine nucleotides, exhibit a clear inhibitory effect also in submitochondrial particles (data not shown) meaning that the protection is mediated by some respiratory chain component. In microsomes either NADH or NADPH are strong inhibitors of both MDA and LOOH formation (Table II). TABLE II: INHIBITION OF VARIOUS SUBSTRATES ON CHP-INDUCED LIPID PEROXIDATION IN MITOCHONDRIA AND MICROSOMES. Additions

None Succinate (5mM) Glutamate (SmM) Isocitrate (5mM) Ρ-Hydroxybutyrate (5mM) Malate (5mM) NADH (O.SnM) NADPH (0.5mM)

Mitochondria MDA LOOH (nmoles/mg protein) 1.15 0.68 0.72 0.82 0.71 0.76

8.82 3.79 2.91 3.00 2.90 3.00

-

-

-

-

Microsomes LOOH MDA (nmoles/mg protein) 12.61 -

28.00 -

-

-

-

-

-

-

-

-

4.60 5.50

8.00 10.00

Rat liver mitochondria (4 mg/ml) and microsomes (1 mg/ml) were incubated as described in the legend of Fig.1 in the presence of 0.5 mM CHP. All the incubations with mitochondria were performed in the presence of 1 mM KCN. Values are the average of 4 experiments. MDA and LOOH were determined according to (9) and (3, 10) respectively.

The activation of CHP by cytochrome P-450 can proceed through either a homolytic or heterolytic cleavage of the 0-0 bond of CHP (11). In the first case the cumyloxyl radical can be the initiator of lipid peroxidation by extracting a hydrogen atom from the unsaturated lipid so being reduced to cumyl alcohol. In addition cumyloxyl radical can undergo a ^-scission pro-

366 ducing acetophenone and methyl radical (12). In the second case the oxyferric form of cytochrome P-450 could be the sparker of lipid peroxidation. In this case only cumyl alcohol should be formed. Anyway, independently of the mechanism, NADH or NADPH can reduce the activated form(s) that spark(s) lipid peroxidation so inhibiting the phenomenon. Nevertheless it is difficult to determine which is the activation mechanism since under our conditions the products of CHP decomposition (namely cumyl alcohol and acetophenone, determined by HPLC analysis) show the same ratio either in presence or in absence of NADH, the only difference being the removal of

CHP

which is stimulated by NADH. Acetophenone formed is about 105o of CHP disappeared, then CHP is mainly reduced to cumyl alcohol (data not shown). Quinones which are present in mitochondria in a large amount, once reduced by the substrates can play a role in the inhibition of lipid peroxidation as already reported by Takanayagi et al. (13); however in our microsomal preparations we did not measure appreciable amounts of quinones, therefore the protection of peroxidation exhibited by NAD(P)H should mainly proceed through the reduction of the activated form(s) deriving from the inter action between CHP and cytochrome P-450.

References 1. Tappel,A.L.: Fed.Proc. 32, 1870-1874 (1973). 2. O'Brien,P.J., Rahimtula.A.: J.Agrie.Food Chem. 23, 154-158 (1975). 3. Cavallini,L., Valente,M., Bindoli,Α.: Biochim.Biophys.Acta 752, 339-345 (1983). 4. Bindoli,Α., Cavallini,L., Jocelyn,P.: Biochim.Biophys.Acta 681, 496-503 (1982). 5. Sato,R., Atsuta,Y., Imai,Y., Taniguchi,S. andOkuda,K.: Proc.Natl.Acad. Sci. USA 74, 5477-5481 (1977). 6. Björkhem,I. and Holmberg,I.: J.Biol.Chem. 255, 5244-5249 (1980). 7. Okuda,K., Ruf,H.H., Ullrich,V.: Hoppe-Seyler's Z.Physiol.Chem. 358, 689-694 (1977). 8. Sato,R., Omura,T.: Cytochrome P-450, Academic Press (1978), p.142. 9. Buege,J.A., Aust,S.D.: Methods in Enzymology 52, 302-310 (1978). 10. Streckert.G., Stand,H.J.: Lipids 10, 847-854 (1975). 11. Cadenas,Ε.,Sies,H.,Graf,H.,Ullrich,V.: Eur.J.Biochem.130,117-121 (1983). 12. Griffin,Β.W., Ting,P.L.: FEBS Letters 89, 196-200 (1978). 13. Takanayagi,R., Takeshige,K., Minakami.S.: Biochem.J. 192, 853-868 (1980).

STUDIES ON THE COMPARTMENTAL RELEASE OF LIPID PEROXIDATION PRODUCTS IN THE PERFUSED RAT HEART

Johan F. Kostera, Regina G. Sleea, Catharina E. Essed*3 and Hans Stam3 a b Depts. of Biochemistry I and Pathology , Medical Faculty, Erasmus University, Rotterdam - The Netherlands

Introduction

Recently (1) we have shown that perfusion of rat heart with eumene hydroperoxide (CuOOH) leads to the release of malondialdehyde (MDA), a breakdown product of lipid peroxidation, and of cellular proteins in the perfusate. Heart tissue revealed an elevation of MDA and fluorescent chromolipids. In this study we used a modified perfusion technique (2), which enabled us to collect separately the vascular/endothelial (Q

) and inter-

stitial fluid (Q ). Furthermore the influence of CuOOH on coronary flow and myocardial contractility was investigated.

Results

Comparing the various hydroperoxides (CuOOH, t-butyl hydroperoxide and H^Oj) in their ability to induce

lipid peroxidation, CuOOH is the most

effective and t-butyl hydroperoxide the least. The extent of MDA release into the perfusate is augmented if the hearts were preperfused with diethylmaleate (5 mM). This indicates that glutathione, as could be expected is involved. Besides cardiac myocytes, heart tissue also consists of endothelial cells, coronary vascular smooth muscle cells and fibroblasts. On cell basis the non myocytes account For 75% of all the cells in heart and can therefore not be neglected in the overall cardiac metabolism. With the modified perfusion technique it is possible to collect separately Q r v and CK. It has been shown (2,3) that the metabolites or products of the coronary vascular system leave the heart in Q

and those of the cardiac myo-

Oxygen Radicals in Chemistry a n d Biology © 1984 Walter de Gruyter & Co., Berlin · N e w York - Printed in Germany

368 Figi

A. CALCIUM MMtADOX

FigS

B. 0.6 πΜ CUMENE HYDMOKKOXDE

cytes predominantly via Q

The result of perfusing with 0.5 mM CuOOH and

the separate collection of 0.i and 0 rv are shovm in Fig. 1. In Q rv there is increase in MDA while in Q MDA appears about 10 min after

an immediate

the addition of CuOOH. These experiments show clearly that lipid peroxidation not only occurs in the coronary vascular system but also in the cardiac myocytes. The addition of CuOOH has an immediate effect on the coronary flow and contractility of the heart (Fig. 2B). There is an increment in the coronary flow which vanishes during the perfusion. The contractility of the heart diminishes slowly and failure occurs concomitant with the release of MDA in Q^. The increment of coronary flow and contractile failure is also seen during Ca^+-free perfusion (Fig. 2a). Restoration of the Fig3

Fig4

369 Ca2 + concentration leads to a decrease in coronary flow and the occurrence of contracture of the heart muscle (the "Ca^+-paradox"). These phenomena are also present in the final stage of the perfusion with CuOOH. For the Ca^+-paradox it is known that during Ca^+-free perfusion the energy state of the tissue is unchanged but upon reperfusion with Ca^ + massive breakdown of high energy phosphates takes place (4). It has been reported (5) that addition of CuOOH to isolated cardiac myocytes resulted in a decrease of ATP content. Fig. 3 shows the adenine nucleotides content and the energy charge (E = y

ATP + J ADP ) of isolated hearts durinq y Fperfusion with ATP+ADP+AMP

CuOOH. The fall in ATP level and the decrease in E occur about simultaneously with the contractile failure and the release of MDA in Q^. In contrast to the Ca^+-paradox there is no concomitant increase in AMP. Also the level of creatinephosphate is decreased at the same time the ATP content started to fall. These data indicate that the mitochondria are affected by CuOOH, possibly by slowly increasing the amount of intracellular Ca^ + .

This prompted us to investigate the effect of CuOOH perfusion

on several mitochondrial membrane enzymes (Table 1). The mitochondria were isolated after perfusion of the heart for the indicated times. It appears that the inner membrane plus matrix enzyme

cytochrome c oxidase, the

outer face of the outer membrane enzymes mono amino oxidase and palmitoylCoA synthetase, and the outer face of the inner membrane enzyme carnitine palmitoyl transferase I were all unchanged by perfusion with CuOOH. It can be concluded that although the ATP and creatinephosphate are decreasing after 10 min perfusion this is probably not the result of failure to oxidize fatty acids. Other possibilities (Ca^+-overload) are under present

Table 1. The activities (nmoles/min/mg protein) of mitochondrial enzyme after perfusion with 0.5 mM CuOOH Cytochrome c oxidase Time

Mono amino oxidase

Carn.palmitoyl transferase I

Palmitoyl-CoA synthetase

Control

Control

CuOOH

Control

CuOOH

Control

5'

1.63± 0.42

1.55± 0. 15

0.964+ 0.18

0.960± 0.029

4.8± 0.2

4.7± 0.2

75. 2± 1.15

69.4± 11.1

10'

1. 43± 0.65

1. 32± 0. 10

0.768± 0.090

0.859± 0.069

4.4± 0.1

4. 7± 0.1

68.7± 1.25

70.0± 2.05

20'

1.40± 0.05

1.20 0.09

0.927± 0.036

0.906± 0.137

4.7± 0.1

5. 0± 0.4

78.3 + 5.2

61. 1± 7.0

CuOOH

CuOOH

370

investigation. The Ca

-paradox and its consequent contracture are 2+

accompanied by a bulk release of protein after reperfusion with Ca Fig. 5 shows that during CuOOH perfusion

the release of protein from the

heart is biphasic. First an increase is observed during initial CuOOH perfusion and second during the occurrence of cardiac contracture, although the release of protein is lower when compared with the Ca^+-paradox. The electronmicroscopy of the control (30' perfusion) and 2 and 10 min perfusion with CuOOH does not show dramatic differences. The 30 min perfused heart shows a destruction of endothelial cell organelles and membranes and loss of intercellular connections (Fig. 5). The myocardial cells show a loss of thick and thin filaments and the mitochondria show a variable loss of cristae (Fig. 6).

References

1. Koster, J.F., Slee, R.G., Stam, H.: Biochem. Int. _2, 525-531 (1981). 2. De Deckere, E.A.M., Ten Hoor, F.: Pflügers Archiv 370, 103-105 (1977). 3. Stam, H., Hülsmann, W.C.: Biochem. Int. 2, 477-484 (1981). 4. Boink, A.B.T.J., Ruigrok, T.J.C., Maas, A.H.J., Zimmerman, Α.Ν.E.: J. Mol. Cell. Cardiol. 8, 973-979 (1976). 5. Noronta-Dutra, A.A., Steen, E.M. : Lab. Invest. _47, 346-353 (1982).

ASCORBIC ACID, LIPID PEROXIDATION AND THE INTERACTIONS OF DRUGS WITH THEIR RECEPTORS IN RAT BRAIN TISSUE PREPARATIONS

Richard E. Heikkila Department of Neurology, University of Medicine and Dentistry of New Jersey-Rutgers Medical School, Piscataway, New Jersey 08854, U.S.A.

Introduction Ascorbic acid i s a commonly used reducing agent that i s often added to dopamine receptor preparations to prevent the autoxidation of unstable dopamine agonists including dopamine, apomorphine, norpropylapomorphine and ADTN (1, 2).

However, in addition to this desired effect, ascorbate

can also promote extensive l i p i d peroxidation in dopamine receptor preparations (3, 4, 5, 6).

This l i p i d peroxidation has been shown to be respon-

s i b l e for the irreversible loss of the stereospecific binding of dopamine receptor antagonists including spiroperidol and domperidone (4, 5, 6). Thus in a s i n g l e experimental system ( i . e . the neostriatal membrane preparation), ascorbic acid acts as both a pro-oxidant and as an antioxidant. I t has become apparent in the l a s t several years that an ascorbic acidinduced l i p i d peroxidation can lead to large decrements in the binding of many other ligands.

For example, the binding of etorphine, an opiate ago-

n i s t ; dihydroergocriptine, an alpha adrenergic agonist; dihydroalprenolol, a beta adrenergic receptor antagonist; serotonin, a serotonin agonist, and haloperidol, a dopamine antagonist are all decreased by an ascorbic acidinduced l i p i d peroxidation (3, 4, 5, 6, 7, 8, 9).

I t should be mentioned

that ascorbic acid can also i n h i b i t the binding of various ligands by a mechanism other than by l i p i d peroxidation (10, 11).

In the present

study, various features about the relationship between the ascorbic acidinduced l i p i d peroxidation and the ascorbic acid-induced loss of binding will be described.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

372

Materials and Methods The binding of ^ - s p i r o p e r i d o l was done in a rat neos t r i atal membrane preparation exactly as described (4).

Sodium ascorbate (0 to 5 mM) was

added to the neostriatal membrane preparation, the samples were allowed to incubate at 37°C for 10 min, the -^-spiroperidol was added to 2 nM and the incubation carried out for another 15 min at 37°C.

Specifically bound ^h-

spiroperidol was then measured by l i q u i d s c i n t i l l a t i o n spectrometry.

Non-

specific binding was determined in the presence of 10~6M (+)-butaclamol. Lipid peroxidation was determined by the thiobarbituric acid (TBA) test in parallel tubes (containing no 3 H) exactly as described (11, 12).

Results and Discussion Sodium ascorbate had very substantial effects on the specific binding of 3H-spiroperidol

(Table 1).

There was a very large decrement in 3H-

spiroperidol binding at 0.05 and 0.5 mM ascorbate with a lesser effect at 0.005 mM and no effect at 5 mM ascorbate.

The dose-response curve for the

sodium ascorbate-induced l i p i d peroxidation was very similar to that observed on ^H-spiroperidol binding (Table 1).

There was a very large

increase in l i p i d peroxide formation at 0.05 and 0.5 mM ascorbate, a smaller increase at 0.005 mM ascorbate and no real increase at 5 mM ascorbate.

This type of U-shaped dose-response curve for ascorbate on

l i p i d peroxidation has been reported several times previously (13, 14). Both the ascorbate-induced loss of ^ - s p i r o p e r i d o l binding and the ascorbate-induced increase in l i p i d peroxidation could be prevented by the iron chelating agents DETAPAC and EDTA and could be enhanced by addition of iron s a l t s (Table 2). without effect.

Other cations including Ca + + , Mg + + and Zn + + were

Several widely used inhibitors of l i p i d peroxidation

including propyl gallate, BHT, cobalt chloride and manganese chloride prevented both the ascorbate-induced loss of ^H-spiroperidol binding and the ascorbate-induced increase in l i p i d peroxidation.

In all of these

373

Table 1.

The effect of sodium ascorbate on the specific binding of 2nM 3 H-

spiroperidol and on l i p i d peroxidation in a rat neostriatal membrane preparation.

Data are from a single experiment which was repeated several

times with similar results.

Ascorbate Cone. (mM)

0

^ - S p i r o p e r i d o l Binding

Lipid Peroxidation

(dpm/mg tissue)

(pmoles TBA/mg tissue)

1017

27

0.005

716

347

0.05

114

736

0.5

150

772

5

1098

51

experiments summarized in Table 2, there was a s t r i k i n g parallelism between l i p i d peroxidation and loss of 3 H-spiroperidol binding.

From all of this

data, one i s led to the conclusion that the l i p i d peroxidation induced by ascorbate was responsible for the observed loss of binding.

3

H-spiroperidol

Washout experiments (preincubation with ascorbate and then

removal by several centrifugations and resuspensions in fresh buffer) showed that t h i s loss of 3 H-spiroperidol binding was irreversible (4). Other ascorbate derivatives (e.g. ascorbic acid hemicalcium, isoascorbic acid) were also able to cause l i p i d peroxidation and loss of ^ - s p i r o peridol binding while others (e.g. ascorbic acid sulfate) had no such effects (15).

Other reducing agents (e.g. d i t h i o t h r e i t o l , glutathione)

were able to induce both a l i p i d peroxidation and loss of

3

H-spiroperidol

binding but were considerably weaker than ascorbate in both regards.

And

in fact under some conditions, these other reducing agents were almost inactive in the absence of added iron.

In other experiments i t was

discovered that the method used for the tissue preparation was a c r i t i c a l factor (15).

Considerably greater effects on both ascorbate-induced loss

of 3 H-spiroperidol binding and ascorbate-induced l i p i d peroxidation were obtained when tissue was o r i g i n a l l y homogenized in isotonic sucrose and subjected to low speed centrifugation than when tissue was i n i t i a l l y

374

Table 2.

The effect of various agents on the ascorbic acid-induced

decrement in ^ - s p i r o p e r i d o l binding and on the ascorbic acid-induced enhancement of l i p i d peroxidation.

Agent

Decrement in

Enhancement of

Binding

Lipid Peroxidation

EDTA

Prevents

Prevents

DETAPAC

Prevents

Prevents

Propyl Gallate

Prevents

Prevents Prevents

Butylated Hydroxytol uene

Prevents

Cobalt Chloride

Prevents

Prevents

Manganese Chloride

Prevents

Prevents

Ferrous Sulfate

Potentiates

Potentiates

Ferric Chloride

Potentiates

Potentiates

Calcium Sulfate

No Effect

No Effect

Magnesium Sulfate

No Effect

No Effect

Zinc Sulfate

No Effect

No Effect

homogenized in hypotonic Tris and then subjected immediately to high-speed centrifugation (15).

We are currently seeking explanations for these

observations. Many of these same experiments with ascorbate have been done with other ligands and other sources of tissue. those described above have been made.

And many observations similar to We have for example, done extensive

work with ^H-dihydroalprenolol, a beta adrenergic receptor antagonist, and have found very similar r e s u l t s .

All of these experiments demonstrate

that the binding of many ligands to their receptors can be markedly affected by an ascorbate-induced l i p i d peroxidation.

The data further

suggest that l i p i d s are extremely c r i t i c a l in determining the nature of a drug-receptor interaction.

375

Acknowledgement Supported by a grant from Hoffmann-LaRoche and NIH grant NS 18849.

The

author wishes to thank F.S. Cabbat and L. Manzino for experimental assistance and G. Bitkower for manuscript preparation.

References 1.

Leff, S . L . , Sibley, D.R., Hamblin, M., Creese, I . : Life S c i . 29, 20812090 (1981 ).

2.

Arana, G.W., Baldessarini, R.J., Kula, Ν.S.: Neuropharmacol. 21^, 601604 (1982).

3.

Cox, Β.M., Leslie, F.M., Dunlap, C.E. I I I . : J. Receptor Res. 1, 329354 (1980).

4.

Heikkila, R.E., Cabbat, F.S., Manzino, L.: J. Neurochem. 38, 1000-1006 (1982).

5.

Coughenour, L.L.: Soc. Neurosci. Abst. ]_, 209 (1981 ).

6.

Chan, B., Seeman, P., Davis, Α., Kalifon Madras, B.: Eur. J. Pharmacol. 81_, 111-116 (1982).

7.

L e s l i e , F.M., Dunlap, C.E. I I I . , Cox, B.M.: J. Neurochem. 34, 219-221 (1980).

8.

Muakkassah-Kelly, S.F., Andresen, J.W., Shih, J.C., Hochstein, P.: Biochem. Biophys. Res. Comm. 104, 1003-1010 (1982).

9.

Weiner, N., Arold, N., Wesemann, W.: J. Neurosci. Meth. 5, 41-45 (1982).

10.

Kayaalp, S.O., Rubenstein, J . S . , Neff, N.H.: Neuropharmacol. 20, 409410 (1981).

11.

Heikkila, R.E., Manzino, L., Cabbat, F.S., Gershefski Hanly, J . : Neuropharmacol. 22, 135-137 (1983).

12.

Kovachich, G.B., Mishra, O.P.: J. Neurochem. 35, 1449-1452 (1980).

13.

Sharma, O.P., Krishna Murti, C.R.: J. Neurochem. 27, 299-301 (1976).

14.

Svoboda, P., Mosinger, B.: Biochem. Pharmacol. 30, 427-432 (1981).

15.

Heikkila, R.E., Cabbat, F.S.: J. Neurochem. In Press (1983).

HLLIPTICINES AND CARBAZOLES AS ANTIOXIDANTS

A.J.F. Searle*; C. Gee and R.L. Willson Biochemistry Department, Brunei University, Uxbridge, Middlesex, U.K.

Introduction Various carbazole, e l l i p t i c i n e and indole derivatives have been i n v e s t i gated f o r both antioxidant and f r e e radical scavenging a b i l i t y .

The

potencies of both 9-hydroxy e l l i p t i c i n e and 6-hydroxy 1,4-dimethyl carbazole (1IDC) have been described previously ( 1 , 2 ) .

We now report f o r

HDC i t s inhibition of the loss of unsaturated f a t t y acids from peroxidising r a t l i v e r microsomes and i t s a b i l i t y to i n h i b i t chemiluminescence in peroxidising human erythrocyte ghosts.

IC 50 values [concentrations of

compound necessary to i n h i b i t l i p i d peroxidation by 501) have been assessed by measuring y i e l d s of TBA-reactive material produced in peroxidising microsomes a t various concentrations of drugs.

The technique of pulse

r a d i o l y s i s has been employed to measure the reaction r a t e constants of these compounds with radical species such as OH· and CC1302· .

Results and Discussion The Methods used are described in the legends t o each t a b l e or f i g u r e . Table 1 shows the e f f e c t i v e n e s s of a range of antioxidants.

Among these

compounds e l l i p t i c i n e shows very l i t t l e inhibitory action, but both 9hydroxy and 9-amino e l l i p t i c i n e have IC 50 values below ΙΟμΜ suggesting t h a t s u b s t i t u t i o n at the 9 position is required f o r good antioxidant activity.

6-hydroxy 1,4-dimethyl carbazole (HDC) i s a s t r u c t u r a l analogue

of 9-hydroxy e l l i p t i c i n e which shows much g r e a t e r i n h i b i t i o n of peroxidation than any other compound so f a r t e s t e d in t h i s system, including propyl g a l l a t e and α-tocopherol.

In the erythrocyte ghost experiment

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

378 only lOnM HDC was required to protect against peroxidation whereas ΙμΜ propyl gallate was required to elicit the same response (fig.l). In the microsomal system ΙμΜ HDC protects completely against loss of unsaturated STRUCTURE

NAME

9 hydroxy

9

JfsoJpM)

ellipticine

omino

1 - 5

elliptic!ne

10 - 100

Ellipticine

6 hydroxy

1 Λ dimethyl

0 3

I6>

carbazole

100

Carbazole 1 , 4 dimethyl

10 - 100

carbazole

6 hydroxy

1Λ.9 t r i m e t h y l

5

indole

carbazole

1 - 10

HO hydroxy

Butylated

hydroxytoluene

Butylated

hydroxyanisole

Propyl

30

7 5

gallate

Promethazine «-tocopherol

3 11 5-

50

Table 1 Inhibition of Peroxidation by Ellipticines, Carbazoles and Wellknown Antioxidants Microsomes (lmg protein/ml) peroxidised by 500μΜ cysteine and 5μΜ FeSO^ as previously described (2,4). IC5o values obtained from 7 concentrations of test compound; except where insufficient measurements made then range is given.

fatty acids (fig.2).

Clues to the antioxidant action of carbazoles and

379 ellipticines may be found from activity studies of the related compounds 5-hydroxyindole (HIN) , carbazole, dijnethylcarbazole (DMH) and 6-hydroxy 1,4,9-trimethylcarbazole (HTC).

Carbazole is a poor antioxidant but the

more lipid soluble DIC inhibits peroxidation substantially.

HDC differs

from DNC only in the hydroxyl substitution at the 6 position and yet the latter is approximately 100-fold less effective as an antioxidant.

Sub-

stitution by hydroxy groups at equivalent positions in the heterocyclic ring system may be important for the antioxidant action of ellipticines and carbazoles.

HIN, which resembles HDC, is a relatively good anti-

oxidant despite being more water soluble than HDC.

It is possible that

when the two properties of good radical scavenging and lipid solubility are joined in one molecule (HDC) then the overall effectiveness is amplified. The importance of the hydroxyindole portion of HDC is indicated by the lower activity of HTC.

HTC is N-methyl substituted and despite being more

soluble in non-polar media than even HDC is about 10-fold less effective as an antioxidant. O

GHOSTS + FERROUS * CYSTEINE

•

AS Ο · ΙΟΟηΜ P R O P Y L

GALLATE

•

AS O »

GALLATE

ΙμΜ P R O P Y L

380

Figure 1 Effect of HDC and Propyl Gallate on Peroxidising Erythrocyte Ghosts Measured by Chemiluminescence Human erythrocyte ghosts (2.5mg protein/ml) obtained by the method of Dodge et a l . (5). These were irradiated (200 kJ k g " ) by a 60Co source, diluted to 80ug protein/ml with Tris:KCl (pH=7.4) and peroxidised by 125μΜ cysteine and 12.5μΜ FeSO., at 37°C. Chemiluminescence was measured by the method of Trush et al (7), using a Packard Tricarb Scintillation counter. Error bars show deviation.

0

C18:2

•

C20*

140 -

g

C 22 6

i i

120100

80

1

J L i

3-

F

60 «

20

0 CONTROL

AS

1 +

FERROUS

and

AS

2 •

I j j M HDC

AS 2 • 1 0 0 n M HOC

CYSTEINE

Figure 2 Prevention of the Loss of Unsaturated Fatty Acids from Microsomes by the Addition of HDC Microsomes peroxidised as in Table 1. Fatty acid determinations according to the method of McDonald-Gibson and Young (3). Error bars show standard deviation. For HDC rate constants with OH· and CC1302· of k = 1.5 χ IO10 M ' V and 8.3 χ 10e M s » respectively, have been determined by pulse radiolysis. These values are not markedly different from those found for promethazine (PMZ) and propyl gallate (PG). OH· + PMZ = 8.0 χ IO9 M~ ' s - ' ;

381

OH· + PG = 1.1 χ ΙΟ10 M 1 s

CC1 3 0 2 · + PMZ = 4.5 χ IO8 M 1 s ' and

CCI3O2· + PG = 1.3 χ IO9 M"1 s " 1 · Although i t has been shown that HDC i s an e f f e c t i v e l i p i d soluble antioxidant in a wide range of systems i t s striking inhibitory activity cannot be explained solely by radical scavenging a b i l i t y .

I t may be that HDC i s

able to interact with a particular " c r i t i c a l s i t e " in the membrane. We have been unable to block the action of HDC at this supposed s i t e using an inactive structural analogue, namely DNC.

The antioxidant mode of action

of e l l i p t i c i n e s and carbazoles remains unclear, but probably i t can be attributed partly to high rates of reaction with hydroxyl and peroxy r a d i c a l s , a substituted indole-type structure and relatively high l i p i d solubility.

Acknowledgments :

We wish to thank the Cancer Research Campaign for their

continued financial support.

References 1. 2.

Malvy, C . , P a o l e t t i , C., Searle, A . J . F . and Willson, R.L.: Biochem. Biophys.Res.Commun. 95, 734-737, (1980). Searle, A . J . F . and Willson, R.L.: Biochem.J. in press, (1983).

3.

McDonald-Gibson, R.G. and Young, M. : Clin. Chim. Acta. 53, 117-126, (1974).

4.

S l a t e r , T.F. and Sawyer, B.C.: Biochem.J. 123, 805-814, (1971).

5.

Dodge, J . T . , Mitchell, C. and Hanahan, D.J. : Archs .Biochem.Biophvs. 100, 119-130, (1963).

6.

Lowry, O.H., Rosebrough, N . J . , Farr, A.L. and Randall, R . J . : J . Biol. Chem. 193, 265-275, (1951).

7.

Trush, M.A., Wilson, M.E. and van Dvke, K.: Methods in Enzvmology 57, 462-494, (1978). ' '

HERBICIDE-INDUCED LIPID PEROXIDATION IN HIGHER PLANTS: THE ROLE OF VITAMIN C Herbicides, Lipid Peroxidation, Antioxidants

Karl Josef Kunert Lehrstuhl für Biochemie und Physiologie der Pflanzen, Universität Konstanz D-7750 Konstanz, Germany

Introduction Many herbicides act by overtaxing or destroying protective systems that control deteriorative reactions in biological tissues. Among these highly toxic reactions peroxidation of polyunsaturated fatty acids has been identified. In both plants and algae, there is considerable evidence that herbicides such as bipyridylium salts or p-nitrodiphenyl ethers induce lipid peroxidation via free radicals in the presence of an active photosynthetic electron-transport system (1,2). The peroxidation process is usually investigated by measuring hydrocarbon gases (3). Recently, it has been reported that vitamin E prevented membrane damage when added to diphenyl ether-treated cucumber cotyledons (4). However, detailed experiments have not been done to elucidate the action of natural antioxidants, such as vitamin C, in vivo. Vitamin C, an antioxidant synergist with vitamin E, is present in substantial amounts in higher plants.

Experimental Design Mustard seedlings (Sinapis alba) were grown for 6 days at 20 °C in flower pots. Ethane evolution was measured by gas chromatography by a modified procedure of Dillard et al. (5).

O x y g e n Radicals in Chemistry a n d Biology © 1984 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

384

6 -

_

-400

•

·

Ethane

a

a

Vitamin C

2·

Days

after

treatment

Fig. 1. Rate of ethane evolution and vitamin C concentration after treatment of mustard seedlings with 660 mg paraquat/m2. Data shown represent the mean value + standard error of 6 samples. Ethane evolved by about 150 seedlings was accumulated for 30 min, collected onto a gas-sample loop and analzyed. Vitamin C was quantitated by paired-ion chromatography according to Finley and Duang (6) .

Results and Discussion After treatment of seedlings with the bipyridylium salt paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride), ethane production increased significantly (p [-S-Fe 1 1 1 -^·] 2 + /4/

This structure which is unique for the P450 proteins can explain the hydroxylating and epoxidising properties of this oxo species which is believed to act by a radical abstraction process at a CH bond or by addition to a double bond (11,12).

399

Another type of interaction of peroxides with metal centers is observed in hemocyanin chemistry.

There the two copper I

centers can add dioxygen to yield a μ-peroxo species, which can also be obtained from H 2 0 2 and the oxidized enzyme.

Very

similar are the interactions of binuclear cobalt II centers with dioxygen (13) which are, however, not used in nature for oxygen transport.

Finally, the bonding of the peroxide can also occur to one metal atom in a side-on fashion leading to a cyclic structure (14) : FeITV Ν

1

0 Again, this possibility of peroxide bonding so far has not been found in biological systems.

We observed a special case of metal catalysis with peroxides recently in the case of prostaglandin-endoperoxides.

The 0-

0 bond of this compound is split by thromboxane A 2 synthase and prostacyclin synthase to yield the two corresponding prostanoids with potent physiological activities (15).

We

could establish that both enzymes act through a heme-thiolate (Cytochrome P450) prosthetic group which allows the activation of one oxygen atom under attack of the side chain (16, 17).

400 COOH

Arachidonic acid Cyclooxygenase

(PGH2)

COOH

-s-Fe-o TxA2 Synthase

PGIj Synthase

p0 O

>D,

COOH COOH

Singlet oxygen Certainly the singlet state of dioxygen not only has a higher oxidation potential compared to the triplet ground state, but it also reacts more readily because the restrictions of spin conservation are not existent.

Free singlet oxygen therefore

is strongly toxic in biological systems and has no function in physiological reactions.

However, it has been speculated

whether it may be formed in side reactions as a toxic

1,

Indeed, 1, we recently reported on a pathway that could lead to pathway that could lead to

or OH radicals.

401

formation (18).

It was found that in cytochrome P450-

catalyzed decompositions of peroxides singlet oxygen is evolved.

Since iodosvlbenzene gave even better yields we

proposed the following sequence of reactions: RI-0 Fe (III)

RI-0

^

[Fe0] RI

3+

Ν

^

•

1

0 2 + Fe (III)

/5/

RI

Interestingly, cyclooxygenase reacted in an analogous way, indicating that not only cytochrome P4 50 but also other ironoxo species can react with oxene donors like iodosyl benzene 1 1 under 0 2 formation (19). Cyclooxygenase generated 0 3 also from PGG 2 , the 15-hydroperoxy endoperoxide of arachidonic acid indicating that this reaction may occur under physiological conditions.

Generally, lipid hydroperoxides could serve in

the presence of hemoproteins as sources of singlet oxygen accordinq to the eauation: R00H R00H 3+ Fe (III) ^ ^ • [Fe0] » λ 0 2 + Fe (III) /6/ ROH ROH It remains to be established whether this is a general and significant reaction in oxygen toxicity with a promoting effect on lipid peroxidation.

Conclusions There is no doubt that the biochemistry but also the major part of the chemistry of oxygen is intimately linked to metal bonding involving the d-orbitals of a transition metal. Biological iron complexes, especially those with porphyrins are among the better investigated, less is known about the corresponding copper, manganese or vanadium complexes.

All

402

oxygen species, like dioxygen itself, the 07

radical, H 2 0 2 ,

the OH radical and atomic oxygen can be bound and stabilized at the metal center.

It is inevitable that on rare events

these species may be liberated or that the metal-oxygen species react with other constituents of the cell leading to uncontrolle oxidation reactions.

The defense mechanisms against these

reactions also involve transition metal enzymes like dismutases, catalases and peroxidases.

References 1.

2.

Reed, C.A. in Dunford, H.B., Dolphin, D., Raymond, K.N., Sieker, L., eds.: The Biological Chemistry of Iron, Reidel Publishing Company, Dordrecht, Boston, London, 25-41 (1982) Gersonde, K. in Sund, Ullrich, eds.: Biological Oxidations, Mosbach Colloquium (1983}

3.

Fong, K.L., McCay, P.B., Payer, J.L., Keele, B.B., Misra, H.: J. Biol. Chem. 248, 7792, (1973)

4.

Cederbaum, A.L., Dicker, E., Cohen, G.: Biochemistry V7 3058 (1978) Fee, J.A. in Rodgers, M.A.J., Powers, E.L. eds.: Oxygen and Oxy-Radicals in Chemistry and Biology, Academic Press, 205-217 (1981)

5.

6. 7.

Ullrich, V., Hey, D., Zubrzycki, Z., Staudinger, Hj.: Z. Naturforschung 206 1 185-1 191 (1965) Ullrich, V., Kuthan, H. in Rodgers, M.A.J., Powers, E.L., eds.: Oxygen and Oxy-Radicals in Chemistry and Biology, 497-505

8.

DiNello, R.K., Dolphin, D.H.: J. Biol. Chem. 25J5 69036912 (1981)

9.

Wagner, G.C., Gunsalus, I.e. in Dunford, H.B., Dolphin, D., Raymond, K.N., Sieker, L., eds.: The Biological Chemistry of Iron, Reidel Publishing Company, Dordrecht, Boston, London, 405-412

10.

Ullrich, V. : J. Molec. Catalysis, 7 159-167 (1980)

403 11.

Groves, J.T., McClusky, G.A., White, R.E., Coon, B i o c h e m . B i o p h y s . C o m m u n 8J_ 1 5 4 - 1 6 0 (1978)

12.

Ortiz de M o n t e l l a n o , P.R., Beilan, H.S., Kunze, M i c o , Β . Α . : J . B i o l . C h e m . 256 4 3 9 5 - 4 3 9 9 (1981) T., this

M.J.: K.L.,

13.

Matsuura,

14.

McCandish, E., Miksztal, A.R., Nappa, M., Sprenger, Valentine, J., Stany, J.D., Spiro, T.G.: J. Amer. C h e m . S o c . J £ 2 4 2 6 9 - 4 2 7 0 (1980)

volume

15.

Samuelsson, Β., Folco, G., Granström. E., Kindahl, H., Malmsten, C.: Adv. Prostaglandin and Thromboxane Res. 4 1 - 2 5 (1978)

16.

Graf., H., Ruf, H.H., Ullrich, V . : Angew. Chemie E d . 22 487 (1983)

17.

U l l r i c h , V . , C a s t l e , L . , H a u r a n d , M . in N o z a k i , M . , Yamamoto, S., Ishimura, Y., Coon, M.J., Ernster, L., Estabrook, R.W.,eds.: Oxygenases and Oxygen Metabolism, A c a d . P r e s s 4 9 7 - 5 0 9 (1982)

18.

Cadenas, E., Sies, H., Graf, H-, Ullrich, V. : Eur. B i o c h e m . j n O 1 1 7 - 1 2 1 (1983)

19.

Cadenas, E., Sies, H., Nastaincyzk, W., Ullrich, V.: H o p p e - S e y l e r ' s Ζ. P h y s i o l . C h e m . _364 5 1 9 - 5 2 8 (1983)

A.Q.,

Int.

J.

DISCUSSION SINGH: About singlet oxygen formation in one of the reactions, is there strong evidence that this type of system does produce singlet oxygen? ULLRICH: Definitely, this type of system generates singlet oxygen. The question you may ask is, do we have evidence that in an intact cell singlet oxygen formation follows that mechanism. If you add organic peroxides to cytochrome P-450, for instance, you do see singlet oxygen formation. We have shown this occurs. The question is whether the level of peroxide is sufficiently high. I could think that under conditions of oxidative stress, where a lot of peroxides, especially from the arachidonic acid cascade are formed, there is a good chance that those cells form singlet oxygen. This may be a physiological way of producing singlet oxygen. One could ask, what are the defence mechanisms against it? SINGH:

What was your evidence for singlet oxygen?

ULLRICH: This is actually the work by E. CADENAS. He characterized the emission spectra of the species formed.

404 BIELSKI : I just wanted to add that you can fill in one mote space in your table. Similar to M n + + , C u + + also forms an adduct with HO2/ 05. The corresponding absorption maxima are near 220 and 270 nm respectively. The complex CuOj can also be formed from C u + upon addition of O2. WESER: You have shown copper(V) , copper(IV) and copper(III). To my knowledge there is no evidence in biochemical systems that copper, as copper (III) or copper(IV) or (V) would be present. ULLRICH: You are right, this table also contains non-biological complexes.

ACTIVATION OF DIOXYGEN SPECIES WITH TRANSITION METAL COMPLEXES

Teruo Matsuura, Akira Nishinaga Department of Synthetic Chemistry, Faculty of Engineering, Kyoto University, Kyoto 6 06, Japan

Introduction Oxygenases catalyze the oxygenation of substrates by incorporating either two atoms of oxygen (dioxygenases) or one atom oxygen (monooxygenases or mixed function oxidases).

Most of

the enzymes contain iron or copper in their active site.

The

catalysis by these metal complexes in enzymes is considered to involve the activation of dioxygen by forming a reactive metal-dioxygen complex of the types of Fe-0-0 and Cu-O-O-Cu, which are formally equivalent to those of natural dioxygen carriers, hemoglobin and hemocyanin respectively (1).

In the

case of cytochrome P-450, organic hydroperoxides are known to act as an oxygen donor in their metal-complex form in biological monooxygenations (2). As models for natural oxygen carriers, many cobalt(II) complexes coordinated with nitrogen-bases have been shown to reversibly interact with molecular oxygen to form C0O2 or CO2O2 complexes depending upon the nature of ligands.

More

than ten years ago, we started model studies for biological oxygenations using cobalt-dioxygen complexes as oxidizing agent to various types of organic substrates including biomolecules (3).

In this paper we summarize the reactions of

these cobalt-dioxygen complexes as well as cobalt-organic hydroperoxides complexes. Typical cobalt(II) complexes used in this work are shown below. There are two types of cobalt-dioxygen complexes.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

406 Co(Salpr),

[Co(CN)5J

3

and Co(MeO-Salen) with pyridine

(Pyr)

ligand give a superoxo-Co (III) complex as Eq. [1] and Co(Salen) with dimethylformamide

(DMF) ligand give a y-peroxo-

Co(III) complex as Eq. [2] (4). [Co(CN) 5 ] 3 ~:

For Co(Salpr), Co(MeO-Salen) (Pyr) and Co(II)

+

o2

—

Co(II)02-

»

Co(III)0 2 7

[1]

For Co (Salen) (DMF) : Co(II)0 2

+

Co (II)

Co(II)0 2 Co(II) 2-

Co (III) O ^ C o (III)

[2]

„3-

uo Co(Salpr)

NC CN

Co(Salen): R=H

[Co(CN) 5 ] 3 "

Co(MeO-Salen): R=0Me

Results IIl_Reaçtivities_of_Çobal^^

.

It has been shown that various organic substrates undergo selective oxygenation in the presence of a Co(II)-Schiff base complex such as Co(Salpr) and Co(Salen), and that a cobaltdioxygen complex, superoxo-Co(III) complex Co(III) complex

(CoC>2) or μ-peroxo-

(CoC>2Co), plays an essential role in these

oxygenation reactions.

Reactions of cobalt-dioxygen complexes

with organic substrates are highly depending upon the nature of the cobalt complexes and the substrates. The results are classified in terms of an initial process occurring between the cobalt-dioxygen complex and the substrate as the following equations, where SH, S- ans S denote a substrate.

407 (I)

Radical reaction. (a)

Hydrogen abstraction: SH

(b)

+

S·

+

Co(III)OOH

Radical recombination: S·

(II)

Co0 2

+

Co0 2

»-

S-OOCo(III)

Anionic reaction. (a)

(b)

Proton transfer: SH

+

Co0 2

S~Co(III)

+

· 00H

SH

+

Co0 2 Co

S~Co(III)

+

Co(III)00H

S"00·

Co (III)

Nucleophilic addition: S

(III)

+

Co0 2

+

Electron-transfer reaction.

(a)

Reduction: S

(b)

+

CoC>2

Sr

+

Co (III)

S*

+

Co(III)0 2 2 "

+

o2

Oxidation: S

+

Co0 2

»>

As seen from the above equations, superoxo-Co(III) complexes (CoC>2) display most of the important reactions of superoxide ion. Ia - Hydrogen abstraction. A superoxo-Co(III) complex such as Co(Salpr)C>2 abstracts hydrogen from 4-substituted 2,6di-i-butylphenols

(1_) to finally give an ortho- (2)or para-

ti) peroxyquinolato-Co(III) complex depending upon the nature of 4-substituent as in· Eq,[3] (5-11). derivatives give and

and

Thus, 4-aryl and 4-alkyl

respectively.

The formation of 2_

provides a model for the enzymic, aromatic ring-clea-

vage of Eq. [4] (12).

In the presence of a protic hydrogen

donor such as methanol, compound 3 undergoes reductive cleavage of the peroxide bond to give a para-quinol 4_ as Eq. [3], implying a possible model for the enzymic, aromatic hydroxylation.

A mechanism of Eq. [5] involving the initial hydrogen

abstraction from a phenol, which was confirmed from the e.s.r. spectroscopic detection of the corresponding phenoxyl radical, has been proposed

(11,13).

408 OH

0

poco(III)

0

Co(Salpr)/0, or

Ψ

Co(MeO-Salen)(Pry) R OOCo(III)

Oo 2 (R=Aryl)

3 (R=A1kyl ) MeOH

[3]

σ

.OH

Enz-Fe-O0 s

OOFe-Enz

Co (II)

[4]

Co(III)O ·

Co(III)0 2 r

ArOH

ArO· Co(III)ArO~

0

OOH CHO

Co(II) Κ

0.

Co(III)OOH

ArO-

Co(III)ArO~ 0=Ar00Co(III) (2 and 3)

[5]

The Co(Salpr)-mediated oxygenation reactions of the following substrates are also interpreted by the initial hydrogen abstraction with C0O2·

Thus, the Co(Salpr)-catalyzed oxygenation

of thyroxine analogs results in the cleavage of the diphenyl ether linkage, providing a model for the peroxidase-catalyzed oxidation of thyroxine (14).

In the presence of Co(Salpr),

4-(N-arylmethyleneamino)-2,6-di-t-butylphenols

Q , R=-N=CHAr)

(15), 4- (N-alkylimino)methyl-2,6-di-t-butylphenols

(1,

R=-CH=NR) (16) and other 2,6-di-t-butylphenols bearing an electron-withdrawing group (1, R=-COR, R=-CR=N0Me, R=-CN) (17,18) and arylhydrazones (19) are readily oxygenated.

409 Ib - Radical recombination.

This particular reaction

occurs between the pentacyanocobalt-dioxygen complex and 4-substituted 2,6-di-t-butylphenoxy radicals to give mainly peroxy-p-quinols 6_, indicating the initial formation of paraperoxyquinolato-Co(III) complexes 3_ as shown in Eq. [6] (20). In case of the 2,4,6 —tri-t—butylphenoxy radical, the formation of an ortho-peroxyquinolato-Co(III) complex 2_ is predominant.

3Ila - Proton transfer.

[Co (CN)^(0^)]

complex exhibits

a different reactivity from that of Co(Salpr) (02) to 4-substituted 2,6-di-t-butylphenols

where the superoxo species acts

as a base to produce the corresponding phenolate anions which undergo direct oxygenation with molecular oxygen to give the known base-catalyzed oxygenation products (21). Co (Salen) catalyzes the oxygenation of 3-substituted indoles 7 and flavonols £ via a proton transfer process, representing a model for tryptophan 2,3-dioxygenase or indoleamine 2,3dioxygenase (heme enzyme) and quercetinase (Cu enzyme). 3-substituted indoles as Eq. [7] (22,23).

The

give formylkynurenine-type products In the stoichiometric reaction, the same

crystalline Co(III) complex (most probably structure 10^ among several possible ones) is formed from skatole (7_, R=Me) with Co(Salen)/02 and Co ( III) (Salen) (t-BuO")/C>2.

This and kinetic

results led us to propose a mechanism of Eq. [8], involving the initial proton transfer followed by the oxygenation of the oxygenation of the substrate anion with molecular oxygen (23) .

410

or"

OC

Co(Salen)/0,

COR

9 (S0 2 H)

7 (SH)

H+

R=Me, -CH 9 CH(NHAc)C00Me, etc.

Me R=Me; Co(Salen)/0,

[7] .O'Co(III)

0

or C o ( I I I ) (Saler)(t-Bu0~)/0,

¿J 10 (Co(III)S0 ? ~)

Co(III)0 2 2 Co(III) SH — Co(III)C>2

SH 2-,

Co(III) + +

S Co(III) Co(III)so ~

SH — C o (III)0 2 2 ~Co (III) S~Co(III)

+

Co ( III)00H

Co(III)SO ~ Co(III)

S0 2 H

[8]

Flavonols £ undergo cleavage of the pyrone ring in a unique manner to give depsides

and carbon monoxide as Eq. [9] (24)

the latter of which is further oxidized to carbon dioxide under the reaction conditions.

The oxygenation reaction is

interpreted in terms of the initially occurring proton transfer followed by the oxygenation of a Co(III) flavonolate complex intermediate with molecular oxygen.

In fact, the

base-catalyzed oxygenation of flavonols £ gives a depside 11^ and carbon monoxide in excellent yield (25)

R Co(Salen)/0, DMF

1 2

R =R =H

1 2

R - R -OH R^=H, R 2 = 0 M e , etc.

XX^ 11

19] CO

411

IIb - Nucleophilic addition. derived from Co(Salpr) and

Superoxo-Co(III) complexes 3_

[Co(CN)g]

react with 2,6-di-t-

butyl-p-benzoquinone methides 12 to give the corresponding p-benzoquinones 1^3 and epoxides

which are proposed to

result from the nucleophilic addition of the superoxo species to the exo double bond of 12 as Eq. [10] (26).

12

13

Ilia - One-electron reduction. complex

[Co (CN) 5 (C>2 ) ]

quinones.

14

Pentacyanocobalt dioxygen

acts as a reducing agent toward

One-electron reduction occurs with o-quinones to

give the corresponding semiquinone radicals, while successive one-electron transfer reactions occur with p-quinones to give hydroquinone dianions as Eq. [11] (27).

Illb - One-electron oxidation.

Abel, et al. reported that

Co(MeO-Salen) acts as an oxidizing agent toward some organic substrates such as

Ν,Ν,Ν',Ν 1 -tetramethyl-p-phenylenediamine

which give the corresponding cation radicals as Eq

[12] (28).

412

NMe

NMe

2

+

]

Co(MeO-Salen)(Og)

[12]

2

2

In view of the importance of the activation of organic hydroperoxides by cytochrome P-45 0 leading to monooxygenation of a substrate (2), the Co(Salen)-catalyzed oxidation of various organic substrates with t-butyl hydroperoxide in methylene chloride solvent has been investigated (29-32).

Under these

reaction conditions, 4-substituted 2,6-di-t-butylphenols

(y

give the corresponding i-butylperoxy derivatives 3J5 and Eq. [13].

Some selected results are shown in Table 1.

as Other

transition metal complexes exhibit similar catalytic activity. Their effectiveness as catalyst is approximately the following order:

Mn(Salen) >Co(Salen) >CuCl>Cu(Pc) >Cu(Salen) >

Fe (Salen) >Co(Salpr).

15

16

Under the similar oxidation conditions, the ρ-nitrophenylhydrazones Y]_ of aromatic ketones give ¿-butylperoxyazo compounds 18^ and the corresponding azoxy compounds

as

major products, in addition to the formation of the parent ketones as a minor product as Eq. [14].

Other transition

metal complexes such as Co(Salpr), CuCl, Cu(Pc), Fe(Pc), Fe(MeO-Salen) and MoO«(acac) act as catalyst.

413

Table 1.

Oxidation of 1 with t-BuOOH/Co(Salen). Yield (%) 15 16

R in 1

Me t-Bu

94 81

6 19

CHO COMe

Ph

R in 1

89

11

Mesityl

7

93

CH=NOMe

11

CMe=NOMe

56

89 44

CEt=NOMe

84

16

i-Pr^ C=

Yield (%) 16_ 15 a —

95 N

5 100

—

Me

N C= or-Me Ph-- w M e

20 b

40

•90%)

Ri

_ W

R ^

R \ /00í"Bu

t-Bu00H

V=N-NH-^-N0

* ¿

Co(Salen)

2

R

¿

A

N = N N

N

.

R1

A r

A r

2

R

¿ /

00t-Bu

X

• 0 17 R

18

1

R2

[14]

N=N-Ar 19

τ R1 +

V

= n

Yield

Ph

Me

87%

13%

Me

Me

76%

24%

4-Me-Ph

Me

84%

10%

A mechanism involving the initial formation of a t-butylperoxyCo(III) complex, Co(III) (Salen) (i-BuOO-), has been proposed as shown in Eq. [15].

In accordance with this mechanism,

reaction of two moles of the 2,4,6-tri-t-butylphenoxy radical with t-butyl hydroperoxide resulted in the formation of the parent phenol ^ (R=t-Bu) and a mixture of 1_5 and 16^ (R=t-Bu) . in a similar ratio to that shown in Table 1.

The peroxy

complex Co(III)(Salen)(t-BuOO ) can be obtained as stable crystals from the stoichiometric reaction of Co(Salen) with t-butyl hydroperoxide, and oxidizes PPhj rapidly to give 0=PPh^ in quantitative yield.

414

2 Co(II)

+

t-BuOOH

»- CO (III) (í-BuO )

Co(III)(t-BuO~)

+

Co (III) (0H~)

t-BuOOH

+

Co(III) (t-BuOO~)

t-BuOOH

+

ArOH

Co(III) (ArO~) 2 Co(II) (ArO·)

+

t-BuOOH

+

Co(III)(OH )

Co(III) (t-BuOO")

+

t-BuOH

Co(III) (t-BuOO~)

+

H20

·- Co (III) (ArO~)

+

t-BuOOH

Co (II) (ArO·) »- ArOH + 0=Ar00t-Bu + 2 Co(II) (15 and 16) [15]

Reaction of t-butylperoxy-Co(III)(Salen) complex with an excess of benzyl alcohols 20_ gives the corresponding benzaldehyde 21^ quantitatively as Eq. [16].

The rate of the

oxidation depends upon the nature of the 4-substituent and is in the order R=N0 2 >CI >H >OMe, suggesting that hydrogen bond between the alcohol substrate and the peroxy group in Co(III)(Salen)(t-BuOO ) may play an important role in the oxidation.

XX

CH ? 0H C o ( III ) (Salen) (t-BuOO")

XT 20

Λ

CHO [16]

21

References 1.

(a) Hayaishi, 0.: ed., "Molecular Mechanism of Oxygen Activation", Academic Press, New York and London 1974; (b) Hayaishi, 0., Asada, Κ.ί eds., "Biochemical and Medical Aspects of Active Oxygen", University of Tokyo Press, Tokyo 1977; (c) Nozaki, Μ., Yamamoto, S., Ishimura, Y., Coon, M. J., Erster, L., Estabrook, R. W.i Academic Press, Tokyo-New York-London 1982.

415

2.

White, R. E., Coon, M. J.: Ann. Rev. Biochem., (1980) .

315

3.

(a) Nishinaga, Α., Tomita, H., Shimizu, T., Matsuura, T.: Fundam. Res. in Homogeneous Catalysis, 241 (1978); (b) Nishinaga, Α., Tomita, H.» J. Mol. Cat., ]_, 179 (1980); (c) Matsuura, T., Nishinaga, A.:, Ref. le, p. 561.

4.

Cf. Vaska, L. , Accounts Chem. Res., 9_, 175 (1976); Jones, R. D. , Summerville, D. Α., Basolo, F.: Chem. Rev., 79_, 139 (1979) .

5.

Nishinaga, Α., Watanabe, Κ. , Matsuura, T.: Tetrah. Lett., 1291 (1974).

6.

Nishinaga, Α., Nishizawa, Κ., Tornita, Η., Matsuura, T.: J. Am. Chem. Soc., 99, 1287 (1977).

7.

Nishinaga, Α., Tomita, Η., Matsuura, T.: Tetrah. Lett., 2893 (1979).

8.

Matsuura, T. Watanabe, Κ. Nishinaga, Α.: Chem. Commun., 163 (1970) .

9.

Nishinaga, Α., Shimizu, T., Matsuura, T.: Tetrah. Lett., 21, 1261 (1980).

10. Nishinaga, Α., Tomita, Η., Matsuura, T., Hirotsu, Κ., Ooi, S.: 14th Symp.Oxidn.Reactions, Nov. 4-5, 1980. 11. Nishinaga, Α., Tomita, Η., Nishizawa, Κ., Matsuura, T.: J. Chem. Soc. Dalton Trans., 1504 (1981). 12. Hamilton, G. Α.: Ref. la, p. 443. 13. Nishinaga, Α., Itahara, T., Shimizu, T., Tornita, H., Nishizawa, Κ., Matsuura, T.: Photochem. Photobiol., 28, 687 (1978). 14. Nagamachi, T., Nishinaga, Α., Matsuura, T.: Chem. Lett., Ill (1972). 15. Nishinaga, Α., Shimizu, T., Matsuura, T.: Tetrah. Lett., 21, 1265 (1980) . 16. Nishinaga, Α., Shimizu, T., Matsuura, T.: Tetrah. Lett., 21, 4097 (1980) . 17. Nishinaga, Α., Shimizu, T., Matsuura, T.: Tetrah. Lett., 22, 5293 (1981). 18. Nishinaga, Α., Shimizu, T. Toyoda, Y-, Matsuura, T., Hirotsu, K.: J. Org. Chem., AJ_, 2278 (1982). 19. Nishinaga, Α., Oda, M., Tomita, H., Matsuura, T.: Tetrah. Lett., 2¿, 339 (1982). 20. Nishinaga, Α., Tomita, H., Matsuura, T.: Tetrah. Lett., 21, 3407 (1980). 21. Nishinaga, Α., Tomita, H., Matsuura, T.: Tetrah. Lett., 21, 2833 (1980).

416

22. Nishinaga, Α.: Chem. Lett., 273 (1975). 23. Nishinaga, Α., Ohara, E., Tomita, H., Matsuura, T.·· 43th Ann. Meeting of Chem. Soc. Japan, March 30-Apr. 2, 1981, 1E37. 24. Nishinaga, A. Tojo, T., Matsuura, T.: J. Chem. Soc. Chem. Commun., 896 (1974). 25. (a) Nishinaga, Α., Matsuura, T., Chem. Commun., 9 (1973); (b) Nishinaga, Α., Tojo, T., Tomita, H., Matsuura, T.: J. Chem. Soc. Perkin I, 2511 (1979). 26. Nishinaga, Α., Tornita, Η., Matsuura, T.; Tetrah. Lett., 21, 4849 (1980). 27. Nishinaga, Α., Tomita, Η., Matsuura, T.: Tetrah. Lett., 21, 4853 (1980). 28. Abel, E. W., Pratt, J. M., Whelan, R., Wilkinson, P. J.: J. Am. Chem. Soc., 96, 7120 (1974). 29. Nishinaga, Α., Tomita, Η., Ohara, E.: 32th Symposium on Chemistry of Metal Complexes, Oct. 13-15, 1982, 2D04. 30. Nishinaga, Α., Shimoyama, T., Iwasaki, Η., Matsuura, T.: 47th Ann. Meeting of Chem. Soc. Japan, Apr. 1-4, 1983, 1F45. 31. Nishinaga, Α., Iwasaki, Η., Matsuura, T.: 47th Ann. Meeting of Chem. Soc. Japan, Apr. 1-4, 1983, 1F46. 32. Nishinaga, Α., Sugimoto, I., Kondo, T., Matsuura, T.·· 47th Ann. Meeting of Chem. Soc. Japan, Apr. 1-4, 1983, 3B30.

DISCUSSION

von SONNTAG: ïou showed us an interesting reaction, i.e. 0^ adding to carbonyl compounds. On what experimental grounds is the assumption based that O^ adds to a C=0 double bond to produce an intermediate which you then considered to be reduced to the hydroxy hydroperoxide? MATSUURA: Such a mechanism has been postulated for the reaction of certain carbonyl compounds by several workers, but the intermediate peroxy radical has never been proven. von SONNTAG: We know that if you form such a hydroxy peroxyl radical, especially in basic solution, it immediately decomposes to give 0-> and the carbonyl compound. So the reverse reaction is extremely fast (BOTHE et al., Photochem. Photobiol. (1978) 26, 639), and I wonder whether you have any evidence for the forward reaction? MATSUURA: In our case (reaction of quinone-methides and C0-O2) , we did not examine the kinetics of the nucleophilic attack of O2, but focused on the final product. Thus, we put a catalytic amount of cobalt (salpr) in the substrate solution and just bubble with oxygen, then we obtain our final products.

417 von SONNTAG: And that also goes through hydrogen peroxide as an intermediate. It is well known that if you have hydrogen peroxide and carbonyl compounds, they form hydroxyl hydroperoxides which, in aqueous solution, are in equilibrium with the starting materials. MATSUURA: I see. In our case we did not examine the first equilibrium reaction, but only analyzed the final products. SINGH: When your cobalt complex reacts with tryptophan, do you see any product other than formylkynurenine. MATSUURA: No, only product.

in most

cases the formylkynurenine-type

compound

is

the

ULLRICH: Have you observed reactions where the cobalt oxygen complex behaves differently from the biological iron oxygen complex? I am thinking in terms of the electronic structure. You know, that in the case of cobalt, at least by EPR, the proposed structure according to the experiments by WITTENBERG, is cobalt (III) -2' whereas in the iron-oxy complexes the electronic structure may be different. MATSUURA: Of course, the electronic structure of the cobalt dioxygen complex is different from the iron-oxygen complex. For example, when the cobalt oxygen 1:1 complex is reduced with sodium borohydride, we obtain only cobalt (OOH), the hydroperoxide. This is a stable compound and we don't have anything corresponding to oxenoid species from cobalt oxygen complexes. ULLRICH: It seemed to me also that the reaction of the hydroperoxide with cobalt giving you a stable complex is different from iron. I would think that the corresponding iron complex would decompose. MATSUURA: When we use an iron(II) complex and a hydroperoxide, this system will undergo some FENTON-type reaction.

DISMUTATION CATALYSED BY WATER SOLUBLE PORPHYRINS. A PULSE RADIOLYSIS STUDY.

Moshe Faraggi Nuclear Research Centre - Negev P.O.B. 9001, 84 190 Beer-Sheva, Israel

Introduction Search for low molecular weight metallo-compounds which could mimic the catalytic a c t i v i t y of the superoxide dismutase began after i t s discovery in 1969 (1). Most reactive i s C u ( I I ) aquo complex (2). Other amino acid complexes of C u ( I I ) have been reported to be less reactive (3,4). However, these compounds have a limited a c t i v i t y at physiological pH due to their limited s o l u b i l i t y in that pH region. Recently, the F e ( I I I ) complex of tetrakis-(4-N-methylpyridyl) porphyrin (Fe(III)TMPyP), a water soluble porphyrin, was found to be an effective catalyst for the dismutation of Og (5-7). Other metal!oporphyrins such as Co(III)TMPyP and Fe(III)TPPS 4 (tetra(4-sulphonatophenyl)porphyrin) have some 0^ dismutation a c t i v i t y (10" 2 to 10" 3 less effective) (8). Cu(II)-TMPyP and Zn(II)TMPyP did not show any reaction with 0^ (5). In this paper we present results of the reaction of 0" with different water soluble metal!oporphyrins of TMPyP,TPPS^, TAP (tetra(4-N,N,N-trimethylanilinium porphyrin), and the picket-fence porphyrin,PFP,(a,a,a,ß-tetra-ortho(N-methyl-isonicotinamide

phenyl) porphy-

rin)using the pulse r a d i o l y s i s technique.

Results and Discussion The reaction kinetics of a metalloporphyrin with 0^ ([0^] 5xlO~^M to 2.4x 10~5M) could be followed by (a) the decay of the superoxide absorption at 254nm. (b) the decay and formation of absorptions of the oxidized and reduced porphyrins at the Soret, and e bands. Details on the properties of

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

420 the porphyrins (stability constants of the complexes, absorption spectra of the oxidized and reduced metal!oporphyrins, sample preparation, measurments and data analysis)have been described elsewhere (7,9,10). Decay of 00· Oxygen-saturated solutions containing 2mM phosphate buffer(pH 5.6 and 8.0) and 5mM sodium formate were pulse irradiated. Under the experimental conditions the only radical left after less than 100ns after the -5 pulse is 0 2 (2.4x10

M) (11). In the absence of reactants reactive towards

0 2 its decay follows a pH and concentration dependent mechanism in good agreement with the results of Bielski and Allen(12). Addition of some of the metalloporphyrins was followed by a change of the rate law of the control solutions to a pseudo-first-order kinetics. When this happened it was associated with an increase of the rate of the reaction. The exponential decay of 0^ and linear dependence of the pseudo-first-order rate constants on the concentration of added porphyrin gave second-order rate constants higher than lO^M'^s"^

(see Table 1.). Within experimental error, the decay

traces of 0^ showed no residual absorption. Some metalloporphyrins

(Cu(II),

Ni(II),Ζη(ΙΙ) complexes of TMPyP.TAP and TPPS 4 together with Mn(III) and Co(III) complexes of TPPS^) had small catalytic activity. All of them however showed a tendency to convert the second-order self-decay of 0 2 to a pseudo-first-order rate law. This was especially true when the metalloporphyrin concentration was the highest used (1.2xlo 5 M). The second-order -1 -1 rate constant calculated was of the order of 5x10 M

s

and represents an

upper limit. Reduction of some of the metalloporphyrins by the 0^. Table 1 shows that the Fe(111),Mn(III) and Co(III) porphyrins have catalytic activity for the disproportionation reaction of 0 2 with Fe(III)TMPyP

aquo complex being

the most efficient. Two mechanisms were proposed for the catalytic action of the metalloporphyrins (3). The first in which the metal cation is reduced and reoxidized (reactions(l) and(2)) M(n)Por + 0~ + M(n-l)Por + 0~

M(n-l)Por + 0 2

(1)

+

(2)

+ 2H

+

M(n)Por + H ^

The second mechanism suggests the formation of an intermediate, a metalsuperoxide complex (reaction 3), which can further react with another superoxide radical in a dismutation step (reaction 4). M(n)Por + 0~ M(n)Por

02

M(n)Por +

0~ + 2H

+

0~ + M(n)Por + 0 2 + H 2 0 2

(3) (4)

421 Table 1: Catalytic effect of some metalloporphyrins on the rate of disproportionation of the superoxide radical and the reduction potential (vs.SCE) of the catalyst k (M - 1 . - 1 Porphyrin

Metal

TMPyP

Fe ( 11 Fe(II

pH=5.6

pH=8.0

2.2x10 8

3.0x10 8

0.85(V> (b) -0.050

1.6xl0 6

-0.300(d)

(c)

E

DicmV1) 2.2x10®

Mn( 11

5.1xl0 7

4.0xl0 7

-0.180^ e )

Co(II

1.4x10

7

5

0.165^

TAP

Mn( II

1.3x10 7

2.9x10®

-0.310(b)

TPPS 4

Fe( 11

8x106

1.2x10®

-0.240(b)

1.9x10

Mn( II

i6xl0 5

s7xl0 4

-0.425(e)

2.0x10

Fe {11

3.5xl0 7

2.4xl0 7

-0.165

2.4x10 5

-0.260

PFP

Fe( II

(c)

9.0xl0

2.2x10"

-6 -6

(b) (a)

(a) Rate constants are within + 10%. The solutions with TPPS^

porphyrins

contained 0.1 M NaCl. (b) Buffer phosphate, pH=5.6, 0.1M KCl. (c) Dicyano complex at pH=10.2. (d) Buffer carbonate pH=10.2, 0.1M KCl. (e) Buffer phosphate, pH=6.7, 0.1M KCl, (f) 1 0 " 2 M HNO,

0.5M NaN0 3 (Ref. 13).

The overall catalytic rate of disproportionation of 0^ by metalloporphyrins under certain conditions (3) is given by: -d[0~]/dt = 2 k l j 3 [catalyst] [0~]

=

k o b s d [0^]

(I)

thus, both mechanisms predict a pseudo-first-order decay of 0^ giving second order rate constants for the first step only and kinetics cannot distiguish between them. One way to distinguish between the mechanisms is to observe an absorption due to the formation of the complex (reaction 3). The first step (reaction 1 or 3) could be followed directly at the absorption bands of the metalloporphyrins when the concentration ratio of [MfnJPorj/tO^] is high enough to prevent contribution of the second step of either mechanisms. Normally ratios of 10 to 100 were used and pseudo first order kinetics were observed for the formation and decay of the reduced and oxidized porphyrin. Table 2 summarizes the results obtained. At the end of the reaction of

difference spectrum recorded showed that the

yield of the reduction of the metal ion was 100% for all

metalloporphyrins

422 except that of Fe(III)TMPyP aquo complex. The reaction of

with this

porphyrin was composed of two fast consecutive steps. The f i r s t , the normal bimolecular process followed by a f i r s t order reaction (t, = 1ms). The Λ

spectrum at the end of the f i r s t step was similar in i t s shape to that of the difference spectrum between Fe(II)TMPyp and Fe(III)TMPyP. However, the optical density values at the Soret band (λ = 445 mm) were twofold r max higher than that expected from the concentration. The normal difference spectrum was observed at the end of the second step. These observations suggest that the product of the f i r s t reaction of

with Fe(III)TMPyP i s

not the Fe(II)TMPyP but rather a complex between Fe(III)TMPyP and Og (reaction 3), Fe(II)TMPyP being formed during the second step. Reaction (3) is also supported by high rate of ligand exchange of the aquo complex of Fe(III)TMPyP(14). Reduction and oxidation with the superoxide radical. These experiments were also performed in oxygen-saturated solutions, but, contrary to the solutions previously described, the porphyrin concentration in these solutions was at least tenfold lower than that of superoxide radical. Under these conditions pseudo-first-order reaction kinetics were also observed. However, while the calculated rate-constant value was always higher than that previously determined (k -j 3 ) , the y i e l d at the end of the reaction was always s i g n i f i c a n t l y lower assuming a total convertion of the oxidized form. This behavior seems to indicate that the oxidized form is regenerated during the formation of the reduced form. I t can be shown that the observed pseudo-first-order rate constant is equal to the sum of these of both steps ( k Q b s d = k-j ^ + k 2 ^)(7). Since we know k-j

3>

k^ ^ i s

established (Table 2). The main conclusion from these results i s that the second-order rate constants of the oxidation process i s faster than that of the reduction, a property similar to that found in superoxide dismutase Manganese porphyrins represent a case where oxidation i s much faster and i s probably related to their redox potentials. The catalytic a c t i v i t i e s of the water soluble metalloporphyrins for the disproportionation of

as presented in the tables show that the most

efficient prophyrin i s Fe(III)TMPyP. I t also shows that the other F e ( I I I ) , M n ( I I I ) and C o ( I I I ) porphyrins have some catalytic a c t i v i t y and that C u ( I I ) , N i ( I I ) and Z n ( I I ) do not show any a c t i v i t y . This behaviour i n d i cates the importance of having a second, energetically accessible, oxi-

423 Table 2: Second-order rate constants of the reaction of metal 1oporphyrins with OZ. 2

k (M"1 s" 1 )

Porphyrin1

Metal

1igand

pH

salt

Reduction

Oxidation

TMPyP

Fe

H2O

5.6

y + 0

1.7x10®

4. 2x10

Fe

M

5.6

O.IM HCOO"

3. lxl O 8

7. 6x10 8

Fe

CN"

10.2

0.1M HCOO"

1.5x10®

1. 7x10®

6

3. 1x10®

2

Σ

Fe

His

7.9

0.1M HCOO"

1.0x10

Fe

Im

7.9

0.1M HCOO"

1.9x10 6

3. 8x10®

4.3x10

7

3. 3x10 9

7

6. 5x10 7

Μη

H,0

Μη

2 OH"

Co

H2O

6.7

0.01 M HCOO"

9.3

0.01 M HCOO"

3.8x10

5.6

0.01M HCOO"

1.2xl0 7

Ν. fast

6

2. 0x10 9

Μη

H2O

6.7

0.01 M HCOO"

6.5x10

Μη

OH"

9.3

0.01 M HCOO"

1.5x10®

5. 6x10 8

TPPS 4

Fe

H20

5.6

0.01 M HCOO"

7.0x10®

9. 0x10 7

PFP

Fe

5.9

0.01 M HCOO"

3.5x10 7

3.,7xl08

Fe

H„0 2 1-Me Im

7.9

0.01 M HCOO"

3.6x10®

9..6x10®

Fe

CN"

9.1

0.01 M HCOO*

1.4x10®

2..5x10®

TAP

dation state available to the metal ion for catalysis. As to the mechanism of their catalytic action it seems that only Fe(III)TMPyP aquo complex follows the one with an intermediate formation (reactions 3,4) whereas all the others metal 1oporphyrins are governed by an outer-sphere mechanism. Marcus

(15)

has shown, that the rate constants of an outer-sphere elec-

tron transfer reaction depend on the reaction driving force (redox potential) and on the electron self-exchange rate constants for both the oxidant and reductant. Co(III)TMPyP represents a special case since it has the highest reduction potential yet is not the most reactive metalloporphyrin. The factor which compensates the reduction potential is its low self-exchange rate constant which is 20 M~^s"^(13). The catalytic effect of other metal 1oporphyrins follow their redox potential. This together with the fact we cannot see the formation of transients, (superoxide radical metalloporphyrin complex) seems to indicate that these metal 1oporphyrins react with Ol via reaction 1 and 2.

424 References

1.

McCord, J.M., Fridovich, I.: J. Biol. Chem. 244, 6049-6055

2.

Rabani, J., Klug-Roth, D., Lilie, J.: J. Phys. Chem. 77, 1169-1175 (1973).

(1969).

3.

Weinstein, J., Biel ski, B.H.J.: J. Am. Chem. Soc. 102, 4916-4919 (1980).

4.

Fee, J.Α.: Metal Ion Activation of Dioxygen, (Spiro, T.G. Ed.) WileyInterscience New York, (1980), pp. 209-237.

5.

Pasternack, R.F., Halliwell, B.: J. Am. Chem. Soc. 1M_, 1026-1031 (1979).

6.

Ilan, Y., Rabani, J., Fridovich, I., Pasternack, R.F.: Inorg. Nucl. Chem. Lett. 17_, 93-96 (1981 ).

7.

Solomon, D., Peretz, P., Faraggi, Μ.: J. Phys. Chem. 86, 1842-1849 (1982).

8.

Pasternack, R.F., Skowronek, Jr., W.R.: J. Inorg. Biochem. 11, 261-267 (1979).

9.

Weinraub, D., Peretz, P., Faraggi, M.: J. Phys. Chem. 86, 1839-1842 (1982).

10. Peretz, P., Solomon, D., Weinraub, D., Faraggi, M.: Int. J. Radiat. Biol. 42, 449-456 (1982). 11. Draganic, I.G., Draganic, Z.D.: The Radiation Chemistry of Water, Academic Press New York (l971). 12. Biel ski, B.H.J., Allen, A.O.: J. Phys. Chem. 81, 1048-1050

(1977).

13. Rohrbach, D.E., Deutsch, E., Heineman, W.R., Pasternack, R.F.: Inorg. Chem. 16^, 2650-2652 (1977). 14. Ostrich, I.J., Lim, G., Hunt, J.P.: Inorg. Chem. 1£, 619-621 15. Marcus, R.A.: J. Phys. Chem. 67, 853-857

(1980).

(1963).

DISCUSSION

WESER: What is your estimate iron-porphyrin complexes.

of

the

thermodynamic

stability

of

these

PARAGGI: Our starting porphyrins contained metal ions in their +3 oxidation state. They are stable compounds at all pH's. The +2 metalloporphyrins are stable in the dark in the absence of oxygen. However, they are oxidized by oxygen to the +3 state. The aquo complex is more reactive than those with other axial ligands.

THE

ACTIVATION

OF

ΝADPH-CYTOCHROME AND

NADPH

Max

E.

OXYGEN P-450

BY

BLEOMYCIN

REDUCTASE

IS C A T A L Y Z E D

IN T H E

PRESENCE

BY

OF

IRON

IONS

Scheulen

D e p a r t m e n t of I n t e r n a l M e d i c i n e ( C a n c e r W e s t German Tumor C e n t e r , U n i v e r s i t y of D-4300 Essen, F.R.G.

Hermann

Research), Essen Medical

School,

Kappus

D e p a r t m e n t of D e r m a t o l o g y (FB 3, WE 1 5 ) , F r e e U n i v e r s i t y of B e r l i n , D - 1 0 0 0 B e r 1 i η ,

F.R.G.

I n t r o d u c t i on

The

bleomycins

isolated

by

vertiaillus tumors

and

activity strand

binds

due

in

to

8.3

result

are

be

with

of

to

active

may

stant

nary

and

malignant

mycin

to

a family et

breaks

correlate

are

UMEZAWA

χ

from

DNA 10 the

of m e t a l 1 o - g l y c o p e p t i d e (1) in

to

the

the

by ®M

The

duction

of m o l e c u l a r

superoxide either Fe(III)

been and

or

as

hydroxyl

produced

of

bleomycin

to

the

by

radi c a i - g e n e r a t i n g

system

DNA

Thus,

the

oxidase

Bleoconappears

via

a

ter-

similarity

catalyzing

vitro

of

Fe(II)

species this

agents

such

(8)

by

xanthine

or

has

oxygen

in

with

to

(3).

Fe(11)-bleomyci η

by

(7).

or

by

which

solid

double-

shown

breakage

radicals

addition

reducing

and

been

a dissociation of

(5)

reactive

ethanol , dithiotreitoi , ascorbate

several cytotoxic

single-

with

free

(6).

of

Their

has

antibiotics Streptomyaes

proliferation

complex

radicals

by

of

which

cell

a ferrous

oxygen

(2).

mechanism

oxygenases

function

of

intercalation (4).

of

chemotherapy

in m a n

of

production

may

cultures

production

o x y g e n - F e ( 11 ) - b l e o m y c i η

heme-containing

the

number

reduction

complex

from

lymphomas

DNA

the

al.

or as

the

complex formed

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

has

from

2-mercapto-

a superoxide

oxidase

re-

like

and

anion

hypoxan-

426 TABLE I : Influence of various f a c t o r s on DNA chain breakage as measured by malonaldehyde-formation and on NADPH-consumption i n the standard reaction mixture after 10 minutes (x + S.O.; η = 4-12) MALONALDEHYDE-FORMATION

NADPH-CONSUMPTION

ηηιο1θ5/200μ9 DNA/10 min

nmoles/ml/10min

+

15

COMPLETE SYSTEM

+

- 50Mg/ml Bleomycin

1,,0

- 150μΜ Fe(N0 3 ) 3

0:,92

- 500μΜ NADPH

0,,72

0,25U/ml boiled Reductase

0,,37

Nitrogen Atmosphere

1,,1

+ 150μΜ CuS0 4 ( - F e ( N 0 3 ) 3 )

0,,48

+ 50mM Mannitol

16

+ 100U/ml SOD

4 :,4

+ lOOU/ml boiled SOD

14

+ 1000U/ml Catalase

19

+ 1000U/ml boiled Catalase

16

+ ΙΟΟμΜ Mitomycin C

6,,5

+ ΙΟΟμΜ Paraquat

11

thine of

(9).

We h a v e f i r s t

Fe( 1 1 ) - b l e o m y c i η

further vity

3

+ + + +

+ + + +

5.2 χ Ι Ο " 5

6.,7%

5,2

6,,1%

2,2

0 ,20*

4,,8%

0 ,21*

2.,4%

0 ,52*

7.,3%

0 ,26* 1 ,5

by t h e

classical

11 2,7

3,,2% 106

%

73

29

%

64

2 ,5

93

%

68

1 ,9 *

127

%

65

1 ,5

107

%

70

0 ,65*

36

t

298

1 ,8 *

43

%

262

1 ,2 *

80

%

82

1 ,8 *

73

% 239

on t h e

release

of

6,1 *

+ + +

7,,5%

0,41*

3,,2%

+

2,6 * 16

+

0,33*

+

6,3

+ + + + + + + +

4,,5%

0,82*

3.,9% 106

%

8,0

93

%

9,3

99

%

7,2

94

%

5,3

101

%

*432

%

28

*380

%

7,2 *119 24

enzymatic

acid-soluble

and a V m a x

of

%

* 346

%

formation

P-450 reductase

DNA and by t h e

%

38

as

radioacti-

consumption

Michaelis-Menten-kinetics

M bleomycin

0,83*

—

3,1

0 ,63*

reported

H-thymidi ne-1abeled

NADPH f o l l o w i n g KH of

+

0 ,35* 0 ,23*

by N A D P H - c y t o c h r o m e

demonstrated

from

+

+

5.,4 12

+

+

+ ΙΟΟμΜ Adriamycin + ImM Misonidazole

+

+

69

1 ,7 *

with

2.0 χ I O - 5

of a

M/min

(10) . DNA c h a i n of

breakage

low m o l e c u l a r

is

accompanied

weight

compounds

turic

acid

(11).

As t h e s e m a l o n a l d e h y d e - 1 i k e

amounts

to form chromophores

approximately

by t h e that

simultaneous

react

absorbing

equivalent

products

with

maximally

at

are formed

t o t h e number o f

release

2-thiobarbi-

DNA

532nm in breaks

427 at

neutral

pH ( 5 ) ,

we have d e t e r m i n e d

yÎtro-system

with NADPH-cytochrome

characterize

the enzymatic

respect in

the

vivo,

to

its

course

activation

of

which might of

this

formation

P-450 reductase

activation

stoichiometry of

their

in

to

an

further

Fe(111)-bleomyciη be o f m a j o r

potent

cytotoxic

in with

importance drug

in

as wel 1 .

Results In

agreement

vestigations from

3

with

our

earlier

on t h e f o r m a t i o n

Η-thymidi ne-1abeled

and c o n s u m p t i o n

of

DNA

findings of

as d e t e r m i n e d

acid-soluble

(10)

formation

NADPH a r e d e p e n d e n t

by t h e

in-

radioactivity of

on e a c h

malonaldehyde active

enzyme,

FIGURE 1: Gel f i l t r a t i o n on Sephadex G-10 of products obtained from

3

H-thy-

midine-label ed DNA after incubation with bleomycin, NADPH-regenerating system, F e ( I I I ) , and NADPH-cytochrome P-450 reductase under aerobic condit i o n s . Arrows indicate the c a l i b r a t i o n peaks of standard markers.

428 oxygen,

NAOPH,

bleomycin,

H a l o n a l d e h y d e - f o r m a t i on demonstrated n i f i c a n t l y

and

rad i o s e n s i t i z e r s (13)

The

simultaneous

and

Using

3

the

plete

catalase,

by

mannitol.

such

G-10

by

gel

Kinetics

system a f t e r

active

as

augmented

by

adriamycin

of

02:MDA

=

5,3

-

(x

-

0,16

which

system

of

other

dismutase and

not

other

and

as

sigis

redox-

mitomycin

substances

C,

such

oxygen-consumption,

A

1,0;

the of

times Δ

revealed

NADPH:MDA η

=

as

of

on

preincubation

30 m i n ,

added up t o

the

f o l l o -

4,7

-

1 , 1 ;

thymine

could

mixture

each

be

on

constituent

above.

of malonaldehyde of

=

incubation

dependent

NADPH-

the

7).

release the

mentioned

15 m i n , was

-

S . D . ;

was

the formation

different

bleomycin

DNA

f i l t r a t i o n

( F i g . l )

incubation

2:

i o n s ( T a b . I ) .

M a i o n a l d e h y d e - f o r m a t i on

is

m a l o n a l d e h y d e - f o r m a t i on

0,88

(no p r e i n c u b a t i o n ) , fresh

superoxide

by

Η-thymidi ne-1abeled

Sephadex

FIGURE

by

determination

and

=

demonstrated

of

inhibited

mi s o n i d a z o l e , a n d

stoichiometry: NADPH:

iron

( T a b . I ) .

-consumption, wing

of

augmented

drugs

as

presence

is

NADPH-consumption

cytostatic

paraquat

the

(12),

influenced

inhibited -cycling

e a r l i e r

and

O

60 m i n . initial

from

DNA

without After

in

DNA: 15

the •

com0

minutes

concentration

min

429 Bleomycin

is

destruction mycin

may

ditions

(14).

lead

unless

-formation system

amenable

DNA

stroyed

in t h e

depending

The

ability

as

in

activation

of

DNA

Fe(II)-bleo-

under

aerobic

con-

, we m e a s u r e d

malonaldehyde-

preincubation

in t h e

2 demonstrates with

duration

fresh

to

and

of

active

that

preincubation

from

DNA

complete

bleomycin

NADPH-cytochrome

of

to f o r m m a l o n a l d e h y d e

addition

activation

(6)

vitro-system on t h e

of

inactivation

times

Figure

cycles

its

is p r e s e n t

various

DNA.

ductase further

However,

to c o n s i d e r a b l e

after

without

to m u l t i p l e

can

be

is

P-450

dere-

without

DNA.

restored

by

bleomycin.

C o n c i usi on

Our

results

bleomycin plex

in

which

the

has

role

of g e n e r a l toxic

and and

findings

situation

in

have

Assuming of

in t h e

quinone

of

vivo

a common above,

combination crosomes

activation additive,

results

the

activation

cin

and m i s o n i d a z o l e

as

of

of

under

clinical

for or

be

cytoother

impact

mi-

in

vitro

of on

however.

the At-

on t h e m e c h a n i s m s

level

of

(17). the

cytotoxic

inhibitory

interest

with

to f i n d i n g s

a competitive

by m i t o m y c i n aerobic

of

and

to

including

The

carefully,

mechanism

re-

radiosensitizer

activation

In c o n t r a s t

of b l e o m y c i n

(15)

the

(13,16).

synergistic

com-

P-450

seems

a number

drugs

cellular

support

reductase

adriamycin

investigations

a r e of g r e a t our

such

anticancer

on t h e

of

(10).

P-450

compounds

chemotherapy.

(18)

proposed

be d e t e r m i n e d by

activation

NADPH-cytochrome

activation

the mechanism must

been made

cytostatics

by

neocarcinostatiη,

related

adriamyciη-resi stance mentioned

been

bleomycin

C and m - A M S A ,

misonidazole,

tempts

already

of

an o x y g e n - F e ( 1 1 ) - b l e o m y c i η

of N A D P H - c y t o c h r o m e

besides

anthracyclines

the m e c h a n i s m of

maintained

importance

agents

tomycin these

support

formation

a redox-cycle

ductase Thus,

further

by t h e

regard in

lung

inhibition

C as well

conditions.

drugs

effects

as

to miof

adriamy-

430 Turning

towards

the

DNA damage c a u s e d

biochemical

by b l e o m y c i n ,

action

of the

by t h e

oxygen-Fe(11)-bleomyciη

stoichiometry mannitol

action

radicals

be e x c l u d e d ,

Supported

anion

and d e s p i t e

the d i r e c t

and h y d r o x y l cannot

superoxide

the of

(21,22)

mechanism(s ) r e s p o n s i b l e our

results

radical complex. lack the

of

the

However,

direct released

according

an i n h i b i t o r y

activated

formed

favour

on DNA ( 1 9 , 2 0 )

for

effect

to of

bleomycin-complex

by a F e n t o n - t y p e

reaction

NRW, Düsseldorf,

F.R.G.

respectively.

by Landesamt

für

Forschung

References 1. Umezawa.H. ,Maeda,K..Takeuchi ,T. ,0kami ,Y. : J . A n t i b i o t . lj?.200-209(1966). 2. Carter,S.K..Crooke.S.T.,Umezawa.H.(Edts.):Bleomycin:Current Status and new Developments, Academic Press, New York-San Francisco-London 1978. 3. Kohn.K.W.,Ewig,A.G.: Cancer Res.36,3839-3841(1976). 4. Chien,M.,Grollman,A.P.,Horwitz,37B.¡Biochemistry 16,3641-3647(1977). 5. Burger,R.H. ,Pei sach,J. ,Horwitz,S.B. : L i f e Sei .28,7T5"-727( 1981 ). 6. Burger,R.Μ.,Peisach,J..Blumberg,W.E.,Horwitz,S.B.: J.Biol.Chem.254, 10906-10912(1979). 7. Caspary.W.J.,Niziak,C.,Lanzo,D.A..Friedman,R.,Bachur,N.R.: Mol.Pharmacol -26,256-260(1979). 8. Sausvi1 le,Ε.Α.,Peisach,J.,Horwitz,S.B.:Biochem.Biophys.Res.Commun.73, — 814-822(1976). 9. Sausvi 1 l e , E . Α . , P e i s a c h , J . . H o r w i t z . S . B . : Biochemistry 17,2740-2746(1978). 10. Scheu!en,M.E. ,Kappus,H. ,Thyssen,D. ,Schmidt,C .G. :BiocTiëm.Pharmacol .30, 3385-3388(1981). ~~ 11. Giloni ,L. .Takeshi ta,M..Johnson,F.,Iden.C.,Grollman ,Α.Ρ. : J.Biol.Chem. 256,8608-8615(1981 ). 12. Gal van,L.,Huang,C.-H.,Prestayko,A.W.,Stout,J.T.,Evans,J.E.,Crooke,S.T. : Cancer Res.41,5103-5106(1981 ). 13. Kappus,H.,Sïës,H.: Experi enti a 27,1233-1241(1981 ). 14. Povirk,L.:Biochemistry 18,3989^995{ 1979). 15. Bachur,N.R.,Gordon,S.L.TGee,M.V..Cancer Res.38,1745-175011978). 16. Favaudon.V.¡Biochimie 64,457-475(1982). 17. Scheulen.M.E..Hoensch.H.P.,Seeber,S.,Schmidt,C.G.:Das Resistenzproblem bei der Chemo- und Radiotherapie maligner Tumoren - Grundlagen und K l i n i k . Beiträge zur Onkologie. Karger, Basel-München-Paris-LondonNew York-Tokyo-Sydney ( i n p r e s s ) . 18. Trush,M.A..Himnaugh.E.G.,Ginsburg,E.,Gram,Τ.E.:J.Pharmacol.Exp.Ther. 221,159-165(1982). 19. Lesko,S.Α.,Lorentzen.R.J.,Ts'o,P.O.P.:Biochemistry 19,3023-3028(1980). 20. Brawn,Κ..Fri dovi c h . I . : Arch.Biochem.Biophys.206,414-ÍT9(1981). 21. Oberley.L.W..Buettner,G.R.:FEBS Lett.97,47-45T1979). 22. Lown,N.R.,Joshua,A.V.:Biochem.Pharmacol.29.521-532(1981).

431 DISCUSSION CADET: You measured the release of thymine. Have you any evidence for the release of other bases such as cytosine, guanine or adenine, and if so, is there any specifity in this release? SCHEULEN: We only labelled our DNA with radioactive thymidine, so we cannot comment on that. But the HORWITZ group has demonstrated that all four bases in DNA are released by Fe(II)-bleomycin (SAUSVILLE et al. Biochemistry (1978) 17, 2746-54). They found cytosine as well as thymine. RALEIGH: I would like to have clarified the effect of the nitro-aromatics added to the system. Did they enhance the destruction of bleomycin? SCHEULEN: No, we did not look for the effect of those substances like misonidazole or nitrofurantoin on the integrity of the bleomycin molecule. We only looked for the influence of bleomycin on NADPH consumption and malonaldehyde formation. From the fact that the malonaldehyde formation was inhibited and the NADPH consumption was enhanced, we can only speculate that there may be a competitive inhibition by those substances. BHUYAN: Is it known whether microsomes can activate bleomycin, and did you confirm whether iron or other metal ions are really necessary? Did you try to see the effect of GSH in your system with NADPH-cytochrome P-450 reductase? SCHEULEN: YAMANAKA and coworkers have demonstrated that bleomycin can be activated by microsomes (YAMANAKA et al.. Cancer Res. (1978) 38» 3900). Our results clearly demonstrate that the presence of iron ions is essential for the activation of bleomycin by NADPH-cytochrome P-450 reductase. The iron ions cannot be replaced by copper ions. This does not rule out any possible role of metal ions other than iron in other activation systems. GSH in a concentration of 1 mM has no effect on the formation of acid-soluble radioactivity from 3 H-thymidine-labeled DNA in an in vitro system with NADPH-cytochrome P-450 reductase as demonstrated earlier by us (SCHEULEN et al., Biochem. Pharmacol. (1981) 30, 3385-88). BORG: You leave open the question of what the attacking oxidant - the ultimate oxidant - might be. If I recall correctly from Peter CERUTTI's work, one might expect to find thymine glycol if there is hydroxyl radical attack. Or if tritiated thymine is used, release of tritium to water can be detected. Did you look for these products? SCHEULEN: No, we didn't look for base-propenals, the phosphate ester of glycolic acid (GILONI et al., J. Biol. Chem. (1981) ¿56, 8608-15), aldehyde sugars (DIZDAROGLU et al., J. Am Chem. Soc. (1975) 9T_, 2277) or other intermediates, which may be formed by hydroxyl radical attack. We only checked the ability of mannitol to inhibit the reaction. I think this does not exclude a major role of the hydroxyl radical, as this reaction may be in close vicinity to the DNA, because of the intercalation of bleomycin. Negative inhibition experiments with mannitol or other hydroxyl radical scavengers are not able to rule out any effect of hydroxyl radicals in the mechanism of destruction of DNA by activated bleomycin.

432 BORG: I agree with what you say about the effect of inhibitors. I believe that what you've described is what BACHUR calls the "site-specific hydroxyl radical formation" associated with bleomycin bound to DNA. That is why I thought looking for the products of hydroxyl radical attack might be of some use in separating out the possibilities. SCHEULEN: I quite agree, but X can only say again that we looked for the formation of malondialdehyde and thymine to demonstrate the activation of oxygen by bleomycin as catalyzed by NADPH-cytochrome P-450 reductase in the presence of iron ions and NADPH. ULLRICH: I wonder why nobody ever considers the intermediate peroxide state, also as reactant. Almost everybody suggests either O2 or OH as active species. But in this case the bleomycin-iron complex is so similar to the active oxygen complex of P-450, that you should consider also either Fe=0, Fe-peroxide or Fe-peroxo complexes. SCHEULEN: That's what I left open, that the activated complex itself may be the ultimate substance which reacts with the DNA. von SONNTAG: We are presently studying the formation of malondialdehyde formed in DNA after OH attack (using ionizing radiation). We haven't progressed far enough to know details in the case of DNA, but with model systems like nucleosides, we are now on the way of understanding this system a little bit better. 05 is apparently not needed for this reaction. It really doesn't play a role in this system. The mechanism most likely involves the peroxyl radical at C-4' and other DNA radicals to help this reaction to proceed. All reactions would involve the hydrogen atom at C-4" irrespective of whether OH radicals or any other active oxygen species are intermediates. Therefore, such radiation chemical studies would not help, unfortunately, to differentiate between the various possible mechanisms. SCHEULEN: That's very interesting, but I think there's one great difference between radiation and bleomycin. That is the intercalation of bleomycin, and I think it could be possible that by this intercalation you get a stereochemical situation where perhaps the superoxide radical may bè active enough to make strand-breaks. It's only a speculation, of course. von SONNTAG: Well, I fully agree, because it is the high specificity as you mentioned with respect to thymine release that indicates that it all happens at a very specific site in the bleomycin case. This will not be so in the case of OH attacking DNA. So, from the radiation chemical results, we can draw only very few conclusions with respect to the bleomycin case. SRIDHAR: tion?

Did you say that nitrofurantoin enhanced malonaldehyde

SCHEULEN: Yes, the malonaldehyde formation was enhanced. We measure the NADPH consumption, it was not possible.

forma-

couldn't

SRIDHAR: There is a problem with the TBA assay, because nitrofurantoin decomposes to give 5-nitro-2-furaldehyde. This aldehyde reacts with TBA

433 to give a condensation product w h i c h has a strong absorption around 532 nm. We reported on this at an Araerical Chemical Society meeting. I c a n send y o u the details of this reaction. Thus the TBA assay is not reliable in the presence of nitrofuraldehyde derivatives such as nitrofurantoin, nifuroxime, nifuraldezone and nitrofurazone. SCHEULEN: Oh, that's the reason. I thank y o u very much for this comment, because we did not have an explanation for this finding. WINTERBOURN: Just a quick comment that m i g h t explain the SOD effect that y o u get. It is possible that the metal oxygen complex is the active s p e cies, b u t the SOD c o u l d drag the O2 off the iron complex and hence inhibit the reaction.

MECHANISM OF CO-OXIDATION REACTIONS DURING PROSTAGLANDIN BIOSYNTHESIS

Hubert Groß, Wolfgang Nastainczyk, Volker Ullrich Institut für Physiologische Chemie, Universität des Saarlandes D-6650 Homburg-Saar

Introduction Recently, we have demonstrated singlet oxygen formation elicited by PGG2 or iodosobenzene both in microsomes from ram vesicular glands or with purified prostaglandin-endoperoxide synthase (1,2). The mechanism underlying the singlet oxygen formation might be of importance for the generation of an active oxygen species during PG biosynthesis leading to the oxidation or oxygenation of xenobiotics, the so-called co-oxidation reaction (2,3). For this reaction we have postulated a cleavage of the 0-0 bond of PGG2 in the presence of ferric PG synthase (Fe + ) and formation of a transient (FeO) + species analogous to the monooxygenase reaction of cytochrome P450 (1,2,3). The existence of such an oxo-ferryl complex, similar to Compound I of HRP (4), is also supported by a 'spectral enzyme intermediate' (at 430 nm) formed in reactions of PG synthase with PGG2 or iodosobenzene (2). Here, hydroxylation reactions of the postulated 1 active oxygen complex' were investigated with various oxene donors and substrates whose hydroxylation reactions are wellknown from catalysis by cytochrome P450.

Methods Ram vesicular gland microsomes were prepared and PG synthase was purified according to (5). PGG2 was synthesized as described in (6) and iodosobenzene was prepared according to

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

436

tfillgerodt (7). The hydroxylation of cyclohexane were done in 5 ml Tris/HCl buffer (pH 8.1) containing various concentrations of oxene donors, 30 mg microsomal protein or 2.5 nmol purified PG synthase (in presence of 5 nmol hematin) and 50 μΐ cyclohexane. After preincubation (2 min) the reactions were started by addition of oxene donors. After 5 min the reactions was stopped with 100 μΐ HC10 4 (17 %) and 2-heptanole was added as standard. The products were extracted 2 times with ether (4 ml) and analysed by GC or GC-MS.

Results Cyclohexane, a substrate with saturated CH-bonds, was hydroxylated to cyclohexanol by ram vesicular gland microsomes or purified PG synthase in the presence of arachidonic acid with 02 or PGGj. After 1-2 min cyclohexanol was formed no longer, a behaviour paralleled by the activity of prostaglandin-endoperoxide synthase as measured by oxygen consumption and PGG2 or PGH2 formation which also occurs within 1-2 min (6). The rapid decreaOXENE DONORS

CYCLOHEXANOL (nmol χ min ' m g p r o t e i n

PCC 2

( 1,7 χ 10 \ l )

Arachidonic Acid + 0 2 (1,6 χ 10~\λ) Ph - J

= O + 02

Ph - J

= 0

( 1 χ 10" 4 M)

anaerob (4 χ 10~ 4 M)

0,45

+0,02

0,31

+ o,02

0.83

+ 0,05

0.83

+ 0,05

Cumene h y d r o p e r o x i d e ( v a r i o u s c o n c . ) m - C I - P e r b e n z o i c acid Endoperoxides

')

(various conc. )

(various conc. )

Fig.1: Effect of various oxene donors on the formation of cyclohexanol in ram vesicular gland microsomes (Incubation as described in Methods)

437

sing activity seems to result from the rapid inactivation of the enzyme. Iodosobenzene, an oxene donor which can hydroxylate cyclohexane in a cytochrome P450-dependent reaction, very likely via a (FeO) species, was also used in the PG synthase system. In incubations with cyclohexane supplemented with iodosobenzene cyclohexanol was formed at a rate of 0.83 nmol/mg microsomal protein/min or 12 nmol/nmol enzyme/min. Other potent oxene donors like endoperoxides (ascaridole, anthracene(9,10)endoperoxide) hydroperoxides (H20J, eumene hydroperoxide, m-Cl-perbenzoic acid) or N-oxides (pyridin-N-oxide, N,N-dimethylaniline-N-oxide) could not hydroxylate cyclohexane with microsomal suspensions (Fig.1). The cyclohexanol formation was dependent on protein- and iodosobenzene-concentration.

I . Intermolecular Kinetic Isotope E f f e c t of the Formation of Cyclohexanol

Π. I n t r a m o l e c u l a r Kinetik Isotope E f f e c t of Undeca Deutero C y c l o h e x a n

11 1

16

110/11

Fig.2:

Deuterium isotope effect of the hydroxylation of cyclohexane by cyclooxygenase and iodosobenzene in ram vesicular gland microsomes (Incubation as described in Methods)

438

Cooxidation substrates, such as phenol, hydroquinone and reduced glutathione inhibited the iodosobenzene-induced hydroxylation of cyclohexane. This corresponded to the inhibition of arachidonic acid-induced singlet oxygen formation (1) or the spectral enzyme complex formation (2) during the PG synthase reaction. The intermolecular kinetic isotope effect for cyclohexane was 7.9 and the intramolecular kinetic isotope effect was 16.1. From the magnitude of kinetic isotope effects it was concluded that the breaking of the C-Η bond in cyclohexane was the rate determining step in the hydroxylation reaction of cyclohexane by PG synthase and iodosobenzene (Fig.2). The great difference between the inter- and intramolecular isotope effect can be explained by an additional second isotope effect.

Conclusions These results show that prostaglandin-endoperoxide synthase forms an oxidizing species capable to hydroxylate cyclohexane. The hydroxylation of cyclohexane by PG synthase in the presence

Proposed Reaction-Cycles of PG-Synthase

ROH

439

of iodosobenzene or PGG2 suggest the formation of a common active oxygen species. Since the hydroxylation and the formation of singlet oxygen or the spectral enzyme intermediate'proceeds under the same conditions with iodosobenzene or PGG2 a common pathway seems to exist for the PG synthase-catalysed reactions. For this common intermediate we have postulated an oxo-ferryl complex (1,2) (scheme). The involvement of a (FeO) + intermediate in the reaction cycle of the enzyme explains the various reactions of the PG synthase in the presence of arachidonic acid, PGG2 or iodosobenzene: (a) Formation of singlet oxygen, (b) Direct hydroxylation or initiation of a radical chain, (c) Reaction with electron donors.

Acknowledments We thank Professor Dr. M.R. Möller for the GC-MS spectra. This work was supported by Deutsche Forschungsgemeinschaft, Schwerpunkt "Mechanismen toxischer Wirkungen von Fremdstoffen" Na 127/2-1 and Sonderforschungsbereich 38 "Membranforschung".

References 1. 2. 3.

4. 5. 6. 7.

Cadenas, E., Sies, H., Nastainczyk, W., Ullrich, V., Hoppe Seyler's Z.Physiol.Chem. 364, 519-528 (1983) Nastainczyk, W., Ullrich, V. , Cadenas, E., Sies, H., III. ICOR, München 1983 Kuehl, F.Α., Humes, J.L., Ham, Ε.Α., Egan, R.W., Dougherty, H.W.: Prostaglandin and Thromboxan Research (Samuelsson, B. et al., eds.) 77-86, Raven Press, New York 1980 Hoffmann, M.B.: The Biological Chemistry of Iron (Dunford,H.B., eds.) pp. 391-403, D.Reidel Publishing Comp., Dordrecht 1982 Roth, G.J., Machyga, E.T., Strittmatter, P., J.Biol.Chem. 256, 10018 (1981) Graff, G., Stephenson, J.M., Glass, D.B., Haddox, M.K., Goldberg, N.D., J.Biol.Chem., 253, 7662 (1978) Willgerodt, C., Chem.Ber. 25, 3495 (1982)

MECHANISM OF ARACHIDONIC ACID-STIMULATED SINGLET OXYGEN FORMATION BY PROSTAGLANDIN—ENDOPEROXIDE SYNTHASE

Wolfgang Nastainczyk, Volker Ullrich Institut für Physiologische Chemie, Universität des Saarlandes D-6650 Homburg-Saar Enrique Cadenas, Helmut Sies Institut für Physiologische Chemie I der Universität Düsseldorf D-4000 Düsseldorf

Introduction Prostaglandin-endoperoxide synthase-catalysed conversion of PGG2 to PGH2 is associated with the formation of a potent oxidant (1,2,3). With respect to the nature of this oxidant, recently it has been reported that PGG2 , the product of dioxygenase reaction, gives rise to singlet oxygen in the presence of isolated PG synthase under anaerobic conditions (4). These findings and similarities of the PG synthase to cytochrome P450 led to the hypothesis that the first step -in the reaction of PGG2 and PG hydroper— oxidase is the formation of an (FeO)"^+ intermediate (4). Then this active oxygen complex can react with a second molecule of PGG2 and yield singlet molecular oxygen (5). Here we attempted to find additional evidence for the formation of an oxo-ferryl complex as the initial oxidant by using iodosobenzene as an oxene donor (6). We report the formation of a spectral intermediate and

0 2 associated with the interaction of iodosobenzene

or PGG2 with PG synthase.

Methods Microsomal fractions were prepared frömram vesicular glands and PG synthase was purified as described (7). Hematin was added

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

442

to the isolated enzyme (1 to 2 mole/mole), a requirement for enzyme activation (8). Low-level chemiluminescence was measured as described (9,4). Chemiluminescence assays contained ram vesicular gland microsomes or PG synthase which were suspended in 02saturated 0.1 M Tris/HCl buffer, pH 8.1. Difference spectra were recorded on an Aminco DW-2 spectrophotometer using 10 mm cuvettes each containing 1 ml of a microsomal suspension or purified PG synthase in 0.1 M potassium phosphate buffer, pH 8.1, and 40 % glycerol.

Results Microsomes from ram vesicular glands as well as purified PG synthase supplemented with either arachidonic acid or PGG2 formed a spectral intermediate with a peak at about 430 and a trough at about 411 nm (Fig.1). At -20°C the peak at 430 nm disappeared within 4 min whereas the trough at 410 nm increased 3-fold. Subsequent addition of PGG2 or arachidonic

acid did not produce

the peak again, obviously due to the destruction of PG synthase. At higher temperatures PG synthase was so rapidly destroyed that only the trough at 410 nm was observed. The nature of the enzyme intermediate was further elucidated by the use of the oxene donor iodosobenzene which could provide one oxygen atom and therefore would favor an (FeO)

+

intermediate. Iodosobenzene induced

the rapid formation of an enzyme intermediate in microsomes as well as with the purified enzyme similar to the complex formation observed with PGG2 or arachidonic acid (Fig.1). Substrates for the co-oxidation, such as phenol, hydroquinone or glutathione, inhibited the complex formation induced by arachidonic acid, PGG2 or iodosobenzene. Assuming the formation of an (FeO)3+species and its subsequent interaction with a second molecule of PGG2 or iodosobenzene it could be expected that iodosobenzene as well as PGG2 or arachi1

donic acid (4) should produce

0 2 which could be observed by its

chemiluminescence. An analogous reaction is postulated for iodoso

443 430

Fig.1:

Difference spectra of ram vesicular gland microsomes with arachidonic acid, PGG2 or iodosobenzene The incubation mixture contained 4 mg/ml of microsomal protein, 0.6 ml of 0.1 M potassium phosphate buffer, pH 8.1, and 0.4 ml glycerol. 15 μΐ of substrate (in acetone)were added to the microsomal suspension and 15 μΐ of acetone to the reference cell. The spectra were immediately recorded.

benzene and cytochrome P450. Actually, iodosobenzene when added to microsomes or the purified enzyme, induced low-level chemiluminescence which could be assigned by its spectrum to the di— ι mol emission of Oa (Fig.2). The light emission did not depend on oxygen concentration and proceeded also under N 2 . Hence θ 2 was not required for the production of ^02 , even when iodosobenzene was used which could donate one oxygen atom only, the ι formation of 0 2 could be explained by the reaction of the presumed (FeO)"^+ intermediate with a second molecule iodosobenzene

444

Fig. 2: Emission spectrum of araahidonic acid- or iodosobenzene-induced chemiluminescence of PG synthase. Isolated PG synthase (58 μg/ml) and 1 μΜ hematin in 0.1 M Tris/HCl buffer, pH 8.1, were supplemented with 54 μΜ arachidonic acid ( — · — ) or 0.5 mM iodosobenzene (—•— ) .

—1

600

1

650

1

700

750

Light emission intensity was corrected for photomultiplier efficiency and transmission.

WavalvngthÎnm)

Conclusions Our present results suggest the formation of two oxidizing species, singlet oxygen and an oxo-ferryl complex

(FeO)

+

during

the PG hydroperoxidase-catalysed conversion of PGG 2 to PGH 2 . The ι spectral intermediate is suggested to be the precursor of 0 2 and also responsible for the oxidation and oxygenation reactions of PG synthase. In analogy to horseradish

peroxidase Compound I, we propose that

the first step in the reaction of PGG 2 and PG hydroperoxidase is the formation of an oxo-ferryl or oxenoid complex analogous to the 'active oxygen complex' of cytochrome P450

(10). The

(FeO)

intermediate may react with a second molecule or PGG 2 by formation of

1

0j or can pick up two electrons from co-oxidation sub-

strates as known from other peroxidase reactions

(scheme next page

445

Enzymatic Cycle for the Formation of Singlet Oxygen by Prostaglandin Endoperoxide Synthase PG Synthase

PGG-3 Fe(lll)Por

Fe(lll) Por

HOOR

2e + 2H* GSH, Phenols, Amines

PGH.

e-Oil • F e V_$»^Fe l v -ö—-• FFe-Oil Por

Por

Port J

Compound I

References 1. Marnett, L.J., Bienkowski, M.J., Leithauser, Μ. , Pageis, W.R., Panthananickal, Α., Reed, G.A.: Prostaglandins and Related Lipids (Powells, T.J. et al., eds.) 2, 97-111, A.R.Liss, Inc., New York 1982 2. Rahimtula, Α., O'Brien, P.J.: Biochem.Biophys.Res.Commun. 70, 893-899 (1976) 3. Kuehl, F.A., Humes, J.L., Ham, E.A., Egan, R.W., Dougherty, H.W.: Prostaglandin and Thromboxane Research (Samuelsson, B. et al., eds.) 6, 77-86, Raven Press, New York 1980 4. Cadenas, E., Sies, H., Nastainczyk, W., Ullrich, V. : Hoppe Seyler's Ζ.Physiol.Chem. 364, 519-528 (1983) 5. Cadenas, E., Sies, H-, Graf, H., Ullrich, V.: Eur.J.Biochem. 130, 117-121 (1983) 6. Lichtenberger, F., Nastainczyk, W., Ullrich V. : Biochem.Biophys. Res. Commun. 70, 939-946 (1976) 7. Roth, G.J., Machuga, E.T, Strittmatter, P.: J.Biol.Chem. 256, 10018-10022 (1981) 8. Ogino, Ν-, Ohki, S., Yamamoto, S., Hayaishi, 0.: J.Biol.Chem. 253, 5061-5068 (1978) 9. Cadenas, E., Sies, H.: Methods Enzymol. 105, 00-00 (1983) 10„Ullrich, V. , Ahr, H.J., Castle, L., Kuthan, Η., Nastainczyk, W., Ruf, H.H.: The Biological Chemistry of Iron (Dunford,Η.Β. e d s J pp.413-425, D.Reidei Publishing Comp.,Dordrecht 1982

THE INTERACTION

Kevin

D.

OF HYDROGEN

PEROXIDE

the reactions

with H202 details

of the ferri

to produce

ferryl

of the analogous

overlooked. generate

The reaction

The analogous

reactions

gous monomeric myoglobin

of H202

have

of myoglobin been

with

oxyhemoglobin

(7).

derivatives

myoglobin,

of Hb02

In order

to provide

the reaction

(1,2) , largely

reported

to

solutions

with H202

stated

reliable

of membrane

been

(4,5). report-

the structurally

(MbO^i has been briefly

decomposition

examined

has been

(6), while

hemoglobin

have

(3) and in purified

of the a- and E- subunits

oxyhemoglobin

and

rigorously

of the oxyhemoproteins

in erythrocytes

to the catalyzed

sarcoplasmic

derivatives

derivatives

hemichrome

(ferrylMb)

University

reactions

ferrihemoglobin

edly form low-spin

ground

OXYMYOGLOBIN

Whitburn

Department of Chemistry, Boston Boston, MA, U.S.A. 02215

While

WITH

mechanistic

lipid

of 1¡202 with Mb02

to form

analoferrylback-

hydroperoxides has been

by

examined

in

detail.

Materials

and

The method

Methods

of purification

of bovine

as is the spectrophotometric ted myoglobin 10-50

solutions.

vM in aqueous

in quantities

Results

and

The reaction [H.O.iof

muscle

porcedure

is described

for compositional

All reactions

solutions

Mb02

were performed

at pH 7.3.

Additions

elsewhere

analysis using

of solid

of

(8), aera-

[myogiobin] reagents

of

were

of < 1 mg.

Discussion

of

H

0.1 to 3.0.

w

a

s

Characteristic

exam ne

^ ^

over a range

of the results

of R = [MiO^ ] :

obtained

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

for R < 1.0

are

448 the

spectral

rity

product

rum

of

rum

ferroMb

upon

reagents,

R > 1.0, is

for

R < 1.0.

and

604

nm.

absorbance version

In

ferryl

After

after

Na^S^O^.

with

the

and

nm,

630

product

properties

Effect of H20

forms

of

at

is

of

of

two

527,

of change with time.

the to

spect-

that

of

of

solid

to a

over

the

this

esa C nm D

(65 vM) on Mb02

to of

derivative

The 588

first

nm,

as

isosbestics significant

ferriMb.

stage seen

at

521

growth Total

of

con-

hours.

+ H^O^

derivative

and

with

^ 1 hr

several

MbO^

stages.

560

characteristic

of myoglobin.

see WAVELENGTH

has

reaction,

were After

compared converting

the

absorption

with

those

the

MbO.

of

ese

(25 \iM) at pH 7.3. Absorption

measured 1.0, 2.8, 8.2, 51.2 min after addition of reagents. Hons

to

spect-

Na^S^O^.

addition hrs)

g]

The

converted of

integ-

K^lFefCN)

solution,

slowly

proceeds

within

thereafter

hemichrome

H^O^

reaction

at

redox

addition

protocal

very

of

ferriMb.

product

which

this

isosbestics

leads

the

the

appears,

process

500

Addition

isosbestic

ferriMb.

by

occurs

ran.

which

s p e c t r u m of

of

converts

of MbO^

t 5 min,

588

in

a subsequent

Without

to

R = 0.4)

the

aliquot

solution

caa

Figure 1.

and

second

to identify

and

560

10 min)

characterized

The

and

appears

reaction

(for

generates

similar

the

1

557,

to another

product

peaks

spectrum

527,

slowly

to ferriMb

order

the

KCN

spectrally

(< 5 min)

Figure

quickly

adding

the

is

in

quickly of

CNferriMb

ferroMb

For

at

solution

addition of

that

shown

is maintained

the

Upon

data

Arrows

show

spectJ di red

449 solution to ferriMb with K^[Fe(CN) A, ferrylMb was subsequently generated J

Ό

by addition of excess (3:1) ^

^^ '

hemiMb was produced by addi-

tion of benzoate (2M) to the ferriMb solution (9). Although the spectra of these derivatives are similar in the 530-600 nm region, only that of ferrylMb crosses the spectrum of MbO^ at the isosbestic wavelengths observed previously for the product of the HbO^ + fi^O^ reaction. Also, ferrylMb behaves the same towards the protocol of reagent additions as observed for the latter product.

For hemichrome, this procedure produces hemo-

chrome upon additions of Ka^S^O^.

Clearly the first product of the reac-

ierr

tion of MbC>2 with Η2°2

y 1Mb, and not hemiHb or ferriMb.

Using the previously described method of compositional analysis (8) , the changes in the percentage compositions of MbO^, ferriMb and ferrylMb were monitored as a function of time for t 1 hr after addition of reagents over the range of R = 0.1-3.0. The observed compositional changes are illustrated in Figure 2 for values of R = 2.7, 1.0 and 0.33. Several important features of the MbO^ + ^2^2

react on

^

are

apparent from this analysis.

For R > 1.0, MbO2 is converted to ferrylMb while the percentage of ferriMb remains essentially constant for the first 5-10 min.

This conversion

is gradually replaced by a concerted process in which losses of MbO2 and ferrylMb having the same rate, are attended by a growth of ferriMb at double the rates of loss.

This behavior suggests the occurrence of reac-

tions [J] and [2] over the observed time duration;

MbO2 +

—

MbO, + ferrylMb

•> ferrylMb >

2 ferriMb

[J] [2]

The synproportionation on this timescale designated in reaction [2] was separately demonstrated by mixing ferrylMb and MbO^ solutions; the ferrylMb was generated by addition of excess fl^O^ to ferriMb solution, with a later addition of catalase to remove the remaining Ji^O^· A correlation of the overall changes in [MbOand

[ferriMb] as a function of initial

[h^02] for R < 1.0 indicates η - 2 in reaction [J]. The kinetics of this reaction are complex.

For F < 1.0, three obvious differences arise.

First, a fast conversion of

450 ferriMb to ferrylMb is observable for t < 2 min.

Also, the conversion of

Mb02 to ferrylMb in reaction [J] is not replaced by reaction [2], Thirdly, the rate of the later growth of ferriMb is reduced at increased concentrations of H^O^.

These observations, illustrated in Figure 2c,

are consistent with the participation of reaction [3] in the presence of excess H.O., for which k. > k,. 2 ¿ —J ^ —1

The inclusion of this reaction

ferriMb + H0

>

ferrylMb

[3]

ferriMb

>

ferrylMb

[4]

prolongs the duration of growth of ferrylMb, and decreases the rate of later formation of ferriMb.

Over a period of several hours another pro-

cess must be considered ; this is the very slow conversion of ferrylMb to globin-modified ferriMb (10) in reaction [4].

Several important features emerge from this study of the reaction of resu

-^ts

MbC>2 with

show that this reaction is complex, involv-

ing several processes, and is mediated by ferrylmyoblobin. The nature of the observed product depends both on the ratio of the reagent concentrations and on the time elapsed between the addition of reagents and the

Figure 2.

Compositional changes with time of MbO2 (—) , ferri Mb (- - -)

and ferrylMb (

) upon addition of HC>2 to MbC>2 (12 vM) at pH 7.3 (a) R =

2.7; (b) R = 1.0; (c) R = 0.33.

451 qualitative spectral analysis. If this elapsed time is long (> 1 hr), ferriMb is observed as the major product, especially if [MbO>

[H^O^].

If [MbO^] < [HjCy and analysis of the product is immediate upon addition of reagents, ferrylMb is observed as the major product.

Ko evidence for

the formation of hemiMb is obtained for this reaction. Details of the kinetics and mechanism of the reaction of MbO2 with H^O^ will be presented elsewhere.

Acknowledgments

This research was supported by the U.S. Army Research Office through contract No. DAAG-29-82-K-0132 to Boston University. The active interest of Prof. M.Z. Hoffman in this work is gratefully acknowledged.

References

1.

Fox, J.B., Nicholas, R.A. , Ackerman, S.A. and Swift, C.E.: Biochem., 13, 5178-5186 (1974), and references therein.

2.

Dalziel, K. and O'Brien, J.R.P.:

3.

Aebi, Η., Heiniger, J.P. and La über, E.: 1428-1440 (1964).

4.

Eyer, P., Herthe, Η., Kiese, M. and Klein, G. : Mol. Pharmacol., 11_, 326-334 (1975) .

Biochem. J. 54, 648-659 (1954). Helv. Chim. Acta, 47,

5.

Tomoda, A. and Yoneyama, Y.:

6.

Tomoda, Α., Sugimoto, Κ. , Suhara,. Μ., Takeshita, M. and Yoneyama, Y.: Biochem. J. 171, 329-337 (1978).

7.

Kobayashi, K. and Hayashi, Κ.:

8.

Whitburn, K.D., Shieh, J.J., Sellers, R.M., Hoffman, M.Z. and Taub, I.A.: J. Biol. Chem., 257, 1860-1869 (1982).

9.

Tsushima, K.:

10.

Experientia, 35, 15-16 (1979).

J. Biol. Chem. 256, 12350-12354 (1981).

J. Biochem. 41_, 215-237 (1954).

King, N.K. and Winfield, M.E.:

J. Biol. Chem. 238, 1520-1528 (1963).

BIOLOGY AND FHOTOBIOLOGY OF SINGLET OXYGEN

Norman I. Krinsky Department of Biochemistry and Pharmacology, Medicine, Boston, MA 02111 (USA)

Tufts

University

School

of

Introduction

The potential

role of

singlet

oxygen

(102)

in biological

tracted many investigators over the last 15 years.

systems has

at-

It was the first review

b y Foote (1) that began focusing the attention of biochemists and biologists on

this

unique

electronically

excited

species

of

oxygen.

Wilson

and

Hastings (2) speculated on the possible role of ^0. in bioluminescence and 1 also reviewed the possible role of 0~ in a variety of enzymatic reactions. L ι However, the problem in attempting

to relate

02

to biological systems has

not been a shortage of speculation but rather In the difficulty of confirming or denying the various speculations that have been put forth.

A n early

attempt to evaluate the role of ^0. in biological systems (3) focused on the ¿

1

various chemical reactions that had been reported to generate ing

that

these

reactions. logical

reactions might

serve

as potential models

consider-

for

biochemical

In addition, the criteria available for identifying

systems

focused on product

analysis, D 2 0 enhancement

in bioof

02

times, inhibition by specific quenchers, and specific chemilumlnesence The

reactions

which have been reported

the pattern of a clock in Figure 1. rect evidence

for

the involvement

to generate

life(3).

*0 2 are represented

in

Although there is some direct or indiof all of

these reactions

in biological

systems, we will concentrate only on the reactions designated 1 o'clock, 2 o'clock, 5 o'clock and proven extremely valuable which reactions

11 o'clock. with respect

A careful series of calculations has to clarifying

our

understanding

are thermodynamlcally possible w i t h respect to

tion (4,5).

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

^0 2

of

genera-

454

Photosensitized Generation of The process of generating method

by photosensitized reactions still remains the 1 of choice with respect to introducing 0^ in a biological system.

Sometimes the introduction is deliberate as in the case of adding exogenous sensitizers whereas at other times it is accidental, either through errors in diet or treatment plans or by genetic defects such as occur in certain porphyrias.

The generation of

is basically a Type II photosensitized

reaction as described by Gollnick and Schenck (6). is shown below:

The reaction leading to

455 O2 was

clearly

comparison of

demonstrated

to

arise

from

photosensitized

reactions

by

the products formed by photosensitized oxidations and those

formed by a chemical system for generating ^Oj (7).

The topic of the photosensitized generation of

has been reviewed many

times in the past with respect both to the chemistry of the reactions

(8)

and

de-

their

biological

implications

(9).

Only a few examples will be

scribed of photosensitized generation of The widespread

use

of

psoralens

in biological systems.

as photosensitizing

agents using

light as the radiation source has been shown to generate Ι cently, a report has appeared indicating that both from Irradiated psoralens (11).

near-UV

0, (10). -

Κε-

0^ and 0· are generated

There are also examples of damage that can

occur to a variety of systems when appropriate sensitizers are either applied

or

generated

ija vivo.

For

example,

the

cataracts

that appear

in

elderly individuals have been attributed to the photosensitized formation of a tryptophan derivative, which in turn generates

0^ leading to cross-link-

ing of lens proteins which are comparable to those seen in cataracts (12). There are also many examples cells and organisms.

in the literature

of

^2

acting

to

destroy

A recent report indicates that the nematicidal action

of dithiophenee is light-dependent and the reaction appears to generate (13).

Finally,

the widespread

use

of derivatives

of

hematoporphyrin

as

photosensitizing agents in the treatment of several different types of tumors appears to be associated with the ability of this compound to generate *0 2 when illuminated in situ (14). In most of these cases, However

has been determined by an independent technique.

it should be noted that photosensitized oxidations

can also

occur

via electron transfer reactions which in many cases mimic those seen with the generation of

(15, 16).

456 Chemical Generation of ^Oj. For màny years, the reaction of hydrogen peroxide and hypochlorite, depicted below, has been the reaction of choice for the chemical generation of

H202

+

OCl"

^

+

HCl

+

0H~

In fact, many investigators have compared the effects of this reaction with respect to its impact on chemical or biological systems, as a means of determining whether

had been produced in these systems.

The biological

significance of this reaction was first suggested by Allen et al. (17) the possible bactericidal agent generated from activated (PMN) leukocytes.

as

polymorphonuclear

The supporting evidence was the appearance of chemilumi-

nescence when the PMN were activated.

Indirect support for this hypothesis

was presented by Krinsky (18) who observed that carotenoid-containing bacteria were killed more slowly by human PMN than were a carotenoidless mutant strain of the same organism.

There are now several reports suggesting that

^C>2 is produced either by PMN or by myeloperoxidase,the enzyme that plays a central role in the oxygen-dependent bactericidal mechanism of PMN (19, 20). Other peroxidases, such as lactoperoxidase, have also been proposed as producing

, based on the appearance of chemiluminescense as well as by the 1 Isolation of products characteristic of 0 2 reactions (21, 22).

All of these observations have been received somewhat skeptically.

A ra-

tional basis for this skepticism was presented (23, 24) when two groups of 1

workers observed that many of the chemical reactions attributed to actually be duplicated by the H0C1 myeloperoxidase/l^C^/halide

that was known

system (25).

This

0 2 could

to be produced by the

included

the appearance

of

red chemiluminescence when the myeloperoxidase system was functioning (24).

At about this same time, we were attempting to make a simple light meter for detecting the major emission bands of ^0 2 that occur in the red region spectrum and had been referred

to as

"dimoi" emission

(26).

These

emission

bands, which center at 634 nm and 703 nm, were readily detected in a simple device we constructed (27) but the instrument was not sensitive enough to detect emission from biological systems.

During the course of

evaluating

457 the efficiency of this instrument, we observed (28) that DABCO bicyclo[2.2.2]octane) Figure 2. nism

is

actually

enhanced

the

(1,4-diaza-

634 nm emission, as shown

in

We offered an explanation at the time (28) but the exact mechastill

not

clear.

However,

this

observation has been used by

a

number of workers since then, particularly with respect to the appearance of chemiluminescence from biological systems as will be discussed below.

The Conversion of

to ^0 2 >

The hypothesis that ported

and rejected

can be converted to since

it was

has been simultaneously sup-

first proposed

20 years ago

(29).

The

arguments for and against the hypothesis were reviewed in 1979 (9) and additional evidence has been presented to support the hypothesis via quenching and trapping experiments (30) as well as by direct spectral evidence of chemiluminescence characteristic of (31). In addition, other reports conL 1 tinue to present evidence that O2 is not a major product of the dismutation of

(32).

It would appear that we must bide our time until this particu-

lar issue is resolved.

Peroxy Radical Dismutation Yielding *0 2 · In 1957, Russell proposed that the dismutation of two peroxy radicals, as demonstrated below, would result in one of the products being formed in a singlet configuration (33):

2

R2HCOO·—^[R2HCOO-OOCHR2]

•R2C=O

+

R 2 CHOH

+

o2

The mechanism was amended by Howard and Ingold (34) who concluded that

458

WAVELENGTH (nm) Figure 2. Corrected emission spectra of the HjO./OCl reaction in the absence or presence of either DABCO or NaN^ (28). (Reproduced by permission of Pergamon Press) would be formed in peroxy radical dismutations.

Since then, many investi-

gators have used the Russell Mechanism as explaining would be indicative that

their findings which

1

C>2 had been generated in a biological system in

which lipid peroxides had been formed (35-38).

In many cases, the evidence

for

0„ dealt with the appearance of either a visible or red chemilumines1 cence or product formation which might occur under conditions of Oj oxidation.

The appearance

systems were

of a red chemiluminescence

treated with

hydroperoxides

when various

biological

has been studied extensively

by

Cadenas and associates, initially with Boveris and Chance (39-42) and more recently in collaboration with Sies and associates (43-47). In the experiments in which organic hydroperoxides were added either to subcellular organelles (39), enzymes (40,42), or intact organs (41), chemiluminescence was detected in the red region of the spectrum.

In many

cases,

the chemiluminescence could be either quenched by the addition of beta-caro-

459 tene or enhanced by the addition of DABCO.

Attempts at characterizing the

emission spectrum in the red were relatively primative in this early series of experiments.

In the more recent studies, Cadenas et al. have refined the

measurement of the emission spectrum under various experimental conditions. So, for example, Cadenas and Sies (44) have demonstrated that liver microsomes treated with tert-butyl hydroperoxide results in the appearance of a red chemiluminescence which is inhibited by a free radical trap and enhanced by the addition of DABCO.

Specific emission at 634 nm and 703 nm, with a

minimal emission at 668 nm has been observed (45) in microsomal fractions obtained following the addition of paraquat, a compound known to generate 0j and other oxygen species.

The same type of red chemiluminescence was seen

when microsomes or cytochrome P-450 were treated with oxene donors (46). Finally, Cadenas et al. (47) have presented very compelling evidence that the red emission observed when prostaglandin-endoperoxide synthase acts on its substrate, arachidonic acid, is characteristic of the dimoi emission of ^Oj.

The spectral analysis of their observation is shown in Figure 3.

In all

of the

cases reported

by

Cadenas

et

al.

(39-47), the

Mechanism has been proposed as the reaction generating ^ 2 ·

Russell

It has been

suggested that the 634 nm and 703 nm bands can only arise in the gas phase and that a more precise index of the direct transition of 1268 nm.

1

production would be the measurement of ^

by measuring the monomol emission at

The application of 1268 nm emission analysis is described below.

0 2 Detection at 1268 nm.

The first demonstration that

could be detected at 1268 nm in solution

was made by Krasnovsky (48, 49) using a photosensitized system to generate .

Other workers have also been able to detect this infrared emission at

1268 nm in both dye-photosensitized and chemically generated solutions at

460 S il II h li 11 11

-I

1

600

1—

650 700 Wavelength (nm)

ï*— 750

Figure 3. Spectral analysis of photoemission of arachidonlc acid-stimulated chemiluminescence of prostaglandin-endoperoxide synthase (reproduced by permission of Cadenas .et al· and Walter de Gruyter & Co.)

room temperature (50, 51).

In fact, the technique is becoming sufficiently

common that three consecutive reports recently appeared in which the 1268 nm emission

was

used

to

detect

produced

either

by

decomposition

of

an

ozonide (5 2) or through photosensitized reactions (53, 54).

A most interesting application of this procedure has been reported by Kanofsky

(55).

In his case, lactoperoxidase

resulted in the appearance of infrared the lactoperoxidase-catalyzed

treated

light at 1270 nm.

recently

with HjOj and

Br

Kanofsky compared

emission in the infrared over the range

1070-

1370 nm with the H 2 0 2 /Na0Cl system and both systems gave comparable infrared chemiluminescence.

In addition, ás seen in Figure 4, the 1270 nm chemilumi-

nescence was very markedly enhanced in the presence of 98% D.O, a character-

istic expected for

1 0,. 1

These experiments raised a number of questions ι

eluding one on the yield of tion system.

in-

that can be measured by the infrared detec-

One of the dangers of Improving the technology for

detecting

461

low-level Infrared chemilumlnescence Is that we may reach a point where we will be able to detect levels of light that may not correspond to physiologically significant concentrations of excited species.

However, concern

about the quantitative aspects of this experiment in no way detracts from the fact that the characteristic infrared emission of ^Oj has now been detected from an enzyme-catalyzed reaction (55).

We should now be able to

apply the same technology to the many biological systems that had been proposed as generating ^C^·

We can look forward to those reports which will be

forthcoming indicating that biological systems do or do not have the capacity

to

generate

at

physiologically

significant

levels.

40 c 20 Ot=L IO οί

I E » 5

2 oΧ 0

30 60 Time (s)

Figure 4. Infrared chemilumlnescence at 1270 nm in the lactoperoxidose (0.1 mg/ml)/H 2 0 2 (20 mM)/Br (20 mM NaBr) system. A. 98% D,0. D 2 0. Β. H,0 C. H,0, senand sitivity" increased by 10 (reproduced by permission of Kanofsky Williams and Wilkins).

References

1. Foote, C. S.:

Science 162, 963-970 (1968).

2. Wilson, T., Hastings, J. W.: in "Photophyslology" (A. C. Giese, ed. Academic Press, New York, 5, ΤΨ-95 (1970). 3. Krinsky, N.I.:

Trends Biochem. Sci. 2,, 35-38 (1977).

4. Koppenol, W. H.: Nature (London) _262. 420-421 (1976). 5. Koppenol, W. H.: Photochem. Photobiol. 28, 431-433 (1978).

462 6. Gollnick, Κ., Schenck, G.O.: fri "1,4-Cycloaddition Reactions" (J. Hamer, ed.), Academic Press, New York, pp. 255-344 (1967). 7. Foote, C.S., Wexler, S.:

J. Am. Chem. Soc. 86, 3879-3880 (1964).

8. Foote, C.S.: lji "Pathology of Oxygen" (A. P. Academic Press, New York, pp. 21-44 (1982).

Autor, ed.),

9. Krinsky, N.I.: fri "Singlet Oxygen" (H.H. Wasserman, R.W. Murray, eds.), Academic Press, New York, pp. 597-641 (1979). 10. Poppe, W., Groesweiner, L.I.: (1975). 11. Joshi, P.C., Pathak, M.A: 638-646 (1983).

Photobiol. 22,

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Biochem. Biophys. Res. Commun. 112,

12. Zigler, J.S. Jr., Goosey, J.D.: (1981).

Photochem. Photobiol. 33, 869-874

13. Gommers, F.J., Bakker, J., Wynberg, H.: 615-619, (1982).

Photochem. Photobiol. 35,

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16. Schaap, A. P., Zaklika, Κ. Α., Kaskar, Β., Fung, L. W.-M.: Chem. Soc. 102, 389-392 (1980). 17. Allen, R.C., Stjernholm, R.L., Stele R.H.: Commun. t£_, 679-684 (1972). 18. Krinsky, N.I.: 19. Allen, R.C.:

217-219

J. Am.

Biochem. Biophys. Res.

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20. Rosen, H., Klebanoff, S.J.:

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FEBS

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34. Howard, J.Α., Ingold, K.U.:

J. Am. Chem. Soc. 90, 1056-1058 (1968).

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J.

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Biochem. Pharm. 32,

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Hoppe-Seyler's Z.

48. Krasnovsky, A. A. Jr.:

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49. Krasnovsky, A. A. Jr.:

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50. Khan, A. U., Kasha, M.:

Proc. Natl. Acad. Sci. 7±< 6047-6049 (1979).

51. Kahn, A. U.: Chem. Phys. Lett. 72, 112-114 (1980). 52. Hurst, J. R. , McDonald, J. D., Schuster, G. B.: 2065-2067 (1982). 53. Parker, J.G., Stanbro, W. D.: 54. Ogilby, P. R., Foote, C.S.: 55. Kanofsky, J. R.:

J. Am. Chem. Soc. 104,

J. Am. Chem. Soc. 1Ό4,2067-2069 (1982). J. Am. Chem. Soc. J04, 2069-2070 (1982).

J. Biol. Chem. 258, 5991-5993 (1983).

464

DISCUSSION

KRALJIC: Since azide reduces hypochlorite, I wonder that it does not pro tect in the H 2 O 2 / O C I - system. If y o u have a big excess of H2O2/ 0C1~, azide will not interfere w i t h the reaction, but it w o u l d q u e n c h the I02 that is produced. Higher concentrations of azide w o u l d interfere with the H2O2/OCIreaction so that less I02 is evolved, and y o u w o u l d see less emission. KRINSKY: The azide concentration in the slide that I d e m o n s t r a t e d w a s 6 m M , the initial concentration of hydrogen peroxide w a s 3.5 M and the hypochlorite w a s 0.6 M. W e really have those compounds p r e s e n t at a m u c h larger c o n c e n t r a t i o n than the azide. LANDS: Y o u raised a very important q u e s t i o n about biological significance of the chemiluminescence and the evidence for singlet oxygen. You p o i n ted o u t that biological m a t e r i a l s m a y generate singlet oxygen. The q u e stion is, "Do they d o so in vivo"? One of the things that is very important to keep in m i n d is that the PG synthase m a y be capable of producing all kinds of signals in the laboratory, but if the lipid hydroperoxide content in v i v o is less than I O - ? M, the probability of ever generating those extra species w o u l d be greatly reduced. I think the conditions that were used to generate that chemiluminescence m a y be very nonphysiological. KRINSKY: Thank you for the comment. I just w a n t to make one o b s e r v a t i o n w h i c h I forgot to point o u t earlier. If y o u remember the clock figure, at 11 o'clock, endoperoxides can also decompose to generate singlet o x y g e n . I wonder, since the b e s t way of making endoperoxides ¿ n vivo is through PG synthase, whether in fact we m a y not be observing some reversal in terms of the endoperoxides that are formed, and whether PGG2 or any of the endoperoxides m i g h t not have the capacity to spontaneously generate singlet oxygen. SIES: M a y I just q u i c k l y respond to the remark of Prof. LANDS. I think the K,,, for the appearance of chemiluminescence w a s similar to the e n z y m a t i c activity. It w a s about 10~® M arachidonate added a t half m a x i m a l activity for chemiluminescence. I d o n ' t think there's m u c h of a d i f f e r e n ce in the profile of the enzyme in isolated form a n d in isolated m i c r o s o mes. W e h a v e n ' t done it in intact cells yet. GROSCH: I have a q u e s t i o n w i t h respect to lipid p e r o x i d a t i o n and the type I photosensitizer. We've tested a number of photosensitizers, also riboflavin, w i t h oleic and linoleic a c i d as substrates and have analyzed the d i s t r i b u t i o n of the hydroperoxides and there was only evidence for type II. My q u e s t i o n therefore is, are there type I p h o t o s e n s i t i z e r s of lipid p e r o x i d a t i o n ? KRINSKY: I c a n n o t recall any specific studies o n lipid p e r o x i d a t i o n using type I sensitizers such as riboflavin or ketones followed by p r o d u c t analysis similar to that d e s c r i b e d by THOMAS & P R Y O R (Lipids (1980), 15, 554) to characterize both type I and type II products.

SINGLET OXYGEN PRODUCTION FROM PHOTODYNAMIC SENSITIZERS

C. S. Foote and D. C. Dobrowolski

Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, California 90024

1 2 Interest in photodynamic sensitizers spans many years. ' There is a wide range of naturally occuring photodynamically active compounds from plant sources.

Examples include the

plant toxin hypericin, 3 the fungal pigment cercosporin, 4 ' 5 and a variety of polyacetylene derivatives.® These compounds probably serve as natural biocides, acting to protect the synthesizing organism from animal (mammal or insect) browsing, or, in the case of cercosporin, to promote the attack of the cercospora fungus on the target cell.

Another interesting

compound for study is 4-thiouridine, a nucleoside found in transfer RNA of certain wild-type ÊJ. Cßl± strains.

This

nucleoside appears to be the sensitizer in an oxygen-sensitive near-UV photodynamic single strand breakage of DNA in these organisms. 7 There are two well-known mechanisms by which these compounds

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

466

might operate, the so-called Type I sensitized photooxygenation, in which the sensitizer acts directly on a substrate, producing electron or hydrogen atom transfer, or the Type II sensitized photooxygenation, in which energy is transferred from the sensitizer to oxygen, to give singlet oxygen.®

3

RADICALS SUBSTRATE

1

SENS 3

02

02

We have recently initiated a program of examining certain photosensitizers (including naturally occurring photodynamic toxins) to measure the yield of singlet oxygen produced in the presence of light and oxygen by detecting the 1268 nm emission 9 from singlet oxygen.

In our apparatus, the sensitizer is

excited by a short pulse from a NdsYAG laser.

If the excited

sensitizer transfers energy to oxygen, singlet oxygen is produced.

The weak luminescence from singlet oxygen can be

detected in a time resolved system using a germanium photodiode and associated computerized digitization and g analysis equipment.

The intensity of the luminescence is

compared with the intensity of singlet oxygen luminescence produced from a sensitizer with a known singlet oxygen quantum yield.

In this way, the quantum yield of singlet oxygen

production from a variety of sensitizers can be readily determined. 10 These quantum yields can be checked by measuring the quantum yield of photooxidation of typical singlet oxygen

467

acceptors;

the results of the control experiments agree well

with those of the luminescence experiments in all cases so far studied.

We report at this time on the singlet oxygen

production from cercosporin and from 4-thiouridine.

Cercosporin

The fungal pigment cercosporin causes lipid peroxidation in plant cells in the presence of light and oxygen.

The lipid

peroxidation is believed to cause the breakdown of plant cell 4 membranes, promoting the entry of the fungus.

This pigment

has been suggested to produce singlet oxygen, since photooxidation of cholesterol sensitized by cercosporin produces the typical singlet oxygen product (the 5-a-hydroperoxide)

11

and its effects are inhibited by 12

0-carotene and other singlet oxygen quenchers.

In our

apparatus, the characteristic 1268 nm singlet oxygen emission is readily observed when its solutions in CgDg are irradiated at 532 nm.

The luminescence intensity was the same under air

and oxygen, indicating that the triplet state of the pigment is the precursor of singlet oxygen;

luminescence is quenched

by addition of 2,3-dimethyl-2-butene, confirming its origin in singlet oxygen.

The absolute quantum yield of singlet oxygen

production from cercosporin was determined by comparison with that produced by meso-porphyrin IX dimethyl ester.

After

correction for sensitizer optical densities, the ratio of the intensity of singlet oxygen luminescence sensitized by the

468

porphyrin to that sensitized by cercosporin was 1.01 + 0.04; the ratio of the rates of photooxygenation of 2-methyl-2-pentene in benzene sensitized by the two sensitizers was 1.00 ± 0.12.

Since the quantum yield of

singlet oxygen production from meso-porphyrin IX dimethyl ester is 0.81, 1 3 the yield of singlet oxygen sensitized by cercosporin is also 0 . 8 1 . 1 4

4-Thiouridine

The quantum yield of singlet oxygen formation sensitized by 4-thiouridine was investigated using methods very similar to those employed with cercosporin.

Oxygen saturated solutions

of 4-thiouridine in CHgCN or DjO, when irradiated at 3 55 nm, gave detectable 1268 nm singlet oxygen luminescence;

in both

solvents the emission was quenched by 2,3-dimethyl-2-butene, a common singlet oxygen trap.

The singlet oxygen luminescence

in CHjCN was quantified by comparison with the known quantum yield of singlet oxygen production from 9,10-dicyanoanthracene (DCA)After

correction for differences in sensitizer

optical densities, the ratio of kinglet oxygen luminescence sensitized by 4-thiouridine to that sensitized by DCA was 0.45.

Since the quantum yield of DCA-sensitized singlet

oxygen formation under oxygen is 1.56, 1 ® the 4-thiouridine quantum yield is 0.70.

No difference was observed in the

4-thiouridine luminescence intensity under air relative to oxygen atmosphere, again suggesting that the reactive species

469

leading to singlet oxygen formation is the triplet.

The efficient production of singlet oxygen from these two sensitizers is a necessary but not sufficient condition for the intermediacy of singlet oxygen in the photodynamic damage sensitized by these compounds.

That is, singlet oxygen

production could be only a side reaction accompanying

the

actual photodynamic processes.

effect

The strong protective

of singlet oxygen quenchers against 1o damage

cercosporin-mediated

makes it nearly certain that singlet oxygen is the

active species with this pigment.

However, further work will

be required for the 4-thiouridine case.

The effects of

inhibitors do not seem to be totally explicable on the basis of a pure singlet oxygen mechanism in this c a s e . 1 5 These studies of two model photodynamic sensitizers are offered as examples of the use of a new technique which is now available for the determination of the mechanism of action of photodynamic pigments.

Similar work is under way in our

laboratories on several polyacetylenes, hypericin, and riboflavin.

Acknowledgement. M.

J.

Grateful acknowledgement is made to Prof.

Daub and to Dr.

compounds studied.

Meyrick J.

Peak for providing the

This work was supported by NSF Grant

CHE80-20140 and NIH grant GM-20080.

470

References

(1) Blum, H.: Photodynamic Action and Diseases Caused by Light, Reinhold, Ine, New York (1941). (2) Spikes, J. and J.

D.s

A.

(3) Seely, G. (4) Daub, M.

in The Science of Photomedicine, Regan

Parrish, eds., Plenum press 113-144 R.s

E.:

Photochem.

Photobiol.

Plant Physiol.

(5) Yamazaki, (1972) . S., Ogawa, T.:

2fL, 115 (1977).

£¿, 1361-1364

Agr.

(1982).

Biol.Chem.

(1982). 2£, 1707-1718

(6) Arnason, T., Towers, G. H. Ν., Philogene, B. J. R., Lambert, J. D. H.: Am. Chem. Soc. Symposium Ser. 208. 139-151 (1983). (7) Peak, M.

J., Peak, J.

Photobiol. (8) Foote, C.

G., Nerad, L.:

21, 169-172 (1983). S.:

Free Radicals in Biology 2, 85-133

(9) Ogilby, P. (1983) R., Foote, C. 3423-3430 . (10) Dobrowolski, D. Phys. (11) M.

E.

Photochem.

Chem.

S.s

C., Ogilby, P.

J.

Am.

Chem.

R., Foote, C.

(1976).

Soc. S.s

105., J.

in press (1983) .

Daub, private communication.

(12) Daub, Μ. E.: Phytopathology 22, 370-374 (1982). (13) Bonnett, R., Charambides, Α. Α., Land, E. J., Sinclair, R. S-, Tait, D., Truscott, G.: J. Chem. Soc. Farad. Trans. I If., 852-859 (1980). (14) Dobrowolski, D. press (1983). (15) M.

J.

C., Foote, C.

S.s

Peak, private communication.

Angew.

Chem.

in

471 DISCUSSION BORG: Chris, I'm very impressed that the 1.27 micron emission is really a good way to learn about singlet oxygen. However, I want to refer not to photochemistry but to dark biochemistry and question how much of the experimental work looking for dimoi emission is really informative regarding singlet oxygen. This leads me to ask you if you can tell us something about the efficiency of detecting dimoi emission at the kinds of concentrations of singlet oxygen to be expected in biochemical reactions, and also please discuss the quantum yield of singlet oxygen dimoi emission to be expected in the aqueous or gel phase as opposed to the gas phase. FOOTEs That's a very good question. First of all, the 1.27 micron emission will be more easily quantitated than the dimoi emission. Nobody really knows how to argue back from the intensity of the dimoi emission to what concentration of singlet oxygen could have produced it. It's a second order process and obviously subject to some very surprising complexities as seen by the increase in dimoi luminescence intensity on treatment with DABCO (DENEKE & KRINSKY, Photochem. Photobiol. (1977) 25, 299). The overall quantum efficiency for production of 1.27 micron luminescence in water is of the order of 1 0 - 6 (it's very inefficient because of the fast quenching of singlet oxygen). It's not quite as bad as previously believed, because according to KRASNOVSKY's results (KRASNOVSKY, Chem. Phys. Lett. (1981) _81, 443) , the radiative lifetime of singlet oxygen is also decreased in solvents, so it's not as inefficient as you might have thought. This low efficiency puts some really stringent requirements on sensitivity. The photodiode systems are not nearly as sensitive as the photomultiplier systems, but KANOFSKY (KANOFSKY, J. Biol. Chem. (1983) 258, 5991) has designed a very good system for steady state work. I don't yet know how much singlet oxygen could be detected. The numbers to do this calculation are all available but I have not yet done it. I think there is absolutely no way of quantitating dimoi emission. In the case of DABCO, you get obviously more emission from less singlet oxygen, so there are effects on this system that make it very difficult to know what yield of singlet oxygen will give what yield of light. Both dimoi and 1.27 /u luminescences can be seen in water solution. SINGH: Just two brief comments. One is, that on reaction with singlet oxygen DABCO does produce damaging species, for example, in ribosomes (SINGH & VADASZ, Biochem. Biophys. Res. Commun. (1977), JA' 391). As far as that emission at 632 nm is concerned, I would remind the audience that at Austin we presented absorption spectra of oxygen under high pressure in water, and at 632 nm we do not see any absorption (Oxygen and Oxy-Radicals in Chemistry and Biology, M.A.J. Rodgers and E.L. Powers, Eds., Academic Press, New York, 1981, pp. 736). Now we have to realize that the efficiency of the absorption is smaller than the efficiency with which one can see emission, but one should watch out for complications. SUTTON: What do you think is the likelihood that singlet oxygen is produced in a biological environment and that it could cause an effect there. You talked, for example, about DNA single strand breakage: do you think it could be produced in such a situation, and could do its job before it is quenched?

472 FOOTE: If it were produced near DNA it w o u l d certainly react with g u a n i ne and I'm told that d e p u r i n a t i o n can lead to single-strand breakage. SUTTON: It just seems d i f f i c u l t to me to visualize how it c o u l d be p r o duced there selectively in just the right place. FOOTE: A c t u a l l y its lifetime is not so short in solution. It lives m i c r o seconds; that's enough for a very long diffusion, on the order of m i c r o n s . SRIDHAR: W h e n 4 or 5 o l e f i n s are used for p r o d u c t d i s t r i b u t i o n studies aimed at establishing the involvement of singlet oxygen, will it be useful to test a non-reactive olefin, such as, 1,4-bis-methylenecyclohexane w h i c h is n o t very reactive towards singlet oxygen? FOOTE: Yes, I think it always helps to have a spectrum of things to work with. If radicals are around, y o u w o u l d g e t benzene from 1,4-cyclohexadiene; that compound will react w i t h singlet oxygen, b u t I think it's very, very slow. CADET: Are y o u suggesting that singlet o x y g e n breaks directly or through secondary processes?

may

induce

DNA

strand

FOOTE: I d i d n ' t really w a n t to g e t involved in that question, m a y b e s o m e body else can c o m m e n t on that more effectively than I can. KRINSKY: I w a n t to c o m m e n t that a t the A m e r i c a n Society for Photobiology meeting held at Madison, W i s c o n s i n o n June 21, 1983, Dr. PEAK d e m o n s t r a ted, using rose bengal as a sensitizer, that he was able to d e t e c t single strand breaks w i t h isolated DNA (Photochem. Photobiol. (1983) 2 2 ' ·

GENERATION OF SINGLET OXYGEN FROM EXCITED SINGLET STATES

R. Stephen Davidson, Dean Goodwin and Jul i e E. Pratt Department of Chemistry, The C i t y U n i v e r s i t y , Northampton Square, London EC1V OHB

Summary Solvent isotope e f f e c t s have been used to show that the d i r e c t photooxidation of h i g h l y fluorescent anthracenes involve the production of s i n g l e t oxygen as a r e s u l t of quenching of t h e i r excited s i n g l e t states by oxygen. Attention i s drawn to the fact that misleading r e s u l t s can be obtained i f there i s a solvent isotope effect upon the photophysical properties of the excited substrate. Excimers and charge t r a n s f e r complexes have been shown to s e n s i t i s e the photo-oxidation of both a sulphide ( d i - t - b u t y l sulphide) and an alkene ( c i t r o n e l l o l ) . The observation of solvent isotope e f f e c t s upon these reactions i n d i c a t e s that s i n g l e t oxygen i s involved, to at l e a s t some extent, i n these o x i d a t i o n s . I t i s proposed that the s i n g l e t oxygen i s generated by energy t r a n s f e r from t r i p l e t s produced via decay of the excited s i n g l e t complexes.

1.

THE DIRECT PHOTO-OXIDATION OF SUBSTITUTED ANTHRACENE DERIVATIVES.

The four most important processes f o r oxygen quenching of the fluorescence and phosphorescence of aromatic hydrocarbons a r e : ArH s

+

3

ArH,

+

\

+

3

ArH T

02

0

1

ArH T 'l

+

\

ArHy ^ ArH3-CPs

adducts

since

deamination

of

5,6-dihydrocytosine

derivatives is a fast process in aqueous solutions. It is also interesting to note that 3-CPs or 8-MOP were able to photoreact to a significant extend with 2'-deoxyadenosine

(dAdo)

in the dry

state.

The

negative

plasma desorption mass spectrum of one of the major products of this reaction exhibited a molecular peak (M-H)

at m/z : 508. This is con-

sistent with the formation of a 1:1 dAdo-3-CPs photoadduct. This photoproduct

displayed

fluorescence

electronic

absorption in

the

320-360 nm range

features which are characteristic of a furan type

and

adduct.

Under the same conditions, 2'-deoxyguanosine was completely unreactive towards photoaddition with either 3-CPs or 8-MOP. 2)

Photooxidation reactions As mentionned above, the presence of oxygen in the aqueous solut-

ions of thymidine containing 3-CPs or 8-MOP inhibited partly the formation of dThdOpsoralen

adducts.

This may be interpreted in terms of

efficient quenching of the triplet excited state of the furocoumarins by molecular oxygen

(6).

reasonable yield ( © T

Singlet oxygen was shown to be produced in a ~ 0.2) as a result of the deactivation reaction of

the triplet excited state of 3-CPs ( k 3 _ 3 C P s + 0 2 = 3 χ IO9 M - 1 s - 1 ) . Thymidine

was

slightly

photodegraded

in

the

presence

of

3-CPs

whereas it was resistant to exposure to near UV light in aqueous aerated solution containing 8-MOP. The two main photooxidation products

were

488

characterized

as

thymine

and

N - ( 2 - d e o x y - β-D-erythropentofuranosyl)

formamide, suggesting that OH" radical may be the reactive species of the

photoreaction.

A

reasonable

mechanism

to

be

considered

would

involve the initial formation of superoxide radicals from charge transfer reaction between O2 and presumably 3-CPs" . Dismutation of Og " would generate hydrogen peroxide which subsequently may undergo homolytic dissociation under UV irradiation or by a Fenton type reaction.

Similar

degradation processes were observed in the photoreaction of 3-CPs with 2'-deoxyuridine in aqueous aerated solutions. However strate

2'-deoxyguanosine

appears to be the more reactive

to the 3-CPs mediated photooxidation

2'-deoxyribonucleosides.

of purine

and

sub-

pyrimidine

It should be pointed out that the extend of the

photodegradation of this nucleoside was about six times higher than the yield of the photocycloaddition of 3-CPs to thymidine in the absence of oxygen.

In fact 2'-deoxyguanosine was used at its

derivative in order to facilitate the HPLC and TLC

3',5'-di-0-acetylated separations of the

rather complex mixture of the polar photooxidation produits.

Irradiations

were made on methanol aqueous solutions (1:1) to minimize possible side effects which could result from the formation of reactive oxygen species such as hydroxyl radicals (vide supra).

Aco

Figure 2 : 3-CPs mediated photooxidation of 3',5'-di-0-acetyl-2'-deoxyguanosine

489 The

photosensitizing

properties

exhibited

by

3-CPs

are

comparable to those of several dyes (methylene blue, proflavine, terms of

efficiency and for

the

photooxidative degradation of accounted

for

by

both

II

type

(11,12).

these two photooxidation pathways approaches oxygen 1

:

deuterium

(^Δ0),

C>2 (12).

I

isotopie

mechanism

and

the

may be

singlet

oxygen

Attempts to distinguish

(Fig.

. . . ) in

In particular

3 , ,5'-di-0-acetyl-2'-deoxyguanosine

radical

oxidation process or type

reactions involved.

quite

between

2) were made using different

solvent

effect,

quenching

of

singlet

chemical and microwave electric discharge generation

Two

main photoproducts

which

were

shown

to

be

of

formed

through type II mechanism were characterized as the 3,5-di-0-acetyl-2deoxy- g -D-erythro

pentofuranosyl

4,8-dihydro-4-hydroxy-8-oxoguanine

derivatives (13).

A

of

cyanuric

reasonable

acid

and

mechanism

for

the formation of the cyanuric acid would involve in the first step of the reaction

the

concerted

addition

of

^C^ across

the

giving rise to an unstable dioxetane derivative.

4,5-ethylenic

bond

It is likely to propose

that the second photoproduct would result from the thermally decomposition

of

initially

produced

4,8-endoperoxide

derivative

(1,4-addition

reaction). However the most important degradation pathway nucleoside

appears

mechanism)

with

reaction

with

to

involve

subsequent

oxygen

charge

formation

would

generate

through peroxy intermediates.

The

labilize

the

disruption

of

N-glycosidic

the

bond.

of

a

80 %) of the (type

I

cation.

Further

photooxidation

produts

the a and g anomers of

may be explained in

imidazole In

(

reaction

radical

several

release of

3,5-di-Q-acetyl-2-deoxy-D-erythropentose photo-induced

transfer

ring

addition

which three

is

terms

of

expected

to

other

nucleoside

derivatives which have an ureid type structure have been isolated. It is interesting

to note

photodynamic

effect

that on

8-MOP which did not exhibit S.

cerevisiae

(6)

was

not

any

able

to

significant sensitize

photooxidation of the purine nucleosides. These results show the diversity of the sensitized photoreactions of purine and pyrimidine nucleosides by psoralen derivatives.

490 References 1.

Scott, B . R . , Pathak, M . A . , Mohn, G.R. : Mutation Res. 39, 29-74 (1976).

2.

Averbeck, D . , Moustacchi, E . , Bisagni., E. : Biochim. Biophys. Acta, 518, 464-481 (1978).

3.

Dall'Acqua, F . , Caffieri, S . , Vedaldi, D . , Guitto, Α . , G. : Photochem. Photobiol., 33, 261-264 (1981).

4.

Song, P . S . , Tapley, K.J. : Photochem. Photobiol., 29, 1177-1197 (1979).

5.

Kanne,

D.,

Straub,

Κ.,

Hearst,

J. E . ,

Rapoport,

Rodighiero,

H.

: J.

Am.

Chem. Soc., 104, 6754-6764 (1982). 6.

Ronfard-Haret, J . C . , Averbeck, D . , Bensasson, R . V . , Bisagni, E . , Land, E.J. : Photochem. Photobiol., 35, 479-489 (1982).

7.

Cadet, J . , Berger, M., Voituriez, L. : J. Chromatogr., 238, 488-494 (1982).

8.

Cadet, J . , Voituriez, L . , Gaboriau, F . , V i g n y , P . , Della Negra,S. : Photochem. Photobiol. 37, 363-371 (1983).

9.

Cadet, J. ; Voituriez, L . , Joshi, P . C . , Wang, S . Y . : Abst. Annu. Congr. Photobiol., Vancouver, June 26 - July 1 (1982).

10.

Bensasson, R . V . , Land, E . J . , Salet, C. : Photochem. Photobiol., 27, 273-280 (1978).

11.

Gollnick, K. : Advances in Photochemistry (Edited by Noyes, Vi.Α., Hammond, G . S . , Pitts, J.N. J r . ) , vol. 6, Interscience, New-York, 1968, pp. 1-122.

12.

Cadet, J . , Teoule, R. : Photochem. Photobiol., 28, 661-667 (1978).

13.

Cadet J . , Decarroz, C . , Wang, S . Y . , Midden, W.R. : Abstr. Annu. Congr. Photobiol., Vancouver, June 26 - July 1 (1982).

DISCUSSION

GOLLNICK: If you do the reaction with the psoralen itself as a sensitizer and as a substrate, do you get any [2+2]cyclo-diraerization in the presence of oxygen? CADET: Yes, the photocycloaddition of psoralen derivatives to thymidine has been observed in the presence of molecular oxygen, at least with 3carbethoxy psoralen. We observed as expected a significant decrease in the yield of this photoreaction due to efficient quenching of the triplet excited state of 3-carbethoxy psoralen by On the other hand no photocycloaddition of 8-methoxy psoralen to thymidine has been observed in the presence of molecular oxygen.

RADIOLYSIS AND PHOTOLYSIS OF AQUEOUS AERATED TRYPTOPHAN SOLUTIONS Ajit Singh, Stephen A. Antonsen, Grant W. Koroll, Walter Kremers and Harvant Singh Medical Biophysics Branch, Whlteshell Nuclear Research Establishment, Atomic Energy of Canada Limited Research Company, Pinawa, Manitoba, Canada, ROE ILO

Abstract Two hydroperoxides have been observed as precursors of N-formyl-kynurenlne (FK) and 3a-hydroxyhexahydropyrroloindole (HPI), subsequent to the reaction of singlet oxygen (^2) with tryptophan (trp) at room temperature· These hydroperoxides are not formed on photolysis of tryptophan at 292 nm and four different hydroperoxides are observed on gamma-radiolysis of tryptophan.

FK formation on 292 nm photolysis is attributed to the inter-

mediacy of trp dloxetane· Introduction Various aspects of the photolysis and radiolysis of tryptophan have been investigated over the last three decades (1-8 and references In them). We have reported on the formation of FK and HPI on radiolysis and photolysis of tryptophan and on its reaction with ''Oj (1,2).

We now present data on

the Internedlacy of hydroperoxides and dloxetane in the formation of FK and HPI. Experimental Details of the techniques and the materials used were as previously reported (1,2), except as follows.

For the high-pressure liquid chromato-

graphic (HPLC) analyses, the detector was Schoeffel SF 770; the two columns used in series at 24°C were Partiell M-9, 10/25 ODS (Whatman) and Lichosorb RP 18, 7 μ (Unimetrics/Knauer); the aqueous eluent (using triple distilled water) contained 4 g/L K^PO^ (Fisher, Certified) and 0.4% each

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlín • New York - Printed in Germany

492 of acetonitrile and tetrahydrofuran (spectroquality) at pH _ 3.5; and the flow rate was 0.8 niL/min. and thiourea

The concentrations of FeSO, (Fisher, C e r t i f i e d ) -4 -3 -3 Analyzed) were 1 χ 10 and 1 χ 10 mol.dm , -3 -3

(Baker,

respectively, and that of trp was 1 χ 10 with

the present HPLC analysis

previously ( 1 , 2 ) , observed.

292 + 5 nm p h o t o l y s i s was 1.4 χ 10 aqueous

.

The product p r o f i l e than that

obtained

but the separations were better and more products were

The dose rate for gamma-radiolysis -4

Detector Technology Power Meter, oxygen,

mol.dm

system was d i f f e r e n t

solutions

of

J/s,

was 30 Gy/min and that for as measured with a United

Model 21A.

trp,

under

For

reaction with

13 MPa oxygen

singlet

pressure,

were

irradiated for 2 h with f i l t e r e d light ( λ >450 nm) from a 1600 W Xe-arc lamp. Results ( i ) Singlet oxygen Peroxy compounds were among the products 0j ( 1 ) ,

formed on reaction of

as shown by the liberation of I j

KI; no I2 was liberated if _ 20 min. addition

the irradiated solution was heated at 60°C f o r

The products seen on HPLC analysis to FK, HP1 and kynurenine

products.

On heating

trp with

from an a c i d i f i e d solution of

(K),

the irradiated

there

solution

are shown in Fig. l a . are

several

In

unidentified

at 60°C f o r 10 min,

the

peaks HP-1 and HP-2 decreased and both FK and HPI increased,

suggesting

that HP-1 and HP-2 might be the hydroperoxides

of FK and

HPI. trp,

Formation of

at low temperature

heating

and precursors

a hydroperoxide on dye-sensitized ( - 7 0 ° to 0°C),

the hydroperoxide

have

been

photo-oxidation

and formation of

reported

(9,10).

of

FK and HPI on On addition

of

FeSO^, which converts hydroperoxides into alcohols (11,12), the peaks HP-1 and HP-2 disappeared with

Increases

in FK and HPI

increases

in FK and HPI were

seen

irradiated

solution

in this

(Fig.

cannot be observed due to

lc);

on a d d i t i o n case,

the overlapping

known to convert hydroperoxides

(Fig. of

changes

thiourea

lb).

to

the

in HP-1 and HP-2

peak.

Sulfides

into the corresponding alcohols

addition of FeSO^, as well as thiourea,

Similar

thiourea

another product increased

Figs, l b and l c , respectively), which has not been i d e n t i f i e d .

are

( 1 3 ) . On (FT-1,

493

HF HF>1 FT-1 1

1P-2 F K< Κ I

5-HT 1

1

1

1

1

Trp

50x α

2χ 50x X

b

2x 2x

Ν

50x

Ν \

c

5 0 x

D P DP c o Q. O U) χι
5) c «I e

I

e

1 50x

f

. 1 50x

•

ι

. . ι

.

,

g

1

h p4

HP

50x

Ιι , 1

,

1

h

R -1 50x

2x

\\ \\ , 1

20

.

i

1 50x

. ι 1 Elution Time (min)

j ^

'00

FIGURE 1. Products seen by HFLC analysis of irradiated aqueous trp -3 -3 (1 χ 10 mol dm ) solutions. Post-irradiation additives were FeSO, -4 -3 -3 -3 (F, 1 χ 10 mol dm ) and thiourea (T, 1 χ 10 mol dm ), as shown: (a)^;

(b) X 0 2 + F;

292 nm, HjO solution; lysis;

(c) X 0 2 + T; (f) 292 nm + F;

(i) 7"-radiolysis + F;

(d) 292 nm, D 2 0 solution; (g) 292 nm + T;

( j) 7"-radiolysis + T.

(e)

(h) ^-radio-

494 (ii) Photolysis at 292 nm The products formed on direct photolysis of trp include FK, HPI and K. The products and their yields were the same in D2O and H2O (Fig. Id and le, respectively).

However, the hydroperoxides HP-1 and HP-2 were not

among the products, and the FK/HP1 ratio was higher (_ 3) than in the case of reaction with ^

(_ 0.7) (Fig. la).

On heating the irradiated solu-

tion, the increases in FK and HPI were somewhat larger than in the case with 1 0 2 ·

On addition of FeSO^ or thiourea to the irradiated solution,

two products increased (DP-1, DP-2) and two new ones appeared (DP-3, DP-4) (Fig. If and lg, respectively).

None of the original products disappeared

and there was no significant change in the yields of FK and HPI.

Thiourea

converts dioxetanes to diols (13), suggesting that DP-3 or DP-4 may be a diol; FeSO^ may be acting similarly.

The yields of FK and HPI were linear

with dose, for up to 10 min of exposure (see Fig. 2).

Thus, our results

are not complicated by secondary photolysis of FK or HPI. The decomposition of trp at the highest dose used in Fig. 2 was < 2%.

(iii) Gamma-Radlolysls The results of gamma-radiolysis are shown in Fig. Ih, li and lj. products include FK, HPI, Κ and 5-hydroxytryptophan (5-HT). FK/HPI was _ 4.

The

The ratio of

The FK peak was separated by HPLC, and its absorption

spectrum compared to the separated FK peaks in the other two cases ( 0^ reaction and direct photolysis) and to a commercial sample of FK. four absorption spectra agreed with each other.

All

The pattern of products

obtained on radiolysis was different than that obtained with ^Oj or direct photolysis, consistent with the previously published work (1,2). hydroperoxides HP-1 and HP-2 were absent in this case. solutions showed a positive

The

The irradiated

test, which could at least partly be due to

the small amount of H^Oj formed radiolytically

(14).

On heating the

irradiated solution to 60°C for 10 min, the FK, HPI, Κ and 5-HT peaks increased, while some of the other peaks decreased.

On addition of FeSO^

(Fig. li) the peaks HP-3, HP-4, HP-5 and HP-6 decreased, while FK, HPI, 5-HT and Κ increased and a large new peak, R-l, appeared. viour was seen on addition of thiourea (Fig. lj).

Similar beha-

The 5-HT peak seems to

be unchanged in this case, but since the region of the peaks HP-3, HP-4, HP-5 and R-l overlaps the thiourea peak, their fate is unknown.

495

5.0

2.5

7.5

10.0

T i m e (min) FIGURE 2 . exposure.

Variation

in

the

yields

of

FK and H P I ,

with

292 nm

Discussion

The peaks HP-1 and HP-2 ( F i g . peroxides

1) are a t t r i b u t a b l e

C3-hydroperoxyindolenine

(A)

pyrroloindole

( B ) ] shown in Scheme I .

formation

FK and HPI,

of

consistent

However, invoking the intermedlacy

of

and

to

These hydroperoxides with

other

the dioxetane

the formation of FK from the hydroperoxides

is

not

formed,

intermediate in this case.

suggesting

since

any i n d i c a t i o n

292 nm p h o t o l y s i s ,

that

the

(5,9). the

On the other hand, in 292 nm photolysis of tryptophan, HP-1 and HP-2 are not

to

justified,

of

discussion

work

for

the presence

(see

hydro-

(C i n Scheme I )

do not g i v e

dioxetane

two lead

published

products obtained with FeSO^ and thiourea of

the

3a-hydroperoxyhexahydro-

of

below).

the hydroperoxides

is

not

an

This i s consistent with Borkman's

where the rate of trp photolysis was found to be s i m i l a r

in

Important

result(15), and D^O.

Our r e s u l t s f o r D2O s o l u t i o n show that the products and t h e i r y i e l d s

are

very similar to those found with H_0 ( F i g . Id and l e ) . Thus, we conclude L 1 that O2 i s not Important in the photo-oxidation of trp at 292 nm ( 2 , 1 5 17).

Sun and Zigman ( 1 8 ) suggested that, in the u l t r a v i o l e t

photo-oxida-

496

Trp

hi/ , Trp*

^ Trp*

2 9 2 nm

Η Η (Β)

SCHEME I tion of trp, ^(>2 was important in their

system.

However, their

results

were perhaps complicated by direct light absorption by the product FK and the resulting FK-sensitized photolysis of trp ( 2 ) . The most obvious peroxy intermediate in 292 nm photolysis is the dioxetane (C in Scheme I ) .

I t s intermediacy is supported by the large y i e l d of FK,

which can be formed by the widely accepted mode of dlcarbonyl formation from dioxetanes (13). the

dioxetane

Formation of HPI suggests that a small portion of

rearranges

mediate (13,19—21).

to

it,

perhaps

through

a zwitterionic

Dioxetane formation in u l t r a v i o l e t

also been suggested by Slawinski et

al.

(16).

Since

inter-

photolysis has

trp

fluorescence

quenching by O2 is n e g l i g i b l e in aqueous solutions (17,22), the dioxetane i s most l i k e l y formed by direct reaction of t r i p l e t excited trp with 0 . In radlolysis,

trp decomposition

is

initiated

by

the hydroxyl

radical

("OH) ( 1 ) , the main reaction being addition to one of the two rings (23, 24), e . g . , D and E in Scheme I I .

The lack of HP-1 or HP-2 (A,Β in Scheme

I ) in this case suggests that hydrogen atom abstraction is not important. Since the

*0H can add to four positions in the benzene ring and two in the

hetero-ring, a large number of hydroperoxides can result.

The four peaks

that decrease on adding FeSO^ are attributable

hydroperoxides.

to these

However, further work is needed to i d e n t i f y these hydroperoxides and to relate them to the various products.

The unknown products also need to be

497

To

•O H /

, Trp \·ΟΗ

CH,-CH-COOH ί I NH,

(XX Η

Oj.RH

CH2-CH-COOH nh, 2

OH

O 2I RH 5+IT

OOH HO/^ η

i-CHg-CH'COOH 'ι NH,

Η (D)

OOH II

1 N^OH Η

^H,

I

(E)

t

DIOLS

FK,HPI SCHEME Π

identified.

Work 011 these is in progress.

However,

"OH addition to the

benzene ring is required for the formation of 5-HT, one of the products.

observed

Also, E in Scheme II is the same as proposed by Hamilton (25)

and Aldrich and Cundall (26), as a precursor of FK.

ACKNOWLEDGEMENTS We thank Dr. Creed for making a copy of the draft of his review on tryptophan available to us.

REFERENCES 1.

Singh, Α., Bell, M.-J., Koroll, G.W., Kremers, W., Singh, H. : Oxygen and Oxy-Radicals in Chemistry and Biology, Edited by M.A.J. Rodgers, and E.L. Powers, Academic Press, New York (1981), ρ 461-470.

2.

Singh, Α., Singh, H. Kremers, W., Koroll, G.W.: Bull. Europ. Physiopath. Resp., Γ7 (suppl.), 31-41 (1981).

3.

Nakagawa, Μ., Kato, S., Nakano, Κ., Hino, T.: J. Chem. Soc., Commun., 855-6, (1981).

4.

Grossweiner, L.I., Brendzel, A.M., Blum, Α.: Chem. Phys., 147-155 (1981) Inoue, K., Matsuura, T., Saito, I.: Photochem. Photobiol. 35^, 133-139 (1982).

5.

Chem.

498 6.

Mialocq, J.C., Amouyal, E., Bernas, Α . , Grand, D.s J. Phys. Chem., 86, 3173-3177 (1982).

7.

Lion, Y . , Kuwabara, M·, Rlesz, P . : Photochem. Photoblol., (1982).

8.

Bazin, M., Patterson, L.K., (1983).

9.

Nakagawa, M., Kato, S . , Kataoka, S . , Rodato, S . , Hatanabe, H . , Okajima, H., Hino, T . , Witkop, B.: Chem. Pharm. B u l l . , 29^ 1013-1026 (1981).

35^, 43-52

Santus, R. : J. Phys. Chem., 87^ 189-190

10. Nakagawa, M., Watanabe, H . , Kodato, S . , Okajima, H . , Hino, Flippen, J . L . , Witkop, B.: Proc. Natl. Acad. S c i . , Hash., 4730-4733 (1977). 11. Salto, I . , Matsuura, 10, 346-352 (1977).

T.,

Nakagawa, M., Hino,

T.:

Acc.

T., 74,

Chem. Res.,

12. Sosnovsky, G., Rawlinson, D.J.: in Organic Peroxides, vol I I Edited by D. Swern, Wiley-Interscience, Ne)w York, 1971, p.153-268. 13. Wasserman, H.H., Lipshutz, B.H.: in Singlet Oxygen, Edited by H.H. Wasserman and R.W. Murray, Academic Press, New York, 1979, p. 429-509. 14. Draganic, I . G . , Draganic, Z.D.: Academic Press, New York (1971).

The Radiation

15. Borkman, R.F.: Photochem. Photobiol.,

Chemistry

of

Water,

163-166 (1977).

16. Slawinski, J . , Elbanowski, M., Slawinska, D.: Photochem. Photobiol., 32, 253-260 (1980). 17. Creed, D. : The Photochemistry of Some Possible Cellular Targets of Near-Ultraviolet Radiation. I . Tryptophan, Photochem. Photobiol., (1983, being submitted). 18. Sun, M., Zigman, S.: Photochem. Photobiol., 29_, 893-897 (1979). 19. S a i t o , I . , Imuta, M., Takahashi, Y . , Matsugo, S . , Matsuura, J. Am. Chem. Soc., 9£, 2005-2006 (1977). 20. J e f f o r d , (1978).

C.W., Rimbault,

21. Ryang, H.-S., Foote, C.S.:

C.G.:

J. Am. Chem. Soc.,

100,

T.:

6437-6445

J. Am. Chem. Soc., 101, 6683-6687 (1979).

22. Santus, R., Bazin, M., Aubailly, M.:

Rev. Chem. Interned., 3^» 231-283

(1980). 23. F a r h a t a z i z , Ross, A . B . : S e l e c t e d S p e c i f i c Rates of Reactions of Transients from Water in Aqueous Solution. I l l , Hydroxyl Radical and Perhydroxyl Radical and Their Radical Ions, NSRDS-NBS59 (1977). 24. Armstrong, R.C., Swallow, A.J.: Rad. Res. 40, 563-579 (1969). 25. Hamilton, G.A.: Adv. Enzymol., 32^, 55-96 (1969). 26. Aldrich, (1969).

J.E.,

Cundall,

R.B.:

Int.

J.

Radiat.

Biol.,

343-358

499 DISCUSSION

LUNEC: How do you anticipate the production of kynurenines from the tryptophan in proteins by this mechanism? SINGH: First there is the question whether it is formed directly or from formylkynurenine. We have some evidence, about which we are not 100% certain, that probably it is a direct product. Looking at the formula that is difficult to see and we have still not resolved it. In the case of proteins where you have the tryptophan moiety, you would still initiate the reaction by hydroperoxide formation on the heterocyclic ring which would then go through a sequence of reactions to give you formylkynurenine and kynurenine. For metabolic formation of kynurenine perhaps I should refer you to papers by SAKIYAMA and by BRCWN in Biochemical and Medical Aspects of Tryptophan Metabolism (Eds: O. Hayaishi, Y. Ishimura and R. Kido, Elsevier, North Holland, (1980) pp 73 and 227) . SCHOLES: In 1954, we showed for the first time the production of formylkynurenine from tryptophan (JAYSON and SCHOLES, Biochem. J. (1954) 57, 386). At that time we did in fact suggest that it was due to OH addition to the indole ring via a peroxy radical, giving what we thought would be an unstable hydroperoxide. I think we tested for hydroperoxide but didn't find it. Are you now reporting that there is a semi-stable hydroperoxide in this system? SINGH: There is a hydroperoxide which is really quite stable at room temperature and stays around for hours in the dark. If you heat the solution or expose it to light, then it decomposes faster. Your work has been cited in our papers on this system. KRALJIC: I would like to know if formylkynurenine is among the primary products in sensitized oxidation. In some experiments, where we had only singlet oxygen and very low tryptophan concentrations, we tried to look for the emission by formylkynurenine but we didn't observe any. You probably treat the solution before you isolate the product, so I would think that maybe for a short irradiation time, formylkynurenine is not produced right after the radiation at room temperature. SINGH: It is a primary product, we analyse our solutions right after irradiation. Please note that the singlet oxygen we produce is not through photosensitization, it is directly produced by light absorption by oxygen in high pressured cells (Photochem. Photobiol. (1978) 28, 607, 611). May I also say that I do not know of a single photosensitized system in which you only produce singlet oxygen. KRALJIC: I would say with some sensitizers at low substrate concentration, you don't have a free radical mechanism, that's what I wish to say. SINGH:

We can discuss that later.

BHUYAN: Dr. SINGH, could the tryptophan radical itself be damaging in a biological system? I asked several people working on tryptophan and they said it doesn't, because of it's short half life. Could you please explain

500 that more. I don't understand why it cannot damage tissue directly? SINGH: You can form many types of tryptophyl radicals as I showed. The radical lifetime is quite long, several microseconds. We have done pulse radiolysis, where you can produce different types of radicals (Radiat. Phys. Chem. (1981) 137). However, products of tryptophan are also toxic, e.g. see STEINHART and KIRCHGESSNER ("Biochemical and Medical Aspects of Tryptophan Metabolism") and papers of ZICMAN et al. in refs. 1 and 2 of our paper. KRINSKY: May I just ask you to comment on the observations and suggestions of ZIGLER and GOOSEY (Photochem. Photobiol. (1981) ¿3, 896), that in fact the products of tryptophan photolysis might themselves act as sensitizers and might lead to further damage? SINGH: Yes, that is quite correct and if you go back in the literature to direct photolysis studies, that is the problem. (For example, see refs. 1 and 2 of our paper).

PHOTODYNAMIC AND REDUCTIVE MECHANISMS OF OXYGEN ACTIVATION BY THE FUNGAL PHYTOTOXINS, CERCOSPORIN AND DOTHI STROMIΝ

Richard J. Youngman and Erich F. Elstner Institut für Botanik und Mikrobiologie, Technische Universität München, Arcisstrasse 21, 8000 München 2, W. Germany.

Introduction

The fungi Cercospora beticola and Doth i stroma pini are responsible for causing leaf spot disease in sugar beet and Dothistroma blight in pine, respectively (1,2).

Both fungi produce red coloured pigments cercosporin

and dothistromin and toxicity is manifested by a breakdown of photosynthetic pigments and lipid peroxidation (1-3).

The requirement for light and 0 2

in

the phytotoxic response suggests that photodynamic mechanisms of 0 2 activation may be responsible for the observed damage (2,3). 1

0 2 , superoxide

Singlet 0 2

and H 2 0 2 have all been proposed to be involved in the

mechanism of pigment breakdown (4,5). The production of

1

0 2 mainly occurs

via photosensitisor-dependent reactions involving energy transfer, whilst 0 2 ~ and H 2 0 2 are products of reductive oxygen activation, dependent upon electron transfer (6,7). Thus it was of interest to determine the oxygen activating abilities of cercosporin and dothistromin and to compare the relative importance of the reductive and photosensitising mechanisms.

Materials and Methods

Cercosporin was a kind gift from Dr. G. Nasini and dothistromin was generously donated by Dr. R.A. Franich. Both compounds were stored as ethanol solutions at 4"C in the dark. The water-soluble carotenoid, crocin was isolated from saffron as previously described (8). NADPH-cytochrome c(ferredoxin)-oxidoreductase was prepared from Euglena gracilis (9) with the modifications given in (10). Crocin bleaching was followed at 440 nm under

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

502 the conditions described recently (11). Oxygen exchange was monitored with a Hansatech 0 2 electrode. 0¡~ was determined by its ability to oxidise hydroxylamine to nitrite (12,13). Crypto-OH" radical was detected by methionine fragmentation under the conditions described previously (13,14).

Results and Discussion The bleaching of the carotenoid crocin can be used as an indicator for the presence of photochemically generated

(11). Table I shows the pattern

of crocin destruction in the presence of cercosporin or dothistromin. Table I. Bleaching of Crocin by Cercosporin and Dothistromin iÜBleaching Fungal Toxin

7.5 uM Cercosporin 37.6 uM Cercosporin 75 pM Cercosporin 50 pM Dothistromin 100 pM Dothistromin

10 min

Illumination Time 20 min

30 min

5 9 18

14 23 30

16 32 40

0 0

0 0

0 0

Reaction mixture contained in 2 ml ; 200 Mmol phosphate buffer pH 7.8 and the indicated toxin concentrations. Crocin concentration was adjusted to give an initial absorbance of 1.0 at 440 nm prior to illumination.

Cercosporin can clearly act as a photosensitisor leading to crocin breakdown in a reaction which was dependent upon the cercosporin concentration and the duration of illumination. In contrast, dothistromin did not result in an extinction decrease at 440 nm indicating that this Phytotoxin does not participate in a Type II photodynamic reaction leading to the formation of 1

0 2 . Even in the presence of tetramethylene diamine which can act as an

electron donor for Type I photodynamic reactions (15), no crocin bleaching was observed. It was thus concluded that dothistromin does not possess any significant photosensitising properties. The generation of

by

503 cercosporin in a light-dependent reaction via a Type II mechanism has recently been confirmed in studies on the photooxidation of unsaturated fatty acids (11). Dothistromin possesses a quinone moiety whits cercosporin is a dihydroxyperylene quinone. Thus, it might be expected that both compounds could undergo reduction followed by autoxidation which would lead to the formatbn of active oxygen species. It was found that both cercosporin and dothistromin could be reduced in a reaction catalysed by NADPH-cytochrome c(ferredoxin)-oxidoreductase.

Oxygen electrode studies in the presence and

absence of catalase showed that the subsequent autoxidation product of both substances was H 2 0 2 (data not shown).

Both dothistromin and cercosporin

also led to the production of Oj" as shown in Table II, although neither toxin was as effective as anthraquinone-2-sulphonate.

Table II. 0'2~ and OH' Radical-Type Formation by Cercosporin and Dothistronin

Additions

None Anthraquinone-2-S Cercosporin (100 uM) + 100 U SOD + 100 U Catalase Dothistromin (100 μΜ) + 100 U SOD a

0;" Formation

1.5 97 35 3 n.d. 30 2

a

0H"-like Production'

0.5 440 62 80 18 0 n.d.

measured as N0¡ formation from hydroxylamine (nmol/30 min),

k measured as ethylene, production from methionine (pmol/45 min). Reaction mixture contained in 2 ml: 10 umol glucose-6-phosphate; 50 ug glucose-6-phosphate dehydrogenase; 1 umol NADP; NADPH-cytochrome c(ferredoxin)-oxidoreductase containing 0.1 mg protein. For 0¡ detection, 1 umol hydroxylamine, for OH'-like radical measurement, 20 μπιοί methionine and 0.2 umol pyridoxal phosphate were added, n.d. = not determined. The fragmentation of methionine forming ethylene has been used to detect OH" radicals and the crypto-OH' radical species (13,14,16). Under normal

504 aerobic conditions, only cercosporin was able to mediate ethylene production (Table II). This reaction was markedly inhibited by catalase but scarcely affected ( or slightly stimulated) by SOD. Clearly, H 2 0 2

is a prerequisite

for the terminal oxidant, which may be formed by a reaction of the semiquinone and H 2 0 2 , via a mechanism similar to those proposed for paraquat (14), nitrofurantoin (13) and adriamycin (-17). The slight stimulation observed in the presence of SOD might indicate that 0 2 " can react with the semiquinone, reducing it further to the hydroquinone. Thus, the inclusion of SOD would tend to conserve the semiquinone while concomitantly ensuring that all the 0 2 " produced was dismutated to H 2 0 2 .

In contrast to cercosporin, dothistromin did not give rise to a species capable of forming ethylene from methionine under normal aerobic conditions (Table II). However, when the flask atmosphere was rendered partially anaerobic (14), ethylene was produced in a catalase-inhibitable (data not shown). Thus, it would appear that under normal

reaction

aerobiosis,

autoxidation was favoured and that the reaction of dothistromin

semiquinone

with H 2 0 2 was insignificant. Upon lowering the partial pressure of oxygen, the autoxidation reaction became limiting, thus increasing the possibility of a reaction between the semiquinone and H 2 0 2 .

Conclusions

The fungal Phytotoxins, cercosporin and dothistromin

are red coloured

pigments which require light and oxygen in order to exert their activities. However, the mechanisms of oxygen activation which could account for their in vivo toxicity have been found to be significantly different, although reactions common to both toxins were observed.

The two compounds were

readily reduced in a NADPH-dependent system and resulted in the formation of 0 Γ

and H 2 0 2 upon autoxidation. The generation of a more toxic species

possibly the crypto-OH" radical was catalysed by both compounds, although in the case of dothistromin, the reaction only occurred under oxygen limiting conditions.

Only cercosporin was able to act as a photosensitisor

and exerted its effect via Type I and Type II reaction mechanisms, according to whether an electron donor was present.

505 In vivo, it seems likely that both Phytotoxins could be reductively activated, possibly via photosynthetic electron transport which could explain the light requirement for toxicity.

The actual species responsible

for damage is probably the OH" radical or a species with similar reactivity, formed via a metal-catalysed Haber-Weiss reaction or from the interaction of cercosporin semiquinone with H J 0 2 . In addition to reductive oxygen activation, cercosporin probably acts a photosensitisor in vivo, producing 1

02

in addition to Oj" and H 2 0 2 .

Acknowledgements

The authors are grateful to Dr. G. Nasini, Milan, Italy and Dr. R.A.Franich Rotorua, New Zealand for gifts of cercosporin and dothistromin respectively. This work was supported by the Deutsche

Forschungsgemeinschaft.

References

1.

Schlösser E.: Phytopathol.Ζ. 44, 295-312

2.

Shain L., Franich R.A.: Physiol.Plant Pathol.

(1962).

3.

Macri F., Vianello Α.: Plant, Cell Environm, 2, 267-271

4.

Youngman R.J., Dodge A.D.: Z.Naturforsch. 34c, 1032-1035 (1979).

49-55 (1981). (1979).

5.

Elstner E.F., Pils I.: Z.Naturforsch. 34c, 1040-1043 (1979).

6.

Gollnick K.: Adv.Photochem. 6, 1-122 (1968).

7.

Foote C.S.: in "Free Radicals in Biology" ed. W.A.Pryor. Vol. II, Academic Press, London, New York, pp 85-133 (1976).

8.

Friend J., Mayer A.M.: Biochim.Biophys.Acta 41^, 422-429 (1960).

9.

Lengfelder E., Elstner E.F.: Ζ.Naturforsch. 34c, 374-380 (1979).

10. Youngman R.J., Osswald W., Elstner E.F.: in "Oxy Radicals and Their Scavenging Systems" Vol. II. Cellular and Molecular Aspects, eds. R.A.Greenwald, G.Cohen, Elsevier Biomedical, New YOrk, Amsterdam pp 212-217 (1983). 11. Youngman R.J., Schieberle P., Schnabl H., Grosch W., Elstner E.F.: Photobiochem.Photobiophys.

in press

(1983).

12. Elstner E.F., Heupel Α.: Anal.Biochem. 70, 616-620

(1976).

506 13.

Youngman R.J., Osswald W., Elstner E.F.: Biochem.Pharmacol. 31_, 3723-3729 (1982).

14.

Youngman R.J., Elstner E.F.: FEBS Lett. 129, 265-268 (1981).

15.

Youngman R.J., Elstner E.F.: Ber.Deutsch.Bot.Ges. in press (1983).

16.

Saran M., Bors W., Michel C., Elstner E.F.: Int.J.Radiat.Biol. 37, 521-527 (1980).

17.

Elstner E.F., Paur E., Youngman R.J.: in "Oxy Radicals and Their Scavenger Systems" Vol.1 Molecular Aspects, eds. G.Cohen, R.A.Greenvald Elsevier Biomedical, New York, Amsterdam, pp 308-311

(1983).

DISCUSSION BIELSKI: I would like to make a comment on the use of hydroxylamine as a diagnostic chemical for OJ. We have tested the reactivity of HO2/ C>2 with hydroxylamine over the entire pH range and found no reaction of consequence. Hence at present we are inclined to agree with BORS et al. (Biochem. Biophys. Res. Comm. (1977) 75, 973-979) who suggested that the initial attack on hydroxylamine is carried out by an OH radical and not OJ. YOUNtMAN: I'm aware of your work and also of BORS' paper, but in all of our reactions, we do the control with and without superoxide dismutase, so we are measuring the difference of a superoxide dismutase-inhibitable reaction. BIELSKI : It is quite possible that in your system you are observing a metal-catalyzed reaction involving hydrogen peroxide in addition to hydroxylamine. BORS: Metal catalysis of hydroxylamine autoxidation was also suggested by KONO (Arch. Biochem. Biophys. (1978) 186 189-195) and 05 was proposed only as a chain carrier, in our experiments several years ago (see ref. above) we did not check for metals involved, since we assumed the pulse-radiolytic system to be pure. However, we found that 05 alone does not produce nitrite from hydroxylamine. However, if you have a mixture of OH radicals and 05, they form the maximal possible yield of nitrite. We have not pursued this matter further. GOLLNICK: Cercosporin is an alpha-hydroxyl-quinone and a singlet oxygen sensitizer. Is dothistromin, which does not act as a ^-Oj sensitizer, also an alpha-hydroxyl-quinone?

507 YOUNOtAN: Cercosporin is a di-hydroxyperylene-quinone derivative. Dothistromin is a five-ring linear system with the quinone in the second ring (see structure)

OCH3 CH 2 CH0HCH 3 H 2 CHOHCH 3 OH

0

OCH,

OH

Cercosporin

0

OH

0

Dothistrorain

GOLLNICK: But there are no alpha-hydroxyls. This is interesting, because I think that with hydroxyl-quinones one may predict which are singlet oxygen sensitizers and which are not. Twenty-five years ago we showed (SCHENK and GOLLNICK, J. Chim. Phys. (1958) 55, 892-900) that alphahydroxyl-quinones produce singlet oxygen (which was called moloxide at that time). But if the hydroxyl is in another position, beta for example, the quinone may become a type I photooxygenation sensitizer. There seems to be a correlation between the structure of a photosensitizer and its capability to produce singlet oxygen. PRYOR: I was intrigued by the simple test device, advertised to detect hydroxyl radicals or singlet oxygen. How do you know that hydroxyl is the only species that can oxidize methionine to ethylene? Any one-electron oxi dant - you have other one electron oxidants there - can do that as well. YOUNCMAN: There is a paper by SARAN et al. (Int. J. Radiat. Biol. (1980) 37, 521-527), where they compared various oxygen radicals produced in a radiation system for their ability to react with methionine to produce ethylene. It was quite clear that OH radicals were the only active species in this system. You can't do it with superoxide, and you can't do it with, for example, a compound I-peroxidase-hydrogen peroxide complex. PRYOR: You can't do radical, but you can showed for instance one-electron oxidants YOUNOIAN:

it with singlet oxygen, which acts like a reducing do it with other one-electron oxidizing species. We that alkoxy radicals can do it, but you have other in your system.

We don't have alkoxy radicals in there.

PRYOR: No, I appreciate that, but you have other one-electron oxidants, don't you or do you? Maybe you don't. YOUNGMAN: ELSTNER:

It's a very simple system, I don't think we do. We are looking at the products and if you are looking for the

508 sulfoxide, you can get the sulfoxide of methionine by different oxidants. But you only get ethylene using OH radicals. That's the difference. We are not looking for oxidation of methionine, we are looking for the production of ethylene. That's a different mechanism. PRYOR:

Yes, I appreciate this.

YOUNCMAN: I think the other point to be made is that there is a big difference between ethylene formation from methionine, from methional or from ketomercaptobutyric acid. With methional and KMB you get ethylene relatively easily; you can do that, for example, with the compound I mixture. Methionine is far harder to fragment, and requires pyridoxal phosphate in our system.

DISSIPATION OF EXCESS LIGHT ENERGY THROUGH H 2 0 2 FORMATION IN PHOTOSYNTHETIC

SYSTEMS,

K. Colbow, W. Vidaver, R. Popovic and D. Bruce, Department of Physics and Bio-Sciences, Simon Fraser University, Burnaby, B.C.

V5A 1S6

Introduction In direct sunlight land plants can in general utilize a large fraction of the available light energy than marine algae. example photosynthesis

Fo

(C0 7 uptake) saturates in some crop

plants at intensities above 400 watts m -2 saturation occurs around 15 watts m

.

- 2

, while in kelps

Given a similar

efficiency for light absorption by the pigment systems of the two types of plants, the largely unanswered question then arises what are the differences in light energy tions.

transforma-

The overall system of photosynthesis is believed to b

similar in all plants

(Fig.l).

NADPH

2H + +50 2

H20

Fig.l Overall system of photosynthesis showing 1 likely site where 0^ removes electrons from the charge carriers in the thylakoid membrane.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

510 Variable Fluorescence

in v i v o

W e h a v e e x a m i n e d d e t a i l s o f the s y s t e m u t i l i z i n g c h l o r o p h y l l - a f l u o r e s c e n c e a n d o x y g e n e x c h a n g e in A t room temperature

variable chloroplasts.

the f l u o r e s c e n c e a r i s e s p r i m a r i l y

a n t e n n a e c h l o r o p h y l l - a a s s o c i a t e d w i t h the

from

water-splitting

system PSII and reaches its maximum value F

when electron max t r a n s p o r t to P S I , the N A D Ρ r e d u c i n g s y s t e m , is p r e v e n t e d by the i n h i b i t o r

DCMU.

time (s)

Fig.2 Fluorescence induction curves. a, b e a n ; b, U l v a sp; c, M . i n t e r g r i f o l i a ; d, a c o m p a r i s o n of the b e a n , U l v a sp., a n d M . i n t e g r i f o l i a f l u o r e s c e n c e i n d u c t i o n c u r v e s in air. For a, b a n d c, F m a x w a s m e a s u r e d in the p r e s e n c e of D C M U , the c o n t r o l c u r v e i s l a b e l l e d air, a n d in the l o w e r t r a c e s the n u m b e r s r e f e r to O2 p r e s s u r e in a t m o s p h e r e s . T h e F 0 (all P S I I t r a p s open) t r a c e in a i r is s h o w n o n a n e x p a n d e d time s c a l e .

511

There is evidence from fluorescence in the presence of selective inhibitors and molecular C^, that O2 can remove electrons from the carriers in the thylakoid membrance from at least two different sites (Refs.1-5).

The variable fluorescence is a

measure of the relative redox state of the primary PSII electron acceptor Q (Ref.6) is thus quenched by C^.

A large difference

may be seen in the variable fluorescence of a typical higher plant, bean (Fig.2a) compared to the algae (Figs.2b,c).

In the

higher plant Q becomes almost fully reduced, but never achieves this level in the algae.

Since only variable fluorescence is

strongly quenched by 0 7 > this suggests that losses in electron

4

3

fm.fp

1

0

0

10 02

20 30 pressure (atm)

40

Fig.3 0? quenching of f m , dashed line, and fp, solid lines, in bean ( Q ) 1 Ulva sp. ( Q ) and M. integrifolia ( A ) •

512

flow to (>2 are much greater in algae than in higher plants. Furthermore, the weak quenching of F by 0 o when electron max ¿ transfer is blocked (Fig.3) distinguishes C^ quenching in PSII antennae from that in the electron transport system (Fig.l). C>2 Exchange in Chloroplasts O2 exchange provides information about both PSI and PSII activity.

We have succeeded in isolating O2 evolving chloro-

plasts from kelps.

Fig.4 shows Oj evolution with KFe(CN)g as

electron acceptor for PSII;

comparison of the rates of 0 9

150 100

!c Ξ 150 o σι ε loo

ε ^ 300 200

100

100

200 300 400 500 W m-2 Fig.4 Light saturating curves of the Hill raction in the presence of ferricyanide in broken chloroplasts of Fucus, Nereocystis, Laminaria, Macroscystis, spinach, and lettuce.

513

Time (min)

Fig.5 Light dependent 0 2 reduction in the presence of methyl viologen and subsequent 0 2 release after the addition of catalase compared to 0 2 release in nonilluminated samples for Fucus, Nereocystis, Laminaria, Macrocystis, spinach, and lettuce. Reaction media of lmL contained: 50mM Hepes (pH 7.6), ImM M n C l 2 , ImM M g C l 2 , 10-15wg Chi, ImM N H 4 C 1 , 0.2mM methylviologen, 6mM isoascorbate and 300 units of commercial catalase where indicated. evolution reflects the greater efficiency of light utilization in higher plants and their saturation at higher light

inten-

sities . In our thoroughly washed thylaKoid preparations endogenous 0 2 and H 2 0 2 scavangers, normally present in whole intact chloroplasts (Ref.7), are absent making it possible to observe the buildup of H 2 0 2 . PSI activity is evaluated through O2 uptake in chloroplasts by blocking electron transport with DCMU, using DCIP as electron donor and methyl viologen as acceptor for PSI.

Fig. 5 com-

pares PSI activity in 4 species of brown algae with the much larger rates in 2 higher plants.

The 0 2 reduced is converted

to H 2 0 2 which then releases 0 2 in the dark on the addition of catalase.

The kelp chloroplasts produced larger amounts of

H o O ? during the isolation procedure under low intensity room

514 light even before the addition of reagents, w h i c h is indicative of electrons being transferred to C^ more readily.

Sub-

sequent chloroplast isolation in the dark has confirmed this observation. Conclusions We suggest that both the fluorescence and

exchange data

indicate that the transfer of electrons to C^ is a significant energy dissipation mechanism in brown algae and may contribute to their low light

saturation.

References 1. 2. 3.

4. 5.

6.

7.

Vidaver, W., R. Popovic, D. Bruce, K. Colbow, 1981, Oxygen quenching of chlorophyll fluorescence in chloroplasts. Photochem Photobiol 34: 633-636. Vidaver, W., K. Colbow, S. Wessel, G. Hall, 1981, Chlorophyll fluorescence quenching by oxygen in plants. Can. J. Bot. 5J3: 190-198. Asada, K., Y. Nakano, 1978, Affinity for oxygen in photoreduction of molecular oxygen and scavenging of hydrogen peroxide in spinach chloroplasts on illumination. J Biol. Chem.28: 917-920. Radmer, R.J., B. Kok, 1976 Photoreduction of 0 2 primes and replaces C 0 2 assimulation. Plant Physiol 58: 336-340. Egneus, H., U. Heber, U. Matthiesen, M. Kirk, 1975, Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide. Biochim Biophys Acta 403: 252-268. Papageorgiou, G., 1975, Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In: Govindjee, ed. Bioenergetics of photosynthesis, Chap. 6, Academic Press, New York, pp 319-371. Elstner, E.F., 1982, Oxygen Activation and Oxygen Toxicity. Ann. Rev. Plant Physiol. 33: 73-96.

515 DISCUSSION

KRINSKY: Several years ago WITT (Quart. Rev. Biophys. (1971) 4, 765) and then KRAMER and MATHIS (Biochim. Biophys. Acta (1980) 593, 319) demonstrated in photosynthetic systems that at light intensities greater than saturation, large amounts of carotenoid triplet species developed, and they both suggested that these mechanisms were useful in terms of the dissipation of the excess light energy. They were both using chloroplasts. I wonder if you know of comparable studies that have been carried out in brown algae and whether you think that might also be a mechanism to dissipate excess radiation energy? COLBOW: I'm pretty sure nothing like that has been carried out in brown algae, for the simple reason that up to now nobody could produce chloroplasts of brown algae, which would be still active with oxygen. SIES: Usually H2O2, if formed, is taken care of in sub-compartments. Is it the case here that you have peroxisomes in these brown algae? COLBOW: SIES:

I have no idea. There would be a likelihood of finding peroxisomes, I think.

EFFICIENCY OF SINGLET OXYGEN PRODUCTION FROM THE 0 ? QUENCHING OF THE EXCITED SINGLET AND TRIPLET STATES OF RUBRENE

Charles Tanielian, Christian Wolff Laboratoire de Chimie Organique Appliquée, Département de Chimie, Université Louis Pasteur, 1, rue Biaise Pascal, 67008 Strasbourg, FRANCE

Introduction 3 Quenching of electronically excited states by molecular oxygen ( 0 2 been the subject of numerous investigations.

ς) has

Several studies indicate

that oxygen quenching of the first excited singlet states of aromatic hydrocarbons, S·,, leads to catalytic production of the lowest triplet 1 1 states, T-j, possibly with singlet oxygen{0 2 ¿Jformation if the S-j-T^ energy separation exceeds the Ο ^ δ excitation energy of 7900 cm" 1 s, + ο 2 3 ς —

—

τ »

+ ο^δ

1 τ

ι

+

m

1

On the other hand quenching of the lowest triplet state of aromatic hydro3 1 carbons by 0 2 ς often results in energy transfer to produce 0 2 δ T1 + 0 2 3 ς

»

S0 + Ο ^ Δ

(3.)

»

Sq + 0 2 3 ς

(4)

There have been many attempts to measure the relative significance of processes (_1_) and {2) characterized by α = k ^ / ( k + k 2 ) and of Ο ^ δ

production accompanying T-| quenching,

ε

the efficiency

= k^kg+k^).

literature results are still in large disagreement.

However

Thus, in the case of

rubrene, reported values vary from 1 to 2 for a + ε and from 0 to 1 f o r « (1 and references cited therein, 2, 3, 4, 5).

These discrepancies must be

ascribed to the conditions of excitation and to the Ο^^δ counting technique employed by the different groups.

This contribution examines fur-

ther the efficiency with which singlet oxygen is generated by quenching of the excited states of rubrene in benzene.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

518 Experimental

procedure

Our study was based on the analysis of the oxygen concentration dependence (air, oxygen and a 1:1 N 2 / 0 2 mixture) of quantum yields of photooxygenation of tetramethylethyl ene (TME) sensitized by rubrene.

The concen-

tration of TME (a reactive singlet oxygen acceptor which does not quench C ^ a physically) was sufficient to capture more than 99% of the singlet oxygen produced in the solution.

Thereby g = γ^, φβ

and γ^being the

quantum yield of photooxygenation and of singlet oxygin formation. The irradiations were performed in an external cylindrical

photoreac-

tor and the reaction was followed by oxygen uptake measurements.

The

535 nm, 546 nm and 577 nm lines of a mercury halogen lamp HANAU TQ 150 Z2 were chosen as the excitation wavelengths. oxygen production for rubrerve

was determined by comparison with methy-

lene blue for which it was shown that γ results).

The quantum yield of singlet

Δ

= 0.50 in methanol

(unpublished

The concentration of the two sensitizers was sufficient so that

more than 99% of the incident radiation was absorbed in the photolysis cell. The solubility of oxygen in benzene at one atmosphere was taken as 9.1X10" 3 M. (6).

Results and discussion

If Y i s denotes the S 1

>

T-| intersystem efficiency in the absence

of dissolved oxygen and p n

the probability that a rubrene singlet state 2 1 is quenched by oxygen, the 0 2 Δ yield γ Δ is given by the equation I Υ

Δ

=

eY

is

+

p

02(o

+

ε

-eYis'

(e(

1·

Π

which describes the linear dependence of γ Δ on p Q

measured independently

from oxygen quenching of sensitizer fluorescence as (FQ-F)/FQ where FQ is the fluorescence intensity in the absence of oxygen or as K[0 2 ]/(1+K[0 2 ] ) where Κ is the Stern-Volmer fluorescence quenching. We find that Κ = 160 M" 1 . data where p Q

Table 1 is a summary of the experimental

is obtained as K[0 2 ]/(1+K[0 2 ]).

519 Table 1 103[02], M

γΔ(ρ0

slope + intercept

%

Δ

9.1

0 90

0 90

0.59

4.6

0 63

0 64

0.42

1.8

0 36

0 36

0.23

Plot of γ Δ against pg intercept =

Υ

give a s t r a i g h t l i n e ( F i g . I ) for which

= 0) = εγ· 5 = 0.03 ± 0.02 2

= i¿(p0

= 1) = α + e = 1.48 ± 0.04

Figure I Quantum y i e l d of Op^formation γ Δ as function of p r o b a b i l i t y Pg that S^ (rubrene) i s quenched by 0 ? .

2

520 This value of α + ε is in significant disagreement with those recently reported by Gurinovich et al. (4) and Darmanyan in toluene (5) (a

- ε ~ 1)

and by Merkel et al. (7) (o = 0, ε = 1), but in very good agreement with that found by Wu and Trozzolo (1) ("r^/Pg (1.4 ± 0.2) in benzene.

= 1.47) and by Stevens et al. (3) ^

In order to separate the values of a and ε we have attempted to use a procedure based on the external heavy atom perturbation effect which is known to enhance the production of triplet sensitizer molecules.

But none

of the used additives was successful in enhancing the triplet quantum yield which may be due to the low value of the rate constant for intersystem crossing (8) M 0 ® s

^ for rubrene in benzene).

However it was recently confirmed that ε = 1 for anthracene, coronene on benzophenone (3) and since α+ε =1.5 for rubrene there is no direct evidence for process (4_). have obtained and

Moreover the values εγ^ $ = 0.03 ± 0.02 that we

= 0.012 ± 0.002 found by Darmanyan in toluene are

not inconsistent with ε = 1.

But anyhow

there is a substantial

efficien-

cy for the direct production of singlet oxygen by the oxygen quenching of the rubrene S 1 excited state.

References 1.

Wu, K.C., Trozzolo, A.M.: J. Phys. Chem. 83, 2823-2826 (1979).

2.

Wu, K.C., Trozzolo, A.M.:

3.

Stevens, B., Marsh, K.L., Barltrop, J.Α.: J. Phys. Chem. 85, 3079-3082 (1981). ~~

4.

Gurinovich, G.P., Salokhiddinov, K.I.: a) Chem. Phys. Letters 85, 9-11 (1982) ; b) Soy. Phys. Dokl. 26, 1071-1073 (1981). ~~

5.

Darmanyan, A.P.: Chem. Phys. Letters, 86, 405-410 (1982).

6.

In Solubility Data Series, Battino, R. Editor, 7, Oxford 1981.

7.

Merkel, P.B., Herksirceter, W.G.: Chem. Phys. Letters, 53, 350-354 (1978). ~~

8.

Patterson, L.K., Rzad, S.J.: Chem. Phys. Letters, 31, 254-256 (1975).

J. Phys. Chem. 83, 3180-3183 (1979).

250, Pergamon Press,

SINGLET OXYGEN REACTIVITY IN MICROHETERQGENEOUS SYSTEMS

Plato C. Lee and Michael A. J. Rodgers Center for Fast Kinetics Austin, TX 78712 U.S.A.

Research,

University

of

Texas

at

Austin

Introduction A kinetic model was derived that describes the effect of phase composition on the lifetime of singlet oxygen and its quenching by azide ions and tryptophan in normal micelles and reverse micelles.

Experimental data

adds confirmation to the model which could be useful for heterogeneous biological systems.

Fran the lifetime studies the equilibrium constant

for C > 2 ( ) partition between

the two phases

was abstracted.

On

the

other hand, the structure of water pools in reverse micelles was indicated in the quenching studies.

Results and Discussions Due to the long lifetime and fast entrance (k+) and exit (k_) rate constants of singlet oxygen

(Δ) in a

microheterogeneous solution, an equi-

librium in the distribution of Δ between the internal (ΔΘΧ|-) phases is established

during its lifetime.

and external Then it can be shown

that: kd = (1 - f m ) W - . + fmkj nt-Kpq d ~ fm) + fir^eq

(1)

where f m is the volume fraction of the interior pihase and Kgq = k+yk-. In the presence of a quencher, Q, solubilized only in the interior phase: kd = fnfegkint + (1 - f m ) W (1 ~ f m) + fmKeq

+

kgKeg [Q] (1 ~ f m) + ^raPeq

(2)

where kq is the bimolecular quenching rate constant in the interior phase

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

522 and [Q]

the

bulk quencher

concentration.

Thus, in a

microheterogeneous

system

the decay rate constant and quenching

rate constant of

singlet

oxygen

are

func-

tions

of

the volume frac-

tions

of

the two

Figure 1A

shews

phases. 02 (f/l-f)

the decay

rate constant as a function of water volume fraction in

Figure 1. Jy as function of water volume fraction, f, in AOT-heptane solutions at ω = 22.2. (A) kj vs. f 2 is formed by one

eq. 1

E

electron-transfer from electron donors to ^ aqueous medium.

in polar

Electron-rich

1/2

2 upon interaction with ^ in highly polar solvents,

1.0

\ o o\ α

-

2 In

fact, p-hydroxythioanisole

0.5

\o

(0.48 V vs^ SCE) undergoes one electron-transfer with

0 2 to

give [¡2 in accompany with the sulfoxide

formation

(13).

o

1

ι

ι ο

CD\ r

ι

(kcal/mol)

Fig. 1. Plot of the logarithm of the relative yield of

against

AG for electron transfer and against E^ of amines.

538 F i g . 2.

P l o t s of the

logarithm of kg a g a i n s t AG f o r electron t r a n s f e r

(lower

scale) and a g a i n s t E,

(upper

scale);

N.N-dimethylanilines

in methanol-water work); phenols benzenes

(O, t h i s

( · ) ; methoxy-

( • ) ; N,N-dimethyl-

a n i l i n e s in methanol

(Δ).

The broken l i n e represents the expected r e l a t i o n s h i p for f u l l electron t r a n s f e r reported by Rehm and Weiler 15

20

25

(kcal/mol)

(9).

References 1. Bel l u s , D. Adv. Photochem.

105

(1979).

2. Foote, C. S . , Dzakapasu, Α. Α . , L i n , J. W.-P. Tetrahedron Lett. 1247 (1975). 3. S a i t o , I . , Matsuura, T . , Inoue, K. J . Am. Chem. Soc. 103, 188 (1981). 4. Peters, G., Rodgers, M. A. J. Biochim. Biophys. Acta 637 (1981). 5. Manring, L. E . , Foote, C. S. J. Phys. Chem. 86, 1257 (1982). 6. S a i t o , I . , Matsuura, T . , Inoue, K. 0. Am. Chem. Soc. in p r e s s . 7. Young, R. H., Wehrly, K., M a r t i n , R. L. J . Am. Chem. Soc. 93, 5774 (1971). 8. Koppenol, W. H. Nature 262, 420 (1976). 9. Rehm, D., Weiler, A. I s r a e l J . Chem. 8, 259 (1970). 10. Thomas, M. J . , Foote, C. S. Photochem. Photobiol. 24, 683 (1978). 11. S a i t o , I . , Imuta, M., Matsuura, T. Tetrahedron 28, 5307 (1972). 12. Young, R. H., M a r t i n , R. L . , F e r i o z i , D., Brewer, D., Kayser, R. Photochem. Photobiol. ]7_, 233 (1973). 13. Inoue, Κ., S a i t o , I . , Matsuura, T. unpublished r e s u l t .

CHEMI LUMINESCENCE OF SCHIFF BASES CATALYZED BY HORSERADISH PEROXIDASE. Etelvino J.H. Bechara and Marisa H.G. Medeiros I n s t i t u t o de Química da USP, C.P. 20780, Säo Paulo, SP, B r a z i l .

Introduction Horseradish peroxidase (HRP) has been shown to catalyze the i n s e r t i o n

of

molecular oxygen into l u c i f e r i n - l i k e substrates (R-CHR'-C0-X;X= Ή,ΟΗ, OR, a l k y l , etc.).follótred by c y c l i z a t i o n of a hydroperoxy anion intermediate to 12 a dioxetane, which in turn cleaves to e l e c t r o n i c a l l y excited products ' . Energy transfer to synthetic and natural compounds, such as xanthene dyes, the 9,10-dibromoanthracene-2-sulfonate ion (DBAS), chlorophyll

and

phytochrome, has also been accomplished^. Of p a r t i c u l a r interest i s

the

HRP-catalyzed conversion of tryptophan to indoleacetamide in the presence 2+ of pyridoxal phosphate and Μη , which should occur via a S c h i f f base i n t e r 3 mediate . We report here our preliminary studies on the kinetics and chemiluminescence (CL) of the 0g consuming reaction of several a l i p h a t i c S c h i f f bases in the presence of HRP. In t h i s respect, McCapra's work^ on the chemiluminescent aerobic oxidation in DMSO/t-butoxide of S c h i f f bases derived from isobutanal with aromatic amines i s relevant.

Resul ts Oxygen uptake. Addition of HRP (2 yM) to solutions of either

N-isobutyl-

idenemethylamine (BMA), N-isobutylidene-i-propylamine (BPA), N - i s o b u t y l idene L - v a l i n e or N-sec-butylidenemethylamine in 0.6 M phosphate buffer, pH 7.4, at 35°C r e s u l t s in complete depletion of the dissolved O2 within a few minutes. In all cases, the rate of

uptake i s zero order with respect

to the O2 concentration and f i r s t order in both substrate (3.0-30 mM) and phosphate (0.1-1.0M). In acetate (pH 3.8-5.6) and T r i s (pH 7.4-9.0) b u f f e r s , no O2 uptake i s detectable. Hydrolysis of the substrates does not occur to a s i g n i f i c a n t extent under the experimental conditions. Addition of

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - FYinted in Germany

540

catalase ( I O - 7 Κ), superoxide dismutase CIO" 6 tf), or EDTA (10~4M)does

not

affect the oxidation kinetics of BPA. Acetone was identified as i t s 2,4-dinitrophenylhydrazone derivative by TLCand by ^HNMR as the spent reaction mixture from BPA. Chemiluminescence. The aerobic oxidation of the S c h i f f bases i s accompanied by l i g h t emission detectable by photon-counting or, in the case of BPA, in a conventional spectrofluorimeter. Figure 1 shows the temporal correlation between Og consumption and integrated l i g h t emission observed with BfA and BPA as substrates. At least two different emitters are evident in the CL spectra: one with a maximum in the carbonyl phosphorescence region (430 to 470 nm) and another with emission in the range 490-520 nm. The emission intensity from BPA and BMA (Fig.2) i s enhanced in the presence of DBAS, a known t r i p l e t carbonyl energy acceptor ("heavy atom e f f e c t " ) , but not by 5 the 9,10-diphenylanthracene-2-sulfonate ion (DPAS) . The CL intensity i s a linear function of the concentration of either BMA or BPA in the range of 3 - 3 0 mK, both in the presence or absence of DBAS. That the main emitter i s a t r i p l e t species i s also suggested by an increase in emission intensity which accompanies 0 2 depletion

Fig. 1. Correlation between oxygen uptake (

(Fig. 2).

) and integrated photon

emission (·) for the systems: (A) BPA/HRP and (B) BMA/HRP).

541

30 SECONDS

90

Fig.2. Time course of the CL emission from 30 mM BMA/(2 μΜ) HRP in 0.6 M phosphate buffer pH 7.4 at 37 C in the absence (- -) and presence of 10 yM DPAS ( — )

and 10 μΜ DBAS (

).

The sorbate ion, a typical triplet quencher, quenches both the direct and DBAS sensitized CL from BKA and BPA. The corresponding Stern-Volmer plot for BPA is linear with a slope (k τ°) of 2 χ ΙΟ 3 M"1

The corresponding

value for quenching of triplet acetone generated by the system (2) M 0 2 /HRP is k τ' = 6 χ 103 „-1

isobutanal/

Discussion The chemiluminescent HRP-catalyzed 0 2 consuming reaction of aliphatic Schiff bases reported here presumably occurs via the following route:

542 RI

NR3

//

N R2H H 02 t U HRP* R, N _ / N H R 3

/ R2

\

RI.

NHR-:

NR,

HCONHRN 3*

^

r^MH

> = 0

R2

H

Phosphorescence emission, triplet-singlet energy transfer to DBAS

and

quenching by Og and the sorbate ion attest to the triplet nature of

the

product. The role of phosphate is probably linked to its ability to catalyze the tautomerism of the substrate to the enolic form 6 , which in turn suffers hydrogen abstraction. Similarly, the HRP-catalyzed oxidation 2 isobutanal

of

requires phosphate as buffer whereas that of 3-methylaceto-

acetone^ which exist in the enolate form in aqueous solutions at high pH, do not show any dependence on the buffer type. The importance of our results is indicated (i) by the widespread formation of Schiff bases in biological processes; (ii) by the fact that these reactions are suitable models for bioluminescent reactions^; and,

finally,

(iii) by their potential as a chemiluminescent analytical tool for

imines

determination, including very basic proteins pretreated with isobutanal. FINANCIAL SUPPORT:

CNPq, FINEP, FAPESP, OAS.

References 1. Cilento, G.: in Chemical and Biological Generation of Excited States, W. Adam and G. Cilento, pp. 277-307, Academic Press, New York, 1982. 2. Bechara, E.J.H., Faria Oliveira, O.M.M., Duran, Ν., Casadei de Baptista, R. Cilento, G.: Photochem.Photobiol. 30, 101-110 (1979). 3. Augusto, 0., Cilento, G.: Biochem.Biophys.Res.Commun. 79, (1977).

1238-1244

4. McCapra, F., Burford, Α.: J.C.S.Chem.Commun. 607-608 (1976). 5. Wilson, T., Schaap, P.:

J.Am.Chem.Soc. 93,

6. Chiang, Y., Kresge, A.J., Walsh, P.A.: (1982).

4126-4136 (1971).

J.Am.Chem.Soc. 104,

6122-6123

7. Shimomura, 0.: in Chemical and Biological Generation of Excited States, W. Adam e G. Cilento, pp. 249-276, Academic Press, New York, 1982.

THE MECHANISM OF THE DIRECT PHOTO-OXIDATIVE DECARBOXYLATION OF α-ΟΧΟCARBOXYLIC ESTERS R. Stephen Davidson, Dean Goodwin and J u l i e E. P r a t t , Department of Chemistry, The City U n i v e r s i t y , Northampton Square, London EC1V OHB.

Introduction Sawaki and Ogata^ have proposed that in the d i r e c t photo-oxidation of methyl phenylglyoxylate the ester underwent a Type I fragmentation reaction to y i e l d a benzoyl radical which was subsequently scavenged by oxygen to form a benzoylperoxy radical (Scheme 1) capable of epoxidising α-methylstyrene. 0

Il Ph—C—COpMe

0

he

0

II 0« -C0?Me + Ph—C· —

II Ph—C—00·

TYPE I Scheme 1 2

However, i t has since been appreciated that alkyl pyruvates and other a. 3 oxo-carboxylic esters fragment via a Type I I and not via a Type I process (Scheme 2). H 0 II c / \ R

\HR' I o / C 0

h,

• TYPE I I

OH -CHR' I I c· o / \ / R C 0

HO p.

^ ^ >==CC==00 »

+ R'CHO s

R—C—CHO II o Scheme 2 We now report upon investigations to elucidate the role played by oxygen i n the d i r e c t photo-oxidative decarboxylation of α-oxo-carboxylic esters.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

544 Results and Discussion

A wide variety of a-oxo-carboxylic esters were found to undergo direct photo-oxidative decarboxylation in oxygenated acetonitrile and benzene solutions (Table 1). There are a number of ways in which oxygen can interact with excited a oxo-carboxylic esters. The high yield of hexaldehyde produced via Type II fragmentation of triplet n-hexyl pyruvate (Table 2) would suggest that the

Table 1 Yields of carbon dioxide from the direct irradiation of a-oxo-carboxylic esters (10~ Z M) under oxygen, for 3 hours.

a-Oxo-carboxylic ester

Yield (%) of carbon dioxide Acetonitrile Benzene

Methyl Pyruvate Ethyl Isopropyl " n-Butyl t-Butyl n-Hexyl Benzyl Ethyl benzoyl formate 2-(l-naphthyl)ethyl pyruvate 2-(2-naphthyljethyl pyruvate 2 - ( 2 - n a p h t h y l )ethyl-a-oxo-octanoate a:

20 hrs irradiation.

b:

18 hrs

37

(83)a

34

20 58

(59)

54 23

63 46

19

h

47

(116)

35 33

33 23

16

18

15

18.5

21

11 17

17

(82)

b

irradiation.

Table 2 Yields of carbon dioxide and hexaldehyde from the direct irradiation of nhexyl pyruvate (5 χ ΙΟ"2 M) in oxygenated and in degassed acetonitrile solution for 3 hours.

Product

Carbon dioxide Hexaldehyde (determined by glc)

Yield (%) Oxygenated Degassed

35 45

7 30

545 3 1,4-di radi c a l , l i k e those formed from α-oxo-octanoic esters , i s riot intercepted by oxygen?

The observation that a higher y i e l d of hexaldehyde

i s obtained from n-hexyl pyruvate under oxygenated than under degassed conditions (Table 2) i n d i c a t e s that oxygen can reduce the e f f i c i e n c y with which the t r i p l e t ester undergoes bimolecular reactions such as reduction 3 by the hexaldehyde . S u r p r i s i n g l y , t - b u t y l pyruvate (which i s photostable under degassed condit i o n s , suggesting that i t does not fragment via e i t h e r a Type I or a Type 2 5 I I process ' ) was found to undergo e f f i c i e n t photo-oxidative decarboxyl a t i o n (Table 1).

Decarboxylation must involve a d i r e c t reaction between

oxygen and the excited t - b u t y l pyruvate.

The competition between the

d i r e c t reaction of oxygen with the excited e s t e r s and the Type I I

process,

f o r the degradation of the e s t e r s , i s i l l u s t r a t e d by the fact that prolonged i r r a d i a t i o n of t - b u t y l pyruvate produces considerably more carbon dioxide (Table 1) than the analogous reaction of methyl pyruvate (which can fragment via a Type I I

reaction).

For a-oxo-carboxyl ic e s t e r s which fragment v i a a Type I I r e a c t i o n ,

inter-

action between oxygen and a product of the fragmentation process may lead to decarboxylation, as in the analogous reaction ofa-oxo-octanoic acid^. The lack of any appreciable solvent isotope e f f e c t s upon the decarboxylat i o n of methyl and ethyl pyruvates 7 i n d i c a t e s that s i n g l e t oxygen plays l i t t l e , i f any, part in these r e a c t i o n s . Since alkyl pyruvates are known to reduce methyl viologen via an electron t r a n s f e r mechanism and 9,10-dicyanoanthracene (a known electron acceptor) has been shown to s e n s i t i s e the photo-oxidative decarboxylation of methyl pyruvate in a c e t o n i t r i l e s o l u t i o n 7 , i t i s conceivable that electron t r a n s f e r from the excited e s t e r molecule to oxygen may take place (Scheme 3).

The r e s u l t a n t ester radical cation could react with the superoxide

anion to y i e l d carbon dioxide or fragment to y i e l d r a d i c a l s which are subsequently scavenged by oxygen to form peroxy species which can ultimately lead to carbon dioxide formation (Scheme 3 ) . The y i e l d of greater than 100% carbon dioxide from prolonged i r r a d i a t i o n of t - b u t y l pyruvate, in a c e t o n i t r i l e s o l u t i o n , i s i n d i c a t i v e of the i n v o l v e ment of species such as 0 = C — O R 1 ® which can undergo d i r e c t or i n d i r e c t

546 O

II

(R—C—COpR')

-

Oí

R—C—C02R'

O II R-O

0=C—OR1

I +

or

0?

Oj

0-

R — C — C O ,2R ' I 0-0-

0=C—OR'

1

V ° 2

per-acids ^

3

+

\ „•

(RCO) +

I

Scheme 3

*

R—C—00·

+

0=C-0R'

CO2 and other products

decarboxylation (Scheme 3).

The involvement of peroxy species is also

supported by the observation of perbenzoic acid in the photo-oxidation of methyl phenylglyoxylate^ and would account for the reported epoxidation of alkenes during this reaction. These results confirm that photochemical α-cleavage of the bond linking the carbonyl groups in 1,2-dicarbonyl compounds is an unfavourable process. References 1. Y. Sawaki and Y. Ogata, J. Amer. Chem. Soc., 103, 6455-6460 (1981). 2. R.S. Davidson and D. Goodwin, J.C.S. Perkin Trans. II, 993-997, (1982). 3. R.S. Davidson, D. Goodwin and Ph. Fornier de Violet, Tetrahedron Letters, 26, 2485-2486 (1981). 4. J.C. Scaiano, E.A. Lissi and M.V. Encinas, Reviews of Chemical mediates, 2 , 139-196 (1978).

Inter-

5. R.S. Davidson, D. Goodwin and J.E. Pratt, J.C.S. Perkin Trans. II, in the press (1983). 6. R.S. Davidson, D. Goodwin and G. Turnock, Tetrahedron Letters, 2^, 4943-4946 (1980). 7. R.S. Davidson, D. Goodwin and J.E. Pratt, Tetrahedron, 39, 1069-1074, (1983).

MENADIONE MEDIATED PHOTOOXIDATION OF PURINE AND PYRIMIDINE 2'-DEOXYRIBONUCLEOSIDES Chantal Decarroz, Jean Cadet L a b o r a t o i r e s de Chimie, D é p a r t e m e n t de R e c h e r c h e F o n d a m e n t a l e , C e n t r e d ' E t u d e s N u c l é a i r e s de G r e n o b l e , 85 X, F.38041 G r e n o b l e France

Cedex,

R i c h a r d Wagner a n d G o r d o n F i s h e r MRC G r o u p in t h e R a d i a t i o n S c i e n c e s , D é p a r t e m e n t de Médecine N u c l é a i r e et de Radiobiologie, F a c u l t é de Médecine, S h e r b r o o k e , Q u é b e c , C a n a d a .

Introduction Menadione

(or

2-methyl-l, 4-naphtoquinone)

which is a n a t u r a l

product

h a s b e e n shown to s e n s i t i z e h y p o x i c cells t o t h e l e t h a l action of i o n i z i n g radiations

(1).

More r e c e n t l y it h a s b e e n r e p o r t e d

t h a t t h i s vitamin is

able to s e n s i t i z e b a c t e r i a l cells t o t h e d e l e t e r i o u s e f f e c t s of n e a r UV l i g h t in oxic c o n d i t i o n s ( 2 ) . An i m p o r t a n t cellular t a r g e t to b e c o n s i d e r e d f o r t h e photobiological action of menadione is DNA. experiments

have

shown

that

efficient charge

Laser

transfer

flash

photolysis

reaction

b e t w e e n t h y m i n e a n d menadione in i t s t r i p l e t e x c i t e d s t a t e ( 3 ) .

ation of t h e formation of a t r a n s i e n t p y r i m i d i n e r a d i c a l cation h a s provided

b y t h e isolation a n d

the characterization

o x i d a t i o n p r o d u c t s of t h y m i n e ( 4 ) .

of t h e

occurs

Confirm-

major

been photo-

In t h e p r e s e n t s t u d y p y r i m i d i n e a n d

p u r i n e 2 ' - d e o x y r i b o n u c l e o s i d e s h a v e b e e n u s e d a s DNA model c o m p o u n d s for a f u r t h e r investigation

of t h e p h o t o s e n s i t i z i n g

ability of

2-methyl-

1,4-naphtoquinone.

Results Typically, carried

out

steady-state on

ImM

near purine

UV and

photolysis pyrimidine

experiments

have

been

2'-deoxyribonucleoside

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

548 aqueous aerated solutions in the presence of 0.5 mM menadione.

The

photoreactivity of the 2'-deoxyribonucleosides decreases in the following order

: thymidine > 2'-deoxycytidine

> 2'-deoxyguanosine

> 2'-deoxy-

adenosine. 1)

Photooxidation of thymidine. The major menadione mediated photooxidation products of thymidine have been separated by either thin-layer chromatography (TLC) on silicagel plates and/or by high performance liquid chromatography (HPLC) on octadecylsilicagel columns. ducts

have

been

characterized

by

The bulk of the

the

comparison

photopro-

of their

and/or HPLC properties and some of their spectroscopic 1 13 ( H and

TLC

features

C NMR, circular dichroism) with those of the authentic

samples. A major class of the photodegradation products consists of the four cis and trans hydrothymidine

(5)

diastereoisomers

of

5,6-dihydroxy-5,6-di-

and of N(2-deoxy- Β -D-erythropentofuranosyl)

formamide ( 6 ) . The presence of the 5R* and 5S* diastereoisomers of l - ( 2 - d e o x y - β -D-erythropentofuranosyl)-5-hydroxy-5-methylhydantoin has been also observed.

It is interesting to note that radia-

tion-induced degradation of thymidine in aqueous aerated solutions gives rise also to the various diastereoisomers of thymidine glycol and other disrupted and rearranged pyrimidine ring compounds (7) as characterized above.

However hydroxyl radicals which are the

main reactive radiolysis species of water (8) are not involved in the menadione photosensitized reaction of thymidine. We note in particular

a complete lack

product

of thymine

of the initial

hydrogen

which

would be

abstraction

the

reactions

expected

within

the

deoxyribose moiety by hydroxyl radicals ( 9 ) . A reasonable products

mechanism

of thymidine

for

the

formation

would involve

of the

the initial

photooxidadion

generation

of

a

pyrimidine radical cation. This would result from a charge transfer reaction

from the

nucleoside

in its

ground

state

to the

triplet

excited state of menadione ( 3 ) .

Similar intermediate has been pro-

posed

biphotonic

to

be

produced

by

a

process

when

thymine

aqueous aerated solution is irradiated with lasers of high intensity (10).

Subsequent reaction of the pyrimidine radical cation with a

549 water molecule would generate the 5-hydroxy and/or 6 - h y d r o x y - 5 , 6 dihydrothymid-6(5)yl

radicals

which

may

be

also

produced

addition of radiation-induced OH radicals across the

by

5,6-ethylenic

bond ( 7 , 8 ) . These intermediates further react rapidly with molecular oxygen giving rise to unstable thymidine hydroxyhydroperoxides through transient hydroperoxy radicals. The thymidine glycols and the formamide deoxyribose derivative are the result of subsequent hydrolytic pathways. The two UV absorbing nucleosides which have been characterized as 5-hydroxymethyl-2'-deoxyuridine and 5-formyl-2'-deoxyuridine derived also from the photo-induced initial pyrimidine radical cation. Competitive deprotonation of this intermediate is

expected

gives

rise

to

produce

5-(2'-deoxyuridilyl)methyl

to

hydroperoxymethyl-2'-deoxyuridine

transient corresponding peroxy radical. that the oxidation

radical through

which the

It has to be pointed out

of the pyrimidine methyl group has not

been

observed in the menadione photosensitized degradation of thymine. This may be explained in terms of a preferential deprotonation of the transient thymine radical cation at nitrogen N ( l ) . The resulting 1-yl radical reacts with a molecule of thymine giving rise to three major types of diadducts ( 4 ) . Sensitized photooxidations of the other 2'-deoxyribonucleosides. The menadione mediated photooxidation of 2'-deoxycytidine is about three times less efficient than that of thymidine.

The major pro-

ducts of the photoreaction have been characterized as the four cis and trans diastereoisomers of

5,6-dihydroxy-5,6-dihydro-2'-deoxy-

uridine. These nucleosides result from the deamination of the corresponding

2'-deoxycytidine

glycols

which

are produced

initially.

The presence of 2'-deoxyuridine has been also observed. This latter "nucleoside is particularly resistant to the menadione photooxidation reaction. This contrasts with the efficient photodegradation of uracil (4) which may be explained by the possibility of a deprotonation reaction within the photo-induced pyrimidine radical cation at position

1.

The

two purine

2'-deoxyribonucleosides

and

particularly

2'-deoxyadenosine are quite unaffected under exposure to near UV irradiation

in

aqueous

aerated

solutions

containing

2-methyl-l,4-

550 naphtoquinone.

It

is

interesting

to

note

that

2'-deoxyguanosine

which is the most reactive nucleoside to the photodynamic effect (11) aspect

is

slightly of

the

photodegradated.

menadione

sensitized

This

emphasizes

photooxidation

of

the

unusual

nucleic

acid

components. In particular neither singlet oxygen oxidation (12) nor type I radical mechanism appear to be significant processes in the photodegradation of 2'-deoxyguanosine.

References 1.

Adams, G.E. : Cancer. A comprehensible treatise. (Edited by Becker F . F . ) , vol. 6, Plenum Press, New-York and London, 1977, pp. 181-223.

2.

Fisher, G . J . , Watts, M.E., Patel, K . B . , Adams, G.E. : B r . J. Cancer 37, 111-114 (1978).

3.

Fisher, G . J . , Land, E.J. : Photochem. Photobiol., 37, 27-32 (1983).

4.

Wagner, J . R . , Fisher, G . J . , Decarroz, C . , Cadet, J. : Abst. Annu. Congr. Photobiol., Vancouver, June 26 - July 1 (1982).

5.

Cadet, J . , Ducolomb, R . , Hruska, F.E. : Biochim. Biophys. Acta, 563, 206-215 (1979).

6.

Cadet, J . , Nardin, R . , Voituriez, L . , Remin, M., Hruska, F.E. : Can. J. Chem., 59, 3313-3318 (1981).

7.

Teoule, R., Cadet, J. : Molecular Biology, Biochemistry and Biophysics (Edited by Hüttermann, J . , Köhnlein, W., Teoule, R . , Bertinchamps, A . J . ) , vol. 23, Springer, Berlin, 1978, pp. 171-203.

8.

Scholes, G. : Photobiology of nucleic acids (Edited by S . Y . Wang), vol. 1, Academic Press, New York, 1976, p. 521-577.

9.

Isildar, M., Schuchmann, M . N . , Schulte-Frohlinde, D . , von Sonntag, C. : Int. J. Radiat. Biol., 40, 347-354 (1981).

10.

Rubin, L . B . , Menshonkova, T . N . , Simukova.N.A., Budowsky,E.I. : Photochem. Photobiol., 34, 339-344 (1981).

11.

Cadet J . , and Teoule, R. : Photochem. Photobiol., 28, 661-667 (1978).

12.

Foote, C.S. : Free radicals in biology (Edited by W.A. P r y o r ) , 2, Academic Press, New York, 1976, pp. 85-133.

vol.

DYE-SENSITIZED PHOTOOXIDATION OF TRYPTOPHAN

Charles Tanielian, Hubert Müller, Lucien Golder Laboratoire de Chimie Organique Appliquée, Département de Chimie, Université Louis Pasteur, 1, rue Biaise Pascal, 67008 Strasbourg, FRANCE

Introduction Due to its particular interest in connection with photodynamic action, photosensitized oxidation of tryptophan (Trp) is a subject of continuous investigation.

However i) the mechanism of the oxidation is not defini-

tively established

ii) there are large differences in the rate constants

reported for the interactions of Trp with the excited states of the sensitizer and with singlet oxygen (0~(^δ ) or e. g

c

iii) the relative ,

importance of physical (kg) and chemical quenching (k R ) of

isa subject

of controversy (see Table I). In this paper, the rate constants for the interactions of Trp with the excited states of rose bengal

(RB) and with singlet oxygen has been deter-

mined using a new method (to be published) which permits accurate results to be obtained.A 1:1 M e O H ^ O mixture was used as solvent for comparison with literature data ; the irradiations were performed in a reactor similar to that described by Gollnick et al. (1).

Results The method consists in measuring quantum yields of oxygen consumption by tryptophan and a reference compound F (dimethylfuran) concentrations of F and Trp.

at various

F reacts with singlet oxygen without physi-

cal quenching and does not interact

with the excited states of the

sensitizer, underthese conditions the following processes take place :

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin - New York - Printed in Germany

552

F0„

S arid S+hvp

quenching

quenching

T r p + 0 2 T r p 0,,

The o v e r a l l quantum y i e l d o f oxygen c o n s u m p t i o n i s g i v e n by (where φ i s t h e p r o b a b i l i t y t h a t 1, Τ , Trp F, Trp i s converted t o oxygenation producta , kp[F] + kR[Trp] i sc Γ 0 2 F,Trp

Trp Ύ-

=

ks+kisc+

k

Trp

ε

Q[Trp]

k 0 [ 0 2 ] + kg [ T r p ] j

k p [ F ] + (kR+kQ) [Trp]+kD

and t h e T r p q u e n c h i n g o f t h e r o s e b e n g a l f l u o r e s c e n c e i s d e s c r i b e d by ,S Γ + k¡j[Trp]/(ks+kiSc)

I0/I = Then,

^(γ^Ρε^Ρ)"

1 + K[Trp]

(1 + Κ [ T r p ] ) (1 + K' [ T r p ] )

1

k i s ^ k „ + k , _ a n d K' k¿/k0[o2]. ISC I n t h e absence o f F , t h e p r o b a b i l i t y t h a t

where

ΎΤ

nation product i s φ - ^ (^p)"

1

• 1

P l o t s Of γ τ ( γ } Γ Ρ

+

0 2 i s converted t o oxyge-

and ^

ι knR

++

-

kn kR[Trp]

ε ^ Ρ ) " 1 versus [Trp] give a s t r a i g h t l i n e f o r which the

i n t e r c e p t i s 1 and t h e s l o p e i s ( 1 0 . 0 ± 0 . 5 ) M \ T h i s r e s u l t may be a s c r i 1 3 bed t o t h e q u e n c h i n g by T r p o f S(K) o r S ( K ' ) . In order t o d i s t i n g u i s h between t h e s e t w o k i n d s o f i n t e r a c t i o n , t h e q u e n c h i n g o f RB f l u o r e s c e n c e by T r p was e x a m i n e d .

The v a l u e

Κ =(10.8±0.4)M"1 which i s obtained from

the slope of the Stern-Volmer p l o t s i s c o n s i s t e n t w i t h t h a t of 10.5 M f - e p o r t e d by I n o u e , M a t s u ^ r a and S a i t o ( 2 ) . prom t h e s e r e s u l t s i t may be c o n c l u d e d t h a t T r p does n o t quench t h e t r i p l e t

s t a t e o f RB s i g n i f i c a n t l y

(kg uracil >> thymine.

In each

case spectra indicated more than one radical site, but resolution in powder spectra was generally insufficient for precise site identification.

Thiol or methyl-mercapto substituents, when present,

provided preferential sites for radical attack by oxidizing lipids, which appear as low-field ESR signal components. 3.

Purines formed stable radicals with difficulty.

nucleoside did not form detectable radicals at all.

Adenine and its

Guanine and

guanosine required long incubation periods (about 20 days) before ESR signals were detected.

The guanine signal was a clean singlet, possibly

reflecting substantial delocalization of the electron.

The guanosine

signal formed somewhat less facilely, and showed at least five lines arising from more than one center,· it is likely that the ribose moiety was also a site for radical attack (11). 4.

That purines did not form stable radicals does not indicate they

are not susceptible to attack by oxidizing lipids.

In aqueous model

systems, incubated for three days at 37°c, TLC of the reacted bases

605 showed formation of p r o d u c t s ( a s y e t u n i d e n t i f i e d ) in the p u r i n e s but no deconqpositicn in the p y r i m i d i n e s ; c o n c e n t r a t i o n s were t o o d i l u t e t o a l l o w ESR d e t e c t i o n of any r a d i c a l s produced.

Two a l t e r n a t i v e e x p l a n a t i o n s are

plausible: a) o x i d i z i n g l i p i d s indeed t r a n s f e r f r e e r a d i c a l s t o p u r i n e s ( i . e . , a b s t r a c t l a b i l e h y d r o g e n s ) , but the r e s u l t i n g p u r i n e r a d i c a l s a r e u n s t a b l e and f u r t h e r i n t r a m o l e c u l a r rearrangement or decomposition o c c u r s q u i t e r a p i d l y ,

or

F i g u r e 1. T y p i c a l ESR s p e c t r a freni s o l i d samples of DNA, p y r i m i d i n e s , and p u r i n e s incubated with o x i d i z i n g methyl l i n o l e a t e f o r p e r i o d s of from 1 day (DNA) t o 3 weeks ( g u a n o s i n e ) .

606 b) LO/LOO* radicals from oxidizing lipids add to purines, e.g., at C-8, rather than abstracting hydrogens.

[This

has been shown in ESR flow studies to be the preferred mechanism of ·0Η reaction with purines (11).] 5.

The powder spectra detected and the patterns of reactivity

exhibited for DNA and pyrimidine and purine bases are all remarkably similar to those reported for gamma and UV irradiation (12-15).

Although

much more definitive information must be obtained regarding the kinetics and reaction mechanisms involved and the specific products formed, it is clear that oxidizing lipids are capable of exerting radiomimetic effects on nucleic acids and bases.

References

1.

"Radloprotectors and Anticarcinogens", M. G. Simic, O. F. Nygaard (eds.). Academic Press, New York (1983).

2.

Borg, D.C., Schaich, K.M., Elmore, J.J.,Jr., Bell, J.A. Photobiol. 28, 887-907 (1978).

3.

Borg, D.C., Schaich, K.M. Oxy Radicals and their Scavenger Systems, Vol. Is Molecular Aspects, G. Cohen and R. Greenwald, Elsevier Science Publishing Co., Inc., New York (1983), pp.122-130.

4.

Schaich, Κ. M., Karel, M.

5.

Karel, Μ., Schaich, Κ. M., Roy, R. B. (1975).

6.

Henriksen, T., Sanner, T.

7.

MiIvy P., Farcasin, M.

Photochem.

Lipids 11, 392-400 (1976). Agrie. Food Chem. 23, 159-162

Rad. Res. 32, 164-175 (1967).

Rad. Res. 43, 320-331 (1970).

8.

Cook, J. Β., Alyard, S. J.

9.

Grasland, Α., Ehrenberg, Α., Rupprecht, Α. Strom, G. Photobiol. 29, 245-251 (1979).

Int. J. Rad. Biol. 11, 357-365 (1966). Photochem.

10. Sevilla, M.D., D'Arcy, J. B., Morehouse, Κ. M., Engelhardt, M. L. Photochem. Photobiol. 29, 37-42 (1979). 11. Schmidt, J., Borg, D. C.

Rad. Res. 65, 220-237 (1976).

12. Singh, Β. Β., Charlesby, Α. 13. Myers, L. S., Jr.

14. Shields, H., Gordy, W. 15. Muller, A.

Int. J. Radiât. Biol. 9, 157-164 (1965).

Fed. Proc. 32, 1882-1894 (1973). Proc. N.A.S. 45, 269-281 (1959).

Progress Biophys. Molec. Biol. 17, 101-147 (1967).

INFLUENCE OP ARACHIDONIC ACID ON IRRADIATION EFFECTS IN A DNA MODEL SUBSTANCE

David T&it, Heidi Martin-Bertram, Ulrich Hagen and Wolf Bors Abt. Strahlenbiologie, GSF Forschungszentrum, 8042 Neuherberg, F.R.G.

Introduction The role of membrane damage in radiation effects on cells has been frequently discussed (1,2). One mechanism which has been proposed involves transfer of radiation induced membrane damage to the cellular DNA. Owing to their sensitivity to radiation (3), the unsaturated fatty acid (UFA) residues of the membrane are considered likely mediators of such damage. This suggestion is supported by the observations that DNA (i) inhibits the spontaneous peroxidation of UFAs in vitro (4), (ii) is damaged in the presence of spontaneously oxidizing lipids (5,6) and (iii) is associated with the membrane (7) on bacterial and probably also manmalian cells. We have therefore studied the interaction of arachidonic acid (AA), with a ENA model compound under different irradiation conditions. Hie model is a double stranded polynucleotide (Hi), poly-deoxy-adenylic-cytidylic acid poly-deoxy-thymidylic-guany1ic acid.

Materials and Methods Arachidonic acid, 20:4ω-6 (99% ex Sigma) was purified by HPLC using a Waters /u Bondapack C^s reverse phase column (7.8 mn i.d. χ 30 cm). AA was eluted after 18 minutes using 70:30 acetonitrile (Baker):5x10"H2SO4, flow rate 1.5 ml/min. Detection was at 214 nm. AA emulsions were prepared by gently sonicating methanolic solutions in 1x10" NaCl (suprapur,Merck) in an ice bath under a stream of N2. the double stranded PN (Boehringer, Mannheim) had a molecular weight of 1x106 D by sedimentation analysis, and was used as received. In all experiments, the FN concentration was 50/ug/ml (= 5xl0 -8 M S 1.6x10"4 M nucleotides). All irradiations were carried out in 1 x 1 0 " M NaCl solutions (pH = 7.2) in a 60co-Ganma-cell, using a dose of 50 Gy at 50 Gy min~l. Before measuring the melting characteristics, the PN was separated fron the reaction mixture by ethanol precipitation method described in ref. (8) and re-dissolved in 2.5x10-3 M NaCl/3xl0"3 M tri-sodium citrate. Melting points (Tm) and the

Oxygen Radicals in Chemistry a n d Biology © 1984 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

608 differentiated melting carves of the FN were measured in a spectrophotometer equipped with heatable cuvettes according to ref 9; (for T ^ , heating was started at 55°C and increased at 0.5°C/min to 85°C. TD get T ^ , the samples were then cooled to 55°C in 2 minutes and kept at this temperature until >80% renaturation was attained. The heating was then repeated). Damage to the FN is indicated by broadening or shifts of the differential melting curves, and changes in the melting point.

Results and Discussion

The melting characteristics of the polynucleotide after incubation with AA, after irradiation in the absence of AA, and after irradiation in the pre-2 -5 sence of 1x10 - 1x10 M M and 24 hours incubation, are conpared in Figs.

Fig. 1: Differentiated melting curves of Hi a) incubated with 1x10"2 m AA, but non-irradiated ( — ) ; elfter 50 Gy in air in presence of lxlO - 3 M (-.-), lxlO" 4 M (..) and lxlO" 5 M (- -) AA, (second meltings). b) after 50 Gy in air, in absence (- -), in presence of 1x10"2 M AA ( — ) , (second meltings). c) after 24 hrs incubation with 2.5x10" 2 M AA (- -); after 24 hrs incubation with 2.5xl0 -2 M AA irradiated with 50 Gy in CH3CI/O2 (80:20) atmosphere and allowed to autoxidze in air, until the 235:215 nm absorption ratio stopped increasing (—), (first meltings).

609 Following irradiation of PN/AA mixtures containing 1x10

_2

M AA, which

should be sufficient to scavenge most of the water radicals produced, the polynucleotide's melting behaviour resembled that of PN irradiated alone. Although the probability of scavenging water radicals by AA decreases greatly with concentration, irradiation of EN in the presence of 1χ10~^-1χ10~5 M AA resulted in melting properties similar to those of non-irradiated PN.

'm CCI

ñ

69

[1

1i

|]

70

Τn 2

Τηn1 ri(•

69

1 68 1

h

68

Í1

67

67

I Fig. 2

h

a 1 11 11 1 1 1 I 1 1 I I

I

I

3

D

I

II

I

π

h

: I 1 1 1 1ì

1 1 1 1

II

Τ,ρΐ and T ^ of FN, a) non-irradiated, b) after 24 hrs incubation with 2.5x10"2 M AA but non-irradiated; c) alone, after 50 Gy in air: d), e), f) & g) after 50 Gy in air in the presence of lxlO -2 , 10 - 3 , Ι Ο - 4 & 1 0 - 5 M AA respectively; h) after 24 hrs incubation with AA (2.5xl0"^l) which had been irradiated with 50 Gy under CH3CI/O2 (80:20) atmosphere and allowed to autoxidize in air until the 235:215 nm absorption ratio stopped increasing.

1t> check if more damage occurs when the PN is incubated with AA which has been irradiated and allowed to autoxidize, a different type of experiment was carried out. AA emulsion was thus irradiated with 50 Gy and allowed to autoxidize in air until the 235:215 nm absorption ratio (a measure of UFA autoxidation) stopped increasing. Hi was then added and incubated for 24 hours. When «nuisions of lower (lxlO-^ M) AA concentration were used, no change in the T^ values or in the differential melting curves were observed. However, repeating the experiments at larger (2.5xl0~2 M) AA concentrations caused much greater changes to the Hi melting characteristics than irradiation of PN alone with 50 Gy (Figs. lb,c & 2c,h). Hie large de-

610 crease in the Ί^ values at the higher AA concentration 'mpiies the presence of unstable hydrogen bonds, probably as a result of base damage. Furthermore, the broadening of the "Adenine-Thymine side" (left hand side) of the differentiated melting curves indicates that it is this base pair which is damaged. In conclusion, these results indicate that products or intermediates of radiation induced autoxidation of AA emulsions can react with DNA. Ulis effect, however, exceeded that of direct irradiation of HH only at high concentrations of AA. It is also interesting that a protective effect of AA is observed when EN/AA mixtures are irradiated under conditions in which most of the water radicals react with the DNA model substance.

References 1. Alper, T: Adv. Radiat. Res. 7, 445-456 (1977). 2. Alper, T: Membrane Toxicity (Adv. Exp. Med. Biol.) Miller, M.W. and Shamoo, A.E., EHs.; Plenum Press, New York, 84, 139-165 (1977). 3. Mooibroek, J., Trieling, W.B., Konings, A.W.T.: Int. J. Radiat. Biol. 42. 601-609 (1982) - and references therein. 4. Pietronigro, D.D., Seligman, M.L., Jones, W.B.G., Demopoulos, H.B.: Lipids U , 808-813 (1976). 5. Pietronigro, D.D., Jones, W.B.G., Kalty, Κ., Demopoulos, H.B.: Nature 267, 78-79 (1977). 6. Reiss, U., Tappel A.L. : Lipids J5, 199-202 (1973). 7. Moyer, M.P.: Int. Rev. cytology 61, 1-61 (1979). 8. Maxam, A.M., Gilbert, W. : in Methods in Enzymology, Grossman, L. and Moldave, Κ., Bäs., ¿fcademic Press, New York, 65, 499-560 (1980). 9. Martin-Bertram, Η., Hagen, U.: Biochim. Biophys. Acta 561, 312-323 (1979).

THE

OXYGEN

RADIATION

EFFECT

DAMAGE

AS

A

PROBE

RESULTING

FOR

CLASSES

IN MUTATION,

GENE

OF

IONIZING

CONVERSION

OR

CELL DEATH

R.E.J. Mitchel and D.P. Morrison Atomic Energy of Canada Limited Chalk River, Ontario, Canada KOJ 1J0

Genetic

alteration

and

points most commonly not clear however

cell

death

are

used to assess

the

biological

radiation harm.

if these end points are a measure

same or different types of damage.

end

It of

The work presented

is the

here

demonstrates an approach to this problem by using oxygen

to

identify classes of lesions which can be responsible for cell death and comparing them to lesions which can lead to genetic alteration.

The fate of an irradiated cell, whether it be no

permanent

change,

a

mutation

or

cell

death

depends

principally upon the dose delivered, the presence or of DNA

O2

a

the

repair.

repair and

nd

cells' genetically In yeast

processes:

error

prone

two repair

either

therefore

repair,

absence

competence

known

types

recombinational

repair

is

for

of

DNA

repair

considered

to

light types of DNA damage, while

processes

are

and

recombinational increases

three

Excision

produced by both UV-light in

are

excision repair.

operate on ultraviolet other

there

determined

thought

ionizing

repair

or

the senstitivity

to

recognize

radiation. error

the

damage

A

defect

prone

repair

of yeast to killing

by

ionizing radiation.

Figure 1 shows the radiation sensitivity

of wild

compared

type

these systems. greatly

yeast

to

yeast

bearing

mutations

While a defect in either DNA repair

increases

the

sensitivity,

repair appears to be more important

process

recombinational than error prone

in ensuring the survival of Ύ-irradiated yeast.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed In Germany

in

type repair

612

DOSE

(krad)

Figure 1. Sensitivity of diploid yeast, Saccharomyces cerevisiae, to Co-γ radiation delivered in the absence of oxygen. Solid squares, RAD+/RAD+, wild type; open diamonds, rad6/rad6, deficient in error prone DNA repair; solid triangles, rad52/rad52, deficient in recombinational DNA repair.

When

any

cell

is

exposed

to

ionizing

radiation,

it

is

virtually certain that a wide variety of DNA damage products are generated, and this spectrum of damage is independent of the DNA repair capacity of the cell. recombinational repair

DNA

process

sensitivity therefore

repair

does

(Figure

deal

different ways.

process

not 1),

with

the

Since a mutation in the or

produce

these

cells

two

spectrum

the

repair of

various

of

prone

equivalent

processes

radiation

DNA must

damage

in

One possible difference could be the ability

of each repair system to recognize and the

error

types

of

ionizing

occurred in an exposed cell.

repair only some of

radiation

damage which

have

This is analagous to the known

ability of the excision repair system to recognize and repair UV type damage but not ionizing radiation type damage. We have tested the idea that different classes of radiation damage

exist

and

are

recognized

by

the

different

repair

613

1

IO

100

OXYGEN CONCENTRATION

1

1000

10

( μ* )

100

1000

OXYGEN CONCENTRATION

( μΗ )

F i g u r e 2. Left panels. Survival of y e a s t after exposure to 1500 Gy of ^^Co-γ rays. Panel A. Survival as a function o f C>2 c o n c e n t r a t i o n . P a n e l B. The protective effects of •OH scavenging by 1M ethanol, as a function of C>2 concentration. + Right panels. Gene conversion (trp to trp ) in yeast following exposure to 20 Gy of rays. Panel C. Gene conversion (trp+/107 cells/Gy) as a function of O2 concentration. P a n e l D. Protection against genetic damage b y · O H s c a v e n g i n g (1M e t h a n o l ) . Copyright Academic Press, ( r e f e r e n c e 8) r e p r i n t e d w i t h p e r m i s s i o n . systems. damage using

as

survival from

We used molecular

classes an or

ranging

genetic

of

techniques were

o x y g e n as a p r o b e

basis

point damage,

bacterial that two

or

(1-10). exposed

concentrations,

two

the

their O2

ie.

induced

the

oxygen

utilizing

spores

to

Figure

components 2A a

classes

shows

Ο2»

alterations

in

effect.

Results

biological

systems

mammalian

cells

oxygen

resolvable dose

damage

the

with

at

have

effect by

yeast

varying

resulting

is

various

that when wild type

constant of

to separate

reactivity

radiobiological

more to

of

the

laboratories

from

composed

the

end

several

demonstrated

cells

on

in

O2 cell

614

killing could be resolved.

The transition between these two

damage classes occurred at about 50 Ρ M 0 2 (7,8). indicates

the

resolution

of

two

classes

contributed

to an increase in a genetic

process

gene

of

repair.

The

conversion,

transition

a

Figure 2C

damage

which

alteration by the

product

between

of

of

these

recombinational

two

classes

also

occurred at about 50 y M O2' giving a result which at first appears very similar to the result obtained when cell death was

monitored

(7,8).

Figure

2

also

shows

however,

the

results of a test to identify the original radical responsible for these classes of damage. figure

2A and

2C were

The experiments shown in

repeated

in the presence

of a

-

oH

scavenger (1M ethanol) and the protection provided (the ratio of survival or gene conversion in the presence and absence of the scavenger) was plotted as a function of 0 2 tion (Figure 2B and 2D). increasing section

as

indicate

Any section of these curves showing

protection

originally

the to

(positive

result

of

hydroxy1

no increasing Figure

2B

slope)

02

protection

shows

identifies

reacting

radicals.

lesion which was not due to damage.

concentra-

the

two

with

Areas

of

that

damage zero

due slope

and therefore a type of reaction

areas

of 0 2

of

w

ith

positive

"OH

slope,

corresponding to the two damage classes resolved in figue 2A, suggesting that both damage classes (or at least components of them) resulting

in cell death are

ΌΗ

initiated.

When

gene conversion was monitored however, one area of positive slope was seen (Figure 2D).

This area corresponded to the

class

with

of

damage

(y

HYDROXYAPATITE FILTRATION

lo« HW product!

polytnorirod DNA

i DNA la I HYDROLYSIS HPLC S.V. PHOSPHODIESTERASE | HYDROLYSIS AoclootMo producta HPLC FIGURE 1. Schematic representation of the method for analyzing base damage in DNA irradiated in aqueous solution or in cells in vitro. The method combines selective enzymatic hydrolysis of DNA to 5'-^mononucleotides, and the separation and quantification of absorbing products by HPLC.

622

RETENTION TIME (m)

FIGURE 2. Typical chromatogram of the four deoxyribonucleotides 5'-CMP, 5'-AMP, 5'-GMP and 5'-TMP from hydrolyzed calf-thymus DNA, using r e v e r s e d phase HPLC. o f UV absorbance in DNA, including r e l e a s e of i n t a c t or bases and n u c l e o s i d e s Experiments

with

and the p r o d u c t i o n

DEAE-Sephadex

of

radiation-damaged

UV a b s o r b i n g

chromatography

(14)

and

fragments.

hydroxyapatite

f i l t r a t i o n (15) have shown that the y i e l d s o f f r e e bases and low molecular weight

fragments

released

from

damage to bases in the i n t a c t presence

and

absence

of

irradiated polymer.

oxygen

are

DNA are much lower

The y i e l d s

of

a non-linear

than

for

fragments in

the

function

of

dose,

i n d i c a t i n g a possible dependence upon macromolecular conformation which i s disrupted at higher doses r e s u l t i n g in r a d i a t i o n 2)

Damage t o

individual

nucleotides.

denaturation.

A typical

chromatogram o f

lyzed unirradiated calf-thymus DNA i s shown in F i g .

2.

Base-line

hydrosepara-

tion of the four 5 ' - d e o x y r i b o n u c l e o t i d e s i s obtained, and the assay procedure i s complete in l e s s than t h i s technique can separate

15 minutes

t i d e s , equivalent to that extracted diated

in

linear

yield-dose

aerated

each n u c l e o t i d e 'survival'

aqueous

solution

p l o t s are obtained can be q u a n t i f i e d

curves,

and are

(7).

Under optimum

conditions

and d e t e c t _ microgram q u a n t i t i e s o f from _ 10^ c e l l s . in

the

(Fig.

absence 3).

from the

shown in F i g .

changed per 100 eV o f energy absorbed).

of

exogenous

The y i e l d s slopes

nucleo-

When DNA i s

of

3 as G-values

of

the (# o f

irrasalts,

damage

to

individual molecules

Because o f the mildness and

623

Hydrolyied calMhymus DNA t 0 . 2 g / L i In Air

G value -9 δ'-ΑΜΡ 0.55

•*5'-TMP 0.81 • 5 l - G M P 0.4Θ 5 ' - C M P 0.64

200

400

600

800

Dose (Gy)

FIGURE 3. Dose-response p l o t s f o r the destruction of individual nucleot i d e s in calf-thyraus DNA i r r a d i a t e d in a i r . G values are given f o r damage to each n u c l e o t i d e , obtained from the slopes o f these l i n e s .

s e l e c t i v i t y of v i t y of

the enzyme treatment and the high r e s o l u t i o n

the HPLC system, changes o f

From these data,

vidual nucleotides

it

(3).

than purines,

sensiti-

as low as a few % to individual DNA

bases can be observed.

sensitive

and

as with the r a d i o l y s i s o f

can be seen that

and thymidine

sites

pyrimidines are the most

are more

indiradio-

radiosensitive.

Another observation that supports previous work (16) i s that the A-T base p a i r s , when in the m a j o r i t y as i s the case f o r calf-thymus DNA (56Ï are more r a d i o s e n s i t i v e than G-C p a i r s . pairs increases with t h e i r

A-T),

The r a d i o s e n s i t i v i t y o f A-T base

r e l a t i v e abundance, suggesting that thymine as

a r a d i o s e n s i t i v e t a r g e t may be able to r e l a y r a d i a t i o n damage to adjacent bases. 3) The oxygen e f f e c t . The o v e r a l l y i e l d of DNA base destruction in a i r , obtained by summing the contributions from the four individual nucleot i d e s , i s G(-DNA) . = 2.H8. This value i s similar to that obtained f o r air an equivalent mixture of f r e e nucleotides ( 5 ) and suggests that the damaging species d i f f u s e to and react with DNA bases in the native h e l i x with a high degree of e f f i c i e n c y , r e l a t i v e l y

and other f a c t o r s determining i t s secondary and t e r t i a r y structure. nucleotide destruction in i r r a d i a t e d a small

initial

oxygen e f f e c t

at high doses, y i e l d - d o s e

DNA, as i l l u s t r a t e d

(oxygen enhancement r a t i o

p l o t s are n o n - l i n e a r ,

Total

in F i g . 4, shows (OER) _ 1 . 6 ) ,

particularly

the curves in a i r and nitrogen appear to converge.

double

unimpeded by H-bonding

in a i r ,

The non-linear DNA

but and

624 Τ

6-

DNA

1

1

Γ

400

600

(0.2g/L)

200

800

Dose(Gyï

FIGURE 4. Dose-response plots for total nucleotide destruction in calfthyraus DNA irradiated in the presence and absence of air. An oxygen enhancement ratio (OER.) of _ 1.6 is obtained from the ratio of the initial slopes.

response at high doses may be due to radiation denaturation as a result of intra-strand

Η-bond

breakage, or

secondary

effects

involving

relatively

unreactive primary or secondary damaging species reacting together or with previously damaged sites in the DNA. 4)

The damaging species and mechanisms.

aqueous

solution

is caused

by

radiolysis species e~ , H· and aq radicals. converted

In oxygenated

the

The majority of damage in dilute

indirect

action

of

the

primary

water

-OH that react with targets forming target

solution, the reducing

species

and

H·

are

to 0^, and oxygen can take part in peroxidation reactions with

target radicals in competition with reductive back reactions Scavenger studies (Table

1) indicate that

damaging species (3,5).

Over 80Í of the DNA damage due to

(5,9,10,17):

-OH radicals are the

principal

-OH is to the

nucleotide bases, with the remain ing damage affecting the sugar-phosphate backbone causing strand breaks (1-5).

The present study indicates that 0^

is not particularly damaging in this system (Table nificant oxygen

effect

c o n s i s t e n t with

is supported

by

the o x y g e n - f i x a t i o n

1). The lack of a sig-

previous work hypothesis

(12,18,19), and

of Alper

and

Flanders (18,19) that requires competition between oxygen and agent unlike

for

target

individual

radicals bases

or

(reaction

3).

nucleotides

In

macromolecules

(3,5,15),

target

is

Howard-

a reducing like

DNA,

radicals

immobile and are not free to disproportionate effecting reconstitution

are of

625 H O

^

, Η· , ·0Η, H,0 + , OH

e 3Q

¿

0 | (+H + )

e~ q (H·) + 0 2

damaging species .. ^ A ^ — reconstitution

DNA target

(1)

.i

Target

(2)

fixation •» Target damage

r a dicals

reductants

(3)

oxygen

the target. This is evident from the relatively high yield of G(-DNA) compared with G(-OH). presence

and

Although the types of damage may be different in the

absence

of oxygen, the

quantitatively similar. effect

overall

yield

of

target

damage

is

Our experiments generally show an enhanced oxygen

for DNA base damage with

added

thiols and

other

reducing

agents,

but the results are variable and further work is needed to quantitate the. effect.

Pulse radiolysis studies the 5,6 double purines

(reaction

disproportionation involving

electron

subsequent

(3,17) indicate

bond of pyrimidines 4). when

Although

can

(22)

-OH preferentially

4,5 and

radicals

in

may

effect

that the

these

immobilized

transfer

hydrolysis

and

DNA,

occur

some

are

unlikely

radical-radical over

attacks

7,8 double bonds

large

reconstitution

to

of

undergo

reactions

distances,

and

(reaction

5),

TABLE 1. Yields of overall base damage and UV-absorbing fragments in calf-thymus DNA irradiated under selective scavenging conditions, to study the action of specific reactive species.

Irradiation ν

conditions

t-BuOH

e~ aq

e

aV

N 2 , formate °2'

t-BuOH

G(--DNA)

G(fragments)

0..2

0.05

0 .,

1

0.02

0 .,15

0.05

2

e" , -OH, H- a aq'

1.,65

_0.15 b

NO

2 »0H, H· a

3.,25

_0.40 b

N

a)

Damaging species

trace amount

b)

dose-dependent

626

+ -OH

JoH

+

j 10 •C CL to

tn

•C CL

cr

IO t/1 1

CM

O O

>>

a .

o

υ~ι

•ι— •ι— s+-> T5 C IO ,—V s:

C J τ—4 O

M O

ΙΛ •1

>> o •a ca s1

>-

φ .c c •1 (Λ Φ 3 IO

>

C3 S-

•f—

φ -d +J

•o c 10 (Λ υ "O O ίο. ι—I Φ 1 .a IO I—

-

O

φ IO -C a. (Λ o -C a.

•C +J φ S-l->

E:

1

>>

a. Ο Φ S- + J CL (0 O JZ ISI Q . •r- tn •i- O S- - E 1 - OL

(/)

σ> «A-

CO

CO

CM

CM

ο

ΙΟ

Φ Ο 10

CM

+->

ί .

+J C φ (Λ -Ο 10

>3LT)

í-

φ ι— +-> >> IO •C - C + J CL φ Vt •R-

O

S- - C h - CL

o 4-

C\J o i-

Ι Ο

IO

LO

co

-

O o

o

c

o

O

CO

Ut . a

LT>

O

(Β

LO

CM

O—

o o o_ o

: E

ι— φ >>•!-> - C IO +-> - C φ CL ε ω •r- O S- J = 1— a .

CO

co

o

σι

O

fH

co

«-i

I—1 O

•

φ +J IO -C a. (Λ o -C CL

T3 ω -P c o •I—

•σ

IO s-

υ 10

•f—

o

-!->

>i .e +-> φ ε

.Q

s-υ

t/J Ό +J O 3 X> O ία .

υ IO 1 IO 4->

O 1—

ίΟ .c ο. ΙΛ O -C Q.

>> ι

10

•Ι-

Ο

φ Ό >1

ir> O o

αϊ "Ο

υ IO

χ ο

φ Ό >1 -C

-σ >> -φ C

Τ3

Τ3

Φ

Φ

S-

αι

ε ίο

φ ο •t

o

c o -O i. o

Ό

>>

χ o

ÍD

í-

Φ

632

®I 01 Xo ι oXι o ©ι 01 f IM 0 011 0 I 011 01 XΝ O oI

©

01 Xor* ©

©

o o^ Üo φa s ® χ 0 1 o ®

®

• oI

bI Xg o-o 0I1

01

oX

•o

® 01 Xrvi υ oιι

o+

01 o o-o X

o+

oX „o I o ®

®

0 oX1 + ?

® oX(NI o X X 0 0 O + O1I + oI1

® ©

oX I I 0=ü--0 I 0 1 X o II ®

633 The products of triethyl phosphate (Table I) indicate that there is some C-C bond fragmentation (products: formaldehyde and formic acid), an observation which is pertinent to the interpretation of the fate of the DNA-(5') peroxyl radical (1, 4, 5). Fragmentation of the C(5)-C(4') bond in DNA will be more significant than the corresponding reaction in triethyl phosphate because of the enhancing effect of the oxygen at C(4') (cf. 6). In the radiolysis of DNA no products have been observed so far which could be attributed to an OH attack at C(3') (1). For this reason the study on triisopropyl phosphate which can serve as a model for the DNA(31) radical is of special interest. Hydroxyl radicals react with triisopropyl phosphate by abstracting H atoms from a and g positions of the phosphate in equal proportions as determined by pulse radiolysis. The a-peroxyl radical is a tertiary peroxyl radical which are known (7) to react much more slowly with one another than the primary and secondary ones such as those derived from trimethyl and triethyl phosphates. They only can follow the decay path with oxyl radicals as intermediates (cf. reaction IV in Scheme I). The presence of the ß-peroxyl radicals in the triisopropyl phosphate system allow, however, reactions between the tertiary peroxyl radicals and the primary peroxyl radicals. The typical products are aceton and formyl-ethyl diisopropyl phosphate (reactions 5 and 6) ch3 0-O-C-O-O· ch3

oo-ch2 +

ch3

» (?)-

ch3

CHg 0-0-C-OH

ch3

HC-0-(?)

CHO

0-C -OH + ( ? ) - 0 - C H + ch3

[5]

ch3

CH3 ?· ( P ) - 0 H +

C=0

[6]

ch3

The rate of reaction 6 has been determined independently by oxidizing the α-radical with tetranitromethane (kg = 3 χ IO 4 s - 1 ). In addition to reaction 5 considerable fragmentation of the carbon skeleton occur in this system as evidenced by the high 5 values of the products containing one or two carbon atoms. These results possibly allow some speculations with

634 respect to DNA radiolysis. A typical product derived from the DNA-(3') peroxyl radical should be the corresponding 3'-oxo compound which is expected to be unstable and will decompose further (8). Furthemore one will have to expect excessive fragmentation especially of the C(3')-C(4') link.

References

1.

von Sonntag, C., Hagen, U., Schön-Bopp, Α., Schulte-Frohlinde, D.: Adv. Radiat. Biol. _9, 109-142 (1981)

2.

Schuchmann, M.N., von Sonntag, C.: (in print).

3.

Bielski, B.H.J.: Photochem. Photobiol. 28, 645-649 (1978).

4.

Dizdaroglu, Μ., Schulte-Frohlinde, D., von Sonntag, C.: Z. Naturforsch. 3θ£, 826-828 (1975).

5.

Isildar, M., Schuchmann, M.N., Schulte-Frohlinde, D., von Sonntag, C. Int. J. Radiat. Biol. 40, 347-354 (1981).

6.

Schuchmann, M.N., von Sonntag, C.: J. Phys. Chem. 86, 1995-2000 (1982).

7.

Furimsky, Ε., Howard, O.A., Selwyn, J.: Can. J. Chem. 58, 677-680 (1980).

8.

Binkley, R.W., Hehemann, D . S . , Binkley, W.W. : Carbohydr. Res. 58, C 10-C 12 (1977). DISCUSSION

FARAGGI: Your and Or. GREENSTOCK's results show that the G-value for single strand breaks in poly U and DNA is higher than previously reported. This seems to indicate that the bases are the primary sites of OH attack followed by hydrogen transfer from the sugar and not a direct OH reaction with the sugar. What would be the consequence of this for your mechanism? von SONNTAG: Well, that would be an interesting point. Eventually we also would end up with a sugar-centered radical, but then we would have to consider only two positions to be of importance. That would be the 4' position and the 1' position, because these are the only positions that are somewhat labile. We have shown, that when you change from an alcohol or ether-type system to the phosphate, the reactivity of the hydrogen atoms is strongly reduced. In the tri-isopropylphosphate system the ratio of OH attack was 1:1 and in the isopropanol system OH attack favours the C(2) position, by 85:15. Thus positions were the phosphate groups are centered appear to be de-activated with respect to radical attack.

635 RALEIGH: In our studies of model nucleotides, one of the things that really impressed us was the pH-dependence; that is the ionization state of the phosphate profoundly affected radiation damage (RALEIGH et al. Radiat. Res. (1976) £5, 414-422). For this reason, I'm wondering why you are looking at trialkylphosphates rather than dialkylphosphates in these model systems? von SONNTAG: Well, you are right. We studied dimethylphosphate and obtained more or less the same result as with the trialkylphosphate. With the analytical techniques we are using, pulse radiolysis with conductivity detection, it's very difficult to work with dialkylphosphates because of the high background conductivity and it is more informative to use the non-conducting trialkylphosphates as model systems for those kinetic measurements. But product studies can be done with the dialkylphosphate and they showed results quite similar to those of the trialkylphosphates. Thus I think some of the ideas that we get from the trialkylphosphates will also relate to the dialkylphosphates. BORS: I appreciate your pointing out the additional intramolecular possibility of alkoxy radical decay. Would this be confined to such phosphoric acid derivatives, is an ether group necessary, or could it also occur with other aliphatic alkoxy radicals? von SONNTAG: Well, we observed the rearrangement of the alkyloxyl into the hydroxyalkyl radical with quite a number of compounds. We observed it in systems which start from the methyl peroxyl radical (SCHUCHMANN and von SONNTAG, to be published) , and we observed it with the peroxyl radical derived from diethylether (SCHUCHMANN and von SONNTAG, J. Phys. Chem. (1982) ji6, 1995). There it plays the major role. Thus it is quite common. In some cases it is the most important route in aqueous solution. CADET: Do you think that the RUSSELL mechanism would be significantly involved in the DNA damage? Especially at low doses, because in this case the probability of obtaining two peroxy radicals would be very low. von SONNTAG: Well, this is quite an important question. One always poses the question whether it is likely for two peroxyl radicals to meet if one considers those large molecules like DNA. Therefore we were not too surprised when we measured the G-value for oxygen uptake from DNA and its high yield indicated that a chain reaction must occur, because the breakdown of the peroxyl radicals that react with each other is slowed down in the high molecular weight compound and other routes are taken up. In aqueous DNA solutions the chain is only short and its nature is not yet sufficiently established (ISILDAR et al., Int. J. Radiat. Biol. (1982) 41, 525)

THE ROLE OF OH RADICALS AND OF ABSORBED ENERGY IN THE MECHANISM RADIOSENSITIZING ACTION OF IODINE CONTAINING COMPOUNDS.

OF

THE

V.Capuano, M.Coppola

E.N.E.A., CRE Casaccia, Lab. Dosimetria e Biofisica, Rome, Italy

M.Quinti]iani C.Ν.R.,Istituto Tecnologie Biomediche, Rome, Italy

Introduction

lodine containing radiosensitizing compounds offer the peculiar feature of bringing about their biological action according to several possible mech= anisms,namely a biochemical mechanism, a radiation chemical and a physical mechanism, according to their structure, to radiation quality and dose and to thp biological system involved. Speaking of "iodine containing sensitizers" for a variety of inorganic and of aliphatic and aromatic organic chemicals, is justified by the experimen= tal evidence that the radiation-chemical and physical mechanism of radio= sensitization mainly depend on the presence of iodine atoms in the molecule

The Biochemical mechanism of sensitization

Iodoacetic acid (IAA) was the first chemical clearly found to be a

radio=

sensitizer. In 1954 it was, in fact, reported (1,2) that the administration of subtoxic doses of the drug to mice or rats prior to radiation resulted in a marked increase of 30 days lethality. This finding was interpreted as

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

638 a result of irreversible blockage of SH groups produced by IAA, resulting in inhibition of SH-enzymes and depletion of SH natural radioprotectors. The above results were confirmed (3) and extended to other SH reagents (4). Further support

to the SH depletion mechanism of sensitization came from

experiments carried out by Kinder, Sinclair and Elkind (5) on mammalian cells in culture. A two component model of cell survival after irradiation was then proposed, postulating that one component involves DNA synthesis and the other some fraction of intracellular sulphydryl groups.

The Radiation Chemical mechanism

In the meantime, studies on iodine containing sensitizers were focussed on simpler systems such as microorganisms and enzyme molecules. It became very soon evident that

many other iodine containing compounds,not at all

re=

acting with SH groups, were able to enhance bacterial radiation lethality or enzyme radiation inactivation (6). Fig.1 shows the effect of 4 iodine -4 compounds, at 5x10

M concentration, on the survival of X ray irradiated

cells of E.coli B/r (7). It is clear that iodopropionic acid (IPA), which reacts with SH groups at least 90 times slower than IAA, is only slightly less sensitizing than this latter. Iodide ions show much lower sensitizing activity. The opposite appears to be true in the case of enzyme inactiva= tion as seen in fig.2, showing that iodide ions display a greater

sensi=

tizing activity than IPA (8). This finding was confirmed on other enzyme molecules (8). Iodine compounds also enhance the radiation induced release of intracellular potassium from mammalian erythrocytes or from E.coli cells With respect to this end point iodide ions are nearly as active as the iod= i n e containing organic drugs (6). Many other reports exist in the litera= ture demonstrating the radiosensitizing activity of many different iodine compounds on a variety of biological radiation responses (6). With regard to the mechanism involved, it has been shown that in most cases, in order

639

Fig.1 Effect of 5x10 ^M iodine compounds on X-ray survival of E.coli B/r IAAM = iodoacetamide Fig.2 Effect of iodine compounds on X-ray inactivation of alcohol dehydrogenase from yeast YADH concentration 1.2xlO~S M. Molar ratio additive/enzyme shown in the figure

\ A ι·

4i

ig

it

i)t

ι:· D O S E ( G Y )

640 to produce their effect, iodine compounds need just to be present at

the

moment of irradiation. The enhancement of the radiation response is in fact exactly the same whether they are added to the cell suspension or

to

the

enzyme solution few milliseconds or several minutes before irradiation (6). Moreover, irradiated solutions of several iodine containing sensitizers exhibit a radiation induced cytotoxic activity which appears to account for the radiosensitizing effect. In the opposite situation, namely by adding irradiated bacteria or enzymes to unirradiated drug, no modification of the response can be detected. These observations, considering the low concen= trations of iodine compounds involved, strongly suggested the interpretF ation that, in

systems such as enzyme solutions or bacterial suspensions,

the enhancement of the radiation response was due to iodinated transient intermediates (free radicals) produced by radiation from iodine containing compounds. As alternative to iodine free radicals, molecular iodine could be consid?= ered. Free iodine can in fact be measured in irradiated solutions of either inorganic or organic iodine compounds, and treating such solutions with thiosulphate completely abolishes the radiation induced cytotoxic effect previously mentioned. The radiolytic scheme of iodine containing tizers is known in full details only for I

_ I

+

OH

—

° I

+

_ OH

: 0

;

sensi=

I

+

_

I

_ I

;

2I~

e

aq

— I" + I" : I" — I" + I 2 3 ' 3 2 has no practical role in the process. It is not yet clear whether the

radiosensitizing effects are due to secondary iodine radicals or to

mol=

ecular iodine, however it is clear that the process is primarily due to the action of OH radicals. Organic iodine containing compounds, either aliphatic or aromatic, all react at fast rate with hydrated electrons releasing iodine in the form of iodide ions ( RI + eaq — —

R° + I ),Jy e t this reaction

cannot account for the radiosensitizing effect. Evidence exists indicating that also with organic compounds such an effect is mainly an OH operated process (9)· However little information is available on the details of OH

641

reactions with organic iodine containing compounds. End product analyses demonstrate that iodide ions and molecular iodine are produced. For in= stance, in the radiolysis of diatrizoic acid (DA), a triodinated derivative of benzoic acid, it was found (10) that the rate of formation of I is about 4 times faster than that of I^ · In addition to the release of I by e , an alternative mechanism through OH addition to an aromatic double aq bond can be envisaged as postulated by Potapova et al.(11) : RI + OH

OH

—- R°0 + H+ +

Other processes have not been identified as yet.

The Physical mechanism Attempts to investigate the radiosensitizing effect of iodine compounds on mammalian cells in culture were initially unsuccessful because of the tox= iclty of IAA or IAM. With less toxic IPA and I some positive results were reported by Vos in 1969 (12), who showed that iodide ions induced in Τ cells a sensitization with a DMF of about 2 at the concentration of 0.5 M . About the same enhancement was obtained in V79 cells with 0.1 M Iothalamic acid (ITA), an analogie of

DA. At such concentration 1evels the increase

in the absorbed radiation dose, due to the presence of the heavy iodine atoms, may become significant in the appropriate energy range of irradiat= ing photons. This was first noted by Matsudayra et al.(13) who observed that iodamide, a similar compound to ITA, at S% concentration enhanced cell killing, frequency of micronuclei and yield of DNA single strand breaks in= duced by 200 kVp X rays whereas no such effect was found after irradiation with 60 Co gamma rays. Fig.3 summarizes the results obtained by the present authors(14) on survival of V79 cells irradiated with 25O kVp X rays or 60

Co gamma rays in the presence or in the absence of 0.1 M ITA. The figure shows that a large enhancement effect is induced by ITA on X-ray irradiation while on gamma ray irradiation no effect of ITA can be detected up to 5 Gy and only above that dose an increased lethality becomes apparent in cells

642

DOSE (OY) Fig. 3 Effect of ITA on X and gamma ray survival of V79 cells. irradiated in ITA containing medium. Calculations of true absorbed dose in a .1 M aqueous solution of ITA indicate that the averaged mass absorption coefficient is about 3-6 times that of water for 250 kVp X-ray and very 60 nearly equal to 1 for

Co gamma rays. Evidence was obtained that X-ray

data could be completely accounted for by the increase in the absorbed radi ation dose due to iodine atoms, by introducing a dose scaling factor of 3.6 at least for cell survival above 30%.At higher dose levels the slope of the survival curve obtained with ITA tended to become steeper than could be expected from the data at lower doses, when fitted with a simple expression -aD-bD such as S=e

. This finding and that observed in gamma-ray survival

indicate that in the presence of ITA two

enhancing

mechanisms are in

operation: a physical increase of absorbed dose, acting at all doses, and a radiation chemical action which becomes evident only above a certain dose 1evel.

643 Aknowledgments Work partially supported by the Radiation Protection Programme of the Commission of the European Communities. Contribution n. 2030

References 1. 2.

Langendorff, Η., Koch, R.: Strahlentherapie 95, 535 (1954)· Feinstein, R.N., Cotter, G.J., Hampton, M.M.: Am. J. Physiol. 177, 156 (1954) 3. Quintiliano, M., Boccacci, M.: Rend. 1st. Sup. Sanità 23, 5 (I960) 4. Moroson, H., Spielman, H.A.: Int. J. Radiat. Biol. .Π, 87 (1966) 5. Kimler, B.F., Sinclair, W.K., Elkind, M.M. : Radiat. Res. 7J_, 204 (1977) 6. Quintiliani,M.: Advances in Chemical Radiosensitization (Vienna IAEA) p. 87 (1974) 7. Sapora, 0.: Ann. 1st. Sup. Sanità 6, 293 (1970) 8. Quintiliani, M., Shejbal, J.: Int. J. Radiat. Biol. _l6, 267 (1969) 9. Simone, G., Quintiliani, M.: Int. J. Radiat. Biol. 31, 1 (1977) 10. Quintiliani, M., Betto, P., Davies, J.V., Ebert, M.: Radiation Biology and Chemistry p. 49, Edwards, E.H. et al. eds., Elsevier Se. P. (1979) 11. Potapova, Z.M., Kharlamov, V.T., Pikaev, A.K., Guslkov, A.P.: Khim. Vys. Energ. 9, 386 (1975) 12. Vos, 0.: Radiation Damage and Sulphydryl Compounds p. 117, IAEA, Vienna (1969) 13- Matsudayra, H., Ueno, M.Α., Furuno, I.: Radiat. Res. 84, 144 (1980) 14· Barile, G., Bertoncello, G., Capuano, V., Coppola, M., Quintiliani, M.: Proc. 8th Symp. on Microdosimetry p. 659, Booz, J., Ebert, H.G. Eds., C.E.C. ( 1 9 8 3 )

RADIATION SENSITIZATION OF BACTERIAL SPORES BY CO

G. L. Gazsó "Frédéric Joliot-Curie" National Research Institute for Radiobiology and Radiohygiene 1775 Budapest, P.O.Box 101, Hungary

Introduction One of the problems in the cancer therapy is the presence of cells inside the tumour which are totally deprived of oxygen. This means that a given dose of radiation would cause greater damage to the normal, well oxygenated tissues and less to the tumour cells. Blood enters the capillary network as arterial blood, and having therefore an oxygen pressure of the order of 12.63 kPa and a carbon dioxide pressure of the order of 5.32 kPa. In the tissues the oxygen pressure decreases and the CO2 pressure increases. The nitrogen pressure is constant in the arterial blood, tissues and even in the hypoxic solid tumours. Considering the above factors, carbon dioxide would play an important role in the development of hypoxy. However, the radiobiological significance of CO2 has not been fully clarified. Under anoxic conditions CO2 can remove hydrated electrons without H2O2 build-up. e~ g + C02

>,C02

k = 7.7 χ 109 M _ 1 sec

_1

Cross, Simic and Powers using pure CO2 concluded that the radiosensitivity of Bacillus megaterium spores is the same seen in N2/ where e is not scanvenged /1/. Ewing obtained a radioprotective effect of CO2 in citric acid-phosphate buffer using similar bacterial spores /2/. In his opinion CO2 is one of the very few e scavengers that is not a radiation sensi-

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

646

tizer /3/. Radiochemical experiments resulted in the reduction of C02 in aqueous solution by cobalt-60 gamma rays leading to the formation of formic acid, aldehydes and small quantities of oxalic acid /4/. In this study, a series of experiments was carried out to investigate the effect of CC>2 and O 2 gas mixtures on the radiosensitivity of Bacillus megaterium spores.

Materials and Methods Bacillus megaterium (ATCC 8245) spores growing and plating technique were described by Tallentire /5/. The gamma radiation facility was ΡΧ-γ-30 apparatus with 96.43-94.42 Gy/min dose rate. Dose-In curves were constructed from four experimental points. Suspended spores were irradiated in dis7 tilled water. The test suspension containing about 5 χ 10 viable spores was equilibrated with nitrogen and oxygen in nitrogen gas mixture or carbon dioxide and oxygen in C0 2 gas mixture. Suspensions were bubbled at 200 ml/min for 30 minutes prior to the commencement of irradiation. Experiments were done by equilibrating suspensions with gas mixtures of 0.8%, 1.6%, 4.2% and 7.9% 0 2 in C0 2 and pure C02 at 20° C. Seven oxygen concentrations in N 2 were used as control/100% N 2 , 0.7%, 1.2%, 2.1%, 3.1%, 5.1%, 21% 0 2 in N 2 and 100% 0 2 /.

Results and Discussion A plot of the value of inactivation constant as a function of the log of 0 2 concentration is depicted in Fig.l. On increasing oxygen concentration up to 1.2% 0 2 in N 2 , there is a gradual increase in the value of k followed by a sharp increase. Further increase in 0 2 concentration causes another gradual rise in k value, reaching a maximum at 100% 0„. ~ 1 - 4 For irradiation in N„ the mean value of k is 11.85 Gy - xlO

647

The mean k value for irradiation of Bacillus megaterium spores in equilibrium with 100% 0 2 in the absence of other agents is 21.35 Gy _1 x 10 -4 .

Figure 1

Changes in the radiation sensitivity of Bacillus megaterium spores at different oxygen concentration (· 0 2 in N 2 , O 0 2 in C02)

Carbon dioxide gives no change in response when cells are irradiated in presence of 100% C0 2 or 0.8% 0 2 in C0 2> With increasing concentration of oxygen (1.6% or 4.2% 0 2 in C02) the sensitivity of spores starts to rise enhancing lethal damage in comparison with 0 2 in N 2 gas mixture response. Further increase of 0 2 concentration shows a decreasing tendency in sensitization. The control response (02 in N 2 ) is reached at 7.9% 0 2 in CO.,. Oxygen operates in more than one way in affecting the radiation sensitivity of the cells. Sensitization of spores by oxygen could be separated into at least three components, one of which involves reactions of hydroxyl radicals and two that apparently

648 do not /6/. Recent work has made it very clear that different concentrations of oxygen can produce different kinds of damage /7/. Chemically, the radiosensitizing effect of carbon dioxide in a certain oxygen concentration can hardly be explained. In anoxic conditions removal of eaq does not effect the H„0~ 2 2 build up. However, over a limited range of oxygen concentration, CO2 sensitizes spores to a small extent. It is possibly due to the reaction of "CO^· radicals with oxygen. It would result in the formation of 0* or t^C^ as a stable product. Further radiochemical experiments would lead to an understanding of how carbon dioxide sensitizes our spore system.

References 1.

Cross, Μ., Simic, Μ., Powers, E.L.: Int. J. Radiat. Biol. 24, 207 (1973).

2.

Ewing, D.: Radiat. Res. £8, 459 (1976).

3.

Ewing, D., Powers, E.L.: Oxygen dependent sensitization of irradiated cells. Radiation Biology in Cancer Research (Meyn, R.E., Withers, H.R. ed.J Raven Press, New York (1980) .

4.

Scholes, G., Simic, Μ. , Weiss, J.J.: Nature 188, 1019 (1960) .

5.

Tallentire, Α., Jacobs, G.P.: Int. J. Radiat. Biol. 21, 205 (1972).

6.

Ewing, D., Powers, E.L.: Science, 194, 1049 (1976).

7.

Ewing, D.: Int. J. Radiat. Biol. 30, 419 (1978).

18

DETERMINATION OF

O TRAPPING IN X-IRRADIATED

AMINO ACIDS BY

MEANS OF NUCLEAR REACTIONS

Claus Wiezorek Institut für Strahlenbiologie der Universität Münster D-4400 Münster

Introduction Studies of radiation induced biological free radicals have been greatly hindered by the chemical complexity of living organisms. ESR experiments have helped to advance this effort but quantitative determinations of the radiation damage are affected by relevant errors which can be made assuming an ideal paramagnetic behaviour of the samples (1) or measuring under power saturation (2) . This paper describes a new method in which the stable oxygen 18

isotope

0 bound as radical scavenger is detected by a nuc-

lear reaction. This technique makes use of molecular oxygen as a high capacity radical scavenger forming peroxides and hydroperoxides of high stability. In contrast to the stable oxygen isotope ^ 0 which represents 99.76% of natural oxygen, the stable isotope 1 80 can undergo low energy proton induced nuclear reactions releasing α-particles of specific energy which are briefly na18 med 0(ρ,α)-reactions. Therefore, the probes are irradiated with X-rays under solid state conditions in an atmosphere of 18

pure

O2 gas, mounted in a nuclear reaction chamber of an ac-

celerator. A beam of low energy protons is focused onto the probes and the emitted α-particles are detected. The low natural abundance of 1 80 of 0.205 % in oxygen containing biological 18

molecules allows determination of small O-labeling indices from the measured α-particle counting rate Ν , the number of

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

650

incident protons N^, and the well known nuclear reaction probability (cross section) o according to N

18 0 Ν. t

Na(-%, Ν,·Ν ·σ t ρ 18

For quantitative analysis of the 0 concentration, the number of biological target molecules has to be determined by weighing the probes. The investigation of biological molecules which contain nitrogen atoms, however, even provides quantitative determination of their 180 content without involving ΝP and the number of target molecules because the stable nitrogen isotope 15Ν also reacts with the incident protons releasing α-particles of higher energy. Since there is no artificial isotope enrich15 ment, the Ν content is given by the natural abundance of 0.36 % , and the counting rate of these α-particles depends only on the product Ν,·Ν . Therefore, in the above mentioned Ρ 15 equation, the product Ν. · pΝ can be substituted by αΝ ( Ν) if the α-particle counting rates of 180 and 15Ν are measured si18 15 multaneously. The ratio of 0 and Ν α-particle counting rates multiplied by the number of nitrogen atoms per molecule re18 presents a measure of 0 trapping per molecule which in turn corresponds to the radical yield.

Method Amino acids were dried onto gold foils and irradiated with 30 18 5 kV X-rays under C>2 gas at constant dose rate of 2.5>10 Gy/ hour. Until the nuclear reaction measurement probes were sto1 18 red under vacuum in order to remove interstial O2 gas. The measurements were carried out at the 350 kv-accelerator at the Institut für Kernphysik der Universität Münster. This machine supplied a 330 2keV proton beam of about 0.1 μΑ which was spread over 1 cm .To prevent heating of the probes the suppor-

651

ting gold foils were cooled to -60 °C. For α-particle counting a silicon surface barrier detector was positioned at an angle of 135° to the beam direction at a distance of 30 mm from the surface of the probes.

Results Figure 1 shows parts of the α-particle spectra of glycine, L-leucine and DL-tryptophan revealing α-particle groups of 15. and

18

0 at channel numbers 51 and 41, respectively.

Figure 1 : α-particle spectra of glycine, LLeucine and DL-tryptophan

c

100-

30

10

50

60

30

10

50

60

30

40

50

channel number ( 63 keV per channel )

18

To obtain a reasonable

0 concentration in the following expe-

riment, the amino acids were irradiated 12 hours to a total dose of 3.0 · 10® Gy. The ratio R of the α-counting rates is multiplied by a numeric factor of 20.5 which reflects the well 18 15 known ratio of the Ο(ρ,α)- and Ν(ρ,α)-reaction probabilities and the ratio of the natural abundances of 180 and 15N. This value has to be multiplied by the number of nitrogen atoms per molecule η to give the resulting mean number Ν of trapped 18

0 atoms per one hundred molecules: Ν = η · R · 20.5 .

652 From these data and the molecular weight MG it is possible to calculate the G-values, defined as the number of free radicals induced by 100 eV of absorbed energy, bearing in mind, that 18 two fixed

0 atoms correspond to one original radical. The re-

sults of the investigated amino acids are summarized in Table 1. Table 1 :

18

O-concentration and derived radical yield of irradiated amino acids

Glycine L-Threonine L-Cysteine

HCL

MG

Ra

Ν

G

75.1

0.11

2.3

0.5

119.1

0.08

3.3

0.4

175.6

0.17

3.5

0.3

L-Cystine

240.3

0.30

12.3

0.8

L-Aspartic acid

133. 1

0.19

3.9

0.5

L-Glutamic acid

147. 1

0.22

4.5

0.5

L-Glutamine

146.2

0.16

6.6

0.7

DL-Serine

105.1

0.33

6.8

1.0

L-Isoleucine

131.2

0.37

7.6

0.9

DL-Valine

117.2

0.50

10.3

1.4

L-Proline

115.1

0.71

14.6

2.0

L-Phenylalanine

165.2

0.72

14.8

1.4

L-Leucine

131.2

0.73

15.0

1 .8 1 .5

L-Arginine

210.7

0.31

19.1

182.7

0.49

20.1

1.8

L-Methionine

149.2

1 .09

22.3

2.4

DL-Tryptophan

204.2

1 .20

49.2

3.9

L-Lysine

HCL HCL

The total error of the R-values amounts to < ίì %. References 1.

Crippa, P.R., Tedeschi, R.A., Vecli, Α.: Int. J. Radiat. Biol. 25, 497-504 (1974).

2.

Rezk, A.M.H., Johnson, R.H.: Int. J. Radiat. Biol. 34, 337-348 (1978).

DETOXICAT ION OF OXYGEN FREE Helmut

RADICALS

Sies

Institut

für

Physiologische

Moorenstrasse

Chemie

I, Universität

5, D - 4 0 0 0 - D ü s s e l d o r f ,

Düsseldorf,

W.Germany

Introduction Although

the l a n d s c a p e

become s i z e a b l e

of the f i e l d

in r e c e n t y e a r s ,

to be o v e r v i e w e d here

still

of w h i t e

patches.

dicals?

C e r t a i n l y not ground s t a t e oxygen,

self;

however,

dical(HOj), organic

and i t s

and o f c o u r s e

oxygen-centered

peroxy(ROO') gen which

ra d i c a l s ,

the h y d r o x y !

radicals

Thus,

and e x c i t e d

there

quantitative scarce. cific

states

This

network pertains,

subcellular

on t h e s e

locations.

n o t a b l y the h y d r o x y !

radical,

tially

its

to a s k i n g f o r

reactivity their

site

radical

is

While f o r this

of f o r m a t i o n .

terms

some of the

problem b o i l s

radicals

large distances

is

low

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

yet spe-

radicals, high which

away from

For example, the s u p e r o x i d e

t h o u g h t to be of c o m p a r a t i v e l y

form and

down e s s e n -

due to i t s

t h e r e are o t h e r

relatively

which

one a s k s a b o u t

r a t e of g e n e r a t i o n

úi loco nascendi,

a p p a r e n t l y may t r a v e l

in b i o l o g i c a l

oxy-

molecules.

oxygen s p e c i e s

if

oxy-

These

and r e a c t i o n p a t h w a y s ,

in p a r t i c u l a r ,

and

of

molecular

of oxygen i n o r g a n i c

of c h a i n s

information

forms

context.

singlet

ra-

and the

such as the a l k o x y ( R O ' )

is a f a m i l y of r e a c t i v e

an i n t e r l i n k e d

radical(HO')

in a b i o l o g i c a l

it-

superoxide

the p e r h y d r o x y

t h e r e are o t h e r r e a c t i v e

s h o u l d be i n c l u d e d

ra-

the d i r a d i c a l , as the

p r o t o n a t e d form,

comprise e x c i t e d forms of oxygen, e . g . gen('Oj)

areas

What i s meant by the term, oxygen f r e e

i n a d d i t i o n to such s p e c i e s

anion r a d i c a l ( O j )

has

t h e r e are numerous

anion

reactivity.

654

As d i v e r s e

as t h e s e

oxygen s p e c i e s a r e ,

o f measures to c o u n t e r a c t t h a t can be i n i t i a t e d levels

non-enzymatic

the term, a n t i o x i d a n t s , enzymes, of c o u r s e , hydroperoxidases a variety

the p o t e n t i a l l y

but a l s o

enzymatic

are the s u p e r o x i d e peroxidases.

localizations zinc,

content,

to b i o l o g i c a l

i.e.

in t h i s

area

is utterly

to p r e s e n t a r e v i e w o f t h i s

would be i n v a i n . n o g r a p h s on t h i s

Fortunately, topic

on the B i o l o g i c a l from o u r s e 1 v e s ( 8 ) .

Basis

nature, major "oxida-

metabolites

its

actuality

in a small

comprehensive early

e.g. 1983,

of D e t o x i c a t i o n

Therefore,

on some comments on m o n i t o r i n g current

in

a c t i v e at p r e s e n t ,

field

are a v a i l a b l e ,

c o v e r the time from 1979 u n t i l

b l e models f o r

way,

selenium.

the s o - c a l l e d of oxygen

sub-

catalysis

i n c o p i n g w i t h the one

the damaging e f f e c t s

in

systems.

As the r e s e a r c h attempt

and

t h e s e enzymes have wide d i s t r i b u t i o n

stress",

and

by s p e c i f i c

i n the

iron(heme)

problem t h a t a e r o b i c m e t a b o l i s m e n t a i l s : tive

various

peroxidase

i n a complementary

involvement

manganese,

essentiality

These

and

They are c h a r a c t e r i z e d ,

which o v e r l a p

including

systems.

dismutases

glutathione

activity

form of metal

their

It

repair.

denoted by

cellular

all

all

interception,

and by a s p e c i f i c

underscoring

reactions

includes

and q u e n c h e r s

by a h i g h c e l l u l a r

Further,

It

scavengers

general,

copper,

repertoire

prevention,

such a s c a t a l a s e ,

of other

the

hazardous

by oxygen m e t a b o l i t e s .

of p r o t e c t i o n :

comprises

so i s

this

and mo-

overview w i l l and on some

selection

and by the p r e d i l e c t i o n s

being

of the

which

a chapter

of Oxygen Free

brief

of t o x i c i t y

study, with their

reviews

Refs.(1-10) including

an

chapter

Radicals center suita-

b i a s e d by

author.

655 Monitoring

The

of

Toxicity:

detection

even

genous were

With

compound

or a l a n i n e These

of

ral,

of t h e

with

the

toxic

is m o r e of

The

likely to

lactate are

to be

Similarly,

generally

because

the

causative in

reticulum.

intracellular

in

reflect

the

associated,

endoplasmic

employed.

change

they

for

exo-

as

dehydrogenase

a late

locale

and

some

to a n

useful(11)

or C a ^ + .

already

effect,

the

of d a m a g e

as

liver)

indicate

membrane.

quite K+

of

such

provided

penetrability

found

release

has

radicals,

geneThere-

membranes

have

been

for.

of t h e

field as

branes

of

biochemical

after

leave

introduced

peroxidation

ethane

ambient

the

or

toxicology.

pentane(and

least

Ethane to

with

at

damage

are

in p a r t , is the

because

pentane.

The

organs

fatty

of

into

from

lipids the

mem-

and

alkane

for

low

rate

of

methodology

with

intact

and

eel 1 s ( 1 4 )

the-

external

preferable its

pro-

hydrocarbons

of

into

as

acids

liberated

fragmentation

probably

isolated

of a l k a n e s

Volatile

others)

organism,

membrane

or

monitoring

and

as c o m p a r e d

mals(13)

the

of p o l y u n s a t u r a t e d

peroxidation

space.

monitoring

has

metabo-

been

ani-

descri-

recently.

Singlet

molecular

cay

of

lipid

has

also

technique and

oxygen(^02)

hydroperoxides

found

radicals. 634

was

enzymes

the m e m b r a n e s

e t al( 12)

bed

the

however,

Riely

lism

of

plasma

ducts the

as

probably

indicators

looked

such

by o x y g e n

toxicity,

cells,

NADH

cellular

development

fore,

cellular as

Methods

exerted

aminotransferase^rom

reactions,

se

such

parameters

status

such

of

New

effects

isolated

ion m o v e m e n t s release

the

toxic

the definition

difficulty.

the

of

Some

increasing

use

B o v e r i s et a 1(15) of

703

monitoring nm)

to

the

intact

as

or

a product

via

an

in the applied

singlet

cells

and

oxene study the

of

the

radical

transfer of

effects

single-photon

oxygen organs.

dimoi The

de-

mechanism of

oxygen

counting

emission(at technique

has

656 been a d a p t e d to a f f o r d measurements w i t h e x p o s e d o r g a n suspensions

of i s o l a t e d

enzyme r e a c t i o n s

cells

or s u b c e l l u l a r

in s o l u t i o n ( 1 6 ) . not i n i t s e l f

is

although there

are good c o r r e l a t i o n s

of malondialdehyde spectral

superoxide dicals.

Singlet

anion radical

This

chemi1uminescence without

not be p r e s e n t e d h e r e .

redox

i n the

which l e a d s

in r e c e n t work u s i n g

red

from the lipid

ra-

has been a m a t t e r o f Suffice

f o r m a t i o n can be d i s s o c i a t e d ;

f o r example,

peroxidation,

intervening it

to s a y

i n d i c a t e d by

this

has been

p a r a q u a t as a

de-

that

to the p r o d u c t i o n

02° and t h a t of membrane d e c o m p o s i t i o n as aldehyde

and to

singlet

accumulation

oxygen may a l s o a r i s e

itself

of redox c y c l i n g

of

of l i p i d

between the

area of oxygen c h e m i s t r y

bate and w i l l the p r o c e s s

indicative

and l o w - l e v e l

region(17).

fractions,

The o c c u r r e n c e

o x y g e n as such

surfaces,

of

malondishown,

one-electron

cycler(18).

The r o l e

of NADPH : q u i n o n e ox i d o r e d u c t a s e ( D T - d i a p h o r a s e )

counteracting carrying

the o n e - e l e c t r o n

out the d i r e c t

reaction(19)

by i t s

reduction of quinones

has r e c e n t l y

In t h i s

work(20),

monitor

the s t e a d y s t a t e

pathway and i t s

cycling

been d e m o n s t r a t e d

low-level

t h r o u g h the

m o d u l a t i o n by d i c o u m a r o l

of

in a t w o - e l e c t r o n

i n the i n t a c t

chemi1uminescence

of f l u x

in

capability

liver.

was f o l l o w e d

to

single-electron

as an i n h i b i t o r

of

DT-diaphorase. When h y d r o p e r o x i d e s

are formed d u r i n g o x i d a t i v e

can be d e t o x i f i e d .

Hydrogen p e r o x i d e as w e l l

peroxides al

can be r e d u c e d

matrix(21)

disulfide,

upon o x i d a t i v e

reaction.

stress,

Cells

so t h a t the e f f l u x

that there e x i s t s

concentration

these hydro-

mitochondriThe

resulting

of flux

were f o u n d to r e l e a s e of GSSG i n t o

space can be used as a parameter ( 2 3 ) ,

servation lic

and i n the

GSH p e r o x i d a s e ( 2 2 ) .

GSSG, can be m o n i t o r e d as an i n d i c a t o r

the GSH p e r o x i d a s e ternal

i n the c y t o s o l

by the s o l u b l e

stress,

as o r g a n i c

a relationship

and the r a t e of t r a n s p o r t

the

based on the

between the out of

the

through GSSG exob-

cytosocell(24).

657

Models

to S t u d y

The r o l e ther tion the

of

the

Detoxication

of

various

detoxication

systems

by d e p l e t i o n of

lenge.

of

that

cells

to

Further,

a loss

note act

of

that with

tion

The

deficiency

oxidative cation was

tective as

of

also

damage

(T.

of

example,

of

cannot

and t h a t

for

upon t h e

led

of to

or

used

out

hold.

be

highacti-

the

re-

interest

to

directly

inter-

prior

reduc-

fatty

acid

lung

to

from

the

other

alpha-

identify

radicals, or

for

example

liver(31).

defense,

on t h e

Fortifi-

other

in

addition

of f l a v o n o i d s

liver

studying

pro-

such

of A n t i o x i d a n t

of oxygen

capable practice

h a n d , an

hand,

the

dama g e ( 32 , 33 ) .

development

drugs

to

example,

detoxication the

such as

attempts

Fortification

in c l i n i c a l

On t h e

in

by o x y g e n

antioxidant

useful,

nitroaromatic

cruzi)(34).

itself to

may n o t

in

chal-

in

not always

antioxidants

a place

perfused

Depletion

has

are

decrease

to o x i d a t i v e

is

peroxidized

r a d i c a l - d e r i ved

lowered c a p a c i t y

nifurtimox)

in

observed in

of

the

to membranes

type

effect

some p r o t o z o a For

found

found q u i t e

cyanidanol

to

GSH p e r o x i d a s e

It

ei-

deple-

shown,

depletion

can c a r r y

hydroperoxides

non-enzymatic

hyperoxia

Applications The

of

this

The

order

GSH p e r o x i d a s e

GSH p e r o x i d a s e , of

studied

requi red(30) .

has e a r l y

studying

susceptible

hydroperoxides(27) .

release

been

in

glutathione

non-Se-dependent

membrane-bound

tocopherol in

the

Se-dependent is

selenium

by GSH S - t r a n s f e r a s e s

organic

phospholipid

that

Se-dependent

because

hydrolytic

has

experiments.

p e r o x i d a t i o n ( 28 , 29 ) seems

of

as e x h i b i t e d

duction

view

Radicals

GSH p e r o x i da s e ( 2 7 ) has

become more

the

lipid

critical,

and o f

Se-dependent

However,

may l e a d

vity

by f o r t i f i c a t i o n

glutathione(25,26)

activity

general,

ly

or

Oxygen

radicals

of

redox-active

of

redox

with

Defense

in drugs.

cycling(e.g.

Trypanosomiasis

increased

capacity

of

658 detoxication protection cation

of

matory types

of

of

oxygen

some

superoxide

responses of

in

diseases

Glutathione

has

found

disease,

a lethal

of

China(39). course,

outside

This

the

brief

several cal

is

of

exposé

in

appli-

decreases and

cystitis to

also

and

in

other 36).

other Keshan

a province

antioxidants in

the

inflam-

several

alleviate

developing chapter

in

(see

shock(37,38)

shown

chemical

topical

in

in

nutrition,

itself

and

is

scope(40-42).

is o n l y in t h e

problems

in

was

important

linkage

detoxication

led to

application the

arthritis(35)

application

use

present

of

has

cardiomyopathy

The

found

radiation

Selenium

a highly

kinds

medical

dismutase rheumatoid

conditions.

has

Interestingly,

including

clinical Red

radicals

organs.

intended now

basic

in c l e a r l y

to

between

indicate the

sciences

defined

that

study

and

of

the

there

oxygen

are radi-

application

to

conditions.

Ac k n o w l e d g e m e n t Work

coming

Deutsche nismen rium

from

the

author's

laboratory

Forschungsgemeinschaft,

toxischer

für

Wirkungen

Wissenschaft

und

von

was

supported

Schwerpunktsprogramm

Fremdstoffen",

Forschung,

and

by

by

the

"MechaMiniste-

Nordrhein-Westfalen.

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3.

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29.

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& S iegers,C.Ρ.: Res.Comm.Path . Ρ harm.27 ,119-128

30.

Grossmann,Α.

31.

Nishiki.K.,

Oshino,N,

160,343-355

(1976)

Slater,T.F.

& Eakins.M.N.

(1980)

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of 33.

Liver

& W e n d e 1 , A. : E u r . J . Β i o c h e m . ( 1 9 8 3 ) in Jamieson.D.

Cadenas,E.,

Müller,Α.,

Sies,Η.:Biochem.J.214, 34.

Docampo.R., Vol.VI in

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in:"New

Diseases"84-89 , Karger

& Chance , Β . : Β iochem . J . Trends

Basel

Βri gel i u s , R . , 419-487

Moreno,S.Ν.J.

press

in

the

Therapy

( 1975 ) Esterbauer,Η.

&

(1983)

in:"Free

(W.A.Pryor,ed.)Academic

Radicals

Press

New

in

York

Biology" (1983)

press

G o e b e l , Κ.Μ . , S t o r c k . U .

& N e u r a t h , F. : L a n c e t

1,1015-1017

(1981) 36.

Puhl.W. und

& Sies,H.,

37.

Reichard,S.Μ.,

38.

Kosugi,I.,

37-45 and

Bailey,N.M.

Tajimi.K.,

Cellular

Keshan

Einsatz,

- Dismutase

per i m e d ,

Erlangen

-

Biochemie (1982)

& Gal ν i η , Μ . J . : A d v . S h o c k

Res.5,

(1981)

Li s s , I n c . N e w 39.

eds. :Superoxid

therapeutischer

Aspects York

Disease

0hmura,A. of Shock

& Okada.K.

i η :"Molecular

and T r a u m a " 2 5 3 - 2 6 9 ,

Alan

R.

( 1983)

R e s . G r o u ρ :Ch i η . M e d . J . : C h i η . M e d . J . ( P e k i η g,

Engl .Ed. ) 92 , 4 7 7 - 4 8 2

( 1979)

661 40.

Ta ppel , A . L . i n : " F r e e

Radicals

Vol.4,1-47,

Press,New

41.

Witting,L.A.

Academic in:"Free

Vol.4,295-319, 42.

Diplock.A.T.

Radicals

Academic

York

in

( 1 980 )

in Β i o 1 o g y " ( W . A . P r y o r ,ed. )

Press,New

in:"Selenium

(J . E .Spall hol ζ , J . L . M a r t i n Avi

in β i o 1 o g y " ( W . A . P r y o r ,ed.)

York

Biology

(1980)

and

& Η.E.Ganther,

Pubi . . W e s t p o r t , C o n n e c t ! c u t

Medicine" e d s .) 3 0 3 - 3 1 6 ,

(1981)

DISCUSSION MICHELSON: Your last slide is a very simplified version, because you stopped at NADPH; to have that you need glucose-6-phosphate dehydrogenase, for that you need glucose-6-phosphate, for that you need ATP, so that you are going to have loops outside of this whole system, to have a selfsustaining approach. I think we should also decide what is a glutathione peroxidase and what is a glutathione transferase. There is a difference in substrate specificity: glutathione peroxidase will handle only a freefloating lipid hydroperoxide; if this is in a membrane, it is not attakked. And GROSSMANN and WENDEL (these proceedings, p. 719) show that even if it is in a simple lecithin, it is not attacked. It can only be attacked after previous action of phospholipase A 2 to liberate the lipid hydroperoxide. Are there any mechanisms for repairing cell membranes? The simplest approach is phospholipase A 2 , which nicks out the damaged fatty acid, another enzyme comes along, hits the lyso-lecithin and puts in a new acyl group. With respect to glutathione transferases the substrates that interest us are not only epoxides, but endoperoxides, di-substituted peroxides (R2OOR2) as well as simple hydroperoxides. The product is not an alcohol, because it is a nucleophilic attack of glutathione with inversion at the carbon center to give a glutathionyl adduct. So before you call something a non-selenium glutathione peroxidase, look at the product: if it is hydroxyl, it is a peroxidase, if there is glutathione in it, it is a transferase. SIES: I fully agree with this and I can only say that because the overview is very short I had to omit a few things. SUTTON: In your talk today and in an earlier one it was suggested that singlet oxygen could be derived from interaction of two superoxide molecules. Is that right? SIES: SUTTON:

Yes. I thought this was thermodynamically impossible.

662 SIES: Well, I think that is the problem of the chemistry of excited oxygen species. We have shown from our point of view that with menadione, for example, we can get a low level chemiluminescence without other parameters of lipid peroxidation showing any sign of membrane damage (Arch. Biochem. Biophys. (1983) 224, 568-578). That is the basis of that statement. SINGH: First, why was vitamin C left out from the antioxidant list? Secondly, how sure are you that hydroperoxides do indeed give singlet oxygen? SIES: Vitamin C is included in the step vitamin Ε-vitamin C-NADPH. (See Dr. NIKI's contribution). Hydroperoxides e.g. t-butyl hydroperoxide as such, added without anything else, do not give us any signal. We need metabolism with microsomes or other enzymes. PRYOR: I would like to comment on the correlation of SOD with lifespan. I have just finished editing CUTLER'S chapter for volume 6 of "Free Radicals in Biology". I think it provides the strongest evidence for a free radical involvement in ageing that I have seen. He shows a very strong correlation between SOD levels and lifespan for different species. However, he does not report data on fluctuations among individuals of a single species. The species dependence, however, is fairly large. I believe CUTLER'S evidence for SOD correlations with lifespan and for free radical involvement in ageing is quite strong. FORMAN: I think it should be pointed out that evidently thiol transferases are extremely important in hepatocytes due to the fact that there is so much conjugation going on there rather than reduction. In other organisms there isn't as much of that enzyme. Secondly, the thiol transferases will not use hydrogen peroxide as a substrate, while the selenium glutathione peroxidase does. SIES: Yes, I think Ray BURK has studied different organs, and it is interesting that some organs seem to have much less of the selenium-containing enzyme and therefore an increase in the non-selenium activity. But one cannot always match this by adding similar antioxidant defenses. Of course, the glutathione transferase Β activity with free-floating hydroperoxides is worth considering as an antioxidant enzyme. BORG: My concern is with attribution of red light emission to the presence of singlet oxygen. We have followed the autoxidation of a number of substances by single photon counting using cut-on filters to get crude histogram spectra. We can find strong red emission - even 'redder' than dimoi emission by looking at just ascorbate and hydrogen peroxide. Your own first slide showed a nice way for peroxyl radical recombination to give rise to blue-green emission from excited carbonyls. We see that, too. KONINGS: I would like to make a comment on the repair possibilities of biological membranes. When we looked at the permeability of tumor cells, after irradiating them with a low dose (5 Gy) of X-rays, we see a permeability change for fatty acids in these cells. This damage of the membrane is repaired within one hour. We wanted to know if the polyunsaturated fatty acids in the membrane were attacked and repaired. We did turnover studies by incorporating radioactive palmitic acid and radioactive arachido-

663 nie acid in the membrane phospholipids. After investigating the turnover rate after radiation we didn't see any difference in turnover of the polyunsaturated fatty acids as compared to the saturated fatty acids. So we assume that in this situation the polyunsaturated fatty acids don't have a function in the damage of the membrane. There indeed is damage to the membrane which is repaired rapidly, but this doesn't seem to be connected with polyunsaturated fatty acid peroxidation.

ROLE OF ACTIVATED OXYGEN SPECIES IN THE MECHANISM OF CYTOTOXICITY OF CYSTEAMINE

R.D. Issels Klinikum Großhadern, Ludwig-Maximilians-universitat, Munich,W. Germany J.E. Biaglow Case Western Reserve University, Cleveland, Ohio L.E. Gerweck Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 U.S.A. Introduction Elucidation of the molecular mechanism of heat-induced killing is important because of the potential useof hyperthermia in cancer therapy and for understanding the basic cellular processes disrupted by heat. The mechanisms by which heat kills cells involve one or more cellular organelles or molecules:the plasma membrane, the lysosome, denatured or otherwise altered proteins and DNA. Also the potential for cell killing in mammalian eel 1 s of a number of widely used chemotherapeutic agents has been reported to be enhanced by heat (1). The exact mechanism of this enhancement is not fully understood and might differ with variant compounds from each other. Based on preliminary results in our laboratory we developed a new hypothesis about the molecular effects of hyperthermia (3). In the cell many reactive intermediates, such as various radicals including activated oxygen species are produced during metabolism. These intermediates are normally either necessary components of metabolic reactions or inevitable side products. However, they are toxic and may damage the functional

integrity of the cell,

if cellular control and protective mechanisms became insufficient. Following hyperthermia the relation between the production of potentially toxic species (e.g. certain radicals) and protective mechanisms may deteriorate. Superoxide radicals (.0J) are reactive intermediates produced within the cells and they are controlled enzymatically by superoxide dismutase

(SOD).

The dismutation of .OJ catalyzed by SOD leads to the generation of hydrogen

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

666 peroxide (H 2 0 2 ) which is removed by either catalase or glutathione dependent peroxidase. Activated oxygen species are also generated during the autoxidation of sulfhydryl compounds (6) and more recently, thermosensi tization by these compounds was observed at different concentrations^,4). The aim of this paper was to address the question of whether or not the generation of activated oxygen species by cysteamine is involved in the interaction between heat and drug treatment.

Methods Chinese hamster ovary cells were maintained in exponential growth at 37 e C. in a 5% C0 2 atmosphere. The population doubling time of those cells was approximately 13 hr and colony forming efficiency 80 - 90%. Twenty-four hours prior to cysteamine exposure or heat treatment, cells were trypsinized, counted and dilutions of known cell numberswere inoculated in four to six replicate flasks. Cysteamine was added directly, and after 2 hr drug exposure at 37° C or 5°C the medium was removed. Cells were washed twice and incubated 7 to 9 days in fresh medium for colony forming ability.In the case of heat treatments flasks were placed in a waterbath at 44° 30 minutes after adding cysteamine and then returned to an incubation at 37°C. The total time of exposure to the drug was 2 hr including the time of heat treatment. Viable colonies ( 50 cells) were fixed, stained and counted. The surviving fraction was determined after correction for cellular multiplicity (approx. 1.8) determined at the time of the heat treatment. Catalase(50jjg/ ml) and superoxide-dismutase (10 jjg/ml) were added as indicated (both obtained from Sigma Chemical Co). Oxygen consumption in complete McCoys 5a medium buffered with 0.02 M Hepes, pH 7.4 without cells was measured using a YSI Model 53 oxygen monitoring system at different concentrations of cysteamine. After adding the drug to the reaction vessel (total volume 3.0 ml) the electrodewas then inserted and the reaction mixture was stirred. The initial rate of oxygen uptake over time was followed on the recorder. For measurements at different temperatures the reaction vessel was equilibrated to the desired temperature by circulating water before adding the drug. In some experiments catalase was added to the reaction mixture after 10-12 min using a Hamilton Syringe introduced into the test chamber through the access slot of the oxygen

667 electrode plunger. The data were expressed as nmol 0 2 uptake/min/ml

from

the initial oxygen uptake (1-2 min).

Results Table I shows the survival of exponentially growing CHO cells exposed to a fixed concentration of cysteamine at different temperatures and the effect of catalase or superoxide dismutase.

Cysteamine

Temperature

conc.

Survival

(° c)

(mM)

Clonogenic

(%)a

no drug

5

100

0,4

5

90

no drug

37

100

0,4

37

1,0

0,4

37

1,0

+ S0D b 0,4 + Cat. 0

37

100

no drug

44

15

0,4

44

0,008

44

0,008

0,4 + SOD 0,4 44

+ Cat. Table I

a

15

The value indicated is the average of 3 experiments. Variants between experiments were less than 10%

b

SOD, superoxide dismutase (10 fjg/ml)

c

Cat., catalase (50 ^g/ml)

Although no significant cell killing was found after 2 hr exposure of cells to 0,4 mM cysteamine at 5° C, substantial toxicity was observed at 37° C.

668 When drug-treated cells were exposed to a 30 minutes 44°C heat treatment (surviving fraction 15% in the absence of drug) toxicity was markedly enhanced. Cysteamine toxicity was not modified by the addition of SOD but completely blocked by the addition of catalase. Modification of toxicity by catalase was observed at 37°C and 44°C. The rate of oxygen consumption at 0.4 mM cysteamine was measured at different temperatures (see Table II). At 22°, 37° and 44° oxygen uptake was higher with increasing temperatures, however at 5° no oxygen consumption was observed. In the presence of catalase (50 ug/ml) the rates of oxygen uptake was substantially reduced at each of these temperatures but relatively increased with higher temperatures. In the presence of superoxide dismutase (10 ug/ml) no reduction of oxygen uptake was found, but slightly higher rates of uptake at elevated temperatures (37° and 44°)were observed as compared to cysteamine alone.

Table II Oxygen consumption in complete McCoys 5a medium buffered with 0.02 M Hepes, pH 7.4 was measured at different temperatures as described in "Methods". For these studies, cysteamine was present at a concentration of 0.4 mM. The values represent the initial rate of oxygen uptake.

Temperature

Oxygen uptake (n mol/min/ml) +Superoxide Cysteamine +Catalase Dismutase (0.4 mM) (50/jg/ml) (10 /ug/ml) 0b

5°

a b

0b

na

0b

3

22°

4.2

1.8

4

3

37°

13.2

8.3

17

3

44°

25

28

3

11

Number of experiments The value indicated is the average of three experiments. Variants between experiments were less than 10%.

669 Discussion and Conclusions The autoxidation of sulfhydryl compounds like cysteamine in the presence of oxygen involves the univalent reduction of oxygen to .02 and the formation of H 2 0 2 by a consequent dismutation reaction (6). Both of these activated oxygen species are produced extracellularly and are freely diffusible across membranes. Concerning the toxicity of cysteamine, we found that the autoxidation of the drug at 0.4 mM accumulates H 2 0 2 in the medium within 15 to 20 minutes of incubation at 37°C (4). Moreover, the absence of cytotoxicity at 37°C and 44°C under identical conditions by adding catalase indicates that the generation of H 2 0 2 plays a key role in the mechanism of thermosensitization. The inability of SOD to inhibit the cytotoxic effects of cysteamine by removing .0j at either temperature may be related to an alternate or competing pathway for the reduction of the metallic compound, which is needed for the generation of hydroxyl radicals (.OH) by a Fenton reaction (2). The following reactions provide a basis for rationalizing the data described here. a.

R - S"

+

b.

RSH

+ RS· + 0 2

c.

.Ol

+ Me11

d. e.

.Oí

+ Me

n

.0¡

1

+ R-S-S-R + H +

Me n " 1 + 0 2

+ .0¡ + 2 H

H202

R - S· + Me11 "

Me"

~

1

+

— - H202 .OH

+ 02 + OH"

+ Me"

At pH 7.4 cysteamine (RS~) might directly reduce a transition metal (Me11) 2

such as Cu

3

+

or Fe

+

(see equation a). For example, the reduction of ferri-

cytochromecatpH 7.4 in the presence of cysteamine was not inhibited by SOD (R.D. Issels and L.E. Gerweck, unpublished results)

It has to be

assumed that these transition metals are mostly present in a complexed form.

Alternatively, the reduction of transition metal can occur indirect-

ly via .02 (equation c) generated by a thiyl radical in the presence of oxygen (equation b). In both competing pathways H 2 0 2 is formed by the spontaneous or catalyzed dismutation of .02 (equation d) and accumulates in the medium (4). The interaction between H 2 0 2 and the reduced form of transition metal leads finally to the production of .OH (equation e), which is the most powerful one-electron oxidant in biological systems and indiscriminately reactive with e.g. DNA or membrane compounds. Since this reaction is not depending on .0j,adding of SOD does not protect against toxicity of

670 cysteamine. In contrast, removal of H 2 0 2 by catalase added to the medium should block the toxicity of cysteamine and this effect was observed in our studies. The role of activated oxygen species might be a common mechanism for the heat potential of other cytotoxic agents which are also known to produce free radicals (e.g. adriamycin, bleomycine and mitomycin). Further investigations are in progress to determine if the mechanism of the interaction between heat and such chemotherapeutic agents are similar to our results with cysteamine.

References 1.

Hahn, G.M.: Potential for therapy of drugs and hyperthermia. Cancer Research 39, 2264 - 2268 (1979).

2.

Halliwell, B.: An attempt to demonstrate a reaction between and hydrogen peroxide. Febs. Lett. 8 - 1 0 (1976).

3.

Issels, R.D. and Lengfelder, E.: Superoxide radical formation in cancer - a new hypothesis of molecular action of hyperthermia. Strahlentherapie J 5 8 , G, 383 (1982).

4.

Issels, R.D., Biaglow, J.E., Epstein, L. and Gerweck, L.: Enhancement of cytotoxicity of cysteamine by heat and its modification by catalase and superoxide dismutase. Cancer res. (paper submitted) (1983).

5.

Kapp, D.S. and Hahn, G.M.: Thermosensitization by sulfhydryl of exponentially growing Chinesehamster cells. Cancer res. 39, 4630 - 4635 (1979).

superoxyde

compounds

(This work was supported in part by grant 300/402/532/2 from the German Foundation for Cancer research and grant Is 31/-1 from the Deutsche Forschungsgemeinschaft.) DISCUSSION

KRINSKY: I might have missed this, but did you say that you tried to measure superoxide production by cytochrome c reduction on the outside of the cells, and did you get any? ISSELS: No. the NBT test cytochrome c not detect a

Superoxide radical production by cysteamine was assayed using because using cytochrome c test you will get immediately a reduction after adding cysteamine. So, by this method you can superoxide-dependent cytochrome c reduction.

671 KRINSKY:

I see. And this is again in the intact CHO cells?

ISSELS:

No, this assay was performed in a cell-free system.

KRINSKY: Then in your survival where you are using the intact cells you find a protection by catalase and the failure of protection by the SOD. I wonder if it is possible that what you are observing is caused by membrane permeability towards hydrogen peroxide, that is being generated in the cell and going into the medium, and possibly the lack of permeability towards the superoxide anion. Under those circumstances could catalase be effective as an extracellular protective agent, whereas the added SOD may not be demonstrating any function. ISSELS: I think this is an important question. We assume that superoxide radical and also hydrogen peroxide are freely diffusible through the cell membrane. Moreover, I should mention some preliminary results we obtained from experiments using an animal system. As soon as the cell density is increased, like in an animal tumour or by adding erythrocytes in ¿n vitro experiments during the cysteamine exposure, we did not find any toxicity. We explained this by an effect of intrinsic enzymatic activity in those feeder-layer cells which are able to scavenge superoxide and hydrogen peroxide generated by the thiol. SINGH: Could the change in the protection by catalase and superoxide dismutase be due to the effect of change of temperature on their reactivity or that of 05 and H 2 0 2 ? ISSELS: Related to this point, we presented some data at the last International Meeting of the European Hyperthermia Group in London, June 1982 (ISSELS and LENGFELDER, Strahlentherapie (1982) 158, 383). In a system like we used for our cell studies, the ability of SOD to scavenge 0 2 was unchanged between 20° and 50°C. So I don't think that the effect of temperature on the activity of the enzyme is important. The second point of your question should be related to the lifetime of 0 2 . We observed in our results at 37° protection by catalase and no protection by SOD. If there exists a temperature dependency I would expect a difference to our experiments performed at 44°, but this was not the case. However, we need more detailed information about the influence of temperature on the molecular mechanism of cysteamine-dependent toxicity.

FORMATION OF S U P E R O X I D E A N D CELL DAMAGE UV-VISIBLE

IRRADIATION

OF

DURING

MELANIN

I . A . Menon, S. Persad, N . S .

Ranadive, H.F.

Haberman

Clinical S c i e n c e D i v i s i o n a n d D e p a r t m e n t o f P a t h o l o g y , Medical B u i l d i n g , U n i v e r s i t y of T o r o n t o , Toronto M5S 1A8, Canada

Sciences

Introduction

It

is

recognized

that

UV

radiation

etiology o f s k i n c a n c e r a n d

is

one

o f the

a g i n g o f the s k i n .

major

These

factors

in

the

effects are

more

common a n d more s e v e r e in people with red h a i r a n d l i g h t - c o l o u r e d

skin.

It is b e l i e v e d that melanin in the s k i n

p r e v e n t s o r d e c r e a s e s the

above

effects

of

irradiation

human

red

UV

radiation.

hair

(RHM)

eumelanin from b l a c k it w a s s u g g e s t e d

UV-visible produces

hair

(BHM).

that t h e s e

more

0*

of

than

pheomelanin the

F r o m t h e s e a n d o t h e r similar

differences

from

irradiation

in the photochemical

of

results

properties

o f the two t y p e s o f m e l a n i n s may c o n t r i b u t e to the harmful e f f e c t s o f the radiation

(1, 2).

In the p r e s e n t

s t u d i e s the p o s s i b l e

role o f O j

in

the

e f f e c t s o f i r r a d i a t i o n o f E h r l i c h a s c i t e s carcinoma cells in the p r e s e n c e o f the melanins was i n v e s t i g a t e d u s i n g an |n v i t r o cellular model.

Results The

melanins

(2).

Ehrlich

vapor

lamp ( 3 ) .

according

to

determined

were

prepared

ascites The

human

cells

release o f

'cr

Ranadive

according

from

carcinoma et to

al. the

hair

were

as

described

irradiated

using

from labelled cells w a s

(4).

Lactic

method

of

dehydrogenase

Bergmeyer

et

al.

mercury

determined (LDH)

was

(5).

The

(6).

The

amount o f H 2 0 2

formed w a s d e t e r m i n e d

amounts of N B T

r e d u c e d w a s d e t e r m i n e d b y m e a s u r i n g a b s o r b a n c e at 560

nm.

This

reduction

of

NBT

spectrophotometrically

previously a

was

inhibited

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

by

SOD

(7).

674 The photobiological effects of eumelanin and pheomelanin are in Table 1.

illustrated

The quantities of ^ C r as well as LDH released from Ehrlich

ascites carcinoma cells were larger when these cells were i r r a d i a t e d

in

the presence o f RHM than in the presence o f BHM. The v i a b i l i t y of the cells determined by t r y p a n blue exclusion test and the

transplantability

o f the cells in mice were decreased a f t e r i r r a d i a t i o n in the presence o f RHM ( 8 ) .

In view of our previous observations t h a t i r r a d i a t i o n o f RHM

produced more

than the i r r a d i a t i o n o f BHM, the possibility t h a t the

above effects on the cells were due to the O^ was i n v e s t i g a t e d .

Table 1.

Effects o f i r r a d i a t i o n of Ehrlich ascites carcinoma cells in the presence of melanine

Melanin

51

added

Inc.

Cr released (%)

LDH released {%)

Irr.

Inc.

Irr.

None

2

4

7

7

BHM

4

13

9

20

RHM

9

59

11

47

The cells were incubated in the d a r k ( i n c . ) or i r r a d i a t e d ( i r r . ) w i t h 200 ug/ml o f BHM or RHM or w i t h o u t e i t h e r melanin. Two

simple

systems,

exogenously added

one

generating

and

the

other

consisting

were employed for comparative purposes.

of The

cells exposed to x a n t h i n e - x a n t h i n e oxidase system u n d e r w e n t lysis (Table 2).

This lysis was s l i g h t l y enhanced by SOD, whereas it was completely

abolished by catalase and was p a r t i a l l y i n h i b i t e d by N B T .

The addition

o f Η ^ 0 2 produced cell lysis which was i n h i b i t e d b y catalase (Table 3 ) . Irradiation of

RHM was f o u n d to produce

H 2 0 2 and cell lysis

whereas

incubation in the d a r k d i d not produce any detectable amounts o f e i t h e r (Table

4).

One mM cysteine enhanced the

cysteine decreased the lysis

(unpublished

lysis whereas

results).

5 or

A d d i t i o n of

20 mM 1 mM

cysteine increased the formation o f H ? 0 7 as well as the cell l y s i s . SOD

675 Table

2.

Release

^1Cr

of

from

Ehrlich

ascites

carcinoma

formation o f H _ 0 ? a n d the r e d u c t i o n o f N B T b y x a n t h i n e o x i d a s e Reagents added

NBT

reduced

^1Cr

H^O^ formed

(UM)

(%)

None

Nil

Nil

SOD

Nil

Nil

7

Catalase

Nil

Nil

5

Nil

5

XO

system

XO

s y s t e m ,. S O D

XO

s y s t e m . catalase

XO

system N B T

The xanthine oxidase unit/ml

xanthine

concentrations

21.3

8.9

38

7.7

12.2

56

19.1

2.8

4

5.5

11

(XO)

oxidase

of

SOD,

units/ml a n d 62 μ Μ

s y s t e m c o n s i s t e d of 0 . 1 mM x a n t h i n e a n d in

3.0

ml

catalase

Medium

and

NBT

199. were

Where 165

the

accumulation

the cell l y s i s d u r i n g and

cysteine,

irradiation.

of

H202

and

eliminated

units/ml,

could

in not

the be

the 3,500

all

conditions

the

effect

Catalase and

decreased

in the p r e s e n c e o f

of

cysteine

during

it d i d not affect the l y s i s o f t h e cells d u r i n g

i r r a d i a t i o n in the p r e s e n c e o f R H M . melanin

under

the i n c u b a t i o n in the d a r k

However

a s well as d u r i n g

0.1

added,

respectively.

d i d not lower the amount o f H ^ O j formed or the cells l y s i s . abolished

system

released

(UM)

NBT

cells,

T h e formation o f H^O^ in the

RHM the the dark

i r r a d i a t i o n w a s d e c r e a s e d w h e n N B T w a s a d d e d to the

absence

o f cells.

determined

because

The

effects

irradiation

b r o u g h t about considerable reduction of

of

NBT

upon

of

NBT

in

cell

medium

lysis 199

NBT.

Discussion The

results

reported

here

show

that

irradiation

of

Ehrlich

ascites

676 Release of

Table 3.

51Cr

from Ehrlich ascites carcinoma cells treated with hydrogen peroxide

H 2 0 2 added (μΜ)

51

Cr released (%)

Without catalase

With catalase

Nil

4

1

6

5

33

5

10

ι»

100

53

5

1000

85

7

Where added, the concentration of catalase was 3,500 units/ml.

Release of ^ C r from Ehrlich ascites carcinoma cells and

Table 4.

formation of H^O^ during irradiation in the presence of RHM

Reagents added

H 2 0 2 formed (μΜ)

51

Inc.

Irr.

Inc.

Irr.

None

Nil

Nil

5

6

RHM

Nil

1.2

RHM, SOD

Cr released (%)

8

41

19

49

11

41

RHM, catalase

Nil

Nil

RHM, N B T

Nil

Nil

RHM, cysteine

2.3

5.5

24

69

RHM, cysteine. SOD

1.6

5.0

54

90

RHM, cysteine. catalase

Nil

Nil

8

42

RHM, cysteine. N B T

Nil

Nil

Where added the concentration of cysteine was 1 mM.

The

tions of other reagents were the same as in Tables 1 and 2.

concentra-

677 carcinoma

cells

percentages

in

the

of the

presence

of

release o f L D H

red

hair 51

and

melanin

Cr,

produces

indicating

cell

larger

lysis.

In

t h e r e f e r e n c e s y s t e m c o n s i s t i n g of x a n t h i n e a n d x a n t h i n e o x i d a s e the cell lysis was slightly enhanced by S O D ;

o n the c o n t r a r y

a b o l i s h e d b y catalase a n d p a r t i a l l y i n h i b i t e d b y these

results

H2O2

that the cell

formed

from

OJ.

concentrations of o f catalase

H2O2.

The

formed Since

OJ

by

the

to t h i s (2).

the

lysis

these

Treatment

system

formation

SOD

under

under

conditions

of

the

by

It

may

present

of

the

completely

this

system

is

cells

therefore

conditions

cells

in

the

is

be c a u s e d b y the H 2 0 2

be

been

in

the

not b y

by

with

various

As

expected

the

effects

that

from

to

the

of be

H202

generated.

presence

SOD,

formed from the O ^ .

from

mediated

reported

concluded

originated

dark

c y s t e i n e w a s i n h i b i t e d b y catalase b u t

eliminated

has

completely

It is e v i d e n t

p r o d u c e d d o s e - d e p e n d e n t cell l y s i s .

addition inhibited

lysis

it w a s

NBT.

this

of

lysis

RHM

and

seems

T h e cell l y s i s d u r i n g

to the

i r r a d i a t i o n in the p r e s e n c e o f R H M w a s not i n h i b i t e d b y S O D or catalase, a l t h o u g h the e n h a n c e m e n t b y c y s t e i n e o f the cell l y s i s d u r i n g was

abolished

involved

in

by

catalase.

This

suggests

that

p h o t o t o x i c e f f e c t s o f the m e l a n i n s .

two

irradiation

mechanisms

While the cell

lysis

are by

the melanin a n d c y s t e i n e in t h e d a r k a n d the additional effect o f c y s t e i n e during

the

irradiation are b r o u g h t

about b y

H^O^ o r i g i n a t i n g

from

O^,

l y s i s b y the i r r a d i a t i o n in the p r e s e n c e o f the melanin d o e s not seem to be mediated b y H^Oj

are

O^ or

formed

melanin-free

H^O^.

under

radicals

This

these

and/or

is i n t r i g u i n g

conditions.

singlet o x y g e n

It

because is

produced

both 0 ^

possible by

UV

that

and the

irradiation

may be i n v o l v e d in the l y s i s o f the cells d u r i n g the i r r a d i a t i o n .

T h e r e is c o n t r o v e r s y

in t h e l i t e r a t u r e w h e t h e r O j b y itself is biologically

t o x i c or w h e t h e r the a p p a r e n t t o x i c i t y o f 0 ^ is d u e to the H ^ O ^ , singlet

oxygen

xanthine additional evidence

oxidase

formed

information for

the

from

system in

toxicity

and this of

the

O^

the

regard. 0|

(9-12).

Our

phototoxicity

per

These se.

On

of

data the

results melanin do

not

other

.OH or

with

the

provides show

hand,

any they

d e m o n s t r a t e that the toxic effects o b s e r v e d in t h e s e s y s t e m s a r e d u e to

678 the

H^Oj

formed

the

toxicity

of

from

due

to

product

H^O^

Oj.

singlet

that

or

due

to

a combination

of

r e a c t i v e o x y g e n species may be r e s p o n s i b l e f o r t h e cell

Acknowledgements :

Supported

by

the

MRC

.OH

whether

present

is also p o s s i b l e

itself

The question or

It

is

primary

o x y g e n w h i c h may be f o r m e d i n s i t u is b e y o n d t h e scope o f t h e investigation.

H^O^

the

more

than

one

lysis.

(MA-5043).

Associate o f the Ontario Cancer Treatment and Research

H.F.H.

is

an

Foundation.

References

1.

Menon, I.A., Persad, S., J . I n v e s t . D e r m a t o l . 80, 202-206

2.

Persad, S . , Menon, 37, 63-68 ( 1 9 8 3 ) .

3.

Menon,

4.

Ranadive, N.S., Menon, I.A., D e r m a t o v e n e r 59, 493-497 ( 1 9 7 9 ) .

5.

Bergmeyer, H.U., Berut, G., Hess, B . : A n a l y s i s , Academic P r e s s , New Y o r k , 1963.

Methods

6.

Wang, R . ,

(1978).

7.

Beauchamp, C . ,

8.

Menon, I.A., Persad, S., C a n c e r Res. in p r e s s ( 1 9 8 3 ) .

9.

Fee, J . S . : Oxygen and Oxyradicals ( M . A . J . Rodgers, E . L . Powers, e d s . ) . p p . 205-239, 1981.

10.

Fee, J . S . :

11.

Halliwell,

B.:

T r e n d s i n B i o c h e m . Sci.

12.

English,

T.,

Lukens,

I.A.,

I.A.,

Haberman,

Haberman, H . F . :

Nixon, B . T . :

Haberman, (1983).

H.F.,

H.F.:

I.:

C a n . J . P h y s i o l . 58, 743-749 Haberman,

H.F.:

of

(1980). Acta

Enzymic

A n a l . Biochem. 44, 276-287 (1971 ) . Ranadive,

T r e n d s in B i o c h e m . S c i .

J.N.:

C.J.:

Photochem. P h o t o b i o l .

In V i t r o J_4, 715-722

Fridovich,

Kurian,

N.S.,

Haberman,

H.F.:

in C h e m i s t r y a n d Biology Academic P r e s s , New Y o r k ,

M a r c h 84-86 August,

J.Immunol.

130,

(1982). 270-272

(1982).

850-856

(1983).

679 DISCUSSION

LANDS: I was interested in the concept that one of the melanins might be more toxic than the other. However, the original idea was that somehow the eumelanin might be more beneficial. Have you ever done mixing experiments to see whether the eumelanin can diminish the effect of the red-haired melanins? MENON: When eumelanin is added to ¿ n vitro systems, it protects against the phototoxicity in several systems. SINGH: Based on the mechanistic studies you have described, can you suggest what sort of properties suntan oils should have to protect skin and reduce ageing? MENON: I do not yet have results on the action spectra of the phototoxic effect of pheomelanin. These results may indicate the compounds which could be effective sunscreens. As it stands, the best thing is to avoid UV radiation.

EFFECTS OF COPPER-DEFINCIENCY ON LIPID PEROXIDATION OF RAT MICROSOMAL MEMBRANES Gianna Maria Bartoli and Silvia Bartoli Istituto di Patologia generale, Università Cattolica S.Cuore Roma, Italia

Introduction It has been well established that tumor

cells have a low level

of oxygen defensive enzymes (1-5). Tumor transformation is accompanied by a loss of superoxide dismutase and glutathione peroxidase, which is proportional to the degree of growth rate; such conditions lead to an enhanced lipid peroxidation of cell membranes, hypothetically due to an increased steady state level of oxygen radicals. Moreover, tumor membranes have a more saturated phospholipid pattern and a different assembly and or ganization (6-8). On the other hand, ±t was suggested that lipid peroxidation products, such as hydroperoxides and aldehydes can inhibit the mitotic activity during cell division (9-13). From the data above considered it is possible to correlate the loss of defensive enzymes in tumor cells and the lipid peroxide production with the growth rate of the tumor. In fact the markedly decreased lipid peroxidative activity observed in tumor cells (4,6) can indicate that in transformed cells lipid peroxides become inadequate to control the mitotic activity in a proportion which is related to the growth rate of the tumor. The aim of the present work is to reproduce a model situation which would mirror that observed in tumor cells by treatment of rats with a Se- and/or Se-Cu-deficient diet for either six or eight weeks. Such treatment causes a deficiency of hepatic

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

682 SOD and GSH-Px (15,16) which might be related to the decrease of lipid peroxidation observed in these conditions.

Materials and Methods Copper and selenium deficiencies have been obtained with a previously described method (14-16). All experimental conditions have been reported elsewhere (5).

Results Table 1 shows the content of copper and cytosolic superoxide dismutase (SOD) in liver from normal, Se-deficient and Se-Cu-de ficient rats. As previously described (15,16), the Cu-deficient diet lowers the hepatic copper content to a value of about 13% of the control. In addition the Cu-Zn SOD in six weeks decrease to a value of 14% of the control. The Se-deficiency does affect neither the copper nor the Cu-Zn SOD. The treatment of the animals with the Cu-deficient diet for eight weeks does not induce a further decrease either of copper content or SOD level. Table1. CONTENT OF COPPER AND CYTOSOLIC SUPEROXIDEDISMUTASE IN LIVER FROM NORMAL,Se-DEFICIENT AND Se-Ou-DEFICIENT RATS Group

Cu ( xi/g w.w. ) 6 weeks

Superoxidedismutase ( Ai/g w.w.)

8 weeks

(5 weeks

8 weeks

Control

4,03±0,12(4)

4,03+0,12(4)

306,5±13,1(6)

306,5±13,1(6)

O-Se

4,23+0,60(3)

4,14+0,60(5)

316,8+46,6(5)

315f2±40,1(9)

CK3e-Cu

0,55+0,09(6)

0,54

43,5± 4,0(9)

65,0+11,3(8)

(2)

Valuesare expressed as mean + S.E.M. (number of observations)

683 Table 2. GLUTATHIONE PEROXIDASE ACTIVITY IN LIVER FROM NORMAL, Se-DEFICIENT AND 3e-Cu-DEFICIENT RATS Group

Se-GSH-Px (H202) (nncles/im-n/mg prot. ) 6 weeks

Control

194,8

(3)

Non Se-GSH-Px (t-BOOH) (nrroles/min/mg prot. ) 6 weeks

8 weeks 194,8

(3)

114,3

(2)

8 weeks 114,3

(2)

O-Se

7,9±0,3(8)

9,6+0,9(4)

113,9±9,0(4)

134,8+13,2(10)

O-Se-Cu

7,5±0,9(11)

8,0±1,1(9)

105,9+19,6(4)

239,2±44,9(7)+

Valves are expressed + S.E.M. (number of observations) + = Ρ 0,05 Copper deficiency was shown to induce an inhibition of Se-gluteathione peroxidase (Se-GSH-Px) (16). In order to differentiate the effect of the lack of SOD from that of Se-GSH-Px, animals were treated with a Se-deficient diet.After six weeks of treatment Se-dependent GSH-Ρχ- -was very low in both groups of animals being less than 10% of the control (Table 2). The treatment for eight weeks does not change the value of this enzyme activ¿ ty. The non Se-GSH-Px is not affected after six weeks of treatment, whereas after eight weeks the Se-Cu-deficiency induces an increase of non Se-GSH-Px, which is significantly higher than that observed in the Se-deficient rats. Fig. 1 shows the effect of Se- and Se-Cu-deficient diet on malon dialdheyde production of

microsomal membranes isolated six we-

eks after the treatment: the decrease of Se-GSH-Px is accompanied by a low ability of microsomal membranes to peroxidize endogenous unsaturated fatty acids in the presence of phenylhydra zine. The loss of both defensive enzymes (Se-GSH-Px and Cu-ZnSOD) causes a further diminution of phenylhydrazine-induced malondialdheyde formation,thus indicating that both enzymes are important in the regulation of the endogenous membrane lipid peroxidation. The loss of Se-GSH-Px for eight weeks is related to

684

Fig. Ί. Lipid peroxidation of liver microsomes from control,Sedeficient and Se-Cu-deficient rats, in the presence of 50 jjM phe nylhydrazine. Microsomal membranes (3 mg protein/ml)were incuba ted at 37°C in 0,15 M KCl, 50 mM Tris-HCl (pH 7,5)saturated with oxygen. Values are means + S.E.M. of 5-11 experiments.

Ì weeks

ε5

r~J Control EzJo-Se-Cu

E2

Fig. 2. Lipid peroxidation of liver microsomes from control, Sedeficient and Se-Cu-deficient rats,in the presence of 50 jiM Phenylhydrazine .För other conditions see legend of fig.1. Values are means + S.E.M. of 7-8 experiments.

685

a still more pronounced incapability of membranes to be peroxidized (Fig.2)¡however, this further decrease in malondialdheyde production is not observed in the membranes isolated from the animals treated with the Se-Cu-deficient diet. This effect can be due to the compensatory increase of non Se-GSH-Px observed in these animals.

Discussion The deficiency of Se-glutathione peroxidase and Cu-Zn superoxide dismutase induced by the treatment with a Se and Se-Cu-deficient diet may be comparable to that observed in tumor celisi 15). We have previously observed that the "in vitro" induced lipid peroxidation of membranes from tumor cells is lower than that from control cells (5-7). A reduced availability of polyunsaturated

fatty acids in membranes from tumor cells (i.e.

the substrate for lipid peroxidation) would account for such a difference (3). The lower content of Cu-Zn SOD and Se-GSH-Px by a Cu and Se-deficiency induces cellular oxidative conditions which can enhance lipid peroxidation "in vivo", with a consequent modification of lipid composition and assembly. If microsomal membranes have been damaged by the oxidative conditions induced by Se and Cu deficiencies in terms of a more saturated pattern in their endogenous phospholipids, the substrate availability for lipid peroxidation may become a limiting factor, as it was assessed in "in vitro" systems. The low level-of malondialdheyde producti on displayed by the membranes isolated from Se-deficient rats and the more

pronounced lowering of MDA formation due to the

làck of SOD, seem to indicate that such deficiencies can have induced a modification in the lipid composition of the microsomal membranes. Moreover the prolonged inhibition of both defensive enzymes Se-GSH-Px and Cu-Zn SOD causes a compensative increase of non Se-GSH-Px probably required for detoxification of an enhanced production of lipid peroxides.

686

The data presented s upport the hypothesis that in tumor cells the loss of defensive enzymes would cause an intracellular oxidative situation which might further influence the chemical and physical status of organelle membranes. In summary the low level of protective enzymes against oxyaen toxic species may be the primary event occurring durina tumor transformation,leading to a higher subcellular concentration of oxygen radicals. A decrease of lipid peroxidative activity by tumor membranes would be hypothesized to take place in a secondary fashion.This low capability of tumor cells to produce lipid peroxides can be, at least in part, one of the factors responsible for the high mitotic activity expressed by tumor cells.

ACKNOWLEDGMENTS The work was supported by CNR grant N. 81.01374.96 of the Special

Project "Control of Tumor Growth".

References 1.

Bozzi,A.,Mavelli,I.,Finazzi Agr$>,A.,Strom,R.,Wolf,Α. Μ., Mondovj. Β.,and Rotilio,G.:Mol.Cell. Biochem. 10,11-16 (1976)

2.

OberieyJi.W.,and Buettner,G.R.¡Cancer Res. 39,1141-1149(1979)

3.

Bartoli,G.M.,Bartoli,S.,Galeotti,T.,and Bertoli,E.SBiochim. Biophys. Acta 620, 205-211 (1980) Galeotti,T.,Bartoli,G.M.,Santini,S.,Bartoli,S.,Neri,G.,Vernole,Ρ.,Masotti,L.,and Zannoni,C.: in Oxygen and Oxy-Eadicals in Chemistry and Biology,Rodgers,M.A.J. and Powers,E.L. eds., Academic Press,New York, 641-644 (1981)

4.

5.

BartolijG.M.,Galeotti,T.,Borrello,S. and Minotti,G.: in Membranes in Tumor Growth,Galeotti,T.,Cittadini,Α.,Neri,G.and Papa,S. eds., Elsevier Biomedical Press,Amsterdam, 461-470 (1982)

6.

BartolijG.M. and Galeotti,T.tBiochim. Biophys. Acta,574r537541 (1979)

7.

Galeotti,T.,Bartoli,G.M.,Bartoli,S. and Bertoli,E.:in Biological and Clinical Aspects of Superoxide an95% C^· Surprisingly, the results described in this report appear to conflict with that hypothesis. Although GSHPX clearly is important in tolerance, the role of this enzyme in the phenomenon of adaptation to hyperoxia is complex.

The Torula yeast diet used by many laboratories to establish Se deficiency in rats is also deficient in sulfur-containing amino acids, methionine and cysteine. We have therefore included in this report the effect of met + cys on tolerance and adaptation to hyperoxia.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

700

Results 1) Diets and exposure. Rats (50-70 g body weight) were divided into dietary groups. Four Torula yeast based diets: + added sulfur-containing amino acids (0.6% D,L-met + 0.2% L-cys) + 0.1 ppm sodium selenite were compared with a standard laboratory diet. All diets were nutritionally adequate in vitamin E. Unsupplemented Torula yeast diets had 0.016 ppm Se. Exposure to 80% O2 at 1 atmosphere began 4 weeks after initiation of the diets. None of the standard diet rats died during 1 week of exposure indicating tolerance to 0^ that is normal for this species. When Se and/or met + cys were deficient significant differences in susceptibility to O2 were observed (Fig. 1). This was reflected in both the percentage and time of onset of mortality.

Fig. 1. There were 10 rats in each of the high sulfurcontaining amino acids (S.a.a.) groups, 20 rats in each of the low S.a.a. groups, and four standard diet rats. To test for adaptation to hyperoxia, survivors from 7 days in 80% O2 were exposed to >96% O2 for 4 days. Except for 1

701

of the 6 high met + cys, low Se diet rats, all pre-exposed rats survived for 96 hours in >96% indicating adaptation. (The

LDÇ.Q

for non-adapted rats is about 72 hours in >96%

°2.> TOLERANCE VERSUS ADAPTATION TOLERANCE DIET

EXPOSURE TO 80%

ADAPTATION EXPOSURE TO >96% 0, i

(7 days) Torula yeast + Se + S.a.a. + Se + S.a.a. Standard

(4 days)

4/20

4/ 4

13/20

13/13

6/10

5/6

10/10

10/10

4/ 4

4/ 4

Table 1. Results are rats surviving over rats exposed. S.a.a. = added sulfur-containing amino acids. * = surviving rats from 80% 0^ exposure. 2) Effect on antioxidant enzymes. Low Se diets produced significant decreases in lung GSHPX activity in rats. This decrease was of similar extent in rats fed diets supplemented or unsupplemented with met + cys. Following sequential exposure to 80% and >96% 0 2 , lung GSHPX activity was elevated in all dietary groups (Fig. 2). Rats fed the low Se diets actually showed a greater relative increase in GSHPX activity due to hyperoxic exposure than did the high Se rats. Although the increase was relatively large in the low Se rats, the absolute specific activity was still only 34-70% of that found in unexposed rats fed a Se-repleted diet. Therefore adaptation to hyperoxia does not correlate with the GSHPX specific activity in rat lungs.

702

^

-AA DIET

F i g . 2. T h e e f f e c t o f d i e t a r y d e f i c i e n c i e s a n d 0 exposure are indicated. AA = sulfur-containing amino acids. The control d i e t (Cont.) w a s a T o r u l a y e a s t b a s e d d i e t w i t h b o t h A A a n d Se added. The o t h e r m a j o r c a t a l y s t in tissues removal

Se d e p l e t i o n a n d h y p e r o x i c e x p o s u r e test whether activity

for hydrogen

is c a t a l a s e . W e t h e r e f o r e m e a s u r e d a compensatory

could occur

(Table

increase

on catalase in this

(units/g dry wt.)

3.00 + 0.33 (4)

+ Se

2.93 + 0.62 (5)

activity

to

2).

Air Torula yeast

of

enzymatic

EFFECT OF SE DEPLETION AND HYPEROXIA ON LUNG CATALASE

peroxide

the e f f e c t

CATALASE Oxygen/air

Oxygen 5.13

+ 0.41*

1.71

(5) 5 . 9 1 + 0 .43 *

2.01

(6)

T a b l e 2. B o t h g r o u p s o f r a t s w e r e f e d d i e t s s u p p l e m e n t e d w i t h cys and m e t . * = ρ < 0 . 0 5 compared w i t h air exposure by S t u d e n t ' s t t e s t . T h e n u m b e r o f a n i m a l s i n e a c h g r o u p is s h o w n in p a r e n t h e s e s .

703 It is clear that exposure to hyperoxia did produce an increase in catalase activity but that Se deficiency had no effect.

Discussion Deficiency of either Se or met + cys decreased tolerance to Oj in agreement with the reports of Cross et al. (1) and Deneke et al. 2). Deficiency of both Se and met + cys caused an even greater decrease in tolerance. Nevertheless, the present studies also showed an unexpected result: almost all the survivors from 80% C>2 survived the subsequent exposure to ]>96% C>2 regardless of dietary deficiencies. Thus while these nutrients have an important role in tolerance, their role in adaptation is unclear. Prolonged hyperoxic exposure causes an increase in lung GSHPX activity (3,4). We observed that the relative increase in GSHPX activity was greater when the activity was initially low due to Se depletion. This occurred even though the low Se diet produced only 15% of normal GSHPX activity in the unexposed rats. Thus at least 2 factors regulate GSHPX activity in lung: Se content and hyperoxic stress. While the absolute GSHPX specific activity does not correlate with adaptation, it is possible that the enzyme activity could increase in particular cells and therefore indirectly contribute to adaptation. Studies in this laboratory have shown that the specific activity of GSHPX in granular pneumocytes is relatively high compared with other lung cells but does not increase in hyperoxic exposure (5). The increase in GSHPX may then reflect the increased percentage of these cells in adapted lungs (6), which then produce a high constitutive amount of the GSHPX apo-enzyme, a high Se affinity protein.

We cannot be certain as to the role of the sulfur-containing

704

amino acids. One possible role for these amino acids may be in maintaining the sulfur balance that affects glutathione metabolism. Glutathione concentration increases in the lungs of oxygen exposed rats (4). Recently, Deneke, et al.

showed

that deficiency of cys in the diet for only a few days before exposure to hyperoxia prevented the increase in lung glutathione and increased mortality. It seems likely that the prolonged cys and met restriction in the present studies worked by a similar mechanism.

References 1. Cross, C.E., Hasegawa, G., Reddy, K.A., Omaye, S.T.: Res. Commun. Chem. Pharm. Path. 16^, 695-704, (1977). 2. Deneke, S.M., Gershoff, S.N., Fanburg, B.L.: J. Appi. Physiol: Respir. Environ. Exercise Physiol. 54_, 147-151, (1983) . 3. Frank, L., Bucher, J.R., Roberts, R.J.: J. Appi. Physiol: Respir. Environ. Exercise Physiol. £5, 699-704 (1978). 4. Kimball, R.E., Reddy, K., Pierce, T.H., Schwartz, L.W., Mustafa, M.G., Cross, C.E.: Am. J. Physiol. 230, 1425-1431, (1976). 5. Forman, H.J., Fisher, A.B.: Lab. Invest. 45, 1-6, (1981) . 6. Crapo, J.D., Peters-Golden, M., Marsh-Salin, J., Shelburne, J.G.: Lab. Invest. 3_9, 640-653, (1978).

Acknowledgements This work was supported by a grant from the National Institutes of Health HL-26710. We thank June Nelson and Eric Rotman for their valuable technical assistance. A more detailed version of this report has been accepted for publication in Laboratory Investigation.

705 DISCUSSION

BHUYAN: Do you see any abnormality 100% o x y g e n after adaptation?

in the

FORMAN: After exposure to 100% oxygen, even are tremendously changed. They are fibrotic, cells, and those rats do not do w e l l in 20% peroxia is not really the solution for being sure. The animal doesn't have a normal lung, thy.

rats w h e n y o u expose

to

in the adapted rat the lungs they have increased type II oxygen. Adapting rats to h y able to sustain oxygen expoit lives but it is not h e a l -

BHUYAN: In the beginning of your talk you said that all enzymes are stimulated w h e n y o u expose to hyperoxia. FORMAN:

them

the

defensive

The ones that have been looked at all increase in the whole lung.

BHUYAN: That's clear, but your c o n c l u s i o n is o n l y o n glutathione dase. Were other enzymes also stimulated?

peroxi-

FORMAN: The granulocyte w h i c h is a cell that d o e s well in high o x y g e n has constitutively high levels of all the antioxidant enzymes that we looked at (FORMAN & FISTER, Lab. Invest. (1981) 45, 1-6), that is Mn-SOD, Cu/Zn-SOD, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. W h e n you take the cells o u t of animals that have adapted, the enzyme level per cell has not changed in any of those, except for the M n - S O D w h i c h about doubles. A n interesting thing is that w h e n you take those cells and p u t them in culture, they start decreasing again in the M n - S O D back to the normal constitutive level. They decrease e v e n if y o u keep them in oxygen, but at a slower rate. SINGH: Do y o u know w h i c h would be more useful?

type of chemical

compound containing

selenium

FORMAN: I haven't done any of that work, but there is a fellow named Orville LEVANDER (J. Nutr. (1981) 111, 2180-87) who has done n u t r i t i o n studies a n d several other people have too. W e used sodium selenite a d d e d to the diet. There have been studies showing that the selenium seems to be first incorporated into methionine, but will end up in cysteine, too. There is quite a biochemistry of selenium that goes on, n o matter w h i c h w a y the selenium comes in. It doesn't have to come in as the amino acid.

GLUTATHIONE PEROXIDASE SYSTEM IN THE LUNGS OF OZONE-TOLERANT AND NONTOLERANT RATS

Ching K. Chow, Department of Nutrition and Food Science, University of Kentucky, Lexington, Kentucky 40506, U.S.A.

Introduction

The health hazard of ozone, the principal oxidant air pollutant of photochemical smog, has been well documented (1,2).

One of the notable

features of ozone toxicity is the phenomenon of tolerance.

W h e n animals

are exposed to a single sublethal dose of ozone they become resistant to subsequent lethal exposure.

This tolerance phenomenon has been

demonstrated in several species of animals, and the effect has been shown to persist well over one month (1-3).

Exposure of experimental animals to

near ambient levels (under 1 ppm) of ozone causes measurable and biochemical

lesions in the lungs (2,4,5).

morphological

The biochemical

alterations

in the lungs are characterized by the augmentation of the overall activity.

metabolic

While the increase in metabolic activity appears to reflect

inflammatory and reparative responses resulting from ozone-induced cell injury, it may also alter cellular susceptibility to further damage

(4,5).

Glutathione (GSH) peroxidase, which utilizes the reducing equivalent of GSH to catalyze the decomposition of toxic hydroperoxides, and metabolically related enzymes GSH reductase and glucose-6-phosphate

(G-6-P)

dehydrogenase, have been suggested to be a key mechanism for cellular protection against the deleterious effects of hydroperoxides

(5,6).

In the

present study, therefore, experiments were conducted to determine the possible role of the GSH peroxidase system in the development of tolerance to ozone.

Materials and Methods

Sixty or seventy days old male Sprague-Dawley rats (chronic

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

respiratory

708 disease free) were initially exposed filtered air for 3 days (to develop described previously

(7).

to either 0.73-0.76 ppm ozone or tolerance) in monitored chambers as

They were then allowed to recover for 8 - 1 9 days.

Animals from each group were then exposed ppm ozone for 8 hours.

to either filtered air or 3.5-4.0

Four to six surviving animals from each group were

killed at 1, 16, 18, 44 or 114 hours following each acute exposure. perfusing through the pulmonary artery with cold saline to remove the lungs were homogenized, fractionated

After

blood,

and assayed for the levels of

protein and GSH, and activities of GSH peroxidase, GSH reductase and G-6-P dehydrogenase as described previously

(8,9).

The significance between

two

mean values were determined using Student's "t" test at 957. confidence interval.

Results and

Discussion

The effect of prior exposure to develop tolerance on animal

mortality

following acute exposure

tolerant

is shown in Table 1.

animals exhibited a markedly

lower mortality

As expected,

rate than that of the non-

tolerant group.

None of the mortality occurred

acute exposure.

All the lungs of acute exposed animals showed marked

Table

Experiment I

1.

Effect of Prior Exposure on Animal

Prior

Recovery

Exposure 0.76 ppm 0^

χ

3 days

Exposure

8 days

4..0 ppm

8 days

4..0 ppm

°3 °3

the

Mortality

Acute

Period

Air χ 3 days II

16 hours following

Animal mortality

X 8 hours

1/7

X 8 hours

5/10

0.73 ppm 0j χ 3 days

11 days

3.,9 ppm 03 X 8 hours

1/13

Air χ 3 days

11 days

3..9 ppm 0

X 8 hours

7/15

X 8 hours

1/12

X 8 hours

4/13

3 III

0.73 ppm 0 3 χ 3 days

19 days

Air χ 3 days

19 days

^Sixteen hours

following

3..5 ppm 0 3

the acute

3,.5 ppm

exposure.

709 haemorrhage and edema, and the lungs of tolerant animals

exhibited

relatively less severe lung injury than the non-tolerant group. mortality or lung lesions were observed in control

No animal

animals.

The biochemical responses in the lungs following exposure to 3.9 ppm ozone for 8 hours are shown in Table 2. As expected, exposed animals had a marked increase in the levels of protein over the control values.

The

increases

averaged over 807. for both tolerant and non-tolerant rats, and the values were relatively smaller for the tolerant animals.

Table 2 also shows that

relative to the control values ozone exposure resulted in a significant Table 2.

GSH Peroxidase System in the Lungs of Rats Following Exposure to 3.9 ppm Ozone for 8 Hours

Biochemical parameter

Time After Exposure

(hours)

Tolerance* 1

Protein (mg/lung)

143+15a(4)** No 132+15 a (4) Yes Control 73 ±o (6)

GSH (nmoles/mg protein)

No Yes Control

GSH Peroxidase (nmoles/min/mg protein)

18

44

156±16®(4) 140±9 a (4) 71+7 (6)

147+12 (4) 7278 (5)

10.9+1.3 3 13.3+1.9® 27.4+2.3

12.6+2.4® 18.4+2.l b 26.1+1.9 C

26.5+1.4 27.9+2.1®

No Yes Control

38.2+5.I a 44.1+6.1® 77.0+8.2

43.5+4.1® 56.2+5.5 80.2+7.4°

75.0+6.8:* 81.478.2

GSH Reductase (nmoles/min/ mg protein)

No Yes Control

17.2+2.7® 20.2+4.5® 34.1+3.2

17.9+2.9® 24.0+2.4 36.2+2.9°

31.9+2.6" 35.674.1

G-6-P dehydrogenase (nmoles/min/mg protein)

21.9+2.1® No 25.0+3.5® Yes Control 45.6+3.6

30.4+4.6® 42.7+4.3 49.0+5.1 C

65.2+4.3" 51.574.8

*Exposed to 0.73 ppm ozone for 3 days and allowed to recover for 11 days. Control animals were exposed to either 0 or 0.73 ppm ozone for 3 days and allowed to recover for 9, 12 or 20 days. * * M e a n ± standard deviation. The number in the parentheses indicates the number of animals per group. The means which do not share the same superscript are significantly different (pv

Increase of prot-SSG content was induced by perfusion with 100 μΜ t-butyl hydroperoxide and the various contents obtained by perfusing the livers for 10,20 and 30 min. (r=0.98). The symbol with SEM indicated reflects control.

? 0,8-

χ Ω CL IO

° 0,6-

0

200 100 prot S S G (nmolxg liver"1)

744

together with the enhanced glutathione mixed disulfide formation in a linear relationship. It might be assumed that an increased enzyme activity is dependent on enhanced prot-SSG content rather than on that of GSSG, since GSSG contents were decreased when GSH falls below 1.5 nmol/g (fig. 1). The direct relationship between glutathione mixed disulfide content and G6P-DH activity indicates that the pentose phosphate cycle activity may be regulated both via the NADPH system (dependence on the substrate concentration, NADP + ) and via changes in the glutathione state with the interconversion of the key enzyme by mixed disulfide formation. Whether both these possibilities could be operative at the same time is under current investigation. Acknowledgements Expert technical assistance was provided by B. Gabriel Supported by Deutsche Forschungsgemeinschaft, Schwerpunktsprogramm "Mechanismen toxischer Wirkungen von Fremdstoffen".

References 1.

Sies, H. in: Glutathione - Storage, Transport and Turnover in Mammals, (Y.Sakamoto, T.Higashi, and N.Tateishi, eds.) pp. 51-76, Japan Scient. Soc. Press, Tokyo (1983).

2.

Mannervik,B. and Axelsson,K.:Biochem.J. 190, 125-130 (1980).

3.

Brigelius,R.: Hoppe Seyler's Z. Physiol. Chem., submitted (1983).

4.

Cadenas,E., Brigelius,R., Akerboom,T. and Sies,Η. in: ^ Biological Oxidations (Sund,Η. and Ullrich,V., eds.) 34 Mosbacher Kolloquium, Springer Verlag, Heidelberg (1983).

5.

Eggleston,L.V. and Krebs,H.A.:Biochem.J. 138,425-435 (1974) . Gilbert,H.F.: J.Biol.Chem. 257, 12086-12091 (1982).

6.

BILIARY TAUROCHOLATE RELEASE DURING REDOX CYCLING IN PERFUSED RAT LIVER

Theo Akerboom, Manfred Bilzer, Helmut Sies Institut für Physiologische Chemie I, Universität Düsseldorf, Moorenstrasse 5, 4000 Düsseldorf

Introduction In intracellular detoxication of reactive oxygen metabolites glutathione plays a crucial role. Glutathione peroxidases present in the cytosolic and mitochondrial compartments of the cell catalyse the reduction of hydroperoxides by GSH leading to the formation of GSSG (see 1 for review). In perfused rat liver, oxidative transitions in the glutathione system have been shown to occur upon infusion of different hydroperoxides and under conditions of increased intracellular 9enerati°n

(2). Changes in the glutathione redox state

may be of regulatory significance for several metabolic processes, including transport, by altering the membrane thiol status and the amount of glutathione-protein mixed disulfides (3-5) . We have investigated here the influence of the intracellular glutathione redox state on the hepatic disposition of taurocholate. Glutathione disulfide formation was induced either by the addition of hydrogen peroxide or by menadione (2-methyl-1,4-naphtoquinone) which causes increased formation of superoxide anion by redox cycling which, in turn, results in the intracellular generation of hydrogen peroxide via the superoxide dismutase reaction (6). Biliary excretion of taurocholate was inhibited, leaving the hepatic uptake of the bile acid from the sinusoidal compartment unaffected. The inhibition proved to be dependent on the presence of the Se-GSH

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

746

peroxidase suggesting that the glutathione system is involved.

Results and discussion Perfusion of rat liver with low concentrations of taurocholate leads to an almost quantitative uptake and biliary release of the bile acid, reaching a steady state after a few minutes. Addition of hydrogen peroxide (250 jiM) causes an 85% inhibition of biliary excretion in control rats (fig 1, open symbols). The inhibition is reversible and is accompanied by an increase in intracellular GSSG amounting to about 250 nmol per g liver. The sinusoidal uptake of taurocholate in the presence of

Practically unaffected, leading to a corres-

ponding intrahepatic accumulation of the bile acid. This may also explain the overshoot observed upon termination of infusion. The inhibition of biliary taurocholate transport appears to be related to the flux through Se-GSH peroxidase as can be deduced from experiments carried out in Se-deficiency. Addition of H2O2 to livers from rats fed a Se-deficient diet for

Taurocholate [3|>*j Hydrogen ReroxrielSOl·/·« Tv 's Fig 1•

Influence of H 2 0 2 on

biliary release of taurocholate. Radioactively labelled taurocholate was infused into the entering perfusate (flow-through perfusion (2)) from 30 to 70 min and hydrogen peroxide from 50 to 56 min at the concentrations indicated. ·• Β OJ 40 60 Perfusion Time (min)

747

Fig 2.

Influence of menadione on the hepatic disposition of

taurocholate and biliary release of glutathione disulfide. Rat liver perfused as described in (2). Hepatic uptake of taurocholate was calculated from influent and effluent perfusate by difference. Radioactively labelled taurocholate was infused from 30 to 85 min and menadione from 50 to 56 min at the concentrations indicated.

six weeks (GSH peroxidase activity less than 5% of controls) results in an inhibition of only 20% (fig 1, closed symbols). The intracellular contents of GSH and GSSG were 5.0 jxmol and 25 nmol per g liver, respectively, which is close to the control values obtained in the absence of ^ 0 2 « Thus, the lack of substantial inhibition by hydrogen peroxide suggests that the release of taurocholate is not affected by the hydroperoxide per se, but rather via metabolic changes mediated by GSH peroxidase i.e. changes in the glutathione redox state. Upon infusion with menadione a response similar to the addition of H i s

observed (fig 2). In livers from normal

rats, the taurocholate efflux is almost completely inhibited,

748

whereas the hepatic uptake remains unaltered. The intracellular GSSG content rose from 18 (controls) to 540 nmol per g liver. GSH decreased from 5.5 to 3.5 jamol per g liver, due to the formation and biliary excretion of GSSG and glutathione conjugate. In livers from Se-deficient rats, the inhibition amounted to 25% and the intracellular GSSG and GSH levels were 48 nmol and 4.6 ^jmol per g liver, respectively. Prom these data we conclude that in normal livers the inhibition caused by menadione is due to the intracellular generation of í^C^. In this respect it is noteworthy that not only menadione itself but also the conjugated form, which is ultimately excreted into the bile, is able to produce redox cycling (7).

Acknowledgements Excellent technical assistance was provided by Maria Gärtner Supported by Deutsche Forschungsgemeinschaft, Schwerpunktsprogramm 'Mechanismen toxischer Wirkungen von Fremdstoffen1 and Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen.

References 1. Sies, Η. , Wendel, Α., Bors, W.: in Biological Basis of Detoxication, Jakoby, W.B., Bend, J.R. and Caldwell, J. eds. Academic Press, New York, pp 307-321 (1982). 2. Akerboom, T.P.M., Bilzer, Μ., Sies, H.: J. Biol. Chem. 257, 4248-4252 (1982). 3. Brigelius, R., Muckel, C., Akerboom, T.P.M., Sies, H.: Biochem. Pharmacol. (1983) in press. 4. Kosower, N.S., Kosower, E.M. : Intern. Rev. Cytol. 54^, 109160 (1978). 5. Mannervik, B., Axelsson, K.: Biochem. J. 190, 125-130 (1980). 6. Thor, H., Smith, M.T., Hartzell, P., Bellomo, G., Jewell, S.A., Orrenius, S.: J. Biol. Chem. 2^7, 12419-12425 (1982). 7. Weiers, Η., Sies, H.: Arch. Biochem. Biophys. 22 4, (1983) in press.

FREE RADICAL INTERMEDIATES IN THE TRYPANOCIDAL ACTION OF DRUGS AND PHAGOCYTIC CELLS.

Roberto Docampo"'" and Silvia N.J. Moreno^ Centro de Investigaciones Bioenergéticas, Facultad de Medicina Universidad de Buenos Aires, Buenos Aires, Argentina, and Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.

Introduction Intracellular reduction followed by autoxidation yielding superoxide anion and hydrogen peroxide has been suggested as the mode of action of several trypanocidal agents (1-4). Moreover, oxygen-reduction products have been implicated in the mechanism of cytotoxicity of phagocytic cells against T. cruzi (5).

Nitroimidazole drugs are widely used in the treat-

ment of protozoal infections, and benznidazole, a nitroimidazole derivative, is effective in the treatment of Chagas' disease (6).

Previous attempts to demonstrate benznidazole

reduction to a nitro anion radical in the presence of

cruzi

intact cells, and mitochondrial or microsomal fractions under conditions similar to those used with the nitrofuran nifurtimox (3) were unsuccessful (7), and it has been speculated that this reduction may not involve a one-electron transfer to this nitrocompound (7).

In this report, we provide evidence

of one-electron reduction of benznidazole by Ί\_ cruzi homogenates and describe differences between the nifurtimox and benznidazole modes of action on

cruzi.

Present address : Laboratory of Molecular National Institute of Environmental Health 12233, Research Triangle Park, N.C. 27709,

Biophysics Sciences, USA.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

, P.O.

Box

750 Results and Discussion

Fig. 1. ESR spectra obtained from incubations of cruzi homogenates. (A) Spectrum from a homogenate containing 1 mM nifurtimox, an NADH-generating system and cruzi homogenate protein (5 mg/ml) prepared as des cribed (3) . The oxygen was displaced by purging with nitrogen, and the reaction was initiated with NADH. The nominal mi crowave power was 20 mW, and the modulation amplitude was 5.0 G. (B) The same as in (A) but with 4 mM benznidazole instead of nifurtimox, and 25 mg/ml homogenate protein. Other conditions as described in (8).

The incubation of

cruzi homogenates with nifurtimox (Fig.

IA) and an NADH-generating system generated a multiline spectrum that could be identified as the nitro anion radical (3,8) No benznidazole radical could be discerned under the same conditions.

However, a weak signal was evident at 4 mM benz-

nidazole in the presence of a high protein concentration (Fig. IB).

The value of

was 13.45 and compared favorably with

the value of 13.75 G obtained in rat liver microsomal incubations of benznidazole (7).

The radical formation depended on

all components of the system, namely homogenate, NADHgenerating system, and nitrocompound.

The spectra of the

anion radicals could not be observed under aerobic conditions. In spite of the many similarities in the mode of action of nitrofurans and nitroimidazoles in several biological systems (10), benznidazole produced an inhibition of

cruzi respira-

751

Fig. 2. Effect of nifurtimox (Ν) and benznidazole (Β) concentrations on oxygen consumption by cruzi epimas ti go tes. The rates reported here were determined during three minutes of incubation of epimas tigotes (4 mg/ml) in 0.1 M potassium phosphate buffer (pH 7.4). Oxygen uptake was measured in the Gilson polarograph using a Clark electrode as described before (2,9) .

tion in contrast to the stimulation produced by nifurtimox under similar conditions (Fig. 2). This ruled out any major role for redox cycling of benznidazole under physiological conditions. Moreover, benznidazole inhibits growth of Τ. cruzi at concentrations which do not stimulate C>2 and generation (7), thus indicating that the trypanocidal action does not depend on the effect of oxygen radicals. In contrast, pharmacological concentrations of nifurtimox are able to produce maximal stimulation of O^ production by T_;_ cruzi mitochondrial fraction, to initiate diffusion of HjOj outside the cells and to inhibit growth completely (2). The difference noted between nifurtimox's and benznidazole's capability to generate oxygen free radicals may be significant in the treatment of Chagas' disease when these drugs are compared for their toxicity.

752 Acknowledgements The authors wish to thank Dr. Ronald P. Mason for his encouragement and stimulating discussions.

This work received fi-

nancial support from the UNDP/World Bank/WHO Special Programe for Research and Training in Tropical Diseases, and from CNPq and FINEP, Brazil.

R.D. is Career Investigator of the CONICET

(Argentina) and S.N.J.M. is Visiting Fellow of the NIH (USA).

References 1.

Docampo, R., Cruz, F.S., Boveris, Α., Muniz, R.P.A., Esquivel, D.M.S.: Arch. Biochem. Biophys. 186, 292-297 (1978) .

2.

Docampo, R., Stoppani, A.O.M.: 197, 317-321, (1979) .

3.

Docampo, R., Moreno, S.N.J. , Stoppani, A.O.M., Leon, W., Cruz, F.S., Villalta, F., Muniz, R.P.A.: Biochem. Pharmacol. 30, 1947-1951 (1981).

4.

Docampo, R., Moreno, S.N.J.: In Free Radicals in Biology, (W.A. Pryor, ed.) Vol. 6, Academic Press, New York, in press.

5.

Docampo, R., Casellas, A.M., Madeira, E., Cardoni, R.L., Moreno, S.N.J., Mason, R.P.: FEBS Lett. 155, 25-30 (1983).

6.

Van den Bossche, H.:

7.

Moreno, S.N.J., Docampo, R., Mason, R.P., Leon, W., Stoppani, A.O.M.: Arch. Biochem. Biophys. 218, 585-591 (1982) .

8.

Docampo, R., Mason, R.P., Mottley, C., Muniz, R.P.A.: J. Biol. Chem. 256, 10930-10933 (1981).

9.

Docampo, R., Moreno, S.N.J., Stoppani, A.O.M.: Biochem. Biophys. 207, 316-324 (1981).

Arch. Biochem. Biophys.

Nature (Lond.) 273, 626-630 (1978).

Arch.

10. Mason, R.P.: In Free Radicals in Biology (W.A. Pryor, ed.), Vol. 5, pp. 196-212, Academic Press, New York (1982).

EFFECT GAMMA

OF FREE RADICAL ACTIVITY

ON T R I P T O P H A N AND

HUMAN

GLOBULIN

David Wickens,

Anthony Norden and Thomas

D e p a r t m e n t of C h e m i c a l L o n d o n N 1 9 5NF

Dormandy

Pathology, Whittington

Hospital

Introduction The r e a c t i o n s extensively recently

of t r y p t o p h a n

the e f f e c t

of free

eins has r e c e i v e d little human

gamma

ble e f f e c t s

(trpH) w i t h free

s t u d i e d i n the l a s t 30 y e a r s

globulin

radicals

on t r p H

attention. Free

(HGG)

solutions

of t h e n a t i v e

results

fluorescence

(3),

(c) g e n e r a t i o n

of a n e w f l u o r e s c e n c e

(3),

(d) g e n e r a t i o n

of free

modification similar, exudates

identical,

nature

of the

icals in trpH and HGG

Results

and

A reverse trpH

in f i v e

characteristics,

(3) a n d

(e)

336nm) Í5Anm)

protein

(c) a r e p r i m a r i l y

Current work

study utilises

in

observa-

(Ex 2 9 6 , Em

suggests

changes may occur in

changes

prot-

due

to

that

proteins

) in

inflammatory

HPLC

to

investigate

b r o u g h t a b o u t by f r e e

rad-

solutions.

Discussion

phase HPLC m e t h o d has

oxidation products.

detected

groups

(b) a n d

been until

activity

(Ex 360, E m

the trpH rich g a m m a g l o b u l i n s

(5» 6). T h e p r e s e n t

chemical

thiol

(a),

of t r p H r e s i d u e s .

perhaps

(particularly the

(4·). E f f e c t s

has

containing

radical

: (a) c h a n g e i n t h e uv a b s o r p t i o n

(b) d e s t r u c t i o n

aggregation

radicals

(1,2). H o w e v e r ,

on t h e b a s i s

cent properties

been developed

o f t h e i r uv a b s o r p t i o n

of w h i c h

to

separate

Some 17 oxidation products only

and/or

3 have been identified

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · N e w York - Printed in Germany

can

be

fluores(fig

1).

754

O

U

8

12 O Elution

time

4. (min)

F i g u r e 1 T y p i c a l HPLC t r a c e s showing uv abs . 240nm ( ) and f l u o r e s c e n c e Ex 326, Em 370-700nm o f lmmol/l trpH s u b j e c t e d t o ( A ) uv i r r a d i a t i o n (254+366nm) f o r 1 8 0 ' , ( Β ) i n c u b a t i o n w i t h 50μπιοΐ/ΐ F e 2 + and 50mmol/l H2O2 f o r 1 0 ' , ( C ) 1 0 ' uv i r r a d i a t i o n i n t h e p r e s e n c e o f 250ymol/l H2°2> ( D ) bench t o p i n c u b a t i o n f o r 1 8 0 d a y s . HPLC c o n d i t i o n s : L i C h r o s o r b RP-18 (5um) column, s o l v e n t w a t e r / m e t h a n o l 70/30, f l o w r a t e 0.75ml/min and i n j e c t i o n volume 2 0 u l . P e a k s , l = n - f o r m y l k y n u r e n i n e ( N F K ) , 2 = 5 - h y d r o x y t r y p t o p h a n ( 5 - H T P ) , 3=kynurenine ( K ) , 4 = t r p H .

755

O

A

8

12 O E l u t i o n time

U (min)

8

12

F i g u r e 2 H P L C t r a c e s of (A) 2 . 5 g / l H G G u v - i r r a d i a t e d f o r 1 8 0 ' t h e n i n c u b a t e d w i t h O . l g / l P r o n a s e a t 3 7 ° C f o r 24-hr to e n s u r e c o m p l e t e h y d r o l y s i s a n d (B) 2 . 5 g / l H G G i n c u b a t e d w i t h 5 0 p m o l / l F e 2 t a n d 5 0 m m o l / l H2°2 f o r 1 8 0 ' , t h e n t r e a t e d as (A). H P L C c o n d i t i o n s as f i g u r e 1. Although ratios ems

some products are

studied

TrpH was

gave different

unaffected

ally hydrolysed the p r e s e n c e

of NFK and Κ

are able

systems

their syst-

of t h e p r i m a r y

products.

chromatographic

5 - H T P . In the l i g h t

(7) t h a t h y d r o x y i n d o l e s to d i r e c t l y (table)

reduce

but not other

cytochrome

indicates

separa-

of t h e

c, i t s

findsubst-

reduction

that this process

gener-

hydroxyindoles.

Analysis ive way ited

studied

the OH' g e n e r a t i n g

anion. The HPLC of enzymatic2+ or F e /HgOg treated HGG reveals

(fig 2 ) . T h e

to d e t e c t

by u v i r r a d i a t e d H G G ates

yields

uv i r r a d i a t e d

ing of A l i v i s a t o s

to a l l

Indeed

by s u p e r o x i d e

tion was insufflent ituents

common

can vary considerably.

of the products to m o n i t o r

since in cases where

the r e l a t i v e

yields

of t r p H o x i d a t i o n m a y p r o v e

oxidative

species. However

the i n i t i a t i n g

of o x i d a t i o n p r o d u c t s

an

effect-

t h i s m a y be

radical is c a n be

the

lim-

same

considerably

756 The direct reduction of cytochrome c by uv irradiated HGG. Irradiation time

(min)

0

100

200

300

400

pmol/l cytochrome c red./lO'

0.02

2.88

4.16

4.73

4-98

SD

0.03

0.33

0.08

0.11

0.29

Assay procedure, test : 1ml 0.2mmol/l cytochrome c in saline + 0.5ml uv irradiated HGG (2.5g/l in ¿Ommol/l tris/HCl containing 0.1mol/l NaCl, pH 7.65) + 50yl lg/1 superoxide dismutase (SOD), blank : as test with uv irradiated buffer replacing HGG. The assay was performed at 37°C in a Pye-Unicam SP8-100 spectrophotometer, λ 549.2nm. SOD was included to inhibit any superoxide dependent reduction of cytochrome c. Results from three experiments. different. If the fluorescence

(Ex 360, Em 454nm) associated

with IgG isolated from inflammatory exudates

(5,6) is due to

the oxidation of trpH residues the measurement of such fluorescence may prove useful in assessing in vivo free radical activi ty.

References 1. Jayson, G. G., Scholes, G., Weiss, J. : Biochem. J. 57, 386-391 (1954). 2. Singh, Α., Bell, M-J., Korcll, G. W., Kremers, W., Singh, H. : in Oxygen and oxy-radicals in Chemistry and Biology, eds Rodgers, M.A.J., Powers, E.L., pp ¿61-473, Academic Press, New York 1981. 3. Wickens, D.G., Graff, T.L., Lunec, J., Dormandy, T.L. : Biochim. Biophys. Acta 7¿2, 607-616 (1983). 4. Wickens, D.G., Graff, T.L., Lunec, J., Dormandy, T.L. : Agents & Actions 11, 650-651 (1981). 5. Wickens, D.G., Dormandy, T.L. (1982).

: Clin. Rheumatol. 1, 151-152

6. Wickens, D.G., Dormandy, T.L. (1983).

: Agents & Actions in press

7. Alivisatos, S.G.A., Williams-Ashman, H.G. Biophys. Acta _86, 392-395 (1964).

: Biochim.

INVOLVEMENT OF OXYGEN RADICALS IN ETHANOL OXIDATION AND IN THE ETHANOL^ INDUCED DECREAPF TN Τ,TVER GLUTATHIONE.

Helena Antébi, Catherine Ribière, Jamal Sinaceur, Carlos Abu-Murad and Roger Nordmann. Service de Biochimie de la Faculté de Médecine Paris-Ouest et INSERM U 72, 45, rue des Saints-Pères 75270 Paris Cedex 06, France

Introduction Recent studies have suggested a possible role for hydroxy! radicals (OH') in the microsomal production of acetaldehyde from ethanol (1-4). The generation of OH" is known to require superoxide (0^') and HgO^ and to involve iron salts as catalyst (5). Some iron-chelators like desferrioxamine (DFO) and diethylenetriaminepentaacetic acid (detapac) have been reported to inhibit OH" formation in vitro by chelating the catalytic iron (5-6) and to reduce microsomal ethanol oxidation in vitro (7-8). Consequently, if OH' dependent systems play a significant role in the elimination of ethanol, one would expect these agents to interfere with ethanol metabolism in vivo. On the other hand, glutathione (GSH) has been shown to react directly with oxygen radicals (9) and probably play a major part as a freeradical scavenger in controling the intra-cellular concentration of 0^· (i0), while acute ethanol intoxication has been shown to cause a drop in the liver GSH content (11). To ascertain the role of oxygen radicals in the overall elimination of ethanol and on liver GSH content, we studied the effects of the administration of DFO and detapac in rats receiving an acute ethanol load (2.3 g/kg, i.p.)

Results DFO (100 mg/kg body wt, i.p.), administered to adult overnight fasted male Sprague Dawley rats 1 hr prior to the ethanol injection as well as

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

758 at 1 ;3 and 5hr after this ethanol load, reduced the rate of ethanol elimination by 19 %. Detapac (250 mg/kg body wt, i.p;), administered once 1 hr prior to ethanol, reduced this rate by 16 % (Table 1). Neither of these two iron-chelators when added in vitro (1 mM) had any effect on ADH and catalase activities. These data show that the reduction of ethanol elimination by either DFO or detapac is not due to a direct effect of these chemicals on either ADH or catalase. As it has been previously shown that cytochrome P450 is also unaffected by DFO (4), one can suggest that the reduction in the ethanol elimination rate following the administration of iron^helators includes the intervention of non-heme iron. So far·, ethanol oxidation via OH' radicals is the only mechanism proposed to be iron-dependent and conseguently inhibited by iron-chelators. Such a mechanism has in fact been detected and subseguently studied in isolated liver microsomal preparations (1-4,8). However this microsomal system has been reported to be only partly sensitive to DFO in vitro (4,7). Furthermore, microsomes are known to play only a minor role in the overall ethanol oxidation under the experimental conditions presently used, namely the administration of a single moderate dose of ethanol to sober rats. Considering our results it can be suggested that the role of OH' radicals in ethanol metabolism is not restricted to liver microsomes, but could be extended to extra-microsomal compartments and eventually to extra-hepatic tissues.

Table 1. Influence of desferrioxamine or detapac on ethanol administration in rats in vivo.

Treatment

Ethanol elimination (mmoles/kg body wt/hr)

Saline

7-58 + 0.32

Desferrioxamine

6.11 + 0.70'

Detapac

,a 6.34 + 0.18'

.a

All animals received ethanol (2.3 g/kg, i.p.) plus saline, DFO or

detapac

as described in the text. The reported values are means + S Ε M with at least 7 animals in each group. a p1M), rapidly mixed with the protein, has no effect on activity. Steady state analysis of the scheme associates V m a x with the reduction of Fe3"1 and K m with the binding of 0 2 _ to Fe 3 + . The

789 proposed mechanism accounts for the dependence of K m (apparent) on pH and the pH-independence of V m a x .

It also suggests

that OH - , like N3 - and other anions, is a competitive inhibitor as observed experimentally (15).

Summary Iron superoxide dismutases are highly helical, two-domain structures in which the active center ferric ion, coordinated by four amino acid ligands, is located at an interface between the two domains.

Cu/Zn- and Fe-dismutases show sub-

stantial differences in their secondary, tertiary and quarternary structures.

Nevertheless, the active centers in both

proteins possess solvent-accessible coordination sites in accord with predicted structural requirements for superoxide dismutase activity.

Unlike the Cu/Zn-protein, superoxide

dismutation catalyzed by the Fe-protein is not diffusion controlled.

Analysis of the steady-state kinetic data in con-

junction with anion binding and spectral information suggests that the rate limiting step is the reduction of the Fe"1"·' by C>2~ within a ferri-superoxo complex.

Alignment of secondary

structural features predicted from sequences for Mndismutases with those observed in the Fe-dismutase structures provides strong supporting evidence for structural homology between the Mn- and Fe-proteins. Acknowledgements. We are grateful to Jane Richardson for allowing us to include her illustrations. We thank A.L. Metzger for preparing some of the figures and D. Rapley for assistance in preparing the manuscript. Research supported by grants GM 16429 and GM 21519 from the National Institutes of Health. References 1.

Michelson, A.M., McCord, J.M. and Fridovich, I., eds.: Superoxide and Superoxide Dismutases, Academic Press, New York (1977) .

790

2.

Steinman, H.M. and Hill, R.L.: Proc. Natl. Acad. Sci. USA, 70, 3725-3729 (1973) .

3.

Sato, S. and Nakazawa, K.J.: Biochera. (Tokyo) 83, 11651171 (1978).

4.

Anastasi, Α., Bannister, J.V., Bannister, W. Η.: Int. Biochem., 7, 541-546 (1976) .

5.

Yamakura, F.: Biochim. Biophys. Acta, 422, 280-294 (1976).

6.

Stailings, W.C., Powers, T.B., Pattridge, K.A., Fee, J.A. and Ludwig, M.L.: Proc. Natl. Acad. Sci. USA. In Press (1983) . Ringe, D., Petsko, G.A., Yamakura, F., Suzuki, K. and Ohmori, D.: Proc. Natl. Acad. Sci. USA. In Press (1983).

7. 8.

Tainer, J.Α., Getzoff, E.D., Beem, K.M., Richardson, J.S. and Richardson, D.C.: J. Mol. Biol. 160, 181-217 (1982).

9.

Richardson, J.S.: Advances in Protein Chem., 34, 167-339 (1981).

10. Gregory, E.M. and Dapper, C.H.: Arch. Biochem. Biophys., 220, 293-300 (1983) . 11. Meier, B., Barra, D. , Bossa, F., Calabrese, L. and Rotilio, G.: J. Biol. Chem. 257, 13977-13980 (1982). 12. McLachlan, A.D.: Int. J. Quantum Chem. 12, Suppl. 1, 371385 (1977). 13. Walker, J.E., Auffret, A.D., Brock, C.J. and Steinman, H.M.: in "Chemical and Biochemical Aspects of Superoxide and Superoxide Dismutase," eds. Bannister, J.V. and Hill, H.A.O., Elsevier/North-Holland, New York, pp. 212-222, (1980) . 14. Villafranca, J.J.: FEBS Lett. 62, 230-232 (1976). 15. Fee, J.A. and McClune, G.J.: in "Mechnisms of Oxidizing Enzymes", Eds. Singer, T.P. and Ondarza, R.N., Elsevier/ North-Holland, Amsterdam, pp. 273-284, (1978). 16. Fielden, E.M., Roberts, P.B., Bray, R.C., Lowe, D.J., Mantner, G.N., Rotilio, G. and Calabrese, L.: Biochem. J. 139, 49-60. (1974). 17. Fee, J.A.: in "Metal Ions in Biology" Volume 13, ed. Sigei, Η., Marcel Dekker, New York, pp. 259-298 (1981). 18. Lavelle, F., McAdam, M.E., Fielden, E.M., Puget, Κ., and Michelson, A.M.: Biochem. J. 161, 3-11 (1977). 19. Fee, J.Α., McClune, G.J., O'Neill, P., and Fielden, E.M.: Biochem. Biophys. Res. Commun. 100, 377-384 (1981). 20. Fee, J.A., McClune, G.J., Lees, A.C., Zidovetski, R., and Pecht, I.: Isreal J. Chem. 21, 54-58 (1981).

791 DISCUSSION

ROTILIO: Is it possible that the pH dependence of your K,,, is due to ionisation of a nearby positively charged amino acid side chain, like lysine? STALLINGS : Using magnetic resonance techniques, VILLAFRANCA (réf. 14) detected water as a ligand in Fe dismutase. We associate the pH dependence of Κ,η with the ionization of a bound water because of the similar pH dependence of Kj and K D for azide. ROTILIO: Yes, but even azide could be attracted by positively amino acid side chains.

charged

STALLINGS: In the crystal structure azide is bound to the iron. The best model would be an inner-sphere coordinaton complex. ROTILIO: In the kinetic model that does not mean that you have other rate-limiting steps before the binding to the iron on the protein surface. What is the effect of ionic strength on that K,,,? STALLINGS: I think that in general, for _EI_ coli Fe dismutase, increasing the anion concentration increases K^. PARNHAM: It is well known that copper complexes of a lot of different compounds exhibit superoxide dismutase activity. What struck me about your structures is that iron-SOD and copper-SOD have almost totally different structures, the only factor in common being a metal ion in the center of the catalytic site. Is this massive protein structure, then, all a confidence trick, just to add a bit of extra protein to the catalytic site consisting of a metal ion and a few amino acids? STALLINGS: The Cu/Zn protein and the Fe protein are found in different places in different cells, and so it is possible that their external morphology simply reflects their need to bind at certain places and at certain times to do their job - they may need to bind at different places in different ways. I am not sure that it is fair to say that the only common feature that they have at their active center is an exposed face. At 3 Angstroms resolution and in the absence of sequence, it is impossible for us to characterize the precise coordination geometry in Fe dismutases; it certainly looks as if the active centers are otherwise different, but I think we have to await the sequence as well as higher resolution data which is coming along. PARNHAM: In your title you say "correlation of structure and activity". You have just shown the structure, but we know absolutely nothing about the relation of that tertiary protein structure to the activity of the enzyme. STALLINGS: I have tried to briefly compare the structure and kinetics of iron SOD with the Cu/Zn protein. Both the Cu/Zn and Fe proteins, for which X-ray structure exists, share one common feature, an exposed coordination site at which solvent-exchangeable ligands can bind. We believe that this

792 is an important three-dimensional aspect of the mechanism of dismutase catalysis. At 3 A resolution and without the Fe dismutase sequence, we can not say more than this. The structures appear to be completely different otherwise, certainly in their backbone fold and quarternary organization. So, if the true role of these proteins is to catalyze the dismutation reaction, then the Cu/Zn and Fe proteins have evolved with the same function but from different genes. SRIDHAR: One way to solve this would be to look for a bacterial SOD that has two identical or nearly identical subunits. If it is possible to dissociate the two subunits, then isolate pure populations of each subunit. Isolate a mutant strain of the bacteria that produces SOD with significantly altered subunits. Reconstitute hybrid enzymes using subunits from different sources. Testing these hybrid enzymes for SOD activity will show how important the subunits are. STALLINGS: units. SRIDHAR: STALLINGS:

Fe dismutases are usually isolated as dimers of identical subDid you dissociate the dimers? No.

MICHELSON: May I answer that question for you? Actually, coli has already done it. There is an iron enzyme and a manganese enzyme, and you always have a hybrid enzyme as well. SRIDHAR:

How reactive is that?

MICHELSON: Just the same. Now, to come to the other point: this nonsense about metal complexes being as good as enzymes. If you take the copper enzyme, and remove the copper and then put in manganese or iron, there is no activity. You can do this with all of the known superoxide dismutases, replace the original metal by another, it is liganded very well, it is just as stable, but there is no activity. So there is something special, perhaps it is channeling by the protein, and the nature of the ligands with each individual metal in the different kinds of superoxide dismutases. The protein is essential. STALLINGS: Thank you for your support. I should correct you on one small point and that is - I believe it is in Propionibacterium shemani (ref. 11) - that the synthesis of the iron and manganese dismutase has been shown to be dependent on the metal supplied in the growth medium, but the protein itself appears to be the same, and both have good activities. MICHE I£ON: The other point is that in eukaryotes the situation is slightly different, and it is very rarely in eukaryotes that you ever find the iron enzyme. Salin has one example of a eukaryotic iron enzyme. So if you are trying to compare a eukaryotic enzyme with a prokaryotic enzyme, which is what you did with copper-zinc bovine enzyme and the iron coli enzyme I don't see why you should have homology or morphological homology at all.

THE PHYLOGENETIC POSITION OF THE CU-ZN-SOD OF P. LEIOGNATHI.

Leopold Flohé, Wolfgang A. Giinzler, Sung-Man A. Kim, Fritz Otting, Gerd-J. Steffens Grünenthal GmbH, Center of Research, D-5100 Aachen, FRG Joseph V. Bannister Department of Inorganic Chemistry, University of Oxford, UK William H. Bannister Nuffield Department of Clinical Biochemistry, University of Oxford, UK

Introduction The proposed role of SOD in the defense against the hazards of aerobic l i f e prompted numerous investigations relating the a c t i v i t i e s and properties

of

superoxide

demand or tolerance

dismutases

in

various

organisms

to

their

oxygen

(1). In this context also the phylogenesis of SODs

attracted interest. Until recently, Cu-Zn-SOD appeared to have developed under the evolutionary pressure of an oxygenated atmosphere in eukaryotes only. The f i r s t exception to the rule, the Cu-Zn-SOD of P. leiognathi,

was correspondingly

photobacterium natural

from i t s

gene transfer

believed to be acquired by the symbiontic

teleost (2).

host,

This

the pony f i s h ,

intriguing

by means of a

idea was corroborated by

comparative determination of amino acid compositions of various SODs (3). The present investigation intended to verify this hypothesis by elucidation of the amino acid sequence of the bacterial Cu-Zn-SOD.

Materials and Methods The Cu-Zn-SOD of

P. leiognathi

was isolated from cultured bacteria as

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

794 described by Puget and Michelson homogeneous

(4). The final product appeared almost

in discontinuous SDS Polyacrylamide gel electrophoresis. It

contained 0.95 g atoms Cu and 1.04 g atoms Zn per subunit, had a specific activity of 3240 units/mg determined according to McCord and Fridovich (5), reacted with antibodies raised against the bacteriocuprein and kindly supplied by Dr. A. M. Michelson, and showed spectral

character-

istics typical for Cu-Zn-SODs.

The purified protein was reduced by mercaptoethanol and carboxymethylarl4 τ ted with

[ CJiodoacetate, cleaved by cyanogen

bromide in 70 % formic

acid or 90 % formic acid/heptafluorobutyric acid, by tryptic digestion or treatment with endoproteinase

Arg-C

(Boehringer

Mannheim), and the

resulting peptides were separated by gel filtration or HPLC and automatically sequenced by means of the liquid phase sequencer 890 C (Beckman) and the solid phase sequencer 4020 (LKB), as described in detail elsewhere (6). PTH amino acid derivatives were identified and quantitated by TLC

or

HPLC, respectively.

Amino acid determination

was

performed

by

means of the amino acid analyzer LC 6001 from Biotronik (Frankfurt).

Results

Within

experimental

limits, the amino acid composition of Cu-Zn-S0D of

P. leiognathi

proved

Steinman

Further,

(7).

to be it

identical with that previously reported by appears

similar

to

the recently

described

amino acid composition of the Cu-Zn-S0D from Caulobacter crescentus (7) and also related to other cupreins analysed so far. The fragmentation procedures outlined above yielded overlapping peptides which, after

sequence determination, allowed the reconstruction of the

entire amino

acid

sequence of the Cu-Zn-S0D of

P. leiognathi.

Fig. 1

compares

this sequence with those of human, equine, bovine and yeast

cupreins

published

from

published

earlier

data

(8, 9)

(1). The human sequence in

a

in fig. 1 differs

few

positions.

These

modifications

are based on a recent reinvestigation

(Grünenthal

GmbH, unpublished).

795 P.leiognathi Human Bovine Horse Yeast

1 Q Ac -A Τ Ac-•A Τ Ac -A L V

D Κ Κ Κ Q

Τ V V V V

V C C C A

ΚM V L V L V L V L

Τ Κ Κ Κ Κ

D G G G G

L Q DG DG DG DA

|FIT]P[1]L0D - L V WG S I Κ G - L )V[T]G S I[Ä)G - L JL Κ G F I E G - L V S Y[Ë]I[Â]G Ν S

P.leiognathi Human Bovine Horse Yeast P.leiognathi Human Bovine Horse Yeast

S [C G C G c G c G£

P.leiognathi Human Bovine Horse Yeast

H H H H H

80 G F G G G G G G G A

P.leiognathi Human Bovine Horse Yeast

V I 1 M F

L E V Κ Κ

-

-

G G G G

Κ R Κ Κ

P.leiognathi Human Bovine Horse Yeast

L A A A A

A Τ Τ Τ ν

G G G G

G V V V V

40 Ρ G Ε G Ε G Κ [g Ν A

Κ P QG QG HG SG M L D D E

0G I I Τ I V I V V

H G H G H G H G RlG

F F F F F

Τ Ν H H Κ

I F F F F

20 E E E E E

L Q Α Q Q

S0N Κ E S Κ G D Q[g]E A S E

H I H H H H H I H Y F F F F

Ρ Ρ Ρ Ρ Ρ

100 A ΝG Κ D G Κ Ν G E Ν G E Ν G

Μ Κ κ κ τ

-

G G G D

S Ν Ν Ν Τ

Τ D D D D

D E E E E

D E E E V

Ν R R R R

H H H H H

G G G G G

D L D L D L D L DIM

Ρ G G G G

A Ν Ν Ν Ν

L V V V V

I I I I

R S S S Κ

L L L L L

Τ S S s I

L 0 G D H G E Y g|k]h G Ρ Τ

EL C I S I S I S V

K I I I V

G G G G G

120 H A I RTL R Τ M R Τ M R S V

D E E E E

M E E E E

Ρ S S s s

Κ Τ Τ Τ L

A Κ Κ κ Κ

-

Κ V V V V

L Τ τ τ τ

140 G G G G Ν A G Ν A G Ν A G Ν A

G A G S G S G S G, Ρ

R R R R R

F Τ Τ Τ Κ

V L L L Ρ

V A A A Τ

A A A A A

S D D D D

Κ Ν G S

D Ν Ν Ν Ν

G G G G G

G Ρ Τ Ρ Ρ

Ρ E H τ Ν Κ Ρ - L s R Κ Ρ - L s Κ Κ Ρ - L s Κ Κ Ρ - F κ κ Τ L A V A V A Κ A V [aj

τ D I D Κ

M O Â~GlG Ε Κ A V V VV Ε Κ Ρ Ε ΚQ V V V[T H Ä G | Q C C C C C

Y G G Ε

D Ν D Ν

60 S s Ε Κ D G Κ V V L G G A A G G H S A G Ρ H s A G Ρ H τ A G A H IsJA G Ρn

A 0 D S V D[P]L D S V D S L -

Τ Τ Τ Τ Ρ

Τ Ρ Ρ Ρ G

V V V V V

I I I I I

151 Q G G G G

A A A Τ

Ν Ν V V G

Ρ s D D S

Ν D D D D

Η L L L L

Q Κ Ρ Ν

F i g . 1: Comparison o f t h e amino a c i d sequence o f P h o t o b a c t e r i u m l e i o g n a t h i Cu-Zn s u p e r o x i d e d i s m u t a s e w i t h e u k a r y o t i c Cu-Zn-SODs f r o m human e r y t h r o c y t e , b o v i n e e r y t h r o c y t e ( 1 0 ) , h o r s e l i v e r ( 1 1 ) , and y e a s t ( 1 2 ) . The d i f f e r e n t amino a c i d sequences were a r r a n ged i n o r d e r t o o b t a i n maximal sequence homology. I d e n t i t i e s as compared t o t h e sequence o f p h o t o b a c t e r i u m b a c t e r i o c u p r e i n a r e i n d i c a t e d by b o x e s . Numbers mark p o s i t i o n s i n t h e sequence o f P. l e i o g n a t h i SOD.

796 The chosen alignment of sequences takes into account both, a maximum of identities

positions

and the conservation of amino acid

residues considered to be essential

in homologous

for activity of the enzymes (1). It

reveals considerable conservation of some parts of the molecule over the entire

tree of phylogenesis.

In particular

the following typical

fea-

tures of Cu-Zn-SODs are retained. The aspartic acid residue 91 and the six histidine residues 45, 47, 70, 79, 88 and 125 of the P. leiognathi sequence corresponding

to Asp 81, His 44, 46, 61, 69, 78 and 118 known

to coordinate the metals in the bovine enzyme, the cysteines forming the only disulfide bridge (52 and 147 in P. leiognathi and 55 and 144 in the bovine

sequence), and

thought

to guide the

the arginine residue superoxide

(144 or 141, respectively)

anion towards the copper during

cata-

lysis.

In

contrast

to

the

vertebrate

SOOs,

the

corresponding

enzyme

of

P.

leiognathi, like those of Caulobacter and yeast, lack acetylation of the N-terminus.

Discussion Based on molecular size, metal content, specific activity and amino acid sequence the bacteriocuprein of P. leiognathi has to be classified as a real

Cu-Zn-SOD homologous to the Cu-Zn-SODs of eukaryotes and structu-

rally

unrelated

relatedness

to

Fe-

or

between

the

homologous

number of identical ding

to

remote

this

of

the

the

and yeast, we

Cu-Zn-SOD

family

different mammals, mammals mammals

SODs.

enzymes

is

The best

actual

degree

estimated

by

of the

amino acid residues in homologous positions. Accor-

criterium,

relative

Mn-containing

of

described

and another

find identical

P. so

leiognathi far

is the most

(Fig. 2).

vertebrate,

Comparing

the swordfish, and

amino acid residues in more than

80, 60 and 50 % of the SOD sequences, respectively.

In contrast, only

less than 30 % identities between the SOD sequence of P. leiognathi and

797

PL P.leiognathi

(PL)

151

Human

(HU)

39

153

BO

Bovine

(BO)

45

125

151

HO

HU

Horse

(HO)

43

123

123

153

SW

Swordfish

(SW)

44

102

108

101

152

Yeast

(YE)

40

84

83

87

82

Fig. 2:

that

of

Matrix of identical amino acid residues in homologous positions of sequenced Cu-Zn-SODs. The numbers are based on the sequences and alignments shown in Fig. 1. The swordfish data were kindly made available by T. Huisman (13).

all

other

species

are

detectable.

In

order to achieve

this

moderate degree of relatedness, we have already to assume several deletions of 1-12 amino acids in the course of evolution. The established rank order of relatedness of the cupreins thus corresponds nicely to that of the cytochrome c family (14) and to the generally assumed relationship

of the species

under consideration.

We therefore

have to conclude that the Cu-Zn-SOD is a genuine bacterial enzyme which, though homologous to eukaryotic cupreins, has not been acquired somehow by

the

bacterium

phylogenesis

of

from

any

superoxide

vertebrate

gene bank.

dismutase, as proposed

In consequence,

the

up to now, e.g.

by

Asada et al. (15), has to be revised (Fig. 3): Not only can the Fe- and Mn-containing life; also

SODs

be

traced

back

to the early prokaryotic

the Cu-Zn-SOD must have developed

forms

some billion years

of

ago,

i.e. in a poorly oxygenated atmosphere. This conclusion is not readily reconciled with present views on the function of SOD as an enzyme detoxifying

oxygen-centered radicals: We have to envoke either the necessity

of cellular oxygen defense despite very low oxygen tension or a change in function of the cupreins during evolution.

798

799 References

1.

Steinman, H.M., in: Superoxide Dismutase, Vol. I (Oberley, ed.), pp. 11-68, CRC Press, Inc., Boca Raton, Florida 1982.

2.

Puget, Κ., Lavelle, F., Michelson, A.M., in: Superoxide and Superoxide Dismutases (Michelson, A.M., McCord, J.M..Fridovich, I., eds.), pp. 139-150, Academic Press, London 1977.

3.

Martin, (1981).

J.P.,

Jr.,

Fridovich,

I.:

J.

Biol.

Chem.

256,

L.W.,

6080-6089

4.

Puget, Κ., Michelson, A.M.: Biochemie 56, 1255-1267

5.

McCord, J.M., Fridovich, I.: J. Biol. Chem. 244, 6049-6055

(1974).

6.

Steffens, G.-J., Bannister, J.V., Bannister, W.H., Flohê, L., Günzler, W.A., Kim, S.-M.A., Otting, F.: Hoppe-Seyler's Ζ. Physiol. Chem., in press.

(1969).

7.

Steinman, H.M.: J. Biol. Chem. 257, 10283-10292

8.

Barra, D., Martini, F., Bannister, J.V., Schimina, M.E., Rotilio, G., Bannister, W.H., Bossa, F.: FEBS Letters 2 2 0 , 53-56 (1980).

(1982).

9.

Jabusch, J.R., Färb, D.L., Kerschensteiner, Biochemistry Jj), 2310-2316 (1980).

1U.

Steinman, H.M., Naik, V.R., Abernethy, J. Biol. Chem. 249, 7326-7338 (1974).

11.

Lerch, K., Ammer, D.: J. Biol. Chem. 256, 11545-11551

12.

Johansen, J.T., Overbaile-Petersen, C., Martin, B., Hasseman, Svendsen, I.: Carlsberg Res. Commun. 44, 201-217 (1979).

13.

Rocha, H., Scott, D., Huisman, T.H.J., Bannister, W.H., Bannister, J.V.: in preparation.

14.

Dayhoff, M.O., in: Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 2, pp. 25-35, Natl. Biomed. Res. Found., Washington, D.C. 1976.

15.

Asada, Κ., Kanematsu, S., Okaka, S., Hayakawa, T., in: Chemical and Biochemical Aspects of Superoxide and Superoxide Dismutase (Bannister, J.V., Hill, H.A.O., Eds.), p. 136, Elsevier/North Holland, New York 1980.

D.A.,

Deutsch,

J.L.,

Hill,

Η.F.: R.L.:

(1981). V,

DISCUSSION

ELSTNER: I think the same thing holds for the iton-SOD in eukaryotes, as Dr. MICHELSON already pointed out. I think there are more than one species of eukaryotes which contain iron-SOD. For example there's Euglena gracilis which is a plant animal, it's a highly organized eukaryote. It only contains Fe-SOD. And as SALIN pointed out, Brassicaceae and Nymphaceae are

800 highly developed plants and they have Fe-SOD. So that doesn't neccessarily mean that in your scheme the Fe-SOD is terminated with the appearance of eukaryotes. If you extend the Cu/Zn-SOD downward, it might also be useful to continue the Fe-SOD upwards (for review see: RABINOVICH and FRIDOVICH, Photochem. Photobiol. (1983), 37, 679-690). MICHELSON: You say that leiognathi is a late bacterium and it's on the edge between prokaryote and eukaryote with respect to these enzymes, but you see, the second enzyme is the copper enzyme. You would expect that the second enzyme in the bacterium would be manganese. It isn't, there is no manganese-enzyme, there is only an iron-enzyme. And the iron-enzyme was there first, because in this case it's the copper-enzyme which is induced by high oxygen. I entirely agree, at least in a few cases, that the phylogeny must be extrapolated downwards and upwards. There is one other bacterium, known to contain a copper-enzyme ... FLOHE:

Caulobacter, very recently published by STEINMAN (ref. 7).

MICHELSON:

Right, and this again has no relationship to mammalian Cu-SOD.

FLOHE: Well, its sequence looks a little bit similar. The partial sequence has been published (ref. 7). I think it's homologous, but with a similar low degree of relatedness. WESER: I think this was a very good example to show that for the low molecular weight copper complexes the active center really is the most important thing, because the reaction with superoxide radicals does not actually require large protein molecules. It's a radical mechanism where you don't need activation energy, and so nature itself shows that the best way is really to produce a very stable copper chelate, surviving all the biological systems and still sharing the good old SOD activity. FLOHE: Well, that isn't particularly what I wanted to demonstrate. Consider the histidines constituting the active center! You need a large proportion of protein to construct this active site. And it is constructed by different loops of the protein. You can make the whole bulk of the protein with different amino acids. There's a large variability. But that doesn't mean that the low molecular weight copper complexes can do all the job. The arginine is conserved. Everything that has been considered essential is conserved. SRIDHAR: To what extent is there a relatedness for proteins such as insulin, cytochromes and superoxide dismutases isolated from different species FLOHE: Well, E. coli and leiognathi don't have a pancreas, so I do not know anything about their insulins. But the cytochrome c sequences have been worked out for quite a number of species and we did compare the numbers of identities of the cytochrome c tree with that of the SODs. The percentages of identities between different Cu/Zn-SODs correspond very nicely to what has been worked out for cytochrome c and agree with the general assumption about the relatedness of species. MARRLUND: First I want to point out that we now have to introduce the new EC-SOD into this scheme, adding a new complication to it. I further want

801 to point out that there appears to be Fe-SOD in mammals as well. There has been a report by an Armenian group (GRIGORYAN & NALBANDYAN, in "Chemical and Biochemical Aspects of Superoxide and Superoxide Dismutase", J.V. Bannister & H.A.O. Hill, Eds., Elsevier/North-Holland (1980) pp. 196-200), that there is Fe-SOD in bovine adrenal medulla. FIX)HE: I agree, but I didn't want to change everything at one time. So I showed the scheme of ASADA and coworkers (ref. 15) and just inserted one additional arrow (see Fig. 3).

ERYTHROCUPREIN (Cu Zn2SUPEROXIDE DISMUTASE) CARRIES 90% OF THE ERYTHROCYTE COPPER

Alfred Gärtner, Margareta Leippert and Ulrich Weser Anorganische Biochemie, Physiologisch chemisches Institut der Universität Tübingen, Hoppe-Seyler Str. 1 7 400 Tübingen 1, GFR

Introduction Since the early days of Mann and Keilin (1) our knowledge on erythrocyte copper proteins other than erythrocuprein (Cu2Zn2superoxide dismutase) is very limited. In 1961 an additional copper protein was reported by Shields et al. (2) . The existence of a pink copper protein was published in 1970 (3) and recently a fourth copper protein called Cu^(haem^) protein of M

= 400 000 was prepared from bovine and human

erythrocytes (4, 5). No quantitive assignment of the copper content to each of these copper proteins is available. Thus, a reexamination of the amount of copper being bound in erythrocuprein prompted this study. Earlier immunochemical quantification studies led to the conclusion that 40% of the red blood cell copper was bound to erythrocuprein (6).

Results and Discussion In the classical isolation technique organic solvents were employed. The final yield of erythrocuprein usually was between 25-30% of intracellular copper. The major portion was lost. An improvement of the yield was expected using the

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin - New York - Printed in Germany

804 strictly aqueous method of Stansell and Deutsch (7) which was modified in that the discontinuous absorption of erythrocyte copper on DEAE-cellulose was replaced by continuous chromatography on DEAE-Sephacel. A typical isolation pattern of erythrocuprein is summarized in the table. Aqueous purification of erythrocuprein Preparation-step

Volume (ml)

Copper Ug)

% of Cu

Haemolysate

Superoxide Dismutase Activity * U/ml 0.94

2 000

1 020

100

DEAE-Sephacel eluate

470

870

85

1 60

G-75

350

850

83

600

second DEAEeluate

240

600

58

1 200

670

635

62

1 300

280

530

50

3 200

CM Sephadex third DEAEeluate *NBT-Assay

Virtually all of the erythrocyte copper is found in erythrocuprein. The elution profile of the G-75 chromatography shows essentially no high M

copper protein. There was

neither a low molecular weight species (Fig. 1) . After each ion exchange step the copper concentrations were high enough to allow EPR detection. Addition of known amounts of separately prepared cuprein (8) served as an internal standard. Erythrocuprein isolated by the present aqueous procedure carries 1.9 Mol copper and 2.0 Mol zinc. The specific superoxide dismutase activity was 3 200 U per mg of protein. Control of superoxide dismutase activity in the course of the isolation procedure coincided with the EPR properties. The chemical environment around the copper and the SOD-activity was exclusively

assigned to erythrocuprein. Thus, it must be

805 concluded that this protein is the major copper binding protein of the red blood cell.

Effluent· (ml)

Figure 1 : Elution profile of the gel chromatography on Sephadex G-75 of Table 1

Gel filtration of the crude haemolysate (step one) was carried out on Sephadex G-75 to exclude the possibility that some high molecular weight Cu-containing proteins were lost during the first DEAE-chromatography

(Table). 91% of copper

and superoxide dismutase activity migrated in the region of M r = 32 000. The amount of high M r proteins including CU2(haem^)2~protein was less than 5%. The earlier conclusions (4, 5) regarding the yield of this high M r protein have to be reconsidered and will have to stand up to rigorous questioning. It cannot be excluded that this protein was formed at a later stage of the isolation. The absorption spectrum of CU2(haem^)2~protein (Fig. 2) can be simulated by either a molar mixture of 1 oxyhaemoglobin and 3 methaemoglobin or 1 oxyhaemoglobin and 3 catalase. As no superoxide dismutase activity is observed in CU2(haem^)^-protein (4) the EPR-signal indicative for type II copper may be attribu-

806 ted to trapped erythrocuprein by polymerized haem proteins. The time consuming preparation procedure may have led to the formation of a Cu^(haem^)2-protein which now appears to join the fate of the other two reported copper proteins (2, 3).

Figure 2: Simulation of electron absorption properties of Cu„(haem, )„-protein

807 Conclusion Intracellular erythrocuprein and serum caeruloplasmin are the major copper proteins of whole blood. Both proteins have one point in common. The discussion on the biological role of either protein continues.

References 1. 2.

3. 4. 5.

Mann, T., Keilin, D.: Proc. Roy. Soc. London Ser Β 126, 303-315 (1939) . Shields, G.S., Markowitz, H., Klassen, W.H., Cartwright, G.E., Wintrobe, M.M.: J. Clin. Invest. 40, 2007-2015 (1961) . Reed, D.W., Passon, P.G., Hultquist, D.E.: J. Biol. Chem. 245, 2954-2961 (1970). Weser, U., Gartner, Α., Sellinger, Κ. H.: Biochemistry 21 , 6133-61 37 (1982) . Gartner, Α., Sellinger, Κ. Η., Weser, υ.: Hoppe-Seyler's Ζ. Physiol. Chem. 363, 959 (1982).

6.

Stansell, M.J., Deutsch, Η.F.: J. Biol. Chem. 240, 4306-431 1 (1965) .

7.

Stansell, M.J., Deutsch, Η.F.: J. Biol. Chem. 240, 4299-4305 (1965) .

8.

Weser, U., Bunnenberg, E., Cammack, R., Djerassi, C., Flohê, L., Thomas, G., Voelter, W.: Biochim. Biophys. Acta 243, 203-213 (1971 ) .

Acknowledgement This work was aided by a grant from the Deutsche Forschungsgemeinschaft DFG We 401/16.

SUPEROXIÜE-DEPENDENT PATHOLOGY AND SUPEROXIDE DISMUTASE TREATMENT.

Leopold Flohê Grünenthal GmbH, Center of Research, D-5100 Aachen

Many findings indicating a crucial events

have

Oxygen

Radicals

related

been

reported

at

in Chemistry

subjects

the

role of "Og" in certain pathological Third

and Biology

International

Conference

and at previous

symposia

on on

(1-6), and it can not be the intention of this short

preface to compile and discuss all aspects of "Og'-dependent pathology. Neither will be attempted to review in any detail the present experience with superoxide dismutase treatment. Instead, I shall try to provide a preliminary rationale of the clinical application of SOD and, then, focus on some pertinent questions.

1)

Disappointments resulting from naive concepts. It is generally accepted that radiation of oxygenated watery fluids yields

"0 2 ~,

chondrial

that increased oxygen tension induces elevated mito-

·0 2 " production,

and microsomal

'Q^

that adriamycin triggers mitochondrial

formation, and that paraquat poisoning is asso-

ciated with excessive O g " production. These insights have prompted numerous attempts to balance the increased '0^

levels by injection

of SOD in the conditions mentioned. A benevolent statistical

evalua-

tion of pertinent published reports yields conflicting results at best. Acute death from a lethal radiation dosage is not consistently prevented adriamycin

by

SOD

in

experimental

cardiotoxicity

animals,

sensitive

to

SOD

nor

is obviously

treatment.

the

Finally,

those patients which survived a seemingly lethal paraquat poisoning in retrospect proved to have paraquat blood levels compatible with survival.

In fact, the by and large disappointing outcome of these

experiments is not as unexpected as it may appear. In most, if not

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

810

a l l examples mentioned so far, '0 2 ~ i s formed and acts intracellular^.

Just to mention one historical example, the *0 2 ~ formation

of mitochondria could only be demonstrated after inside-out particles

largely freed of intramitochondrial

SOD had been prepared

(7). To our knowledge, i t has never been demonstrated that

"0 2 ~

formed by any intracellular organel can pass the SOD-rich cytoplasm and leaks into the i n t e r s t i t i a l f l u i d . An injected SOD, however, i s certainly

distributed in the extracellular

space exclusively and

therefore has no chance to interact with intracellular

2)

Hopes Prevention

of

tissue

damage due to intracellular

*0 2 ~

requires

elevation of intracellular SOD. Theoretically,

this

can be achieved by induction.

Unfortunately,

however, the i n d u c i b i l i t y of SOD in mammalian c e l l s appears poor (2) and i t i s s t i l l

unclarified whether the 50 % increase of SOD

levels in the lung after exposure to high oxygen tension are caused by induction or a change in cell population (2). In consequence, the principle of SOD induction has not yet attracted the interest of therapists. Alternatively, SOD could be smuggled into the c e l l s by means of liposomes.

To some degree

the

liposomal

technique also

allows

tissue targeting. Some promising therapeutic results in auto-immune diseases have been obtained by this approach by Michelson and his colleagues

(see 4 ) ; but a final

judgement about the therapeutic

value of this kind of treatment requires controlled c l i n i c a l

trials

with appropiate numbers of patients.

3)

The r e a l i s t i c approach In inflammatory diseases, PMNs and macrophages release large amounts

811

of O g

into the extracellular space which i s almost devoid of SOD.

formation by activated PMNs and macrophages represents part of the host defense system, but is also triggered by non-infectious stimuli such as any opsonized particle or immune complexes. In the meantime i t

i s widely accepted that '(^"contributes to the mani-

festation and self-maintainance

of inflammation in various ways:

1. I t may directly or indirectly affect cell membranes, enzymes or macromolecules of the extracellular matrix (1, 4); 2. i t generates yet unidentified chemotactic factors (2); and 3. i t modifies macromolecules

which,

then, may further activate PMNs or macrophages

(Lunec and H i l l , this symposium). Injection of SOD should ideally prevent all these "0 2 "-dependent pathological effects. In r e a l i t y , this can be achieved only i f accessible and i f of

adequate SOD levels

considerations,

the s i t e of inflammation i s easily

the anatomical circumstances allow maintainance in

the

inflamed

tissue.

Based on these

the efficacy of SOD applied i n t r a a r t i c u l a r l y was

primarily tested in osteoarthritis and one example of the throughout favourable outcome of these c l i n i c a l

trials

will be presented by

Puhl (this symposium). In principle, however, SOD injected locally ameliorates a variety of inflammatory diseases (5, 6).

4)

Limitations and questions Most problems we are confronted with in SOD therapy have to be attributed to the protein nature of the drug, other

difficulties

result from our scarce pathophysiological knowledge. The protein nature of the drug SOD implies high purification costs, little flexibility

in the mode of application, special pharmaco-

kinetic problems, and steady attention to avoid immunological complications. At present, only bovine SOD i s available and approved for

clinical

preparation

use. The incidence of a l l e r g i c

reactions with

this

(Peroxinorm , Grünenthal) i s very low and thus toler-

able. However, even the extremely low immunogenic potential must

812

not be ignored, and indications which would require repeated intravascular injections of SOD can not be tested before a human SOD of sufficient purity i s available. Some aspects

of

the pathophysiological

role

of

"0^

are

still

poorly defined. In particular, we do not yet know the structure and the precise pharmacodynamic profile of the ^ " - d e p e n d e n t

chemo-

tactic factor(s). We have but vague ideas on the interaction Og"

with

the

intermediates

of

prostaglandin

and

of

leukotriene

biosynthesis, and a clear view of the relevance of Ό 2 " and other mediators of inflammation in a given disease i s only slowly emerging.

This means that therapeutic results obtained in a defined

disease are not easily extrapolated to other c l i n i c a l conditions. A better understanding of the relative contributions of the various mediators to various patterns of inflammation would certainly help optimizing

the

fit

between any rationale

of

treatment

and the

actual therapeutic effect. References 1.

Michelson, A.M., McCord, J.M., Fridovich, I . : Superoxide and Superoxide Dismutases, Academic Press, London 1977.

2.

Ciba Foundation Symposium 65 (new s e r i e s ) : Oxygen Free Radicals and Tissue Damage, Excerpta Medica, Amsterdam 1979.

3.

Autor, A.P.: Pathology of Oxygen, Academic Press, New York 1982.

4.

Bannister, W.H., Bannister, J.V.: Biological and C l i n i c a l Aspects of Superoxide and Superoxide Dismutase, Elsevier North Holland, New York 1980.

5.

The European Journal of Rheumatology and Inflammation 4, No.2 (1981).

6.

Puhl, W., Sies, H.: Abakterielle, artikulare und peri artikulare Entzündungen, perimed Fachbuch-Verlag, Erlangen 1982.

7.

Loschen, G., Azzi, Α., Richter, C., Flohê, L.: FEBS Lett. 42, 68 (1974).

SOD TREATMENT IN OSTEOARTHRITIS OF THE KNEE JOINT

Wolfhart Puhl Orthopädische Klinik und Poliklinik der Universität Heidelberg D-69U0 Heidelberg Leopold Flohe Grünenthai GmbH, Forschungszentrum D-5100 Aachen Gerd Biehl St. Franziskus-Hospital D-5000 Köln Hans Hofer Orthopädische Abteilung der Landeskrankenanstalten A-5020 Salzburg Reinhard Kölöel Orthopädische Universitätsklinik und Poliklinik D-2UOO Hamburg-Eppendorf

Introduction: Definition of the Disease and Rationale of Treatment The most frequently observed inflammation of the knee joint is the secondary synovitis in osteoarthritis. Pathogenetically, cartilage lesions develop in previously healthy joints due to chronic biomechanical problems, acute trauma, hydroxyapatite depositions or other reasons. Hydrolytic enzymes released from damaged chondrocytes, and detritus derived from cells or extracellular matrix will then induce an inflammation of the synovial membrane. Thus, any type of primary degenerative joint disease may lead to a synovitis. This "secondary

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

814

synovitis" resembles the "primary synovitis" characterizing e.g. rheumatoid arthritis in many respects: It causes pain, swelling of the synovium, occasionally effusion, and thus reduces mobility and impairs joint function. At the same time, ongoing synovitis represents a hazard immanent to the cartilage already damaged: Phagocytes evading from the synovial tissue release a variety of hydrolysing enzyme and mediators of inflammation which comprise ·O^ (1). The superoxide radical itself or other oxygen-centered radicals derived therefrom have been implicated in a variety of pathological phenomena which may be relevant to joint destruction, e.g. biomembrane oxidation (2), depolymerisation of hyaluronic acid (3) and collagen (4), and desinhibition of elastase by oxidation of «^-antitrypsin (5). · f u r t h e r takes part in the formation of a potent chemoattractant (6) thereby raising the cell density in the synovial cleft. It is therefore hypothesized that formation by PMNs and macrophages aggravates inflammation, contributes to connective tissue destruction and perpetuates the inflammatory process. In turn, any medication scavenging · should prove beneficial in inflammatory joint disease.

Clinical Trials and Clinical Experience with SOD in Osteoarthritis Based on the outlined rationale supporting evidence from animal experiments (7) and a promising clinical pilot study (b) we started to investigate the efficacy of intraarticular SOD injections in patients with active osteoarthritis about 5 years ago. In a multicenter placebo-controlled double-blind trial 4 mg bovine Cu-Zn SOD or placebo, respectively were injected into the affected knee joint in weekly intervals for eight consecutive weeks.

815 T a b . I; I m p r o v e m e n t of c l i n i c a l s y m p t o m s of o s t e o a r t h r i t i s a f t e r e i g h t w e e k s of S O D t r e a t m e n t . D a t a (x +_ s-) are t a k e n x f r o m ref. 9.

Parameter

SOD

(dimension)

T h i c K e n i n g of s y n o v i a , h y d r o p s (scores, % improvement) Joint circumference (decrease, cm) Pain

(scores, % d e c r e a s e )

Disability scores (% i m p r o v e m e n t ) * SOD vs. Placebo

(n = 45)

Placebo

(n = 40)

46.6 + 6.4

21.4 _+ 7.3*

1.3 + 0.2

0.2 + 0.24*

65.5 + 3.7

19.8 + 6.6*

45.3 + 4.8

11.1 + 6.3*

ρ2 dismutation, which has received some useful comment (16) and support (17) in the literature, pointed out that a basis for the greater activity of small molecular weight Cu complexes when compared to Cu-ZnSOD derived from bovine liver may be due to their ability to cross cell membranes.

This is

consistent with their greater lipid solubility as well.

Anticonvulsant Activity of Copper Complexes

Being aware that Cu deficiency in animals and man is associated with seizures, we submitted Cu complexes to the National Institute of Neurological and Communicative Disorders and Stroke-Antiepileptic Drug Development Program (NXNCDS-ADD) for evaluation as anticonvulsants in their models of seizure.

Cu complexes of salicylates and aminoacids were found to be potent

anticonvulsants following subcutaneous(sc) administration(18).

Since neither

salicylates nor aminoacids are known to have anticonvulsant activity and neither C u C ^ nor Cu acetate, given sc, had anticonvulsant activity, it was suggested that the observed activity was due to the complexed form of Cu. This lead to the hypothesis that the active forms of the anticonvulsant drugs might be their Cu complexes.

Supportive evidence was provided when it was

shown that the Cu complex of amobarbital was more effective than sodium amobarbital as an anticonvulsant(19).

In addition, Cu(II)(amobarbital)

found to be less toxic than sodium amobarbital.

was

We continue to find that Cu

complexes of various antiepileptic drugs which dismutate 0^ (unpublished observation) have anticonvulsant activity(20).

More detailed comparisons, in

progress, are required to fully investigate the possibility that these Cu complexes can be distinguished from their parent drugs.

824 There are no studies that document the relationship of decreased neuronal Cu-ZnSOD activity as an etiology of seizures in epilepsy.

However, epileptic

seizures resulting from brain trauma, infection, and tumors(21,22) are clearly associated with inflammatory lesions wherein Oj release or accumulation may have an etiologic role.

It seems worthwhile to at least consider

the possibility that ideopathic epileptic seizures might also be due to reduced Cu-ZnSOD levels and/or the abnormal accumulation of 0„,

Antineoplastic Activity of Copper Complexes

Following the report by Oberley and Buettner(9) that neoplastic cells were deficient in SOD activity and Cu-ZnSOD decreased the growth of a solid sarcoma 180 tumor and increased survival of the implanted mice(23), a variety of O 2 dismutating Cu salicylates were examined for anticancer activity.

The Cu complex of aspirin, Cu(II)2(acetylsalicylate)^, was found

to produce a rapid and marked reduction of an ascites neuroblastoma tumor in mice(20) and a marked reduction in growth of a solid Ehrlich tumor accompanied by an increase in survival of the implanted mice.

Solvates of this

complex were even more effective than the parent complex in the solid Ehrlich tumor model(23).

Since these solvates were thought to be more lipid soluble

and lipid solubility appeared to increase activity, the ether soluble complex Cu(II)(3,5-diisopropylsalicylate)2[Cu(IX)(dips)2] was evaluated as an anticancer agent.

It was found to be the most effective of all salicylate

complexes tested to date(24-26).

Subsequent mechanistic studies support the

suggestion that O^ dismutation by Cu(II)(dips)2 or induced de novo synthesis of Cu-ZnSOD and production of Η,,Ο,, may account for its antineoplastic activity (26).

Research directed toward the re-establishment of normal SOD levels

in neoplastic cells merits serious consideration as an approach to cancer therapy.

This suggestion is consistent with the recent report that ascorbic

acid plus Cu(II)(gly-gly-his)had a marked inhibitory effect on the growth of an Ehrlich cell ascites tumor and markedly increased survival of the implanted mice(27).

825 Anticarcinogenic Activity of Copper Complexes

The involvement of oxygen radicals in the multistep, initiation followed by promotion, process of carcinogenesis is becoming evident.

Not only are these

species important in the metabolic activation of some carcinogens, but they may be an obligatory component of tumor promotion(28-30),

Recently, Kensler

et al.(31) demonstrated anticarcinogenic activity for Cu(II)(dips) , in mouse epidermis.

Topical administration of Cu(II)(dips)^ markedly inhibited tumor

promotion in mouse epidermis.

Application of 2umol Cu(II)(dips)

30 min.

prior to each treatment with 4nmol 12-tetradecanoyl-13-acetylphorbol to 7,12,dimfethylbenz(a)anthracene initiated mice for 18 weeks resulted in a 60 and 93% inhibition in tumor incidence and the number of papillomas per mouse, respectively, conpared to those initiated mice receiving vehicle and promotor alone.

No significant inhibition of tumor formation was observed with

either Cu(II)^(acetate)^ or 3,5-diisopropylsalicylic acid(3,5-dips) implying that lipophilicity as well as SOD-mimetic activity are essential.

The induction of ornithine decarboxylase(ODC) activity is a prominent, early, and transient event following exposure to epidermal tumor promoters and can be inhibited by a spectrum of antipromoting agents(32).

Cu(II)(dips)^ in-

hibited the phorbol diester mediated induction of ODC in a log-dose response (31).

The concentration of complex that caused 50 percent inhibition was

1 uM while ®u(II)S0^, Cu(II)2(acetate)^, and 3,5-dips, compounds lacking in lipophilic character or SOD-mimetic activity, did not produce 50 percent inhibition at concentrations of 5, 10, and 20 uM respectively.

Thus, the

finding that a low molecular weight, lipophilic, Cu chelate with SOD-mimetic activity can inhibit certain phorbol diester-induced biochemical and biological responses strengthens arguments for an essential role of oxygen radicals in the promotion stage of carcinogenesis and of SOD or SOD-like compounds in the homeostatic prevention of carcinogenesis.

Antidiabetic Activity of Copper Complexes

Gandy, Buse, and Crouch(33) were the first to provide data demonstrating that O. dismutating Cu complexes have antidiabetic activity in the

826 streptozotocin-induced diabetic rat.

Glucose utilization was impaired in

streptozotocin-treated mice as well as streptozotocin and salicylic acid or 3,5-dips-treated mice.

Glucose utilization was improved in the streptozoto-

cin and Cu(II)(salicylate)2 or Cu(II)(dips)^-treated groups as evidenced by the lower plasma glucose values at time zero and at the end of a 60 minute glucose tolerance study.

Improved glucose utilization could not be attribu-

ted to hepatic dysfunction or glycosuria, which were not evidenced at the time of testing.

It is noteworthy that compounds possessing SOD-like chemical reactivity can attenuate streptozotocin diabetes.

This activity is attributed to the com-

bination of O2 dismutating reactivity and lipophilicity of the complexes, which may allow cellular penetration and re-establishment of β cell Cu-ZnSOD This suggestion has merit since it has been demonstrated that streptozotocin decreases Cu-ZnSOD activity in pancreatic 3 cells(34) and intravenous(iv) administration of Cu-ZnSOD attenuated streptozotocin-induced diabetes(35,36). Testing SOD-like Cu complexes for their ability to prevent or attenuate experimental diabetes may be worthwhile since it is possible that the etiology of Type I diabetes involves oxygen radical mediated β cell damage following the loss or reduction of Cu-ZnSOD activity.

Radiation Protection Activity of Copper Complexes

Petkau and his colleagues(37-39) demonstrated that Cu-ZnSOD, given iv, afforded protection against doses of x-irradition ranging from 300 to 1000 rads.

Seventeen percent of the treated mice survived the highest radiation

dose with SOD treatment both pre- and post-irradiation.

Since Cu(II)(dips)2

was thought to penetrate cell membranes and elevate intracellular O^ dismutating reactivity, it was selected for evaluation as a radioprotectant.

Two groups of 24 mice were treated with one sc injection of 5 mg of Cu(II)(dips)2 per mouse.

One group was treated 3 hrs. before irradiation and the

other 24 hrs. before irradiation. injection of vehicle.

A control group of 22 mice was given a sc

All of the mice were bilaterally gamma-irrated with a

total dose of 1000 rads, 40 rads per minute.

All of the control mice died

827 by day 18.

However, 33% and 58% of the respective 3 hr. and 24 hr. pre-

treated groups survived.

Deaths occurred in the treated groups up to day

18 or 19 with no subsequent deaths for the remaining term of the experiment, 30 days.

Even though these data are preliminary, the possibility that small

molecular weight Cu complexes might be useful as radioprotectants necessitates careful evaluation of this observation.

Acknowledgements

We are indebted to the American Cancer Society; Grants SIG-3 and IN-11U; the Arhtur Armbrust Cancer Foundation; the American Diabetes Association; the International Copper Research Association; The Kroc Foundation; the National Cancer Institute; Grant T32-CA-09125; and the National Institute of Environmental Health Sciences; Grants ES07067 and ES00454 for financial support. We are also most grateful to Cmdr. Brian Gray of the Armed Forces Radiobiology Research Institute for his efforts in obtaining the radioprotectant data with Cu(II)(dips) .

References

1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

Powanda, M.C.: Inflammatory Diseases and Copper, Sorenson, J.R.J., ed Humana Press, Clifton, New Jersey P. 31-44 (1982). Flynn, A: Inflammatory Diseases and Copper, Sorenson, J.R.J., ed., Humana Press, Clifton, New Jersey, P. 17-30 (1982). Sorenson, J.R.J. : Inorg. Perspect. Biol. Med., 2_, 1-26 (1978). Sorenson, J.R.J.: Metal Ions in Biological Systems, Vol. 14, H. Sigel ed., Marcel Dekker, Inc., New York P. 77-124 (1982). Hass, Μ.Α., Frank, L., Massaro, D.: J. Biol. Chem. 257, 9379-81 (1982). McCord, J.M.: Science 185, 529-531 (1974). Rister, M.: Trace Elements in the Pathogenesis and Treatment of Inflammation. Rainsford, K.D., Brune, Κ., and Whitehouse, M.W., eds., Birkhauser Verlag, Basel P. 137-143 (1980). Rister, Μ., Bauermeister, Κ., Gravert, U., Gladtke, E.: Lancet, May 20 , 1094 (1976). Oberley, L.W., Buettner, G.R.: Cancer Res. 39, 1141-1149 (1979). Sorenson, J.R.J.: Prog. Med. Chem. 15, 211-260 (1978). Sorenson, J.R.J.: Inflammatory Diseases and Copper, Sorenson, J.R.J., ed., Humana Press, Clifton, New Jersey P. 289-301 (1982). Sorenson, J.R.J.: J. Med. Chem., 19, 135-148 (1976). Sorenson, J.R.J., Hangarter, W. : Inflammation, 2_, 217-238 (1977). Weser, U., Richter, C. , Wendel Α., Younes, M.: Bioinorg. Chem. Q, 201-213 (1978). DeAlvare, L.R., Goda, Κ., Kimura, T.: Biochem, Biophys. Res. Commun., 69, 687-694 (1976).

828 16. 17.

Young, C.L., Lippard, S.J. ; J, Am. Chem, Soc. 102, 4920-4928 (1980). Nappa, M., Valentine, J.S., Miksztal, A.R., Schugar, H.J,, Isied, S.S.: J. Am. Chem. Soc. 101, 7744-7746 (1979). 18. Sorenson, J.R.J., Rauls, D.O., Ramakrishna, Κ., Stull, R.E., Voldeng, A.N.: Trace Substances in Environmental Health XIII. A Symposium, Hemphill, D.D., ed., University of Missouri, Columbia, MO P. 360 (1979). 19. Sorenson, J.R.J., Stull, R.E., Ramakrishna, Κ. , Johnson, B.L., Riddell, Ε., Ring, D.F., Rolniak, Τ,Μ., Stewart, D.L.: Trace Substances in Environmental Health-XIV, Hemphill, D.D. ed., University of Missouri, Columbia, MO. P. 252-264 (1980). 20. Sorenson, J.R.J., Oberley, L.W., Crouch, R.K., Kensler, T.W., Kishore, V., Leuthauser, S.W.C., Oberley, T.D., Pezeshk, Α.: Biol, Trace Elem. Res. #5 in press 1983. 21. Rose, A.L., Lombroso, C.T., Pediatrics, 45, 404-425 (1970). 22. Oxbury, J.M., Whitty, C.W.M., Brain, 94, 733-744 (1971). 23. Oberley, L.W., Leuthauser, S.W.C., Buettner, G.R., Sorenson, J.R.J., Oberley, T.D., Bize, I.B.: Pathology of Oxygen. Autor, A.P., Ed., Academic Press, New York P. 207-221 (1982). 24. Sorenson, J.R.J., Oberley, L.W., Oberley, T.D., Leuthauser, S.W.C., Ramakrishna, Κ., Vernino, L., Kishore, v.: Trace Substances in Environmental Health-XVI, Hemphill, D.D., ed.. University of Missouri Press, Columbia, MO P. 362-369 (1982). 25. Leuthauser, S.W.C., Oberley, L.W., Oberley, T.D., Sorenson, J.R.J., Ramakrishna, K.: J. Nat. Cancer Inst., 66, 1077-1081 (1981). 26. Oberley, L.W., Rogers, K.L., Schutt, L., Oberley, T.D., Leuthauser, S.W.C., Sorenson, J.R.J.: J. Nat. Cancer Inst, in press (1983). 27. Kimoto, Ε., Tanaka, Η., Gyotoku, J., Morishige, F., Pualing, L.: Cancer Res. 43, 824-828 (1982). 28. Goldstein, B.D., Witz, G., Amoruso, M., Troll, W.,: Biochem. Biophys. Res. Comm. 88, 854-860 (1976). 29. Kensler, T.W., Trush, M.A.: Cancer Res. 41, 216-222 (1981). 30. Slaga, T.J., Klein-zanto, A.J.P., Triplett, L.L., Yotti, L.P., Trosko J.E.,: Science 213, 1023-1025 (1981). 31. Kensler, T.W., Bush, D.M., Kozumbo, W.J.: Science 221, 75-77 (1983). 32. Slaga, T.J., Fischer, S.M., Weeks, C.E., Nelson, Κ., Mamrack, Μ., Klein-Szanto, A.J.P.: Carcinogenesis - a Comprehensive Survey, Vol. 7 Hecker, Ε., Fusenig, Ν., Kunz, W., Marks, F., Thielmann, H., eds., Raven Press, New York P. 19-29 (1982). 33. Gandy, S.E., Buse, M.G., Sorenson, J.R.J., Crouch, R.K.: Diabetologia 24, 437-440 (1983). 34. Crouch, R.K., Gandy, S.E., Kimsey, G., Gailbraith, R.A., Gailbraith, G.M.P., Buse, M.G.: Diabetes, 30, 235-241 (1981). 35. Robbins, M.J., Sharp, R.A., Slonim, A.E., Burr, I.M.: 18, 55-58 (1980). 36. Gandy, S.E., Buse, M.G., Crouch, R.K.,: J. Clin. Invest. 70, 650-658 (1982). 37. Petkau, Α., Chelcak, W.S., Pleskach, S.D., Meker, B.E., Brady, C.M.: Biochem. Biophys. Res. Comm. 65, 886-893 (1975) 38. Pektau, Α., Kelley, Κ., Chelack, W.S., Pleskach, S.D., Barefoot, C. Meeker, B.E.: Biophys. Res. Comm. 67, 1167-1174 (1974). 39. Petkau, Α., Chelack, W.S., Pleshach, S.D.: Int. J. Radiat. Biol. 29, 297-299 (1976).

829 DISCUSSION ΡARNHAM: I have just a couple of short, related questions. Firstly, I didn't see any data for a simple copper salt. Have you seen effects in inflammatory conditions, say, with copper sulfate and have you tested this salt in your irradiation and cancer experiments and - more importantly have you measured free copper levels after administration of these copper complexes? What I am getting at: is it copper that's exerting the effect and not the complex? SORENSON: In 1966 we demonstrated that copper complexes were effective anti-inflammatory agents while the ligands and copper acetate and copper chloride were not (ref. 12). Prof. OBERLEY (ref. 25) did show that 3.5-diisopropylsalicylic acid (DIPS) and copper sulfate had no anti-tumour activity. We haven't done that experiment yet in our radiation protection work We are currently measuring copper in tissues after treatment. SINGH: One minor correction and one question. The source of Dr. PETKAU's SOD in earlier studies was Truett Labs (Photochem. Photobiol. (1978) 28, 765). Ceruloplasmin is reported to be a radioprotector (Chem. Abstr. (1981), 94, 96327b); how do your complexes compare with ceruloplasmin? SORENSON:

They don't.

WINTERBOURN: Related to the first question: do you know whether the copper is still bound to your complexes once you inject it into your animals? SORENSON: We don't really know anything about the disposition of the copper or its complexes following administration. If one uses loosely bound forms of copper, like copper acetate, copper sulfate and copper chloride, one does not see these effects. Nor does one see the effects if only the ligands are used, so some of this activity has to be mediated by the copper complex. That's the state of the art. PRYOR: Has anybody measured the binding constants for Cu-DIPS? It apparently is competing with other ligands that are present. What might the most strongly binding, natural ligand for copper be and how would the DIPS ligand compete? SORENSON: If one measures the stability constants for salicylates, the ß-values are about 12-15. Loosely bound forms of copper have no effect due to the complexation at the site of administration, which negates the systemic distribution. Amino acids and albumin are likely to be the most abundant competing ligands. These may form tertiary complexes with Cu-DIPS and further facilitate tissue distribution. BHUYAN: Copper catalyzes the production of hydrogen peroxide from ascorbic acid. Now, you have one slide where I think PAULING is involved, that shows good results, about 50% regression. In that case you mean that copper complex does not react with the ascorbic acid? Or you don't suspect that there will be any free copper in the tissue?

830 SORENSON: Normally, there is no or very little "free" copper in tissues. In plasma the amount of ionic copper is 10"^·® M, that's not measurable - it's a calculated value, because of all the ligands that can bond with it. With regard to the vitamin C and Cu(gly-gly-his) data, that was actually published by Prof. KIMOTO's group and by Prof. PAULING (ref. 27). It seems reasonable that hydrogen peroxide production may facilitate differentiation to the normal cell type or perhaps neoplastic cell killing. Based upon that work we should examine the antineoplastic effect of our copper complexes plus vitamin C.

OXYGEN RADICALS : PHYSIOLOGICAL AND MEDICAL ASPECTS, WITH SPECIFIC REFERENCE TO HIGH ENERGY IRRADIATION. A.Michael Michelson, Krystyna Puget Institut de Biologie Physico-Chimique, Service de Biochimie-Physique 13, rue Pierre et Marie Curie, 75005 Paris, France.

Introduction Although superoxide anions are probably the most important activated oxygen species produced primarily by high energy irradiation of biological systems, they can also give rise to a cascade of other radicals such as hydroxy, carbonate and lipoperoxy radicals. Other enzymic systems for protection against uncontrolled oxidative processes exist and may have a future medical utility in certain cases. We will consider some aspects with respect to attack of different enzymes with different kinds of active centers by various kinds of radical species, the effects of irradiation on protective enzyme levels in humans, and the development of possible treatments for radioinduced damage in humans, whether accidental or as post radiotherapeutic sequels, based on superoxide dismutase.

Comparison of the deactivation of various enzymes by different oxygen containing free radicalo : effects of carbonate anions. The relative effects of superoxide, hydroxyl, formate and carbonate radicals for the inactivation of various enzymes (superoxide dismutases, catalase, ribonuclease, glucose oxidase and glutathione peroxidase) were examined (1) using γ irradiation under suitable conditions to produce the different radicals (Table I).

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

832 TABLE I + CO3 2 -

t 1/2 (min) o2-

co2-

835 867

491

49

794

Fe SOD bacterial >300 Mn SOD human 2040

>300

Cu SOD bovine Cu SOD human

RNAse pancreatic Glucose oxidase Catalase Glutathione peroxidase

HO·

02τ

C0 2 ~

HO

33

9

32

13 22

19

50

7

5

2053

19

18

26

193

68

13

158

90

>300 >500

>300

30

>300

>500

52 92

>500 24

>300 >500

25

10.5

12

7 10 1. 54 43 .6 42 .5 61 121

Exposure to γ rays ( 6 0 C o ) ; 6Ö8 rads/min. Since the yield of production of the different radicals under various buffer conditions (presence or absence of formate, N2O, anaerobic or saturated with O2) is not identical, the above figures for halflives of enzymic activity represent the kinetics of loss of activity for a given energy input. With respect to C>2~ it is evident that superoxide dismutases are extremely resistant. Glucose oxidase and catalase are also extremely resistant whereas glutathione peroxidase is rapidly inactivated. Ribonuclease is only relatively resistant. A very similar pattern is shown when formate radicals are used. When hydroxyl radicals (equivalent energy input) are considered it is evident that the superoxide dismutases show no particular resistance and are as rapidly inactivated as the other enzymes. It may be noted however, that glutathione peroxidase, the least stable enzyme with respect to C>2T and CÛ2 T is the most resistant to hydroxyl radicals, by a two fold factor com-

833

pared with catalase and SOD, three times as stable as glucose oxidase and seven fold with respect to ribonuclease. Comparison of the effects of O2 7 , C02 T and HO· on the various enzymes in presence of carbonate anions shows a completely changed situation. Whereas

has little or no effect on the

rate of deactivation of ribonuclease, catalase, glucose oxidase or glutathione peroxidase (active sites are peptide, heme, flavin and selenium respectively) by O2"7, the stability of the various SODs is remarkably decreased ded C u

2+

, Mn

3+

(active sites are amino acid ligan-

or Fe^+J. This is undoubtedly due to formation

of carbonate anion radicals : 2 H+ + 0 2 T + C 0 3 2 -

H 2 0 2 + CO3"

These radicals show the same reactivity as 0 2 ~ with respect to peptide, flavopeptide, heme bound iron, or peptide bound selenium, but are much more specific with respect to active centres containing C u 2 + , Mn3+ or Fe-^ + in simple liganded form. It may be supposed that 003" occupies the binding site of 02 T in the enzyme and then forms an irreversible metal complex by electron transfer between the metal and CO3"7. Since CO3· ¿ s

not

a substrate for superoxide dismutases, it is

clear that the autoprotection of these enzymes with respect to 0 2 ~ is no longer effective when C 0 3 2 - anions are present (or HC0 2 ") .

The various superoxide dismutases (Cu, Μη or Fe at the active site) are even more rapidly inactivated by HO· + C O 3 2 -

(i.e.

C0 3 ~) than by hydroxyl radicals alone, despite the fact that C03 T itself is very much less reactive than HO·. In contrast, with enzymes such as ribonuclease, catalase, glucose oxidase and glutathione peroxidase, none of which have a simple metallo active centre, a certain measure of protection is afforded in

834 accord with the r e l a t i v e carbonate

radicals

p a r e d w i t h HO·, active

centres

a r e much l e s s

an i n c r e a s e d increases

s i n c e most amino a c i d attacked time of lable cals

by 0 0 3 " . C03T,

for

This, at

the a c t i v e

proportion

is

residues

of

implicated

directly

in the

hand, role

where t h i s it

sence than

is

of

catalytic

that

of

free

In t h e

case

mechanism.

attack

are

long

not

life

radicals of

by n o n - s p e c i f i c

avai-

HO·

radi-

attack

at

necessarily On t h e

other

by CO31" c a n n o t p l a y

enzymic a c t i v i t y

carbonate anions

presumably

relatively of

com-

metallic

simply because

is

protected

HO· i s

by

more

a

pre-

reactive

003".

Given the clear

sensitivity

that

in vivo

to protection The p r e s e n c e fect

the

centre.

dispersed

at

by HO·,

the p r o t e i n which are not

selectivity

evident

attack

inactivation,

concentration

Although

in p a r t i c u l a r

of

while attacked

coupled with

amino a c i d

HO· and CO3" 7 .

reactive,

the r a t e of

the

of

specificity

residues,

increases

attack

a large

reactivities

of

of

glutathione

injected

this

carbonate

ions

on s y s t e m s

involving

activated

This aspect since

of

003" r a d i c a l s

is

almost

always

effect

oxygen s p e c i e s ,

due

radicals. ef-

possible

carbonates.

in b i o l o g i c a l to

is

significant

or t o peroxy

present

it

by s u c h

thus has a v e r y

s h o u l d n o t be o v e r l o o k e d

carbonate

t o 02T

SOD may h a v e an i n d i r e c t

enzyme f r o m d e s t r u c t i o n

of

due t o p r o d u c t i o n

peroxidase

systems

a greater

or

lesser

of

the

extent. It

is

free

possible radical

carbonate of

that

mechanisms

radicals,

the b a c t e r i a l

anions

could well

tions

reported

reported

superoxide into

explain

in the

in t h i s

involved

dismutase.

a certain

literature. it

is

part

killing for

concern

inactivation

The p r e s e n c e at various

number o f

of

to

divergent

inactivation

that postulation

of

carbonate

concentra-

Given the b i o c h e m i c a l

respect clear

least

species

consideration)

study with

enzyme s y s t e m s ,

at

in b a c t e r i a l

t h e most e f f i c i e n t

(seldom taken

tions

rent

during phagocytosis

of

observaresults diffe-

hydroxyl

835

radicals as a most lethal possibility is subject to considerable caution. Perhaps of much greater importance than chemical reactivity is a decreased reactivity coupled with a longer lifetime and increased specificity when toxic biological effects of free radicals in general are considered. The polemic on the supposed lack of reactivity of C>2T (contradicted in fact by many chemical publications) to deny toxicity of this radical becomes meaningless. Superoxide radicals are themselves toxic ; they can give rise to other toxic products ; they are the primary biological free radical ; they are the unique substrate for superoxide dismutases which are the first line of defense (supported by glutathione peroxidase and glutathione transferases) against uncontrolled oxidative damage which can lead to a wide range of pathological inflammatory conditions.

Enzyme levels after high energy irradiation : accidental or otherwise In four surviving members of a family which received massive overdoses of γ irradiation

we have observed reduced le-

vels of erythrocyte Cu-SOD (396-448 ug SOD/g Hb) and increased glutathione peroxidase values (closely grouped at an average of 11.5 units compared with normal values of 7-8 units). This family was followed over several months and the severity of the clinical symptoms correlated closely with levels of Cu-SOD. Indeed recovery can be predicted once the increase in SOD is established (see Table II). This was confirmed in another case, where after an initial drop following the accident, the level of SOD was rapidly increased over two months (Table III).

836 TABLE II Blood (erythrocyte) levels of SOD and GPX in γ-irradiation subjects as a function of time after the accident. ug SOD/ml blood

GPX/mg Hb

05.10 78

56 4

12. 3

30.11 78 12.02 79

66 0

12. 6

71 5

11. 4

16.05 79

77 7

FAT Recovery

NUA 05.10 78 30.01 78

63 4 62 6

12. 5 10. 4

23.02 79

67 6

10. 8

05.10 78

61 1

9. 7

30.11 78

63 7

10. 8

12.02 79

65 9

10. 5

05.10 78

63 4

11. 5

30.11 78 23.02 79 16 . 05 79

55 6

9 9

57 4

9. 9

Slow recovery

FAH Slow recovery

DJA

55 5

-

TABLE III Date

Hb/100 ml lysate

SOD μ g/ml

SOD ug/Hb

R.B. 13. 03.79

15.40

78 .84

512

21.03 .79 27.03 .79

13.65 14.55

65 . 53 79 .20

480 544

11. 05.79

14.10

84 . 00

597

837 Similarly, dosage of erythrocyte SOD and GPX in six technicians involved in another radiation accident allowed a simple classification of the intensity of radiation received (and of the clinical gravity) particularly when the variation of these parameters with time was studied, the two most severe cases presenting a continued drop in both enzymes several weeks after the irradiation (Table IV). TABLE IV Hb(g/100 ml) ug SOD/ml pg SOD/g Hb blood

GPX/mg Hb

ANC 09.04 81

16.75

93.1

556

27.04 81

13.60

67.9

499

09 . 04 81

13.45

70.0

520

27.04 81

11.90

57.8

486

7.8 7.2

FAF 10.6 8.7

Finally, radiotreatment (35s) of cancer patients can also destroy both SOD and GPX as shown in the following table, in which after treatment an inverse correlation of changes in these enzymes can again be seen (Table V). TABLE V 35

R.L.

S treatment

1 Hb SOD SOD g/100 ml sang ng/ml lysate yg/g Hb

_L Before treatment

GPX I nmoles NADPH/ min/mg Hb

15.1

66.2

444

20.65

36.10.30

13.6

47.3

332

9.75

05.01.81

14.35

43.5

281

12.26

11.09.00 After treatment

838 Other effects of high energy irradiation Since radiation induced chromosome breaks are probably due to the action of free radicals, it is of interest that γ or Xirradiation also gives rise to a plasmic clastogenic factor (2). Examination of the blood in the case of an industrial accident in which the subject received a very high dose of 192j r γ-írradiation showed both a marked decrease of erythrocyte SOD and presence of a clastogenic factor. Similarly, in four surviving members of a family which received massive doses of γ irradiation (60co) a clastogenic factor was again present in the sera. In vitro experiments showed that gamma-irradiation of human (or bovine) whole blood (but not of plasma) produces or liberates such a factor at relatively low levels of irradiation

(400-1200

rads ) .

Such accidents are relatively infrequent but since SOD inhibits (3) the activity of the chromosome breaking factor, this provides another reason for the use of SOD to combat secondary effects of irradiation. Use of the enzyme could inhibit the cascade of events leading to the diverse manifestations and symptoms. The cell itself is normally protected. However, the extra-cellular medium is much less well endowed. For example human plasma contains about 30 ngs Mn-SOD per ml ( 4 )r compared with an average of 60 yg SOD per gm of tissue (3900 mgs for the total human body) (5). The exterior cell membrane is thus vulnerable to free radical attack, which can initiate a chain reaction in the matrix (phospholipids,proteins, etc...) which destroys the membrane and causes lysis, even when production of the external free radicals is stopped, as has been shown with human erythrocytes using the photoreduction of riboflavin as producer of activated oxygen species (6). Cytolytic products from the solubilized membranes thus pass in the extra-cellular medium, with increase in vascular permeability as one of the effects. These irradiation-produced hydroperoxides, endoperoxides, epoxides, etc... are in themselves cytotoxic as a pri-

839

mary phenomena. However, during the sequence of events more O2 - is produced either chemically or due to phagocytotic activity. It is at this second wave of C>2~ production that exogenous SOD (free or liposomal) can be efficient rather than the first, and thus reduce radio-induced sclerosis and inflammatory lesions. There is thus a rational at several biological levels for the possible application of superoxide dismutase. Medical application Superoxide dismutase (bovine copper enzyme) has already been considerably exploited in certain countries for the treatment of inflammatory disorders such as osteoarthritis (7), rheumatoid arthritis (8) and radiation cystitis (9). We decided to look at the possible use of liposomal preparations of the enzyme. The preparation, characterisation and properties of different kinds of liposomal SOD have been documented elsewhere, as have the pharmacokinetic comportments (10-14). The advantages of liposomal encapsulated SOD (or glutathione peroxidase and transferases) are multiple. Thus, they do not attach to bacteria or penetrate and therefore would not increase the resistance of pathogens to phagocytic action. Secondly if liposomal SOD attaches to PMNs or macrophages, this fixation is followed by fusion with the membrane with interior liberation of the encapsulated protein (12). Membrane bound enzyme is attached to the inner surface since if the outer surface was involved there would be loss of enzyme to yield the low levels of fixation of the free enzyme (few external sites appear to exist). This does not occur with SOD, glutathione peroxidase or glutathione transferase liposomes (15-17) and hence it may be concluded that essentially all the membrane bound enzyme is interior and not exterior. Phagocytic activity would thus not be inhibited arid protection of ingested bacteria would not occur, the enzyme being on the outside surface of the vacu-

840 ole. Even within the first hours when the liposome is fixed to the outer membrane surface of the cell during endocytosis, protection would not be afforded to ingested bacteria since the liposomal encapsulated SOD is not active or not available to O2 - as shown by in vitro tests of liposomal suspensions. In view of possible medical applications these conqlusions are of importance since exterior bound enzyme could inhibit the killing action of free radicals during phagocytosis and hence increase the sensitivity of a patient to microbial infection. However, interior bound enzyme in fact protects the polymorphonucleophils against suicide by over excitation and excessive production of superoxide ions or lipohydroperoxides. This phenomenon thus increases the possible value of liposomal enzymes for treatment of inflammatory conditions. Apart from organ specificity which liposomes confer on the SOD, with both extracellular material held in the structure and cellular bound (intracellular) material, which could be of considerable importance, with liposomal SOD (non-active) there is a slow continual liberation of active free enzyme. This avoids a pulse of free SOD in the organism which could be detrimental. At the same time, smaller amounts of total enzyme can be used due to the pharmacokinetic advantages. It may be noted that in a model system, free SOD had no effect on surface potential disorders of mitochondria treated with UV light exposed methyl linoleate (formation of peroxides, etc...) or on respiratory responses (18). Liposomal SOD alleviated the membrane disorders and thus could be useful for preventing cellular damage induced by lipid peroxidation. Three cases of post-radiotherapeutic necrosis, due to overf dosage, were treated during one year, two months and twenty days respectively. In all three cases, the results were dramatic. The radio-induced sclerosis regressed as judged by biopsies X-ray picture of uretus and surgical procedures (19). The liposomal SOD preparations were well tolerated. Another case of facial radionecrosis has also been treated by

841

intramuscular injection and topical application of SOD liposomes. After one week the intensity of inflammation was considerably reduced together with considerable improvement in skin texture of the cheek. Again the results may be considered extremely promising. Radionecrosis and inflammation due to an accident in which the case suffered serious radio-induced damage to one hand was also successfully treated with local application and intramuscular injection of liposomal SOD. The result suggests a therapeutic effect of SOD (encapsulated in liposomes) in radiation induced sclerosis. Various other cases of irradiation damage due to X-rays, or γ-rays in which the lesions were minor to severe have also been successfully treated. In several instances the clinical symptoms appeared some 6-12 months after radiotherapy or the irradiation incident. None of the patients have returned for subsequent treatment suggesting that not only has the immediate pathology been cured, but the tardive sequels which often arise from a delayed outburst have also been presented or eliminated.

References 1.

Michelson, A.M., Maral, J. : Biochimie 65, 95-104 (1983).

2. 3.

Scott, D. : Cell. Tis. Kinet. 2, 295-298 (1968). Emerit, I., Michelson, A.M. : Acta Physiol. Scand. Suppl. 492, 59-65 (1980).

4.

Baret, Α., Schiavi, P., Puget, K., Michelson, A.M. : FEBS Letters 112, 25-29 (1980).

5.

Marklund, S. : Acta Physiol. Scand. Suppl. 492, 19-23 (1980) . Michelson, A.M., Durosay, P. : Photochem. Photobiol. 25, 55-63 (1977).

6. 7. 8.

Beckmann, R., Flohé, L. : Bull. Europ. Physiopath. Resp. Suppl. Γ7, 275-285 (1981). Menander-Huber, K.B. : Developments in biochemistry, vol. 11 b : Biological and clinical aspects of superoxide and

842

superoxide dismutase, 408-423, ed. Bannister, W.H. and Bannister, J.V., Elsevier, Amsterdam, Oxford, 1981. 9.

Marberger, H., Bartsch, G., Huber, W., Menander, K.B., Schulte, T.L. : Curr. Ther. Res. 18, 466-475 (1975).

10.

Michelson, A.M., Puget, K., Durosay, P. : Molecular Physiology 1, 85-96 (1981) .

11.

Michelson, A.M., Puget, K., Perdereau, B., Barbaroux, C. : Molecular Physiology 1, 71-84 (1981).

12.

Michelson, A.M., Puget, K. : Acta Physiol. Scand. Suppl. 492, 67-80 (1980).

13.

Michelson, A.M., Puget, K., Durosay, P., Rousselet, A. : Biological and clinical aspects of superoxide and superoxide dismutase, 348-366, ed. Bannister, W.H. and Bannister, J.V., Elsevier/North-Holland, New-York, Amsterdam, Oxford, 1980.

14.

Michelson, A.M. : Metalloproteins structure, molecular function and clinical aspects, 88-116, ed. Weser, U., Thieme Verlag, Stuttgart, 1979.

15.

Michelson, A.M., Dangeon, 0., Puget, Κ., Durosay, P., Perdereau, B., Barbaroux, C. : Mol. Physiol. 3, 27-34 (1983) .

16.

Dangeon, 0., Michelson, A.M. : Mol. Physiol. (1983).

17.

Puget, K., Michelson, A.M., Durosay, P., Perdereau, B., Barbaroux, C. : Molecular Physiology 3, 43-51 (1983)

18.

Ogura, R. , Murakata, Μ., Sakata, T., Chiba, R. : Kurume Med. J. 28, 1-8 (1981).

19.

Emerit, J., Loeper, J., Chomete, G. : Bull. Europ. Physiopath. Resp. Suppl. 17, 287 (1981).

35-41

CHARACTERIZATION OF THE 02"/H202-GENERATING SYSTEM IN HUMAN NEUTROPHILS

Mie Ν. Hamers, René Lutter, Rob van Zwieten, Dirk Poos Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam. Margriet L.J. van Schaik Department of Pediatrics, Academic Medical Centre, Amsterdam. Ron Wever Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Amsterdam, The Netherlands.

Introduction The nature of the superoxide/hydrogen-peroxide-generating system of human neutrophils is the subject of considerable controversy. From several studies it has been concluded that a flavoprotein might be involved in the production of superoxide (1-3). Solid evidence for this conclusion is lacking, however. In addition to a flavoprotein, a b-type cytochrome is also thought to be a component of this system (4). This cytochrome-b is undetectable in the neutrophils of some patients with chronic granulomatous disease (CGD) (3), a syndrome in which the neutrophils fail to mount a respiratory burst. Furthermore, after stimulation of neutrophils under anaerobic conditions, cytochrome-b is reduced, and it is re-oxidized upon subsequent introduction of air to the cuvette, indicating a role for cytochrome-b in a redox reaction with oxygen (5). Cytochrome-b has been reported to react with carbon monoxide (CO) (4,6); and from this observation. Cross et al. (7,8) concluded that cytochrome-b is directly involved in the reduction of oxygen as a terminal oxidase. However, we (9,10) and others (2,11) have failed to observe CO binding to (purified) cytochrome-b in the absence of hemoglobin contamination. Most investigators have reported a low midpoint potential for cytochrome-b (Em 7.0 of about -245 mV) (2,8,10,11), which is sufficiently low to reduce oxygen to superoxide. This low midpoint potential, however, does not warrant the conclusion that cytochrome-b will indeed reduce oxygen to superoxide. Recently, a quinone has been suggested as an essential part of the system (12,13). This finding is inconsistent with the insensitivity of the respiratory burst to amytal (14) and to antimycin A (15). Furthermore, contamination with mitochondrial quinones (16) derived from either the neutrophils or contaminating cell types was not ruled out in these studies. In the present paper, we describe a further characterization of the so-called cytochrome-b and other possible components of the system.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

844 Results and Discussion Search for a flavoprotein Fig. 1A shows the absolute spectrum of human neutrophils under aerobic conditions at the resting state; thus, all components of the oxidase system are in the oxidized form. The large peak at 417 nm is composed of cytochrome-b at 411 nm and myeloperoxidase at 428 nm, and perhaps some eosinophil peroxidase at 414 nm. The smaller peaks at 499, 568 and 627 nm are derived from oxidized myeloperoxidase. The peak at 527 nm is of unknown origin, and the shoulder at 450 nm could represent a flavoprotein. Upon stimulation of the neutrophils with phorbol-myristate acetate (PMA), no changes in this spectrum were observed. However, after anaerobic stimulation of the neutrophils with PMA, a rapid reduction of cytochrome-^ was found. Fig IB shows the spectrum of intact neutrophils stimulated with PMA under anaerobic conditions minus that of stimulated cells under aerobic conditions.

Fig. 1A

Fig. IB

Reduced cytochrome-b is formed (peaks at 424, 527 and 555 nm), and the trough at 453 nm could represent the difference between fully reduced and oxidized (shoulder at 450 nm in Fig. 1A) flavoprotein. If we assume £453 nm = 11.3 mM~l. cm~l for the flavin absorbance (17), a ratio of flavin to cytochrome-b of 1 is found. Upon admittance of air into the anaerobic cuvette, the signals of cytochrome-b and the "flavoprotein" disappeared immediately. The ratio of flavin to cytochrome-b was confirmed in Triton-X-100-solubilized neutrophil membranes. The fluorescence emission and excitation properties resembled those of FAD, and equal amounts of FAD and cytochrome-b were found; this is in agreement with the work of Cross et al. (18). However, partially purified cytochrome-b (see below) showed also a trough in the redox spectrum at 453 nm, as in Fig. IB indicating the possible presence of a flavin group, but non-covalently bound FAD was not detectable in this preparation. Thus, the trough at 453 nm in the redox spectrum of cytochrome-b might represent a signal of a covalently bound flavin. In our opinion, a flavocytochrome-b, as

845

suggested by Cross et al. (18), is indeed possible. The relation of the FAD found in solubilized membranes to the b-cytochrome has to be established yet. Search for quinones We failed to extract appreciable amounts of quinones from large amounts of isolated neutrophils. Moreover, the respiratory burst activities (02 consumption and H202 production) of UV-light-treated neutrophils were equal to those of untreated neutrophils, even after extreme UV treatment (45 min, 1.5 mW/cm^). Furthermore, the PMA-stimulated anaerobic reduction of cytochrome-b was undisturbed after treatment of the neutrophils with the quinone inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). The resistance of the respiratory burst to treatment with amytal and antimycin has already been mentioned in the Introduction of this paper. Thus, we consider it unlikely that quinones are involved in the respiratory burst. CO fixation by cytochrome-b As mentioned in the Introduction, we never found any CO fixation to cytochrome-b unless contamination with hemoglobin was present (9,10). Whenever CO fixation was found, it was found that the dissociation constant (K¿) for CO was about 0.001 mM. This is in contrast with the reported K^ for CO of 1.18 mM with cytochrome-b (8); but it is in accord with the of about 1 μΜ for CO with hemoglobin. Because the reported ^dCO f o r cytochrome-b is quite high, and CO fixation to a small portion

Fig. 2

846 of the b-cytochrome could have escaped our observation, we performed the following experiment. Highly purified neutrophils without any detectable hemoglobin contamination were incubated in Thunberg cuvettes under anaerobic conditions with 1 mM dithionite to reduce the cytochrome-b and to prevent reoxidation. Thereafter, the sample was treated with pure CO-gas (80 mm Hg) at 0°C for 20 min. This treatment results in a saturated CO concentration of 1.6 mM, well above the reported and at least 50% of the cytochrome-b should be in the CO-adduct form. To get a better resolution between the CO-fixed fraction of the cytochrome and the native fraction, we recorded the redox spectrum at 77°K. The result is shown in Fig. 2. No evidence, whatsoever, for more than one species of cytochrome-b (the native form) was found, indicating that no CO fixation had occurred at this (saturated) CO concentration. To the best of our knowledge, heme-containing oxidases with such a low affinity for CO and a high affinity for 0 2 are unknown (the Kj,, for 0 2 is about 10 μΜ in intact neutrophils). Thus, we conclude that the cytochrome-b does not function as a terminal oxidase in the oxidase system. Partial purification of cytochrome-b Cytochrome-b was partially purified from human neutrophils according to the following scheme. Neutrophils were isolated from about 60 "buffy coats" (the leukocyte-rich fraction) of human blood. The isolated neutrophils were suspended to a concentration of 10 8 cells/ml in ice-cold 100 mM potassium phosphate buffer (pH 7.2) containing 0.34 M sucrose, 5 mM EGTA, and 1 mM PMSF. Thereafter, the cells were homogenized for 1 min with a Sorvall omnimixer at 16,000 rpm and centrifuged for 20 min, 23,000 χ g at 4°c. The resulting pellet was homogenized with the Sorvall omnimixer in ice-cold 100 mM potassium-phosphate buffer (pH 7.2) containing 5 mM EGTA, 1 mM PMSF and Triton X-100 in a ratio of Triton X-100 to protein of 4 to 1 (vol/wt). The homogenate was centrifuged again. The pellet was discarded, and the supernate was incubated with Blue Sepharose (Pharmacia). The flow-through fraction of the Blue Sepharose was discarded and the Sepharose was washed with 100 mM potassium-phosphate buffer (pH 7.2), containing 0.1% (vol/vol) Triton X-100 and 0.4 M NaCl. Thereafter, the Blue Sepharose was eluted with the same medium as mentioned above, containing 1 M NaCl. The different purification steps were monitored for the SOD-inhibitable NADPH-cytochrome-c reductase, for cytochrome-b and for protein. Results of a typical experiment are shown in Table I. This procedure rendered almost identical results when PMA-stimulated cells were used. Table I. NADPH-cyt-c reductase 100% homogenate sucrose supernate 122% Triton X-10 0 homogenate 3% Triton X-100 supernate 6% Triton X-100 pellet 1% flow-through of Blue Seph. 6% 1 M NaCl eluate of Blue Seph. 0%

cytochrome-b

protein

100% 0% 100% 95% 3% 9% 80%

100% 68% 49% 29% 21% 15% 6%

847

It is evident from this table that the SOD-inhibitable NADPH-cytochrome-c reductase was entirely separated from cytochrome-b, indicating that this reductase activity (which is considered to be the superoxide-producing activity) is not intrinsic to cytochrome-^. This is in contrast to the suggestion that cytochrome-b is the terminal oxidase that generates superoxide. The partially purified cytochrome-b preparation did not contain any non-covalently bound FAD, as mentioned above. Preliminary characterization of cytochrome-b Polyacrylamide gel electrophoresis (5%) showed that the cytochrome-b has a high molecular weight. Cytochrome-b was detected by scanning the gels before and after reduction with dithionite and with a specific heme staining (10). The high .molecular weight of cytochrome-b was verified with a calibrated Sephacryl S J Q Q column (Pharmacia), equilibrated in 100 mM potassium-phosphate buffer (pH 7.2) containing 0.1% (vol/vol) Triton X-100 and 10% glycerol. First, an aggregated form of cytochrome-b was eluted, followed by the bulk of the cytochrome-b with an apparent molecular weight of 240 kD in the top fraction. Polyacrylamide gel electrophoresis of this top fraction in sodium dodecyl sulfate (SDS) showed that the 240 kD cytochrome-b complex consisted of a 98-100 kD protein and a 88-90 kD protein. Because the heme signal of cytochrome-b is rather labile in buffers containing SDS, it is extremely difficult to determine which of the two protein bands carries the heme group. However, using lithium dodecyl sulfate (LDS) instead of SDS, we succeeded to show, only once, that the 100 kD protein carries the heme group, whereas the 90 kD protein did not contain heme under these conditions. These preliminary results suggest that the b-cytochrome of human neutrophils has a molecular weight of at least 100 kD, but most probably consists of a protein complex with a 90 kD subunit plus a 100 kD subunit to which the b-heme is bound. Thus, the so-called cytochrome-b of neutrophils probably is not a simple low-molecular-weight electron carrier as e.g. the microsomal cytochrome-b5 or the mitochondrial ^-cytochromes. The neutrophil "cytochrome-b" is a high-molecular-weight enzyme, carrying a b-type heme as prosthetic group and possibly a covalently bound flavin group and/or a non-covalently bound FAD. The function of this b-heme-carrying (flavo)protein, however, has yet to be elucidated. Our feeling is that this enzyme is involved in the hydrogen-peroxide-generating system, although not as a terminal oxidase and not as a simple electron carrier. Further purification and characterization of this enzyme is in progress. Acknowledgements This study was supported in part by the Foundation for Medical Research, FUNGO, which is subsidized by the Netherlands Organization for the Advancement of Pure Research (grant nr. 13-39-40) and by a grant from Beecham Farma B.V., Amstelveen, The Netherlands.

848

References 1. 2. 3.

4. 5. 6. 7. 8. 9.

10.

11. 12. 13. 14. 15. 16. 17. 18. 19.

Babior, Β.M. and Kipnes, R.S.: Blood _50, 517-524 (1977). Light, D.R., Walsh, C., O'Callaghan, A.M., Goetzl, E.J. and Tauber, A.I.s Biochemistry 20, 1468-1476 (1981). Segal, A.W., Cross, A.R., Garcia, R.C., Borregaard, N., Valerius, H., Soothill, J.F. and Jones, O.T.G.: New Eng. J. Med. 308, 245-251 (1983). Segal, A.W. and Jones, O.T.G.: Nature 276, 515-517 (1978) Segal, A.W. and Jones, O.T.G.: Biochem. Biophys. Res. Commun. 88, 130-134 (1979). Shinagawa, Y., Tanaka, C., Teraoka, Α., and Shinagawa, Y.: J. Biochem. (Tokyo) 59, 622-624 (1966). Cross, A.R., Jones, O.T.G., Harper, A.M. and Segal, A.W.: Biochem. J. 194, 599-606 (1981) Cross, A.R., Higson, F.K., Jones, O.T.G., Harper, A.M. and Segal, A.W.: Biochem. J. 204, 479-485 (1982). Hamers, M.N., Wever, R., Van Schaik, M.L.J., Weening, R.S., and Roos, D.: In Developments in Biochemistry, Vol. IIB, Bannister, W.H. and Bannister, J.V., eds., Elsevier/North-Holland, New York, pp. 242-251 (1980). Hamers, M.N., Wever, R., Lutter, R., Van Zwieten, R., Van Schaik, M.L.J, and Roos, D.: In Proc. III. Int. Conf. on Superoxide and Superoxide Dismutases, New York, 1983, Elsevier, New York, in press. Gabig, T.G., Schervisch, E.W. and Santinga, J.T.: J. Biol. Chem. 257, 4114-4119 (1982). Sloan, E.P., Crawford, D.R. and Schneider, D.L.: J. Exp. Med. 153, 1316-1328 (1981). Crawford, D.R. and Schneider, D.L.: J. Biol. Chem. 257, 6662-6668 (1982). Rossi, F. and Zatti, M.: Biochim. Biophys. Acta. 121, 110-119 (1966). Reiss, M. and Roos, D.: J. Clin. Invest. 6^, 480-488 (1978). Cross, A.R., Garcia, R., Segal, A.W. and Jones, O.T.G.: Eur. J. Clin. Invest. 13, A34 abstract 200 (1983). Beinert, H. In: The Enzymes, 2nd ed., Vol. 2, part A, p. 339 (1960). Cross, A.R., Jones, O.T.G., Garcia, R. and Segal, A.W.: Biochem. J. 208, 759-763 (1982). Thomas, P.E., Ryan, D. and Levin, W.: Anal. Biochem. 75, 168-176 (1976).

DISCUSSION

MICHELSON: What happens to the system in chronic granulomatous disease (CGD) patients, for example? What modifications occur to cut the whole chain? HAMERS: The problem in CGD is that it is really inhomogeneous. There are people with cytochrome b and there is no function, and there are people without cytochrome b and there is also no function. There is a recent pub-

849 lication in New Engl. J. Med. (308, (1983) 245-251), stating that the cyto chrome b is missing in X-linked patients, but we have some patients who are autosomal recessive and they lack the cytochrome b. However, the fatype cytochrome is definitely involved. It is part of the system. If you look at the spectrum, it looks like sulfite oxidase. But it is certainly not sulfite oxidase. It only looks like it. FORMAN: May I comment on your question of what's wrong in CGD. Both Harvey COHEN'S and John GALLIN1 s group (SELIGMANN & GALLIN, J. Clin. Invest. (1980) 66, 493-503; WHITIN et al., J. Biol. Chem. (1980) 255, 1874- 1878) have published independently, that the granulocytes in CGD are incapable of depolarizing the plasma membrane potential, which seems to be important for activation of the process. HAMERS: But the study, as they do it, observes also the depolarization of the mitochondria inside the cells, and especially John GALLIN has refrained from this method. What does it mean? CGD patients are not a homogeneous group. FORMAN: The plasma depolarization has been shown in macrophages with the micro-electrode (CAMERON et al., Proc. Nat. Acad. Sci. USA (1983) 8£, 3726-3728) . HAMERS: Yes, you are right. The depolarization defect has been shown in a few CGD patients. Of course there will be CGD patients whose cells can't be triggered. We call them variants. FARAGGI: Regarding the nature of the FAD binding to the protein, could it be electrostatic as it is in the case with the chicken egg white riboflavin-binding protein and flavodoxins? HAMERS: The FAD we find in the membrane comes off when you put it in perchloric acid. That's the assay, in fact. When you purify the b-heme enzyme you still have this trough, which could mean a flavin signal. But there is no FAD coming out when you put it in perchloric acid. So it has to be covalently bound if it is there and then it is very difficult to get it out. You have to digest the enzyme with trypsin and it could still be a FAD which will not give a fluorescence. ELSTNER: Almost all flavoproteins either bound to membranes or in the isolated form have one specific site, this is the NAD/NADPH site, and a more or less unspecific site, this is the acceptor. This unspecific site couples to different quinones, if they just have the appropriate redox potential. We have a series of these quinones, almost all of these quinones couple to most of the diaphorases or flavoproteins we know of. Does your flavoprotein or flavoprotein-b complex couple to any of the artificial additions you have and does that stimulate oxygen uptake. HAMERS: The problem is, we didn't try it with this purified form. But people have put quinones into these homogenates, all work was with very crude homogenates. Then you see an effect: you see more cytochrome c reduction, but it could be the quinone itself. What I said about the SCHNEIDER group (J. Exp. Med. (1981) 153, 1316; J. Biol. Chem. (1983) 25J, 6662) who found quinones, well, there are other groups who didn't find it, and

850 we don't either. I have put in the quinone inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) and it does not inhibit the burst. I have exposed the cells to very rough UV-treatment and it doesn't disturb the burst at all. It was shown years ago, that you could put in antimycin A or rotenone which will not disturb the burst at all. We think that they have isolated mitochondrial quinones from contaminating cells or even from the PMN themselves. EISTNERs Well, I wouldn't be surprised that antimycin A doesn't do anything because if you put in antimycin A, it blocks behind the b-type and it would more or less increase oxygen uptake. But since the cytochrome b autoxidation, like the non-heme iron protein oxidations, is kinetically limited but the quinones are more or less not, you should have a stimulation, let's say by anthraquinone sulfonic acid or other low potential types.

The participation of the hypoxanthine-xanthine oxidase system in t h e generation of free radicals a f t e r intestinal ischemia 1 M.H.Schoenberg ,

2 M.Younes ,

5 U.Haglund ,

4 B.Fredholm ,

5 D.Sellin ,

1 H.Jung ,

F.W.Schildberg1 1

2

D e p t . of Surgery,

University

5

D e p t . of T o x i c o l o g y ,

D e p t . of P a t h o l o g y ,

Lübeck

' D e p t . of Surgery, M a l m ö G e n e r a l H o s p i t a l Sweden 4

D e p t . o f P h a r m a c o l o g y , K a r o l i n s k a I n s t i t u t e , S t o c k h o l m , Sweden

The

damages

subject that

to

these

shock

of

the

small

particular lesions

state

research

are of

itself.

intestine

activities

high

The

during in

prognostic

pathogenesis

ischemia the

value

of

the

last for

and

shock

decades. the

It

have is

been

believed,

f i n a l o u t c o m e of

mucosal

damages,

the

however,

is

s t i l l a m a t t e r of c o n t r o v e r s y . Circumstantial

evidence

occur

during i s c h e m i a and h y p o t e n s i o n but also in the

not only

supports

the

theory

that

intestinal

mucosal

lesions

posthypotensi-

ve phase. D u r i n g p r i m a r y t h e r a p y , superoxide r a d i c a l s are g e n e r a t e d and i n i t a t e e x t e n s i v e tissue damage (1,2). P r e s u m a b l y , t h e source of the s u p e r o x i d e

radical

is the oxygen dependent h y p o x a n t h i n e - x a n t h i n e oxidase s y s t e m . In hypoxic tissues energy r i c h phosphate are degraded v i a adenosine and inosine to

hypoxanthine.

Further

occur

due

to

tissue

and

diffuses

hypoxia.

Under

normal

metabolism

of

Hypoxanthine

passively

into

conditions catabolism

hypoxanthine

therefore

the

to

uric

accumulates

interstitial

of h y p o x a n t h i n e

and

acid in

does

the

hypoxic

intravascular

to u r i c

acid is

not

space.

catalyzed

by the N A D + - r e d u c i n g x a n t h i n e dehydrogenase. It

was

shown

converted

to

shock

The

(3).

in

intestinal

xanthine

tissue,

oxidase

reaction

of

however,

that

immediately

hypoxanthine

after

and

xanthine

dehydrogenase

induction

xanthine

of

oxidase

is

hypoxia

or

consumes

02

and generates superoxide r a d i c a l s . It

is t h e r e f o r e

tempting

to

speculate,

supply, the o x i d a t i o n of h y p o x a n t h i n e centered acids,

free

radicals.

These

radicals

thus l e a d i n g to e x t e n s i v e

that

during

restitution

of t h e

leads to f o r m a t i o n of c y t o t o x i c may

react

with

oxygen oxygen-

polyunsaturated

lipid peroxidation, consequently

brane d é s i n t é g r a t i o n and c e l l death.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

to cell

fatty mem-

852 The

cell

is

not

left

unprotected.

The

enzyme

superoxide

dismutase

(SOD)

c a t a l y z e s the d i s m u t a t i o n of 0 2 * to H 2 0 2 . G l u t a t h i o n e peroxidase ( G P O ) reduces H202

converting

the

cosubstrate

reduced

glutathione

(GSH)

into

its

oxidized

form. These

"protection

If, h o w e v e r , 0 2

r

systems"

are

present

intracellularly

in high

concentrations.

is g e n e r a t e d e x t r a c e l l u l a r l y , it can initiate lipid

peroxidation

without any h i n d r a n c e . If

superoxide

stem,

are

radicals,

of

generated

significant

by

the

importance

for

hypoxanthine-xanthine the

pathogenesis

of

oxidase the

sy-

intestinal

m u c o s a l lesions a f t e r p r i m a r y therapy then, 1.

additional tissue d a m a g e should occur after r e p e r f u s i o n .

2.

hypoxia

should

3.

as

of

sign

have

oxidative

lead

to

high

mechanisms,

hypoxanthine

the

levels

cosubtrate

the

tissue.

red.glutathione

in

should

be partly o x i d i z e d during the r e p e r f u s i o n period. T h i s m i g h t be paralleled by lipid peroxidation. 4.

Competitive

inhibition

by allopurinol and m u c o s a l

should

of

the

hypoxanthine-xanthine

conseguently

lesion w h i c h m i g h t

prevent

oxidase

those b i o c h e m i c a l

system

alterations

o c c u r after r e s t i t u t i o n of the

intestinal

circulation. M a t e r i a l s and M e t h o d s Cats

weighing

anesthesized

1.5-3

with

the p e r c e n t a g e

kg were

150 m g

of C 0 2

fasted

24h prior

Ketamin-HCL

to the e x p e r i m e n t . T h e y

and r e s p i r a t e d a r t i f i c i a l l y

were

controlling

in the e n d e x p i r a t o r y air. A s e g m e n t of the s m a l l

inte-

stine w a s isolated and p e r f u s e d by the superior m e s e n t e r i c a r t e r y ( S M A ) alone. T h e s p l a n c h n i c n e r v e s s u r r o u n d i n g the S M A were c o n n e c t e d to a platin e l e c t r o de and s t i m u l a t e d 30

mmHg

was

with 6 H Z

induced

by

for

6 msec

stenosizing

In addition, an e l e c t r o m a g n e t i c

with

the

10 m A .

SMA

with

Local hypotension an

adjustable

of

clamp.

flow probe was placed around the S M A

distal

to the s t e n o s i z i n g c l a m p for blood flow r e c o r d i n g s . After

control

over

a period

were

observed

10

min.

and

measurements, of for

120

min.

one

1 hour

the

more

after

superior

Thereafter hour.

the

Before

reperfusion,

mesenteric

artery

clamp

released and the

was

stenosis,

various

tissue

2

hours samples

was

stenosized

after were

cats

stenosis, excised.

853

Some w e r e gen

for

taken

the

for

histological

determination

as

the

concentration

of

lipid peroxidation

of

of

examinations,

reduced

conjugated

(5). A t

and

others

oxidized

dienes,

in

t h e same t i m e ,

frozen

in l i q u i d

glutathione

order

to

(4)

assess

the c o n c e n t r a t i o n

nitro-

as

the

of

well

amount

the

purine

m e t a b o l i t e s in the i n t e s t i n a l mucosal - possible s u b t r a t e of t h e r a d i c a l generat i n g s y s t e m - w e r e measured. Treatments: 6 cats received orally to

the e x p e r i m e n t .

50 m g / k g body w e i g h t (BW) a l l o p u r i n o l f o r 2 days p r i o r

During

the

experiment,

again 50 m g / k g BW of

allopurinol

w e r e a d m i n i s t e r e d i n t r a v e n o u s l y b e f o r e p a r t i a l o b s t r u c t i o n of the S M A . Histological examination: Intestinal eosin.

samples

The

tissue

were

fixed

samples

in

were

5%

formalin

coded

and

and s t a i n e d

examined

for

with the

hematoxylin shock-specific

mucosal lesions of the s m a l l i n t e s t i n e . R e s u l t s and Discussion: After

r e p e r f u s i o n , the mean a r t e r i a l pressure ( M A P ) of u n t r e a t e d c a t s

sed s i g n i f i c a n t l y after

from

110

to

65 m m H g .

release of the stenosis, but

The

intestinal

blood

flow

did not reach c o n t r o l f l o w s . This

decrea-

increased significant

decrease of t h e M A P and blood f l o w of the gut a f t e r r e p e r f u s i o n , is b e l i e v e d to

be

due

to

liberation

of

cardiodepressive

shock

factors

from

the

hypoxic

damaged i n t e s t i n e (Fig.1). D u r i n g h y p o t e n s i o n and i s c h e m i a the A T P c o n c e n t r a t i o n dropped in the nal tissue elevated

to 40% of during

times

higher

at

level.

Apparently

its

initial

ischemia. the

end

of

clamping

value. In c o n t r a s t

Consequently the of

hypotensive

the

SMA

the A M P

hypoxanthine period

leads

to

l e v e l was

concentration

compared

severe

to

hypoxia

intesti6-fold is

20

prestenotic of

intesti-

ne, i n d u c i n g t h e c a t a b o l i s m of A T P v i a A M P t o h y p o x a n t h i n e . A f t e r t h e release of the stenosis the h y p o x a n t h i n e l e v e l e x h i b i t e d a m a r k e d decrease,

remained,

h o w e v e r , s t i l l e l e v a t e d 1 hour a f t e r release of the s t e n o s i z i n g c l a m p . O b v i o u s ly, hypoxanthine

was degraded to u r i c a c i d a f t e r r e p e r f u s i o n ; only l i t t l e

hypo-

x a n t h i n e c o u l d be r e u t i l i z e d o v e r the "salvage p a t h w a y " t o r e p l e n i s h t h e energy s t o r e s of t h e c e l l . A T P , a l t h o u g h g r a d u a l l y

i n c r e a s i n g , did not r e g a i n p r e s t e n o -

t i c c o n c e n t r a t i o n 1 hour a f t e r end of stenosis.

854

M «on ArUrigl P r » « u f M of Untr»afd and Allopurinol Trootod Cots

Fig. 1

The d e v e l o p m e n t o f t h e blood pressures o f u n t r e a t e d and a l l o p u r i n o l - t r e a t e d c a t s

Fig. 2

Changes in g l u t a t h i o n e (GSH) and o x i d i z e d g l u t a t h i o n e (GSSG) (in %) of u n t r e a t e d cats

Neither

stenosis

nor

reperfusion

had

any

effect

on

the

tissue

activities

of

SOD or G PO. The

concentration

stenosis and

was

likely Parallel dienes,

and

of

reperfusion.

accompanied

H202 to as

a

the

by

and

lipid

the

increase

measure

cosubtrate

GSH showed

The

fall

main

a significant

hydroperoxides

of

in

oxidized

lipid

of

GSH

increase evoked GSH

peroxidation,

a decrease

the

the was

occured

of

its

of

after

oxidation

of

than

during

reperfusion

oxidized

concentration more

30%

form. GSH

of

Most (Fig.2).

conjugated

doubled

(Fig.3).

855 It w a s a u g m e n t e d f r o m 2 . 4 5 + 0 . 5 t o 5 . 5 + 1 . 2 umol/g Accordingly, such

as

Already in t h e

10

min.

villi

lifting after

at

These

the

the

tops

of

epithelial

examination

surface

Allopurinol

showed

to

treated

stenosis

reperfusion, although showed

often the

the no

and

of

sign

GSH.

did

led

to

désintégration

the

control

further

decrease cats

of

further

allopurinol

not

complete

Moreover,

before take

low,

deterioration

level

and a f t e r place

and,

of

the of

alterations of

edema

the

oxidized

GSH

reperfusion.

less

the

an

cats

propria

prior

systemic

pressure.

After

significant to

70

pressure

mmHg.

The

postreperfusion

drop

in t i s s u e

remained indicate

GSH

period.

concentra-

that

and

fall, MAP

constantly

was oxidized

T h e d e v e l o p m e n t o f c o n j u g a t e d d i e n e s l e v e l s in u n t r e a t e d allopurinol-treated

the

lamina

120 130 w> 2h of IO'attor Ih attor Stono» RofMffuston Roforfusior

3

end

of

lower

This may

conseguently,

after

initially

the

minimized

hour

lesions

injection

80

during

of

period

no

from

1

denudation

Conjugotol Díanos of Untroatod and Alopurinol Trootod Cats

Fig.

ischemia. formation

allopurinol

the

showed

ranging

treatment the

2 hours

aggravated

treated

rather

after

little

impressive

changes

a

was

an

revealed

during

cats.

villi

only

and e v e n s o m e h e m o r r h a g i c

allopurinol

Moreover

unchanged

damage

untreated

MAP

Concomitantly, tion

cats

to

the

clamp,

epithel ium,

ulcerations.

compared

revealed

lifting

and m u l t i p l e h e m o r r h a g i c

MAP

of

the

morphological

Histological from

examination

release

pronounced

observed.

stenosis.

entire

histological

epithelial

mucosa,

were of

the

tissue.

low

oxidative (Fig.4).

856

Treat«d Cols

~~2h oflio 13010-aritf 1«hC afttf Sttnosis Repwfusion Refusion F i g . 4 Changes in G S H and G S S G in % of allopurinol-treated cats Furthermore,

in 5 out of 6 cats no increase

in conjugated dienes as that

seen in untreated cats was observed. The conjugated dienes did not exceed prestenotic levels; thus, lipidperoxidation seems not to have taken place during reperfusion (Fig.2). The

of

histological

examinations

the biochemical

parameters

after

of

results the

intestine

before

confirm

treatment.

reperfusion

are

the

The

almost

observed

ischemic

identical

changes

mucosal

in the

in

lesions

untreated,

S O D treated (1) and allopurinol treated cats. In all groups, we found

little

damage, such as moderate epithelial lifting at the tops of the villi. 10 min. after

reperfusion

no significant

additional

damage,

as edema formation, he-

morrhagic lesions or extensive loss of epithelium, was observed. Also 1 hour after reperfusion the epithelial

layer of the villi

remained unchanged in 4

out of 6 cats. The villi were broadened, however, by enhanced edema formation. In 2 cats, the degree of mocosal lesions aggravated significantly in the late reperfusion phase. One of the animals, although under treatment, exhibited an uncommonly

high conjugated

diene

concentration

after

reperfusion.

The

other suffered from a slow deterioration of the M A P during this period for unknown reasons which ended in a new shock state. From the results it is concluded: 1.

The main mucosal lesions become apparent after reperfusion.

857

2.

After

2 hours

thine

are

of

stenosis

and

20 t i m e s higher

low

flow

than n o r m a l

the

tissue

levels

of

and are c a t a b o l i z e d

hypoxan-

upon

reper-

fusion. 3.

At

the

to

oxidative

same t i m e GSH is consumed in the i n t e s t i n a l mechanism.

Parallely

lipid

tissue m a i n l y

peroxidation

takes

due

place

as

assessed by the increase in c o n j u g a t e d dienes. 4.

Allopurinol and

treatment

oxidative

tissue

primarily

consumption

damage seen a f t e r

of

prevents GSH.

enhanced

Consequently

lipid it

peroxidation

minimizes

the end of stenosis. A l l o p u r i n o l

does not

local pro-

t e c t the tissue to the e x t e n t observed by SOD (1). These as

findings

indirect

tissue

damage

to

mainly

be

together

proof of

with

observation

that

superoxide r a d i c a l s

the

small

generated

by

intestine. the

in are

These

hypoxanthine

SOD

treated

involved

superoxide xanthine

cats

in the

serve

ischemic

radicals

oxidase

seem

system.

References 1.

SCHOENBERG, Scavenger

M.H.,

Systems,

YOUNES,M., Volume

II,

Ed.

MUHL,E.:

Oxy

Radicals

R.A.Greenwald,

and

G.Cohen,

Their

Elsevier

B i o m e d i c a l 154-158 (1983) 2.

PARKS,D.A.,

BULKLEY,G.B.,

GRANGER,D.N.:

Gastroenterology

82,

9-15 (1982) 3.

ROY,R.S., Volume

II,

McCORD,J.M.: Ed.

Oxy

Greenwald,

Radicals

R.A.,

and

Cohen,G.,

Their

Scavenger

Elsevier

Systems,

Biomedical

Y o r k 145-153 (1983) 4.

G R I E E I T H , O . W . : A n a l B i o c h e m . 106, 207-212 (1980)

5.

B U E G E , J . A . and A U S T , S . D . : M e t h o d s E z y m o l . 52, 302-310 (1978)

New

858 DISCUSSION

MICHELSON: In the secondary effects after hypoxia, when you restore circulation, you have a pulse of oxygen, and at that moment all systems of defense are overloaded. So my question is, when you open the clamp, do you open it slowly or quickly? Because it might give better results if you open it slowly to avoid the sudden pulse of oxygen sweeping through the system. SCHOENBERG: Well, it is very difficult to define 'slowly'. We open it as fast as or as slow as, for instance, a surgeon would open the clamp around an aorta after reconstructive aortic surgery. That is to say, we opened it fast. MICHELSON: perhaps they should start opening it over a period of 3, 4, 5 minutes, it might make a difference. BORG: Last autumn, in Ellenville, when Joe McCORD and Ranjan ROY presented their findings (Oxy Radicals and Their Scavenger Systems, Vol II: Cellular and Medical Aspects. R.A. Greenwald, G. Cohen, eds, Elsevier, New York, (1983) pp 145) they indicated that the conversion of xanthine dehydrogenase to xanthine oxidase in different tissues differs both in time and in mechanism. As I recall, they found conversions in a matter of seconds from the dehydrogenase to the oxidase in the intestine, and these appeared to depend on proteases. I believe that for other tissues they found periods of time ranging from tens of minutes in the myocardium to some hours in the brain, and that the conversion outside the gut required glutathione transferases. What is the present understanding regarding the nature of the conversion from the dehydrogenase to the oxidase? SCHOENBERG: Well, as far as I know, nothing has been added to that, that is where we are. I even recall that the conversion in the myocardium was much faster than you say and, interestingly enough, in the muscle tissue, there was no conversion whatsoever. So I think it depends very much on the organ. Kidney, myocardium, intestine show conversion, normal muscle tissue does not. AFANAS'EV:

Did you try to use vitamin C and E?

SCHOENBERG: No, we didn't. But I recall experiments done in Uppsala in 1979, when kidney ischemia was induced just by clamping the renal artery for 1 hour. The experimental animals were rats, and we injected vitamin E intraperitoneally, but saw no beneficial effect. The peritoneum looked terrible afterwards. AFANAS'EV:

And ubiquinone?

SCHOENBERG: No, we did SOD, allopurinol, and the last experimental series just finished with inosine, to see if that has any beneficial effect.

MODULATION

OF M A C R O P H A G E

CHEMILUMINESCENCE

BY

ARACHIDONIC

ACID

PRODUCTS Michael

J. P a r n h a m ,

A. N a t t e r m a n n Postfach

Christine

& Cie. GmbH,

350120,

D- 5000

Bittner

Pharmakologische

Köln

30,

W.

Forschung,

Germany

Introduction Stimulation agents

of m a c r o p h a g e s

leads

enzymes,

by both s o l u b l e

to t h e s e c r e t i o n

including

reactive

of a r a c h i d o n i c

While many of these

products

these

activation that

differ

secretory

in t h a t

products

in v i v o

prostaglandin

products

(PGE^)

inhibits

arachidonic

acid

15-HPETE has

elicited macrophages measure PGE^

inhibits

macrophages, and

Using

while

lipoxygenase

arachidonic

inhibitors

resident mouse macrophages carried acid

out

in o r d e r

products

phages

states

of

of of

for s o m e

b e e n s h o w n to from

time

burst

of

release

mouse (CL) as

previously

and a c t i v a t e d

shown

CL g e n e r a t e d

present

a possible

the oxidative

further

r o l e of burst

activation.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

a that

mouse

(as t h e p r e c u r s o r

inhibit

(6). We n o w

in c o n t r o l l i n g

in d i f f e r e n t

acid

to c l a r i f y

amounts

chemiluminescence

also

3).

lipoxygenase-generated

burst, we have

CL f r o m b o t h r e s i d e n t

(1, 2,

neutrophils,

the o x i d a t i v e

the

(chemiluminescence)

(5).

of t h e o x i d a t i v e

and

oxidative

with their state

It h a s b e e n k n o w n

recently,

product

by

the type and r e l a t i v e

(4) a n d m o r e

oxygen products

secreted

vary m a r k e d l y

macrophages reactive

of t h e

acid metabolites

are also

(1, 2, 3 ) . E^

particulate

of a h o s t o f m e d i a t o r s

oxygen

burst and a variety macrophages

and

of

of

PGE^ )

from studies

arachidonic macro-

860 Methods M a c r o p h a g e s w e r e o b t a i n e d by p e r i t o n e a l CBA m i c e or m i c e c h a l l e n g e d macrophage described

stimulant

i.p. 8 days earlier with

Corynebacterium

previously

triene

parvum

(6). R e s i d e n t and

macrophages were stimulated zymosan

l a v a g e of n o r m a l

C.

(LTB^) and l u m i n o l - e n h a n c e d

orally with indomethacin preincubated

for

All c o m p o u n d s ,

except

opsonized

(PMA) or

before

were obtained

5 min

treated

in 1 % Tylose or m a c r o p h a g e s

LTB

leuko-

CL m e a s u r e d a f t e r

(6). Either a n i m a l s w e r e

10 m i n w i t h i n h i b i t o r s

as

parvum-activated

(OpZ), 20 n M p h o r b o l m y r i s t i c a c e t a t e B^

the

(C. p a r v u m ) ,

in v i t r o w i t h 0.17 m g / k g

also using described methods

male

were

stimulation.

commercially.

Results 1) E f f e c t s of e x o g e n o u s p r o s t a g l a n d i n s

and l e u k o t r i e n e

B^

Two of the m a j o r c y c l o - o x y g e n a s e

p r o d u c t s of s t i m u l a t e d

p h a g e s are

(PGI^)

f o u n d for

PGE^ and p r o s t a c y c l i n PGE2

OpZ-stimulated

(6), P G I 2

inhibited

(3, 7). As

previously

the g e n e r a t i o n of CL by

C. p a r v u m a c t i v a t e d m a c r o p h a g e s

(Fig.

1). The

e f f e c t of FGI^ is p r o b a b l y l e s s than m i g h t be o b s e r v e d c o n d i t i o n s of c o n t i n u a l rapid breakdown

synthesis

in a q u e o u s

under

(as in v i v o ) b e c a u s e of

its

media.

When a t t e m p t s w e r e m a d e to r e p r o d u c e PGE^ in vitro on O p Z - i n d u c e d macrophages, marked

macro-

the i n h i b i t o r y a c t i o n

CL from m o u s e

C. p a r v u m

variation was observed.

activated

R e p e t i t i o n of

the

e x p e r i m e n t at 2 w e e k l y i n t e r v a l s r e v e a l e d

cyclic variation

the i n h i b i t o r y

This v a r i a t i o n

independent

a c t i v i t y of PGE2

of the b a t c h of the a n i m a l s and a p p e a r e d

r e l a t e d to the time of the y e a r . tested

in p a r a l l e l ,

variation

(Fig. 2 ) .

In c o n t r a s t ,

SOD

f a i l e d to s h o w any s e a s o n a l

in its a b i l i t y

to i n h i b i t

CL (data n o t

(60

of

in

was

to be U/ml),

dependent shown).

861

-

PGI-

100

O c o o

-50

80-

-rao

.o

ai o c O) o £ c E D E αι

"I'll .

60-

•π

r

Γ

i t [j ρ

'[

-50

40 -100

PGEj 2.8 χ10-7M

2 0

O 0J

7

6

5

PGEj

4

-log c o n c (M)

Nov

Dec

2.8 x10

M

Jan

Feb

Mar

Apr

Mai

Fig. 1. Inhibition by prostacyclin (PGI2) of chemiluminescence generated by OpZ-stimulated mouse peritoneal C. parvum activated macrophages. Values are means of 3 duplicate observations, expressed as percentages of the response to 0.17 mg/ml OpZ alone . * ρ < 0.02 (vs. control; t test after log transformation). Fig. 2. Seasonal variation in the inhibitory effect of in vitro PGE2 on chemiluminescence generated by OpZ-stimulated mouse peritoneal C. parvum activated macrophages. Bars are means of duplicate measurements carried out regularly at 2 weekly intervals. The shading indicates different batches of mice. Values are expressed as percentages of the response to 0.17 mg/kg OpZ alone.

In view of the reported generation of CL by

thioglycollate-

elicited mouse peritoneal macrophages exposed to 15-HPETE

(5)

and the inhibitory effect of lipoxygenase inhibitors on CL generated

by mouse resident peritoneal macrophages exposed

to

OpZ (6), it was of interest to study the effects of leukotriene B^ (LTB^) on macrophage observe stimulation

by LTBn

CL. Unexpectedly, we failed

to

(1.5 χ 10 ^ Μ), isomers I, II or

862 III, of CL from mouse C. parvum activated macrophages

in vitro

(data not shown ).

2) Effect of indomethacin in vitro and in vivo —7 Following preincubation with indomethacin vitro, CL generated

of OpZ-stimulated

—S

-10

M) ^Ln

by PMA and OpZ from C. parvum-activated

macrophages was unaffected resident macrophages

(10

as was CL generated

by PMA from

(data not shown), however,

resident macrophages with

indomethacin stimulated

preincubation

10"^ M and

10"^ M

CL by 41 % (mean, η = 3, ρ < 0 . 0 1 ) and

16 % (mean, η = 3, P < 0.01), respectively. Enhancement OpZ-induced macrophage

CL was also obtained following

treatment of mice for 2 days with indomethacin; treatment of C. parvum-challenged CL (Table

mice inhibited

of pre-

similar OpZ-induced

1).

Table 1. Effect of indomethacin from mouse peritoneal

in vivo on OpZ-induced

CL

macrophages.

Chemiluminescence

Pretreatment

Resident

C.Ρ.-activated

Control

460 +

39,064 + 8,522 (6)

Indomethacin 3

844 + 175*

2 mg/kg/day

82

(6) (9)

1 1 ,702 + 5,814* (4)

p.o.

Data are means _+ s.e.m. of the no. of observations in brackets a

G i v e n once daily for the 2 days before cell

* ρ2 , -OH, 1 0 2 , H 2°2'

oc1

~ ) » which can be detected by luminol

amplified chemiluminescence (CL) in whole blood (3, 4). In whole blood, specific granulocyte activation may be mediated by immunoglobulins and components of the complement system due to Fc- and C3b-receptors of the neutrophils. Therefore, three different signals were used to challenge granulocytes in diluted whole blood from healthy volunteers and aplenéctomized patients (assay cp. fig. 1): 1) Zymosan as a potent non-specific activator of the alternative complement pathway (Sigma, Munich, FRG). 2) Latex-beads as a non-specific, not complement dependent stimulus of phagocytosis (Sigma, Munich, FRG), and 3) Pneumococcal antigens obtained from a 14-valent pneumococcal vaccine (Merck, Sharp & Dohme, Westpoint, USA) as a potentially specific antibody-mediated granulocyte activator. The resulting CL was recorded as counts per minute per 100 2 polymorphonuclear granulocytes (CPM/10 PMN). lOO pi blood 400 pi Dulbeccos MEM +

10 pi Luminol (40pg/l)

+

10 pi Zymosan (1000mg/l) or Latex (2000mg/l) or Pneumococcal antigens (560mg/l)

Figure 1; Summary of the CL-assay (final concentration)

869

Results ρ Five healthy volunteers were vaccinated with Pneumovax (Merck, Sharp, Dohme, Westpoint, USA), a commercial preparation of polysaccharide antigens from 14 pneumococcal strains. CL was measured in whole blood samples prior to vaccination as well as 1, 3, 9 and in one case 14 weeks thereafter (fig.2). Figure 2 : CL of healthy volunteers following pneumococcal vaccination.

CL (%)

•—»Pneu Zym 500- o—o Lot

Signals used: latex, zymosan and pneumococcal

400·

antigens. n=5, at 14 weeks n=l. 300-

200

01 3

9

1¿t (weeks)

One week after the vaccination, zymosan, latex and pneumococcal antigen stimulated CL were increased just above the preoperative values. This stimulation of phagocytosis was interpreted as a non-specific consequence of vaccination. However, one and two weeks after the vaccination CL stimulated by pneumococcal antigens was significantly increased over both zymosan and latex induced CL. The increase in specifically

870

induced CL (pneumococcal antigens) at normal values of nonspecifically stimulated CL (zymosan, latex) suggests a significance of specific antibodies. Furthermore, the kinetics of the reaction (normal activity at one week, but increased activity three weeks after vaccination and later) would fit with immunoglobulin synthesis but is no proof for an involvement of immunoglobulins. Evidence that the CL induced by pneumococcal antigens depends on specific immunoglobulins would, however, require to demonstrate a corresponding increase of the respective antibody titres. These measurements are presently under way by radioimmuno assays. Parallel with the experiment described so far, CL was measured in both splenectomized patients and controls (fig.3). Compared to

healthy controls, the pneumococcal antigen stimulated CL

was significantly lower in splenectomized patients. There was no difference, however, for zymosan- and latex-induced CL between these two groups. In splenectomized patients, who in addition were treated by partial replantation of splenic tissue and a postoperative pneumococcal vaccination, the pneumococcal antigen induced CL was comparable to normal. Again, nonspecifically induced CL did not yield any significant changes. signal η

zymosan

latex

pneumococcal antigens

Controls

35

102+ 9

317+ 29

89+33

Splenectomy only

16

98+17

348+ 50

57+11

8

129+32

330+ 55

92+19

Splenectomy + Replantation + Vaccination

Figure 3 : CL stimulated by zymosan, latex and pneumococcal antigens, χ + SEM.

871 Discussion and

conclusions

These results in human whole blood can neither prove the ability of CL to detect specific antibodies directed pneumococcal antigens

against

(correlation with antibody titres not

yet established) nor assess the clinical efficacy of splenic replantation and pneumococcal vaccination in preventing

post-

splenectomy sepsis. However, the comparison between CL induced by non-specific and specific granulocyte activation

indicates

some specificity of these measurements. Moreover, the CLincrease following pneumococcal vaccination mimics the kinetics of proliferative antibody response. As granulocytes are known to carry Fc-receptors, an immunoglobulin mediated

granulo-

cytes' response to pneumococcal challenge is likely.

If this were true, the whole-blood-method

might provide

clinically important advantages over the highly specific body titre determination in whole

some anti-

blood:

1, CL in whole blood measures an overall response to pneumococcal challenge depending on both specific and non-specific factors

(complement system,

(irirmunglobulin) granulocytes).

The method would detect a patient's global defense against the 14 most relevant strains of pneumococci. 2. The method is simple, quick and cheap. It is therefore potentially useful for screening purposes : A depressed CL-response in whole blood probably further exploration by more specific methods.

justifies

872 In summary, p n e u m o c o c c a l antigens can induce CL in w h o l e The C L - r e s p o n s e p r o b a b l y bears antibody m e d i a t e d According

specifity.

to p r e l i m i n a r y clinical results, the m e t h o d

allow the evaluation of both an i n d i v i d u a l p a t i e n t ' s against p n e u m o c o c c i and his response to m e a s u r e s such as splenic replantation

blood.

may defense

prophylactic

and

vaccination.

References

1. Conney, D.R., Dearth, J.D., S w a n s o n , S.E., Dawanjee, M . K . , T e l a n d e r , R.L. Surgery 86^, 561-569 (1979). 2. G e l f a n d , J.A., Grabbe, J.P. (1983) .

N.Engl.J.Med.

308,

3. H e b e r e r , Μ . , E r n s t , Μ . , Diirig, Μ . , A l l g ö w e r , M., K l i n . W o c h e n s c h r . 60, 1443-1448 (1982).

1212-1218 Fischer,H.

4. Kato, T., Eggert, H., W o k a l e k , H., S c h ö p f , E., E r n s t , M . , R i e t s c h e l , E . T h . , F i s c h e r , H. Klin.Wochenschr. 203-211 (1981) . 5. M o r r i s , D.H., B u l l o c k , F . D .

Ann.Surg.

70, 513-521

(1919).

DISCUSSION

PARNHAM: I would like to offer an alternative explanation for your findings. It is well known that activation or elicitation ¿n vivo by a number of factors will result in marked enhancement of the oxidative burst of macrophages. When one takes these macrophages out, between three to five days later, and stimulates them iji vitro with a particulate or non-particulate stimulus, their oxidative response is much greater than that of resident macrophages. What I suggest you may be observing, since you are measuring chemiluminescence of whole blood, is simply an activation of your monocyte population and this you may well obtain with any other stimulus besides a vaccine. HEBERER: But if this were true, I would not be able to explain the difference observed between the zymosan and latex stimulation and the so-called specific stimulation by pneumococci. If this were true, one would expect also an enhanced chemiluminescence production following stimulation by latex or, particularly, by zymosan.

873 RISTER: When you used the whole blood, did you assess always the same white cell number. HEBERER: White and differential blood cell count was always performed. The chemiluminescence counts (CPM) were related to the number of polymorphonuclear leukocytes (PMN) in the system. The activity was expressed as CIM/10 2 PMN. RISTER: What was the chemiluminescence? Was total count, or just the T m a x ?

it the overall count, the

HEBERER: It is the integrated count over a 30 min period following the stimulation. Chemiluminescence kinetics normally reaches a peak about 15 min after the stimulation and we usually measure the integral over 30 min. ALLEN: On the last slide that you presented, IgM opsonification was shown to go through an Fc mechanism. That is not correct, IgM can serve as an opsonin by activating the classical pathway of complement. The other comment is that this approach to opsonic measurement is good as a screening technique; however, for quantitative results, the serum should be titrated to avoid the rate limiting effect of phagocyte number. HEBERER: Yes. I must say we are aware of this fact and this work is in progress.

QUANTIFICATION OF POLYMORPHONUCLEAR LEUKOCYTE OXYGENATION ACTIVITY BY CHEMILUMINIGENIC PROBING

Robert C. Allen U.S. Army Institute of Surgical Research and Department of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, Texas 78234, U.S.A. William C. Gilmore Department of Veterinary Pathology, Texas A & M University, College Station, Texas 77843, U.S.A.

Introduction Activation of 02~redox metabolism is required for polymorphonuclear neutrophil leukocyte (PMNL) mediated microbicidal action. The metabolic events, i.e. non-mitochondrial 0^ consumption with a corresponding increase in hexose monophosphate shunt activity, are collectively referred to as the respiratory burst (1), and are necessary for the generation of oxygenating agents that participate in microbe killing (2).

The resulting native

oxygenation reactions yield electronically excited products that relax by photon emission, and are responsible for the native chemiluminescence

(CL) of stimulated PMNL (3).

Single photon counting of native PMNL luminescence can be employed for measuring oxygenation activity in response to stimulation.

However, native substrate oxygenations are of

relatively poor quantum yield, and variability in the composition of the substrates oxygenated limit the interpretation and comparison of results to a given microbe or stimulus (4).

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

876

Chemiluminigenic probing is based on measuring the CL resulting from oxygenation of high quantum yield substrates introduced as probes, i.e. chemiluminigenic probes (CLP's).

Using CLP1 s of

high quantum yield results in a proportional increase in sensitivity of detection, and has the additional advantage of substrate uniformity (4-6).

Furthermore, by considering the CLP

as substrate and CL as product, stimulated PMNL oxygenation activity can be kinetically analyzed using the Michaelis-Menten approach.

Materials and Methods 1)

Bovine PMNL preparation and stimulation.

Bovine neutrophils

were prepared by modifications of previously described procedures (7,8).

Heparinized venous blood was collected and

the granulocyte-rich erythrocyte pellet was separated from mononuclear cells by centrifugation through ficoll-hypaque. After hypotonic lysis of erythrocytes, the neutrophils were purified by continuous gradient centrifugation on Percoli (Pharmacia).

The 1.09 specific gravity fraction was iso-

lated, washed twice, counted, adjusted to the desired concentration, and added to sterile, siliconized counting vials (8 ml capacity) containing complete Veronal buffer (CVB) pH 7.2 and luminol (9).

The concentration of luminol

was determined using a millimolar extinction coefficient of 7.63 in H 2 0 at 347 nm (10). was 2.0 ml.

The final volume per vial

Phorbol-12-myristate-13-acetate

(PMA), a chem-

ical stimulus extracted from croton oil, was added to the cell suspension at time zero.

The final concentration of

PMA was 0.3 μΜ. 2)

Single photon counting.

Luminescence intensity was quanti-

fied at ambient temperature (22 C) using the single photon counting capacity of a liquid scintillation counter equipped with bialkali spectral response photomultiplier

877 tubes (EMI 9829A). coincidence mode.

The counter was operated in the out-ofThe measured CL intensity in relative

counts per minute (CPM) values were converted to photons per minute by multiplying the relative CPM by a photon conversion factor.

This factor was established by daily cali-

bration of the counter with an established blue photon emitting standard prepared by H. H. Seliger.

Results and Discussion The oxygenation of luminol by stimulated PMNL is analogous to an enzyme reaction, and can be treated as a one substrate oxygenation reaction with photon emission as the energy product. As such, the rate of photon emission will be dependent on the concentration of luminol available for reaction.

Figure 1 de-

picts the CL response of purified bovine PMNL stimulated by the addition of PMA at time zero.

The concentrations of luminol

employed range from 40 μΜ (top curve) to 5 μΜ (bottom curve).

o

o

m

o

co

o

en

o

oj

TIME, minutes 4 Figure 1. The CL responses from 4 χ 10 PMNL in 2.0 ml of CVB using luminol at 40 μΜ, 20 μΜ, 10 μΜ, and 5 μΜ from top to bottom respectively. Stimulation was by addition of PMA (0.3 μΜ final) at time zero.

878

ID o o1

O ID O o o ^h o o o 1/ [Luminol]. juM

I T-) ·* ι o

O cu o

Figure 2. Double reciprocal plot of CL velocity against the concentration of luminol. The data were taken from figure 1 plus additional runs at different luminol concentrations. It should be appreciated that the CL response is measured as photon intensity (photons/min) and is a velocity or first derivative value.

At the higher luminol concentrations, the reac-

tion approaches a zero order condition with respect to this substrate.

As such, the maximum velocity (Vmax) of PMA-stimulated

PMNL oxygenation of luminol yielding photons can be extrapolated based on the Michaelis-Menten relationship: v = k

. [PMNL] · [CLP] [CLP]

+

Ks

where ν is the measured CL in photons/min, k is a constant depending on the stimulus-CLP combination employed, and Ks is the concentration of CLP yielding one-half the vmax. In the experiment described, when [luminol] >> Ks, the reaction approaches zero order with respect to luminol, and ν approaches Vmax.

Therefore, Vmax = k · [PMNL].

This relationship is de-

picted in figure 2 as the double reciprocal plot of CL velocity against luminol concentration.

Using the statistical method of

Wilkinson (11) the value of Vmax ± standard error is estimated

879 as 167 ± 4 photons/min/PMNL, and the value of Ks ± standard error is estimated as 6.8 ± 0.5 μΜ. Chemiluminigenic probing provides an ultrasensitive, nondestructive approach for investigating stimulated phagocyte oxygenation activity.

Although only one CLP-stimulus combination is des-

cribed herein, use of different stimuli in combination with CLP's of different physical-chemical characteristics allows differential investigation of the nature of the oxygenation activities of stimulated phagocytes.

References 1.

Sbarra, A.J., Karnovsky, M.L.: J. Biol. Chem. 234 , 13551362 (1959).

2.

Allen, R.C.: in The Reticuloendothelial System, Vol. 2, Biochemistry and Metabolism (Sbarra, A.J., Strauss, R. eds) pp. 309-338, Plenum Press, New York (1980).

3.

Allen, R.C., Stjernholm, R.L., Steele, R.H.: Biochem. Biophys. Res. Commun. £7, 679-684 (1972).

4.

Allen, R.C.: in Chemical and Biological Generation of Excited States (Adam, W., Cilento, G. eds) pp. 309-344, Academic Press, New York (1982). Allen, R.C., Loose, L.D.: Biochem. Biophys. Res. Commun. 69, 245-252 (1976).

5. 6.

Allen, R.C., Pruitt, B.A., Jr.: Arch. Surg. 117, 133-140 (1982) .

7.

Mottola, C., Gennaro, R., Marzullo, Α., Romeo, D.: Eur. J. Biochem. Ill, 341-346 (1980).

8.

Riding, G.A., Willadsen, P.: J. Immunol. Methods £6, 113119 (1981).

9.

Madonna, G.S., Allen, R.C.: Infect. Immun. 32^ 153-159, (1981).

10. Lee, J., Seliger, H.H.: Photochem. Photobiol. 15:227-237, (1972) . 11. Wilkinson, G.N.: Biochem. J. 80, 324-332 (1961).

COMPARISON OF LUMINOL- AND LUCIGENIN-AMPLIFIED CHEMILUMINESCENCE (CL) OF PHAGOCYTES

Reiner Müller-Peddinghaus Kali-Chemie Pharma, Dept.Experimental Pathology, Postfach 220, D-3000 Hannover 1, F.R.G.

Abstract CL determines cell activation via reactive oxygen species (ROS). Inflammatory cells (phagocytes) generate 0· which is 1 converted to H 2 ° 2 ' 0 H " a n d °2' T h e s e R 0 S c o n t r i b u t e to diverse defense mechanisms and noxious events in inflammation. Native CL from ROS can only be detected from activated phagocytes. Amplifiers like luminol (1) and lucigenin (2) react with certain ROS and enhance the amount of light emission. The question to answer was, which amplifier seems superior? Mainly murine (3) thioglycollate-induced peritoneal exudate cells (PEC), harvested 24 resp. 96 hours thereafter and residential macrophages are used to characterize the CL response. 2 χ lO^cells/vial in a total volume of 0.5 ml thermotatized at 37°C in a shaker are stimulated by human complement-opsonized zymosan particles (final conc. 1.6 mg/ml). Parameters are the integral of spontaneous (15 min) and induced CL. Inhibition experiments with maximally stimulated phagocytes comprise a 15 min period with and without the test compound respectively. Both amplifiers are effective at 1.54 χ 10

-4 mol/1 final

concentration to enhance native ROS CL. Both amplifiers are sensitive to detect ROS generation from unstimulated taneous

(spon-

CL) 24h PEC (mainly neutroph. granulocytes = PMNL)

96h PEC (mainly macrophages = M0) and even residential M0.

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

882 Luminol more effectively amplifies ROS from 24h PEC

(PMNL).

Lucigenin is more effective to amplify ROS from 96h PEC (M0) or res. M0. The respective kinetic of induced C L differs. Luminol-amplified C L exerts a distinct peak 10 to 15 m i n after stimulation, whereas lucigenin-amplified CL remains fairly constant in 24h PEC and absolute constant in 96h PEC up to 40 min. Dilution or agitation of the test samples barely influences lucigenin-amplified CL, but characterizes nol-amplified CL as sensitive to technical artifacts. bition experiments with superoxide

lumi-

Inhi-

dismutase, diethyldithio-

carbamate, azide, cyanide, catalase, aminotriazole and cysteamine using 24h PEC and 96h PEC (data not shown) reveal

that

lucigenin seems to react with the primary ROS the super oxide anion radical

(0·) whereas luminol appears to react with 1 H 2 ° 2 ' P r o b a b l Y singlet oxygen ( O2)i but unlikely with OH*. Thus lucigenin seems to be superior as amplifier of phagocyte CL as it detects the primary ROS (0~*) and in contrary to luminol appears to be independent from the m a i n Ü2°2

catal

yts

myeloperoxidase, catalase and glutathione peroxi-

dase. References 1. Allen, R.C., Loose, L.D.: Biophys.Res.Commun. 245 - 252 ( 1976) .

69,

2. Allen, R.C.,: In: DeLuca,M.A., McElroy, W.D. (eds). Bioluminescence and chemiluminescence. Basic chemistry and analytical applications. Academic Press, New York, 63 -73 ( 1981) . 3. Müller-Peddinghaus, R., Hoppe,G., Schumacher, W.: Zbl. Vet. Med. Β (in press).

CORRELATION BETWEEN OXIDATIVE STRESS OF THE HEART, EVALUATED BY C H E M I L U MINESCENCE EMISSION AND CONTRACTILE

FUNCTION.

R.Barsacchi, P.Camici, U.Bottigli, P.Salvadori, Institute of Clinical Physiology C.N.R. Pisa

G.Pelosi.

(Italy).

M.Maiorino and F.Ursini. Institute of Biological Chemistry, University of Padova

(Italy).

Introduction L i p i d peroxidation, suggested

to be

as a mechanism

important

of free radical pathology,

in causing

irreversible

membrane

has

been

damage

fol-

lowing ischemia and/or

ischemia and reflow (1). In a previous paper

we

the

have

the

evaluated,

photon

stress, since

emission,

and

the

on

the

perfused rat heart,

related

to

morphological

infused h y d r o p e r o x i d e s

a and

the correlation

hydroperoxide functional

dependent

between

oxidative

derangement.

may act b o t h as p e r o x i d a t i o n

agents and glutathione depletors, we sought to characterize more ly the role of glutathione

(2)

However, sparking direct-

depletors on the spontaneous lipid p e r o x i d a -

tion, evaluated as p h o t o n emission.

M a t e r i a l s and Methods The

rat h e a r t

were

perfused according

to L a n g e n d o r f f

in a light

box to collect the p h o t o n emission by a single p h o t o n counting tus

as previously

described

(2). The perfusion buffer was

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

tight

appara-

supplemented

884 with

0.1

to 0.5 mM

l-chloro-2,4 dinitrobenzene

(CDNB) or 1 to 5 mM

phorone or 0.2 to 0.8 mM diamide (3). The perfusion with GSH depleting agents

started

after

a

20

min period

alone. The electrocardiogram dial

electrode.

The

of equilibration

was continuously

reduced

(GSH)

and

with

buffer

monitored by an epicar-

oxidized

(GSSG)

glutathione

content of the hearts was evaluated by HPLC as described by (4) . For each experimental condition some hearts were processed for light microscopy using hematoxylin-eosin staining.

Results and Discussion In Fig. 1 the GSH and GSSG content of the hearts during perfusion with the depleting agents is reported. The concentrations of GSH depletors in

the

minimal

experiments required

to

reported obtain

in the Figure

are

those

chosen

as

the

a complete disappearance of GSH within 5

min. However this was not obtained with phorone even at 5 mM concentration.

Fig. 1. GSH and GSSG content of the rat hearts perfused with GSH depleting agents. The results are the mean of three independent determinations.

885 The chemiluminescence increases

in

maximum 70% used.

The

heart

cases

and

during p e r f u s i o n w i t h GSH depletors

with

all

h i g h e r than the control time

disappearance of

all

of the hearts

course

of GSH.

perfused

of

the

the

concentrations

used,

level no matter w h i c h

increase

is

roughly

F o l l o w i n g GSH d e p l e t i o n signal progressively

CDNB

is

reported

as

a

a

is the agent

proportional

In Fig. 2 the trace of chemiluminescence

with

to

typical

to

the

emission

experiment.

in all cases the amplitude of e l e c t r o c a r d i o g r a m

declined and a block of electrical and contractile

activity took place w i t h i n 20 min.

0 Fig. At

2.

time

Spontaneous a

the

10

20

30 mia

chemiluminescence

shutter

in

front

to

of

isolated

perfused

the photomultiplyer

rat

heart.

w a s opened;

at

time b the p e r f u s i o n w i t h 0.125 mM CDNB started. For details see ref. 2.

The hystological

examinations

carried out after 20 m i n of p e r f u s i o n

all cases showed a contraction band necrosis in

the

it

is

30-40%

concluded

increased on

of the observed

the

that

spontaneous

aerobic

GSH

tissue

depletion

(5).

The

myocitolysis)

(Fig. 3). From the above in the

lipid peroxidation

metabolism

(coagulative

GSH

heart

is

results

associated

w h i c h is apparently indeed

appears

in

to

to

an

dependent

play

a

key

role in the p r o t e c t i o n of the myocardial cells against such a challenge. As

in the case of the p e r f u s i o n w i t h hydroperoxides the p e r o x i d a t i o n of

heart

membranes,

even

at

very

small extent,

produces

the typical

pat-

886

Fig.

3.

Light

microscopy

r a t heart perfused w i t h 0.125 mM CDNB

m i n showing the "coagulative

tern of the

for

20

myocitoltsis".

"stone heart" w h i c h seems to be related to a sudden increa-

se of cytosolic free calcium

(6).

References 1.

Meerson,

F.Z., Kagan, V.E., Korlov, Y.P., Belkina, L.M.,

ko, Y.V.: Basic Res. Cardiol. 77, 465-485 2.

Barsacchi, G.,

R.,

Camici,

P., Bottigli,

Arkhipen-

(1982).

U., Salvadori,

Maiorino, M., Ursini, F.: Biochim. Biophys.

P.A.,

Pelosi,

Acta 762,

241-247

(1983). 3.

Plummer, J.L., Smith, B.R.,

Sies, H., Bend, J.R.: in M e t h in E n z y -

mol. vol.

and Kaplan, N.O. Editors)

77

(Colowich S.P.

p.

50 Acad.

Press, New York 1981. 4.

Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, A.E., Ellis, W.W., Potter, D.W.: Anal. Biochem. 106, 55-62

5.

6.

Boveris,

(1980).

Α., Cadenas, E., Reiter, R., Filipkowski, M., Nakase, Y.,

Chance, Β.: Proc. Natl. Acad. Sci. 77, 347-351

(1980).

Ganóte, C.E.: J. Mol. Cell. Cardiol. 15, 67-73

(1983).

AGE-DEPENDENT MODULATION OF SUPEROXIDE A N D H Y n R O G E N PEROXIDE SECRETION A N D CHEMILUMINESCENCE BY IN VITRO CULTIVATED MACROPHAGES

Volker Klimetzek and H. Dieter Schlumberger Institut für Immunologie und Onkologie, B A Y E R AG, D-5600 WuDpertal, FRG

Introduction

Activated macrophages (M$) have been shown to exhibit potent bactericidal and tumoricidal activities, which are attributed largely to the secretion of reactive intermediates arising from the incomplete reduction of molecular oxygen (O^) (1). These intermediates include superoxide (O"), hydrogen peroxide ( 1 ^ 0 2 ) and hydroxyl radical (OH·)· The enzymes involved in their generation are largely unknown. It is not yet clear if Mi^ can only reduce molecular oxygen by a one electron mechanism to O " , which then dismutate to the other species, or if M^ can also reduce O^ directly to

^

a

t w o

e'ec1:ron

mechanism without the lib-

eration of O " . Crude extracts of the enzymes are able to produce O " as well as

not

known if these activities are only typical for the

isolated complex or also found using whole cells. Since no detailed studies on the simultaneous release of O " and H^O^ of M^ are available, we investigated the secretion of the two products during their in vitro culture. First results indicate that the secretion of O " and

independently during in

vitro differentiation of M^. Simultaneous studies on the luminol-dependent chemiluminescence (LdCL) revealed that the kinetik of LdCL response follow that of the O " secretion but not of H O . secretion.

Methods

Animals.

2-3 month old DBA or Balb/c mice were used.

Macrophages.

Peritoneal exudate cells were harvested from mice which were in-

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

888 jected i.p. with Corynebacterium parvum (0.35 mg/mouse, Wellcome, Beckenham, U K ) 14 days b e f o r e s a c r i f i c e , or 4 days b e f o r e with 1 ml 10 % proteose peptone ( D i f c o , Michigan, USA). Exudate cells were plated on petri dishes (0 35 mm N U N C , 5 2 Roskild, Danmark) at a density of 2-4 χ 10 /cm

and cultured as described earlier

(3). These cultures were used f o r experiments with adherent M

2 %. Moreover, the excess of periodate or borohydride was tho-

roughly removed after each treatment and had no effect on spectrofluorometric measurement of NADP concentrations. The treatment of human unstimulated PMNs by an optimal concentration of _3 sodium periodate (1 χ 10 at 10

M) or by a reducing agent (sodium borohydride

M) induced a significant enhancement of NADPH oxidase activity

(Table I). These results compelled us to compare differences between enzymic activities, before and after stimulation, for each PMN treatment. In these conditions oxidation of human PMN membranes by 1 χ 10

M perio-

date strongly inhibited the cellular response to opsonized zymosan. The _5 treatment of periodate-treated cells by a reducing agent (6 χ 10

M boro-

hydride at 25° C) prevented this periodate-induced suppression. Complete _5 reversal was obtained from 6 χ 10

M. In contrast, a borohydride treat-

ment, performed before periodate oxidation, did not affect the periodateinduced suppression.

925 Table I : NADPH oxidase activities (nmol/min/105 cells) in PMN treated by periodate (1 χ 10-3 m) or/and borohydride (6 χ 10 - 5 M)

Cells treated with (n = 11) periodate borohydride + + borohydride periodate

PBS

periodate

borohydride

Unstimulated

2.4 i 0.9

4.3 - 1.4

4.9 - 2.0

5.2 - 1.3

5..5 - 1.4

Stimulated by zymosan

8.0 - 1.0*

5.7 - 0.4

7.1 - 1.2

10.3 - 1.1*

6..5 i 1.0

* Significant difference with unstimulated cells

(p 2 production.

Although most of the C^

and f^C^ produced by

activated neutrophils i s farmed an the external surface of the c e l l s and i s a c c e s s i b l e t o the medium, superoxide diamitase and c a t a l a s e in the i n cubation medium gave no inhibition of i n t r a c e l l u l a r ascorbate oxidation, which a l s o implies that

and H w e r e not responsible.

I f ascorbate were t o play an active r o l e in microbial k i l l i n g by neutrop h i l s , i t should be released i n t o the phagolysosomes on a c t i v a t i o n .

PMA.

causes s p e c i f i c degranulation only, opsonized zymosan r e l e a s e s both s p e c i f i c and azurophil granules.

Cytochalasin Β prevents closure of the phago-

lysosomes so any ascorbate released should be l o s t into the incubation medium.

The absence of s i g n i f i c a n t l o s s of ascorbate from neutrophils

treated with PMA or opsonized zymosan and cytochalasin Β implies, theref o r e , that ascorbate r e l e a s e i s not associated with activation or degranulation. References 1.

Bigley, R . , Stankova, L . , Roos, D., Loos, J . : (1980) .

2.

Stankova, L . , Rigas, D.A., Keown, P . , Bigley, R . : t h e l i a l . Soc. 21, 97-102 (1977).

3.

Miller, T.E.:

4.

Drath, D.B., Karnovsky, M.L.: (1974).

Enzyme 25, 200-204 J . Reticuloendo-

J . B a c t e r i o l . 98, 949-955 (1969). Infection and amunity 10, 1077-1083

5.

Cox, B.D., Whichelow, M . J . :

6.

Winterbourn, C.C., V i s s e r s , M . C . M . : B i o c h i m . B i o p h y s . A c t a

Biochem. Msd. 12, 183-193 (1975).

7. .

Baehner, R . L . , Boxer, L.A., Allen, J . M . , Davis, J . : (1977) .

8.

Roos, D., Vfeening, R . S . , Voetman, A.A., van Schaik, M . L . J . , Bot, A.A.M, Meerhof, L . J . , Loos, J . A . : Blood 53, 851-866 (1979).

9.

Spielberg, S . P . , Boxer, L.A., Oliver, J . M . , Allen, J . M . , Schulman, J . D : B r . J . Haematol. 42, 215-223 (1979).

10. Voetman, A.A., Loos, J . A . , Roos, D.:

(in

pres^

Blood 50, 327-335

Blood 55, 741-747 (1980).

ROLE OF OXYGEN RADICALS IN PULMONARY VASCULAR INJURY

Arnold Johnson, Frank A. Blumenstock, Asrar B. Malik Department of Physiology, Albany Medical College, Albany, New York 12208

Introduction The interest in oxygen radicals as mediators of lung injury has arisen from the observations

that many forms of lung

injury are neutrophil dependent (1,2,3) and that neutrophil activation is an important source of oxygen radicals (4,5). Neutrophils are found sequestered within the lung of patients with the Adult Respiratory Distress Syndrome (6-8).

Oxygen-derived free radicals have been shown to inujure endothelial cells (9-12).

Oxygen-derived free radicals have

been implicated as etiologic agents in mediating pulmonary vascular injury and edema in a variety of conditions, i.e. complement activation (13,14), hyperoxia (15-17), pulmonary embolization (18), and immune complex deposition in the lung (19) .

Role of Oxygen Radicals in Lung Injury Oxygen radical generation induced by xanthine-xathine oxidase and glucose-glucose oxidase systems, producing and H2O2, respectively, has been used to study the role of oxygen radicals.

Xanthine-xanthine oxidase instilled intra-

tracheally in rabbits increased lung vascular permeability which was preventable by SOD (20).

However, vascular injury

induced by intravascular xanthine and xanthine oxidase in

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

932 isolated-perfused rabbit lungs was prevented by catalase, DMTU, and DMSO but not by SOD suggesting that it was mediated by f^C^ and OH" (21,22).

Glucose-glucose oxidase

instillation increased permeability which was preventable by catalase (20).

Lung injury caused by

WaS

P otent:: '- ate ^

by the simultaneous treatment with myeloperoxidase, suggesting that OHCl or ^ ^ generation aggravates the injury (20). Intrapulmonary activation of leukocytes is important in oxygen radical generation.

Tracheal instillation of leuko-

cyte-activating agent phorbol myristate acetate (PMA) in rats increased lung vascular permeability which was prevented by catal ase but not by SOD (23).

Lung vascular injury induced

by intratracheal instillation of immune complexes was also prevented by catalase, while SOD only prevented the early rise in permeability (11,19). injury may have two phases: latter mediated by

an
> +->

•H m ci u •cρ c 40 CD

O e α) υ co αϊ h o 3 i-Η Ρη

,Η

20

4 f ί i

á t ini 10

20

30

40

50

60

Time of incubation (37°C)

Figure 2. Fluorescent IgG aggregates produced by normal human neutrophils ^ ·) . Enhancement by phorbol myristate acetate ( o — o ) and inhibition by superoxide dismutase ( • — • ) .

943

•I« 1%

Vi

s

J*

ν

Figure 3 Complement activation products induced by incubating (a) heataggregated IgG, (b) native IgG, (c) UV-irradiated IgG (90 min) and (d) UV-irradiated IgG (30 min) with normal human serum. Activation was demonstrated by two dimensional electrophoresis against anti C3c antibody.

used as a positive control.

Complement activation products

could be detected in solutions of IgG which had been irradiated for 60 min. while solutions irradiated for 30 min., or less, lacked the ability to activate complement. The ability to activate complement therefore appeared to be a property of the fluorescent aggregation products and not the fluorescent monomer (Figure 3).

944

Discussion Aggregated IgG complexes consisting of IgG and anti-IgG have been detected in the joint fluid of patients with RA. These complexes are generally thought to cause the severe chronic inflammation associated with this disease, and also to perpetuate its course. Although it is believed that IgG undergoes denaturation in order to give rise to these complexes, the mechanism, by which this change could occur in RA has remained elusive. Free-radical reactions generated by UV light, lipid peroxides, or activated neutrophils can alter the nature of IgG so that it becomes fluorescent and aggregates.

This damage to human

IgG is related to observed changes in tryptophan and other constituent amino acids of the molecule (14).

Such changes

in the nature of human IgG are identical to those found in IgG isolated from rheumatoid serum and synovial fluid by high performance liquid chromatography. Mechanical or heataggregation alone cannot account for these observations. The results reported here show that IgG not only undergoes freeradical denaturation but that in so doing it can become a further stimulus for neutrophils to generate more free radicals. This observation may be of considerable importance since it describes a possible self-perpetuating mechanism of radical release and tissue damage mediated by the neutrophil. Conversion of complement induced by the free-radical alteredIgG could be seen to further amplify this destructive mechanism, an observation which is consistent with findings of complement depletion in rheumatoid serum and synovial fluid.

945 References 1

Johnson, P.M., Watkins, J. and Holborow, E.T.: Lancet, 1, 611-613 (1975).

2

Watkins, J. and Swannell, A.J.: Ann. Rheum. Dis., 31, 218-223 (1972).

3

Cats, Α., Lafeber, J.M. and Klein, F.: Ann.Rheum. Dis., 34, 146-155 (1975) .

4

Johnson, P.M., Watkins, J., Scopes, P.M. and Tracy, B.M.: Ann. Rheum. Dis., 33, 366-370 (1974).

5

Johnson, R.B. and Lehmeyer, J.E.: J. Clin. Invest., 57, 836-841. (1976).

6

Goldstein, I.M., Roos, D., Kaplan, H.B. and Weissman, G.: J. Clin. Invest., 56, 1155-1163 (1975).

7

McCord, J.: Science, 185, 529-530 (1974).

8

Lunec, J. and Dormandy, T.L.: Clin. Sci., 56, 53-59 (1979).

9

Lunec, J. and Dormandy, T.L.: J. Rheumatol., 8, 233-245 (1980).

10 Blake, D.R., Hall, N.D., Bacon, P.A., Halliwell, B. and Gutteridge, J.M.C.: Lancet, 2, 1142-1144 (1981). 11 Desai, I.D. and Tappel, A.L.: J. Lipid Res., 4, 204-207 (1963). 12 Cohen, H.J. and Chovaniec, M.A.: J. Clin. Invest., 61, 1081-1096 (1978). 13 Shadforth, M.R. and McNauton, D.C.: Ann. Rheum. Dis., 37, 18-23 (1978). 14 Wickens, D.G., Norden, A.G., Lunec, J. and Dormandy T.L.: Biochim, Biophys. Acta. 742, 607-616 (1983).

SUPEROXIDE PRODUCTION BY POLYMORPHONUCLEAR LEUKOCYTES IN RHEUMATOID ARTHRITIS ( R . A . ) AND ANKYLOSING SPONDYLITIS

(A.S.)

Catherine P a s q u i e r , Saddek L a o u s s a d i , Bernard Amor I n s t i t u t de Rhumatologie, U. 5 INSERM, Hôpital Cochin, P a r i s , France

Introduction T h i s work d e a l t with the behaviour of p e r i p h e r a l

polymorphonuclear

leukocytes (PMN) i n R.A. and A . S . by determining Og' production

correc-

t i v e l y to superoxide dismutase (SOD) a c t i v i t y . Thus O g ' , Cu Zn (SOD·^) and Mn SOD (SOD2) were a s s e s s e d i n 18 healthy c o n t r o l s , 17 a d u l t d e s t r u c t i v e R.A. and 17 A . S . . Rheumatoid p a t i e n t s were s e l e c t e d according to ARA c r i t e r i a and t r e a t e d with non s t e r o i d a l a n t i i n f l a n m a t o r y drugs (NSAID) or second l i n e drugs such as g o l d s a l t s , D - P e n i c i l l a m i n e or h y d r o x y c h l o r o q u i n e . A . S . were o n l y r e c e i v i n g NSAID while c o n t r o l s were untreated.

Methods 1) PoiymonpkonucJÎeM

Lboùvbœn.

PMN were obtained from h e p a r i n i z e d venous

blood according to modified Boyum t e c h n i c (1) and e i t h e r

stimulated

with o p s o n i s e d zymosan f o r 0 ^ ' assay or f r a c t i o n e d f o r SODj and SOD^ measurement. 2) ()"· cu>iay, Og" p r o d u c t i o n was measured according to B a b i o r modified t e c h n i c (2) in equal PMN samples c o n t a i n i n g 3.10^/0.3 ml : a) unstimul a t e d PMNs ; b) s t i m u l a t e d PMNs by opsonised zymosan (1.5 mg) ; c) s t i m u l a t e d PMNs added with 20 p g o f Cu Zn SOD. a, b and c were incubated a t 37°C with 60 nMoles o f f e r r i cytochrome C. The r e a c t i o n was stopped i n melting i c e (0°C) a t 10, 20 and 30 minutes. F e r r i cytochrome C r e d u c t i o n was determined by measuring the absorbance o f the incubated supernate at 550 nm on a ACTA I I I r e c o r d i n g spectrophotometer

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - FWnted in Germany

(Beckman)

948 using the unincubated blank as reference. ΔΕΜ at 550 ran was taken as - 1 - 1

20000 M

cm

and the r e s u l t s expressed as nMoles of 0 2 ' produced by

10 6 PMNs. 3) SOD cL&iay. SOD was assayed spectrophotometrically according to S a l i n and Mc Cord (3) and detected by i t s a b i l i t y to i n h i b i t the superoxide mediated reduction of f e r r i cytochrome C by xanthine-xanthine oxidase system. Assays were performed at 25°C. Cytochrome C reduction was monitored at 418 nm. Total SOD was measured without cyanide while the Mn SOD was assayed with 1 mM KCN added. SOD^ was calculated from the difference between the two a s s a y s . The r e s u l t s were expressed i n pMoles of SOD reported to 10 6 PMNs. 4) Statiitiaxi

anatyiZi

was performed using t Student and c o r r e l a t i o n

tests.

Results The r e s u l t s are given in tables 1 and 2. 1) 0 } · production. After 30 minutes incubation rheumatoid PMNsproduce probably more 0 ^ ' than do PMNs of healthy c o n t r o l s or A , S . . T. „ „ I ™

~

"rZIIHi, i:!"™!

in lu minii+oe minutes

4

_ nη += 318 >42

on ¿u minutes

?>14

, i-niltec jun m minutes

8>44

η = 18 ±

3 ¿ 5

η = 17 ±

3

J3

~

"

Rheumatoid arthritis 4>4g

η >8β

1 0 2 3

η = 18 ±

3

22

η = 18 ±

4>46

n = 15 ±

4>g3

Ankylosing SBondylitis____ 4

gg

η

g8

8 g 4

η+ = 17 3 83 η = 17 ±

4

g2

n = 9 + g 2g

Table 1 : 0 2 " production expressed as nMoles/10 6 PMNs (mean - SD). 2) SOP activity.

In R.A. and A.S. SODj i s not d i f f e r e n t from control

value

while S0D 2 i s s i g n i f i c a n t l y decreased i n both diseases (R.A. : ρ < 0 . 0 0 1 , A.S. : ρ = 0.025).

949

SOD

Controls

RRiûmitôïa arthritis

1

η = 17 2.86 - 1.57

η = 19 2.81 ± 1.66

η = 17 2.94 ± 1.54

SOD 2

η + 10 0.77 - 0.62

η + 11 0.268 - 0.224

η + 5 0,246 - 0.124

ςηη 5υυ

ÁñkylSsTñg" spondei i t i s_

Table 2 : S0D1 and S0D2 a c t i v i t y expressed as pMoles/106 PMNs (mean - SD), 3) CofUiíZation bztmen 0^* and S0V. a) In the control group we f a i l e d to evidence c o r r e l a t i o n between SOD^, S0D2 and 0 2 " production (r = - 0,18 N.S. and r = 0.38 N.S.) ; b) In R.A. SOD-j^ is correlated with 0^' production ( r = 0.51 ρ = 0.025) while no c o r r e l a t i o n between S0D2 and 0 2 " was found ; c) In A.S. S0D2 is s t r i k i n g l y correlated with 0 2 " production ( r = 0.88

ρ < 0.001) while no c o r r e l a t i o n was found with

SOD,.

0 2 " ηMoles/10

cel.

0 2 ' nMoles/10

15

eel

15'

PR

SpA

r = 0.51

r = 0.88

10

r = 0.77 20mn ρ < 0.001

10

N·

I

I

5

10

30mr

ρ < 0.001

S ρ = 0.025

*

SODj pMoles/106 cel.

I

•

OJ5

W

•

S0D2pMoles/106 cel.

950 Comments SODj level modification has been reported only in adult R.A. (4) (5) and chronic juvenile arthritis (6). In this latter,

production by PMNs

is markedly enhanced after stimulation by opsonised zymosan. Our previous data have not established any SOD^ level modification either in R.A. or in A.S.. On the contrary low SODg level in both diseases has been observed. The present study confirms these results and shows that 30 minute stimulated PMNs of adult R.A. produce more

than do control or A.S.

PMNs. Moreover in R.A. the correlation between PMN 0^' production and SOD^ suggests that this enzyme might be more specifically implicated in rheumatoid inflammation regulation than does SOD.,. Furthermore in A.S. this role might be attributed to S0D 2 ·

References 1. Boyum, A. : Scand. J. Immunol. 5, suppl., 9-15 (1976). 2. Babior, B.M., Kipnes, R.S., Curnuttes, J.T. : J. Clin. Invest. 52, 741-744 (1973). 3. Salin, M.L., Me Cord, J. : J. Clin. Invest. 54, I005-I009 (1974). 4. Igari, T., Kaneda, H., Horiuchi, S., Ono, S. : Clin. Orthop. & Rei. Research 162, 282-287 (1982). 5. Banford, J.C., Brown, D.H., Hazleton, R.A. & al : Ann. Rheum. Dis. 41, 458-462 (1982). 6. Rister, M., Bauermeister, Κ. : Klin. Wochenschr. 60, 561-565 (1982).

Thanks are due to Mrs Sylvie Courant for her excellent technical assistance.

EFFECTS OF IMMUNOSUPPRESSIVE THERAPY IN SYSTEMIC LUPUS ERYTHEMATODES 1 Manfred Rister, Annette Schneider Universitäts-Kinderklinik D-5000 Köln 41 Dieter Mitrenga Medizinische Universitätsklinik D-5000 Köln 41

Introduction Since monocytes and polymorphonuclear leukocytes (PMNs) are essential in the removal of opsonized antigens from insoluble antigen-antibody complexes, the importance of phagocytic cells as an effector of immune response has led to increasing studies of its role in autoimmune disease (1). Furthermore, it has become increasingly clear that the PMNs should be viewed as a secretory cell (2). Immune complexes and other membrane active agents cause PMNs besides discharging neutral lysosomal proteases to generate toxic oxygen-derived products such as superoxide anion

(Op,

hydroxyl radical, hydrogen peroxide and singlet oxygen (3). The proteases are antagonized by various serum proteases. Protection against the highly reactive 0^ is provided by the metalloenzyme superoxide dismutase (SOD) (4). This generation of potent biologic products by stimulated PMNs appears to be necessary for the development of the inflammatory manifestations of tissue injury in systemic lupus erythematodes (SLE). This disease remains of unknown origin characterized by major alterations of both the humoral and the cellular arms of immunity (5). Though especially lymphocytic abnormalities have been seen in SLE, we investigated the effect of immunosuppressive therapy on PMN function, by estimating the capacity of patient and control PMNs to produce 0* and to release the lysosomal enzyme ß-glucuronidase. In addition we determined the intracellular content of SOD in PMNs.

1

Supported by DFG Ri 275/6-1

O x y g e n Radicals in C h e m i s t r y a n d Biology © 1 9 8 4 Walter d e Gruyter & Co., Berlin · N e w York - Printed in G e r m a n y

952 Methods Neutrophils were obtained from the v e n o u s blood of n o r m a l h e a l t h y v o l u n t e e r s and patients w i t h SLE as previously described

(4). P M N s w e r e

isolated by Percoli density gradient centrifugation and suspended in Krebs-Ringer Phosphate buffer

(KRP)

(6).

Degranulation. D e g r a n u l a t i o n of PMNs w a s quantitated by measuring

the

release of ß-glucuronidase u p o n exposure to opsonized zymosan for 5, 15 and 30 m i n u t e s (3). At the end of the experiments the cells w e r e c e n t r i fuged, the supernatants w e r e removed for the determination and expressed as per cent of total ß-glucuronidase activity released be 0,2% T r i t o n X-100. Superoxide a n i o n release. A m o d i f i c a t i o n of the assay by Babior et al., w a s u s e d (7). 2,5 χ 10^ phagocytes w e r e preincubated for 5 m i n u t e s at 37°C w i t h KRP buffer and 0,1 m l of o,6 m M ferricytochrome C. To start the r e a c t i o n 10 m g zymosan w a s added. After 25 m i n u t e s the r e a c t i o n w a s stopped. Optical density of the supernates at 550 n m w a s determined and converted to n a n o m o l e s OX dependent cytochrome C reduction using the 4 - 1 extinction coefficient of 2,1 χ 10 M (1). SOD-assay. SOD w a s spectrophotometrically detected by its capacity to inhibit the cytochrome reduction m e d i a t e d by 0*, generated during the oxidation x a n t h i n e catalysed by x a n t h i n e oxidase. The change of optical d e n s i t y was read at 550 n m (8).

Results Patients: W e studied 15 p a t i e n t s ,

14 female and one male, w i t h confirmed

SLE. A l l patients showed four or m o r e criteria for diagnosis of SLE, as recommended b y A m e r i c a n R h e u m a t i s m Association. The m e a n E S R w a s 21/41. In all patients antinuclear antibodies have b e e n detected by FAT^

in

serum diluted 1:5 or greater. In six patients the anti-DNA-values w e r e greater than 25 units. Only three patients demonstrated a h y p o c o m p l e m e n t e m i a in C^ and C^. At the time of the "neutrophil study" 14 p a t i e n t s received 10-20 m g / d steroids. Fourteen patients were additional

Fluorescence Absorbence

Technique

treated

953 with 2-3 mg/kg/d azathioprine, to reduce their major clinical manifestations (e.g. nephritis, CNS disease, polyserositis, hematological). One patient with a mild SLE was treated with corticosteroids and hydroxychloroquine. Superoxide anion. Control cells generated in rest and during phagocytosis 2,28 and 16,7 nmoles 0*/2 ,5 χ 10° cells/25 minutes, respectively. Before starting the therapy PMNs of patients with SLE generated similar amounts. But PMNs obtained from patients on immunosuppressive therapy produced in rest and

during phagocytosis only 1,96 and 5,99 nmoles

0~/2,5 χ 106/25 minutes, respectively (X + SX). Degranulation. Upon stimulation to opsonized zymosan control cells and PMNs obtained from patients before starting the therapy released

12+2,

1 5 + 2 and 1 6 + 2 % of total ß-glucuronidase by 5, 15 and 30 minutes, respectively (X + SX). But PMNs from patients with SLE on immunosuppressive therapy released only 4 + 2, 4 + 2

and 5 + 2% of total ß-

glucuronidase by 5, 15 and 30 minutes, respectively (X + SX). SOD activity. The SOD-content of control PMNs and before the onset of the immunosuppressive therapy was 1,48 + 0,07 U/mg cellprotein compared to 1,78 + 0,33 U/ml cellprotein in PMNs obtained from patients on immunosuppressive therapy (X + SX).

Discussion PMNs represent one of the principal effector cells observed in numerous inflammatory diseases. In response to a variety of stimuli, PMNs release oxygen byproducts as part of the metabolic burst and discharge the content of their cytoplasmic granules into the extracellular space (2, 3, 9). In Behcet's disease and juvenile rheumatoid arthritis oxygen intermediates seem to contribute to tissue damage more than lysosomal enzymes, since the 0^ production by PMNs of these patients was markedly increased (4). The elevated 0^ generation in Behcet's disease may be attributable to circulating immune complexes, which are present in about 70% of Behcet's disease patients though elevated 0* levels were also noted in patients without circulating immune complexes (10). But in an untreated patient with SLE we observed neither elevated 0' nor increased

954 ß-glucuronidase release from PMNs u p o n stimulation w i t h opsonized zymosan. This is confirmed by several studies, w h i c h have also

found

n o r m a l granulocyte functions in patients w i t h u n t r e a t e d SLE (5). Granulocyte d y s f u n c t i o n w a s noted only in patients w i t h active SLE combined w i t h depressed complement

(5). Hence, m o s t evidence implies that the

qualitative abnormalities of granulocyte function in untreated

patients

w i t h SLE are the results of altered humoral factors rather than a defect of phagocytic cells themselves (11). But in patients w i t h SLE on immunosuppressive therapy w i t h corticosteroids and azathioprine PMNs d e m o n strated a severely impaired 0* and ß-glucuronidase release. The inhibition by antiinflammatory d r u g s of the p r o d u c t i o n of superoxide anion is w e l l documented. N e u t r o p h i l s from h e a l t h y volunteers and patients receiving steroids have a decreased capacity to reduce NBT, a reaction known to depend u p o n 0* (12). Corticosteroids c a n interfere w i t h the release of superoxide a n i o n from PMNs, m o n o c y t e s and eosinophils

(13).

The a d d i t i o n of steroids to p h a g o c y t i z i n g PMNs depressed the activation of the hexosemonophosphate shunt, iodination of ingested particles, o x y g e n u p t a k e and hydrogen peroxide generation (14). The

corticosteroid

therapy prevents the r e c o g n i t i o n o n the membrane and interferes w i t h degranulation by inhibiting the rupture of granule m e m b r a n e s

(1). The

m e c h a n i s m s by w h i c h antiinflammatory drugs inhibit leukocyte

function

are largely speculative. Antiinflammatory agents are m e m b r a n e a c t i v e and as m e m b r a n e s play an important role in b o t h the formation of the p h a g o cytic vacuole and the fusion of lysosomes w i t h the vacuole the

suggestion

has b e e n m a d e that these drugs cause defects in n e u t r o p h i l function secondary to a m e m b r a n e effect

(15, 16, 17, 9). Thus the inhibition by

antiinflammatory drugs of the production of 0* is probably caused by blocking of cell m e m b r a n e associated m e c h a n i s m s to produce

superoxide

a n i o n (1, 17, 18). Moreover agents k n o w n to increase intracellular

levels

of cyclic AMP have b e e n shown to decrease activation of h e x o s e m o n o phosphate shunt activity a n

g e n e r a t i o n in cells stimulated by phago-

cytosis (13). It is not clear how antiinflammatory agents that elevate c - A M P influence the oxidative m e t a b o l i s m of phagocytic cells. One m e c h a n i s m could be t h r o u g h the interference in the binding of cells to complexes t h r o u g h phagocytic FC-receptors (19). This m a y finally raise

955 the possibility that immunosuppressive drugs as well as corticosteroids act directly on the enzyme responsible for the respiratory burst or on a membrane associated trigger mechanism.

Summary These findings demonstrate a suppression of the 0* generation and lysosomal enzyme release by stimulated PMNs obtained from SLE patients on corticosteroids and azathioprine, in contrast to the unchanged intracellular SOD activity. Further observation will be required to determine if the clinical course of the patients will also be favorable effected.

956 References 1. McCord, J.M., Fridowich, I.: Biol. Chem. 244, 6049-6055 2. Olsson, I., V e n g e , P.: Allergy 35, 1-13

(1969).

(1980).

3. Weissmann, G.: J. Reticuloendothel. Soc. 26, 687-800

(1979).

4. Rister, Μ . , B a u e r m e i s t e r , Κ.: Klin. W o c h e n s c h r . 60, 561-565

(1982).

5. Budmann, D.R. , Steinberg, A.D.: Ann. Intern. M e d . 86^, 220-228 6. Böyum, Α.: J. Clin. Invest. 9]_, 77-89

(1977).

(1967).

7. Babior, B.M., Kippnes, R.S., Curnutte, J.T.: J. Clin. Invest. 52, 741-744 (1973). 8. M o l i n , L., Stendahl, 0.: A c t a Med. Scand. 206, 451-457

(1979).

9. W o l a c h , B., D e B o a r d , J.E., Coates, I.D., Baehner, R.L., Boxer, L.A.: J. Lab. Clin. Med. J_00, 37-44 (1982). 10. Niwa, Y . , M i y a k e , S., Sakane, T., Shingu, M . , Yokoyoma, M.: Clin. Exp. Immunol. 49, 247-255 (1982). 11. Turner, R., M a r t i n , M . , Schroff, J., Treadway, W . , DeChatelet, L.: Inflammation 2 , 319-327

(1979).

12. Chretien, J.H., Garagusi, V.F.: J. Reticuloendothel. Soc. JJ_, 358-367 (1972). 13. Lehmeyer, J.E., J o h n s t o n Jr. R.B.: Clin. Immunol. Immunopathol. 9_, 482-490 (1978) . 14. Gudewicz, P.W.: C i r c . S h o c k 8^, 95-103

(1981).

15. Chwalinska-Sadowska, H . , Baum, J.: J. Clin. Invest. 58, 871-879 (1976). 16. H ä l l g r e n , R., H a k a n s s o n , L . , V e n g e , P.: A r t h . Rheumat. 21:1, 107-1 13 (1978). 17. Oyanagui, Y.: Biochem. Pharmacol. 25., 1473-1480

(1976).

18. Puig-Parellada, P., Planas, J.M.: Biochem. Pharmacol. 535-537 (1978).

27,

19. Johnston, R . B . , Lehmeyer, J.E.: J. Clin. Invest. 57, 836-841 (1977). 20. W h i t t a k e r , J.Α., H u g h e s , H.R., K h u r s h i d , M . : Br. J. H a e m a t o l . 29, 273-278 (1979) . 21. Norris, D.A., W e s t o n , W . L . , Sams Jr. W.M.: Lab. Clin. M e d . 90:3, 569-580 (1977) .

957 DISCUSSION

PARNHAM: I missed a very important control which I suspect makes all your data totally meaningless. You have compared normal patients with systemic lupus erythematosus (SLE) patients on immunosuppressive therapy, but you have not controlled for the effect of the disease process on the PMN function itself. Since many anti-DNA antibodies are present in SLE plasma, it's highly likely that the neutrophils may be affected. RISTER: You probably missed the beginning of my talk. We didn't test in all patients, but in five patients we did find normal C>2 generation before the onset of therapy in patients with SLE and we did find a normal degranulation before onset of immunosuppressive therapy in PMN's. So we made controls. Another point is, there are a lot of papers and it's in the textbook of rheumatoid arthritis, that in patients with SLE you find normal granulocyte function. If there is a depression of granulocyte function then there is a hypercomplementemia and that's the reason why I showed this horrible slide where only three patients showed a slight decrease in complements C3 and C4. MICHELSON: I think your textbooks are wrong. I don't know whether you are familiar with the work of NIWA in Japan (in press), but he finds increased neutrophil radical production in patients with lupus, with rheumatoid arthritis, with Behçet's disease and others. RISTER: No, I'm sorry. NIWA showed an increased superoxide anion generation in FMN's of Behçet's disease, but he didn't study it in this paper. MICHELSON: He has three other papers in press. He has studied lupus, rheumatoid arthritis, Kawasaki disease, the subcutaneous lymph-node syndrome, and there is a whole group of diseases which are turning out to have superactive neutrophils which are over-producers of superoxide radicals. RISTER: We showed the over-production of superoxide anion in children with rheumatoid arthritis too. That's not the point of our paper; the point of our paper is that we generate qualitative leucocyte function defects by treating patients with steroids and azathioprene. MARKLUND:

Which method did you use to analyse superoxide dismutase?

RISTER: I used the McCORD method by generating superoxide with xanthine and xanthine oxidase and measuring the inhibition of cytochrome c reduction. MARKLUND: Then you seem to get about four times more SOD activity compared to what we get, so I would like to explore a little into why we get so different results. How did you compensate for the interference by myeloperoxidase in that assay? RISTER: MARKLUND:

We add one millimolar azide to it. That will wipe out the activity of myeloperoxidase.

958 RISTER:

Yes it does.

MARKLUND:

Next, how pure were your preparations of PMN's?

RISTER: We isolated with a percoli gradient, and there is usually a purity of 98% of mature PMN's. There are no bands and immature PMN's in it. MARKLUND: But you didn't count them exactly, you guess that you had something like 98%. RISTER: No, what we do is, we count the PMN's to adjust our final suspension to 10^ cells per ml and then we sonicate our PMN's and use 2000xg-supernatant for the assay and after this we do the LOWRY of the 2000xg-supernatant. So we don't correlate our data with the cell count because, I think, it is not accurate enough, we use the LOWRY assay. MARKLUND: I asked about the contamination by the other cells, because I think much of the superoxide dismutase activity you measure actually comes from lymphocytes and other contaminants. RISTER:

No, I think that's not possible, if you use a gradient.

MARKLUND: If you have a contamination of say 3 to 4 percent lymphocytes you have almost fifty percent of the superoxide dismutase activity coming from these cells. Because if you have pure PMN preparations, or count and compensate for lymphocytes or other cells, you will get much lower superoxide dismutase activities. It is possible that part of what you measure comes from other cell types. SCHOENBERG: Just a clinical question. Since your treatment was very effective in the glass tube, how effective was it in patients? Was there a correlation between the well-being of the patients and your data? RISTER:

The data is fresh. We have to follow up on this.

SCHOENBERG: So you don't use it also as a clinical assay of how much corticosteroids you use in this particular individual patient? RISTER: Not yet. But what we do, we use the superoxide anion generation for the detection of rheumatoid arthritis. If patients show a very high increase of superoxide anion release and liver and spleen enlargement without any bacterial infections, it's like a mosaic, we think it might be Still's syndrome. We use it as a diagnostic tool.

INVESTIGATION OF PHAGOCYTE OXIDATIVE METABOLISM IN MICROAMOUNTS OF WHOLE BLOOD FROM PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS Béatrice Descamps-Latscha, Marie-Noëlle Feuillet-Fieux, André Baruchel, Paul Jungers, Maxime Dougados", Ann-Thu Nguyen, Robert Golub INSERM U25, Hôpital Necker and "Clinique Rhumatologique, Hôpital Cochin, Paris, France

Introduction During the past ten years evidence has accumulated that the oxygen-derived radicals produced during phagocyte oxidative metabolism activation could be implicated in other biological fields than solely bactericidal activity (for review see 1 and 2) . Of great interest were more recent reports suggesting that the superoxide anion 0^ and/or H202 could contribute to modulating host inflammatory and immune reactions via their ability to generate substances such as neutrophil-derived chemotactic factor (3) or macrophage-derived suppressor factor (4). It has also been established that this membrane metabolic activation can be initiated by biological compounds more physiological than microorganisms or inert particles, i.e., complement components (5), alloantibodies (6) and immune complexes (7). Altogether these observations strongly suggest that immune reactions might contribute to phagocyte oxidative metabolism activation and that this pathway could in turn be implicated in immune regulatory mechanisms and clinical immune disorders. The present study proposes to investigate the oxidative metabolism of mononuclear (MN) and polymorphonuclear (PMN) phagocytes in systemic lupus erythematosis (SLE), a disease where immune complexes play a large role in tissue injury and in which the immunoregulatory aberrations mainly ascribed to Τ cell subsets (8) could also involve monocytes and/or their secretion products (L. Chatenoud et al., unpublished data).

Oxygen Radicals in Chemistry and Biology © 1984 Walter de Gruyter & Co., Berlin · New York - Printed in Germany

960 Figure

CL PRODUCTION IN IO 1 DILUTED BLOOD FROM PATIENTS WITH SLE

1a

I

I Notmal subjects (44) Patients with S I E (18)

30-

M Lie

1-

20-

100-

0.5-

50-

25·

5-

25-

HBSS

Figure

Latex

1b

Zymosan

PMA

ConA

CL PRODUCTION IN 10-2 DILUTED BLOOD FROM PATIENTS WITH SLE

I—)

Normal subjects (29)

ES3

Patients with SLE (18) 150

MLIc 20

HBSS

200-

1000-

200-

100-

500

100-

Latex

Zymosan

100-

50

50-

PMA

ConA

961

Subjects, materials and methods Eighteen CL determinations were performed in 15 adult female SLE patients

with definite symptoms as defined by the Ameri-

can Rheumatism Association.

All but 2 had an inactive form of

SLE at the time of study and received no (5 cases) or less than 20 mg daily corticosteroids; 6 patients were given Danazole alone (3 cases) or associated with corticosteroids (3 cases).

Forty-four age-matched healthy volunteer blood donors

were used as controls. One ml of heparinized blood was drawn and immediately diluted -1

-2

10 and 10 in phenol red-free Hanks balanced salt solution (HBSS). Luminol-dependent CL determination was performed as previously described (9) in 100 μΐ diluted blood samples after a 10-min incubation with 10 μΐ HBSS (resting CL) or the following stimulating agents: latex, serum-treated zymosan, phorbolmyristate acetate (PMA), concanavalin A (Con A) -7 (according to preparation and concentrations in Ref. 9) and 10

diluted N-

formyl-methionyl-leucine-phenylalanine (FMLP). maximal light intensity (MLI) was corrected per The 10 3 measured PMN +MMN.

Results Figures la and lb show that when compared to control subjects, 1) Resting CL from SLE patients did not significantly differ -1

m

10

-2

diluted blood whereas it was markedly reduced in 10

diluted blood (p