Biochemical and Biological Effects of Organotins 9781608053674

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Biochemical and Biological Effects of Organotins Editors

Alessandra Pagliarani, Fabiana Trombetti and Vittoria Ventrella University of Bologna-Department of Veterinary Medical Sciences, Italy

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MEMORIAL Professor Laszlo Nagy, one of the coauthors of Chapter 2 of this e- book, was born on 3rd October 1950 in Zámoly, Hungary. His career developed through the following steps: 

M.Sc. 1970-75 A. József University, Szeged



Ph.D. 1975-79 A. József University, Szeged



Candidate of Science 1989, Hungarian Academy of Sciences



D.Sc. 1996, Hungarian Academy of Sciences



Ph.D. Thesis: Decomposition of hydrogen peroxide catalyzed by osmium-tetraoxide



C.Sc. Thesis: Electronic structure of metal complexes formed with carbohydrates



D.Sc. Thesis: Metal complexes of carbohydrates and related compounds: equilibrium and structural studies

During his long teaching activity he spaced along the topics: 

Introductory course in analytical chemistry



Foundation laboratory course in inorganic chemistry



Advanced course in inorganic chemistry



Special course in bioinorganic chemistry

His scientific interests were devoted to metal complexes of biologically active molecules: carbohydrates, amino acid-carbohydrate adducts, nucleic acids, flavonoides etc. Thermodynamics of complex formation; Solution and solid state structure of complexes. EXAFS studies of metal complexes; Kinetic studies of oxidation of bioligands. During his scientific activity, he collaborated with many scientists in foreign laboratories in Argentina, Canada, Czech Republic, Germany, Japan, Italy, and Norway, many of which he visited for long periods, everywhere he was appreciated for his great human charge and scientific merits. He was the co-author of hundreds of publications, many of which in collaboration with the Bioinorganic group at the Chemistry Department “S. Cannizzaro”of University of Palermo. We regret that Prof Laszlo Nagy died, in Szeged, Hungary on February 24, 2011.

CONTENTS Foreword

i

Preface

ii

List of Contributors

iv

Abbreviations of Organotin Compounds

vii

CHAPTERS 1. Ecotoxicological Impacts of Organotins: An Overview

3

Toshihiro Horiguchi 2. Biological Activity of Organotin(IV) Compounds: Structural and Chemical Aspects

25

László Nagy, Claudia Pellerito and Lorenzo Pellerito 3. Covalent Interactions of Organotins with Nuclear Receptors

53

Felix Grün 4. Biomembrane Perturbation Induced by Organotin in Model and Biological Membranes

70

Giancarlo Falcioni 5. Organotins as Endocrine Disruptors: An Examination of Tributyltin-Induced Imposex in Neogastropods

75

Robin M. Sternberg 6. Lipid Homeostasis Perturbation by Organotins: Effects on Vertebrates and Invertebrates

83

Miguel M. Santos, Maria A. Reis-Henriques and Luis F. C. Castro 7. Genotoxicity and Immunotoxicity of Organotins

97

Francesca Cima and Loriano Ballarin 8. Organotins as Mitochondrial Toxins

112

Sabrina Manente, Alessandra Iero and Marcantonio Bragadin 9. Organotins and Hydromineral Homeostasis in Aquatic Animals

125

Mark G.J. Hartl 10. Mechanisms of Organotin-Induced Apoptosis

149

Željko Jakšić 11. Organotins and Humans: Threat and Risk

164

Asha Giriyan and Sangeeta Sonak 12. Organotin Effects in Different Phyla: Discrepancies and Similarities

174

Alessandra Pagliarani, Salvatore Nesci, Fabiana Trombetti and Vittoria Ventrella Index

197

i

FOREWORD The e-Book “Biochemical and biological effects of organotins” represents a due contribution and fills a gap in the scientific literature on environmental contaminants. Organotins are widespread contaminants, especially of water environments. Their chemically versatile structure makes organotin compounds able to bind to a variety of biomolecules, thus widely affecting biological functions. Especially trisubstituted species, namely tributyltin and triphenyltin, widely employed in the past in antifouling paints and still employed as plastic stabilizers, display unwanted harmful effects. Even if increasingly banned by European and non-European governments, due to their persistency and bioaccumulation, organotins still represent a global threat of unpredictable duration. The lipophilicity of the organic moiety not only favours organotin bioaccumulation in animal tissues but also facilitates their crossing plasma membranes and entering the cytoplasm. Once embedded in membrane lipid bilayers, organotins can act both at the plasma-membrane level and intracellularly by disrupting processes depending on compartmentalization. In its 12 chapters written by experts in their respective fields, this e-Book unravels the chemical properties and the biological and biochemical effects of organotins by a careful analysis of the most recent findings, with special focus on contaminant action mechanisms at cellular and molecular level in a variety of different organisms, including humans. The various hypothesized mechanisms at the cellular and molecular level are deeply reviewed in order to provide a quite exhaustive pattern of organotin toxicity as well as of their potential beneficial or therapeutic use. Such a comprehensive review on organotins was deeply needed: besides furnishing an up-dated and excellent overview of organotin effects, the e-Book provides a intriguing insight into the molecular mechanisms involved, thus representing a unique tool to biochemists, biologists, ecotoxicologists and life science students and researchers.

Giorgio Lenaz, MD Professor of Biochemistry University of Bologna, Italy

ii

PREFACE From the synthesis of the first organotin, in the mid of nineteenth century, and the amazing discovery of a wide spectrum of exploitations and industrial uses of these man-made chemicals in the subsequent century, the dark side of organotins increasingly came out in the second half of the twentieth century. Organotins now represent a matter of environmental concern, for the toxic effects displayed on a wide spectrum of organisms, from bacteria to humans, and for the global contamination, especially of water basins which act as reservoirs. Unfortunately, but realistically, contamination and worries are fated to persist, due to the environmental persistency and high bioaccumulation potential of these pollutants. In spite of the wealth of studies, the intimate mechanisms of the toxic action of organotin compounds are not entirely established and our knowledge needs to be continuously reviewed and updated. This e-book aims at gathering the emerging data on organotin effects on animals, with special focus on their biochemical interactions with biomolecules, on which toxicity mechanisms are often founded. Chapter by chapter, the biological and biochemical effects of organotins are unravelled by a careful analysis of the most recent findings, paying special attention to the contaminant action mechanisms at cellular and molecular level. In the first chapter, the legislation on organotin-based antifouling paints and organotin contamination levels of aquatic environments are carefully reviewed. Special attention is paid to the most known toxic effect, namely imposex, which is a irreversible superimposition of male-type genitalia in female gastropod mollusks. Imposex mirrors environmental concentrations of the most toxic organotins. The structural and chemical properties of toxic organotin compounds are carefully examined in Chapter 2, with special focus on their biological and pharmacological activities. In Chapter 3 the recent discovery of direct high affinity interactions between trialkyltins and the family of nuclear hormone receptors which function as ligand dependent transcription factors for lipophilic endocrine hormones in vertebrates and invertebrates is presented. Studies on the interactions of organotins with model and natural biological membranes are reported in Chapter 4. The ability of organotins to act as endocrine-disrupting chemicals is considered in Chapter 5, focusing on TBTinduced imposex in neogastropods. Chapter 6 embraces the most recent knowledge on the molecular and biochemical mechanisms involved in lipid homeostasis disruption within several metazoan phyla, from mollusks to amphibians, teleosts and mammals. In Chapter 7 the organotin-driven genotoxicity and immunotoxicity, which among toxic effects are the most important in affecting survival, are reviewed in mammals, fish and aquatic invertebrates. Effects on mitochondria, one of the main targets of organotins are considered in Chapter 8. The impairment of mitochondrial functions and the consequent ATP synthesis depletion may play a crucial role as molecular basis of several toxic effects of organotins. Chapter 9 reviews the impact of organotin exposure on fresh- and seawater organisms of various phyla by examining the histophathological, physiological and molecular interactions of organotin compounds with relevant enzymes, membranes, the endocrine system, and the consequential ramifications for individuals, populations and community structure in aquatic ecosystems. Recent research on the mechanisms of organotin-induced apoptosis is reviewed in Chapter 10. The modulation of apoptosis, involved in ontogenesis, development and cell turnover, is a critical step of cellular toxicity. Chapter 11 reviews the impacts of organotins on humans and presents major applications of organotins, their entry in the food chain, human exposure and effects of organotins.

iii

Once overviewed the broad susceptibility to organotins in literature data, Chapter 12 underlines the astonishing analogies of the toxic action of these chemicals in animal kingdom on the one hand and on the other, the variegated toxicity displayed in different cells, tissues and species. The long work to combine different competences as well as the complementary approach to intersecting topics mirror different standpoints and skills and provide an updated knowledge on organotin toxicity mechanisms, other than a significant contribution to detect the directions in which current research on organotins is moving. We want to dedicate this e-book in the memory of our unforgotten biochemist Prof. Romano Viviani, who taught us that biochemistry can be a helpful tool to understand biological phoenomena. Preparing this e-book was a sort of collective adventure which gave us the opportunity to increase our reciprocal knowledge. We are most grateful to all contributors for their competent efforts and patience in the editing process. We would like to thank Prof Giorgio Lenaz for writing the foreword, and, last but not least, Bentham Science Publishers, particularly the Assistant Manager Asma Ahmed, for the continuous assistance and support.

iv

List of Contributors Ballarin Loriano Department of Biology, University of Padova, Via U.Bassi 58/ B, 35121 Padova, Italy. E-mail: [email protected]

Bragadin Marcantonio Department of Environmental Sciences, Cà Foscari University, Calle Larga S. Marta 2137, Dorsoduro 30123 Venice, Italy. E-mail: [email protected]

Costa Castro Luis Filipe Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. E-mail: [email protected]

Cima Francesca Department of Biology, University of Padova, Via U.Bassi 58/ B, 35121 Padova, Italy. E-mail: [email protected]

Falcioni Giancarlo School of Pharmacy and Health Products, University of Camerino, 62032 Camerino, MC, Italy. E-mail: [email protected]

Giriyan Asha The Energy and Resources Institute (TERI), Western Regional Centre, Bambolim – Goa, India. E-mail: [email protected]

Grün Felix Center for Complex Biological Systems, University of California, Irvine, California 92697-2300 US. E-mail: [email protected]

Hartl Mark Centre for Marine Biodiversity and Biotechnology, School of Life Sciences, Heriot-Watt University Riccarton, EH14 4AS Edinburgh, Scotland UK. E-mail: [email protected]

Horiguchi Toshihiro Center for Environmental Risk Research, National Institute for Environmental Studies 16-2, Onogawa, Tsukuba, Ibaraki, 305-8506, Japan. E-mail: [email protected]

Iero Alessandra Institute of Applied Ecology, Health Science and Design, University of Camberra, Bruce, ACT, 2601 Australia. E-mail: [email protected]

Jakšić Željko Ruđer Bošković Institute, Center for Marine Research G. Paliage 5, 52210 Rovinj, Croatia. E-mail: [email protected]

v

Manente Sabrina Department of Environmental Sciences, Cà Foscari University, Calle Larga S. Marta 2137, Dorsoduro 30123 Venezia, Italy. E-mail: [email protected]

Nagy László Department of Inorganic and Analytical Chemistry, University of Szeged, H-6701, Szeged, P.O. Box 440, Hungary. E-mail: [email protected]

Nesci Salvatore Department of Biochemistry “G. Moruzzi”, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, BO, Italy. E-mail: [email protected]

Pagliarani Alessandra Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, BO, Italy. E-mail: [email protected]

Pellerito Claudia Department of Analytic and Inorganic Chemistry "Stanislao Cannizzaro", University of Palermo, Viale delle Scienze, Parco d’, Orleans, 90128 Palermo, Italy. E-mail: [email protected]

Pellerito Lorenzo Department of Analytic and Inorganic Chemistry "Stanislao Cannizzaro", University of Palermo, Viale delle Scienze, Parco d’, Orleans, 90128 Palermo, Italy. E-mail: [email protected]

Reis-Henriques Maria Armanda CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. E-mail: [email protected]

Sonak Sangeeta Centre for Natural Resource Management, Srujan, A-G/26, Kamat Arcade, Caranzalem, Goa, 403002, India. E-mail: [email protected]

Santos Miguel Machado CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, Laboratory of Environmental Toxicology, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. E-mail: [email protected]

Sternberg Robin Michelle United States Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention Office of Science Coordination and Policy 1200 Pennsylvania Ave. NW (7203M) Washington, DC 20460. E-mail: [email protected]

Trombetti Fabiana Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, BO, Italy.

vi

E-mail: [email protected]

Ventrella Vittoria Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, BO, Italy. E-mail: [email protected]

vii

Abbreviations of Organotin Compounds A ACT

azocyclotin (Peroral)

B BTs

butyltins

D DBT DBTC DBTSCN DET DMPhT DMT DOT DOTC DPhT DPT

dibutyltin dibutyltin dichloride dibutyltin isothiocyanate diethyltin dimethylphenyltin dimethyltin dioctyltin dioctyltin chloride diphenyltin dipropyltin

F FBTO

fenbutatin oxide (Lexitin, Torque)

M MBT MBTC MDPhT MET MMT

monobutyltin monobutyltin trichloride methyldiphenyltin monoethyltin monomethyltin

O OTC

organotin compounds

T TBT TBTA TBTB TBTC TBTF TBT-MMA TBTN TBTO TCT TET TeET THT THTC TMT TMTC TOT

tributyltin tributyltin-acetate tributyltin benzoate tributyltin chloride tributyltin fluoride tributyltin methyl methacrylate copolymer tributyltin naphthenate bis-(tributyltin) oxide tricyclohexyltin (Cyhexatin, Plictran) triethyltin tetraethyltin trihexyltin trihexyltin chloride trimethyltin trimethyltin chloride trioctyltin

viii

TOTC TPhT TPhTA TPhTC TPhTH TPT

trioctyltin chloride triphenyltin triphenyltin acetate (Fentin acetate, Brestan) triphenyltin chloride (Fentin chloride) triphenyltin hydroxide (Fentin hydroxide, Super Tin, Duter) tripropyltin

Biochemical and Biological Effects of Organotins, 2012, 3-24

3

CHAPTER 1 Ecotoxicological Impacts of Organotins: An Overview Toshihiro Horiguchi* National Institute for Environmental Studies, Japan Abstract: The legislation on organotin-based antifouling paints, including the International Convention on the Control of Harmful Anti-fouling Systems on Ships (AFS Convention) is here summarized. Concentrations of TBT and TPhT in the marine environment and toxicities of TBT and TPhT to mainly marine organisms are also overviewed, as is the relevant legislation. As one of the most typical toxicological effects of TBT and TPhT, imposex in gastropod mollusks is reviewed. Imposex is a superimposition of male-type genitalia (penis and vas deferens) in females and is considered an irreversible pseudohermaphroditic condition. It is typically induced by very low concentrations (~1 ng/L) of TBT, TPhT, or both. Reproductive failure occurs in the severe stages of imposex, either because of oviduct blockage by the formation of vasa deferentia or because of ovarian disorder (spermatogenesis as well as suppressed production of matured oocytes), and eventually results in population decline or mass extinction. Globally, approximately 200 species of mesogastropods and neogastropods are affected by imposex. Imposex among gastropods has been recognized as a clear manifestation of endocrine disruption. Five main hypotheses of the mechanisms by which organotins induce imposex in gastropods have been proposed: (1) an increase in androgen (e.g., testosterone) levels due to TBT-mediated inhibition of aromatase; (2) TBT-mediated inhibition of the excretion of androgen sulfate conjugates; (3) TBT interference in the release of penis morphogenetic/retrogressive factor from the pedal/cerebropleural ganglia; (4) an increase in the level of alanine-proline-glycine-tryptophan (APGW)amide neuropeptide in response to TBT; and (5) involvement of the retinoid X receptor (RXR), a nuclear receptor. Each hypothesis is critically reviewed.

Keywords: AFS Convention (International Convention on the Control of Harmful Anti-fouling Systems on Ships), bioconcentration factor- ADI (acceptable daily intake)- imposex, aromatase inhibition- APGW amide (alanineproline-glycine-tryptophan amide)- RXR (retinoid X receptor)- 9CRA (9-cis retinoic acid). POLLUTION BY ORGANOTIN (BUTYLTIN AND PHENYLTIN) COMPOUNDS FROM ANTIFOULING PAINTS IN THE MARINE ENVIRONMENT: A SUMMARY OF THE HISTORY OF POLLUTION AND LEGISLATION LEADING TO THE AFS CONVENTION More than 800 organotin compounds are known, and most of them are of anthropogenic origin, with the exception of methyltins, which can also be produced by biomethylation [1]. There are a larger number of organotin derivatives in commercial use. An increase in the variety of commercial applications markedly increased the worldwide production of organotin compounds from less than 5.000 tons (t) in 1955 to about 50.000 t in 1992 [1]. The major application of organotin compounds (approximately 70 %) is the use of mono- and di-alkyltin derivatives as heat and light stabilizer additives in polyvinyl chloride (PVC) processing [1]. It is well known that mainly tri-substituted organotin species have biocidal properties [2, 3]. Therefore, these tri-substituted organotins have been used as fungicides, miticides, molluscicides, nematocides, ovicides, rodent repellants, wood preservatives, and antifouling paints, primarily containing TBT, TPhT, and TCT as toxic additives. Biocidic products make up about 20% of the total annual organotin production [1, 4]. TBT is the main organotin species used in antifouling paints worldwide. In Japan, however, TPhT as well as TBT was used in antifouling paints for vessels and fishing nets from the mid-1960s to 1989 [5]. The total annual production and import of TBT and TPhT in Japan was 6 340 t in 1989; approximately 70% was used in antifouling paints for vessels and 20% as antifouling for fishing nets [5]. The rest was used for agriculture, wood preservation, and other industrial purposes in Japan [5]. As a consequence of this worldwide use of TBT- or TPhT-based antifouling paints on vessels and fishing nets, contamination of the aquatic environment by these compounds became a concern. There are many reports on the *Address correspondence to Toshihiro Horiguchi: Center for Environmental Risk Research, National Institute for Environmental Studies 162, Onogawa, Tsukuba, Ibaraki, 305-8506, Japan; E-mail: [email protected]

4 Biochemical and Biological Effects of Organotins

Toshihiro Horiguchi

levels of contamination by TBT and TPhT, including their metabolites, detected in the aquatic environment (e.g., a review by Maguire [6]). For example, TBT at over 1 µg/L has been detected in freshwater and seawater near marinas, harbors, and shipyards where severe contamination by antifoulants released from ships’ hulls and old paint stripped from the hulls by surface blasting with water or abrasive slag fines has been observed [6, 7]. A number of papers have also reported butyltin and phenyltin contamination in aquatic invertebrates and vertebrates (e.g., Alzieu [8]; Takeuchi [9]). One study detected TBT at 750 ng/g and TPhT at 1 770 ng/g (wet wt. basis) in the soft tissues of rock shell (Thais bronni) collected at Aburatsubo, Japan, in 1990 [5]. Legislation on tri-organotin-based antifouling paints started in France in 1982, the U.K. in 1987, and the U.S.A. in 1988: Vessels shorter than 25 m (excluding those made from aluminum) were prohibited from using tri-organotinbased antifouling paints [10, 11]. Vessels longer than 25 m were permitted to use tri-organotin-based antifouling paints if the maximum release rate of tri-organotin was less than 4 µg cm -2 day-1 [10, 11]. Similar legislation was introduced in Canada, Australia, and New Zealand in 1989 [10]. Environmental quality standards (EQSs) were established for TBT and TPhT in the U.K. in 1989: 20 ng/L for both TBT and TPhT in freshwater and 2 ng/L (TBT) and 8 ng/L (TPhT) in seawater [10, 12]. Ambient water quality criteria were also established for TBT in the U.S.A. in 1988 [13]. In Japan, the regulatory system for TBT and TPhT compounds is different from those in other countries, such as the U.K. and U.S.A. Since 1990, regulations for TBT and TPhT compounds have been implemented for each chemical species of TBT and TPhT in accordance with the law concerning the Examination and Regulation of the Manufacture etc. of Chemicals in Japan. Since January 1990 the manufacture, import, and use of TBTO has been completely prohibited by law. As of September 1990, however, other TBT (13 substances, including TBT-MMA) and TPhT (seven substances) compounds were allowed to be used, manufactured, or imported if their expected amounts were reported to the Ministry of International Trade and Industry (MITI). Although it was permissible to use TBT- or TPhTformulated antifouling paints on fishing nets and on any kind of ship or boat (including those shorter than 25 m) at that time, the sale of TPhT products in the Japanese domestic market had essentially ceased in June 1989 under the administrative guidance of MITI. However, administrative guidance by Ministries and Agencies of the Government imposes no penalties and therefore differs from legal regulation. Such guidance systems are typical of regulatory systems in Japan. Similarly, the manufacture, import, and use of TBT compounds (excluding TBTO) had also been controlled by the administrative guidance of MITI, the Ministry of Transport, and the Government’s Fisheries Agency from July 1990, but the mass media reported that the sale of TBT products to the Japanese domestic market had completely ceased by April 1997. No ambient water quality criteria have been established for TBT and TPhT in Japan. Meanwhile, in other Asian countries, for example, in Korea since March 2000 the use of antifoulants that contain triorganotins at more than 0.1% has been prohibited on fishing nets and on small vessels (including fishing boats) using coastal waters and harbor facilities (Cho et al., personal communication). The efficacy of organotin legislation has been examined in many countries on the basis of improvement of water quality—namely, a decrease in the concentrations of TBT in water, sediment, and biota—and recovery of the marine ecosystem. Although water quality improvements have been reported for Europe, the U.S.A., Canada, and Australia, there are also reports of no improvement or little improvement in several areas of Europe, Canada, and Australia (for example, Maguire [6]; Batley [7]; Stewart [10]; Rato et al. [14]; Horiguchi et al. [15]; Gibson and Wilson [16]). On the other hand, in Asian countries such as Japan and Korea, trends in water quality have not improved. In a nationwide survey of imposex and contamination by TBT and TPhT in the rock shell Thais clavigera collected between January 1999 and November 2001 from 174 locations along the Japanese coast, imposex was observed at 166 of the locations, whereas no, or rare, instances were found at the remaining eight sites. The percentage occurrence of imposex was still as high as, or close to, 100% at approximately half of the locations surveyed. On the basis of the relationships among relative penis length (RPL) index, vas deferens sequence (VDS) index, and the percentage occurrence of oviduct blockage (i.e. by vulval blockage) in females (i.e., sterile females), it is expected that spawning obstruction will occur in more than half the population of females when the (RPL) index exceeds 40 [17]. RPL index values exceeding 40 were found at 41 of the 174 locations. High RPL and VDS indices were generally observed in the western part of Japan. Compared with the results of a survey conducted previously (from 1996 to 1999), these indices seemed to have decreased but they remained almost unchanged in some locations [17]. TPhT concentrations in the tissues of the rock shell showed a decrease over time but varied among locations, with relatively high pollution levels in a few locations. Decreases in TBT concentrations were also apparent, but the

Ecotoxicological Impacts of Organotins

Biochemical and Biological Effects of Organotins 5

degree of decrease was lower than in the case of TPhT. Changes in TBT concentrations over time were not observed in several locations, and increases were observed at two locations near fishing ports [17]. In the first imposex survey conducted in Korea (1995-1997), the percentage occurrence of imposex in T. clavigera populations was 100% at nearly all sites surveyed. Observed RPL indices were generally high, and 60% or more of the females in the eastern South Sea were sterile. No sterile females were observed in open-sea areas. In the second imposex survey in 2001-2002, the percentage occurrence of imposex in T. clavigera populations was still 100% at most of the sites surveyed along the South Sea coast, whereas the corresponding values in T. clavigera populations from the Jeju coast were in the range of 0% to 100%. The geographical distributions of RPL index values and the incidence of sterile individuals along the South Sea coast were similar to those in the first survey. No sterile females were found at five of eight sites in Jeju. Concentrations of butyltins, including TBT, in the tissues of T. clavigera collected in 2002 were higher than those in the 1995-1997 survey. Despite legislation banning the use of TBT as an antifoulant, these results indicated that there had been no recovery from imposex in T. clavigera populations in Korea, suggesting the possible continued illegal use of TBT (Cho HS, Chonnam National University, et al. in preparation). To introduce effective international regulation of the use of tri-organotin-formulated antifoulants, a first proposal was made at the 29th Session of the Marine Environment Protection Committee (MEPC 29) of the International Maritime Organization (IMO) in March 1990. Following MEPC 29, a resolution on measures to control potential adverse impacts associated with the use of TBT compounds in antifouling paints was adopted at MEPC 30 in November 1990. The resolution indicates that MEPC agrees to: 1) Recommend that Governments adopt and promote effective measures within their jurisdictions to control the potential for adverse impacts on the marine environment associated with the use of TBT compounds in antifouling paints and that, as an interim measure, Governments specifically consider the following actions: a)

Eliminate the use of antifouling paints containing TBT compounds on non-aluminum-hulled vessels shorter than 25 m;

b)

Eliminate the use of antifouling paints containing TBT compounds that have average organotin release rates of more than 4 µg cm -2 day-1;

c)

Develop sound management practice guidelines applicable to ship maintenance and construction facilities to eliminate the introduction of TBT compounds into the marine environment as a result of painting, paint removal, cleaning, sandblasting, or waste disposal operations, or in run-off from such facilities;

d)

Encourage the development of alternatives to antifouling paints containing TBT compounds, giving due regard to any alternative formulations; and

e)

Engage in monitoring to evaluate the effectiveness of the control measures adopted and provide for the sharing such data with other interested parties.

2) Consider appropriate moves toward the possible future total prohibition of the use of TBT compounds in antifouling paints for ships [18]. Unfortunately adoption of the resolution at MEPC 30 in November 1990 did not result in the establishment of a new treaty or convention toward the total prohibition of the use of TBT compounds in antifouling paints for ships. At MEPC 38 in July 1996, Japan, the Netherlands, and some Northern European countries then proposed the need to establish a new treaty or convention aimed at worldwide total prohibition of the use of tri-organotin-formulated antifoulants, such as TBT compounds. In this proposal they took into account the temporal trends in contamination by TBT and TPhT in the marine environment, the adverse effects of TBT and TPhT as endocrine-disrupting chemicals, and the current status of development of alternatives to antifouling paints containing TBT compounds. MEPC set up a Correspondence Group for the Reduction of Harmful Effects of the Use of Antifouling Paints for Ships (chaired by the Netherlands) for the investigation. The results of the investigation performed by the Group were considered at MEPC 41 in March 1998. Finally, at its assembly in November 1999, the IMO decided to phase out TBT in antifouling paints over the period from 2003 to 2008. An International Convention on the Control of Harmful Anti-fouling Systems on Ships (AFS Convention: 21 Articles) was then adopted by the IMO on 5 October 2001 [19]. According to the AFS

6 Biochemical and Biological Effects of Organotins

Toshihiro Horiguchi

Convention, all ships shall not apply or re-apply organotin compounds that act as biocides in antifouling systems after 1 January 2003, and all ships either (1) shall not bear organotin compounds that act as biocides in antifouling systems on their hulls or external parts or surfaces, or (2) shall bear a coating that forms a barrier to such compounds leaching from the underlying non-compliant antifouling systems after 1 January 2008. Because it had taken more time than expected for the AFS Convention to be ratified by member states, it finally came into force on 17 September 2008 (http://www.imo.org/). Continued monitoring is needed for marine/aquatic ecosystems to recover from the impacts of organotin pollution and to protect the marine/aquatic environment. TOXICITIES OF ORGANOTIN (BUTYLTIN AND PHENYLTIN) COMPOUNDS TO AQUATIC ORGANISMS Organotin-based antifouling paints are very effective in preventing the settling of barnacles, oysters, and other sessile organisms on the hulls of vessels and the surfaces of fishing nets. This effectiveness, however, means that organotin compounds, such as TBT and TPhT, are strongly toxic to aquatic organisms. There are numerous papers and several reviews on the toxicities of TBT and TPhT to aquatic invertebrates and vertebrates (e.g., reviews by Alzieu [8]; Hall and Bushong [20]). Lethal, developmental, behavioral, and reproductive toxicities as well as other various kinds of physiological toxic effects have been reported in aquatic invertebrates and vertebrates. For example, toxicity of TBT and TPhT to the embryonic and larval stages of aquatic invertebrates has been detected at a few micrograms per liter or even lower concentrations [21, 22]. Imposex and intersex in neo- and mesogastropods—an irreversible syndrome of masculinization of female gastropod mollusks (see below), leading to sterility in severe cases—is induced by TBT (or even by TPhT in some species) at a few nanograms per liter or even lower concentrations (e.g., Smith [23]; Bryan et al. [24]; Gibbs and Bryan [25]; Gibbs et al. [26]; Horiguchi et al. [27]). The persistence and fate of TBT and TPhT have also been well studied (e.g., reviews by Seligman et al. [28] and Kawai and Harino [29]). TBT and TPhT in marine, estuarine, and freshwater environments are decomposed mainly by microbial degradation; photolysis and chemical degradation are not significant in the degradation of TBT [28]. Although TBT and TPhT are not highly persistent in freshwater and marine environments, they are fairly persistent in sediments, especially under anaerobic conditions. Because their half-lives can be several months or longer, contaminated sediments may act as secondary sources of these compounds [28, 30]. The accumulation of TBT and TPhT (including their metabolites) as well as TBT and TPhT concentration and magnification factors, the metabolism of TBT and TPhT by aquatic organisms, and the tissue distributions of these compounds in aquatic organisms have been reported and reviewed (e.g., Bock [31]; Laughlin [32]; Roberts et al. [33]; Lee [34]; Yamada and Takayanagi [35]; Suzuki et al. [36]; Matsudaira et al. [37]; Oshima et al. [38]; Horiguchi et al. [39]). Laboratory experiments conducted over a few months in flow-through exposure systems indicate that bioconcentration factors of TBT by marine organisms are more than 10 000, whereas those of TPhT by marine fish are less than 10,000 [35, 40, 41]. However, high concentrations of TPhT have been detected in marine organisms (fishes, crustaceans, mollusks, and algae) collected in coastal waters where TPhT concentrations in seawater are below the detection limit [42]. In fact, the ability to metabolize TPhT seems to be generally low in many kinds of organism, although the ability to metabolize TBT differs among species [31, 36, 39, 40, 43-45]. Therefore, TPhT is considered to be concentrated via the food web in aquatic ecosystems [41, 46-48]. Biomagnification factors of TPhT by aquatic organisms, however, are estimated to range from 2 to 3; this is lower than those of PCBs [46-48]. The biological half-life of TBT was estimated at 22 days and the ecological half-life of TPhT was estimated at 347 days in the rock shell T. clavigera [59]. The biological half-life of TBT was estimated to be between about 50 to more than 100 days in the dog-whelk Nucella lapillus, depending on the conditions [40]. In the ivory shell Babylonia japonica, higher tissue burdens of TBT and TPhT have been observed in the reproductive organs (ovary, oviduct, and testis) and head (including the central nervous system ganglia) as well as in the muscle and tissues adjacent to the mantle (such as the ctenidium, siphon, and heart) [49]. A similar accumulation pattern has also been observed in T. clavigera [39], whereas a slightly different pattern has been found in Ocenebra erinacea in which approximately half of the total body TBT burden accumulated in the capsule gland [50], suggesting a difference in organotin accumulation patterns among species. Although concentrations of TBT and TPhT in the ganglia were quite high in T. clavigera, the total tissue burden of those organotins was not high because of the small amount of ganglia tissue in that species [39]; this may also be the case with B. japonica. Similar concentrations of TBT and TPhT have been detected in the ganglia of the European whelk Buccinum undatum [51].

Ecotoxicological Impacts of Organotins

Biochemical and Biological Effects of Organotins 7

Because TBT and TPhT are also very toxic to mammals, acceptable daily intake (ADI) values have been designated at 1.6 µg kg -1 (body wt.) day-1 for TBT and 0.5 µg kg -1 (body wt.) day-1 for TPhT [2, 52]. IMPOSEX IN GASTROPOD MOLLUSKS: AN ENVIRONMENTAL ISSUE CAUSED BY ORGANOTIN POLLUTION One of the typical adverse effects caused by organotin compounds, such as TBT and TPhT, in aquatic organisms is imposex, represented by masculinization of females, in prosobranch gastropod mollusks. There have been numerous reviews of imposex caused by organotin compounds in prosobranch gastropods (e.g., Gibbs and Bryan [53]; Gibbs and Bryan [54]; Horiguchi [55]; Horiguchi [56]). The first report of masculinized female gastropods was made by Blaber [57] who described a penis-like outgrowth behind the right tentacle in spent females of the dog-whelk N. lapillus around Plymouth, U.K. The term imposex, however, was coined by Smith [23] to describe the syndrome of superimposition of male-type genital organs, such as the penis and vas deferens, on female gastropods. Imposex is thought to be irreversible [24]. Reproductive failure may occur in females with severe imposex, resulting in population decline or even mass extinction [25, 53, 54]. In some species, imposex is typically induced by TBT and TPhT from antifouling paints used on ship hulls and fishing nets [26, 27, 40, 58, 59]. As of 2005, approximately 200 species of mesogastropods and neogastropods had been reported to be affected by imposex worldwide [60-68]; many of these gastropod species belong to the families Muricidae (e.g., N. lapillus, O. erinacea, T. clavigera, and Urosalpinx cinerea), Buccinidae (e.g., B. japonica, B. undatum, and Neptunea arthritica arthritica), Conidae (e.g., Conus marmoreus bandanus and Virroconus ebraeus), and Nassariidae (e.g., Ilyanassa obsoleta and Nassarius reticulatus) of the Neogastropoda [62, 63]. Numerous studies have examined the incidence or severity of imposex, investigated the use of certain gastropod species as biological indicators of TBT contamination, and surveyed TBT contamination using gastropods [e.g., 14-17, 26]. Only a few reports, however, have presented evidence, based on either morphological or histological methods, for population-level effects of reproductive failure due to imposex [5, 24, 25, 49, 50, 69, 70-74]. Below, a case study [49] of imposex and population decline in the ivory shell Babylonia japonica (Reeve, 1842), one of the species targeted by commercial fisheries in Japan, is presented. Effects of Organotin Pollution on Gastropods at the Population Levels Collapse of commercial fisheries for B. japonica (Neogastropoda: Buccinidae) was observed in Japan, Fig. (1), and an ecotoxicological study was conducted to examine whether reproductive failure caused by imposex had brought about the drastic population decline [49]. B. japonica, which inhabits sandy or muddy sediments in shallow water (approximately 10-20 m depth) from the south of Hokkaido to Kyushu, Japan, is a scavenger in inshore ecosystems and has traditionally been targeted by commercial fisheries in Japan. Imposex seems to have been observed in B. japonica since the 1970s (Kajikawa A. (Tottori Prefectural Fisheries Experimental Station), personal communication), and the total catch decreased dramatically all over Japan in the late 1970s or early 1980s [75]. Much effort has been made to enhance the ivory shell stocks. Seed has been produced using adult ivory shells reared in hatcheries, with subsequent release of seeds/juveniles into the sea. Most of the ivory shell seeds/juveniles released into the sea (approximately 90% of the total production in Japan) has been produced at a hatchery in Tomari, Tottori Prefecture, western Japan [49]. In Tottori Prefecture, however, not only the total catch but also the numbers of egg capsules spawned by adult shells at the hatchery and the numbers of seeds/juveniles artificially produced/released into the sea has decreased since the mid-1980s [49], Fig. (1). The total catch drastically decreased after 1984, 2 years after the first observation of imposex-affected female ivory shells in Tottori Prefecture; this involved an increase in both the percentage occurrence of imposex individuals and mean penis length in females [76-79], Fig. (1). Introduction of adult ivory shells from another prefecture (Niigata Prefecture, Japan) to compensate for insufficient numbers of the normal brood stock since 1992 also has resulted in failure of the release of seeds/juveniles into the sea owing to their high mortality at the hatchery before release, Fig. (1). Recovery of the total catch of the ivory shell was not observed, despite such efforts to enhance ivory shell stocks. Finally, operation of the ivory shell hatchery for stock enhancement in Tottori was stopped, and the hatchery was closed in 1996, Fig. (1). Reproductive failure caused by imposex in the ivory shell was suspected as the reason for the failure of the population to recover.

8 Biochemical and Biological Effects of Organotins

Toshihiro Horiguchi

60 Egg capsule weight (g) 50

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Figure 1: Temporal trends in average weight of egg capsules spawned by adult ivory shell, Babylonia japonica, at the Tottori hatchery, as well as numbers of seeds/juveniles released into the sea and the total catch off Tottori Prefecture, Japan.

In a previous study, I and others [49] examined the incidence of reproductive failure accompanied by imposex in B. japonica on the basis of histopathological observations of gonads. We investigated the relationship between organotin compounds and imposex in B. japonica on the basis of chemical analysis of organotin concentrations in the tissues of B. japonica. The occurrence of imposex in B. japonica specimens collected in a representative declining population in Tottori Prefecture, western Japan, was 82.6% from December 1988 to November 1989 and 88.9% in June 1991. Both the penis and the vas deferens were found to be well developed in imposex-exhibiting females [49]. No oviduct blockage (i.e., occlusion of the vulva) by vas deferens formation, however, was observed in imposex-exhibiting female B. japonica [49], a finding that differs from the imposex symptoms observed in N. lapillus, Ocinebrina aciculata, and T. clavigera [5, 25, 26, 73]. Histological examination of the gonads showed that ovarian maturation seemed to be suppressed in females, unlike testicular maturation in males, Fig. (2), although the fact that the spawning season of B. japonica is late June to early August may have partly explained the presence of immature females throughout the spawning season [49, 79]. However, during the spawning season, clearer ovarian maturation and spawning of much more egg capsules than in the Tottori population were observed in B. japonica females in a population from a reference site at Teradomari, Niigata Prefecture, Japan [80-82]. Testicular maturation in males from Tottori was clear in July and August, the spawning season for B. japonica [49], Fig. (2). Thus, the reproductive cycle was unclear in females but was clearly observed in males [49], Fig. (2). The suppressed ovarian maturation during the spawning season could be the direct reason for the decreased number of egg capsules spawned by adult B. japonica at the hatchery and might accompany imposex in B. japonica [69]. Ovarian spermatogenesis (i.e., ovo-testis formation) was observed in six (one normal female and five imposex individuals) of 92 female or imposex B. japonica specimens examined—a frequency of about 6.5% [49]. It is well known that most prosobranch gastropods (including B. japonica) are dioecious, although there are few hermaphroditic prosobranchs in which the gonad produces eggs and sperm simultaneously [83, 84]. Ovarian spermatogenesis has been observed in neogastropods (e.g., N. lapillus, O. aciculata, and T. clavigera) and archaeogastropods (e.g., Haliotis madaka and Haliotis gigantea) exposed to TBT or TPhT, although no penis formation is involved in spermatogenesis by the ovaries of female abalone [69, 73, 75, 85-87].

Ecotoxicological Impacts of Organotins

Biochemical and Biological Effects of Organotins 9

5

4

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0

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Jan. 1989

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Figure 2: Reproductive cycle of the ivory shell (Babylonia japonica) in 1989, represented by population reproductive development scores. Female (F) reproductive cells were scored on the basis of five categories, and those of males (M) were based on four categories. The female curve includes imposex-exhibiting females [49].

Ovarian spermatogenesis was even observed in a normal female B. japonica without any penis or vas deferens formation, although the frequency was low (one of six, 16.7%). The development of male-type genital organs (penis and vas deferens) and ovarian spermatogenesis in females exposed to TBT or TPhT might be controlled through different physiological pathways. This ovarian spermatogenesis may be one of the reasons why the spawning ability of female B. japonica decreased [49]. Tissue concentrations of organotin compounds, such as butyltins and phenyltins, were determined by gas chromatography with flame photometric detection (GC-FPD), and different tissue distributions were observed in different tissues [49], Fig. (3). Marked accumulation of TBT was observed in the ctenidium, osphradium, and heart in both males and females, whereas the highest concentrations of TPhT were detected in the ovaries of females, Fig. (3) and the digestive glands of males [49]. On the basis of the total body burden of TBT in B. japonica, more than one-third of the total TBT accumulated in the digestive glands of both males and females, followed by the testis, ctenidium, muscle, and heart in males and the muscle, ovary, ctenidium, and head (including the central nervous system ganglia) in females [49]. On the basis of the total body burden of TPhT, approximately three-quarters accumulated in the digestive glands of males and more than one-half in those of females. The second-highest tissue burden of TPhT was observed in the gonads of both males and females, followed by the muscle, ctenidium, and heart in males and the muscle, oviduct, and head in females [49]. Mortality of larvae and seeds or juveniles might also be due to the accumulation of TPhT and TBT in the ovaries as well as the contamination of seawater with TPhT or TBT [88-94]. A survey of imposex and organotin concentrations in the tissues of T. clavigera [5] revealed that levels of contamination with TBT and TPhT were relatively high along the coast of Tottori Prefecture, especially in Miho Bay, where the B. japonica specimens used in this study were collected, compared to those in other sites of Japan.

10 Biochemical and Biological Effects of Organotins

Toshihiro Horiguchi

1868.1 1000 900 MBT

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Figure 3: Tissue distributions of organotin compounds in ivory shell (Babylonia japonica) from Yodoe, Tottori, Japan (June 1991): top, butyltins in females (including imposex individuals); bottom, phenyltins in females (including imposex individuals) [49]. MPhT: monophenyltin

Concentrations of TBT and TPhT were high in the ovaries of females [49], Fig. (3). Both TBT and TPhT concentrations in the gonads were positively correlated with penis length in females [49] (Fig. 4), as was the case with T. clavigera [5, 95]. Laboratory experiments revealed that both TBT and TPhT induced or promoted the development of imposex in T. clavigera [59, 27]; therefore, imposex could be caused by TBT or TPhT in B. japonica as well. However, laboratory flow-through exposure experiments with B. japonica, using TBT and TPhT, are needed to estimate the threshold concentrations for the development of imposex. The estimated threshold concentration of TBT (in whole body tissues) inducing the development of imposex has been reported to be

Ecotoxicological Impacts of Organotins

Biochemical and Biological Effects of Organotins 11

approximately 20 ng Sn/g dry wt. (corresponding to approximately 10 to 12.5 ng TBT/g wet wt., assuming that the concentration on a dry-weight basis is four to five times that on a wet-weight basis) in N. lapillus [26]; in T. clavigera it is estimated at 10 to 20 ng/g wet wt. [5]. Because there are limited experimental and analytical data for B. japonica, it is difficult to compare the sensitivities to TBT and TPhT between B. japonica and other gastropod species, such as N. lapillus, O. erinacea, U. cinerea, and T. clavigera [5, 26, 40, 50, 59, 70]. 16 14

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Figure 4: Relationship between triorganotin (sum of TBT and TPhT) concentrations in gonads and penis length in female Babylonia japonica [49].

Previously, I and others [49] discussed the possibility that the marked decline in B. japonica populations in Japan could have been brought about mainly by reproductive failure accompanied by imposex induced by TBT and TPhT from antifouling paints. The planktonic stage of B. japonica is estimated to last approximately 4 or 5 days [76, 77]. This means that recruitment of veliger larvae from other populations inhabiting remote less-contaminated areas is unlikely. Reproductive failure accompanied by imposex in females could result in extirpation of the B. japonica population within several years because the number of offspring produced by adult B. japonica in the population is likely to continue to decrease. The existence and duration of a free-swimming phase during larval development is an important factor in determining the linkage between impaired reproductive ability caused by imposex and population decline [24, 25, 49, 50, 69, 70]. In conclusion, reproductive failure (suppressed ovarian maturation and ovarian spermatogenesis) in adult females with imposex, possibly induced by TBT or TPhT from antifouling paints, could have brought about the marked decline in B. japonica populations that has been observed. Mechanism of Development of Imposex Caused by Organotins in Gastropods The mechanism on the development of imposex caused by organotins as well as the basic endocrinology of gastropods has been reviewed by Horiguchi [56] and Sternberg et al. [96]. Endocrinology of Gastropod Mollusks Because of a lack of information on the basic endocrinology of mollusks, our understanding of the reproductive physiology and/or endocrinology of gastropods is very limited. Knowledge has been obtained mainly from research on the Opisthobranchia (e.g., Aplysia californica) and Pulmonata (e.g., Lymnaea stagnalis); in these gastropods

12 Biochemical and Biological Effects of Organotins

Toshihiro Horiguchi

several neuropeptides released from the visceral ganglia, the cerebral ganglia, or the prostate gland act as hormones to promote ovulation, egg laying, or egg-release [97-99]. There is very little understanding of the reproductive physiology and/or endocrinology of the Prosobranchia (including the Archaeo-, Meso-, and Neogastropoda). A review by LeBlanc et al. [100] has suggested that gastropods have both peptide and steroid hormones, but it remains unclear exactly what type of sex hormone gastropods have (see below). Because sex steroid hormones, such as testosterone and 17β-estradiol, play physiologically important roles in the development of sex organs and the maturation of gonads (i.e., in oogenesis and spermatogenesis) in vertebrates, similar sex steroid hormones might also regulate the reproduction of invertebrates, such as gastropods [100]. After removal of the hermaphroditic organ, oogenesis and spermatogenesis were observed in the gonads of 17β-estradioltreated females and testosterone-treated males, respectively, of the slug Limax marginatus; egg laying was also induced by 17β-estradiol in female slugs, implying the existence of vertebrate-type sex steroid hormones in this species [101, 102]. The in vitro metabolism of androstenedione and the identification of endogenous steroids (androsterone, dehydroepiandrosterone, androstenedione, 3α-androstanediol, estrone, 17β-estradiol, and estriol) by gas chromatography with mass spectrometry (GC-MS) have been reported in Helix aspersa [103]. Several vertebrate-type sex steroids (androsterone, estrone, 17β-estradiol, and testosterone) and a synthetic estrogen (ethinylestradiol) have also been identified by high resolution GC-MS in the gonads of T. clavigera and B. japonica. The ethinylestradiol detected in the gonads was presumably of environmental rather than endogenous origins, indicating that contamination of the habitat of B. japonica had occurred [104]. It is therefore likely that the presence of other vertebrate-type sex steroids in T. clavigera and B. japonica may have been due to environmental exposure, as opposed to synthesis in vivo. In contrast, biotransformation of testosterone has been characterized in the mud snail I. obsoleta [105]. However, as there has been no scientific verification of the presence of an androgen receptor (AR) in gastropods (see below), we should perhaps interpret with caution the biological significance of the transformation of testosterone in I. obsoleta exposed to a high dose (1.0 μM) [105]. Further evidence of steroid-producing cells and synthetic/metabolic enzymes for steroid biosynthesis needs to be obtained to clarify the existence of vertebrate-type sex steroid hormones in gastropods. Aromatase-like activity has been measured and reported in several gastropod species [106, 107]. However, the measured aromatase-like activity does not necessarily confirm the existence of vertebrate-type aromatase in gastropods. To the best of our knowledge, there has not yet been a scientific report of the successful isolation of aromatase protein from invertebrates. Although an estrogen receptor (ER)-like cDNA has been isolated from A. californica, and the protein it encodes functions as a constitutively activated transcription factor, estrogen cannot bind this protein [108]. Similarly, an ERlike protein has also been isolated from T. clavigera, although this is not bound by estrogen either [109, 110]. This T. clavigera protein is also a constitutively activated transcription factor [110]. To the best of our knowledge, no scientific report has described the successful cloning of an AR from the tissues of invertebrates, including gastropods. In the absence of direct evidence for ERs and ARs, their physiological role in mollusks remains in doubt, even if estrogens and androgens are detected in tissues. A study of fully sequenced invertebrate genomes failed to find homologues of ER and AR in invertebrates [111]. It therefore remains unclear whether gastropods have ARs and ERs. Further studies are necessary to identify steroid receptors and clarify their functions in gastropods. Mode of Action of Organotin Compounds in the Development of Imposex Several hypotheses on the mechanism of induction of imposex have been proposed. They can be summarized as follows, in terms of the steroid and neuroendocrine pathways: 1) increased levels of androgens, such as testosterone, owing to aromatase inhibition by TBT [112-114]; 2) TBT-mediated inhibition of the excretion of sulfate conjugates of androgens [115]; 3) disturbance of the release of penis morphogenetic/retrogressive factor from the pedal/cerebropleural ganglia by TBT [116]; and 4) TBT-mediated increase in the level of a neuropeptide, alanineproline-glycine-tryptophan amide (APGWamide) [117, 118]. There is also another hypothesis that differs completely from those concerning both the steroid and neuroendocrine pathways; it involves retinoid X receptor (RXR), a nuclear receptor [119-124].

Ecotoxicological Impacts of Organotins

Biochemical and Biological Effects of Organotins 13

Experimental evidence is weak for the four hypotheses related to steroid and neuroendocrine pathways. In the aromatase inhibition hypothesis, there is a lack of correlation between the time course of the increase in testosterone titers and penis growth in females [112, 114]. In regard to hypotheses 1) and 2), Spooner et al. [114] reported that testosterone levels were significantly higher in TBT-exposed dog-whelks (N. lapillus) on days 28 and 42 than in the controls, although the penis length of female N. lapillus started to increase on day 14. On the other hand, Iguchi et al. [110] reported that a combination of the aromatase inhibitor fadrozole (5 µg/g wet wt.) and testosterone (0.1 µg/g wet wt.) had little effect on the induction and/or promotion of imposex in T. clavigera, as indicated by the incidence of imposex and penis growth. Consequently, the mechanism by which organotins induce imposex in gastropods seems uncertain, assuming that vertebrate-type steroid hormones are involved. It is unknown whether aromatase-like activity is actually inhibited by TBT concentrations in the tissues of gastropods collected at natural sites slightly contaminated by TBT. There is also contradictory evidence of the relationship between reduced aromatase-like activity and advanced imposex symptoms in the gastropod Bolinus brandaris [106]. Santos et al. [113] suggested the involvement of the AR, besides aromatase inhibition, in the development of imposex in N. lapillus, although gastropods may not have ARs [111]. There is no evidence that gastropods have membrane receptors for steroids. However, if gastropods, like vertebrates, do have ARs, then it may be profitable to consider the potential for activation of androgen receptor-mediated responses by TBT or TPhT in these organisms, as enhancement of androgen-dependent transcription and cell proliferation by TBT and TPhT has been reported in human prostate cancer cells [125]. It is possible that the results given in support of the inhibition of testosterone excretion hypothesis [115] reflect a phenomenon that is at least partly short term and/or associated with acutely toxic TBT concentrations [126]. Several neuropeptides released from the visceral ganglia, cerebral ganglia, or prostate gland of gastropods (e.g., A. californica and L. stagnalis) act as ovulation, egg-laying, or egg-releasing hormones [97, 98]. Féral and Le Gall [116] suggested that TBT-induced imposex in O. erinacea might be related to the release of neural morphogeneticcontrolling factors. Their study used in vitro tissue cultures derived from a presumed penis-forming area in the immature slipper limpet Crepidula fornicata as well as the isolated nervous systems of male or female O. erinacea in the presence/absence of TBT (0.2 μg/L) [116]. The accumulation of TBT or TPhT in the central nervous systems of H. gigantea [86], N. lapillus [43], and T. clavigera [39] indicates the potential for toxic effects of TBT and TPhT on neuroendocrine systems. Oberdörster and McClellan-Green [117] reported that APGWamide, a neuropeptide released from the cerebral ganglia of gastropods, such as L. stagnalis, induces marked development of imposex in female I. obsoleta. The effect of APGWamide in inducing and/or promoting the development of imposex, however, appears weak in light of experimental results on imposex and penis growth [117, 118]; the incidence of imposex was higher, and penis growth much longer, in gastropods exposed to TBT and/or TPhT in the laboratory [127]. Thus, at present, the four hypotheses related to steroid and neuroendocrine pathways cannot be fully supported as mechanisms of induction of imposex in prosobranch gastropods. Nishikawa et al. [119] proposed a unique mechanism of action of TBT or TPhT in the development of imposex in gastropods. This hypothesis was completely different from other hypotheses already proposed for the imposex induction mechanism. They showed that organotins (both TBT and TPhT) bound to human RXRs (hRXRs) with high affinity and that injection of 9-cis retinoic acid (9CRA), a natural ligand of hRXRs, into female T. clavigera induced the development of imposex, Figs. (5) and (6). Cloning of an RXR homologue from T. clavigera revealed that the ligand-binding domain of the rock shell RXR was very similar to that of the vertebrate RXR and bound to both 9CRA and organotins [119]. I and others [122] treated female T. clavigera with three different concentrations (0.1, 1, or 5 μg/g wet wt.) of 9CRA or with a single concentration (1 μg/g wet wt.) of TBT; with TPhT (as positive controls); or with fetal bovine serum (as a negative control) to confirm the effectiveness of 9CRA in inducing the development of imposex in T. clavigera. 9CRA induced imposex in a dose-dependent manner, Fig.(7). Imposex incidence was significantly higher in rock shells that received 1 μg (P < 0.05) or 5 μg (P < 0.001) 9CRA than in the controls. After 1 month, the rock shells treated with 5 μg 9CRA exhibited substantial growth of a penis-like structure. The length of the structure differed between the 0.1-μg and 5-μg 9CRA treatment groups (P 0.05). Compared with the controls, the vas deferens sequence (VDS) index increased significantly in the 1-μg (P < 0.05) and 5-μg (P < 0.001) 9CRA groups. A light

14 Biochemical and Biological Effects of Organotins

Toshihiro Horiguchi

microscopic histological observation revealed that the penis-like structures behind the right tentacle in female rock shells treated with 5 μg 9CRA were essentially the same as the penises and vasa deferentia of normal males and of TBT-treated or TPhT-treated imposexed females [122], Fig. (8). 100

**

90

*

80

Incidence (%)

70

**

60 50 40 30 20 10 0 Control

RA 9CRA

TPhT

Figure 5: Incidences of imposex in female rock shells (Thais clavigera) 1 month after treatment with fetal bovine serum (control), 9-cis-retinoic acid (9CRA) at 1 μg/g (wet wt.), or triphenyltin chloride (TPhT) at 1 μg/g (wet wt.). *P < 0.05; **P < 0.01 [119]. ov

ov

cg

p

ov

cg

p

cg PL=6.06 mm

Control

9CRA

PL=6.50 mm

TPhT

Figure 6: Substantial penis growth observed in female rock shells (Thais clavigera) after a month of 9-cis retinoic acid (9CRA) injections. cg: capsule gland; ov: ovary; p: penis; PL: penis length (Left). Neither penis nor vas deferens was observed in the control female (after shell removal). (Center) Substantial penis growth as well as vas deferens development in a female that received 9CRA injection at 1 μg/g (wet wt.) (after shell removal; penis length: 6.06 mm). (Right) Substantial penis growth as well as vas deferens development in a positive control female that received TPhT injection at 1 μg/g (wet wt.) (after shell removal; penis length: 6.50 mm). Signs of imposex symptoms, based on penis length and the vas deferens sequence (VDS) index of females that received 9CRA injections, were clearly promoted and were similar to those in females receiving TPhT injections [119].

I and others [121] investigated RXR gene expression and measured the RXR protein content in various tissues of wild male and female T. clavigera to further elucidate the role of RXR in the development of organotin-induced imposex in gastropod mollusks. By using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry with a commercial antibody against human RXRα, we revealed that RXR gene expression was significantly higher in the penises of males (P < 0.01) and in imposexed females (P < 0.05) than in the penisforming areas of normal females, Fig. (9). A polyclonal antibody against RXR of the rock shell (T. clavigera) has recently been established [124]. Immunoblotting has demonstrated that this antibody could recognize T. clavigera RXR. In males and imposex-exhibiting females, immunohistochemical staining with the antibody revealed nuclear localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and in the interstitial and epidermal cells of the penis. These results suggest that the polyclonal antibody against T. clavigera RXR can

Ecotoxicological Impacts of Organotins

Biochemical and Biological Effects of Organotins 15

specifically recognize RXR protein in the tissues of T. clavigera and therefore is useful for evaluating RXR protein localization. From the results of these studies as well as their previous studies, we [121, 124] suggested that RXR is involved in organotin-mediated induction of male-type genitalia (penis and vas deferens) in female rock shells. 100 90

*

80

***

***

***

Incidence (%)

70 60 50 40 30 20 10 0 Control

9CRA　0.1μg 9CRA　1μg

9CRA　5μg

TBT 1μg

TPhT 1μg

Figure 7: Incidence of imposex in female rock shells (T. clavigera) 1 month after treatment with fetal bovine serum (Control), three different concentrations of 9-cis-retinoic acid (9CRA), tributyltin (TBT) chloride, or triphenyltin (TPhT) chloride. *P < 0.05; ***P < 0.001 [122].

A

vd

B p vd p

Figure 8: Histology of the penis-like structure (7.00 mm long) that developed behind the right tentacle of a female rock shell (T. clavigera) 1 month after treatment with 9CRA at 5 μg/g (wet wt.). The sections in A and B were stained with hematoxylin and eosin. Scale bars represent 0.5 mm. p, penis; vd, vas deferens [122].

16 Biochemical and Biological Effects of Organotins

Toshihiro Horiguchi

3.5

**

*

3

RXR / 16S rRNA

2.5 2 1.5 1 0.5

di ge t st est is iv e gl an d he pe ad ni ga s ng lia ct en id iu m di ge ov st pe iv ary ni e s gl fo rm and i he ng ad are ga a ng lia ct en id iu m di ge ov st iv ary e gl an d he pe ad n ga is ng lia

ct en id iu m

0

male

female

imposex-exhibiting female (n = 3, *; p < 0.05, **; p < 0.01)

RXR/18S rRNA

Figure 9: RXR gene expression in various tissues of male, normal female, and imposex-exhibiting female rock shells (T. clavigera) [121].

0.3 0.25

A

0.25

0.2 0.15 0.1 0.05 0

TPhT

C

0.25

0.2

0.2

0.15

0.15

0.1

0.1

0.05

0.05

0 -

-

Exposure period(months) 0

+

-

1

-

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-

-

0

-

Exposure period(months) 0

-

**

+ 1

-

-

-

+

+

-

3

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-

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+ 1

-

+ 2

-

+ 3

D

-

+ 3

-

2

1.4 1.2 1 0.8 0.6 0.4 0.2 0

*

+ 2

+ 1

B **

**

0

+ 3

RXR/18S rRNA

0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 TPhT

+

E

0

+ 1

-

+ 2

-

+ 3

Figure 10: Normalized RXR gene expression in (A) ctenidium, (B) ovary, (C) digestive gland, (D) penis-forming area or penis, and (E) head ganglia of female rock shells exposed to TPhT chloride at 500 ng/L for 3 months in a flow-through system. TPhT: triphenyltin; -, control females; +, TPhT-exposed females; * P < 0.05; ** P < 0.01. [123].

To further examine the role of RXR in the development of imposex in gastropods, I and others [123] investigated the time course of expression of the RXR gene in various tissues (ctenidium, ovary or testis, digestive gland, penis-forming

Ecotoxicological Impacts of Organotins

Biochemical and Biological Effects of Organotins 17

area or penis, and head ganglia) of female and male T. clavigera exposed to TPhT in a flow-through exposure system for 3 months. Accumulation of TPhT in the tissues was clearly observed in exposed individuals, whereas no accumulation of TPhT was observed in the control groups. In females, a 3-month exposure to TPhT resulted in the development of imposex, and penis lengths in imposex-exhibiting females were significantly longer in small females (shell height R3Sn > R2Sn > RSn > Sn all for tin(IV) compounds. This is consistent with general trends and long alkyl chains such as n-octyl (due to the low solubility) are generally biologically inactive with respect to bacterial inhibition. CHEMISTRY AND USES OF OTC OTC are classified as R4Sn, R3SnX, R2SnX2, and RSnX3. In industrial compounds, R is usually a butyl, octyl, or phenyl group and X, a chloride, fluoride, oxide, hydroxide, carboxylate, or thiolate. So far, monosubstituted OTC (RSnX3) have had a very limited application, but they are used as stabilizers in poly(vinyl chloride) films. Disubstituted OTC (R2SnX2) are mainly used in the plastics industry, particularly as stabilizers in poly(vinyl chloride). They are also used as catalysts in the production of polyurethane foams and in the room-temperature vulcanization of silicones. Trisubstituted OTC (R3SnX) have biocidal properties that are strongly influenced by the R-groups. The most important of these compounds are the TBT, TPhT, and TCT, which are used as agricultural and general fungicides, bactericides, antihelminthics, miticides, herbicides, molluscicides, insecticides, nematocides, ovicides, rodent repellents, and antifoulants in boat paints. Tetrasubstituted OTC (R4Sn) are mainly used as intermediates in the preparation of other OTC [8]. OTC have one or more carbon-tin covalent bonds that are responsible for the specific properties of such molecules and all are essentially of the SnIV type. The only well established compound with tin in the oxidation state + 2 is the tin(II)cyclopentadienyl, C10H10Sn. There are four series of organotin(IV) compounds depending on the number of carbontin bonds. These series are designated as mono-, di-, tri-, and tetraorganotin(IV) compounds with the general formula: RnSnX4-n where R = an alkyl or aryl group, Sn = the central tin atom in the oxidation state +4, X = a singly charged anion or an anionic organic group. The chemical nature of the R group has a strong influence on the biological properties of these compounds. The Xgroup, on the other hand, influences their solubility and volatility. Tetraalkyl- and tetraaryltin(IV) compounds are primarily used as intermediates in the preparation of other OTC. Tetraalkyltin(IV) compounds are colourless and the compounds of lower molecular weight are liquids at room temperature. The tetraaryltin(IV) compounds are solids. Tetraorganotin(IV) compounds possess typical covalent bonds and are stable in the presence of air and water. Tetrabutyltin(IV), Sn(C4H9)4 is a colourless oily liquid with a distinct odour. Tetraphenyltin(IV), Sn(C6H5)4, is a white crystalline powder, soluble in organic solvents and insoluble in water. OTC DEGRADATION IN THE ENVIRONMENT The Sn–C bonds are stable in the presence of water, atmospheric O2 and heat. They are reported to be stable at temperatures up to 200°C [9], so thermal decomposition has no significance under environmental conditions. UV radiation, strong acids and electrophilic agents readily cleave the Sn–C bonds. The number of Sn–C bonds and the length of the alkyl chains have a profound effect in the chemical and physical properties of the organotins. In general, the solubility of organotin(IV) compounds in water decrease with increasing number and length of the organic substitutes but it also depends on the particular X. The carbon-tin bond is susceptible to nucleophilic and electrophilic attack, e.g., hydrolysis, solvolysis, acidic and basic attack, and halogenation. Water has little effect on symmetrical saturated OTC. Dialkyltin(IV) compounds react spontaneously with moisture and air to form dialkyl hydrated oxides. Photochemical reactions of OTC are mentioned in connexion with environmental transport and transformations. The OTC degradation in the environment may be defined as a progressive loss of organic groups from the Sn cation R4Sn→R3SnX→R2SnX2→RSnX3→SnX4.

Biological Activity of Organotin(IV) Compounds

Biochemical and Biological Effects of Organotins 27

The removal of the organic groups can be caused by various processes which include: (1) Ultraviolet (UV) irradiation; (2) Biological cleavage; (3) Chemical cleavage. (1) Photolysis by sunlight appears to be the fastest route of degradation in water. The mean bond dissociation energies for Sn–C bonds are in the range of 190–220 kJ mol−1. UV radiation of wavelength 290 nm corresponds to an energy of approximately 300 kJ mol−1. Consequently, provided that absorption of light takes place, Sn–C bond cleavage could occur [10]. Due to the attenuation of sunlight with depth in the water column, photolysis is probably not important at greater depths in water, nor in sediments or soils. However, TPhT- and TCT species are rapidly dealkylated by UV irradiation while the TBT derivatives show much lower degradation rates. Degradation is due to the ability of some bacteria, like Pseudomonas aeruginosa, Pseudomonas putida C, and Alcaligenes faecalis, which are able to degrade OTC under certain conditions. The microalgae species Skeletonema costatum is also capable of degrading TBT even at 4°C [11]. Reported half-lives of OTC are often related to laboratory experimental conditions and thus not directly comparable with natural circumstances, under which the actual rate of breakdown depends on numerous factors, e.g. the intensity of sunlight. Experiments using both 14C-labelled and unlabelled TBT in harbour and estuarine waters, with ambient microflora, yielded half-lives in water between 4 and 14 days [12]. (2) Beside the anthropogenic sources, methyltin(IV) compounds can stem from biomethylation processes. Several biotic and abiotic methylation agents are known. Methylcobalamin (CH3B12, the methyl coenzyme of vitamin B12) is a carbanion donor and is able to convert inorganic Sn(IV) to the several methyltin(IV) species [13]. The methylcobalamin can be demethylated by SnCl2 in aqueous HCl solution, in the presence of an oxidizing agent (Fe3+ or Co3+), to form MMT species. Methyliodide (CH3I) which is produced by certain algae and seaweeds, can methylate inorganic Sn (II) salts in an aqueous medium to produce monomethyltin(IV) species, whereas tin(IV) compounds do not react [14]. Certain Pseudomonas bacteria are able to form various methyltin(IV) compounds [15]. The transmethylation of methyltins with other heavy metals is of great ecological relevance because some methylated metals have a higher toxicity to aquatic organisms than the inorganic metal (e.g. methylmercury) [16,17]. From these studies it can be suggested that both inorganic Sn(II) and Sn(IV) compounds and methyltin(IV) derivatives can be methylated by chemical or biological processes under simulated environmental conditions. (3) The capability of sediments to act as sinks for OTC poses a permanent risk of OTC for water contamination due to desorption processes and particle ingestion by bivalves. Speciation of various triorganotins (TOT) in aqueous solution has been investigated. In natural water, these compounds are present predominantly as neutral TOT-OH species or as TOT+ cations depending on the pH-value [18]. At pH < 4 the predominant species of DMT is the cation Me2Sn2+, while under environmental conditions (pH 6–8) the species mainly found is Me2Sn(OH)2. TMT compounds at pH < 5 primarily occur as the TMT cation Me3Sn+, and at pH > 5 as Me3SnOH [19]. OTC USES AND APPLICATIONS OTC have biocidal properties and are used as; (a) agricultural fungicides [TPhTA, TPhTOH]; (b) general biocides [TBTO] in paints; in the preservation of manila and sisal ropes, leather, textiles; to make mildew-resistant fabrics; for the jute protection and jute bags; in wood preservatives, slimicides, and in the production of paper; (c) bactericides and biostatics such as disinfectants for hospitals and stables [TBTB]; (d) helminthicides in poultry [DBTdilaurate, tetraisobutyltin(IV)]; (e) nematocides [p-bromophenoxytriethyltin(IV)]; (f) herbicides [vinyl-tin compounds, e.g., trivinyltin(IV) chloride]; (g) rodent repellents [TBTC, TPhTC and acetate] eg; (h) molluscicides [TPhT- and TBT] compounds; (i) ovicides [trialkyl- and triaryltin(IV) chlorides in combination with DDT or pyrethrins]; (j) antifoulants in ship paints and underwater coatings [TPhT- and TBT- compounds]; and (k) miticides [TCTOH]. Furthermore, TPhTcompounds have been suggested as insect chemosterilants [8]. Ecotoxicological effects occur at all levels of the biological organization, from the molecular to the ecosystem level. Not only may certain organisms be affected, but the ecosystems as a whole in its function and structure. OTC

28 Biochemical and Biological Effects of Organotins

Nagy et al.

bioavailability is dependent on the pH and on the organic matter content. Organotins accumulate in sediments, but remobilization occurs during disturbance and dredging. Except for methyltin(IV) possibly engendered by methylation during biological activities, organotin(IV) has been introduced into the environment exclusively through anthropogenic sources. The long degradation period of OTC in sediments creates a long-term pollution source to the water column, resulting from the release of accumulated OTC through resuspension and/or desorption [20, 21]. The presence of butyltin(IV) species in sediment thus directly affects its surrounding aquatic ecosystem. These aspects are previously treated in Chapter 1. The relative persistence of butyltins, combined with their affinity for biological tissues, has led to their widespread occurrence in fish, snails, mussels, seals and dolphins [22, 23]. One criterion for OTC environmental persistency is their lipophilic character. Most studies concerning OTC uptake by aquatic organisms deal with TBT because of its extreme and widespread toxicity. Some marine bacteria display a remarkable ability to accumulate this contaminant. Research on TBT accumulation by aquatic invertebrates has been mostly confined to mollusks (bivalves) and crustaceans (decapods) because these groups are important seafood resources and are ecologically dominant in many habitats. The adsorption behavior of organotin(IV) species is important in determining the transport processes as well as their bioavailability specially to aquatic organisms. Dissolved species are more likely to be expelled into the sea or directly inserted in the food chain by bioaccumulation. Some effects on non human organisms may have deep implications to human health, or viceversa, when mechanism of effects and levels and exposure rank are taken into account. Organotin(IV) compounds can be found in some foods, such as seafoods, meat, farm products and liquid foods. No obvious degradation of BTs occurs in the cooking process [24-26]. In higher species, including mammals, organotin(IV) compounds tend to accumulate in certain organs, namely the liver, kidneys and brain [23,27]. Organotin(IV)s efficiently penetrate through the skin and easily cross the placenta and blood–brain barrier [28, 29]. TOXICITY TOWARDS MAMMALS INCLUDING HUMANS Since a number of organotin(IV) compounds have been demonstrated to be toxic, increasing concern is rising about their widespread use. Besides carcinogenic, neurotoxic effects, endocrine and reproductive effects, the most obvious effects of both di- and trisubstituted organotins in mammals were found on the immune system. Specific effects of organotin(IV) compounds on the biological systems and health include disturbance of the structure and function of the central nervous system (CNS) (interstitial edema of white matter), inhibited mitochondrial oxidative phosphorylation, thymus and thymus dependent lymphoid tissue atrophy resulting in the disfunction of T cells, inhibited enzyme activity, liver and bile duct lesions etc., although specific effects were observed among animal species. In addition to wildlife, OTC have undesirable effects on human health. In 1954 a widespread poisoning occurred in France, where a pharmacon used for the oral treatment of staphylococcal skin infections led to at least 100 deaths and over 200 intoxications [31]. The proprietary formulation was based on DET diiodide (Stalinon) and linoleic acid and substantial amounts of highly toxic triethyltin iodide occurred as an impurity. Other cases of occupational exposure, including two deaths, were reported by Blunden and Evans [19]. OTC may enter water from antifouling paints or molluscicides in which, to be effective, they attain concentrations of about 1 mg/L. Food is the main source of tin for humans. A diet composed principally of fresh meat, cereals, and vegetables, is likely to contain a mean tin concentration of about 1 mg/kg. OTC may be introduced into foods through pesticides and, to some extent, through tin migration from poly(vinyl chloride) materials [8]. In general, OTC are more readily absorbed from the gut than inorganic tin compounds even if wide variations occur among different compounds and species. As a rule, tin compounds with a short alkyl chain are more readily absorbed from the intestinal tract. The trialkyltin(IV) compounds are usually well absorbed through the skin. The highest OTC concentrations in rats, guinea pigs, rabbits, and hamsters were mainly detected in the liver. Trisubstituted OTC were found in the brain of various species even if the tin form was not satisfactorily identified [8]. Many OTC are transformed, to some extent, within tissues. Apparently the dealkylation and dearylation of tetra-, tri-, and disubstituted organotin(IV) compounds occurs in the liver, while the dealkylation of diethyltin compounds takes place both in the gut and in other organs. The excretion mode of OTC largely depends on the compound. Ethyltin(IV) trichloride seems to be mainly excreted with the urine, but diethyltin (DET) is eliminated with the faeces, urine, and the bile. Triethyltin (TET) is not only excreted with the urine, but, at least in lactating sheep, also

Biological Activity of Organotin(IV) Compounds

Biochemical and Biological Effects of Organotins 29

with the milk. The excretion route of many compounds is not known. OTC exhibit various biological half-times and many compounds disappear slowly from the organs; half-times are generally longer in the brain than in other organs [8]. The threat and risk of OTC to humans are reviewed in detail in Chapter 11. OTC INTERACTIONS WITH BIOMOLECULES When OTC enter living organisms, they may interact with biological macromolecules or low molecular compounds in biological fluids. Such interactions significantly alter OTC speciation, thus leading, to biological effects (usually toxicity). The biological activity of organotin(IV) compounds may be due to the presence of easily hydrolysable groups (easily dissociable chelating ligands) yielding intermediates such as [RnSn(4−n)+] (n = 2 or 3) moieties, which may bind with DNA, proteins, carbohydrates and lipids. A number of studies on the triorganotin(IV) compounds, R3SnX, and diorganotin(IV) compounds, R2SnXY, indicated that the marked biological activity of the organotin(IV)s may be due to the transport of either more active species ([RnSn(4-n)+], where n = 2 or 3) or of the whole molecule across the cellular membrane, and X or XY group influences only the readiness of delivery into the cell of the active part, namely [R3Sn]+/[R2Sn]2+ [32]. OTC Interactions with Aminoacids and Proteins Including Enzymes The most widely studied interactions between biologically active ligands and organotin(IV) cations deal with aminoacids and derivatives (N- or S-protected amino acids and peptides), as reviewed in detail by Molloy [33] and Nath[34], though new data on several of the most commonly occurring aminoacids are still being published. This is especially true for aqueous speciation studies. Cysteine and histidine residues are the primary coordination sites for organotin(IV) compounds [35] and vicinal dithiols rather than monothiols constitute a general target for organotin(IV) [36]. This effect is originated from “chameleon” nature of organotin(IV) cations. In spite of “hard” character of cations (very easily hydrolyzed in water), the complexes formed with ligands containing “soft” –SH donor group have considerable higher stabilities than the complexes with oxygen or nitrogen atom. The study of binding the TET moiety to rat-liver mitochondria led to the assignment of two binding sites in the membrane: one of high-affinity, the other of low-affinity. These binding sites are considered to involve histidine and thiol groups. Most notable amongst the protein systems to which triorganotin or Me2Sn(IV) fragments are known to bind are the haemoglobins and ATP-ase system. Both histidine (via the imidazole ring nitrogen) and cysteine (via the sulphydryl group) residues have been implicated in the binding of trialkyltin moieties to cat and rat haemoglobins, with two triorganotin residues bound at identical sites on the alpha-chain sub-unit of the haemoglobin tetramer, and also in the low-affinity site of the ATPase system. In both cases the coordination number of tin was considered from 119Sn Mössbauer data to be five in a distorted Tbp arrangement with equatorial alkyl carbon and axial nitrogen and sulfur atoms SnR3(Sthiol)(Nhet). However, the binding at the high-affinity ATP-ase site appears to involve only a histidyl imidazole residue resulting in four-coordination for tin, although a cis-trigonal bipyramidal geometry, could not be unequivocally excluded. Such studies, however, concerning organotin binding to biological macromolecules are still very rare in the literature and they are far from the quantitative description [34]. Okada et al. examined the effects of TBT on cellular content of GSH in rat thymocites using a flow cytometer. 5chloromethylfluorescein diacetate was used as fluorescent probe for determining the change in the cellular content of GSH. TBT at nanomolar concentrations reduced the cellular content of GSH. There is an important implication on the TBT-induced depletion of cellular GSH since GSH has an important role in protecting the cells against oxidative stress and chemical and metal intoxications. TBT-induced decrease in cellular content of GSH in thymocytes may increase the vulnerability of immune system [37,38]. Organotin(IV) compounds affect cell signaling: they activate protein kinase C [39] and increase free arachidonic acid through the activation of phospholipase A2 [40]. Billingsley et al. [41] have identified a small membrane protein, stannin (Snn), containing vicinal dithiols at the membrane interface, which mediates the selective neurotoxic activity of TMT in mammalians by triggering neuronal apoptosis in the hippocampus. OTC are highly specific neurotoxins [42]. TMT and TET cause damage in the CNS. While TMT causes lesions in specific regions of the hippocampus and neocortex, TET damage is localized within the spinal cord [43]. Interestingly, in mammalian brain, liver and kidneys, organotin(IV)s are progressively dealkylated to inorganic

30 Biochemical and Biological Effects of Organotins

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Sn(IV) [44]. This reaction probably also occurs in environment. Astrocytes may represent an important link in the TNT-driven CNS damage since prominently swollen astrocytes without neuronal involvement were detected after in vivo exposure [45]. In vivo exposure to TMT also produced a transient increase in the specific cell marker glial fibrillary acidic protein (GFAP) [46]. TMT in vitro disrupts the glutamate transporter and stimulates cytokine release from astrocytes [47]. Eukaryotic DNA is packaged into chromatin, whose basic subunit is the nucleosome, which consists of DNA and a core histone octamer. Histone acetylation, catalyzed by histone acetyltransferase (HAT), is important for the regulation of gene expression. Some OTC – TBT and TPhT – enhanced HAT activity of core histones in a dosedependent way while other endocrine-disruptin compounds (EDCs) did not affect HAT activity.Organotin compounds have various influences on physical function including the hormone and immune systems, embryogenesis, and development. DBT and DPhT, metabolites of TBT and TPT, respectively, also promoted HAT activity, but monobutyltin, monophenyltin(IV), and inorganic tin had no effect. Further, TBT and TPhT enhanced HAT activity when nucleosomal histones were used as substrates. These data indicate that the organotin compounds have unique effects on HATs independent of their EDC activities and suggest that the varied toxicities of the organotin compounds may be caused by aberrant gene expression following altered histone acetylation [48]. TBT and TPhT – according to the X-ray diffraction and Mössbauer spectroscopic measurements have distorted Th structure in the solid state – enhanced HAT activity of core histones. As it was suspected, the action is dosedependent. The other endocrine disrupting chemicals (EDCs) did not affect HAT activity. Organotin(IV) compounds have various influences on physical function including the hormone and immune systems, embryogenesis, and development. DBT and DPhT, metabolites of TBT and TPhT, respectively, also promoted HAT activity, but MBT, MPT and inorganic tin salts had no effect. Further, TBT and TPhT enhanced HAT activity when nucleosomal histones were used as substrates. These data indicate that the organotin(IV) compounds have unique effects on HATs independent of their EDC activities and suggest that the varied toxicities may be caused by aberrant gene expression following altered histone acetylation [48]. OTC Interactions with Nucleotides and Nucleic Acids Nucleic acids including DNA are probable targets of citotoxic activity of OTC. Barbieri et al. [49-51] investigated the interactions between ethanolic [R2SnCl2(EtOH)2 and R3SnCl(EtOH), R = Me, Et, Bu, Oct or Ph] and aqueous [Me2Sn(H2O)n]2+ and [Me3Sn(H2O)2]2+ species with aqueous DNA from calf thymus by 119Sn Mössbauer spectroscopy. In particular, the latter data showed that the solids obtained by adding solvated [R2SnCl2(C2H5OH)n] and R3SnCl(EtOH) to DNA (R = Me, Et, Bu) were possibly R2Sn(DNA phosphate)2 and R3Sn(DNA phosphate). In these complexes, the environment of the {Sn} atoms would be trans-Oh, with a linear CH3–Sn–CH3 skeleton [dialkyltin(IV)], and [Tbp (trialkyltin(IV) derivatives], respectively. According to Barbieri et al. it cannot be excluded that some of the coordination sites are occupied by {N} atoms of the nucleic acid constituents [49,50]. The precipitate obtained from [Ph2Sn(IV)]2+ would contain both the [R2Sn(IV)]2+-DNA complex and distannoxane [(Ph2SnCl)2O]. The main products of the interactions of aqueous DNA with n-Oct2SnCl2(EtOH)2 and R3SnCl(EtOH) [R = Oct, Ph] were possibly stannoxanes [(R2SnCl)2O] and hydroxides [R3SnOH]. Furthermore, no interaction at all was detected when the water-soluble hydrolysed species [Me2Sn(OH)(H2O)n]+, Me2Sn(OH)2 and Me3Sn(OH)(H2O)2 were added to native DNA. Such results imply that organotin(IV) moieties may interact in vivo with cellular DNA only if they are weakly solvated [50]. The interactions between ethanolic solutions of Me2SnCl(SPy) or Me2SnCl(SPym) and aqueous calf-thymus DNA have been the subject of a report [52]. It was concluded that the 1:1 complex/DNA condensates are derived from an electrostatic interaction between the cations Me2SnCl(SPy)+ and Me2SnCl(SPym) and the phosphate {O} of phosphodiester groups. The reactions with DNA of two antitumor active organotin(IV) compounds, the dimer of bis[(dibutyl 3,6-dioxaheptainato)tin (C52H108Sn4O*2H2O] compound I., and tributyltin 3,6,9-trioxodecaonate (C19H40SnO5*1/2H2O), compound II., were investigated by different methods [53]. The main chemical features of the two compounds were also previously published [54]. These complexes are moderately water-soluble and very slowly hydrolyze towards the corresponding tin hydroxides or oxides. Both complexes exhibited strong antitumor activities on several human tumor cell lines, even stronger that those induced by some classical antitumor drugs.

Biological Activity of Organotin(IV) Compounds

Biochemical and Biological Effects of Organotins 31

IC50 values were found in 1-5 nM range for compound I and in the 50-200 nM range for compound II were found [55]. Both compounds also produced slight effects on DNA conformation and significantly decreased the DNA melting point. No significant effects on gel mobility of plasmidic DNA samples were detected. This work also suggests that the interaction of organotin(IV) parent compounds or complexes with DNA is not sequence- or basic specific and therefore most likely occurs at the level of external phosphate groups. Semiempirical calculations on the interaction between [Me2Sn(IV)]2+ and a dinuclide triphosphate duplex (DD), mimicking a DNA model system, performed by the semiempirical PM3 method and published by Barbieri et al. [56], showed that the [Me2Sn(IV)]2+ moiety binds to two adjacent phosphate groups. Small-angle X-ray scattering (SAXS), circular dichroism (CD) and UV spectroscopy at different temperatures were used to investigate the nature of calf thymus DNA in aqueous solution, in the presence of [MenSn(IV)](4-n)+ (n = 1-3) species [57]. The results demonstrated that the [MeSn(IV)]3+ moiety does not influence the structure and conformation of the DNA double helix, and does not degrade DNA, as indicated by agarose gel electrophoresis. Inter alia, the radii of gyration, Rc, of the cross-section of native calf thymus DNA, determined by SAXS in aqueous solution in the presence of [MenSn(IV)](4-n)+ (n = 1-3) species are constant and independent of the nature and concentration of the [MenSn(IV)](4-n)+ species. Model DNA nucleotides were also studied. The coordination of [Me2Sn(IV)]2+ to 5’-GMP, 5’-ATP, 5’-AMP and 5’-[d(CGCGCG)2] and to their sugar constituents (Rib and Derib) was investigated in aqueous solution by pHmetric titration and 1H and 31P NMR spectroscopy. The results showed that the phosphate groups can provide suitable sites for metal ion coordination only in acidic medium, while in the higher pH range the –OH groups of the sugars or the sugar moieties of the two nucleotides are involved. The base moieties of 5’-GMP, 5’-AMP’ and 5’-ATP were not coordinated to [Me2Sn(IV)]2+. The stability constants revealed a stronger coordination ability of the triphosphate compared with that of the monophosphate groups. The observed chemical shift changes of the 31P NMR resonances, compared with those measured for the metal-free systems, demonstrated that the phosphate groups of the DNA fragment [5’-d(CGCGCG)2] chains act as binding sites for [Me2Sn(IV)]2+ between pH 4.5 and 7. The 1D and 2D 1H NMR spectra indicated that the base and sugar moieties do not participate in the coordination process under these conditions [58]. Similar studies carried out on the interactions of [Me2Sn(IV)]2+, R-5P, G-6P and G-1P led to conclude that at low pH (pH < 4) the organotin(IV) cations interact with pyrophosphate {O}. At intermediate pH values (4-9.5) no interaction occurs, while at pH > 9.5 the sugar O’2 and O’-3 atoms are the preferred coordination sites. Additionally one oligomeric complex in the solid state, containing {Sn} centre in Tbp geometry is obtained [59]. Formation constants for complex species of mono-, di, and trialkytin(IV) cations with some nucleotide-5’monophosphates (AMP, LIMP, IMP and GMP) were evaluated [60] in the light of speciation of organometallic compounds in natural fluids (I = 0.16-1 mol·dm –3). As expected, owing the strong tendency of organotin(IV) cations to hydrolysis (as already pointed out) in aqueous solution, the main species formed in the pH-range of interest of natural fluids were the hydrolytic ones [61]. The complexes of AMP, ATP, adenosine-N’-oxide, 1-methyladenosine, pyridoxal-5-phosphate and NADP with R2SnO and/or R2SnCl2 (R = Me, Bu) were prepared in the solid state. The vibration spectra demonstrated that R2SnO reacts with the Rib moiety of the ligands, while R2SnCl2 (after dissociation of Cl– ion) is coordinated by the deprotonated phosphate group. The basic part of the ligands does not participate directly in complex formation. The Mössbauer ∆exp values revealed that the organotin(IV) moiety has Tbp, Oh, or in some cases Th geometry [61]. The local structure of these complexes and adducts of calf thymus DNA with different organotin(IV) moieties were determined by means of EXAFS. The EXAFS data were analysed by using multishell models up to 300 pm. These results are the first structural data (bond lengths) on complexes formed with organotin(IV)-DNA and related compounds. The diorganotin(IV) adduct proved to be Oh, while the triorgano(IV) complex has Tbp geometry [62]. Very similar conclusions were obtained earlier on the complexes formed between Bu2SnCl2 or Bu3SnCl and 5’-AMP, 5’-GMP and their 3’-5’ cyclic analogues. The stoichiometry of the compounds of 5'-AMP and 5'-GMP with Bu3SnCl was 1:2, while that with Bu2SnCl2 was 1:1. Only 1:1 compounds were formed with the 3'-5' cyclic nucleotides. Most of the compounds are polymeric and differ in the environments of the two Bu groups. For all the [Bu3Sn(IV)]+ complexes, 31P NMR chemical shifts indicate that the {Sn} is bonded to the phosphate group via oxygen atom, but probably not chelated by it. A much larger 31P NMR upfield shift in (Bu3Sn)2(5'-GMP) is an indication of phosphate chelation. The Rib conformation is different within the [Bu3Sn(IV)]+ and [Bu2Sn(IV)]2+ complexes [63].

32 Biochemical and Biological Effects of Organotins

Nagy et al.

Li et al. [64] investigated the interaction of Et2SnCl2(phen) with 5’-dGMP in aqueous medium, using [transen2Os(η-H2)](CF3SO3)2, a versatile 1H NMR probe. These authors also studied solid mixtures of the same Et2SnCl2 complex with 5’-AMP, 5’-CMP, and 5’-GMP dissolved in DMSO, using 1H and 31P NMR and UV spectroscopy. Recently, Hadjiliadis et al. [65] studied the interactions of purine nucleotides 5’-IMP and 5’-GMP with Et2SnCl2, using various techniques. Finally, one paper reported the interactions between Et2SnCl2 + 5’-CMP, 5’-dCMP, and 5’-UMP, using multinuclear (119Sn, 15N and 31P) 1D and 2D NMR techniques. These studies were combined with electrospray mass spectrometry, IR spectroscopy, solid-state 13C, 31P and 117Sn CP-MAS NMR and elemental analysis [54]. The interactions of [Me2Sn(IV)]2+ 5’-AMP, D-ribose-5-phosphate, D-glucose-6-phosphate and Dglucose-1-phosphate [59] were also investigated. These complementary approaches lead to conclude that at low pH values (pH < 4) the organotin(IV) cations interact with pyrophosphate {O}, at intermediate pH values (4-9.5) no interaction takes place, while at pH > 9.5 the sugar O’-2 and O’-3 atoms are the preferred coordination sites. Additionally two oligomeric complexes were obtained, containing {Sn} centre in Tbp or Oh geometry. OTC Interaction with Sugars Carbohydrate ligands are known to modify the biological properties of OTC. In general, the carbohydrate– organotin(IV) complexes have been studied by 1H and 13C NMR [66,67] or, especially by multinuclear NMR (119Sn, 1 H, and 13C) spectroscopy [68-71]. The effect of the conformation of the sugar –OH groups on metal complexation and complex formation of eight saccharides with [Me2Sn(IV)]2+ were investigated in aqueous solution by pH-metric measurements, 13C NMR, polarimetry and Mössbauer spectroscopy. The findings revealed that deprotonation of Dfructose and L-sorbose is caused by the coordination of [Me2Sn(IV)]2+ in the unusually low pH interval 4–6, in contrast with the other saccharides, which are deprotonated in an analogous way at pH>8. The pH increase resulted in the formation of further complexes that differed from each other only in deprotonation state. 13C NMR measurements led to the assignment of the sugar –OH groups involved in the processes. Mössbauer spectroscopic investigations of the quick-frozen solutions permitted determination of the stereochemistry of {Sn} in the complexes [72]. Evaluation of the pH-metric and 13C-NMR titration curves of [Me2Sn(IV)]2+ polyhydroxyalkyl carboxylic acids revealed that the equilibria in aqueous solutions are also rather complicated. Dialkylstannylene acetals (or, more properly, 2,2-dialkyl-1,3,2-dioxastannolanes, if the ring is five-membered; 2,2-dialkyl-1,3,2-dioxastannanes, if the ring is six-membered; and 2,2-dialkyl-1,3,2-dioxastannanes, if the ring is sevenmembered) are easily prepared by the reactions of vicinal diols with R2SnO or R2Sn(OH)2, or R2Sn(IV) diethoxide [73] under conditions of azeotropic dehydration in benzene, methanol, or toluene. Molecular weight measurements in solution indicated that the main reaction product is a dimeric species [73,74]. Where more than one unprotected –OH group is available for reaction, both partially and fully stannylated products are formed [66, 68, 75-77]. Early work established the regioselectivity of tributylstannylation of carbohydrates, which is related to the easy reaction with which {Sn} can coordinate a neighboring {O} atom. Thus, C-6(O) is the most reactive, followed by the secondary –OH groups, all of which are capable of coordinating to {Sn} by a second, cis-O [77]. POSSIBLE THERAPEUTIC USE OF OTC Antitumor Activity of Organotin(IV) Compounds When the antitumor activity of cisplatin, cis-Cl2Pt(NH3)2, was discovered, several research groups started to investigate possible therapeutic applications of other metal-based, often organometallic, compounds. The OTC that were first tested were those that were available or easily synthesized, like tri- or diorganotin(IV) halides. The in vivo pre-screenings against these two leukemias used initially by the National Cancer Institute (NCI) were later replaced by in vitro pre-screenings against a panel of human tumor cell lines [78-89]. This is also the procedure that was used when organotin(IV) compounds were tested by the Rotterdam Cancer Institute. Seven human tumor cell lines were chosen for the panel that was used: MCF-7 and EVSA-T (two mammary cancers), WiDr (a colon cancer), IGROV (an ovarian cancer), M19 (a melanoma), MEL A498 (a renal cancer) and H226 (a lung cancer). The main disadvantage of organotin(IV) halides for antitumor testing is that, when they are dissolved in water, the pH of the solution dramatically decreases because the X–Sn bonds are converted into water(oxygen)-tin bonds; the formed compounds then lose protons, yielding first organotin(IV) hydroxides that are afterwards possibly converted into insoluble bis(triorganotin) oxides or diorganotin(IV) oxides. Because di- or triorganotin(IV) carboxylates do not

Biological Activity of Organotin(IV) Compounds

Biochemical and Biological Effects of Organotins 33

suffer from the same disadvantage and remain intact for long periods, many series of these compounds were synthesized in order to determine their cytotoxic or antitumor properties. The antitumor properties of organotin(IV) compounds were widely considered [90-95]. Many organometallic compounds exhibit antitumor activity against several human cancer cell lines [96,98]. The well-known complex cisplatin, Pt(NH3)2Cl2, with square planar structure, clinically used in cancer chemotherapy, covalently interacts with the N-7 atoms of two adjacent guanines in the same DNA strand (intrastrand cross-link) [99-101]. The similarities in chemical features of high active metal complexes at the molecular level are suggestive of structural similarities. Drug requirements can be summarized as follows: (i) The molecular structure of anticancer metallic drugs must have hydrophilic (“living” groups) and hydrophobic (“keeping” groups) parts. Such features should make the molecules dissolved in water, transported to the surface of the cell membranes and easily cross the membranes through the lipid bilayers. (ii) The interaction between the drug molecule and its target molecule forms an active intermediate which electrocovalently binds with phosphate oxygen and covalently with purine or pyrimidine nitrogen sites. (iii) The drug molecule should be chiral and left hand enantiomer in order to form a complex with the right hand DNA molecule [102]. In contrast with platin compounds, which follow the rules listed above, little is known of the structural requirement for the antitumor activity of organotin(IV) compounds. OTC are known to bind to thiol groups of proteins [103]. However DNA was repeatedly suggested as the probable target for the cytotoxic activity of organotin(IV) compounds. The most important literature data published to date on this topic is here reviewed, also embracing the interaction of organotin(IV) cations or compounds with native DNA and its model compounds. The composition and antimour activity relationship was summarized by Hubert and Saxena based on literature data available until 1989 [104] (Table 1). Tetraorganotin(IV) compounds were inactive in vivo, whereas organotin(IV) halides and their complexes with amines and other ligands showed borderline activities against P388 or L1210 leukemias [105-110] (Table 2). Table 1: Composition and antitumor activity relationship of organotin(IV) compounds [111]. No. represents the number of the compounds tested. Composition

No.

P338

P338 %

L1210

L1210 %

Total No.

1554

680

25

696

1.0

R4Sn(IV)

339

166

2

144

0.4

R3Sn(IV)X

358

132

9

203

0.0

R2Sn(IV)X2

327

129

48

136

1.0

RSn(IV)X3

33

11

9

11

0.0

Sn(IV)X4

45

15

7

10

0.0

R2Sn(IV)X3 R2Sn(IV)X4

160

143

50

35

0.0

P338 and L1210 are two types of leukemia cell lines.

Among {Sn} derivatives, [R2Sn(IV)]2+ compounds generally exhibited higher antitumor activity than the corresponding mono-, tri- and tetraorganotin(IV) or the inorganic {Sn} derivatives and within the diorganotin(IV) class, by the [Et2Sn(IV)]2+ and [Ph2Sn(IV)]2+ complexes, with coordination number five or six, exerted the highest activity. Three primary factors are involved in the structure/antitumor activity relationships for organotin(IV) derivatives (L)xRnSnX4-n: the natures of the organic group {R}, of halide or pseudohalide {Xn}, or other donor atom of ligands. The average Sn–N bond lengths were > 239 pm in the active complexes and < 239 pm in the inactive complexes. Structural data of some promising complexes are collected in Table 2. Predissociation of the ligand may be an important step in the action mechanism, while the coordinated ligand may favour transport of the active species to the site of action within cells, where they are released after hydrolysis.

34 Biochemical and Biological Effects of Organotins

Nagy et al.

Table 2: Sn–N bond lengths in selected organotin(IV) compounds containing {N} donor ligands. No.

Compound

Sn–N (pm)

Refs.

1

Cl2Sn(CH2CH2CH2)2Nme

244.0

112

2

n

254.2, 259.7

112

Pr(Et)Sn(quin)2

3

Ph2SnCl2.SC7H5N

254.8

113

4

[3-(2-py)-2-C4H2S]2SnPh2

256.0

114

5

MeSn(CH2CH2CH2)3N

262.0*

115

6

Ph3SnSC5H4N

262.0*

113

7

[3-(2-py)-2-C4H2S]Sn(p-tolyl)3

284.1

114

8

(Ph2SnCl2.pyz)n

296.5, 278.2

115

9

(Cys3Sn)2N3(OH)

2.436

116

Abbreviations: py = pyridine, bipy = bipyridyl, Hquin = 2-methylquinolin-8-ol, pyz = pyrazine. Cys = cyclohexyl, *Reported as intermolecular Sn–N distance.

In the Bu2SnL (L = is the dianion of dipeptides) complexes all the ligands act as dianionic tridentate ligands coordinating through the –COO–, NH2 and N-peptide groups, whereas in Ph3SnHL complexes the ligand acts bidentate mode, coordinating by –COO– and NH2 groups. The Bu2SnL complexes are monomeric, and the polyhedron around the {Sn} is a Tbp with the Bu groups and Npeptide in the eq positions, while the ax positions are occupied by a carboxylic {O} and the amide {N} atom. In Ph3Sn(HL) the structure is intermediate between pseudotetrahedral and cis-Tbp, with the N-amino and two phenyl groups in ax positions. All the complexes were tested against seven cancer cell lines of human origin, viz. MCF-7, EVSA-T, WiDr, IGROV, M19, MEL A498 and H226. Ph3Sn(HL) displays the lowest ID50 values of the compounds tested. Its activity is comparable to those of methotrexate and 5-fluorouracil [117] (Table 3). All the [Bu2Sn(IV)]2+ compounds exhibit lower in vitro activities than [Ph3Sn(IV)]+ derivatives, but provide significantly higher activities than that of etoposide and cisplatin [118]. A review of the extensive literature in this field reveals two classes of organotin(IV) compounds with exceptionally high antitumor potency. [Ph3Sn(IV)]+ benzoates exhibit an in vitro antitumor activity higher than that of cisplatin and comparable with that of mitomycin C [119]. The most active of these compounds have been patented [120]. However, Ng et al. reported that, while [Ph3Sn(IV)]+ esters have a greater in vitro activity against four human tumor cell lines. This activity is independent of the structure of the ester moiety and comparable with that of Ph3SnOH, suggesting that hydrolysis product is a common, cytotoxic intermediate[121]. A large number of structurally diverse [Bu2Sn(IV)]2+ carboxylates and other Sn–O bound [Bu2Sn(IV)]2+ derivatives exhibit consistently high in vitro antitumor activity and some possess low mammalian toxicity and greater in vivo activity than those of cisplatin (selected examples can be found in [78, 107, 122-126]. This antitumor potency is, in general, structure-dependent, although for some compounds there is evidence of prior hydrolysis to a common [Bu2Sn(IV)]2+ equivalent species which is responsible for the comparable activity [119]. Di-n-butylstannylene alkoxydes also exhibited greater in vivo antitumor activity than that of cisplatin in a variety of human tumor cell lines. The first step in the mechanism of action is hydrolysis to a common cytotoxic intermediate which targets mitochondria [126]. The [Bu2Sn(IV)]2+ dihydroxybenzoates are also active against different human tumor cell lines [127]. All dihydroxybenzoates with an ortho-hydroxo group are more active against MCF-7 cells than the substituted salicylates [128]. Organotin(IV) derivatives of 2,2’-bisimidazole [129], N-methyl-2,2’-bisimidazole [130] and N,N’-dimethyl-bis-imidazole [131,132] were studied. The spectroscopic data suggest that all the complexes have analogous pseudo-octahedral geometry, with bidentate ligands, and the {R} groups are in trans positions. The [Bu2Sn(IV)]2+ derivatives proved to be the most active compounds against the well-established cell line KB. By reacting with tetraalkylammonium halides, hydrated [Me2Sn(IV)]2+, [Bu2Sn(IV)]2+ [133], [Ph2Sn(IV)]2+ [134] and [EtPhSn(IV)]2+ [135], ester derivatives of 2,6-pyridinedicarboxylic acid yield tetraalkylammonium diorganohalogeno(2,6-pyridinedicarboxylato)stannates. Both classes of compounds exhibit high in vitro antitumor activity.

Biological Activity of Organotin(IV) Compounds

Biochemical and Biological Effects of Organotins 35

Table 3: In vitro anti-tumor activities (ID50 ng/ml) values of compounds 1-7, in comparison with some clinically used reference compounds. Abbreviations are in footnotes. Cell line

1

2

3

4

5

6

7

DOX

TAX

MTX

DDPt

5FU

ETO

MEL A498

138

196

336

155

134

332

30

90

[X]A are used. In the case of (alkyl)3Sn- compounds, the response is cyclosporine-sensitive (csa) (dotted line).

 

Organotins as Mitochondrial Toxins

Biochemical and Biological Effects of Organotins 119

Figure (7) shows an example of spectrophotometrical monitoring of mitochondrial swelling. It is necessary to remark that the absorbance quenching is time-delayed and progressive (not linear). This behaviour cannot be quantified as dose/response relationship and therefore, it is not possible to verify if MTP opening is responsible for ATP synthesis inhibition. However, a help can arise from the fact that in many cases the phenomenon is cyclosporine A sensitive, since the cyclosporine inhibits the pore opening and the consequent swelling. In this case it is possible to ascertain if the swelling is responsible for the ATP synthesis inhibition, by comparing the ATP synthesis obtained in the absence, as in the experiment illustrated in Fig. (2), and in the presence of cyclosporine. If a difference is observed, it allows undoubtedly to conclude that the MTP opening is the key step responsible for the ATP synthesis inhibition. In conclusion, when more steps are inhibited, the investigation of the step responsible for the ATP synthesis inhibition, is complicate. This is the case of the organotin compounds, as we will see. ORGANOTINS TARGET MITOCHONDRIA In the last years, organotin compounds have been largely studied as a consequence of a chemical-theoretical scientific interest, and as a consequence of their practical applications. Organotin compounds are classified as R4Sn, R3SnX, R2SnX2, and RSnX3,where in industrially relevant compounds R is usually a butyl, octyl, or phenyl group, instead X is a chloride, fluoride, oxide, hydroxide, carboxylate, or thiloate function. Generally, mono-substituted organotin compounds (RSnX3) have had a very limited application, but they are used as stabilizers in poly(vinyl chloride) films. Twice substituted organotin compounds (R2SnX2) are mainly used as stabilizers in polyvinylchloride, but also as catalysts in polyurethane foams production and in silicone vulcanization processes performed at room-temperature. Tri-substituted organotin compounds (R3SnX) have biocidal properties that are strongly influenced by the toxicological valence of R-groups. The most important of these compounds show tributyl-, triphenyl- and tricyclohexyltin as substituted groups: they are used as fungicides, bactericides, antihelminthics, miticides, herbicides, molluscicides, insecticides, nematocides, ovicides, rodent repellents, and antifoulants in boat paints. The tetrasubstituted organotin compounds (R4Sn) are mainly used as intermediates in the preparation of other organotin compounds. Among the wide range of organotin compounds which have been synthesized and investigated, the (alkyl)3Sncompounds (almost all organotin have a tetravalent Sn4+ structure) have been largely utilized as antifouling compounds. Despite their efficacy, they have been substituted by the (phenyl)3Sn- compounds (TPhT) before to be definitively banned. Bans were due to the high toxicity of the (alkyl)3Sn- and of the (phenyl)3Sn- compounds and to their high environmental persistence. The (phenyl)3Sn- compounds have been also utilized as agricultural fungicides before to be banned. Therefore, the toxicological problems of organotin compounds involve mainly the (alkyl)3Snand the (phenyl)3Sn- compounds. Since the toxicity of organotin compounds, and especially of trisubstituted species, is an acute toxicity (which manifests its effects within 48 hours after exposure), there are many evidences that, as in many cases of acute toxicity, the mitochondria are the preferential target. Low doses of (alkyl)3Sn- compounds inhibit the ATP synthesis in isolated mitochondria. For TBT (tributyltin), TPT (tripropyltin) and TET (triethyltin), the concentration which 100% inhibits ATP synthesis is around 10-7 M. For (methyl)3Sn- (TMT), the inhibition is attained at around 10-6 M [11-14]. As regards the mitochondrial function responsible for the ATP synthesis inhibition, Stockdale et al. found that the (alkyl)3Sn- compounds inhibit phosphorylation more than oxidation [15]. This finding lead to exclude the RC inhibition as the step responsible for ATP synthesis inhibition. This conclusion was confirmed by the fact that the RC inhibition is a delayed effect, while the ATP synthesis inhibition is not a delayed effect [15-19]. It was also reported [15] that, in a sucrose medium, the (alkyl)3Sn- compounds had an oligomycin-like effect. The doses necessary to inhibit the ATPase were about 2 M and were close to those producing inhibition of the oxidative phosphorylation. This behaviour was subsequently confirmed by Bowman [20] who, accounting for the

 

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protein concentration, found a similar inhibitory effect at around 3 M TET. Taking into account the two experimental data, it can be supposed that the inhibitory effect of (alkyl)3Sn- compounds is due to an inhibitory effect on the ATPase (oligomycin-like effect). In a previous paper [21] Aldridge and Street observed that the ATP synthesis inhibition occurred with trialkyltin doses similar to those promoting mitochondrial swelling. In this case, a quantitative appraisal is not easy as illustrated in Section 2.2. In addition, those experiments were performed in 1964 [21] when the process of MPT opening and its cyclosporine-sensitivity were not known yet. Furthermore, the authors emphasized that the observed phenomena strongly depended on the resuspending medium. On considering the whole of data, it is possible to exclude an organotin effect on the RC; it remains the possibility of separate effects on the ATPase and on MPT opening as possible inducers of ATP synthesis inhibition. Further investigations, however, allow to predict if the MTP opening is the step responsible for ATP synthesis inhibition. Trialkyltin compounds were found to induce apoptosis in various cell types by a cyclosporine-sensitive mechanism. Since mitochondria are involved in all kinds of apoptosis, and since mitochondrial swelling is cyclosporine-sensitive [22], the hypothesis that the MPT opening is the crucial step responsible for the trialkyltin toxicity is strongly supported. Although this process should be the most probable key event, a possible role of an uncoupling effect cannot be excluded. The hypothesis that organotins may act as uncouplers was firstly advanced by Selwin et al. [23] in order to explain phenomena occurring in concomitance with mitochondrial swelling, but the molecular details to support this hypothesis were not provided. The molecular mechanism and its demonstration were given by Bragadin et al. [7,8,24] who depicted a model based on the consideration that the (alkyl)3Sn- compounds are weak acids [25]. Therefore, in solution the two forms are at equilibrium: (alkyl)3Sn-OH ⇆ (alkyl)3Sn+ + OHBeing this the situation, in respiring mitochondria,  (which is negative-inside) drives the uptake of the (alkyl)3Sn+ cation. In the matrix, the electroneutral (alkyl)3Sn-OH compound is extruded, leading to a cyclic mechanism, which gives rise to the transport of a proton in the matrix, at any cycle. The protonophore mechanism, which has been demonstrated following the same procedure utilized for DNP and other compounds as illustrated in Fig. (7), does not provides a threshold. Therefore, it can be always present, since the transport of the compounds (alkyl)3Sn-OH and (alkyl)3Sn+ occurs, like all uncouplers, through the phospholipid bilayer and it is not possible to quantify the uncoupling role on ATP synthesis inhibition. As regards other possibilities and other mechanisms, taking into account the effects found in the medium containing anions, a proposal regarding the role of (alkyl)3Sn- compounds as Cl-/OH- electroneutral exchangers was advanced by Skilleter [26] who suggested that the trialkyl compound, in the form of (alkyl)3-Sn-Cl is present in the cytoplasm and enters the mitochondria as electroneutral undissociated (alkyl)3-Sn-Cl compound. Since an equilibrium between (alkyl)3-Sn-Cl and (alkyl)3-Sn-OH exists (alkyl)3-Sn-Cl ⇆ (alkyl)3-Sn-OH the (alkyl)3-Sn-OH form is extruded from the matrix. The balance of the consequent cyclic mechanism is an Cl-/OHelectroneutral exchange. Although the mechanism, which was firstly proposed to explain transport mechanisms in isolated phospholipids [27], has some weak points (the existence in solution of the undissociated trialkyl-Sn-Cl is not reported in the literature and therefore it can be only postulated), it is important because it suggests not only a mechanism which could contribute to explain the toxic effects, but also a transport mechanism. In general, this is one of the most important and preliminary topics in the in vitro study of the toxicity of all compounds. Indeed, before studying toxicant interactions with mitochondria, it is indispensable to know, or to postulate, a transport mechanism to justify the toxicant presence in the cytoplasm and its possible transport into mitochondria. Since non physiological compounds cannot have a specific carrier (!), in order to be transported inside the cell, these compounds must utilize a physiological carrier in the phospholipidic bilayer. Both the electroneutral and the protonophore models suggest a transport mechanism without the utilization of a physiological carrier.

 

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Biochemical and Biological Effects of Organotins 121

The inner mitochondrial membrane possesses an anion uniport, known as the inner membrane anion channel (IMAC). Experiments performed by Powers and Beavis [28] evidenced that (alkyl)3Sn-compounds inhibit the IMAC channel. The efficacy of the IMAC inhibition increases with the hydrophobicity in the following order: trimethyltintriethyltintripropyltintriphenyltintributyltin In the case of tributyltin (TBT), a complete inhibition is obtained at 0.9 nmoles/mg of protein. The above efficiency order suggests that the binding site of IMAC may be accessible from the phospholipid bilayer. The same investigation [28] confirmed that the ATPase was inhibited by 0.75 nmoles TBT/mg of protein. The comparison between the IMAC and ATPase inhibition data lead to exclude that the effects on mitochondria are due to the IMAC inhibition. Toxicity of Bis(tributyltin) Oxide (TBTO) The interactions of trialkyltin oxide compounds (used in wood preservation, marine antifouling, disinfection of circulating industrial cooling waters) with mitochondria were repeatedly studied [29,30]. All the trialkyl compounds inhibit the mitochondrial respiration [29]. The effects of TBTO and of the dibutyl diisooctyl thioglycolate are localized in the terminal step of the RC. Other alkyltins inhibit before the cytochrome c. The ATP synthesis inhibitory effect of TBTO was found to be similar to that exerted by oligomycin, a potent ATPase inhibitor. These data are in agreement with those obtained by Penninks et al. [31,32]; who suggested that the TBTO target is the mitochondrial ATPase which is inhibited by 0.5 M TBTO. Toxicity of Trialkyltin Derivatives Although the trialkyltin compounds are persistent in the environment (and this is one of the reasons for their banning), they undergoes a slow decomposition to form, after some weeks, the di- and monoalkyl derivatives: (alkyl)3SnCl  (alkyl)2SnCl2  (alkyl)SnCl3 This situation offers the possibility that, like other toxic compounds, the decomposition products can be toxic and, in some cases, more toxic than the starting compounds. The acute toxicity of (butyl)2SnCl2 (DBTC) was in vitro studied. The pattern is complicate, since, as in the case of the precursor TBTC, DBTC inhibits many mitochondrial functions. An uncoupling effect was observed from a level of 8.3 nm DBTC/mg of protein [31], but the oxidation of the -ketoglutarate was inhibited by 0.8 nm DBTC/mg of protein. This finding lead the authors to conclude that the uncoupling effect, although present, was not quantitatively crucial to inhibit ATP synthesis. Penninks and Seinen [32] found that all examined dialkyltin compounds, namely dimethyltin (DMT), diethyltin (DET), dibutyltin (DBT) and dioctyltin (DOT) inhibited the pyruvate dehydrogenase system in mitochondria. Analogously, Cain et al. [33] found, in addition to the -ketoglutarate and pyruvate dehydrogenase complex inhibition which leads to the inhibition of the pyruvate and -ketoglutarate oxidation, an inhibition of the oligomycin-sensitive complex at 2.8 n mol DBT/mg protein. The authors suggested that this was the effect responsible for the ATP synthesis inhibition. The inhibiting concentration value, if compared to that reported for the ATPase inhibition by TBT (about 3 M), would suggest that DBT formed from TBT decomposition could be even more toxic than the starting compound. The ATPase inhibition was confirmed by Cain et al. [34], while Aldridge and Cremer assessed that DBT inhibited oxidative phosphorylation as a consequence of the inhibitory effect of the dihydrolipoate cofactor in pyruvate and ketoglutarate dehydrogenase complexes [35]. It was observed that DBTC induces apoptosis [36]. Since mitochondria are involved in all kinds of apoptosis, this situation supports the possibility that the mitochondrial permeability pore (MTP pore) is the target of DBTC. This hypothesis is not supported by the results of Tomiyama et al. [37] who suggested that TBT induces apoptosis, while DBTC induces necrosis. Toxicity of Triphenyltin (TPhT) As regards the (phenyl)3Sn- compounds (TPhT), as in the case of (alkyl)3Sn- compounds, many effects were observed. The pattern is complicate and, surprisingly, in some cases, the effects (RC inhibition), even if slightly, depend on the species utilized for the mitochondrial preparation [38].

 

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RC inhibition was first observed by Stockdale et al. [15], but the effect was time-dependent. Furthermore, since RC inhibition did not depend on the organotin compound, it was probably a consequence of the swelling. This lead to the conclusion that RC inhibition was a secondary effect. RC inhibition at the level of the cytochrome oxidase, was also observed by Barranco et al. [39]. Low doses of TPhT inhibit ATP synthesis [40] and induce apoptosis. As far as we are aware detailed data regarding specific interactions with the RC ase are not reported in the literature. Probably, as in the case of (alkyl)3Sn- compounds, the ATP synthesis inhibition is correlated with the MPT pore opening [40]. Low doses of TPhT were found to inhibit the ATPase in submitochondrial particles which are obtained by ultrasound treatment of mitochondria [41]. Probably the swelling observed by Stockdale et al. [15] and by Zazueta et al. [42] in isolated mitochondria (although not cyclosporine-sensitive [43]), could be correlated with the MPT opening. The in vitro induced cell apoptosis by treatment with TPhT [40,43] was probably also due to the same phenomenon. The whole of data induce to suppose that the toxic effect of TPhT is due to the MPT opening in mitochondria and consequent induction of apoptosis. CONCLUSIONS Generally, toxicity of organotin compounds is due to an impairment to biological functions largely common to animal and plants, thus justifying the fact that many biological structures in different tissues and species exhibit the same sensitivity to the compounds. The fact that mitochondria are functionally and structurally similar in all eucaryots, as discussed in the first part, is an indispensable requisite in order to explain the widespread toxicity of organotin compounds. A transport mechanism should be necessarily demonstrated or proposed to justify the presence of organotin compounds in cytoplasm and mitochondria. The utilization of physiological carriers has never been demonstrated, but, taking into account the properties of the biological membranes (even if the composition of the lipid bilayer can be different), organotin transport can be explained by means of the uncoupling mechanism or the Cl-/OH- exchange mechanism. In isolated mitochondria, the (alkyl)3-Sn- compounds inhibit all the steps involved in the ATP synthesis mechanism. The individuation of the step requiring the lowest effective dose is further complicated by the dependence of the responses in the presence of anions. A solution of the problem may come from experiments in isolated cells, where the induced apoptosis is cyclosporine-sensitive. Since an analogous cyclosporine-sensitivity was found in isolated mitochondria, the opening of the MPT pore is thought to represent the crucial step responsible for the ATP synthesis inhibition. This statement does not exclude the concomitance of other mechanisms, such as a protonophore effect and an Cl-/OH- electroneutral exchange, but at this stage it is impossible to quantitatively evaluate their effect. Finally, the toxicity of the products formed by decomposition of the (alkyl)3Sn- compounds, namely of partially dealkylated derivatives, has been undoubtedly assessed. Unfortunately contradictory results and controversial interpretations were provided and the mechanisms involved in their effects on mitochondrial functions are still a matter of debate. REFERENCES [1] [2] [3] [4]

Brush SG. The origin of the planetesimal theory: Origi Life Evol B 1977; 8:pp. 3-6. Miller SL. Production of amino acids under possible primitive earth conditions. Science 1953; 117: pp. 528-9. Mitchell P. Keilin’s respiratory chain concept and its chemiosmotic consequences. Science 1979; 206:pp. 1148-55. Nishimura M, Ito T, Chance B. Studies on bacterial photophosphorylation III A sensitive and rapid method of determination of phosphorylation. Biochim Biophys Acta 1962; 59: pp. 177-84. Moreno A, Madeira VM. Mitochondrial bioenergetics as affected by DDT. Biochim Biophys Acta 1991; 1060:166-74. Shannon RD, Boardman GG, Dietrich AM, et al. Mitochondrial response to chlorophenols as a short-term toxicity assay. Environ Toxicol Chem 1991;10: pp. 57-66. Bragadin M, Argese E, Orsega E. A simple in vitro method for selective detection of phenols in water using the mitochondrial membrane from rat liver. Environ Technol 1991; 12: pp. 777-81.

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Bragadin M, Marton D, Manente S, et al. A bioanalytical method for the monitoring of metalalkyls in solution. Anal Biochem 1999; 268: pp. 420-3. Zoratti M, Szabo I. Mitochondrial permeability transitions: how many doors to the house? J Bioenerg Biomemb 1994; 26: pp. 543-53. Scorrano L, Petronilli V, Bernardi P. On the voltage dependence of the mitochondrial permeability transition pore. A critical appraisal. J Biol Chem 1977; 272: pp. 12295-9. Aldridge WN. Liver and brain mitochondria. Biochem J 1957; 67: pp. 423-31. Aldridge WN. The biochemistry of organotin compounds. Biochem J 1958; 69: pp. 367-76. Aldridge WN, Therefall CJ. Trialkyltins and oxidative phosphorylation. The [32P]phosphate-adenosine triphosphateexchanges. Biochem J 1961; 79: pp. 214-21. Moore KE, Brody TM. The effect of triethyltin on oxidative phosphorylation and mitochondrial adenosine triphosphatese activation. Biochem Pharmacol 1961; 6: pp. 125-33. Stockdale M, Dawson AP, Selwin MJ. Effects of trialkiltin and triphenyltin compounds on mitochondrial respiration. J Eur J Biochem 1970; 15: pp. 342-51. Rose MS, Aldridge WN. The effect of anions on the inhibition by triethyltin of various mitochondrial functions and relationship between this inhibition and binding of triethyltin. Biochem J 1972; 127: pp. 51-9. Cremer JE. Biochemical studies on the toxicity of tetraethyllead and other organo-lead compounds. Br Indust Med 1959;16: pp. 191-201. Ueno S, Kashimoto T, Susa N, et al. Effects of butyltin compounds on mitochondrial respiration and its relation to hepatotoxicity in mice and guinea pigs. Toxicol Sci 2003; 75: pp. 201-207. Moore AL, Linnet PE, Beechey RB. Dibutylchloromethyltin chloride, a potent inhibitor of electron transport in plant mitochondria. J Bioenerg Biomemb 1980; 12: pp.309-32. Bowman EJ. Comparison of the vacuolar membrane ATPase of Neurospora crassa with the mitochondrial and plasma membrane ATPases. J Biol Chem 1982; 258:pp. 15238-15245. Aldridge WN, Street BW. Biochemical effects and properties of trialkyltins. Biochem J 1964; 91: pp. 287-297. Nishikimi A, Kira Y, Kasahara E, et al. Tributyltin interacts with mitochondria and induces cytochrome c release. Biochem J 2001; 356: pp. 621-6. Selwin MJ, Dawson AP, Stockdale M, et al. Chloride-hydroxide exchange across mitochondrial, erythrocyte and artificial lipid membranes, mediated by trialkyl- and triphenyltin compounds. Eur J Biochem 1970; 14: pp.120-6. Bragadin M, Marton D. A proposal for a new mechanism of interaction of trialkyltin (TAT) compounds with mitochondria. J Inorg Biochem 1997;68: pp.75-8. Tobias RS. The Chemistry of Organometallic Cations in Aqueous Media. In: Organometals and Organometalloids. Brinckman FE, Bellama JM (eds) Washington ACS Symposium Series 82, 1978; pp.130-148. Skilleter D. The influence of adenine nucleotides and oxidizable substrates on triethyl-mediated chloride uptake by rat liver mitochondria in potassium chloride media. Biochem J 1976; 154:pp. 271-6. Karniski LP. Hg2+ and Cu2+ are ionophores, mediating Cl-/OH- exchange in liposomes and rabbit renal brush border membranes. J Biol Chem 1992; 267: pp. 19218-25. Powers MF, Beavis AD. Triorganotin inhibit the mitochondrial inner membrane anion channel. J Biol Chem 1991;266: pp. 17250-6. Rotemberg IS, Mazaev VT, Shlepina TG. Peculiarities of alkyl tin effects on respiration and oxidative phosphorylation of rat liver mitochondria. Ukr Biokhim Zh 1978;50: pp. 695-700. Veiga A, Pinto AF, Loureiro-Dias MC. Tributyltin oxide affects energy production in the yeast Rhodotorula ferulica, a utilizer of phenolic compounds. Can J Microbiol 1997; 43: pp.683-7. Penninks AH, Vershuren PM, Seinen W. Di-n-butyltindichloride uncouples oxidative phosphorylation in rat liver mitochondria. Toxicol Appl Pharmacol 1983; 70: pp. 115–20. Penninks AH Seinen W. Toxicity of organotin compounds. IV. Impairment of energy metabolism of rat thymocytes by various dialkyltin compounds. Toxicol Appl Pharmacol 1980;56: pp. 221-31. Cain K, Hyams RL, Griffiths DE. Studies on energy-linked reactions: inhibition of oxidative phosphorylation and energylinked reactions by dibutyltin compounds. FEBS Lett 1977; 82: pp.23-8. Cain K, Partis M, Griffiths DE. Dibutylchloromethyltin chloride, a covalent inhibitor of the adenosine triphosphatase. Biochem J 1977; 166: pp. 593-602. Aldridge WN, Cremer JE. The biochemistry of organotin compounds. Diethyltin dichloride and triethyltin sulphate. Biochem J 1955;61: pp.406-18.

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Gennari A, Viviani B, Galli CL, et al. Organotins induce apoptosis by disturbance of [Ca2+]i and mitochondrial activity, causing oxidative stress and activation of caspases in rat thymocytes. Toxicol Appl Pharmacol 2000;169: pp. 185-90. Tomiyama K, Yamaguchi A, Kuriyama T, et al. Analysis of mechanisms of cell death of-lymphocytes induced by organotin agents. J Immunotoxicol 2009;6: pp. 184-93. Van Graft M, Leeuwangh P. The action of the fungicide triphenyltinchloride on respiration in fish liver mitochondria. Meded Fak Landbouwwet R.U Gent 1977; 42: pp. 1705-14. Barranco J, Darszon A, Gomez-Puyou A. Extraction of mitochondrial protein-lipid complexes into organic solvents: Inhibition of cytochrome oxidase electron transport by dicyclohexylcarbodiimide and triphenyltin chloride. Biochem Biophys Res Commun 1981;100: pp. 1402-8. Robertson JD, Orrenius S. Role of mitochondria in toxic cell death. Crit Review Toxicol 2000;30: pp. 609-27. Byngton KH Effects of triphenyltin compounds on adenosine triphosphatase activity of beef heart submitochondrial particles. Biochem Biophys Res Commun 1971; 42: pp.16-22. Zazueta C, Reyes-Vivas H, Bravo C, et al. Triphenyltin as inductor of mitochondrial membrane permeability transition. J Bioenerg Biomembr 1994; 26: pp. 457-62. Robertson JD, Orrenius S. Molecular mechanisms of apoptosis induced by cytotoxic chemicals. Crit Rew Toxicol 2000; 30: pp. 609-27.

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125

CHAPTER 9 Organotins and Hydromineral Homeostasis in Aquatic Animals Mark G.J. Hartl* Centre for Marine Biodiversity and Biotechnology, School of Life Science, Heriot-Watt University, Edinburgh, Scotland, UK Abstract. Enzymes, non-enzymatic proteins and other organic molecules are vital components in living cells. Their respective function depends on specific spatial configurations which are linked to intracellular conditions. Any fluctuation of these conditions, beyond certain threshold values, such as a disruption of ionic regulatory mechanisms, can lead to the destabilisation of a finely balanced intracellular dynamic physiological equilibrium or homeostasis. Hydromineral homeostasis in aquatic organisms is maintained by a complex endocrine controlled array of specialised cross-membrane ion transport systems and the regulation of membrane water permeability. Depending on how aquatic organisms maintain hydromineral homeostasis, they can be roughly divided into two groups: osmoconformers and osmoregulators; the former are mostly invertebrates with high water permeability, the latter include some invertebrates and most fish species, whose permeable external epithelia are usually restricted to the gills. Other important organs involved in hydromineral regulation include the intestine and the various phyla-specific organisational types of renal systems. Environmental concentrations of organotin compounds, such as tributyltin and triphenyltin, have been shown to interfere with the maintenance of hydromineral homeostasis by inhibiting ATPases and affecting membrane permeability for water. The present chapter reviews the impact of organotin exposure on fresh- and seawater organisms of various phyla by examining the histophathological, physiological and molecular interactions of organotin compounds with relevant enzymes, membranes, the endocrine system, and the consequential ramifications for individuals, populations and community structure in aquatic ecosystems.

Keywords: Hydromineral regulation - gills - membranes- osmosis - ionic flux - organotins – ATPase. INTRODUCTION The vast majority of the Earth’s water is found in the oceans, but also exists on landmasses as glacial ice, groundwater, freshwater and saline lakes, and as vapour in the atmosphere. Despite the very dynamic nature of water, giving rise to a complex hydrological cycle, discrete hydrological domains can be distinguished: seawater, freshwater and, where the two meet and mix, brackish water. These domains have very distinct characteristics in terms of ionic composition and pH. The most important anions in freshwater are CO32- and HCO32-; SO42-, Cl- and NO3- are of lesser importance. Among the cations Ca2+ dominates, followed by Mg2+, Na+ and K+. Over eons, the easily soluble salts (e.g. NaCl and Na2CO3) have been washed out and are now in the _oceans, leaving behind the poorly soluble salts, such as CaCO3. In seawater, the most important ions are the anions Cl , SO42-, HCO32-, Br- and the cations Na+, Mg2+, Ca2+, K+ and Sr2+. Together these ions constitute 99% of the dissolved salts in seawater and, because they maintain a nearly constant proportionality, independent of the salinity, are often referred to as the “conserved” elements, as opposed to the variable or “non-conserved” elements, such as nutrients and dissolved gases (Table 1). The water content of living organisms approximately ranges between 40 and 99%. The physico-chemical characteristics of water covey a remarkable capacity as a solvent of both inorganic and organic substances, and provide the medium for the chemical reactions associated with life. Intraorganismal water contains solutes involved in cell metabolism and structural components, such as proteins, the solubility of which can be influenced by the presence of particular ions, while certain metals may act as co-factors in enzymatic reactions. Solutes may also provide chemical gradients used as potential energy stores and signal transduction, as well as in the regulation of the osmotic mobility of water. *Address correspondence to Mark G.J. Hartl: Centre for Marine Biodiversity and Biotechnology, School of Life Sciences, Heriot-Watt University Riccarton,EH14 4AS Edinburgh, Scotland UK; E-mail: [email protected]

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Table 1: Ionic composition and osmotic concentrations of selected environmental and biological media

1)

Seawater Osmotically important ions (mMol L-1) Ca2+ K+ ClNa+

Mg2+

468

10

10

545

53

SO42-

Species

Refs

Mnemiopsis leidyi (Ctenophora)

[7]

-

102

94

103

100

77

Aurelia aurita (Cnidaria)

[8]

-

96

106

104

97

47

Ruditapes philippinarum (Bivalvia)

[9]

-

-

-

-

-

-

Epinephelus coioides (Pisces)

[10]

-

-

-

-

-

-

Platchthys flesus (Pisces)

[11, 12]

150

-

3

151

-

-

Scophthalmus maximus (Pisces)

[13]

161

-

6

145

-

-

Salmo salar (Pisces)

[14]

148

-

-

128

-

-

Fundulus heteroclitus (Pisces)

[15]

260

-

-

-

-

-

Freshwater1 Osmotically important ions (mMol L-1) Ca2+ K+ ClNa+

Mg2+

SO4-

0.22

0.32

0.06

0.2

0.16

0.11

Craspedagusta sowerbyi

[16]

14.5

-

0.6

-

-

-

Dreissena polymorpha (Mollusca)

[17]

14

5

0.5

15

-

-

Lymnea truncata (Mollusca)

[18]

49

16.6

2.4

32

8.4

-

Cambarus diogenes (Crustacea)

[19]

190

-

-

250

-

-

Salvelinus sp (Pisces)

[20, 17]

-

-

-

117

-

-

Platchthys flesus (Pisces) Salmo salar (Pisces) Fundulus heteroclitus (Pisces)

[21, 11] [22, 14] [15]

123 132 180

2.7 -

2.9 -

148 135 -

-

-

world average river water[23].

Cells are the basic units of all living beings and consist of a water-insoluble phospholipid structure, the plasma membrane that encloses a space separating it from the surrounding media. The plasma membrane is an approximately 75Å thick complex, highly organised and dynamic structure made of lipid and protein molecules that are in constant motion (see Chapter 5). The plasma membrane and its associated proteins allow for the maintenance of a nearly constant internal (intracellular) environment essential for many life-supporting biologically important processes, by regulating the passage of water and material in and out of the cell. In addition, most tissue and blood cells are bathed in fluids that serve as osmotic buffers for cells, preventing potentially harmful fluctuations to the intracellular milieu. Some of the materials transported across the plasma membrane possess electrical charges and are selectively accumulated in either the intracellular or extracellular fluids, thus affecting a charge or potential difference across the membrane. Such charges may aid or prevent the passive movement of charged materials, such as ions, across the membrane. Whereas passive transport of ions occurs mainly through ion co-transporters, active transport against either electrical or concentration gradients, is an energy-consuming process facilitated by specialised membrane bound ion pumps, mainly ATPases [1]. As will be shown later, many of these processes are under endocrine control. Triorganotin compounds, such as tributyltin (TBT) and triphenyltin (TPhT) are highly toxic man-made biologically active substances [2]. In an aquatic context, they have been widely used as active ingredients in self-polishing copolymer antifouling paint formulations and as such, represent one of the most toxic substances intentionally introduced into the aquatic environment[3]. TBT and TPhT have been shown to interfere with the hydromineral regulation of aquatic organisms through endocrine disruption, ATPase inhibition and plasma membrane destabilisation. The present chapter reviews the impact of triorganotin exposure on the hydromineral regulation of fresh- and seawater organisms of various

 

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Biochemical and Biological Effects of Organotins 127

phyla, by examining the histophathological, physiological and molecular changes induced by interactions of TBT and TPhT compounds with relevant enzymes, membranes, the endocrine system, and the consequential ramifications for individuals, populations and community structure in aquatic ecosystems. HYDROMINERAL HOMEOSTASIS In fresh water the blood of most fish species is hyperosmotic with respect to the external medium, causing an increase in water influx and a loss of electrolytes. In seawater the blood is hyposmotic with respect to the external medium inducing the loss of water and a diffusional influx of electrolytes through the intestinal epithelium. Osmoregulating organisms, as opposed to osmoconforming organisms, employ a variety of mechanisms to regulate osmotic balance and body fluid volume, thereby maintaining constant intracellular conditions enabling efficient cellular function. Such mechanisms include, as well as strategies for ionic regulation, the control of drinking rates, urine production and membrane permeability [4-6]. The normal osmolarity of the body fluids of aquatic species can range between ~20 mOsmol L-1 in freshwater cnidarians to 1,100 mOsmol L-1 in seawater molluscs [24, 25] (Table 1). Thus, in freshwater, all organisms are hypertonic in relation to the osmolarity of the surrounding medium and maintain this condition through hyperosmoregulation. In seawater, in terms of osmolarity, two conditions can be distinguished: hypoosmotic osmoregulators, whose body fluid osmolarity is hypotonic in relation to the surrounding medium, and osmoconformers, whose body fluids are isosmotic towards seawater. Some species have developed considerable tolerance to fluctuating external osmotic conditions, usually encountered during changes to salinity, such as those seen in estuaries over a tidal cycle, and are referred to as euryhaline; conversely, stenohaline species require relatively static osmotic conditions and are therefore unlikely to be found in environments that are prone to frequent and often wide changes in salinity, such as estuaries and tide pools. As indicated above aquatic organisms are subject to constant osmotic exchanges with the environment, which are driven by physico-chemical gradients between the body fluids and the surrounding medium, and affect the movement of water and solutes (nutrients, metabolites, ions) in and out of the body. Many of these exchange processes are obligatory and, left unchecked, may lead to the disruption of internal homeostasis: freshwater organisms tend to accumulate water and lose ions, while the opposite occurs in seawater organisms. Consequently, aquatic organisms need to be able to control these processes in order to develop a dynamic steady state between internal and external fluxes, thus facilitating the maintenance of internal homeostasis. The degree of intervention required will depend on the osmotic anatomy and will vary with species. Generally, osmotic exchange occurs across those surfaces exposed to the external medium, the integument, and is related to surface area and its permeability for water and solutes. The forces facilitating the exchanges of water and solutes across the integument of aquatic organisms include diffusion, osmosis, facilitated diffusion and active transport, the manipulation of which forms the basis for the regulation of hydromineral homeostasis in aquatic organisms. Ionic Regulation Ionic regulation in fish has been extensively reviewed [26-30]. Under hypoosmotic conditions (freshwater) aquatic organisms may lose ions to and gain ions under hyperosmotic conditions (seawater) from the external medium. The net passive ion exchange occurs as a consequence of physico-chemical gradients across the integument and through the process of eating and drinking. The European flounder, Platichthys flesus is a highly euryhaline catadromic fish that can tolerate rapid fluctuations in external salinity over a wide range. Although juveniles will tend to avoid freshwater where possible, because of the increased permeability of their integument [31] and the need to channel as much energy into growth rather than ionic regulation, adults spend much of their time in estuaries, often way beyond the tidal limit, in what is essentially freshwater, but can also be found in seawater where they spawn [32, 33]. Consequently, ionic regulation has been extensively studied in Platichthys flesus [34-36] and other euryhaline fish species, such as Fundulus heteroclitus [37-39] and invertebrates such as Tigriopus brevicornis [40]. Integument permeability can vary greatly between phyla and even between species within phyla and during species onthogeny [31, 32, 41]. Whilst the regulation of integument permeability plays a major role in volume regulation of euryhaline osmoconforming invertebrates [42-49], the intact skin of most adult fish species is a complex layered structure

 

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containing numerous mucus glands, often covered with scales, and is considered to be largely impermeable [31, 50]. The majority of research points towards gills of fish, rather than the skin, as the principle pathway for hydromineral exchange [51-54]. Fish gills consist of delicate, highly vascularised internal epithelia, with the primary function of gas exchange [5557]. Three distinct elements make up the gill structure: the skeletal elements of gill arch and filaments that support the epithelia, and the gill epithelium itself whose surface area is greatly increased by folds branching out either side of each filament, the so-called lamellae. The secondary epithelium is a two layered structure, characterised by highly differentiated pavement cells, that rests on the basal laminar of pillar cells which stabilise the epithelial folds, forming the lamellae. The large surface area and relative thinness contributes to the maximised permeability of the secondary epithelium for gases and at the same time less permeable for ions, particularly in seawater species. The primary epithelium is multi-layered and surrounds all the filaments and the interlamellar spaces [58]. Interspersed within the primary epithelium are various functionally distinct cell types whose frequency and distribution may change with species and external conditions, such as mucus secreting goblet cells, non-differentiated cells and mitochondria-rich cells, the latter constituting the basic element of branchial ionic regulation. Mitochondria-rich cells, first described by Keys and Wilmer [59] as chloride secreting cells (hence-forth referred to as chloride cells) occupy less than 15% of the total area of epithelial cells exposed to the environment [60], although under osmotic challenge, the gas exchange may be compromised by the proliferation of chloride cells and subsequent increase of blood-to-water diffusion distance in favour of osmoregulatory adjustment [60, 61]. Chloride cells are characterised by an abundance of mitochondria associated with a densely branched tubular system that communicates with the basolateral membrane, an array of sub-apical vesicles and a large ovoid nucleus [62-65]. The apical membrane of each cell is characterised by the presence of microvilli and is firmly connected to neighbouring pavement cells forming a tight junctional complex. In seawater, the apical chloride cell membrane is buried in a crypt that appear as holes in the pavement cell layer. Furthermore, in seawater-adapted species, chloride cells develop an association with accessory cells, which develop rapidly during seawater adaptation, forming chloride cell complexes [66-68]. The membranes of chloride and accessory cells form a much weaker junctional apparatus than the tight junctions found in freshwater adapted species and form the leaky junctions of the paracellular pathway [68]. In euryhaline species, such as European flounder [69, 70], tilapia [71], guppy [72] and Fundulus heteroclitus [73], chloride cells undergo morphological and physiological changes and the differentiation of various subtypes has been observed [72]. Whereas in seawater adapted fish, large chloride cells are found at the base of the lamellae, in some species, freshwater adaptation is accompanied by a reduction in size of these interlamellar chloride cells and the development of chloride cells within the lamellar epithelium [69, 70, 74, 75]. There is an increasing amount of evidence pointing towards the hormonal control [68; 76-78] of chloride cell dynamics occurring during seawater or freshwater adaptation: cortisol [79-81] and growth hormone [79, 80] play a role in the differentiation (proliferation) of seawater chloride cells and prolactin (PRL) [82] that inhibits the proliferation of seawater chloride cells and promotes the differentiation of freshwater chloride cells. This notion may be complicated by the fact that the degree of responsiveness to hormonal control may vary in different chloride cell types within the same species, possibly due to differential upregulation of cortisol receptors [78, 83]. More recent work has shown that cortisol also has a regulatory effect on V-type H+-ATPase (see below) and may therefore also be an important endocrine component of ion uptake in smolting salmon [84] and other freshwater-adapted species [85]. In seawater, the driving force teleost gills is provided by a basolateral Na+/K+-ATPase [86], _ for NaCl secretion by seawater _ + + co-transporter enabling Cl entry across the basolateral and an anion channel in_ the apical a basolateral Na , K , 2Cl _ membrane permitting Cl to flow down its electrochemical gradient out in to seawater. Na+ follows that of Cl passively down its electrochemical gradient via a cation-selective paracellular pathway located in the tight junctions between mature chloride cell complexes formed by chloride cells and accessory cells [87]. The rapid increase of Na+/K+-ATPase activity following transfer of juvenile Platichthys flesus to seawater [88, 89] suggests that a post-transcriptional activation process may be involved as has been observed in killifish [90, 91]. In freshwater, the application of bafilomycin, a specific inhibitor of V-type H+-ATPase proton pumps [92], to the exterior medium reversibly inhibited the activity of V-type H+-ATPase and Na+ uptake in carp [93], thus confirming V-type H+ATPase as the driving force for NaCl uptake in freshwater teleosts [94, 95]. There is also evidence that the enzyme is involved in acid-base regulation [26]. V-type H+-ATPase has been immunolocalised to the apical areas of gill lamellae, in particular chloride and pavement cell membranes [96, 97], although the precise apical cellular location appears to be species-specific [26, 98]. Sodium is then driven through an epithelial sodium channel down its electrochemical gradient, provided by the V-type H+-ATPase into the cell. Sodium uptake across the basal-lateral membrane of the chloride cell is then facilitated by Na+/K+-ATPase [97].

 

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Biochemical and Biological Effects of Organotins 129

Divalent ions, such as calcium, play important roles in metabolic homeostasis in both freshwater and seawater organisms [26, 60, 99-101]. Much like terrestrial vertebrates, teleost fish require calcium, which is deposited in bones and scales, to grow. Calcium ions can be taken up directly from water through the gills or through the intestine by drinking water or absorbed from food [99]. In seawater fish, up to 30% of Ca2+ uptake can occur through the intestinal epithelia, especially in females during reproduction [102]. In freshwater species that do not drink under often hypocalcic conditions, Ca2+ is actively taken up across the gill epithelia in a multistep process, involving passive entry of Ca2+ through apical chloride cell membrane channels, followed by the energy-dependent movement across basolateral membrane via a high-affinity Ca2+-ATPase or a lower-affinity Na+/Ca2+-exchanger [60]. Any surplus Ca2+ taken up by seawater adapted species is excreted through the extrabranchial routes of the renal system and/or the intestinal tract [99, 103]. Water Balance As described above, with respect to their external environment, hyperosmotic organisms tend to take up water and hypoosmotic organisms will lose water through diffusion via permeable membranes. Water flux between organisms and their environment is tightly regulated by the balance between membrane permeability, drinking and urine production rates and are under endocrine control [78, 104]. According to their speed of action, osmoregulatory hormones can be divided into two groups: fast-acting hormones, secreted immediately following a change in external salinity, have rapid onset effects and half-lives of only minutes and regulate channels and target transport epithelia through phosphorylation or dephosphorulylation. These include amongst others catecholamines, natriuretic peptides, urotensins, arginine vasotocin (AVT) and angiontensin II (ANG II). ANG II in particular is active in the renin-angiotensin system (RAS) that acts as a powerful vasoconstrictor increasing arterial blood pressure through activation of the sympathetic nervous system [105], and relates to water balance, playing an important role in inducing spontaneous drinking behaviour in fish [106, 107]. AVT on the other hand reduces renal water loss in teleost fish by reducing the number of filtering glomeruli which leads to a reduction in urine production during adaptation to seawater [108]. The second group are slow or long-term active hormones whose level gradually increases and remains elevated for extended periods of time following a change in external salinity and include steroid hormones, such as cortisol, and protein hormones, including growth hormone (GH) and prolactin (PRL). PRL regulates the number of channels in transport epithelia and also the morphological transformation of osmoregulatory tissues during adaptation to a changed osmotic environment. _ PRL is associated with freshwater adaptation, by decreasing the osmotic permeability to water and increase Na+ and Cl uptake (see above) across transport epithelia in euryhaline fish species and is particularly enhanced in stenohaline freshwater fish. Gills, as the main permeable epithelia exposed to external environment, are particularly important in regulating water balance in aquatic organisms [109]. As outlined above, the outer-most layer of gill epithelia is constituted by the plasma membranes of pavement cells. According to the fluid mosaic model [110], plasma membranes consist of a fluid lipid bilayer of phospholipid molecules in which the proteins are embedded or otherwise associated, the position of which changes constantly. The fluidity of the membrane depends on the types of lipids present: a higher proportion of saturated lipids will create a more rigid membrane, whereas an increase in unsaturated and polyunsaturated lipids results in a more fluid membrane. An important constituent in the regulation of the membrane fluidity is cholesterol, a slightly amphiphatic largely hydrophobic steroid. Owing to the presence of a single hydroxyl group, cholesterol is able to associate with the hydrophilic heads of the phospholipids with the hydrophobic remainder of the molecule fitting between the lipid hydrocarbon chains. By acting as a spacer between the lipid hydrocarbon chains, cholesterol’s condensing effect contributes to plasma membrane viscosity [111, 112]. The phenomenon of membrane viscosity and its opposite, membrane fluidity, is of great significance in determining the water permeability of plasma membranes, because the degree to which a phospholipid membrane is permeable for water and other small hydrophilic molecules is directly proportional to its fluidity. In addition, aquaporins, a specialised group of membrane-integrated proteins that let water and small solutes pass through the phospholipid membranes of transport epithelia [113-115], can account for a large amount of observed water permeability [113], and in teleosts their expression has been shown to be regulated by cortisol [116]. ORGANOTINS Organotins are organometallic compounds where Sn (in the majority of cases Sn+4) is covalently bound to one or more carbon atoms of any aryl or alkyl group (R), an inorganic derivative, such as a halide or carboxylate, and are represented by the chemical structures RSnX3, R2SnX2, R3SnX and R4Sn. Organotin compounds have three main uses: as catalysts to stabilize PVC polymers, antifungal agents and as biocides to protect plants from insects and immersed structures from fouling [2, 117]. Triorganotin compounds consist of a Sn (IV) atom covalently bound to three organic moieties and an

 

130 Biochemical and Biological Effects of Organotins

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associated anion. They are bioactive at very low concentrations [118] and therefore were marketed in the early 1970s as the perfect biocides, finding heavy application in the maritime industry [117] (see Chapter 2). The impact of triorganotins, in particular tributyltin (TBT) and triphenyltin (TPhT), on non-target organisms in the marine environment, such as imposex in female dogwhelks (Nucella lapillus) [119] and shell-thickening in Pacific oysters Crassostrea gigas [120-122], led initially to an International Marine Organization (IMO)-initiated partial ban at the end of the 1990s and a recently internationally ratified total ban on the use of these compounds as active ingredients in antifouling paint formulations1 (see Chapters 1 & 5). TBT consists of three n-butyl chains covalently bound to a single Sn (IV) via Sn-C bonds and a moiety conveying a univalent positive charge. The main commercial formulations are bis(tributyltin) oxide (TBTO), tributyltin-acetate (TBTAc), tributyltin halides, namely fluoride (TBTF) or chloride(TBTC), and as a co-polymer with methymethacrylate (TBT-M) [123]. Similarly, TPhT contains three convalently bound Sn-C phenyl groups, with the main industrial TPhT derivatives being acetate (TPhTA), chloride (TPhTC) and hydroxide (TPhTH) [124, 125]. Organotin Compounds in Aqueous Environments The potential environmental risk of a substance to organisms is a function of exposure and toxicity. Underpinning these factors, however, are bioavailability, bioconcentration and accumulation, pathways of exposure and persistence. The persistence of any xenobiotic substance is a function of the sum of removal mechanisms acting upon them, biotic: adsorption, uptake and transformation by biota [126-128]; physical: volatilization, freezing and adsorption to suspended particulate matter [129, 130]; chemical: chemical and photochemical reactions [131]. The speed of degradation and removal can be influenced by a number of factors, such as the density of suspended particulate, dissolved organic and colloidal matter, pH, CO2, salinity and hydrological conditions [132, 133]. The fate of organotin compounds in particular in the natural environment is complicated by the large number of possible species [134, 135]. Of the species found in aqueous solution, the oxide, chloride and carbonate forms are of particular significance in the aquatic environment [123]. The solubility of the various species in water is very different and plays an important role in determining their fate [136], with the oxide species being generally more water soluble than the chlorides [137]. The occurrence, persistence and general fate of TBT and TPhT in freshwater [132, 138-140], seawater [130, 133, 141-143] and estuaries [144-146] has been extensively reviewed elsewhere. IMPACT OF ORGANOTIN ON HYDROMINERAL REGULATION Biochemical activity of various triorganotin compounds varies depending on organ system and structure under exposure, but generally, TBT and TPhT are the most potent [147] (see also Chapter 2 of this volume). The general mechanistic considerations of TBT and TPhT toxicity have been widely reviewed in the literature: endocrine disruption [122, 148150], immunotoxicology [151-155], cytotoxicity [156-159], genotoxicity [160, 161] and enzyme inhibition [122, 148-150, 162, 163] (see Chapters 1, 5, 7). Many of these modes of toxicity can interfere with the physiology of hydromineral regulation, whereby the main routes of exposure are the interaction of organotin compounds with gill membranes and intestinal mucosa. Furthermore, cell shrinkage observed in many tissues following organotin exposure is an important effect of TBT as it is likely to cause the indirect inhibition of many cellular membrane-bound ion pumps (ATPases) [164]. Ionic Regulation As outlined above, membrane bound transport enzymes, such as ATPases and membrane ion channels are key elements in ionic regulation. The toxicity of triorganotin compounds to ion translocating ATPases has been known for many years [165]. In fact, trialkyltin compounds have been instrumental in the unravelling of the biochemistry of oxidative phosphorylation as they inhibit electron transport phosphorylation in mitochondria [166, 167] (see Chapter 8). For membrane-bound ATPases the binding site for alkyltins has been shown to be on the membrane-bound components leading to an inhibition of the electron flow through these components [166, 147]. The V-type H+-ATPase, that couples ATP hydrolysis to transmembrane proton translocation through a rotating proton carrier subunit, provides the driving force for NaCl uptake in freshwater teleosts, is organotin susceptive. In vitro studies on subcellular components from various species have demonstrated the inhibitory capacity of triorganotin compounds on V-type H+ATPase (Table 2) [168, 169].

                                                             1

 

International Maritime Organisation – Conventions (http://bit.ly/c6zF5J; access date: November 2010).

Organotins and Hydromineral Homeostasis in Aquatic Animals

Biochemical and Biological Effects of Organotins 131

Table 2: In vitro effects of organotin compounds on ATPase activities. Species Bacteria Tapes philippinarum Mytilus galloprovincialis Mytilus galloprovincialis Botryllus schlosseri Bovine

Cell type

Structure

Enzyme

subunit a

Na+ATPase Na+/K+ATPase

Gill, mantle

mitochondria

Mg2+ATPase

Hemocytes

cytoskeleton

Heart

submitochondrial particles mitochondria

Gill, mantle

Adrenal gland

Mouse

Thymus Brain

Rabbit

Muscle

Rat

Heart

adrenal chromaffin granules

thymocyte membrane synaptosomes

sarcoplasmatic reticulum sarcoplasmatic reticulum

Concentration

Effect

Refs

200 nM 0-34 µM

↓ ↓ ↔

[192] [193]

TBTC

0–34 µM

↓

[194]

Ca2+ ATPase

TBTC

10 µM

↓

oligomycinsensitive ATPase F0F1-ATPase

Triorganotin

1.2 nMol mg-1 protein

↓

[195; 196] [197]

DBTCl

2 nMol g-1 protein 82 nMol g-1 protein 0-20 μM

↓

[198]

↓↔

[168]

TBTC, TETC, TMTCl, TPhTC TBT

10 µM

↓

[169]

100 nM-1mM

↓

[199]

TMT, DMT, MMT, TET, TPT, TBT, TPhT TPhT

1-10 µM

↓

[200]

0.5-10 mM

↓

[201]

TBT

0.6 µM

↓

[202]

V-type H+ATPase F0F1-ATPase

Ca2+ ATPase Na+/K+ATPase

Ca2+ ATPase Na+/K+ATPase Mg2+-ATPase

Organotin species TBTC TBT, DBT, MBT, TeET

DBT

Ca2+ ATPase Liver Brain

homogenates

F0F1-ATPase Na+/K+ATPase

Erythrocytes Squalus acanthias

Morone saxatilis Fundulus heteroclitus

Epithelial cells

Gill

isolated rectal gland

Na+/K+ ATPase; H+ ATPase

Na+/K+ ATPase; Mg2+ ATPase Na+/K+ ATPase; Mg2+ ATPase

↔

0.4 µM

↓

[203]

DBTBr TBT

0.9-56 µM

↓

[204] [205]

DBTC

64 - 252 µM

↓

DBTSCN

132 - 399 µM

↓

[206]

TBT

1.2-66 µM

↓

[205]

TBT

10-100 nM

↓

[207]

TMT; TET, TPhT TBT

↔ 106 µg L -1 53-106 µg L -1 25-50 µg L -1 5-50.5 µg L -1

↓ ↓ ↓ ↓

[208]

TBT: tributyltin; TBTC: tributyltin chloride; DBT: dibutyltin; MBT: monobutyltin; TeET: tetraethyltin; TBTBr: tributyltin bromide; BDTBr: dibutyltin bromide; DBTC: dibutyltin dichloride; DBTSCN: dibutyltin isothiocyanate; TMT: trimethyltin; TET: triethyltin; TPhT: triphenyltin; ↓: inhibition; ↔: unchanged; ↕: inhibited & stimulation;

 

132 Biochemical and Biological Effects of Organotins

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Although there are a number of in vivo studies involving the inhibitory effects of metals and other organic xenobiotics on V-type H+-ATPase [168-170], to my knowledge, there is no available published data on the effects of organotin compounds on V-type H+-ATPase in aquatic organisms following in vivo exposure. The mechanism by which organotins inhibit V-type H+-ATPases involves a decoupling of the ATPase component from the membrane-bound H+porter components [147, 168]. More recent investigations have revealed that organotins arrest the elementary steps for rotary catalysis of a V-type ATP-driven rotary motor by reducing the rotational rate of the peripheral subunit (V1), without influencing the binding affinity for ATP [171]. The consequence of this decoupling process is the secession of ATP hydrolysis by the ATPase component that cannot function unless protons are pumped through the membrane. Proton movement, on the over hand is restricted unless it is driven by the ATPase reaction. Furthermore, in vitro studies on rainbow trout, O. mykiss, erythrocyte membranes demonstrated a sensitivity of adrenergically activated Na+/H+ exchanger to 0.1 – 1 µM TBT [172]. As the Na +/H+ exchanger of the basal pavement cell membrane is an integral constituent of the Na+ uptake process in freshwater fish [26], inhibition of this ion exchanger is likely to affect the ionic regulatory capacity of exposed fish. As outlined above, many substances have been demonstrated to inhibit the Na+/K+-ATPase: Pb [173-175], NaNO3 [176], Cd [177], Cu [178, 179]; Hg [36, 180]; Cr [181]; Ag [19]; Zn [180]; NH3 [180], including triorganotins [70, 88, 89, 182, 183]. Ouabain, a steroid glycoside, specifically inhibits Na+/K+-ATPase activity by binding to specific isoforms of the E2P (membrane open/cytoplasm closed state) α-subunit, and is consequently used in Na+/K+-ATPase activity assays [184-187]. The ion gradients maintained by this protein are responsible for actively pumping (active transport) Na+ and K+ ions across cell membranes, driven by the hydrolysis of the terminal phosphate of ATP [188]. It is one of the most studied P-type ATPases, mainly because it contains core sequences that are conserved throughout most higher eukaryotes and has consequently been extensively reviewed [184]. Briefly, Na+/K+-ATPase is a hetero-dimeric protein consisting of an α and β subunit present in 1:1 stoichimetry. The αsubunit with approximately 1000 amino acids and a molecular mass of about 110 kDa is the catalytically active subunit as the binding sites for cations are primarly located here [188; 189]. It has 10 transmembrane segments with 5 extracellular loops, one of which, between M7 and M8 provides the important contact region with the β subunit [189]. In contrast to the α-subunit, β subunits have only been described in Na+/K+-ATPase and intestinal H+/K+-ATPase [184; 189]. The β subunit is a 55 kDa glycoprotein consisting of 330 amino acids and is thought to be involved in the structural stabilisation of the Na+/K+-ATPase in associated membranes [189]. Na+/K+-ATPase with previously bound ATP and Mg2+ works by binding 3 intracellular Na+ ions and hydrolysing ATP which leads to the phosphorylation of the Na+/K+-pump at a highly conserved aspartate residue on the α-subunit and subsequent release of ADP [184]. A conformational change in the Na+/K+-pump exposes the Na+ ions to the extracellular milieu where they are released as the phosphorylated form of the Na+/K+-pump has a low affinity for Na+ ions. The Na+/K+-ATPase then binds 2 extracellular K+ ions, which results in the dephosphorylation of the ATPase, thereby returning it to its original conformational state, and transporting the K+ ions into the cell. The unphosphorylated form of the pump has a higher affinity for Na+ ions than K+ ions, so the two bound K+ ions are released. ATP binds, and the process starts again [190, 191]. The relative ineffectiveness of triethyltin and trimethyltin compounds at inhibiting Na+/K+-ATPase suggests that the hydrophobic properties of these compounds are very important in the interaction with the enzyme. Moreover, inhibition does not occur instantaneously, but is rather a gradual process taking several minutes. Indeed, it has been suggested that it may take up to 60 TPhT moieties to inhibit Na+/K+-ATPase in vitro, although the actual number may be less, as some may be bound to inactive subunits or non-specific sites or in fact dissolve in membrane lipids [147]. The bulk of the available published data concerning the underlying mechanics of the inhibitory effects of organotin compounds on the activity of Na+/K+-ATPase involves in vitro studies on primary cell extracts and cell lines from a variety of species, including mammals (Table 2). TBTC has been shown to inhibit the ATP hydrolysis by the Na+translocating ATP synthase of the bacteria Ilyobacter tartaricus and the H+-translocating counterpart of Escherichia coli and also the binding of Na+ to the α-subunit of Na+/K+-ATPase, suggesting that the α-subunit ion channel may be the site of inhibition [192]. In the search for human analogues, much research on Na+/K+-ATPase-triorganotin interaction has been carried out on mammals. In rats, the Na+/K+-ATPase in cardiac membrane fraction, prepared from heart ventricles, was inhibited by 0.6 µM TBT [202]; in brain homogenates and erythrocyte preparations, the Na +/K+-ATPase was inhibited by 0.9-56 µM and 1.2-66 µM TBT, respectively [205]; in brain synaptic membranes the Na +/K+-ATPase was significantly inhibited by 64-252 µM dibenzyltin dichloride and 132 - 399 µM dibenzyltin diisothiocyanate [206]. In vitro studies into the impact of organotin compounds on the Na+/K+-ATPase in aquatic species have yielded more relevant data. In bivalves

 

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Biochemical and Biological Effects of Organotins 133

Tapes semidecussatus and Mytilus galloprovincialis 34 µM TBT significantly inhibited Na +/K+-ATPase in both gills and mantle, although the gills were more sensitive. Furthermore additional organotin species were also tested showing that TBT was more toxic to Na+/K+-ATPase than DBT and that MBT and TeET had no significant effect [193]. In isolated perfused rectal glands of the elasmobranch Squalus acanthias Na+/K+-ATPase was significantly inhibited by 10-100 nM TBT [207]. Na+/K+-ATPase activities in gill homogenates of the estuarine fish species Morone saxatilis and Fundulus heteroclitus were significantly reduced following exposure to 106 µg L -1 and 5-50 µg L -1, respectively [208]. In vivo exposure of 5-day old neonate rats to triethyltin (TET) inhibited the Na+/K+-ATPase in mitochondrial preparations of liver and brain homogenates, at concentrations ranging from 1-260 µM depending on age [209], and liver Na +/K+ATPase was also inhibited following intraperitoneal exposure to dibutyltin chloride (DBTC) (10 or 30 mg kg-1d-1) [210]. Organotin compounds are also known to inhibit Na+/K+-ATPase in vivo in aquatic organisms at environmentally relevant concentrations through a variety of exposure routes (Table 3). In fish, juvenile European flounder, Platichthys flesus, experienced a significantly reduced gill Na+/K+-ATPase activity following sub-chronic non-lethal exposure to sedimentassociated TBTC and TPhTC (150 ng g-1). This was accompanied by a concomitant reduction in active Na+-efflux during adaptation to seawater following rapid transfer from freshwater [70, 89]. In seabass, M. saxatilis exposed for 14 days to aqueous TBT (0.1-1.09 µg L -1) and DBT (22-53 µg L -1) leached from tributyltin metacrylate painted panels gill Na+/K+ATPase activity experienced a significant stimulation at the lowest TBT concentration (0.1 µg L -1). Na+/K+-ATPase activity was also increased at 0.34 µg L -1 and reduced at the highest recorded concentration (1.09 µg L -1), albeit, nonsignificantly. These fluctuations in Na+/K+-ATPase activity did not lead to any changes in plasma Na+ levels [208]. Juvenile Oreochromis niloticus (=Tilapia nilotica) exposed to 0.1, 1 and 10 ng L-1 TBT for 15 days showed a dosedependent inhibition of gill Na+/K+-ATPase [211]. Similarly, subacute (7-28 d) exposure of adult O. niloticus to 4.1 µg L -1 TBTO caused a significant inhibition of gill Na+/K+-ATPase. In the same study acute (3-96 h) exposure of to 10-90 µg L -1 TBTO also caused a significant inhibition of gill Na+/K+-ATPase. The extent of inhibition appeared to be a function of time, with the highest degree of inhibition (52%) occurring after 96h exposure under acute conditions. During subacute conditions the peak of 55% inhibition was reached after 7 days, after which a partial recovery to 14% inhibition was observed [212]. However, it is unclear whether this represents a true recovery or is merely the reflection of TBTO degradation, as other studies observed a 20% degradation of sediment-associated TBTC over a five weeks incubation period [183]. Red tilapia displayed a similar degree of gill Na+/K+-ATPase inhibition following subchronic exposure to 5 µg L -1 TBTO [213]. Unlike fish, most euryhaline invertebrates are volume-regulating osmoconformers. Nevertheless, there is a surprising lack of data on the effect of organotin compounds on the Na+/K+ATPase activity of in vivo exposed euryhaline aquatic invertebrates. An example is the inhibition of Na+/K+ATPase in Artemia salina [214] exposed to 0.02µg L -1 fenbutatin oxide. However, Na+/K+ATPase activity in gill and epipodite membranes of Penaeus japonicus was not affected by 100 and 200 µMol TBTO [215]. As outlined above, maintenance of divalent ion concentrations, such as Ca2+ and Mg2+, are important for growth, muscle contraction, oxidative phosphorylation and therefore general metabolic homeostasis. Organotin compounds may disrupt calcium homeostasis by several means: TBTC has been shown to interact with calmodulin (CaM), a calcium modulating protein, by non-covalent hydrophobic interaction between the aliphatic chains of TBT and the hydrophobic regions of Ca2+-activated CaM. The consequence of this is an inhibition of CaM-dependent Ca2+ATPase at TBTC concentrations ranging from 01 to 1 mM, leading to a detrimental increase in cytosolic Ca2+ concentration in vitro [151, 216]. TBT is also known to inhibit sarcoplasmic-endoplasmatic reticulum Ca2+-ATPase (SERCA), thus triggering the release of Ca2+ stores from the endoplasmic reticulum and the activation of the storedependent Ca2+ influx. The massive accumulation of Ca2+ observed is a result of the activation of the Ca2+-influx pathway, especially following prolonged inhibition of SERCA [217]. Similar results have been reported for isolated mouse SERCA exposed to 2 µM TBT, 63 µM TET and 280 µM TMT [203], as well as isolated rabbit SERCA exposed to 0.5-10 mM TPhT [201]. Experiments with mouse thymocytes showed that 1 µM TBT increased the membrane permeability of the intracellular organelles for Ca2+ and decreased the membrane Ca2+ pump activity, again resulting in a sustained increase in the intracellular Ca2+ concentration [199]. TBT has been shown to cause a rapid depletion of thiols, suggesting that organotin compounds may be interacting directly with Ca2+ pumps by binding to its thiol groups, especially as TBT induced effects can be prevented by various thiol reducing agents [218]. Interestingly, DBT appears to be a more potent inhibitor of Ca2+-ATPase than TBT, possibly because of the hydrophobicity of TBT facilitates its insertion into the membrane and retention within lipidic bilayers [219], leaving less free TBT available in the cytoplasm to interact directly with Ca2+-ATPase [220].

 

134 Biochemical and Biological Effects of Organotins

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Table 3: In vivo exposure to organotin compounds and their effects on ATPases. Species

Deveopmental stage

Duration

Tissue

Enzyme

Organotin species

Rats

neonatal/adult

?

Brain/liver mitochondria

Na+/K+ATPase

TET

Route of Exposure

?

Concentration

Effect

Refs

↓

[209]

1-260 µM

↔

DET, MET,TMT Artemia salina

Nauplii

Penaeus japonicus

adult

?

Liver

DBTCl

intraperitoneally

10 or 30 mg kg-1d-1

[210]

?

?

fenbutatin oxide3

Aqueous

0.02 µg L -1

[214]

Platichthys flesus

Gills

Na+/K+ ATPase

TBTC, TPhTC

sedimentassociated

150 ng g-1

↓

[88; 183; 89; 70]

Morone saxatilis

juvenile

Gill

Na+/K+ ATPase

TBTO

Aqueous

0-1.09 µg L -1

↕

[208]

juvenile

Gill

Mg2+ ATPase

TBTO

Aqueous

0-1.09 µg L -1

?

[208]

Oreochromis niloticus1

juvenile

Gill

Na+/K+ ATPase

TBTO

Aqueous

0.1-10 µg L -1

↓

[211]

Oreochromis niloticus

adult

3-96h

Gill

Na+/K+ ATPase

TBTO

Aqueous

10-90 µg L -1

[212]

adult

7-28d

Gill

Na+/K+ ATPase

TBTO

Aqueous

4.1 µg L -1

[212]

adult

3-96h

Gill

Mg2+ ATPase

TBTO

Aqueous

10-90 µg L -1

[212]

Red Tilapia2

2+

adult

7-28d

Gill

Mg ATPase

TBTO

Aqueous

4.1 µg L

?

?

Gill

Na+/K+ ATPase

TBTO

Aqueous

5 µg L -2

-1

[212] [213]

1

) Tilapia niloticus; 2) hybrid of several other Oreochromis species; 3) bis[tris(2-methyl-2-phenylpropyl)tin] oxide; TBTC: tributyltin chloride; TBTO: tributyltin oxide; DBTCl: dibutyltin chloride; MBT: monobutyltin; TMT: trimethyltin; TET: triethyltin; DET: Diethyltin; MET: Monoethyltin; TPhTC: triphenyltin chloride; ↓: inhibition; ↔: unchanged; ↕: inhibited & stimulation;

Mg2+ balance which is important for oxidative phosphorylation and anion transport has been shown to be compromised by organotin compounds. In vivo exposure of F. heteroclitus to 5-50 µg L -1 TBT yielded significant inhibition of gill Mg2+-ATPase, whereas, Mg2+-ATPase activity was unchanged in the gills of M. saxatilis exposed to the far lower concentration of 1.09 µg L -1 TBT, although the same concentration was enough to significantly stimulate the Na+/K+-ATPase activity. The latter probably reflected the increased metabolic activity due to osmotic stress as a result of organotin exposure [208]. Water Balance Being able to control the integumental water fluxes, especially across delicate permeable membranes, such as those of gill and intestinal epithelia, is of paramount importance for the regulation of the water balance in aquatic, particularly estuarine, species. As described earlier, one way of doing this is to regulate the fluidity of the membrane by adjusting the fatty acid composition and cholesterol concentration [221, 222]. In fact gills, often the first target of water-borne contaminants, can experience a significant change in lipid composition in epithelial membranes following exposure [223, 221]. Interaction of organotin with membranes leads to membrane-associated tin-containing aggregates, which appear intercalated between the inner and outer membranes of human erythrocytes, and may cause physical alterations to the state of membrane proteins and lipids, including membrane fluidity[224] (Table 4). Triorganotin compounds and their degradation products interact with phospholipid membranes according to their hydrophobicity and also steric structure [225-227], whereby the membrane surface charge appears to play an important role [219]. It has been observed that hydrophobic butyl and phenyl moieties of TBT and TPhT align themselves with the prevailing directions

 

Organotins and Hydromineral Homeostasis in Aquatic Animals

Biochemical and Biological Effects of Organotins 135

of the phospholipid acyl chains [228]. This is likely to disrupt the packing of the membrane, thereby increasing its fluidity and permeability [229]. The all important permeability of biological membranes has been found to be highly sensitive to TBT [230-233] and TPhT [234]. Indeed, changes to membrane fluidity can affect the permeability of the membrane for water and solutes. In in vitro preparations, the permeability of model membranes for dimethylarsinic acid (DMA) was found to increase following exposure to TBT but decreased after treatment with DBT, which may be a reflection of the hydrophobicity and steric structure of the two compounds and their subsequently different mechanics of interaction with phospholipid membranes [231]. Similar observations have been made for TPhT that associates with the headgroup region of the lipid bilayer vesicles, whereas DPhT caused changes to the hydrophobic regions[233]. The perturbations of model phosphatidyl choline membranes following exposure to 10-100 nM TBTC and TPhTC has caused the depolarisation of these membranes [235]. TBTC and TPhTC, albeit at the much higher exposure concentration range of 30-200mM, are also known to significantly increase the fluidity of model membranes [228, 236, 237], which is likely to be the underlying cause of changes to membrane permeability (see Chapter 4). A change of membrane fluidity and permeability has demonstrably increased hemolysis of erythrocytes exposed in vitro to TBTC [238] and TPhTC [233, 239]. Haemolytic potency appears to depend on the location of the compound in the bilayer, as TPhT adsorbed on the surface in one study caused a much higher degree of hemolysis than DPhT that had penetrated much deeper [233]. As the perturbations of the membranes by butyl groups disrupt the packing of the lipid alkyl chains, the shape and flexibility of erythrocytes can be affected, caused by an increase in motion and disorder of the erythrocyte membrane [240]. Table 4: Organotin-induced membrane perturbations in vivo, in vitro and in model membranes Species

Organ/ type

Platichthys flesus

Salmo irideus

Human

cell

Effect

Organotin species

Route of Exposure

gills

reduced water permeability

TBTC, TPhTC

sedimentassociated

erythrocyes

increased membrane fluidity

TBTC

in vitro

hemolysis

TBTC; DBTC

in vitro

hemolysis

TBTC, TPhTC

in vitro

erythrocyes

Porcine

TPhTC; DPhTC

Duration 35d

-

Concentration

Refs

150 ng g-1

[88]

20 µM

[229]

10-90 µM

[238]

-

2-10 µM

[239]

0-4h

100 µM

[233]

Model membranes in vitro depolarization

in vitro

1

increased membrane fluidity

phosphatidyl choline

increased membrane fluidity

TBT; TPhT

in vitro

EYL2

decreased membrane fluidity

TeMT; TeET

in vitro

increased membrane fluidity

DPhC; TPhTC

DPPC

1)

TBTC, TPhTC

dipalmitoylphospatidylcholine;

2)

TPTC;

[231] -

10-100 nM

[235]

-

30-50 mM

[236]

100-400 mM

[228]

180h

[237]

Egg yolk lecithin; TBTC: tributyltin chloride; DBTC: dibutyltin chloride; MBT: monobutyltin; TMT:

trimethyltin; TeMT: Tetramethyltin; TeET: Tetraethyltin; TPTC: tripropyltin chloride; TPhTC: triphenyltin chloride; DPhTC: diphenyltin dichloride;

 

136 Biochemical and Biological Effects of Organotins

Mark GJ Hartl

Data concerning the in vivo exposure of aquatic organisms to organotin compounds and the effects on membrane permeability are sparse and restricted to euryhaline fish. Freshwater-adapted juvenile European flounder exposed to 150ng g-1 sediment-associated TBTC and TPhTC showed a significantly increased half-time of exchange of tritiated water (THO) across the permeable gill membranes, which is consistent with a reduced gill membrane permeability, in particular a shift from diffusional towards osmotic permeability [88, 183, 241]. A reduction in membrane ‘fluidity’ has also been caused by other lipophilic compounds, such as cholesterol [242] and alpha-tocopherol [243]. In addition, plasticisers and petroleum hydrocarbons, also lipophilic pollutants, have been found in the gill membranes of the amphipod Gammarus duebeni and have caused alterations to the fatty acid composition of the gill phospholipids and changes to membrane permeability [221, 223]. There are also a number of in vivo studies concerning the permeability of gill membranes following exposure to other metals, such as Cd, Cu and Al. For example, freshwater O. mossambicus exposed to 100 and 1000 µg L -1 CdCl2 experienced a concentration dependent increase in gill permeability [244]. This increased permeability was most likely caused by direct interactions of metals on the gill surface, particularly competitive interactions with Ca2+ binding sites on the gill membrane and in tight junctions [245, 246]. These data were supported by the observation of a protective effect of high water Ca2+ levels against metal toxicity by competing with metals for binding sites on the gill membrane [246]. Many of the above physiological aberrations have observable histopathological origins or consequences, particularly in gill epithelia, blood cell constitution and blood composition of fish (Table 5). Acute exposure of juvenile O. mykiss to 5.85 mg L-1 resulted in significant damage to gills, characterised by a separation of the gill epithelium from the basal membrane and pillar cells. In addition, a swelling of the secondary lamellae and dilation of blood vessels was also observed [182]. Atlantic salmon, Salmo salar, held in TBT-treated nets for up to four weeks showed lamellar hyperplasia and lateral lamellar branching, suggesting interference of TBT in gill epithelial growth and development [247]. 0-group European flounder, Platichthys flesus exposed to 150 ng g-1 sediment-associated TBTC and TPhTC for 35 days showed significant interference in gill lamellar chloride cell dynamics following rapid transfer from freshwater to seawater [89]. The effects of aqueous exposure of freshwater-adapted 0-group Platichthys flesus to 32 µg L -1 for up to 15 days resulted in serious histopathological damage to gill epithelia, including swelling and budding of epithelial cells, epithelial proliferation and fusion of lamellae [248]. Although no changes to the surface morphology of gill epithelia were observed, acute exposure of the mummichog, Fundulus heteroclitus to 17.2 µg L -1 TBT led to hypertrophy of the gill lamellar epithelium and at 35.6 µg L -1 caused 40% reduction in the volume of lamellar blood channels. However, a 42 d exposure to sublethal concentrations (up to 2 µg L -1) showed no histopathological changes to the gill epithelia [249]. During sub-chronic exposure (1-4 months) to much higher concentrations of tributytin hydroxide (TBTH 1-3 mg-1), the gill epithelia of Oreochromis nilotica had developed hyperplasia, leading to congestion of the lamellar blood vessels and the aneurysmal formation of lamellar capillaries [250]. Acute (12h) and sub-chronic (7-14d) exposure of Chinese rare minnow (Gobiocypris rarus) to 50-5,000 ng L-1 TBTC caused a series of ultrastructural pathological changes to the gills, including fractured nuclei [251]. In addition, histopathological damage has been recorded in other osmoregulatory organs, such as the kidney. O. mykiss yolk sac fry showed hydropic degeneration of the tubule segments of the pronephros following a 10-day exposure to 5 µg L -1 TBTO [252]. The hemopoietic interstitial tissues in the head kidney of juvenile guppies, Poecilia reticulata, exposed for up to 3 months to TBTO (0.01-32 µg L -1) and for 1 month to TBTC (320-3,200 µL -1), developed hyperplasia [253]. At comparable concentrations of TBTH, Oreochromis nilotica, during subchronic exposure (1-3 months) developed hydropic degeneration and accumulation of hyaline droplets in tubular epithelial cells [250]. In embryos and larvae of the minnow, Phoxinus phoxinus, gills were not yet fully developed, but 3 to 10 day exposure to 0.8 - 19.5 μg L-1 TBTC and 1.8 - 15.8 μg L-1 TPhTC, lead to degenerative alterations in renal tissue [254, 255]. Disruption of osmo- and ionic regulatory mechanisms following exposure to organotin compounds has indirect implications for blood constituents and the osmoregulatory capacity of exposed organisms of various phyla (Table 6). The osmoregulatory capacity of the shrimp Penaeus japonicas, was significantly reduced following acute exposure to 50-400 µg L -1 TBTO [215]. Subchronic exposure of Crassotrea virginica to 0.5-2 g L-1 TBTO caused a significant delay in osmotic adaptation of haemolymph in terms of osmotic pressure and chloride ion concentration [256].

 

Organotins and Hydromineral Homeostasis in Aquatic Animals

Biochemical and Biological Effects of Organotins 137

Table 5: Histopathological damage caused by exposure to various organotin compounds Species

Developmental stage

Tissue

Effect

Organotin species

Route of Exposure

Duration

Concentration

Refs

Oncorhynchus mykiss1

0+ group

Gills

swollen mitochondria; disorganised cristae

TBTO

Aqueous