Culture Techniques: Applicability for Studies on Prenatal Differentiation and Toxicity. Fifth Symposium on Prenatal Development, May 1981, Berlin 9783110858242, 9783110087543

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Culture Techniques

Culture Techniques Applicability for Studies on Prenatal Differentiation and Toxicity Fifth Symposium on Prenatal Development May, 1981, Berlin

Edited by D. Neubert and H.-J. Merker With the assistance of J. Klein-Friedrich and R. Kreft

w DE

G Walter de Gruyter • Berlin • New York 1981

Editors: Professor Dr. D. Neubert Professor Dr. H.-J. Merker Institut für Toxikologie und Embryonal-Pharmakologie, Freie Universität Berlin, Garystraße 9, D-1000 Berlin 33, West-Germany

CIP-Kurztitelaufnahme der Deutschen Bibliothek Culture techniques: applicability for studies on prenatal differentiation and toxicity/ 5. Symposium on Prenatal Development, May, 1981, Berlin. Ed. by. D. Neubert and H.-J. Merker. With the assistance of J. Klein-Friedrich and R. Kreft. Berlin; New York: deGruyter,1981. ISBN 3-11-008754-5. NE: Neubert, Diether [Hrsg.]; Symposium on Prenatal Development [05,1981, Berlin, West] Library of Congress Cataloging in Publication Data Main entry under title: Symposium on Prenatal Development (5th: 1981: Berlin, Germany) Culture techniques. Includes index. 1. Embryology - Research - Methodology - Congresses. 2. Teratogenic agents - Congresses. I. Neubert, Diether. II. Merker, H.-J. (Hans-Joachim), 1929 - . III. Title [DNLM: 1. Toxicology - Congresses. 2. Abnormalities, Druginduced Congresses. 3. Organ culture - Congresses. 4. Cells, Cultured Congresses. 5. Embryology - Congresses. W3 SY542N 5th 1981c/ QS 679 S9881981c] QM601.S891981 612'.64 82-8 ISBN 3-11 -008754-5 AACR2 © Copyright 1981 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Karl Gerike, Berlin. - Binding: Lüderitz & Bauer, Buchgewerbe GmbH, Berlin. - Printed in Germany. The quotation of registered names, trade names, trade marks, etc. in this copy does not imply, even in the absence of a specific statement that such names are exempt from laws and regulations protecting trade marks, etc. and therefore free for general use.

Dedication We wish to dedicate this book to the memory of the late professor and chairman of the Department of Anatomy, University of Virginia, Charlottesville, Virginia, USA, Dr. Jan Langman, with whom we enjoyed many years of productive collaboration. He was an active participant at our many symposia. His thoughtful consideration as co-editor of our third Symposium book was greatly appreciated. In 1978/79 we had the privilege of having Professor Langman as Guest-Professor at our institute in Berlin. We all learned so much from him; the year he spent with us will be faithfully remembered. Professor Langman is commemorated, not only as an outstanding embryologist and pioneer in the field of morphologically oriented prenatal toxicology, but also as a worthy collegue and a good friend. A man who understood a positive way to live.

Preface The applicability of in vitro methods in studies on prenatal differentiation and toxicology has gained increasing attention during the last few years. Therefore, we felt that this was a highly suitable topic for the 5th Symposium on Prenatal Development which, just as its predecessors, was organized by the Institut f ü r Toxikologie und Embryopharmakologie of the Freie Universität Berlin (Sonderforschungsbereich 29 - Embryonale Entwicklung und Differenzierung). During the days of May 7th through 10th, 1981, approximately 50 scientists from 8 countries met in Berlin to discuss problems involved with the applicability of culture techniques for studies on prenatal differentiation and toxicity. The papers presented and relevant parts of the discussions are compiled in this book. We have also included some papers which could not be presented at the symposium due to lack of time, some of which were presented as posters, and one paper from an author who was unable to attend the May meeting. As on previous occasions, the active cooperation of many members of the Institute of Toxicology was essential in making the Symposium more successful . Donations from the Amersham Buchler GmbH & Co. KG. (Braunschweig); Lange & Springer, Wissenschaftliche Buchhandlung GmbH & Co. KG. (Berlin); Paul-Martini-Stiftung der Medizinisch-Pharmazeutischen Studiengesellschaft e.V. (Mainz); and F. Winkelmann, Versuchstierzucht GmbH & Co., KG. (Borchen) greatly helped to organize the meeting. The financial support we received from the Freie Universität Berlin and the Deutsche Forschungsgemeinschaft is highly appreciated.

D. Neubert H . - J . Merker

Table of Contents INTRODUCTION

13

I.

17

STUDIES ON WHOLE-EMBRYO CULTURE

HERKEN and ANSCHUTZ: Differentiation of embryonic tissues in whole-embryo culture as compared to the development in vivo

19

BECK: The differentiation between teratogenic action on mother, fetal membranes and embryos using the whole-embryo culture method

37

GULAMHUSEIN: in vitro

43

Trypan blue teratogenesis in the rat

ANSCHUTZ et al.: Studies on embryotoxic effects of thallium using the whole embryo culture technique

57

KLEIN et al.: Evaluation of serum teratogenic activity using rat embryo cultures

67

HSU:

Regulation of mouse embryogenesis by exogenous growth factors

83

KLEE-TRIESCHMANN and NEUBERT: Preliminary communication on the feasibility of culturing whole embryos in a chemically defined medium

97

DAVIS et al.: In vitro development of the heart under influence of retinoic acid

101

II.

117

STUDIES ON ORGAN CULTURES A.

Differentiation of the Skeleton; predominantly Extremities

MERKER et a l . : in vitro

Simulation of limb bud skeletogenesis

119

BARRACH et a l . : Comparison of the differentiation of muscle and connective tissue of mouse limb buds in culture and in vivo: A morphological study by indirect immunofluorescence

135

DUSEMUND and BARRACH: Quantification of collagen types I and II in mouse limbs during differentiation in vitro and in vivo

161

NEUBERT and BLUTH: Feasibility of storing embryonic tissues for subsequent use in organ culture

171

10

NEUBERT and BLUTH: Limb bud organ cultures from mouse embryos after apparent induction of monooxygenases in utero. Effects of cyclophosphamide, dimethylnitrosamine and some thalidomide derivatives

175

BLANKENBURG et al.: On the significance of ascorbate and of cysteine on differentiation of limb buds in organ culture

197

STAHLMANN et al.: Effects of some "indirectly" alkylating agents on differentiation of limb buds in organ culture

207

BOCHERT et al.: Comparison of effects on limb development in vivo and in vitro using methyl(acetoxymethyl)nitrosamine

223

KWASIGROCH et a l . : Development of limb buds in organ culture: Examination of hydroxyurea enhancement of bromodeoxyuridine toxicity using image analysis

237

BROWN et al.: Biochemical characterization of mouse hereditary chondrodystrophies in organ culture

255

RA J AN et a l . : Effect of teratogens on human embryonic skeletal tissue in vitro

269

SCOTT and Klein: Aspirin induced Polydactyly in rats mediated by inhibition of prostaglandin synthesis

277

B.

Differentiation of other Organ Anlagen (kidneys, lungs, heart, prostate gland, haematopoetic system, and intestine)

289

SAXEN and EKBLOM: The developing kidney as a model system for normal and impaired organogenesis

291

MERKER et al.: The embryonic lung as in vitro model for testing teratogenic substances

301

EVANS et al.: The toxic effects of metal dusts on human foetal lungs in vitro

319

RISO and ZIMMERMANN: Effects of substances influencing glycosaminoglycan synthesis on lung development in culture

331

ZIMMERMANN et a l . : Effect of L-a2etidine-2-carboxylic acid and ß-D-xylosides on lung development in vitro

341

LASNITZKI and MIZUNO: Interaction of epithelium and mesenchyme in the induction of foetal rat and mouse prostate glands by androgens in organ culture

359

11

GROPP et al. : In vivo and in vitro assays of trisomie cells isolated from the fetal organism or rescued by transfer to non-trisomic hosts

371

SILANO et a l . : Effects of gliadin-derived peptides from bread and durum wheat on small intestine cultures from rat fetus and coeliac children

385

III.

395

STUDIES ON PRE-IMPLANTATION EMBRYOS

SURANI et a l . : Differential adhesiveness as a mechanism of cell allocation to inner cell mass and trophectoderm in the mouse blastocyst

397

Inefficient capacity for NILSSON and MAGNUSSON: oxidative phosphorylation of the trophoblast as a cause of delayed implantation in the mouse

415

BAUNACK and GÄRTNER: Influence of micromanipulation of ova on the postnatal development of mice (AKR)

423

SPIELMANN et al. : Investigations on the mechanism of action and on the pharmacokinetics of cyclophosphamide treatment during the preimplantation period in the mouse

435

PEDERSEN: Benzo(a)pyrene metabolism in early mouse embryos

447

IV.

455

STUDIES ON CELL AND TISSUE CULTURES

GRUNDMANN et a l . : Influence of different antisera on mouse epiphyseal chondrocytes in monolayer culture

457

ZIMMERMANN: Cell movement during blastema formation. Effect of cytochalasin B on cartilage development in vitro

465

SCHARLACH and ZIMMERMANN: Cell-substrate adhesion of fibroblast-like cells under different in vitro conditions. A scanning and transmission electronmicroscopic study

477

NAU and GANSAU: Development of cytochrome P 4 5 0 dependent drug metabolizing enzyme activities in mouse and human tissues in vitro

495

12

KOPF-MAIER and KRAHL: In vitro treatment with metallocene dichlorides: Determination of the intracellular distribution of metal atoms by use of electron energy loss spectroscopy

509

NAU et a l . : Morphological changes induced by 2 , 3 , 7 , 8 tetrachlorodibenzo-p-dioxin (TCDD) in epithelial monolayer culture using human and rodent fetal tissues

519

V.

527

Studies on Non-Mammalian Tissues

SUMMERBELL and HORNBRUCH: The chick embryo: A standard against which to judge in vitro systems

529

Experimental manipulation leading STEDING et a l . : to cardiac malformation in chick embryos

539

JELINEK and PETERKA: Morphogenetic systems and in vitro techniques in teratology

553

VI.

559

CULTURE TECHNIQUES FOR "SCREENING" ON TERATOGENICITY

FLINT: An assessment of the available in vitro techniques for detecting teratogens

561

NEUBERT: On the predictability of developmental toxicity - especially prenatal toxicity - on the basis of culture experiments

567

VII. SOME METHODS USED FOR CULTURING EMBRYONIC TISSUES

585

A.

Whole-Embryo-Culture:

587

B.

Limb Bud Method:

C.

Trowell Method:

D.

"Micro-Mass Culture":

E.

Preimplantation-Embryo Culture:

598

F.

Teratogenicity Testing Method (Chick)

598

G.

CHEST I and II (Chick):

599

Beck; Anschiitz

Blankenburg Zimmermann Zimmermann

Jelinek and Peterka

590 594 595

Glossary

607

List of Participants

608

Index

612

Introduction D. Neubert, Berlin Although in vitro methods, including culture methods, have been used for a long time, techniques which can supplement or possibly even replace whole-animal experiments have rapidly gained interest during the last decade - also in fields of research in which they have been used very little, or not at all, such as toxicological studies on environmental chemicals or substances used for medication. Several reasons may be responsible for this trend of growing interest: 1)

an increasing, and more or less emotionally expressed uneasiness among scientists and in the public opinion on the necessity and the moral justification of performing large scale animal experiments, as is done today;

2)

the acknowledgement that whole-animal experiments often do not provide sufficient information on the mode or mechanism of the toxic action;

3)

an awareness that the 10,000s of compounds to which the environment, man and animals are exposed, and the excessive number of new chemicals produced every year, can hardly be adequately tested in whole-animal experiments as practised now;

4)

recessions in search funds cal research the necessity

many industrial countries, the resulting shortages of reand the fact that whole-animal experiments in toxicologiare exceedingly expensive and time consuming call forth of finding less expensive methods.

The general public believes that in vitro tests are often considered as "alternative" methods for toxicological evaluations. It must be stressed that in principle in vitro techniques should not be regarded as an alternative for in vivo methods. Such systems have been extensively used as research tools since the 2nd and 3rd quarters of this century. The tremendous progress in biochemistry and in molecular biology has been achieved predominantly by the use of in vitro techniques. Biochemists prefer to experiment with cell-free systems, mostly of mammals (homogenates, isolated cell organelles, multi-enzyme complexes e t c . ) . Furthermore, isolated organ preparations (isolated heart, intestine, uterus preparations e t c . ) , for many decades, have been, and still are, the main test systems in physiology and pharmacology. In developmental biology, in vitro methods and especially culture techniques (organ cultures, cell cultures e t c . ) have also been used for decades to a considerable extent and most of the fundamental principles, such as induction processes, have been elucidated with such techniques. Thus, many of the "in vitro" techniques (including culture techniques) are old, time-proven tools of the scientist. But toxicological risk evaluations with relevance to humans, using techniques other than those which rely on the whole experimental animal, have not been, so far, accepted as routine procedure. The reason for this, although possibly not quite evident for persons without a medical and toxicological education is simple. This problem will be discussed later in more detail (NEUBERT et a l . , this book). It has been agreed upon today that in testing for possible mutagenic hazard, methods using bacteria (AMES test)

14 or cell cultures are valuable. The situation is even less clear in the case of an evaluation of a carcinogenic hazard. Although some encouraging approaches, such as cell transformation assays, have become available recently, these have not, up to now, been used for routine testing. Bacterial tests for this kind of toxicological evaluation have, so far at least, caused as much confusion as they have been helpful. In vitro tests are today still considered inadequate to assess a carcinogenic risk for humans. There is very litte experience available today on the possibility of replacing routine whole-animal tests in reproductive toxicology by in vitro techniques. This is not too surprising since reproductive toxicology is a much more complex field of toxicology than mutagenicity is. Therefore, it can hardly be expected that a single in vitro method or technique will prove adequate to assess a risk of a chemical to the reproductive processes. Whereas today there is no doubt that in vitro techniques are extremely valuable tools for research in developmental biology and even developmental toxicology, it cannot be expected that such methods are equally applicable and suited for all research purposes. Thus, it is essential to specify the scientific problem and the aim of intended studies. There are a number of problems scientists like to tackle in the field of reproductive-biology and -toxicology. These include: 1)

the elucidation of basic biological principles in developmental and reproductive biology.

2)

the more detailed analysis of an action caused by a chemical or physical agent with regard to dose-response relationships, interference with other agents or components - in a synergistic or antagonistic way - or elucidation of the response of a system to a substance with a known or assumed biochemical mode of action.

3)

the elucidation of the mode of a toxic action of a chemical on a reproductive organ system.

4)

the elucidation of a toxic potential of a chemical - in this respect on reproduction and pre- or perinatal development - as part of a risk evaluation with relevance to humans or animals.

It is obvious that the spectrum of these problems is so broad that it is unlikely that only a few standard methods suffice to provide satisfactory answers to all the pertinent questions. We have no doubt whatsoever, that at our present stage of knowledge, all suitable methods available, in vitro as well as whole-animal tests, must be used to serve the purposes anticipated. But it is equally obvious that an approach using predominantly cell or organ cultures will be more rewarding with some of the problems mentioned than with others. The evidence available indicates that organ and even cell cultures will greatly assist and facilitate an evaluation of the problems 1 ) , 2 ) , 3 ) . It is to be expected that such techniques will be less satisfactory in evaluating chemicals for their ability to interfere with reproductive and developmental processes with relevance to the situation possibly existing in man. This is especially true if new chemicals are to be evaluated and no clues exist on a certain toxic potential with regard to reproductive toxicity as is later discussed in more detail (NEUBERT, this book).

15 It was the goal of this Symposium to bring together scientists of various fields of developmental biology, with divers interests and intentions, in order to obtain a survey and a compilation of the available techniques of cell or organ cultures of a variety of tissues and of various organisms. Only on such a broad basis, was it possible to discuss techniques offering the suitability for research or for the "screening" of chemicals for certain types of toxic potential. Since these methods are not necessarily confined to mammalian tissues, several papers on the possible usefulness of organisms other than mammalian, i . e . chicken embryos, have been included.

I. Studies on Whole-Embryo Culture

Differentiation of E m b r y o n i c Tissues in W h o l e - E m b r y o Cultures as C o m p a r e d to the D e v e l o p m e n t In Vivo R. H e r k e n a n d M . A n s c h ü t z Institut für T o x i k o l o g i e u n d E m b r y o p h a r m a k o l o g i e d e r Freien Universität Berlin, G a r y s t r a ß e 9, 1000 Berlin 33

Summary: 10.5-day-old rat embryos (4 - 14 somites) were grown in human serum for 48 hours. The in vitro and in vivo development of the embryos were compared. The somite number, dry weight and protein and DNA content of the embryos served as criteria for in vivo and in vitro growth. Furthermore, the embryos were investigated macroscopically and histologically. It was observed that the in vitro development of the embryos during the first 36 hours of the culture period proceeds almost parallel with the in vivo development. However, after 48 hours the in vitro development falls behind the in vivo development. Based on our morphological findings we established criteria according to which rat embryos (between days 10.5 and 12.5) should be investigated macroscopically and microscopically. The possibilities and limitations of the whole-embryo-culture system for teratological investigations are discussed. Introduction: The whole-embryo-culture technique has reached a standard which makes it possible to employ this system for teratological investigations (NEW, 1978). Using this culture system it has to be kept in mind that apart from drug effects there exist culture conditions which lead to abnormal development in vitro. STEELE and NEW (1974) described double heart formation after cultivation of rat embryos in rat serum centrifuged after clotting. Cultivation of rat embryos under hyperthermia (41° C) resulted in microcephalus, pericardial edema, open neural tube and anterior-posterior fusion of the neural tube (COCKROFT and NEW, 1978). Microcephalus, eye defects, edema and fusion of the anterior and posterior neural tube have been described after cultivation of rat embryos in a culture containing 12 - 15 mg/ ml D-glucose (COCKROFT and COPPOLA, 1977). Abnormal morphogenesis of the cranial neural folds of rat embryos occurred after cultivation under 0 2 pressure of over 20% (MORRIS and NEW, 1979). In fact, it has not yet been possible to clarify why certain embryos of certain developmental stages only develop well under certain culture conditions. When growing rat embryos at the stage of organogenesis, for example, some authors obtained good results with the cultivation in homologous rat serum (COCKROFT, 1977); other authors used a mixture of rat and human sera (FANTEL et al., 1979). Some other investigators reported good results after cultivation in human serum (CHATOT et a l . , 1980). Each team employing the whole-embryo culture system has its own "ideal" medium, and everyone knows, although it is not necessarily published, that even under so-called "ideal conditions" the embryos may suddenly not grow in a normal way, without having an explanation for this phenomenon.

Culture Techniques © 1981 W a l t e r d e G r u y t e r & Co., Berlin • N e w Y o r k

20

These general, still unclarified cultivation problems are the reason why teratological investigations on whole-embryo cultures cannot be performed by simply employing the methods set up by other investigators. Every laboratory has to find out the culture conditions under which the embryos grow most satisfactorily. Having a reproducible whole-embryo-culture system, the question arises what criteria should be used to determine whether a substance added to the culture medium is able to induce abnormal development. The aim of the present study is to compare the in vivo and in vitro development of 10.5-day-old rat embryos for a period of 48 hours. On the one hand, this serves to test the suitability of our in vitro system; on the other hand, after macroscopic and microscopic investigations of the development of rat embryos, criteria are to be established which help decide which organ anlagen develop well during the 48 hour culture period and can therefore possibly be influenced by drugs. Materials and Methods: Wh°te-Embryo-£ulture Female Wistar rats were, kept at a normal day/night cycle and received standard food (Altromin ) and water ad libitum. The day when vaginal plugs were detected after a 2-hour mating period (6 - 8 a . m . ) was designated as day 0 of gestation. On day 10.5 of gestation, the embryos and egg membranes were removed from the uterus. Subsequently, Reichert's membrane was opened and the egg cylinders were grown in a roller culture according to COCKROFT (1977). The embryos were grown in human serum, to which tyrode buffer (6 + 2) had been added. The bicarbonate was replaced by phosphate buffer. Moreover, the culture medium contained 4 mg/ ml glucose. During the first 24-hour period the embryos were grown in an atmosphere of 30% O , , and for the following 24 hours at 95% O,. The culture techniques used in our laboratory are described in detail by M. ANSCHÜTZ (Chapter V I I , this book). Starting with 10.5-day-old embryos, three embryos each were removed from the culture after 12, 24, 36, and 48 hours, and were fixed in Bouin's fluid and embedded in Paraplast. We evaluated only embryos which had beating hearts. 5 vim thick serial sections were cut in longitudinal and transverse direction, stained with haematoxylin-eosine and embedded in Eukit. Other embryos were fixed in a mixture of 3% paraformaldehyde and 3% glutaraldehyde in cacodylate buffer (pH 7.2). After postfixation with 1% 0 s 0 4 in cacodylate buffer, the preparations were embedded in Mikropal (Ferak). Subsequently, 1 yim thick serial sections were cut in transverse direction and stained with alkaline Giemsa solution before being covered with Euparal. Embryos at corresponding stages were also taken from prepared mice and processed in the same way.

P®?®™??^??_2i _DNA_and_ Protein_ Somite number, dry weight, protein content and DNA content of embryos (starting with day 10.5 of gestation) grown in vitro for 12, 24, 36, or 48 hours and of embryos which developed in vivo were determined. The DNA content was determined according to the method of BURTON (1956) and the protein content using the Biuret method.

21

Results: Macroscoj££ The rat embryos placed into culture on day 10.5 were either shortly before the stage of turning or had already turned. The earliest developmental stage before turning corresponded approximately to the neural fold stage ( 4 - 5 somites), (Fig. 3). The most advanced embryos were approximately at the 12 - 14-somite stage. Until day 11.5, size and shape of the embryos grown in vitro corresponded to those grown in vivo. When evaluated on day 12 of gestation, development of the embryos in vivo as well as in vitro showed some variations. There were embryos grown in vitro that were more advanced after a 36-hour culture period than in vivo embryos of the same age. In general, however, it can be stated that there were no macroscopically visible developmental differences between in vitro and in vivo embryos. On day 12.5, however, the development of the in vivo embryos was clearly more advanced than that of the in vitro embryos. The higher growth rate in vivo compared with that in vitro was also evident by the clearly higher dry weight, the higher somite number and the higher protein and DNA content of the in vivo embryos (Tables 1 and 2). During a 48-hour culture period (starting with 10.5-day-old embryos) the following developmental steps could be followed macroscopically: -

Turning of the embryos (between days 10.5 and 11); Closure of the neural tube and development of the telencephalon, mesencephalon and rhombencephalon; Development of the eye and ear vesicle; Development of the olfactory placode; Development and closure of the 1st and 2nd branchial arches; Beginning of the heart action (between days 10.5 and 11); Development of the cardiac tube and formation of the truncus arteriosus, conus (bulbus) arteriosus, ventricles, atria (left and right auricular appendix) and the sinus venosus; Development of upper and lower limb buds; Development of the tail.

From these developmental steps visible during the 48-hour culture period, the criteria listed in Table 3 were derived which should be considered when employing the whole-embryo-culture for teratological investigations of rat embryos between days 10.5 and 12.5. Histology The earliest of the embryos placed into culture on day 10.5 were still in the so-called neural fold stage. They had not yet turned, i . e . , the ectoderm was still located at the inside and the entoderm at the outside of the egg cylinder (Fig. 4).

22

The neural tube below the region of the heart anlage was already closed (Fig. 4). In the cranial and caudal regions, however, it was still open. The development of the entodermal tube proceeds reversely. The entodermal tube was already closed in the cranial and caudal regions of the embryos, whereas it was still open in the region of the closed neural tube (Fig. 4). The only organ that could clearly be identified at this developmental stage was the heart. The coelom cavities also started to develop at this stage (Fig. 4). Although macroscopical inspections did not reveal any developmental differences between in vivo and in vitro embryos on day 11, there were certain histological differences. Although the fixation conditions were the same for all embryos, the fixation of the in vitro embryos seemed to be less satisfactory. The tissue often appears somewhat shrivelled and the cell s t r u c t u r e s were not in all cases as well preserved as those of in vivo embryos. This suggests that at this stage the embryos are possibly in a critical phase of adaptation to the culture conditions. Histological investigations did not reveal any differences in the developmental state between in vitro and in vivo embryos on day 11 either. On day 11.5 and 12, the development of in vitro and in vivo embryos proceeded almost parallel. The differences in the development between in vivo and in vitro embryos on day 12 have already been mentioned. Macroscopic investigations already showed that on day 12.5 the in vivo development was clearly ahead of the in vitro development. Apart from the more advanced course of development and differentiation of the organ anlagen of the in vivo embryos, the in vivo embryos showed structures ( e . g . in the region of the urogenital system) that in vitro embryos did not show at this stage. While on day 12.5 in vitro the mesonephric duct was about to develop, in vivo even the uteric buds and the genital ridge were already clearly visible. Investigations on the developmental course of the eye and ear between days 10.5 and 12.5 have shown that the cranial-caudal development of the embryos is not subject to strict temporal regularities, either in vivo or in vitro. Thus, the developmental stage of a caudally located organ, e . g . the liver, cannot necessarily be taken as an indicator of the developmental stage of the ear vesicle of the same embryo. So, on day 11.5 we observed in vitro embryos whose ear vesicles were still open towards the surface, but whose eye cup had already clearly invaginated. However, in most cases the invagination of the eye cup proceeded almost parallel to the closure of the ear vesicle. On day 12.5 in vivo embryos were observed whose eye lenses had not yet separated from the surface, but whose genital ridge was already clearly identifiable. Normally, on day 12.5 in vivo, the eye lenses had already been separated from the surface. During the 48-hour culture period, the developmental steps of the organs, which were not visible macroscopically, can be followed histologically. Moreover, histological investigations provide a vast amount of additional information, such as: -

Details on eye development, e . g . invagination of the eye cup; Invagination of the lense placode; Details on ear development, e . g . invagination of the ear placode;

23 -

Closure of the ear vesicle, development of the labyrinth system (ductus endolymphaticus, sacculus, utriculus);

-

Development of the Rathke's pouch (adenohypophysis);

-

Course of the closure of 1st and 2nd branchial arches;

-

Development of the thyroid gland anlage;

-

Development of the laryngotracheal duct;

-

Differentiation of the cardiac tube (formation of the ventricle septa and the myocardium);

-

Development of the spinal ganglia;

-

Development of the stomach and the duodenum anlage in the entodermal tube is recognizable, but not clearly identifiable, at all developmental stages;

-

Development of the liver anlage;

-

Development of the mesonephric duct.

Discussion: Compared with the great number of publications on technical problems involved in the whole-embryo culture technique, there are only few publications on teratological investigations using this culture technique. This discrepancy in the practical application of the whole-embryo-culture technique becomes understandable if we look at the broad spectrum of abnormal embryonic development which can be induced by certain culture conditions. This culture system seems to be so sensitive that it may be disturbed by a number of external factors, be it by drugs or other changes of the culture conditions. The question arises whether an abnormal embryonic development, occurring after the addition of the drug, is due to a specific attack of the drug on embryonic structures or, whether the addition of the drug changes the culture conditions in such a way that normal growth and differentiation of the in vitro embryos are impossible. It has been shown, for example, by BROWN et al. (1979) that the addition of alcohol to the culture medium disturbs the development of rat embryos in vitro. Based on this finding, the authors made statements on the "teratogenicity" of alcohol. It is also very likely that the addition of milk, water, urine, salt or other substances to the culture medium also disturbs the development of rat embryos in vitro. However, it would be absurd to claim that milk or water are "teratogenic". This type of consideration certainly throws too negative a light on the whole-embryo-culture system. As already mentioned in the introduction, the reason why embryos only grow in certain media and under certain culture conditions are not as yet fully elucidated and are still extensively based on empiric findings. The fact that embryos do not grow in a normal way after the addition of certain substances to the culture medium is, in our opinion, as such and by itself, no criterium for the teratogenicity of a substance. When employing the whole-embryo-culture system for teratological investigations, it has first to be excluded that the observed effects are induced by general growth disturbances in the culture. If general growth retardation or growth retardation and subsequent unbalanced growth occur in vitro, the corresponding findings should be evaluated very carefully.

24

If, however, the addition of a substance in vitro leads to morphologically demonstrable disturbances of certain embryonic structures during an otherwise normal development of the embryo, this can be taken as a clue for a possible teratogenic potency of the added substance. It could, for example, be shown that after the addition of cyclophosphamide (CPA), embryonic malformations only occur if a drug metabolizing enzyme system is added simultaneously (FANTEL et a l . , 1979). The fact that embryonic development is normal after the sole addition of CPA or the enzyme system may indicate that a CPA metabolite has to be formed in vitro which is responsible for the abnormal development. However, the possibility cannot be excluded that substances added in vitro do not primarily attack the embryo but influence embryonic development by changing the culture conditions. This holds true until we are able to grow embryos reproducibly in chemically defined media. And even then it would be unjustified to expect, for a routine testing, more of in vitro methods than of in vivo investigations, just because one does not want to realize that even teratogenicity testing in vivo involves numerous still unsolved problems. For the elucidation of the mode of teratogenic action, in vivo methods will become indispensible in the f u t u r e . The f i r s t step towards the application of the whole-embryo-culture system for the evaluation of abnormal embryonic development can be made when we succeed in achieving a relatively constant and reproducible embryonic growth and differentiation in vitro. Moreover, one must know the developmental steps of the embryo during the culture period to be able to evaluate developmental disturbances. It appears essential that the method must be much more standardized than it has been so far in almost any of the published studies. Such a standardization may be achieved by culturing only embryos which have nearly the same somite stage at the beginning of the culture. Determination of the somite number, dry weight, protein and DNA content of the in vitro embryos and comparison with in vivo findings yielded reliable data which characterize our in vitro system. It was shown that on day 12.5, i . e . , after 48 hours in vitro, the development of the rat embryos was behind the in vivo development. Although the embryos were grown at 30% and later at 90% O z p r e s s u r e , we were not able to observe the malformations which MORRIS and NEW (1979) described. Histological findings on day 11, i . e . , after 12 hours in culture, indicated that the embryos go through a phase of adaptation to the culture conditions. These f i r s t 12 hours of the culture period are possibly decisive for the fate of the embryos and whether they die or develop in a normal way in vitro. During the following 24 hours, in vitro and in vivo development proceed mainly parallel. In general, we found that it is difficult to make statements on the respective developmental state of the embryos. One problem is the great biological deviation in the intrauterine development of the rat embryos. On day 10.5, we found embryos with 4, and other embryos with 10 somites, in one u t e r u s . The spectrum of the somite number on day 10.5 ranged from 4 to 14 somites. Hence, considerable deviations may also be expected during the culture period and the comparable developmental phase in vivo unless a rigid standardization is performed. A comparison of the individual developmental stages is additionally aggravated by the fact that the cranial-caudal development of the embryo is - as our histological findings have shown - not subject to strict regularities. Thus, an evaluation of the development of a certain organ does not necessarily allow statements on the developmental state of other organs. Only the sum of histo-

25

logical findings obtained with several embryonic organs enables us to make clear-cut statements as to whether the development of one embryo is retarded compared with other embryos. This stresses the need for routinely performed histological studies, even with routine culture testing. Using our culture system for investigations on abnormal development, the macroscopically recognizable developmental steps of the embryo are to be taken as criteria for a f i r s t macroscopic inspection of treated and untreated embryos. If we confine ourselves to a macroscopic inspection, a number of information gets lost that can be obtained with this test system. We have to remember that a 48-hour culture period, for example, is very short compared with the overall developmental period of the embryos d u r ing which malformations may occur after the application of a substance in vivo. Only part of the processes occurring in vivo will proceed during the short culture period. Therefore, it is quite possible that during this short culture period, abnormal development is initiated in such a way that it cannot clearly be detected macroscopically and may thus be overlooked. Assuming a dose-response relationship of the substance to be tested might tempt us to solve this problem at least partially by increasing the dosage and thus increasing the embryonic defect. However, as mentioned above, we are convinced that it is only advisable to test substances in vitro at dosages that do not disturb the whole-culture system. A negative example for this would be the occurrence of malformations in vitro after high doses of D-glucose (COCKROFT and COPPOLA, 1977), although it is rather unlikely that D-glucose is a teratogen. Histological investigations reveal abnormal development caused by certain substances at a level which is far below that revealed by macroscopic inspection. Therefore, the risk that malformations may be overlooked can largely be excluded when the cultured embryos are investigated histologically. The histological findings we obtained with cultured rat embryos are to indicate the possibility to increase the informational value of the wholeembryo-culture system. But owing to the great number of histological information, it is, in contrast to macroscopic investigations, with this kind of technique and at this stage of embryonic development, not possible to make a "check list" for teratological investigations.

ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d b y g r a n t s g i v e n to the S o n d e r f o r s c h u n g s b e r e i c h 29 b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . W e w i s h to t h a n k Ms. H. W o h l f e i l P a r t s of this p a p e r w e r e d e a n d Ms. H. S t ü r j e for t h e i r t e c h n i c a l help. r i v e d f r o m the thesis of M. A n s c h ü t z .

26 LITERATURE Brown, N. A . , Goulding, E . H . , Fabrio, S . , 1979, Ethanol embryotoxicity: direct effects on mammalian embryos in vitro. Science, 206, 573 - 575. Burton, K . , 1956, A study of the conditions and mechanism of the reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J . , 62, 315 - 323. Chatot, C . L . , Klein, N.W., Piatek, J . , Pierro, L . J . , 1980, Successful culture of rat embryos on human serum: use in the detection of teratogens, Science, 207, 1471 - 1473. Cockroft, D . L . , 1977, Postimplantation embryo culture, m: "Methods in Prenatal Toxicology", Teratology Workshop April 1977, Berlin, (D. Neubert, H . - J . Merker, T . E . Kwasigroch, e d s . ) , Georg Thieme Verlag, Stuttgart, pp. 231 - 240. Cockroft, D . L . , Coppola, P . T . , 1977, Teratogenic effects of excess glucose on head-fold rat embryos in culture. Teratology, 16, 141 - 146. Cockroft, D.L. and New, D . A . T . , 1978, Abnormalities induced in cultured rat embryos by hyperthermia, Teratology, 17, 277 - 284. Fantel, A . G . , Greenaway, J . C . , Juchau, M . R . , Shepard, T . H . , 1979, Teratogenic bioactivation of cyclophosphamide in vitro, Life S e i . , 25, 67 - 72. Morris, G.M. and New, D . A . T . , 1979, Effect of oxygen concentration on morphogenesis of cranial neural folds and neural crest in cultured rat embryos, JEEM, 54, 17 - 35. New, D . A . T . , 1978, Whole embryo explants and transplants, in: "Handbook of Teratology", Vol. 4 ( J . G . Wilson, F . C . Fräser, e d s . ) . Plenum Press, New York and London, pp. 95 - 133. Steele, C.E. and New, D . A . T . , 1974, Serum variants causing the formation of double hearts and other abnormalities in explanted rat embryos, JEEM, 31, 707 - 719.

27 Table 1:

V9 DNA/E

^jg protein/E in vivo S3 in vitro

1SOO

in vivo in vitro

60

SO

ieoo

30

I

500

nh 10.5

12.5

28 Table 2:

29

Table 3: Criteria for Macroscopic Investigations of Rat Embryos Between Days 10.5 and 12.5 In Vivo and In Vitro

1.

Posture and shape of the embryos;

2.

Closure of the neural tube;

3.

Development of the head;

4.

Development of the eye vesicles;

5.

Development of the ear vesicles;

6.

Development of the olfactory discs;

7.

Development of the branchial arches;

8.

Development of heart and circulatory system;

9.

Development of the limb buds;

10.

Length and shape of the tail.

30

Fig. 1:

Course of development of rat embryos on days 11, 11.5, 12.5 in vivo.

and

Fig. 2:

Course of development of rat embryos placed into culture on day 10.5 after 12, 24, 36 and 48 hours in vitro.

31

Fig. 3:

Egg cylinder of a rat embryo on day 10.5. Dorsal view. Ec = ectoplacental conus, Al = allantois. The line approximately indicates the cutting line of Fig. 4.

Fig. 4:

Cross-section of an egg cylinder from a day 10.5 rat embryo before its turning. The entoderm is still at the outside and the already closed neural tube (N) at the inside of the egg cylinder. The entoderm of the head region (H) is still open. Caudally an entodermal tube is already recognizable. C = coelom cavity.

32

Fig. 5:

Cross -section of a rat embryo on day 11 in vivo in the region of the first branchial arch ( * ) . N = neural tube, M = region of the prospective mouth. Ear = ear vesicle, open at the surface.

Fig. 6:

Cross-section of a rat embryo on day 11 in vitro, cut slightly before that of Fig. 5. Arrow = thyroid gland anlage in the region of the oral plate. N = neural tube. Ear = ear vesicle, open at the surface.

33

Fig. 7:

Magnification of the ear vesicle on day 11 in vivo (see Fig. 5). * = open connection to the surface of the embryo.

Fig. 8:

Corresponding magnification of the ear vesicle on day 11 in vitro (see Fig. 6). * = open connection to the surface of the embryo.

Fig. 9:

Cross-section of the region of the laryngotracheal duct (Lt) of a rat on day 12.5 in vivo.

Fig. 10:

Cross-section of the region of the laryngotracheal duct (Lt) of a rat on day 12.5, i . e . , after 48 hours in vitro.

35 DISCUSSION Langman:

Your embryos in vitro were generally smaller than those in vivo after a 48-hour culture period. The number of somites may be the same, but the size of the somites may be much smaller. Did you measure the DNA and protein contents, measurments which I find better than dry weight?

Herken:

The embryos were not smaller after a 24-hour culture period but after a 48-hour culture period. We measured the DNA and protein contents, but as a morphologist, I do not feel that these data give better information on the development of the embryos in vitro than the somite number and the dry weight do.

Klein:

You observed that development in vitro is slower than in vivo. I do not understand how this statement can be made because of the great range in developmental stages found at any one time in any one animal. That is to say, if one tries to compare in vitro with in vivo one should find both slower as well as faster development.

Herken:

If you know for example that at the beginning of the culture period, day 10.5, the rat embryos have between 8 and 14 somites and 48 hours later the somite number in vivo is between 34 - 38 somites compared with only 28 - 32 somites in vitro, it seems to be adequate to call it a slower development in vitro. In my opinion, the somite number of an embryo, is an excellent criterion for the development of the embryo.

Klein:

Your in vitro dry weight after 48 hours in culture is less than the corresponding in vivo dry weight: D . A . T . NEW has published that in vitro protein content equals in vivo content. Please explain the difference.

Herken:

As far as I can remember, New describes that the development in his culture system is parallel to the in vivo development during 24 hours and that there is a considerable variability in the results after a 48-hour culture period. If you have seen the pictures of embryos cultured by NEW and COCKROFT at the beginning of their culture period ( i . e . "Handbook of Teratology 4 " ) , you will have noticed that these embryos are in different developmental stages (cf. COCKROFT, 1977). This agrees with our findings. We tried to avoid this variability in development in the culture system by a better staging of the embryos at the beginning of our culture period.

Cockroft, D . L . , 1977, in: "Methods in Prenatal Toxicology", Teratology Workshop April 1977, Berlin, (D. Neubert, H . - J . Merker, T . E . Kwasigroch, eds. ) , Georg Thieme Pubi., Stuttgart, pp. 231 - 240. New, D . A . T , 1978, in:"Handbook of Teratology 4 " , ( J . G . Wilson and F . C . Fräser, e d s . ) , Plenum Press, New York and London, pp. 95 - 133.

The Differentiation Between Teratogenic Action on Mother, Fetal Membranes and Embryos Using the Whole-Embryo Culture Method F. Beck Department of Anatomy, University of Leicester, University Road, Leicester LE1 7RH / U.K.

Introduction: A logical approach to teratogenesis leads inevitably to the conclusion that an embryotoxic agent may act at one or more of t h r e e sites - the mother, the embryo and the materno-embryonic interface. There are some clinical examples of teratogenesis which suggest a primary action at the maternal level. These include diabetes, myxoedema and to a certain extent some inborn e r r o r s of metabolism such as phenylketonurea. Sometimes an e x t r a neous substance such as cyclophosphamide will require metabolism by maternal tissues prior to the acquisition of teratogenic potency (FANTEL et a l . , 1979), alternatively, the teratogenic activity of a particular agent can be considerably enhanced or depressed by the maternal dramatype. T h u s , KIMMEL et al. (1971) have shown that the teratogenicity of sodium salicylate reflected in maternal serum levels can be considerably enhanced by the concommitant presence of high levels of serum benzoic acid which int e r f e r e with salicylate excretion by the mother and keep serum levels high. The majority of teratogenic agents successfully isolated so f a r seem to act primarily upon the embryo or fetus itself. These include physical agents in the form of X - r a y s (BRENT, 1969), chemicals, ranging from antimetabolites such as 6 aminonicotinamide (CHAMBERLAIN and NELSON, 1963) to synthetic progestins (CAHEN, 1966) and also infective agents such as r u bella and syphillis, which produce embryonic or fetal infections. A t h i r d , little studied possibility, is that a teratogenic agent may act at the materno-fetal interface. In the early post-implantation period this may be due to an inhibition of histiotrophic nutrition either by inhibition of endocytosis (WILLIAMS et a l . , 1976) or by inhibition of lysosomal enzymes (LOWY, 1981). After the establishment of a chorio-allantoic placenta, int e r f e r e n c e with the maternal circulation supplying it has been shown to be embryopathic. Other theoretical possibilities are abnormalities at the site of implantation with consequent alteration in the quality of histiotroph or abnormalities in the process of implantation. It seems at least possible that the local microenvironment at the implantation site may be abnormal as suggested by CLARKE and his associates (GARDINER et a l . , 1978). In Vitro Studies: The whole embryo culture method developed by NEW (1976) can be used to determine whether a teratogenic agent is capable of acting directly upon the conceptus. It does not formally exclude an additional action on the mother b u t if all the relevant factors (such as teratogenic serum concentra -

Culture Techniques © 1981 Walter de Gruyter & Co., Berlin

New York

38

tion compared with serum levels in the culture medium (MCGARRITY et al., 1981) are taken into account it is usually possible to make a judgement as to which is the more important. The in vitro technique also does not invariably differentiate between a primary site of action on the embryo or on the yolk sac placenta. Nevertheless, it is often possible to manipulate the system sufficiently to allow one to make an appropriate judgement. For example, action on the inverted yolk sac placenta can be demonstrated morphologically following the addition of leupeptin (a specific inhibitor of cathepsins B, H and L ) to the culture medium (BECK and GULAMHUSEIN, 1980). In this case specific inhibition of the digestion of histiotroph has been demonstrated by unequivocal electronmicroscopic changes in the yolk sac (the organ responsible for breakdown of histiotroph) following dose dependent addition of leupeptin to the culture medium. Trypan blue has been shown to cause malformations not only in the rat but also in the ferret (BECK et al., 1978). In this case disturbance of histiotrophic nutrition is due - at least in the rat - to inhibition of endocytosis by the yolk sac. Trypan blue has other actions, some of which affect the mother (see BECK 1979 for review) and some the embryo directly (GULAMHUSEIN, this book); nevertheless, it seems most likely that, in the rat at least, the action on yolk sac endocytosis is the most important one; in other species such as the ferret the question is still an open one since the nature of the in vivo malformations differ in both nature and timing to those of the rat here indeed the effect on either mother or embryo may be important and the precise inter-relationship between the three possible areas of action must await the development of appropriate techniques. Given a standard maternal drama type (which will, therefore, give rise to a repeatable serum level of salicylate following administration of the optimum teratogenic dose) one can perform experiments which indicate that salicylate teratogenicity is probably the result of the direct action of the drug on the embryo. This conclusion is based upon the fact that the drug causes no apparent morphological changes in the yolk sac, while producing congenital malformations both in vivo and in vitro (MCGARRITY et al., 1981). Furthermore, it can be shown that the drug, rather than one of its metabolites, is of prime importance because the type and level of malformations is almost identical when rat embryos are grown either in serum from a treated mother or in normal rat serum to which the same levels of sodium salicylate have been added (MCGARRITY et al., 1981). Biochemical and morphometric studies concerning the rate of digestion of histiotroph (WILLIAMS et al., 1975; GUPTA et al., 1980) have not been performed. One would expect them to be unaffected by salicylate if the drug acts entirely at the embryonic site. The 'Giant Yolk Sac' If the rat embryo cultured by the New Method is left in vitro after 11.5 days the circulation ceases and the embryo dies. Recently we have discovered, however, that the yolk sac itself continues to grow, so that after 8 days of culture with 4 changes of media and daily gassing with 20% 0 2 , 5% C0 2 and 75% N 2 a diameter of about 2 cm containing about 600 pi of fluid, is obtained. We consider this to be a very good system for studying the function of the yolk sac. Using lanthanum staining Al ALOUSI (personal communication) has shown that the junctional complexes between the epithelial cells remain intact and that the vacuolar system is essentially normal. Analysis of the yolk sac fluid by 2-way chromatography appears to show the presence of all the essential amino acids and polyacrylamide gel electrophoresis demonstrates the presence of at least three major and six minor protein bands. It seems that some proteins are synthesized by

39 the yolk sac (DZIADEK and ADAMSON, 1978, for an account of synthesis of an alpha-feto-protein in the mouse) and others transported intact, presumably in coated vesicles (HUXHAM and BECK - in press). The model is an interesting one because it can be used to investigate lysosomal function as well as the nutritional requirements of the growing embryo and early fetus. Conclusion: I have endeavoured to show how the whole embryo culture technique can be used in experiments to locate the principal site of action of teratogens. Recently KLEIN and his co-workers (CHATOT et al., 1980) have used glucose supplemented human serum as culture medium and this raises exciting possibilities such as the testing of sera from infertile women. In our hands it has been impossible to grow normal embryos on pure glucose supplemented human serum, but we have been successful in producing completely satisfactory embryos when 10% normal rat serum is added to 90% human serum with appropriate glucose supplementation, (RETI, BULMAN and BECK - in preparation). Al ALOUSI (personal communication) has shown that 10% rat serum with added glucose and vitamins is unable to support growth when diluted with 90% buffered saline solution and we are, therefore, confident that the human serum is largely used by the embryo in our experiments.

REFERENCES Beck, F . , 1979, Trypan blue induced teratogenesis, in: "Advances in the study of birth defects", (Persaud, e d . ) , M.T.P. Lancaster, pp. 37 - 51. Beck, F . , Gulamhusein, A . P . , 1980, The contrast between mouse and ferret limb buds in culture - possible advantages of comparing results from a limb culture system with whole embryo explantation, in: "Teratology of the Limbs", ( H . - J . Merker, H. Nau and D. Neubert, eds.), Walther de Gruyter, Berlin, pp. 117 - 127. Beck, F . , Swidzinka, P . , Gulamhusein, A.P., 1978, The effect of trypan blue on the development of the ferret and rat. Teratology, 18, 187 192. Brent, R . L . , 1969, Direct and indirect effects of irradiation upon mammalian zygote embryo and fetus, in: "Method for Teratological Studies in Experimental Animals and Man", (H. Nishimura and J . R . Miller, eds.), Igaku Shoin, Tokyo, pp. 63 - 73. Chamberlain, J . G . , Nelson, M., 1963, Multiple congenital abnormalities in the rat resulting from acute maternal niacin deficiency during pregnancy, Proc. Soc. Exp. Biol. Med., 112, 836 - 840. Chatot, C . L . , Klein, N.W., Piatek, J . , Pierro, L . J . , 1980, Successful culture of rat embryos on human serum: Use in the detection of teratogens, Science, 207, 1471 - 1473.

40

Cahen, R . L . , 1966, Experimental and Clinical chemoteratogenesis, Advanc. Pharmacol., 263 - 334. Dziadek, M. ( Adamson, E . , 1978, Localisation and synthesis of alphafoetoprotein in post-implantation mouse embryo, J . Embryol. Exp. Morph., 43, 289 - 313. Fantel, A . G . , Greenaway, J . C . , Juchau, R . , Shepard, T . H . , 1979, Teratogenic bioactivation of cyclophosphamide in vitro, Life Sci., 25, 67 72. Gardiner, A . , Clarke, C . A . , Cowen, J . , Finn, R . , McKendrick, 1978, Spontaneous abortion and fetal abnormalities in subsequent pregnancy, Brit. Med. J . , 1016 - 1018. Gulamhusein, A . P . , 1981, Further observations on trypan blue teratogenesis in vitro, in: "Applicability of Culture Techniques for Studies on Prenatal Differentiation and Toxicity", - this book. Gupta, M., Gulamhusein, A. P . , Beck, F . , 1980, Studies of endocytosis in the yolk sac endoderm of post-implantation rat embryos in vivo and in vitro, J . Anat., 131, 761 - 762. Huxham, M., Beck, F . , 1981, Receptor mediated coated vesicle transport of rat IgG across the 11.5-day in vitro yolk sac endoderm. Cell Biol. Int. Rep., (in p r e s s ) . Kimmel, C . A . , Wilson, J . G . , Schumacher, H . J . , 1971, Studies on metabolism and identification of the causative agent in aspirin Teratogenesis in the r a t . Teratology, ^ 15 - 24. Lowy, A . , 1981, The effect of an inhibitor of lysosomal proteolysis on early development in the r a t , J . Anat., 132, 449 - 450. McGarrity, C . , Samani, N . , Beck, F . , Gulamhusein, A. P . , 1981, The effect of sodium salicylate on the r a t embryo in culture: an in vitro model for the morphological assessment of teratogenicity, J . A n a t . , 133, 257 - 269. New, D . A . T . , Coppola, P . T . , Cockroft, D . , 1976, Improved development of rat head fold embryos in culture resulting from low oxygen and modification of culture serum, J . Reprod. F e r t . , 48, 219 - 222. Williams, K . E . , Kidston, E.M., Beck, F . , Lloyd, J . B . , 1975, Quantitative studies of pinocytosis. II. Kinetics of protein intake and digestion by the r a t yolk sac cultured in vitro, J . Cell Biol., 64, 123 - 134. Williams, K . E . , Roberts, G., Kidston, M.E., Beck, F . , Lloyd, J . B . , 1976, Inhibition of pinocytosis in the r a t yolk sac by trypan blue. Teratology, 14, 345 - 354.

41

DISCUSSION: Klein:

Why do the lysosomal vacuoles in the yolk sac decrease d u r ing development when the total protein requirement of the embryo must increase?

Beck:

Because the total number of cells increases.

Klein:

You compare a smoker with a non-smoker and observed r e sults that are comparable to ours. However, how do you know that the smoker doesn't do other things that might contribute something harmful to the serum (for example, excess alcohol, tea, etc.)- As I will show, in our smoking study we actually made a "before - after" comparison with the individuals.

Beck:

I agree.

Klein:

Might lupeptin be like trypan blue and block not only proteolysis but also protein uptake?

Beck:

No.

This has been proven by

125

I PVP uptake.

Trypan Blue Teratogenesis in the Rat In Vitro A. P. Gulamhusein Department of Anatomy, The Medical School, University of Leicester, University Road, Leicester LE1 7RH / U.K.

Introduction: The teratogenic effects of the dye trypan blue in the rat and other mammalian species are well documented (BECK, 1979). It has been shown that in the r a t the dye inhibits pinocytosis as well as lysosomal hydrolytic enzymes in the cells of the visceral yolk sac where it accumulates after maternal injection (WILLIAMS et a l . , 1976; BECK et a l . , 1967); the visceral yolk sac endodermal cells act as an organ of embryonic nutrition during much of the period of organogenesis prior to the establishment of the chorio-allantoic placenta. However, the apparent presence of the dye in the embryonic endodermal gut cells as reported by DENCKER (1977) leaves open the possibility that trypan blue may also directly affect the embryonic tissues. In order to investigate f u r t h e r the possible mechanisms of the effects of trypan blue on r a t embryonic growth and differentiation, TURBOW (1965, 1966), used an in vitro method based on the technique developed by NEW and STEIN (1964). From his observations he concluded that the dye acted directly on the embryonic cells because malformations were produced when it was injected through the visceral yolk sac endoderm into either the yolk sac cavity or into the amniotic cavity where it was in direct contact with the embryo. However, malformations were also produced when the 10 - 11-day embryos were exposed to trypan blue outside the visceral yolk sac at a time when the dye has low teratogenicity in vivo (WILSON et a l . , 1959). Moreover, static in vitro technique used by Turbow was to some extent unphysiological and has now been much improved. In this communication the results of experiments to study the effects of trypan blue on r a t embryos grown in vitro by culture methods developed by NEW and his co-workers (NEW, 1976), are reported. This technique involves the culture of rat embryos in a specially prepared serum from 9.5- to 11.5-days of gestation at growth and differentiation rates similar to that found in vivo (NEW et a l . , 1976). Materials and Methods: Male and female Wistar rats weighing between 300 - 500 g were used to obtain serum. 9.5- or 10.5-day embryos were obtained from pregnant females; pregnancy was timed from midnight of the night preceding the morning when sperm was detected in the vaginal smear. Embryos were explanted together with their extra-embryonic membranes using the method described by NEW (1976). The Reichert's membrane was carefully

Culture Techniques © 1981 Walter de Gruyter & Co., Berlin • New York

44

removed and the explants were then cultured in immediately-centrifuged, heat-inactivated rat serum. The glass culture bottles were placed in a roller tube incubator maintained at 37° C. The following experiments were performed: 1.

9.5-day conceptuses were cultured for 48 hours in serum to which trypan blue was added at concentration of 150 yig/ml or 300 yig/ml or 450 ng/ml; these serum levels are teratogenic in vivo (BECK and LLOYD, 1966).

2.

9.5-day conceptuses were cultured for the first 24 hours in serum to which trypan blue was added at the concentration as in ( 1 ) and then transferred to fresh serum for the remainder of the 24 hours of culture.

3.

10.5-day conceptuses were cultured for 24 hours in serum to which trypan blue was added at concentrations as in ( 1 ) .

In the experiments ( 1 ) , ( 2 ) and ( 3 ) there is a possibility that the dye may have direct action on the embryonic cells because until about 11 days, the embryonic cells are directly exposed to the culture medium, more so at 9.5-days when the embryonic endoderm is continuous with the visceral yolk sac endoderm. By day 11 the embryo lies completely within the expanded yolk sac. In order to exclude the possibility of direct contact of the dye with the embryonic tissue, trypan blue was added to the culture medium after the embryo had completely isolated itself from the surrounding medium; this developmental stage was reached at 36 hours after the initiation of culture of explanted 9.5-day conceptuses. 4.

Trypan blue was added at a concentration of 450 ng/ml to culture serum containing cultured 11-day conceptuses. The latter were cultured further for 12 hours.

5.

9.5-day conceptuses were cultured for 36 hours in fresh serum. Trypan blue (0.5 yd of 0.5% stock solution) was injected into the yolk sac cavity using a microneedle. Control conceptuses were either sham-treated or injected with 0.5 pi of sterile distilled water. A further group of conceptuses were injected with 0.5 yil of 0.5% solution of a non-teratogenic dye, azo blue (WILSON, 1954). After treatment, the conceptuses were cultured further for 18 hours.

After culture the conceptuses were assessed using the following criteria: heart beat, vitelline circulation, fusion of the allantois with the chorion, normal turning, normally closed neural tube, normal optic vesicles, presence of fore-limb buds, normal tail, normal somites, somite number, yolk sac diameter (not in experiment 5) and crown - rump length. A certain number of embryos from experiment ( 1 ) , ( 2 ) and ( 3 ) were assayed for protein content (LOWRY et al., 1951). Results: Table 1 shows the results of culture of 9.5-day conceptuses for 48 hours in trypan blue-treated serum. Various criteria were affected and the effect was dose-dependent. Figure 1 illustrates control embryos. Abnormalities produced in the embryos at various concentrations of trypan blue included abnormal turning (Fig. 2 ) , neural tube defects (Figs. 3, 4) and tail defects (Fig. 5 - embryo on the l e f t ) ; the tail defects manifested as

45

fluid-filled blebs on the side and/or tip of the tail. At all concentrations, the mean somite number and mean crown - rump length of the embryos was significantly affected. Culture of 9.5-day conceptuses in trypan blue-treated serum for the first 24 hours and then transferring the explants to fresh serum for the remainder of the culture period produced results generally similar to those obtained in the previous experiment except that the tail was less severely affected (Table 2). Culture of 10.5-day conceptuses for 24 hours in treated serum produced tail defects (spherical fluid-filled bleb at the tip of the tail - Fig. 5, centre and right embryos) which were often associated with an open posterior neuropore. None of the other criteria were significantly affected (Table 3). Protein content of the embryos from experiments 1 - 3 exhibited close correlation with the overall teratogenic activity of the dye. Only the embryos in the 48 hours and the first 24 hours exposure to trypan blue experiments showed a significant decrease in the protein content (see Tables 1, 2 and 3). Addition of 450 pg/ml trypan blue to serum containing 11-day conceptuses (Table 4) and then culturing the conceptuses further for 12 hours produced embryos with tail defects associated with open posterior neuropore similar to those seen in experiment 3. One other criteria that was affected in this experiment (4) was the normal turning of the embryos. In experiment 1 - 4 there was a massive accumulation of dye in the visceral yolk sac endodermal cells. Injection of trypan blue into the yolk sac cavity of 11-day conceptuses resulted in the embryos exhibiting turning, neuropore and tail defects after a further culture period of 18 hours (Table 5); all other criteria remained unaffected. Sham-treatment, injection of sterile distilled water and non-teratogenic dye (azo blue) did not affect development and differentiation of the embryos. It must be pointed out that the abnormalities produced in this experiment were identical to those seen in experiments 3 and 4. Discussion: The results of these experiments indicate that trypan blue is more teratogenic during the first 24 hours of culture and confirm the observations by BECK and LLOYD (1966), WILSON et al., (1959) and BERRY (1970) that its teratogenic activity decreases after 10.5-days of gestation in vivo. The abnormalities produced in the embryos at various concentrations of the dye in the culture serum were identical to those reported by BECK (1963) in vivo experiments. Injection of the dye into the yolk sac cavity of the cultured conceptus at 11 days produced a specific type of abnormality, i.e. a spherical fluidfilled bleb at the tip of the tail often associated with an open posterior neuropore. This defect was only produced with trypan blue (whether injected into the yolk sac cavity or added to the culture serum); injection of non-teratogenic azo blue or sterile distilled water into the yolk sac cavity produced no such defect. Thus abnormalities produced in the

46

r a t embryos by injection of trypan blue into the conceptus are specific to this dye and cannot be due to mere presence of particulate material next to the embryo (TURBOW, 1966). The types of abnormalities reported in this communication are somewhat different to those observed by TURBOW (1966). The embryos in his study exhibited mainly oedemic syndrome. The only oedemic abnormality observed in this study was the fluid-filled bleb. Neural tube defects were not observed in Turbow's work. The differences in the types of abnormalities may be due firstly to the type of culture technique. The static in vitro method used by TURBOW (1966) has been greatly refined by NEW and his associates (NEW, 1976) and added to this, the rate of growth and differentiation of the embryos using this method is identical to that in vivo (NEW et a l . , 1976). Secondly, TURBOW used a commercial sample of trypan blue containing 20% dye s t u f f , the remainder probably being water and NaCl. The dye sample used in this study was desalted and converted into the neutral form by dialysis against tap wat e r , dried over phosphorus pentoxide and then made into an 0.5% solution in sterile distilled water; very little coloured impurity was seen on chromatography (BECK et a l . , 1978). GRABOWSKI (1963) has reported that in the chick, embryo treatment with trypan blue results in the formation of fluid-filled blebs. His suggestion that these blebs are probably the result of ionic imbalances of embryonic fluids is f u r t h e r supported by KERNIS and JOHNSON (1969) who have reporf^d that +H the dye causes a significant increase in the absorption of Ca , S0 4 , and Na ions in the r a t visceral yolk sac. Furthermore, r a t embryos pretreated with trypan blue have altered embryonic blood pressure (GRABOWSKI, 1971). Thus it appears that the tail blebs seen in the embryos reported in this study may well be a manifestation of this phenomenon. As to the mode of action of trypan blue, there is evidence that the dye prevents the visceral yolk sac endoderm from performing its role in histiotrophic nutrition (WILLIAMS et a l . , 1976). In the culture protocol employed in this study, the rat embryo relies mainly on this type of nutrition as the chorio-allantoic placenta does not function in vitro. In spite of this, the teratogenic effect of the dye was found to be much milder in the second 24 hours of culture period (i.e. from 10.5 to 11.5 days). One possible explanation could be the differentiation of the embryonic cells at 10.5 days to a stage where the susceptibility to disturbances by teratogens is reduced. In vivo the decrease in the teratogenic potential of the dye after day 10 is partly attributed to the change in the mode of nutrition as the chorio-allantoic placenta is established (BECK, 1979). The possibility that trypan blue has a teratogenic effect by direct action on the embryonic cells has been suggested with the view that during early organogenesis embryonic endoderm is continuous with the visceral yolk sac endoderm. However, apart from DENCKER'S (1977) observations of dye particles in the embryonic endodermal gut cells no other part of the embryo appears vitally stained. It has been suggested that the presence of the dye in the embryonic gut cells may be caused by some of the embryonic endoderm (exposed to the dye during early organogenesis) moving into the developing gut region as the embryo folds in from the visceral yolk sac (BECK, 1979). It is also worth noting that the types of abnormalities obtained after injection of trypan blue into the yolk sac cavity are virtually identical to those obtained when conceptuses were cultured at equivalent times in serum containing the dye. Based on these observations and those of TURBOW (1966), it seems that the

47

dye does have some restricted action on embryonic cells. However, the massive accumulation of the dye particles in the visceral yolk sac endoderm seen in this study when it was added to the culture serum, suggests that most of its teratogenic effects are probably the result of visceral yolk sac disturbance. In conclusion, it is worth noting that in this study two mechanisms have been identified as to the mode of action of trypan blue. The f i r s t is the disturbance of histiotrophic nutrition probably up to 10.5 days of gestation; here there is evidence of a significant decrease in the protein content of the embryos cultured from 9.5 to 10.5 days. Secondly, the possible disturbances in the embryonic fluid balances which result in the formation of blebs. A more detailed report of this investigation entitled "Trypan Blue Teratogenesis in the Rat - Further Observations In Vitro" (Authors: GULAMHUSEIN, MOORE, GUPTA and BECK) has been submitted to Teratology.

ACKNOWLEDGEMENTS T h i s w o r k h a s b e e n s u p p o r t e d b y a g r a n t from the M e d i c a l R e s e a r c h C o u n cil. M r . W. M o o r e and Dr. M. G u p t a p a r t i c i p a t e d in the e x p e r i m e n t a l w o r k . I am also g r a t e f u l to Mr. C.R. d ' L a c e y for e x c e l l e n t t e c h n i c a l a s s i s tance .

REFERENCES Beck, F . , 1963, Aspects of the teratogenic action of trypan blue. M.D. Thesis, University of Birmingham, United Kingdom. Beck, F . , 1979, Trypan blue induced teratogenesis, in: "Advances in the Study of Birth Defects", Vol. 1, Teratogenic Mechanisms, (T.V.N. Persaud, e d . ) , MTS Press, International Medical Publishers, p p . 3 7 - 5 1 . Beck, F . , Lloyd, J . B . , 1966, The teratogenic effects of azo dyes, in: "Advances in Teratology", Vol. 1, (D.H.M. Woollam, e d . ) , Academic Press, London, pp. 131 - 193. Beck, F . , Lloyd, J . B . , Griffiths, A . , 1967, A histochemical and biochemical study of some aspects of placental function in the rat using maternal injection of horseradish peroxidase, J . Anat., 101, 461 - 478. Beck, F . , Swidzinska, P . , Gulamhusein, A . , 1978, The effect of trypan blue on the development of the f e r r e t and r a t , Teratology, 18, 187 - 192.

48

B e r r y , C . L . , 1970, The effect of trypan blue on the growth of the rat embryo in vivo, J . Embryol. Exp. Morph., 23, 213 - 218. Dencker, L . , 1977, Trypan blue accumulation in the embryonic gut of rats and mice during the teratogenic phase. Teratology, 15, 179 184. Grabowski, C . , 1963, Teratogenic significance of ionic fluid imbalances, Science, 142, 1064 - 1065. Grabowski, C . , T s a i , E . N . C . , Chernoff, N . , 1971, The effects of t r y pan blue on blood pressure in rat embryos. Teratology, 4 , 69. Kernis, M.M., Johnson, E . M . , 1969, Effects of trypan blue and Niagra blue 2B on the in vitro absorption of ions by rat visceral yolk s a c , J . Embryol. Exp. Morph., 22, 115 - 125. Lowry, O . H . , Rosebrough, N . J . , F a r r , A . L . , Randall, R . J . , 1951, Protein measurement with the Folin phenol reagent, J . Biol. Chem., 193, 265 - 275. New, D . A . T . , 1976, Comparison of growth in vitro and in vivo of postimplantation rat embryos, J . Embryol. Exp. Morph., 36, 133 - 144. New, D . A . T . , Stein, K . F . , 1964, Cultivation of post-implantation mouse and rat embryos on plasma clots, J . Embryol. Exp. Morph., 12, 101 - 111. New, D . A . T . , Coppola, P . T . , Cockroft, D . L . , 1976, Improved development of head fold rat embryos in culture resulting from low oxygen and modifications of culture serum, J . Reprod. F e r t . , 48, 219 222.

Turbow, M . , 1965, Teratogenic effect of trypan blue on r a t cultivated in vitro. Nature (London), 206, pp. 637.

embryos

Turbow, M . , 1966, Trypan blue induced teratogenesis of r a t embryos cultivated in vitro, J . Embryol. Exp. Morph., 15, 387 - 395. Williams, K . E . , R o b e r t s , G . , Kidston, M . E . , B e c k , F . , 1976, Inhibition of pinocytosis in rat yolk sac by Teratology, 14, 343 - 354.

Lloyd, trypan

J.B., blue,

Wilson, J . G . , 1954, Withdrawal of claim that azo blue causes congenital malformations, Proc. Soc. Exp. B i o l . , 81, pp. 1. Wilson, J . G . , Beaudoin, A . R . , F r e e , H . J . , 1959, Studies on the mechanism of teratogenic action of trypan blue, Anat. R e e . , 133, 115 128.

49 Table 1: 9.5-day Conceptuses cultured for 48 hours in trypan blue-treated serum (t.b.s.).

Trypan blue (yig/ml) Control n = 34

150 n = 22

300 n = 29

450 n = 32

Heart Beat %

97

95

93

86

Vitelline circulation %

97

95

86

84

Fused allantois %

100

100

100

94

Normal turning %

94

73

41

25

Normally closed neural tube %

94

50

48

31

Normal optic vesicles %

100

100

90

91

Fore limb buds %

100

100

90

81

Normal somites %

100

91

69

66

Normal tail %

100

27

31

16

Mean Y . S . dia. (mm) ± S . E . Mean C . R . lgth. (mm) ± S . E .

3.81

3.93

3.73

± 0.076

± 0.070

± 0.084

3.07 ± 0.068

3. 59* ± 0.,079

2.78*

2.83*

2..56*

± 0.045

+ 0.050

± 0..065

22.9

20.9*

19.7*

18..7*

± S.E.

± 0.29

+ 0.34

± 0.76

± 0..74

Protein

164.3

142.5*

137.6*

110,.6*

Mean somite No.

(yig/embryo)

± 5.4

± 6.3

± 3.6

± 7,.4

± S.E.

n = 12

n = 10

n = 10

n = 9

n = number of embryos; C . R . = Crown-Rump; Y . S . = Yolk sac; * = P < 0.05.

50

Table 2: 9.5-day Conceptuses cultured for 24 hours in t . b . s . and for 24 hours in fresh serum

Trypan blue (yig/ml) Control n = 34

150 n = 20

300 n = 29

450 n = 24

Heart Beat %

97

100

79

83

Vitelline circulation %

100

100

72

79

Fused allantois %

100

100

79

100

Normal turning %

85

70

52

21

Normally closed neural tube %

91

80

38

38

Normal optic vesicles %

100

100

76

92

Fore limb buds %

100

100

69

66

Normal somites %

100

100

69

63

Normal tail %

100

90

69

42

Mean Y . S . dia. (mm) ± S.E. Mean C.R. lgth.

3..95

4.15

+ 0.,069

± 0.079

3.,14

2.97

3.62*

3.55*

± 0.087

± 0.095

2.83*

2.77*

± 0..040

± 0.070

± 0.087

± 0.066

23..6

23.3

21.0*

20.2*

± S.E.

± 0..33

± 0.51

+ 0.63

± 0.91

Protein

170,.0

150.3*

130.1*

114.4*

(pg/embryo)

+ 6,.9

± 5.8

± 9.7

± 6.0

± S.E.

n = 13

n = 10

n = 10

n = 10

(mm) ± S.E. Mean somite No.

n = number of embryos;: C.R. = Crown-Rump; Y . S . = Yolk sac; * = P < 0.05.

51 Table 3: 1 0 . 5 - d a y Conceptuses cultured for 24 hours in trypan blue-treated serum.

Trypan blue (ng/ml) Control n = 21

150 n = 18

300 n = 23

450 n = 24

Heart Beat %

100

100

100

100

Vitelline circulation %

100

100

100

100

Fused allantois %

100

100

100

100

Normal turning %

95

94

96

96

Normally closed neural tube %

95

50

39

33

Normal optic vesicles %

100

100

100

100

Fore limb buds %

100

100

100

100

Normal somites %

100

100

100

100

Normal tail %

100

44

26

21

Mean Y . S . dia. (mm) ± S . E . Mean C . R . lgth. (mm) ± S . E .

3.89

3..97

3..90

3.85

± 0.069

± 0..064

± 0.,075

± 0.075

3.11

3..06

3..07

3.10

± 0.081

± 0,.051

± 0..059

± 0.064

22.6

22,.8

22..5

21.9

± S.E.

± 0.29

± 0..31

± 0..40

± 0.33

Protein

166.7

170..1

165,.0

146.4

(lag/embryo)

± 9.8

± 8,.5

± 7,.3

±10.3

± S.E.

n = 10

n = 10

n = 10

n = 10

Mean somite No.

n = number of embryos; C . R . = Crown-Rump; Y . S . = Yolk sac;

52

Table 4: 11-day Conceptuses cultured for 12 hours in trypan blue-treated serum. Trypan blue (yg/ml) Control n = 16

150 n = 0

300 n = 0

450 n=24

Heart Beat %

100

—

Vitelline circulation %

100

—

92

Fused allantois %

100

—

100

Normal turning %

94

- -

75

Normally closed neural tube %

88

- -

58

Normal optic vesicles %

100

- -

100

Fore limb buds %

100

- -

92

Normal somites %

94

- -

96

—

33

Normal tail %

100

100

3..78

3..90

(mm) + S . E .

± 0..07

± 0..05

Mean C . R . lgth.

3..18 ± 0..04

± 0..04

Mean Y . S . dia.

(mm) ± S . E . Mean somite No. ± S.E.

3..10

25..63

25 .75

± 0..29

± 0 .20

Protein (jig/embryo) ± S.E. n = number of embryos; C . R . = Crown-Rump; Y . S . = Yolk sac;

53 Table 5: Injection of water* or trypan blue** or azo blue** into the yolk sac cavity of 11-day cultured conceptuses. Control n = 24

Water n = 18

Heart Beat %

100

100

88

96

Vitelline circulation %

100

94

88

96

Fused allantois %

100

94

96

100

Normal turning %

92

94

75

100

Normally closed neural tube %

96

94

75

100

Normal optic vesicles %

100

100

100

100

Fore limb buds %

100

100

92

100

Normal somites %

96

100

100

100

100

100

42

100

Normal tail % Mean C . R . lgth. (mm) ± S . E . Mean somite No. ± S.E.

Trypan blue n = 24

Azo blue n = 22

3.10

3.09

3.00

3.14

± 0.04

± 0.05

± 0.05

± 0.04

26.83

27.00

26.75

25.18

± 0.16

± 0.16

± 0.18

± 0.18

* = 0.05 ul distilled water; ** = 0.05 yil of 0.5% solution; n = number of embryos; C . R . = Crown-Rump; Y . S . = Yolk sac;

54

Fig. 1:

11.5-day control embryos explanted at 9.5 days and cultured for 48 hours in normal serum. X 20.

Fig. 2:

11.5-day embryos explanted at 9.5 days and cultured for 48 hours in serum containing 300 |ig/ml ( l e f t ) and 450 pg/ml ( r i g h t ) showing abnormal turning and marked growth retardation. X 17.

55

Fig. 3: 11.5-day embryo explanted at 9.5-days and cultured for 48 hours in serum containing 300 pg/ml trypan blue showing kinking and opening in the neural tube. X 17.

Fig. 5:

Fig. 4: 11.5-day embryo explanted and cultured for 48 hours in serum containing 450 vig/ml trypan blue showing exencephaly and abnormal turning. X 17.

11.5-day embryos showing fluid-filled blebs. The embryo on the left was explanted at 9.5 days and cultured for 48 hours in serum containing 450 pg/ml trypan blue. Note this embryo is abnormally turned and its neural tube is open in the rhomencephalic region. The embryos in the centre and right were grown in serum containing 450 pg/ml and 150 pg/ml trypan blue respectively during the last 24 hours of culture period. X 18.

56 DISCUSSION Scott:

Some of your slides indicated that trypan blue affected forelimb development. Were these specific limb abnormalities or just a retardation of their appearance?

Gulamhusein:

No specific malformations were seen in the limbs. In the affected embryos the limb buds were either present (similar to the controls), or poorly developed or altogether absent in grossly retarded embryos.

Herken:

How do you know that you see teratogenic effects in your experiments and not only growth retardation of the embryos?

Gulamhusein :

In all the experiments reported here, the malformations observed were similar to those observed in BECK's (1963) in vivo work. Using our criteria for determining normal growth and differentiation in vitro, I am confident that malformations observed are teratogenic effects and not merely growth retardation.

Pedersen:

I am intrigued by the results of DENCKER and others that you quoted, because they bear on the issue of whether primitive endoderm contributes any cells to the definitive endoderm of the fetus. The results of SKRET and GARDNER suggest that the primitive endoderm does not contribute, but there are other possible interpretations of these results. Do you confirm DENCKER's observation that some definitive endodermal cells are labelled with t r y pan blue that is administered early in culture, when primitive endoderm is still exposed to the culture medium?

Gulamhusein :

It is very difficult to determine this in the early conceptus at 9.5-days. However, fluorescence studies of 11.5day conceptuses exposed to trypan blue at 9.5-days show the presence of dye particles in the endodermal gut cells as well as the gut lumen.

Beck, F . , 1963, J . Embryol. exp. Morph., 11, Dencker, L . , 1977, Teratology, 15,

179 - 184.

175 - 184.

Studies on Embryotoxic Effects of Thallium Using the Whole-Embryo Culture Technique M. Anschütz1, R. Herken and D. Neubert Institut für Toxikologie und Embryopharmakologie der Freien Universität Berlin, Garystraße 9, 1000 Berlin 33

Introduction: It has recently been claimed t h a t thallium is highly teratogenic in mice (ACHENBACH, et a l . , 1979 a , b ) . Skeletal abnormalities have been r e ported to occur in exposed mouse f e t u s e s . However, t h e s e publications considerably d i f f e r from the usual reliable scientific communications since they do not give any information on the number of t r e a t e d animals or on other important experimental details. Since the data published so f a r , do not give any clue to t h e exceptionally high occurrence of thallium teratogenicity, and studies p r o v e d t h a t the metal does not easily cross the placental b a r r i e r in r a t s (GIBSON and BECKER, 1970) we investigated the capacity of thallium for inducing abnormal development in whole embryo culture of r a t s . The advantage of this t e c h nique is t h a t the teratogenic potential of a chemical directly affecting the embryo can be evaluated with maternal and placental f a c t o r s excluded. First r e s u l t s of studies on the possible i n t e r f e r e n c e of thallium with embryonic development in c u l t u r e are p r e s e n t e d in the following. Material and Methods: 10.5-day-old r a t embryos were cultured for 48 h o u r s in human serum (for details on the culture technique cf. ANSCHUTZ, section V I I , this b o o k ) . The culture medium was supplemented with thallium sulfate at concentrations of 3 - 100 iag thallium/ml. The embryos were cultured for 48 h o u r s with thallium p r e s e n t in the medium d u r i n g the entire c u l t u r e period. A f t e r the culture period, h e a r t beat and yolk sac circulation of the explants was examined and the embryos were s t r i p p e d of their membranes. A f t e r stereo-microscopic evaluation they were fixed in a mixture of 3% p a raformaldehyde and 3% glutaraldehyde in cacodylate b u f f e r (pH 7 . 2 ) . Aft e r postfixation with 1% 0 s 0 4 in cacodylate b u f f e r , the p r e p a r a t i o n s were embedded in Mikropal ( F e r a k ) . S u b s e q u e n t l y , 1 pm thick serial sections were cut in t r a n s v e r s e direction and stained with alkaline Giemsa solution before being covered with Euparal. Results: When general development of the embryos was examined by stereo-microscopy a concentration of 100 pg thallium/ml was found to i n t e r f e r e drastically with growth and differentiation in c u l t u r e . The h i g h e s t concentration

Culture Techniques © 1981 Walter de Gruyter & Co., Berlin • New York

58

where no obvious deviation from normal development could be detected was 10 yig thallium/ml. Further careful histological studies of serial sections of the embryos exposed to 3 - 30 yig/ml thallium in the culture medium revealed clear-cut structural alterations which, after macroscopic inspection, appeared normal in size and development. After a 48-hour culture period in the presence of thallium, with doses ranging from 3 pg thallium/ml to 100 pg thallium/ml, the r a t embryos showed a dose-dependent growth retardation (Fig. 1). 100_yg_ thalHum/ml ^ After cultivation with 100 yig thallium/ml we observed a complete growth inhibition in r a t embryos (Fig. 1). After the 48-hour culture period the embryos had not even reached the developmental stage of the control animals cultured for 12 hours (HERKEN and ANSCHUTZ, this book). Owing to this complete growth inhibition of the embryos cultured in the presence of 100 jig thallium/ml we refrained from doing f u r t h e r investigation on these embryos. 30_¡jg_ thallium/ml According to the check list for the macroscopic inspection of cultured r a t embryos (HERKEN and ANSCHUTZ, this book) the only abnormality which could be detected was a minor disturbance in the shape of the tail of embryos cultured with 30 pg thallium/ml. Striking was a growth retardation of the embryos compared with the development of the control embryos and the embryos cultured with lower thallium doses (Fig. 1). After 48 hours in vitro the embryos reached a developmental stage which corresponded to the development of control animals cultured for 24 - 36 hours, indicating a growth retardation of 12 - 24 hours. Although the macroscopic inspection of the embryos cultured with 30 yig/ml thallium only showed a growth retardation of 12 - 24 hours and minor abnormalities, the histological pictures indicated that the embryos were extremely damaged. In all parts of the central nervous system (CNS), the majority of the neuroepithelial cells were necrotic. We always observed more necroses in the dorsal part of the brain anlage or the spinal cord than in the corresponding ventral p a r t . The caudal region of the spinal cord was so destroyed that no ventricular lumen was visible (Figs. 2, 3). The only region of the CNS where unaffected cells could be observed were the marginal zone and the mantle zone. The histology of the embryonic mesodermal structures did not look much better than the histology of the CNS. In the paraxial mesoderm (Figs. 2, 4), the somitic mesoderm, the mesoderm of the limb anlagen and the mesoderm of the branchial arches numerous cell necroses were visible. When compared with the CNS and the mesodermal structures of the embryos, the entodermal structures were seen to be less affected. In the entodermal tube only single necroses could be seen. Only in the region of the thyroid gland anlage and the laryngotracheal duct were cell necroses visible to a greater extent. In general, except for the skin, the notocord and the wall of the heart and the vessels, all other developing organs of the embryos cultured with 30 jig thallium/ml for 48 hours, were affected, some more and some less.

59 10 _ y cj_ thajlium/ml i Macroscopic inspection gave no indication of malformations in the treated embryos. Compared with the controls, some embryos cultured in the prescence of 10 yig thallium/ml seemed to be somewhat smaller in size and had 2 - 4 somites less than the corresponding control embryos, indicating a minor growth retardation. Although only a minor growth retardation could be detected macroscopically, microscopically strong embryotoxic effects on the CNS of the treated embryos were observed. In the dorsal part of the brain anlage and the spinal cord numerous necroses of neuroepithelial cells were visible (Figs. 5, 6 ) . These cell necroses were mainly localized in the intermediate zone of the CNS (Fig. 6 ) . Compared with the embryotoxic effects on the CNS, the mesoderm of the embryos cultured with 10 yig/ml thallium was much less affected. Evident were cell necroses in the limb anlage and the somites (Fig. 7 ) . In contrast to the effects observed after 30 yig thallium/ml, after cultivation with 10 yig/ml only single necroses could be seen in the paraxial mesoderm and in the mesoderm of the branchial arches. In the entoderm, cytotoxic effects were not detectable. 3_

thalhurn/ml

No differences could be observed macroscopically between control embryos and embryos cultured for 48 hours in the presence of 3 yig/ml thallium. There was no indications of malformations or growth retardation, which could be taken as an indication of embryotoxic effects of the treated embryos . Although the embryos looked absolutely normal, microscopic inspection r e vealed the cytotoxic effect of thallium on the CNS. In contrast to the effects after cultivation with higher thallium doses only some necroses of neuroepithelial cells were visible in the CNS (Figs. 8 - 10), but in comparison to higher doses these necroses were mainly localized in the dorsal part of the brain anlage (Figs. 9, 10) and the spinal cord (Fig. 8 ) . It was striking that in those regions of the CNS where numerous necroses could be observed after higher thallium doses, wide intercellular spaces occurred after cultivation with 3 yig thallium/ml (Figs. 8, 10). These intercellular spaces and the occurrence of macrophages containing cell fragments in their cytoplasm (Fig. 10) could be taken as an indication that numerous necroses had already been removed by phagocytosis during the 48-hour culture period. After cultivation of rat embryos in the presence of 3 yig thallium/ml the cytotoxic effects were limited to the CNS. In contrast to the effects observed after cultivation with higher thallium doses, the mesoderm and the entoderm were not affected. Discussion : Using the whole embryo culture system for the study of embryotoxic and teratogenic effects, it has to be kept in mind that we are only able to culture embryos for a relatively short period of time when compared with the whole period of gestation of the animals. Testing drug effects, for example in a 48-hour culture period, means that the cultured embryos have only

60 48 hours to develop malformations. Many malformations induced by substances in vitro possibly need a longer period than the 48 hours to become macroscopically visible and can therefore not be detected at the end of the culture period. The data presented indicate that thallium is able to interfere with embryonic development in culture. Growth and development of the rat embryos are drastically inhibited in culture at concentrations of 100 pg thallium/ml. After cultivating rat embryos for 48 hours in the presence of 30 pg thallium/ml we observed a growth retardation of 12 - 24 hours, as compared with the development of control animals. Addition of 10 jag thallium/ml to the culture medium resulted in smaller growth retardation (about 2 - 4 somites less). Addition of 3 yig thallium/ml had no effect on the growth and the development of the embryos during the 48-hour culture period. In the embryos cultured with concentrations of 3 or 10 pg thallium/ml no abnormalities could be detected macroscopically. After cultivation with 30 >ig thallium/ml, minor tail abnormalties, later possibly leading to kinky tail development, could be observed. After histological inspection of these embryos it seemed to be unbelievable that macroscopically only minor abnormalities could be detected. The CNS of these embryos cultured with 30 pg thallium/ml, was drastically destroyed and even the mesodermal structures were extremely affected. In the embryos cultured with 10 yig thallium/ml strong cytotoxic effects in the CNS could be observed histologically, although these embryos looked rather normal macroscopically. Even in embryos cultured with 3 pg thallium/ ml, which developed absolutely normal in culture, the histological investigations showed cytotoxic effects in the CNS. These histological findings indicate that it is possible to detect embryotoxic or teratogenic effects in the embryos far below the level of macroscopically visible abnormalities. It has been claimed that thallium is teratogenic in mice (ACHENBACH et al., 1979 a, b ) . Skeletal abnormalities have been reported after oral application of 8 mg thallium/kg to pregnant mice on day 9 of pregnancy. The corresponding thallium concentrations which lead to skeletal abnormalities were given as 2.6 x 10- 5 moles thallium/1 (about 5.3 pg/ml) in the "uterus plus embryo". These teratogenic effects could not be repoduced (CLAUSSEN et al., 1981) when thallium was given during the entire period of organogenesis . If we assume an even distribution of the thallium administered in these studies between the maternal tissue and the embryo, a maximum of about 5.3 ]ag/ml thallium could have reached the embryo. In reality this even distribution between the mother and the embryo is rather unlikely in the light of the finding of a limited placental passage of thallium in rats (GIBSON and BECKER, 1970). Therefore we can assume a thallium concentration in the embryo of much less than 5.3 pg/ml. So far no reliable data on the exact concentration of thallium which reaches the mouse embryo, after a dose of 8 mg/kg, are available. Results of our studies suggest that an embryotoxic, and possibly also a teratogenic effect is likely to occur if concentrations of thallium higher than 1 pg/ml are reached within the embryonic compartment. Whether such a concentration may be obtained under conditions not imposing a severe poisoning to the maternal organism is questionable.

61

It is now well established that the toxic effect of thallium is greatly modified by the simultaneous presence of potassium (GEHRING and HAMMOND, 1967; RUSZNYAK et a l . , 1968). If the potassium concentration in human serum is assumed to be 150 ng/ml the ratio K /T1 for 30 ug/ml thallium added to the culture medium would be 5. It seems likely that the toxic effect of thallium may be increased by reduction of the potassium concentration in the medium. We have not yet performed such studies, but plan to do so in the near future. Summary: 10.5-day-old rat embryos were cultured in 6 ml human serum + 2 ml tyrode-phosphate-buffer in the presence of 3, 10, 30 or 100 jag thallium/ml for 48 hours and were investigated macroscopically and microscopically at the end of the culture period. Cultivation of rat embryos with 100 >ig thallium/ml resulted in drastic growth inhibition of the embryos. After cultivation with 30 yig thallium/ml the embryos showed a growth retardation of 12 - 24 hours and minor abnormalities. Histological inspections of these embryos revealed a totally destroyed CNS and strong cytotoxic effects on the paraxial mesoderm, the somitic mesoderm, the mesoderm of the limb anlagen and the mesoderm of the branchial arches. The entoderm was less affected. Although cultivation of rat embryos with 10 ng thallium/ml only resulted in minor growth retardation, strong cytotoxic effects could be detected in the CNS of these embryos. In the mesoderm, numerous cell necroses in the limb anlagen and the somites were visible but, the entoderm was unaffected. After cultivation with 3 pg thallium/ml the embryos developed apparently normal in vitro, but histological inspection of these embryos showed the development of cell necroses in the CNS. The mesoderm and the entoderm of these embryos were unaffected.

62

REFERENCES Achenbach, C . , Hauswirth, O., Heindrichs, C . , Ziskoven, R . , Köhler, F . , Smend, J . , Kowalewski, S . , 1979 a, Toxizität und Teratogenität von Thallium, Dtsch. Ärzteblatt, 3189 - 3192. Achenbach, C . , Ziskoven, R . , Koehler, F . , Bahr, U . , Schulten, H . - R . , 1979 b . Quantitative Spurenanalyse von Thallium in biologischem Material, Angew. Chem. 91, 944 - 945. Claussen, U . , Roll, R . , Dolguer, R . , Matthiaschk, G . , Majeroski, S . , Stoll, B . , Röhrborn, G . , 1981, Zur Mutagenicität and Teratogenicität von Thallium - unter besonderer Berücksichtigung der Situation in Lengerich, Rhein. Arztebl., 469 - 475. Gehring, P . J . , Hammond, P . B . , 1967, The interrelationship between thallium and potassium in animals, J . Pharmacol. Exp. Therapeut. 155, 187 - 201. Gibson, J . E . , Becker, B . A . , 1970, Placental transfer, embryotoxicity, and teratogenicity of thallium sulfate in normal and potassium-deficient rats, Toxitol. appl. Pharmacol., 16, 120 - 132. Rusznyäk, I, György, L, Ormai, S . , Miliner, T . , 1968, On some potassium-like qualities of the thallium ion, Experientia, 24, 809 - 810.

ACKNOWLEDGEMENTS This work was supported by grants given to the Sonderforschungsbereich 29 by the Deutsche Forschungsgemeinschaft. 1

Part of the data presented in this paper will be included in a doctoral thesis to be submitted to the Freie Universität Berlin.

64

Fig. 2:

Cross-section of a rat embryo, cultured for 48 hours in a medium containing 30 yig Tl/ml. P = parietal mesoderm; S = spinal cord; E = entodermal tube; A = paraxial mesoderm.

Fig. 3: Higher magnification of the spinal cord from Fig. 2. S = destroyed spinal cord, no ventricular lumen visible; N = notochord.

Fig. 4: Higher magnification of the spinal cord from Fig. 2. A = many mesodermal cell necroses in the region of the paraxial mesoderm; S = spinal cord.

65

Fig. 5:

Cross-section through the spinal cord of a rat embryo cultured for 48 hours in a medium containing 10 pg Tl/ml. V = ventricular lumen of the spinal cord; N = neuroepithelium of the spinal cord.

Fig. 6: Magnification of the dorsal part of the spinal cord from embryo in Fig. 5. N = many cell necroses, mainly located in the intermediate zone of the spinal cord. V = ventricular lumen.

Fig. 7: Somite of a rat embryo cultured for 48 hours in a medium containing 10 pg Tl/ml. E = epidermis; 4- = necroses of mesodermal cells in the somite region.

66

Fig. 8:

Spinal cord of a rat embryo cultured for 48 hours in a medium containing 3 jag Tl/ml. V = ventricular lumen; + = necroses of neuroepithelial cells.

Fig. 9: Part of a brain anlage of a rat embryo cultured for 48 hours in a medium containing 3 lag Tl/ml. V = ventricular lumen; * = neuroepithelial cell necroses.

Fig. 10: Magnification of a dorsal part of the brain anlage, as Fig. 9. V = ventricular lumen; * = wide intercellular space between the neuroepithelial cells; 4- = macrophage containing necrotic fragments .

Evaluation of Serum Teratogenic Activity Using Rat Embryo Cultures N. W. Klein*, J. D. Plenefisch, S. W. Carey, C. L. Chatot, M. L. Clapper * Dept. of Animal Genetics, University of Connecticut, Storrs, Connecticut 06268, USA

For the past several years we have been working on the hypothesis that the responses of cultured rat embryos to a medium consisting of serum were indicative of the teratogenic activity of the serum as well as of the teratological risk to the organism donating the serum. This hypothesis appeared reasonable because, first, only a whole embryo undergoing rapid growth and development might possess all those events that have the potential of sensitivity to a teratogen. Second, maternal serum has generally been considered a close representation of those substances which might reach the developing embryo. However, from the start, certain limitations of this hypothesis were obvious such as, an organ specific teratogen might not be detected if the organ did not reach structural and functional maturity during the course of the embryo culture period. Another obvious limitation would be the "false positives" generated by serum substances which, although harmful to the cultured embryo, failed to pass the placenta and were therefore m vivo as well as in reality non-teratogens. With these restrictive considerations in mind, we did proceed. The procedures developed by NEW (1978) and his associates were used for the isolation of head fold stage (9.5 days) rat embryos (with Reichert's membrane removed but yolk sac and ectoplacental cone left intact) and for the preparation of serum as a culture medium (immediate centrifugation and heat inactivation). In addition, we used their recommended 30 rpm culture bottle rotation and their changes of O z , CO z , and N2 levels. All of our experiments have lasted 48 hours as embryo viability was reduced after this period. Our initial studies (KLEIN et al., 1980) involved embryos cultured on serum taken from rats that had been previously injected with the well studied teratogens, cadmium chloride and cyclophosphamide. Depending on the time following injection and amounts injected, we observed responses that ranged from embryo death to survival with abnormalities and reduced size as well as reduced size alone. After sufficient time following teratogen injection, embryos were comparable to those cultured on serum from vehicle injected controls. These observations were reproducible and statistical analyses were applied successfully to the quantitative estimates of embryo growth (protein and DNA contents). Thus, we felt that the crucial question had been answered in the affirmative, namely, it was feasible indeed to use rat embryo cultures to evaluate the teratogenic activity of serum. In addition to this, we felt that justification was provided for the use of serum from teratogen treated animals as opposed to adding teratogens directly to the culture medium. Both cadmium and cyclophosphamide gave quite distinct responses when the two methods of embryo exposure were compared. This was particularly striking in the case of cyclophosphamide

Culture Techniques © 1981 Walter de Gruyter & Co., Berlin • New York

68 as the embryos, while being resistant to directly added drugs, were highly sensitive to serum from cyclophosphamide injected animals. An observation was made in this first study (KLEIN et a l . , 1980) which established an important but taxing matter of procedure. Teratologists have always recognized stage related sensitivity but we were surprised to find an example of a very narrow range of sensitivity. Working with directly added cadmium chloride ( 1 . 6 ]oM) early head fold stage embryos failed to survive 48 hours of culture, middle head fold embryos survived but were abnormal and reduced in size, and late head fold embryos were comparable to controls. This large range of responses from a matter of hours in developmental time led us to a narrow selection for our starting stage. We use exclusively mid head fold embryos as we have described (KLEIN et a l . , 1980). The possibility of extending this approach to the testing of human serum, although of potential clinical use, appeared doubtful as NEW (1966) had reported the inadequacy of various heterologous sources of serum for rat embryo culture and we confirmed his observation that human serum was inadequate. We therefore felt fortunate to find that the addition of glucose to human serum greatly improved rat embryo growth and development (CHATOT et a l . , 1980). For example, during 48 hours of culture the addition of 2 to 3 mg/ml glucose increased embryo protein and DNA accumulation by three and five fold respectively when compared to the same serum sample without added glucose. (The glucose level of all human serum as well as monkey serum is now routinely measured and adjusted to 3 mg/ ml). To provide a preliminary demonstration of the potential usefulness of the technique, serum samples from human subjects receiving medication with known or suspected teratogens were tested. With both cancer chemotherapy patients and patients receiving anticonvulsants, serum samples were either embryolethal or caused abnormalities sometimes with reduced embryo size. Following this success with human serum our general objective has been to evaluate and to demonstrate possible applications of the cultured rat embryoteratogenic activity test. Below, we present a progress report and attempt to stress both major accomplishments and major difficulties that we have encountered. The Testing of Drugs Our drug efforts have been concerned primarily with Thalidomide and anticonvulsants. The former drug was studied because of its known embryo and species specificities, and its historical position as one of the most important substances in the validation of any teratological test. The anticonvulsants received our attention because of their extensive usage during pregnancy and implication as teratogens. Serum taken from rats at various times following oral administration of up to 1 mg/kg Thalidomide (generously provided by Prof. L. Flohe and Dr. E. Frankus of Gruenenthal, GMBH) was not harmful to cultured rat embryos. Assuming this might be the result of species specificity in drug metabolism, we next considered incorporating primate metabolism into the "test" through the use of monkeys. We had found that monkey serum would support excellent embryo growth when glucose was supplemented and that the time of serum sampling in relation to the menstrual cycle did not affect cultured rat embryos. (This work was done in collaboration with Dr. R. Parker, University of California, Davis and involved the species

69 M. mulatta.) By feeding different dosages of Thalidomide hidden in a banana and sampling at different times after ingestion, our most consistent responses of yolk sac circulation failure, reduced embryo size, exencephaly and faulty body rotation were observed with dosages over 300 mg/kg and particularly at 6 hours. However, a single sample taken at 100 mg/kg - 2 hours was teratogenic while one sample at 500 mg/kg - 6 hours was not. A clear dose response relationship has not been demonstrated but the responses we have seen we feel have been the result of Thalidomide (it should be noted that pre- and post-drug serum have been compared in all studies). We believe the variation may be the result of individual differences in drug metabolism but we can't exclude other possibilities such as low responsiveness of rat embryos. The establishment of a relationship between blood levels of the teratogenic metabolites and embryo response would certainly help to resolve this issue. Toward this goal we have added various metabolites (or hydrolysis products) as well as Thalidomide itself directly to serum at a concentration of 1 mg/ml or serum saturation levels and have not observed a response. The metabolites were kindly provided by Drs. J . Faigle and G. Dôrfhôfer of Ciba-Geigy Ltd. and Dr. W. Scott of Children's Hospital Research Foundation, Cincinnati, Ohio and have been designated as compounds 2 through 8 (SCHUMACHER et a l . , 1965). The availability of serum from human subjects taking anticonvulsants has made species comparisons possible with the only relevant organism and has added to the frustration of finding an appropriate animal model. Diphenylhydantoin when added directly to human, rat or monkey serum at 30 yig/ml (Fig. 1A, 1C and IE) and up to 100 pg/ml did not adversely affect rat embryos during 48 hours of culture. However, when the same drug was allowed to pass through an organism, striking differences were observed. Serum from a human subject taking only 2 to 5 mg/kg was highly teratogenic (Fig. I B ) , a rat given 1 mg/kg was non-teratogenic (Fig. ID) while a monkey given 500 mg/kg was teratogenic (Fig. I F ) . The serum levels of the parent compound were: human, 18 yig/ml; rat, 21 jig/ml and monkey, 35 pg/ml. These results, similar to Thalidomide, stress species specificity and again stress the need to identify the active metabolite. Preliminary studies have excluded the major metabolite p-hydroxyphenyl-phenyl-hydantoin (kindly provided by Drs. R. Buchanan and M. Black, Warner-Lambert Co., Ann Arbor, Michigan). Another aspect of our anticonvulsant drug studies has been an attempt to compare drug teratogenicities through testing human serum teratogenic activity. Clearly, large numbers of samples from individuals receiving single anticonvulsant drugs must be studied before meaningful conclusions can be made, but our results to date have been sufficiently encouraging to call this approach to your attention. Comparing drugs either by the number of serum samples ( i . e . number of subjects) that showed teratogenic activity or the frequency of abnormal embryos, diphenylhydantoin was clearly more of a problem for cultured rat embryos than phénobarbital (Table 1 ) . Valproic acid and Tegretol were highly teratogenic but possibly less so than diphenylhydantoin. Comparison between serum levels of the parent compounds and serum teratogenic activity suggested that only in the case of Valproic acid might the parent compound be active. We have been quite gratified to find our differences between diphenylhydantoin and Phenobarbitol supported by in vivo mouse studies (SULLIVAN and McELHATTON, 1975) as well as by M.S. GOLBUS (1980) who recently stated "Phénobarbital has been in use for more than 60 years and from a teratologic point of view appears to be the safest antiepileptic drug".

70 Our studies with anticonvulsants were made possible through the generous and most dedicated cooperation of W. Singer and L. Witkowski, Tufts-New England Medical Center, Boston, MA; B. Russman and G. Holmes, Newington Children's Hospital, Newington, CT; R. Mattson, J . Kramer and P. Klein, Veteran's Administration Hospital, West Haven, CT; and S. Resor, Columbia Presbyterian Hospital, New York, NY. The Testing of Occupations In the near f u t u r e we hope to have studies completed on authentic occupational exposures, but at this time we wish to briefly outline our cigarette smoking studies as a model of our approach. First, because of the almost limitless number of variables encountered when considering human subjects, we felt a subject must serve as his own control. Second, our approach has been to establish teratogenic "clearance times" to benefit those working in a hazardous occupation rather than to merely identify an occupation as hazardous and establish a basis for sexual discrimination. With these two issues in mind, our cigarette study became restricted to those subjects willing to "quit", which indeed restricted the population of participants. Furthermore, of the ten that eventually "quit", six were excluded for various reasons and of the remaining four, only one has remained unquestionably pure (the senior author of this presentation). Considering just the four subjects, certain similarities were striking. For example, the f i r s t day after quitting that their serum did not produce an abnormal embryo was either day 4 or 5, yet maximum embryo protein content was not achieved until days 9, 10, 16 or 18. The percentages of increase in embryo protein content, comparing each individuals day 0 smoking sample versus day of maximum growth, were 29%, 59%, 61% and 80%. Testing for Early Fetal Wastages and High Risk Pregnancies Quite by accident we came upon possibly our most striking validation indicating that the cultured rat embryo test may indeed be a teratogenic test. In the course of the Thalidomide studies with monkeys, which have previously been discussed, serum samples from two of a total of 18 different female monkeys gave small abnormal embryos (prior to Thalidomide administration). In checking over their records our colleagues at the U. of Calif., Davis, reported that we had picked (to their amazement) the only two monkeys of the entire group that had failed to become pregnant in over two years of matings. A year later, serum samples from these same two monkeys still gave small abnormal embryos whose abnormalities were characteristic of the donor. Through collaborations with W. Fredrickson, T. Burbacher and G. Sackett of the Primate Center, U. of Washington, Seattle, it has been possible to extend these studies with their group of M. nemestrina "high risk" p r e g nancy monkeys. These monkeys have histories of repeated spontaneous abortions, still births and neonatal deaths. Although we have been able to distinguish in blind experiments, with a high degree of success, the serum of "high risk" animals from those of "low risk" animals (animals with 3 to 5 surviving b i r t h s ) , we have not been able to clearly relate a specific type of reproductive failure with a specific cultured rat embryo abnormality type (Fig. 2). However, particular abnormality types were generally associated with particular monkeys (Table 2). Considering f i r s t our ability to provide a correct identification of risk, this was achieved with initial serum samples for 8 of 12 low risk and 12 of 14 high risk animals (see G and B column of Table 2) (p = 0.009 by Fisher's exact probability t e s t ) . Our ability to repeat the correct designation with additional samples we feel was

71 excellent, but not perfect (only 4 of 17 repeated animals failed to give repeated embryo responses). Abnormality frequency was 29% for all embryos (63) cultured on low risk serum and 72% for the embryos (89) cultured on high risk serum. The most frequent abnormality for the high risk group was the D type (42%) (Fig. 2) characterized by microcephaly, exencephaly, failure of eyes to develop and reduced size. The overall protein difference between the low (104 pg/embryo) and high risk groups (87 yig/embryo) was highly significant (p = < 0.01 for student t t e s t ) . The Cause of Serum Teratogenic Activity:

A Case Study

Although our ability to correctly identify monkeys with reproductive problems has been a most gratifying experience, our ability to determine the cause would be essential to derive benefit from this effort. Could such a complex mixture as serum be successfully analyzed and could cultured rat embryos provide the developmental bioassay essential for the analysis? For our first effort we selected serum from a 20 year old woman who, although lacking a reproductive history, possessed embryo lethal serum. In testing over 100 human subjects, this individual represented the most extreme example of what might be considered serum teratogenic activity. Supplementing her diet with vitamins and taking samples at various times during her menstrual cycle as well as over the course of two years did not alter the embryo response to her serum. Dialysis of her serum failed to improve the situation. Because as little as 10% of her serum, when added to control (serum that supports excellent embryo growth and development) human serum was still teratogenic, we assumed the problem was a toxic component rather than a deficiency. A series of Amicon ultrafiltrations along with trypsin sensitivity supported the contention that the serum contained a toxic component and probably a protein with a molecular weight greater than 100,000 daltons. Comparing the toxicity of proteins precipitated by ammonium sulfate at 30%, 30 - 50% and 50 - 70% saturation directed our attention to the y-globulins (Fig. 3c, d, e and Fig. 3C, D, E ) . DEAE AffiGel Blue chromatography was then used to purify her IgG. As little as 0.2 mg/ml caused reduced embryo size and abnormalities while levels of 0 . 6 mg/ml produced embryos resembling those grown on her untreated serum (Fig. 3a, f , g, and Fig. 3A, F, G). Serum absorption experiments using yolk sacs or embryos without their membranes suggest an extra-embryonic site of action for this antibody.

ACKNOWLEDGEMENTS This work was supported by the U.S. Dept. of Energy under contract EV03139. Scientific contribution number 884, Storrs Agricultural Experiment Station.

72

Table 1: Growth and Development of Rat Embryos Cultured on Serum from Human Subjects Receiving Anticonvulsants

Drug Dilantin

Serum drug concentration (yig/ml)

Abnormal samples

Abnormal embryos

Protein content (yig/embryo)

0-10 11 - 20

14/18 (78)* 6/7 (76)

39/52 (75)* 16/21 (76)

66. 4 ± 82. 1 ±

4 . 0 (42)** 6.6 (15)

20/25 (80)

55/73 (75)

70..6 ±

3.5 (57)

5-10

7/ 9 (78)

18/25 (72)

60..9 ±

6.4 (23)

0 - 50 51 - 100 101 - 150

1/ 2 (50) 2/ 4 (50) 4/ 5 (80)

4/ 6 (67) 7/12 (58) 13/15 (87)

77..7 ± 6.2 ( 6) 78..1 ± 11.8 (10) 61..0 ± 10.4 (12)

7/11 (63)

24/33 (72)

70..7 +

3/ 4 (75) 4/12 (33) 1/ 4 (25)

9/12 (75) 14/36 (39) 5/12 (42)

59.6 ± 4 . 4 (11) 85.0 ± 5.4 (33) 94.1 ± 14.8 ( 8)

8/20 (40)

28/60 (47)

82 .8 ±

Total:

Tegretol Valproic Acid

Total:

Phénobarbital

Total:

0-20 21 - 40 41 - 70

6.3 (28)

4.5 (52)

*The number in parentheses for these two columns represents percentages. **Mean ± standard error with the number in parentheses indicating the number of determinations.

73 Table 2: Growth and Development of Rat Embryos on Monkey Serum (M. nemestrina)

Monkey number

Morphological t y p e s * # Evaluation*

A

B

C

D

F

Protein (pg/embryo)

LOW RISK BREEDERS: 291 128 182 191 184 491 398 446 485 264 278 281 Total

GGGG GG G B GG BB G B G G GB G BB

11 5 2 2 6 2 2 2 5 3 5

1

G = 14; B = 7.

45

4

G BG B GGB BB BB BBB BB BB B BB BB BBB BGB

5 2

1

4 4

1

G = 5; B = 24.

25

7

1

1 3 1

3

1 1

2

4

8

4

2

1 3

2

3 1 6 6 5 3

2 3 1

120. 9 122. 6 101. 8 88. 8 119. 5 86. 4 100. 3 69. 6 107. 3 98. 7 97..3 80.,7

(12)*** ( 5) ( 2) ( 2) ( 5) ( 5) ( 3) ( 3) ( 5) ( 3) ( 7) ( 5)

103.,5

+

3.3

137..8 57..0 118,.0 135..1 70 .7 44 .6 87 .1 83 .9 71 .6 91 .5 73 .0 46 .6 77 .8 96 .4

( ( ( ( ( ( ( ( ( ( ( ( ( (

5) 5) 3) 8) 6) 5) 8) 4) 4) 3) 5) 4) 8) 8)

86 .6

+

3.8

HIGH RISK BREEDERS: 018 193 045 344 413 209 481 578 273 058 094 333 418 184

Total

6 1 2

3 2

1 3

4 5

3

37

3 6

17

•Evaluation based on embryo morphology and size.

G = good; B = b a d .

••Morphological t y p e s a r e described in Fig. 2. ***Mean ± s t a n d a r d e r r o r with f i g u r e s in p a r e n t h e s e s indicating the number of determinations.

74

REFERENCES Chatot, C . L . , Klein, N.W., Piatek, J . , Pierro, L . J . , 1980, Successful culture of rat embryos on human serum: use in the detection of teratogens, Science, 207, 1471 - 1473. Golbus, M.S., 1980, Teratology for the obstetrician: current status, Obstet. Gynecol., 55, 269 - 277. Klein, N.W., Vogler, M.A., Chatot, C . L . , Pierro, L . J . , 1980, The use of cultured rat embryos to evaluate the teratogenic activity of serum: cadmium and cyclophosphamide. Teratology, 21, 199 - 208. New, D . A . T . , 1966, Development of r a t embryos cultured in blood sera, J . Reprod. Fertil., 12, 509 - 524. New, D. A. T . , 1978, Whole embryo culture and the study of mammalian embryos during organogenesis, Biol. Rev., 53, 81 - 122. Schumacher, H . , Smith, R . L . , Williams, R . T . , 1965, The metabolism of thalidomide: the spontaneous hydrolysis of thalidomide in solution, Brit. J. Pharmacol., 25, 324 - 337. Sullivan, F.M., McElhatton, P . R . , 1975, Teratogenic activity of the antiepileptic d r u g s : phenobarbital, phenytoin, and primidone in mice, Toxicol Appl. Pharmacol., 34, 271 - 282.

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ig P e r m l during the whole incubation period. These explants show a drastic impairment of morphogenetic differentiation. 1 section of the scale = 1 mm.

205

components

control

ascorbate

dehydroascorbate

cysteine

methionine + cystine

results

+

-

+

-

Jdr

-

-

+

-

&

-

+

+

-

&

+

-

-

-

#

+

-

-

+

Jer

J0f = no effect =

Table 1:

impairment of differentiation

Effects of some components of the culture medium on limb development . The culture medium, used as a control-medium, contains ascorbate and cysteine. The ommission of ascorbate from the medium, as well as the replacement of ascorbate by dehydro-ascorbate, does not show any effect. The ommission of cysteine from the medium results in an impairment of differentiation which can be overcome by substituting cysteine by a mixture of cystine plus methionine.

206

REFERENCES Blankenburg, G . , Rautenberg, M. ( Neubert, D . , 1980, Ascorbic acid content of limb buds at different stages of mammalian embryonic development, in: "Teratology of the Limbs", ( H . - J . Merker, H. Nau and D. Neubert, e d s . ) , de Gruyter, Berlin - New York, pp. 183 - 190. Hoffman, D . , Bousquet, W., Miya, T . , 1966, Lycorine inhibition of drug metabolism and ascorbic acid biosynthesis in the rat, Biochem. Pharmacol. , 15, 391 - 393. Lessmollmann, U . , Hinz, N., Neubert, D . , 1976, In vitro system for toxicological studies on the development of mammalian limb buds in a chemically defined medium, Arch. Toxicol., 36, 169 - 176. Minesita, T . , Yamaguchi, K . , Takeda, K . , Kotera, K . , 1956, A. Rep. Shionogi Res. L a b . , 6, 131 - 141, (Publication in Japanese). Neubert, D . , Barrach, H . - J . , 1977, Techniques applicable to study morphogenetic differentiation of limb buds in organ culture, in: "Methods in Prenatal Toxicology", (D. Neubert, H . - J . Merker, T . E . Kwasigroch, e d s . ) , Georg Thieme Publ., Stuttgart, pp. 241 - 251. Neubert, D . , Tapken, S . , Baumann, I . , 1978, Influence of potential thalidomide metabolites and hydrolysis products on limb development in organ culture and on the activity of proline hydroxylase, in: "Role of Pharmacokinetics in Prenatal and Perinatal Toxicology", (D. Neubert, H . - J . Merker, H. Nau, and J . Langman, eds. ) , Georg Thieme Publ., Stuttgart, pp. 359 - 382.

DISCUSSION: Beck:

Why do ferret embryo limb buds grow for 18 days in spite of the ascorbate instability (the media is replaced every 3 days)?

Blankenburg:

Limb buds from mouse embryos also grow in the absence of ascorbate for a number of days. It might be that limb buds from ferret embryos contain a substantial concentration of ascorbate at the beginning of the culture period; and the ascorbate may be stable in the culture within the limbs for many days in the ferret. Do you have results of studies in which you omitted the ascorbate from the culture medium?

Beck:

No, we do not.

Effects of Some "Indirectly" Alkylating Agents on Differentiation of Limb Buds in Organ Culture R. Stahlmann, U. Bluth and D. Neubert Institut für T o x i k o l o g i e u n d E m b r y o p h a r m a k o l o g i e der Freien Universität Berlin, Garystraße 9, 1000 Berlin 33

Introduction: In vitro development of rodent limb buds in organ culture has been studied in our laboratory for several years. Approximately 50,000 mouse limb buds have been cultured by our group during the last 8 years. Figure 1 shows mouse limb buds (day 12 of gestation) in vivo and after a culture period of six days. The suspension culture technique we now routinely use (NEUBERT and BARRACH, 1977 a; LESSMOLLMANN et a l . , 1975) has been well standardized, and the morphogenetic differentiation of carilaginous structures of the fore- or hindlimbs was evaluated at the morphological and the biochemical levels. Moreover, we succeeded in demonstrating that abnormal development can be induced in vitro by a variety of teratogenic substances added to the culture medium (NEUBERT and BARRACH, 1977 b; BARRACH et a l . , 1978). Further methodical details are given by BLANKENBURG et al. (this book). In this paper we would like to present the results of studies performed with two types of alkylating agents which both raise problems when studied in vitro as well as in vivo: 1)

The polyfunctional drug cyclophosphamide - a wellknown teratogen extensively studied in vivo,

and 2) some nitrosamine derivatives. Both types of chemicals require metabolic activation in order to reveal their alkylating capacities on the cells. In both cases the embryonic or early fetal tissues of rodents do not develop enough metabolizing capacity to accumulate a toxic concentration of the active metabolites of these chemicals. However, there is a major difference between these two types of alkylating agents (cf. Fig. 2): Cyclophosphamide is converted by the maternal liver into stable, active metabolites which are apparently able to cross the placenta and impair fetal development in vivo. Nitrosamines, on the other hand, have to be activated within the target cell, which means in the embryonic or fetal cell itself because the activation products are unstable and short-lived. Whereas fetal tissues of rodents appear to lack the capacity for the metabolic conversion of nitrosamines, they were not found to be teratogenic in in vivo experiments, the situation seems to be different in primates whose capacity for cytochrome P 4 5 0 - t y p e monooxygenase reactions apparently develop during late embryonic or early fetal life.

Culture Techniques © 1981 Walter de Gruyter & Co., Berlin • New York

208

Results and Discussion: Studjes_wjth _ CyjqlophjDsjDhamjde By now, it is well established that cyclophosphamide has to be converted by a cytochrome P 4 5 0 -type monooxygenase system into one or more active metabolites (Fig. 3). Although there is adequate evidence that hydroxylation at position 4 of the ring structure, and a ring opening, are the initial steps of the activation process, the nature of the final alkylation product is still uncertain. Three possibilities are conceivable: a)

Alkylation occurs by the activated LOST-derivative with the rest of the ring moiety still attached,

b)

alkylation is performed by phosphoramide-mustard after detachment of the ring-C-atoms, or

c)

the toxic, and in our case the teratogenic, effects induced by acrolein which is formed from the C-atoms of the open ring form.

Our first attempt ( c f . BARRACH et al., 1978; BARRACH and NEUBERT, 1980) to reproduce the toxic effects of this compound which are wellknown from in vivo experiments, was to study the effect of 4-hydroperoxy-CP on limb differentiation in culture. This compound spontaneously decomposes to 4-hydroxy-CP which is in equilibrium with the open ring form, i.e. aldophosphamide (Fig. 3). We observed a drastic interference with limb development at concentrations of 4-hydroperoxy-CP as low as 10 jag/ml in the culture medium (Fig. 4). The effect was far more pronounced with limb buds of 11-day-old mouse embryos than with those of 12day-old mouse embroys - thus demonstrating the phenomenon of phase specificity also to occur in organ culture. 2)

Studjes_with_ Acro_l e in

Further studies were performed in order to elucidate the role of acrolein in the induction of abnormal development. A prerequisite for these studies was the development of a medium free of SH-compounds. The solution to this problem has been reported by BLANKENBURG et al. (this book). When adding 1 yig/ml acrolein to the culture medium ( c f . Fig. 5), no effect on limb differentiation could be induced. The limb buds developed normally and exactly resembled controls. With 10 pg/ml an effect occurred that was not seen before with any other substance. All the paws were bent at the wrist forming an angle of about 90°. No other interference with differentiation in vitro could be recognized. When the experiment was repeated the effect was found to be reproducible. With 30 ng/ml only the metacarpalia, with all the phalanges missing, developed (Fig. 6 a ) . At 100 yig/ml differentiation and development of the paw skeleton were completely inhibited, but those of the "long bones" were still recognizable (Fig. 6 b ) . This concentration appears high when compared with the concentration of 4-hydroperoxy-CP required to induce abnormal development in culture. However, since acrolein is unstable in the culture medium we are not yet able to specify the concentrations which may become active inside the cells. Thus we feel that the question for the metabolite "responsible" for the teratogenic effect produced by cyclophosphamide in vivo and in vitro

209

cannot yet be answered. So f a r , acrolein must be considered a possible candidate. This metabolite is certainly very active in the organ culture system used. 3)

Studies with Nitrosamine_Derivatiyes

It is of considerable significance to know if nitrosamines are principally teratogenic in humans. This question cannot be solved by experiments with rodents exclusively since, as mentioned before, differences in the capacity for metabolic activation of nitrosamines are supposed to exist between rodent and primate fetuses. IVANKOVIC et al. (1974) showed that human fetal liver, in principle, has the capacity to demethylate dimethyl-nitrosamine. However, this observation does not give any clue on whether an alkylating intermediate is formed or if embryonic tissues other than liver have the capacity for metabolic conversion of DMNA. At present, studies are being performed in our laboratory with non-human primates (marmosets) in order to reveal the alkylating capacity of DMNA in fetal tissues of these primates. Other ways to overcome this difficulty, at least partly, would be either to attempt the "induction" of the drug-metabolizing capacity in the explant or to supplement the organ culture medium with a drug-metabolizing system. In collaboration with Docent Dr. Wiessler, from the Deutsches Krebsforschungszentrum in Heidelberg, we have attempted another approach: Since the presumed activation products of the nitrosamines, the hydroxy compounds, are extremely unstable it is feasible to use their more stable esters. They would be expected to give rise to the short-lived active hydroxides when ester is split by esterases within the cell (WIESSLER and SCHMÁHL, 1976). It has been shown that such esters, reacting with the esterases present in most cell types, become highly active alkylating agents and powerful carcinogens (HABS et a l . , 1978; CAMUS et a l . , 1978). The acetate ester of the hyroxy form of dimethyl-nitrosamine, N-methylN(acetoxymethyl)-nitrosamine (MAMN) proved to be an extremely active teratogen in our organ culture system. Abnormalities can be induced with concentrations in the culture medium of 0.1 - 0.3 pg/ml (Figs. 7 a and 7 b ) . Up to now, this is the lowest concentration of a substance we found to be active in this system. At 0.1 >ig/ml morphogenetic differentiation of the cartilaginous bone anlagen was only slightly, however, typically, affected. The impairment is more pronounced with the three-fold concentration: Only two of the five metacarpi developed in a regular way (oligodactyly). The other bone anlagen were also impaired. They were short, bent and irregularly shaped. Since Dr. Wiessler prepared several esters of different alkyl-methyl-nitrosamines, we tested the effects of these DMNA-homologues with respect o the chain length of the substances (Fig. 8). At the low concentration of 0.1 - 0.3 pg/ml, none of these showed an effect comparable to that of MANM. The results given in Fig. 9 indicate that the teratogenic effect, as observed in our system, decreased with increasing chain length of the compounds. However, the structure-activity relationship seems to be complex: Dose-response curves as well as the final outcome of the impairment of morphogenetic development seem to be rather different for the various compounds, as can be seen from Figs. 10 a to 10 e. Testing the tertiary butyl derivative (t-BAMN) at the rather high concentration of 10 pg/ml, we found only minor effects on growth.

210 Again, as found with the cyclophosphamide metabolite, in limb buds from 11-day-old mouse embryos, the effect is more pronounced than in those of 12-day-old mouse embryos. However, at both stages of development treatment in culture resulted in obvious abnormal growth and differentiation. Since the esters are expected to be short-lived under culture conditions pH 7.2 and presence of esterases - the effect is apparently triggered within a very short interval of the culture period. Experimental Conditions: Limb buds from NMRI mice were used for these studies. The organ culturing was performed with a suspension culture using a chemically-defined medium. Details of this procedure are given in Chapter VII of this book (BLANKENBURG). For the studies with alkylating agents, the cysteine was replaced in the culture medium with a mixture of cystine (100 mg/1 saturated solution) and methionine (240 mg/1), as described by BLANKENBURG et al. (this book). The DMNA-esters and acrolein were added by injection through the rubber stopper with a Hamilton-syringe to the gased and completed culture system, which already contained the organ anlagen. Conclusions: From the results presented the following conclusions may be drawn: 1)

A prerequisite for the teratogenic action of cyclophosphamide is its conversion by monooxygenases to active alkylating intermediates. Besides the metabolites containing the N-mustard rest, acrolein may be formed. This metabolite is, in our opinion, a serious candidate for the triggering of an embryonic effect - as can be judged from our in vitro studies.

2)

The presumed activation products of DMNA and homologues - as formed after a hydrolysis of the esters within the embryonic cells when studied in vitro, produce a very pronounced teratogenic effect. Therefore, the teratogenic potential of DMNA in pregnant animals or women would critically depend on the capacity of the cytochrome P 4 5 0 -monooxygenase systems in the target cell to "activate" the nitrosamine (cf. NEUBERT et a l . , this book). So far, little is known on the metabolic capacity to convert nitrosamines to alkylating intermediates in humans.

ACKNOWLEDGEMENTS These studies were supported by grants given to the Sonderforschungsbereich 29, "Embryo-Pharmakologie", der Freien Universität Berlin by the Deutsche Forschungsgemeinschaft. We are indebted to Docent Dr. M. Wiessler (Deutsches Krebsforschungszentrum, Heidelberg) for preparing and supplying the nitrosamine derivatives. We wish to thank also Ruth Kreft and Jane Klein-Friedrich for their help in preparing the manuscript.

211 REFERENCES Barrach, H . - J . , Baumann, I . , Neubert, D . ( 1978, The applicability of in vitro systems for the evaluation of the significance of pharmacokinetic parameters for the induction of an embryotoxic effect, irK "Role of Pharmacokinetics in Prenatal and Perinatal Toxicology", (D. Neubert, H . - J . Merker, H. Nau, J . Langman e d s . ) , Georg Thieme Publ., Stuttgart, pp. 323 - 336. Barrach, H . - J . , Neubert, D . , 1980, Significance of organ culture techniques for evaluation of prenatal toxicity, Arch. Toxicol., 45, 161 - 187. Camus, A.-M., Wiessler, M., Malaveille, C . , Bartsch, H., 1978, High mutagenicity of N-(a-acyloxy)alkyl-N-alkylnitrosamines in S. typhimurium: Model compounds for metabolically activated N, N-dialkylnitrosamines, Mutat. R e s . , 49, 187 - 194. Habs, M., Schmähl, D . , Wiessler, M., 1978, Carcinogenicity of acetoxymethyl-methyl-nitrosamine after subcutaneous, intravenous and intrarectal applications in rats, Z. Krebsforsch., 91, 217 - 221. Ivankovic, S . , Schmähl, D . , Zeller, W . J . , 1974, N-Demethylierung des Carcinogens Dimethylnitrosamin durch embryonales menschliches Gewebe, Z. Krebsforsch., 81, 269 - 272. Lessmöllmann, U . , Neubert, D . , Merker, H . - J . , 1975, Mammalian limb buds differentiating in vitro as a test system for the evaluation of embryotoxic effects, in_^ "New Approaches to the Evaluation of Abnormal Embryonic Development", (D. Neubert, H . - J . Merker e d s . ) , Georg Thieme Publ., Stuttgart, pp. 99 - 113. Neubert, D . , Barrach, H . - J . , 1977 a. Significance of in vitro techniques for the evaluation of embryotoxic effects, iru "Methods in Prenatal Toxicology, Evaluation of Embryotoxic Effects in Experimental Animals", (D. Neubert, H . - J . Merker, T . E . Kwasigroch e d s . ) , Georg Thieme Publ., Stuttgart, pp. 202 - 210. Neubert, D . , Barrach, H . - J . , 1977 b . Techniques applicable to study morphogenetic differentiation of limb buds in organ culture, in^ "Methods in Prenatal Toxicology, Evaluation of Embryotoxic Effects in Experimental Animals", (D. Neubert, H . - J . Merker, T . E . Kwasigroch e d s . ) , Georg Thieme Publ., Stuttgart, pp. 241 - 251. Wiessler, M., Schmähl, D . , 1976, Zur carcinogenen Wirkung von N-Nitroso-Verbindungen 5. Mitteilung: Acetoxymethyl-Methyl-Nitrosamin, Z. Krebsforsch., 85, 47 - 49.

212

ture period of six days. The cartilaginous bone anlagen are well differentiated. 1 section of the scale = 1 mm.

SOME PROPERTIES

OF C Y C L O P H O S P H A M I D E

TWO

1 'ACTIVATION" 2

"INDIRECTLY"

BY FETAL RODENT

F O R M A T I O N OF S T A B L E A C T I V E MATERNAL COMPARTMENT

3 TERATOGENIC CAPACITY RODENT T I S S U E S WHEN

Fig. 2:

A)

ORIGINAL

B)

METABOLITE

(CP) AND NITROSAMINES

ACTING

TISSUE

M E T A B O L I T E IN

IN O R G A N ADDED

(NA)-

TERATOGENS

CP

NA

—

—

+

—

—

—

+

+

C U L T U R E OF

AS:

SUBSTANCE

Properties of "indirectly" acting teratogens. Comparison between cyclophosphamide and nitrosamines.

213 METABOLISM OF CYCLOPHOSPHAMIDE

CL-CH2-CH2 CL-CH2-CH2

y

s

r\

NH-

•CH\ 2 CH 2 -CH/ 2

Cyclophosphamide

Cytochrome,

4-Hydroxycyclophosphamide

Aldophosphamide

CI-CH2-CH2

/

CI — C H 2 - C H 2 Phosphoramide

Fig. 3:

N-P-0' NH2 Mustard

Acrolein

Metabolic conversion of cyclophosphamide. The first step in the activation process is assumed to be the hydroxylation of the ring at the 4-position catalysed by a cytochrome P 4 5 0 - t y p e monooxygenase. The activation product, 4-hydroxy-cyclophosphamide, is apparently in equilibrium with its tautomeric ring-open product: aldophosphamide. The nature of the final alkylation product is still uncertain, but aldophosphamide may be cleaved to stoichiometric amounts of phosphoramide mustard and acrolein, both of which are highly cytotoxic.

214

Fig. 4:

Effect of 4-hydroperoxy-cyclophosphamide on the differentiation of mouse limb buds (day 12 — 50 - 52 somites) in organ culture (10 yig/ml). The unstable metabolite 4-hydroxy-cyclophosphamide is liberated spontaneously from the 4-hydroperoxy-derivative added to the culture medium. The unchanged drug cyclophosphamide has no effect on limb differentiation when added to the culture medium.

CONCENTRATION IN CULTURE [ng/ml]

1

EFFECT ON L I M B

DEVELOPMENT

NO EFFECT

10

BENT W R I S T , P A W WELL D E V E L O P E D

30

P H A L A N G E S NOT D E V E L O P E D

100

PRONOUNCED INTERFERENCE WITH DIFFERENTIATION

Fig. 5:

Effect of acrolein on morphogenetic differentiation of mouse limb buds in organ culture.

215

Fig. 6 a:

Limb development in culture (day 12 = 50 - 52 somites) after addition of 30 iag/ml acrolein to the medium. Only the metacarpalia are developed, all phalanges are missing. 1 section of the scale = 1 mm.

Limb development in culture (day 12 = 50 - 52 somites) after addition of 100 ng/ml acrolein to the medium. A drastic interference with differentiation and development of the paw skeleton is evident. 1 section of the scale = 1 mm.

216

Fig. 7 a b:

Effect of the acetate ester of the hydroxyform of DMNA (= MAMN) on differentiation of limb buds from 12-day-old mouse embryos (50 - 52 somites) in organ culture. At the low concentration of 0.1 >ag/ml morphogenetic differentiation of the cartilaginous bone anlagen is slightly, however, typically affected (7 a ) . At the three-fold concentration the impairment is far more pronounced: only two of the five metacarpi developed in a regular way (oligodactyly). The other bone anlagen are short, bent and irregularly shaped (7 b ) . 1 section of the scale = 1 mm.

217 N-ALKYL-N-(ACETOXYMETHYL)-NITROSAMINES Compound

CH3-N-CH2-O-CO-CH3

Abbreviation

MAMN

NO C2H5-N-CH2-O-CO-CH3

EAMN

NO C3H7-N-CH2-O-CO-CH3

PAMN

NO CÌH9-N-CH2-0-C0-CH3

BAMN

NO /-C3H7-N-CH2-O-CO-CH3

i-PAMN

NO ter A - C4 Hg— N—C H 2 — 0 - CO— C H3

t-BAMN

NO

Fig. 8:

Structural formulas and abbreviations of six acetoxy derivatives of different alkyl-methyl-nitrosamines (synthesized by Dr. Wiessler) (alkyl means methyl, ethyl, n-propyl, n-butyl, iso-propyl, or tert-butyl group). The first compound, N-methyl-N(acetoxy-methyl)-nitrosamine (MAMN) can also be described as "acetate ester of the hydroxy form of dimethyl-nitrosamine". The other substances are closely related, with one of the two alkyl-chains being longer than one C-atom.

218 COMPOUND

ALKYL

(Abbreviation)

R=

0,1

0,3

1,0

MAMN

CH3

0 *

EAMN

C2H5

O

0

PAMN

C3H7

O

0

BAMN

C4H9

O

*

i-PAMN

i-C3H7

O

0

* *

t-BAMN

t-C4H9

0

0

0

0 = no effect abnormal development

Fig. 9:

C O N C E N T R A T I O N IN C U L T U R E 10,0 [jug/ml]

O * 9

*

- 0 = slight effect on growth =

development

Effect of six N-alkyl-N(acetoxy-methyl)-nitrosamines on morphogenetic differentiation of mouse limb buds in culture. The teratogenic effect as seen in culture decreased with increasing chain length of the compounds. However, the structure-activity relationship is more complex: with the various esters, the dose-response curves show different steepness and varying morphogenetic outcomes.

Fig. 10 a - e: Effects of five different derivatives on differentiation of mouse limb buds (day 12 = 50 - 52 somites) in organ culture. The compounds, at a concentration of 1.0 pg/ml ( b , c , d) or at 10.0 yig/ml (a, e ) , were added to the medium by injection through the rubber stopper using a Hamilton microliter syringe, immediately prior to the beginning of the culture period. The acetate ester hydrolized and liberated the corresponding short-lived hydroxilated products which can be regarded as the biological activation products. The teratogenic potentials of the different derivatives are not identical. The ethyl-derivative (EAMN), for example, has a rather weak influence on growth: at 10.0 iag/ml the limb buds develop abnormally. One tenth of the following homologue (PAMN, 10 b ) , however, completely inhibits limb development. The lowest activity in this system is seen with the tertiary butyl-derivative (t-BAMN) at 10.0 yig/ml; only a slight effect on growth is observed. 1 section of the scale = 1 mm.

Fig. 10 b: PAMN, 1.0 >ig/ml. 1 section of the scale = 1 mm.

Fig. 10 c: BAMN, 1.0 yig/ml. 1 section of the scale = 1 mm.

Fig. 10 d: i-PAMN, 1.0 ng/ml. 1 section of the scale = 1 mm.

Fig. 10 e: t-BAMN, 10.0 yig/ml 1 section of the scale = 1 mm

222

DISCUSSION: Preufimann:

The hypothesis that nitrosamines can only be activated in the target organ may perhaps not be the only one likely. The postulated extreme instability of a-hydroxyintrasamines might be wrong. OKADA in Tokyo ( p e r s . comm.) recently synthesized free a-hydroxydibutylnitrosamine and he showed that its half-life under "physiological" conditions is long enough to allow transport away from the site of its formation.

Stahlmann:

Thank you for this very interesting comment. However, from in vivo studies it seems to be evident that DMNA or DEMA are not teratogenic. Our studies suggest that the activation products are powerful teratogens. In vivo the active metabolites apparently are not stable enough to cross the placenta at a sufficient concentration.

Pedersen:

What was the strain of mice used in these experiments, in view of likely differences between strain in their capacity to activate nitrosome compounds?

Stahlmann:

We used randomly bred female mice of the NMRI/Han strain (Zentralinstitut f ü r Versuchstiere, Hannover/FRG) for all experiments. The response of adult NMRI mice to dimethyl nitrosamine (DMNA) or diethylnitrosamine was found to be pronounced. DMNA is activated by fetal tissues during the perinatal period (cf. BOCHERT, 1975).

Bochert, G., 1975, in:"New Approaches to the Evaluation of Abnormal Embryonic Development", (D. Neubert and H . - J . Merker, e d s . ) , Georg Thieme Publ., Stuttgart, p p . 554 - 572.

Comparison of Effects on Limb Development In Vivo and In Vitro Using Methyl(acetoxymethyl)nitrosamine G. Bochert*, T. Platzek and M. Wiessler ' Institut für Toxikologie und Embryopharmakologie der Freien Universität Berlin, Garystraße 9,1000 Berlin 33

Introduction: Methyl(acetoxymethyl)nitrosamine (DMN-OAc) was first synthesized by WIESSLER (1975). The compound represents a "masked" hydroxy-derivative of dimethylnitrosamine (DMNA), which is suggested to be the shortlived active metabolite following bioactivation of DMNA. This metabolic activation of DMNA does not seem to take place in the embryo during organogenesis. No teratogenic effects or DNA alkylation of embryonic tissues could be detected when rodents were studied (BOCHERT, 1975). Under in vivo conditions DMN-OAc is hydrolized very quickly by non-specific ubiquitous esterases to the unstable hydroxylated intermediate (FRANK et a l . , 1980). Contrary to DMNA, therefore, it is assumed that DMN-OAc behaves much like a directly acting alkylating agent. This class of substances has been shown to be highly carcinogenic as well as teratogenic. To date, there is increasing evidence that the initiation of teratogenic effects by directly acting monofunctional alkylating agents - similar to the carcinogenic effects - is related to their capacity to attack DNA bases electrophilicaly. In particular, the extent of O e -alkylation of the base guanine has been found to correlate closely with teratogenicity in vivo (BOCHERT et a l . , 1978). In this paper, some studies are presented on the effects of DMN-OAc on limb development in vivo and in vitro and additionally on DNA base alkylation of embryonic tissues. It will be demonstrated that under certain suppositions, results of experiments performed in vitro are comparable to in vivo findings. Furthermore, by comparing data from both studies some new aspects are supplied for the discussion of in vivo investigations. Materials and Methods: The investigations were performed on mice (Han:Mice, Zentralinstitut f u r Versuchstierkunde, Hannover/FRG) kept under a day/night cycle with darkness from 8:00 p.m. - 9:00 a.m. The animals received ALTROMIN 1324 and water ad libitum. The mating period was two hours (7:00 - 9:00 a . m . ) and the 24-hour period following the detection of vaginal plugs was considered day 0 of pregnancy (CHAHOUD and KWASIGROCH, 1977). For teratological studies, mice, day 11 (2:00 p . m . ) of pregnancy, received a single injection of DMN-OAc. The animals were sacrificed on day 18 of pregnancy and skeletal defects were evaluated after fixation and clearing of the soft parts of the fetuses followed by staining of the skeleton with

Culture Techniques © 1981 Walter de Gruyter & Co., Berlin • New York

224

alizarin r e d . Using a probit transformation and the maximum-likelihood estimation, ED 5 0 -values and slopes of the dose-response curves were obtained on a PDP-11/34 computer (PLATZEK et a l . , 1981). The in vitro development of mouse limb buds in organ culture was studied on day 11 of gestation (= 42 - 45 somites). Differentiation was allowed to proceed for 6 days with one change of the culture medium. DMN-OAc was added immediately to the f i r s t culture medium. Methodical details for the suspension culture technique are given by BLANKENBURG (Chapter VII, this book). Carbon-14-labelled DMN-OAc was synthesized as previously described by BRAUN and WIESSLER (1978), at a specific activity of 20 mCi/mmole. Methodical details about DNA isolation, HPLC separation of alkylated DNA bases and counting of radioactivity have been published elsewhere (BOCHERT and WEBB, 1977; BOCHERT et a l . , 1978). Results : 1.

Teratological studjes

Investigations in vivo were performed using three application routes. With single subcutaneous or intravenous applications up to a dose of 30 mg/kg DMN-OAc, no substance-induced malformations could be detected. However, when the substance was administered intraperitonelly, a dose-dependent occurence of malformations could be registered. Besides some other skeletal abnormalities, the main teratological effects on day 11 of gestation were malformations of the limbs. Figure 1 shows the typical effects on fore- and hindlimbs with increasing dosage. At lower dosages ( 5 - 8 mg/kg) oligoand adactyly were found, while with higher dosages, (10 - 20 mg/kg) aplasia of the limbs occurred. Considering limb malformations exclusively, an adapted dose-response r e gression curve, as given in Figure 2, resulted. The computer-calculated ED S0 -value was 9.0 ± 0.2 mg/kg and the slope of the curve 4.9 ± 0.3. No significant difference was found when the given ED 5 0 -value and the curve slope for limb malformations only were compared to those obtained for the sum of all detected skeletal effects (8.6 ± 0.2 mg/kg and 5.0 ± 0.3, r e spectively) . When the teratological data were examined, it was found the malformation occurred more often in the left fore- and hindlimbs than in the right extremities. Figure 3 shows the distribution of malformed paws in each fetus with increasing dosages of DMN-OAc. Over the given dose range from 7 15 mg/kg no single malformation of the right fore- or hindlimb was found. Even in the combination of two or three affected paws only one fetus at 15 mg/kg had a malformed right front paw and less than 10% of the fetuses had malformations of the hindpaws. The frequency of paw malformations as the sum of each paw affected occurring singly or in combination with other paw malformations is given in Figure 4. The data obtained with DMNOAc (first series of columns, Fig. 4) show that over the whole dose range nearly 100% of the affected fetuses were malformed at the left frontpaw and, dose-dependent, 50 - 95% at the left hindpaw.

225

2•

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Limb buds from 11-day-old mouse embryos were grown in organ culture with DMN-OAc added to the culture medium in concentrations from 0.1 0.3 yig/ml. Within this range of DMN-OAc concentration, a significant growth retardation was found when the limb buds were evaluated after 6 days in culture. The abnormalities observed at 0.1 yig/ml are shown in Figure 5a. The existence of the paw skeleton was just identifiable, the differentiation of ulna and radius was suppressed but recognizable. With the three-fold concentration, morphogenetic differentiation of the cartilaginous bone anlagen was drastically affected (Fig. 5b). Ulna and radius were not differentiated and the paw skeleton was practically not recognizable. 3•

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The methylation of DNA bases was determined using [ 14 C]DMN-OAc in the embryo after intravenous and intraperitoneal administration and in limb buds cultured in vitro. In Table 1 the various DNA alkylation data are summarized. Following a single i . v . injection with 10 mg/kg DMN-OAc, the concentration of methylated DNA purine bases expressed as pmole/pmole guanine in embryonic tissues was very low (0 6 -methylguanine 1.2). In contrast, i . p . injection of the same dose caused an approximate ten-fold higher amount of alkylated bases. Following 5 mg/kg i . p . about half of the alkylation rate found with 10 mg/kg i . p . was measured. Independent of the application route, the values of the alkylation rates in embryonic tissues obtained one or two hours after treatment were similar. When limb buds from 11-day-old mouse embryos in culture were exposed one hour to a concentration of 0.3 pg/ml DMN-OAc for 1 hour, an O 6 methylguanine level of 12.6 pmole/vimole guanine was detected in isolated DNA (Table 1). Comparing the in vivo and in vitro alkylation studies, the calculated O e -methylguanine/ N 7 -methylguanine ratios were very similar and in the range of 0.12 - 0.13. Discussion: At least for the class of directly acting monofunctional alkylating agents, to which in our opinion DMN-OAc belongs, a possible correlation between teratogenicity, on the one hand, and carcinogenicity and mutagenicity on the other hand cannot be ruled out (BOCHERT et a l . , 1978; NEUBERT, 1980). Experiments on the carcinogenic properties have clearly shown that the location of tumors induced by DMN-OAc depends on the route of application (HABS et a l . , 1978; BERMAN et a l . , 1979). Tumors were mostly induced locally or found in the neighbourhood of the application site. Our findings on teratogenicity are in agreement with those of the carcinogenic studies. The embryo was only affected after i . p . administration, whereas s . c . and i . v . injections did not lead to teratogenic effects. It can be assumed that for the embryo, i . p . injection represents the nearest of the three application sites studied. The dose-dependent teratogenic properties of methylnitrosourea (MNU) in mice on day 11 of pregnancy were confirmed as reported recently by BOCHERT et al. (1978). Up to now, MNU stands for the most reactive alkylating teratogen; with a molar ED S0 -value of 39 pmole/kg i . p . and a slope of

226

the dose-response curve for limb abnormalities of only 8.5. Measured against MNU, the ED so -value of 65 pmole/kg and the curve slope of 5.0, calculated for DMN-OAc caused limb malformations supports the view that it si very powerful teratogen in vivo (Fig. 1, Fig. 2). STAHLMANN et al. (this book) showed that DMN-OAc together with other alkyl-homologues are active teratogens in vitro when studied on 12-day-old mouse limb buds in organ culture. With concentrations of 0.1 - 0.3 ug/ml DMN-OAc abnormal growth and differentiation were found. This was the lowest concentration of a substance ever found to be active in this system. Using the same concentration rate with limb buds of 11-day-old mouse embryos the impairment was more pronounced, as presented in Figures 5 a and 5 b. This quantitative difference in effectiveness between day 11 and day 12 of pregnancy demonstrates the phenomenon of phase specificity of teratogenic effects in vitro. Similar results were obtained when the occurrence of limb malformations at both stages of development were studied in vivo (unpublished data, PLATZEK and BOCHERT). The quantitative comparison of biological effects obtained under in vivo and in vitro conditions, among other things, is very difficult due to the noncomparable dose ranges. However, in the case of alkylating agents we are able to measure and compare the biochemical effects caused by a substance as a DNA alkylation rate in both systems. We think this procedure is justifiable since our findings show that with various agents an agreement between dose-dependent teratogenicity and concentration of Oe-alkylguanine in embryonic DNA was obvious. In general, our data on DNA alkylation after DMN-OAc injection (Table 1), document a very fast alkylation process with the maximum beginning probably earlier than 1 h after application. Until now, only MNU injected/i.p. showed a comparable short reaction time in the embryo. The ten-fold difference in the extent of DNA methylation in the embryo obtained after i.v. injection, as compared to i.p. injection, confirms the above mentioned property of DMN-OAc to be effective only in organs near the application site. Following i.p. application of 10 mg/kg DMN-OAc, the alkylation rate of embryonic DNA was determined at 12 pmole 06-methylguanine per p i n o l e guanine . Similar rates were obtained with MNU and ethylmethanesulfonate (EMS) using doses of 6 mg/kg and 215 mg/kg, respectively. In teratological experiments, the corresponding i.p. dosages of all these substances proved to be strongly teratogenic. In contrast, the corresponding methylation extent after i.v. administration was evidently far below a postulated "threshold concentration" for the induction of embryonic impairments in vivo (BOCHERT et al., 1978). A correlation of DNA methylation by DMNOAc with carcinogenicity in various rat organs after i.v. and i.p. application has been recently reported by KLEIHUES et al. (1979). When the data obtained from in vitro experiments are compared to those from in vivo studies, they will be found to be in good agreement. A dose of 10 mg/kg i.p. induced limb malformations to an extent of about 60% (Fig. 2), while a concentration of 0.3 yig/ml DMN-OAc, added to the culture medium, caused a pronounced abnormal development in vitro (Fig. 5b). For both studies the resultant DNA methylation rates 1 hour after treatment, including the O e /N 7 -ratios, were quite comparable (Table 1). With one third of the corresponding doses the biological effect, alkylation studies are in progress, in both systems was obviously reduced (Figs. 2 and 5a). For a calculation we postulate that the concentration of 0.3 yig/ml used in vitro stands for the final concentration at the target organ. Furthermore, when theoretically a uniform distribution of DMN-OAc within both

227

the maternal and embryonic compartments without decomposition will be assumed, an average concentration of 10 pg/ml in the organism, with the higher dosage in vivo, would be expected. Including the alkylation data into this calculation, it will be found that after i.p. injection only about 1/30 and after i.v. application 1/300 of the expected concentration was active at the embryonic target. This shows the high rate of decomposition of DMN-OAc caused by esterases dependent on the application site. The phenomenon of limb-side preference induced by teratogenic agents was intensively discussed during the 4th Symposium on Prenatal Development in Berlin (BARRACH and DILLMANN, 1980). No clear explanation to the mode of action of this specific teratogenic effect could be given there. Our findings with DMN-OAc show a clear preference for malformations to occur in left limbs. The distribution of the effects measured is given in Figure 3. The decrease of singly and doubly affected paws and the increase of triple combinations with increasing doses, demonstrates a dosedependent increase in multiplicity of teratogenic effects. The different frequency of left and right frontpaw malformations is obvious, while this effect is less pronounced on the hindpaws (Fig. 4). Considering the data obtained after i.p. application of other alkylating teratogens, such as MNU or EMS, on day 11 of gestation, the frequency of paw malformations, comparing the left and right front- or hindlimbs, does not show this phenomenon of limb-side preference (Fig. 4). Furthermore, when considering the effects with DMN-OAc induced in the limb bud culture, no difference could be found in the extent of abnormalities between left and right front limb buds (Fig. 5a). In view of these results, a difference in the susceptibility of right and left extremities to an alkylating teratogen may be ruled out. For the case of DMN-OAc in vivo a substance specific pharmacokinetic reason may be postulated. The role of differential vascularization in frontlimb development has been discussed in connection with asymmetric response (SKALKO and KWASIGROCH, 1980). This, together with the high enzymatic decomposition of the substance could be responsible for our effect. The possible occurrence of differing alkylation rates in the left and right extremities after in vivo application may prove this experimentally.

ACKNOWLEDGEMENTS The studies presented in this paper were supported by grants given by Deutsche Forschungsgemeinschaft to the Sonderforschungsbereich 29, Freien Universität Berlin. The authors are indebted to Ute Rahm, Jessie Webb, Marianne Weber and sula Bluth for their excellent technical assistance and to Ruth Kreft Jane Klein-Friedrich for their help in preparing the manuscript.

the der Urand

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REFERENCES Barrach, H . - J . , Dillmann, I . , The occurrence of unilateral abnormality of the limb induced by the teratogen 6-amino-nicotinamide, in: "Teratology of the Limbs", ( H . - J . Merker, H. Nau, and D. Neubert, e d s . ) , Walther de Gruyter, Berlin - New York, pp. 171 - 181 Berman, J . J . , Rice, J.M., Wenk, M.L., Roller, P . P . , 1979, Intestinal t u mors induced by a single intraperitoneal injection of methyl(acetoxymethyl)nitrosamine in three strains of r a t s . Cancer R e s . , 39, 1462 1466. Bochert, G . , 1975, Comparative studies on the formation of methylated bases in DNA of adult and fetal mouse tissues by dimethylnitrosamine in vivo, in:"New Approaches to the Evaluation of Abnormal Embryonic Development", (D. Neubert and H . - J . Merker, e d s . ) , Georg Thieme Publ., Stuttgart, p p . 554 - 572. Bochert, G., Platzek, T . , Rahm, U . , and Webb, J . , 1978, Some new aspects in the study of DNA alkylation in embryonic and fetal tissues, in: "Role of Pharmacokinetics in Prenatal and Perinatal Toxicology", (D. Neubert, H . - J . Merker, H. Nau, J . Langman, e d s . ) , Georg Thieme Publ., Stuttgart, pp. 253 - 261. Bochert, G., Rahm, U . , Schnieders, B . , 1978, Pharmacokinetics of embryotoxic direct-acting alkylating agents: Comparison of DNA alkylation of various maternal tissues and the embryo during organogenesis, in:"Role of Pharmacokinetics in Prenatal and Perinatal Toxicology", (D. Neubert, H . - J . Merker, H. Nau, J . Langman, e d s . ) , Georg Thieme Publ., Stuttgart, p p . 235 - 251. Bochert, G . , and Webb, J . , 1977, The use of liquid ion-exchange chromatography for the determination of alkylated nucleic acid bases in maternal and fetal tissues, in ¡"Methods in Prenatal Toxicology", (D. Neubert, H . - J . Merker, T.E. Kwasigroch, e d s . ) , Georg Thieme Publ., Stuttgart, p p . 456 - 464. Braun, H . , and Wiessler, M., 1978, Synthesis of C-14-labelled nitrosamines, 4. Synthesis of C-14-methyl-(l-acetoxy)methyl nitrosamine and l-C-14-ethyl(acetoxy)ethyl nitrosamine, J . labelled Comp. Radiopharm., 887 - 892. Chahoud, I . , and Kwasigroch, T . E . , 1977, Controlled breeding of laboratory animals, in:"Methods in Prenatal Toxicology", (D. Neubert, H . - J . Merker, T.E. Kwasigroch, e d s . ) , Georg Thieme Publ., Stuttgart, p p . 78 - 91. Frank, N . , Janzowski, C . , and Wiessler, M., 1980, Stability of nitrosoacetoxymethylmethylamine in in vitro systems and in vivo and its excretion by the r a t organism, Biochem. Pharmacol., 29, 383 - 387. Habs, M., Schmähl, D . , and Wiessler, M., 1978, Carcinogenicity of acetoxy-methyl-methyl-nitrosamine after subcutaneous, intravenous and intrarectal applications in r a t s , Z. Krebsforsch., 91, 217 - 221.

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Kleihues, P . , Doerjer, G., Keefer, L . K . , Rice, J.M., Roller, P . P . , Hodgson, R.M., 1979, Correlation of DNA methylation by methyl(acetoxymethyl)nitrosamine with organ-specific carcinogenicity in r a t s , Cancer R e s . , 39, 5136 - 5140. Neubert, D . , 1980, Teratogenicity: Any relationship to carcinogenicity?, in: "Molecular and Cellular Aspects of Carcinogen Screening Tests", (R. Montesano, H. Bartsch, L. Tomatis, e d s . ) , I ARC Sei. Publ., 27, 169 - 178. Platzek, T . , Bochert, G., Rahm, U . , and Schneider, W., 1981, Alkylating agents as teratogens. A model for dose-response relationship. Teratology , in press. Skalko, R . G . , and Kwasigroch, response of the developing gy of the Limbs", ( H . - J . Walther de Gruyter, Berlin

T . E . , 1980, An analysis of the asymmetric limb to embryotoxic agents, in: "TeratoloMerker, H. Nau, and D. Neubert, e d s . ) , - New York, pp. 295 - 300.

Wiessler, M., 1975, Chemie der Nitrosamine. II. Synthese funktioneller Dimethylnitrosamine, Tetrahedron L e t t . , 30, 2575 - 2578.

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