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Arch. Toxicol. 45, 161-187 (1980)

TOXICOLOGY 9 Springer-Verlag 1980

Review Article

Significance of Organ Culture Techniques for Evaluation of Prenatal Toxicity* Hans-Jfirgen Barrach and Diether Neubert Institut f~ir Toxikologie und Embryopharmakologie, Freie Universit~it Berlin, GarystraSe 9, D-1000 Berlin 33

Abstract. A discussion of the applicability of in vitro techniques now available for research in prenatal toxicology is presented. A d v a n t a g e s and disadvantages of the various in vitro methods (such as cultivation of preimplantation embryos, whole e m b r y o culture, and organ culture) as applied to various problems of experimental research are described. As a typical example, the experience gained in our laboratory with the organ culture of m a m m a l i a n limb buds is detailed. Various aspects of the research with this type of organ culture - e.g., different techniques of culturing, extent of differentiation achieved in culture, induction of abnormalities in culture, supplementing the system whith drug-metabolizing capacities and means for quantification of the data - are discussed. It is concluded that certain in vitro techniques using m a m m a l i a n embryonic tissues are very suitable tools for elucidating the m o d e of action of teratogenic agents, and that they may serve as a " m o d e l " for several basic processes also for the situation probably existing in humans. Such organ culture, and other in vitro methods, provide little, if any advantage over in vivo experiments if a "mass screening" of a possible teratogenic potential of chemicals (hazards for the h u m a n population) is attempted. Key words: Organ culture - Limb buds - Teratogenicity.

Introduction In recent years, in vitro techniques have been established and used in several fields of biology. A similar trend is beginning to develop in medical research. * Paper presented on the Symposiumof the Toxicology Section of the German Pharmacological Society: "Isolated Cell Systems as a Tool in ToxicologicalResearch" (20th Spring Meeting, March 19, 1979) Offprint requests to." H.-J. Barrach or D. Neubert at the above address

0340-5761/80/0045/0161/$ 05.40

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Thus far, in pharmacology and toxicology little use has generally been made of the advantages that lie in the application of culture techniques. But in studies on mutagenicity and on prenatal toxicity, in vitro methods - although for quite diverse purposes - are increasingly considered an additional tool to investigations employing whole mammalian animals. The general applicability of in vitro techniques in prenatal toxicology has been discussed previously (Neubert 1980; Neubert and Barrach 1980). In this paper, the following specific aspects of the use of culture techniques in prenatal toxicology are summarized and discussed: 1. Some Special Aspects of Embryotoxic Effects. 2. In vitro Systems Available to Studies in Prenatal Toxicology. 3. Significance and Prerequisites of in vitro Systems to be Used in Prenatal Toxicology. 4. " N o r m a l " D e v e l o p m e n t of Mammalian Limb Buds in Organ Culture. 5. Pathological D e v e l o p m e n t of Mammalian Limb Buds in Organ Culture. 6. Possibility of Supplementing Organ Culture Systems with Drug-Metabolizing Capacities. 7. Applicability of Organ Culture Techniques for a "Screening" of Teratogenic Effects. Although the basic principles ruling prenatal toxicology - or embryopharmacology - do not deviate from the principles known to rule other fields of toxicology there are a n u m b e r of unique aspects which have to be considered as is the case in other special fields of toxicology. Some of these basic aspects of prenatal toxicology will briefly be discussed in the following paragraphs since they form the basis for an understanding of the role in vitro methods may play and the kind of contribution they are likely to make, in this special field of toxicology.

1. Some Special Aspects of Embryotoxic Effects E m b r y o - or fetotoxic effects (which means any kind of a toxic effect interfering with prenatal development - cf. Glossary at the end of this paper) may result in a n u m b e r of different outcomes (Fig. 1) as evaluated shortly before, at, or after birth (cf. Neubert et al. 1980). The resulting lesion may be either reversible (normal outcome of pregnancy) or irreversible (pathological outcome). If irreversible, the defect produced may either be incompatible (embryo- or fetolethal effect, abortion, still-birth, early postnatal death) or compatible with life. If compatible, with life the newborn may be retarded (in growth, d e v e l o p m e n t - either as a whole or with respect to certain organ systems) or show an " a n o m a l y " . This anomaly may be a morphological (structural) one, manifesting itself as a " m a j o r " (gross) or a "minor" malformation - whose description is a matter of definition applied by the divers investigators - or a functional one which often manifests itself after birth (sometimes late during postnatal life) and may concern function of brain, hormonal tissues, immune system, sex or other organs. But a prenatally induced defect may manifest itself

Significance of Organ Culture Techniques

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also as (transplacental) carcinogenesis or even as "unspecific" variables such as duration of life (life expectance). It is easily realized that the various forms of manifestation of prenatally induced toxic effects cannot be expected to be detectable by one simple " m o d e l " system, particularly if an evaluation with relevance to the situation possibly existing in man is desired. In recent years attention of "teratologists" has mainly been focused on structural abnormalities, both from the point of view of "screening" as well as of basic research concerned with the mode of action by which such morphological abnormalities - or "teratological effects" - can be induced. It has recently been realized that the significance of functional abnormalities which manifest themselves postnatalty may have been greatly underestimated. Such prenatally induced functional abnormalities may represent the greater problem for humans, although the induction of structural abnormalities still is the main concern of investigators working in the field of prenatal toxicology and of government agencies dealing with safety evaluations.

2. In vitro Systems Available to Studies in Prenatal Toxicology In recent years, a number of culture systems have been developed which allow us to mimic certain stages and events of embryonic development in vitro (for references cf. Neubert and Barrach 1977a). The systems principally available today are compiled in Fig. 2. It can be seen that, with suitable methods almost all periods of mammalian prenatal development can be studied. Preimplantation

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Fig. 3

Fig. 4

Fig. 3. Result of a "whole-embryo culture" of rat embryos (NEW-technique). The anlage was dissected u n d e r the stereo microscope. The embryo develops within the embryonic m e m b r a n e s . The rat e m b r y o s were 9.5 days old at the start of the culture. 100% rat serum is used as a m e d i u m . Note the excessive growth and the pronounced differentiations occurring during a one- or two-day culture period. T h e beating heart can be observed at the second day of the culture period. Experimental details as given by Cockroft 1977 Fig. 4. Result of a "whole-embryo culture" of rat embryos. Experimental conditions were the same as in Fig. 3 but the culture was started with 10.5-day old rat embryos

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Fig. 5. Differentiation of limb blastema cells to cartilage. Organ culture was initiated with limb buds from 12-day-old mouse embryos. In the example shown in the lower part of the picture, only a chunk of cartilage is formed during the culture period. No clear-cut bone anlagen can be discriminated: differentiation at the "cellular level". In the example shown in the upper part of the picture, the cartilage formed represents typical features of the extremity and the characteristic cartilaginous bone anlagen are clearly visible:

"morphogenetic differentiation"

embryos of various species (mouse, rat, rabbit etc.) can be cultured in a chemically defined medium beginning with the one-cell stage up to the blastocyst and even a bit further (cf. Spielmann and Eibs 1977, Eibs and Spielmann 1977). Whole rat or mouse embryos can be cultured for a period of 2 - 3 days from the beginning of organogenesis (day 9 in the rat) and over the whole period of early organogenesis (cf. New 1966; Cockroft 1977). A typical example of such development in culture is given in Figs. 3 and 4. Growth and differentiation is extremely impressive as achieved under these conditions. But a limit is set to this procedure in which the embryo receives nutrition simply by diffusion from the medium by the size the embryo reaches and by placentation, which, so far, cannot successfully be mimicked in vitro. The following periods of embryonic or fetal development (late organogenesis or early fetal development), therefore, can only be studied successfully with parts of the embryo or fetus, thus directing attention only to special events taking place during development. The most efficient method for studying typical developmental events in vitro has turned out to be the technique of organ culture (el. Thesleff 1977; Neubert et al. 1976). With such a technique it is possible to study mammalian embryonic developmental events, such as: closure of the palate, development of bone anlagen in a limb, tooth development, and certain differentiation processes occuring during the development of the kidney, sex organs, liver etc. These organ culture methods exhibit considerable advantages over pure tissue or cell culture techniques since they allow the study of typical morphogenetic differentiation processes. This is quite in contrast to the simpler -

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H.-J. Barrach and D. Neubert

tissue cultures or cell cultures which permit only differentiation on the "cellular level". The "morphogenetic differentiation" is essential for all studies on typical reactions occurring during embryonic development. We would like to distinguish differentiation processes occurring at two different levels: 1) A "precursor cell" may differentiate to a cell having acquired typical additional functions: a myoblast to a myocyte with typical functions of a muscle cell, a chondroblast to a chondrocyte able to form cartilage (including collagen and glycosaminoglycans), etc. We have suggested calling this a differentiation at the cellular level (cf. Fig. 5). Such a type of differentiation occurs, e.g., if blastema cells of limb buds are dissociated and then allowed to differentiate in a "dense" culture (Pennypacker et al. 1978). Chunks of cartilage develop under these conditions, but no typical organ anlagen. Similar differentiations may, under certain conditions, also take place in the adult organism during regeneration or differentiation induced by hormones or drugs. 2) During embryonic development not only is cartilage tissue or muscle tissue, etc., formed, but also these newly formed tissues are arranged in the form of a very special structure. An example is given in Fig. 5 which shows the differentiation of a mouse limb bud in culture. Not only has cartilage formed, but the cartilaginous anlagen are arranged in a very special way typical for the bone anlagen of an extremity (scapula, humerus, ulna, and radius and the hand skeleton). This is a morphogenetic differentiation which may be considered a higher form of (cellular) differentiation. It will be discussed later that an alteration of morphogenetic differentiation is very possible under conditions where, e.g., the total amount of cartilage formed is not changed. This is the essence of one type of a "teratogenic effect". We have studied various aspects of the development of mammalian limb buds (upper and lower extremities) in organ culture very extensively in recent years (cf. Neubert et al. 1974a, b, 1976; Barrach et al. 1975, 1978; Lessm611mann et al. 1975; Neubert and Barrach 1977b; Welsch et al. 1978). Therefore, we will discuss this type of organ culture system in detail here (section 4). Some of the biochemical aspects of limb development in vivo and in vitro have been discussed in detail elsewhere (Barrach et al. 1980). Many of the aspects considered also apply to other organ culture systems using differentiating embryonic tissues.

3. Significance and Prerequisites of in vitro Systems to be Used in Prenatal Toxicology Some of the important prerequisites that have to be fulfilled to make an in vitro system scientifically useful and to allow conclusions with regard to reactions also taking place in vivo are compiled in Table 1. Although generalized, these factors also apply to a system pertinent for studies in prenatal toxicology. Many - if not most - of the basic principles involved with embryonic development are far from being understood today. We are able to study rather

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TaMe 1. Prerequisites making an organ culture system useful for toxicological research 1. The culture allows tissue to be viable for a suitable period 2. The pathway or event to be studied in the organ culture system: a) proceeds similarly as in vivo; b) is regulated similarly as in vivo; c) is not superimposed by too many simultaneously occurring reactions; d) responds to "model drugs" similarly as in vivo 3. The performance of the organ culture procedure must be simpler and faster when compared with corresponding studies in vivo, and highly reproducible 4. The organ culture system may allow studies which can hardly be performed in vivo (e.g. isotope studies etc.) 5. Reactions deviating in culture from in vivo conditions must be known

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successfully events taking place at the beginning of an e m b r y o t o x i c action, e.g., the p r i m a r y interactions at the molecular level: interactions with DNA; interferences with transcription or translation; etc. W e also largely have the capacities and m e t h o d s to recognize the final o u t c o m e of an e m b r y o t o x i c action. B u t b e t w e e n these two points, a rather extensive "black b o x " exists for us (Fig. 6). Until n o w we largely lack the m e t h o d o l o g y to c o m e to an u n d e r s t a n d i n g of the various processes of e m b r y o n i c differentiation. T h e r e f o r e , our ability is quite limited in evaluating the possibilities of an interference with n o r m a l e m b r y o n i c d e v e l o p m e n t and in recognizing the reactions and basic principles during d e v e l o p m e n t which m a y be suspected to be most susceptible to an attack by teratogenic agents. T h e utilization of in vitro m e t h o d s for teratological research has a n u m b e r of basic advantages and m a n y disadvantages which have to be t a k e n into consideration w h e n attempting to integrate in vitro techniques into toxicological research (cf. Karkinen-Jfi~iskelfiinen and Sax6n 1976; N e u b e r t and B a r r a c h

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whole non-human mammals

o~an(s) organ culture

tissue culture cell culture -mammalian organism organisms -organelle system ienzyme complex yme system y of chemical

Fig. 7. Degree of complexity of a model-system used for the evaluation of toxic effects. The very complex systems allow the study of many variables and permit taking into account complex interactions. Simpler models (cf. Fig. 8) only allow the study of a few reactions, but often more easily than in vivo, and with less complicating interferences occurring simultaneously with the reaction to be studied. The simpler systems, therefore, are especially suited for an evaluation of the mechanism of a toxic action. They generally are less - or not at all - suited for a screening of many possible toxic events that might have been triggered by a given agent

1977a, 1980; Neubert 1980; Neubert et al. 1980). Fundamentally, any in vitro method is less "complex" when compared with an intact human organism (Fig. 7). Choosing the right and most suitable "model" for studying a given toxicological problem then means selecting a system which is simple enough to be understood as thoroughly as possible, but at the same time complex enough to allow conclusions valid for the whole organism. There seems to be little doubt that mammalian in vivo models are by far too complicated to serve as a basis for studying basic parameters - or often even simple biochemical reactions - as they proceed during embryonic development. Therefore, for studying certain types of embryotoxic differentiation, less complex systems would have advantages. A schematic presentation of this situation (it generally applies to in vitro systems less complex than an intact organism) is given in Fig. 8. As mentioned above, only the primary reactions and the final results can be readily assessed with our present knowledge in an in vitro system mimicking embryonic developmental processes. The remainder may be considered a "black box" with numerous reactions and interactions taking place. In a less complex in vitro system the number of events taking place is drastically reduced. For basic research this may be considered a great advantage; for "screening purposes" of allpossible kinds of toxic action, this would surely be a great disadvantage. With a situation as complex as the one just described for prenatal toxicology, what usefulness can be expected of in vitro methods which invariably have to be much simpler - or better much less complex - than an intact mammalian organism? A number of applications may be visualized in which culture methods would be of value in studies connected with problems concerning prenatal toxicology. Such applications may include:

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Fig. 8. Scheme of pathways or events occurring in vivo and in organ culture. Accessable to our studies is in general only the initial drug-induced event and the final outcome. These are linked by reactions and events which are only poorly understood and cannot be revealed with our present knowledge and the methodology available: "black box" (cf. Fig. 6). An in vitro system as a tool should be simpler ("black box" is smaller) and less "complex" (cf. Fig. 7), but the major events to be studied should proceed in vitro in a similar way as in vivo. Fewer drug effects and fewer results will be observable. Artifacts (dashed reaction line) leading to results not occurring in vivo (results from dashed line) should be excluded if possible or should at least be recognized. Under these conditions the in vitro system may serve as a convenient "model" for events occurring in vivo which can be studied more easily

1) t h e e l u c i d a t i o n o f basic b i o l o g i c a l p a r a m e t e r s i m p o r t a n t f o r m a m m a l i a n prenatal development; 2) t h e e l u c i d a t i o n o f t h e m o d e o f a toxic ( t e r a t o g e n i c ) a c t i o n - q u a l i t a t i v e l y a n d quantitatively; 3) p o s s i b l y a " s c r e e n i n g " o f c e r t a i n g r o u p s of t e r a t o g e n i c a g e n t s (?). T h u s , t w o p o i n t s can a l r e a d y be c l e a r l y s e e n at t h e b e g i n n i n g of a discussion: in p r e n a t a l t o x i c o l o g y in vitro m e t h o d s can o n l y b e a p p l i e d u s e f u l l y in combination with in vivo t e c h n i q u e s a n d in vitro m e t h o d s a r e o n l y a b l e to h e l p in solving special p r o b l e m s . If t h e s e p o i n t s a r e t a k e n into c o n s i d e r a t i o n , in v i t r o m e t h o d s l m a y b e c o n s i d e r e d to b e v e r y useful tools in " t e r a t o l o g i c a l " research.

4. "Normal" Development of Mammalian Limb Buds in Organ Culture Six y e a r s b e f o r e , w e i n i t i a t e d studies with d i f f e r e n t i a t i n g l i m b b u d s in v i t r o using a T r o w e l l - t y p e s e t - u p ( T r o w e l l 1954, 1959) in w h i c h t h e e x p l a n t s a r e c u l t u r e d at t h e g a s - l i q u i d i n t e r f a c e o n a m e m b r a n e filter. Such a s y s t e m , s u p p l e m e n t e d with calf s e r u m , h a d b e e n successfully u s e d b y A y d e l o t t e a n d K o c h h a r (1972). A l t h o u g h s u i t a b l e f o r a n u m b e r o f p u r p o s e s ( N e u b e r t et al. 1974a, b, 1977) t h e s y s t e m p r o v e d to h a v e a n u m b e r o f d i s a d v a n t a g e s which l i m i t e d its a p p l i c a b i l i t y f o r t o x i c o l o g i c a l studies. T h e r e f o r e , w e m o d i f i e d t h e t e c h n i q u e e x t e n s i v e l y d u r i n g t h e last few years. F i r s t , t h e s e r u m - c o n t a i n i n g c u l t u r e m e d i u m was r e p l a c e d by a c h e m i c a l l y d e f i n e d m e d i u m (Lessm611mann et al. 1976). This

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Fig. 9. Differentiation of mouse limb buds in organ culture. Limb buds of NMRI-mice at various stages (upper row) of gestation (day 10 to 12 of gestation) were allowed to differentiate for 6 days (lower row) in organ culture (suspension technique in roller flasks). The extent of differentiation and the "end point" reached in culture critically depend on the stage at which the culture was initiated. A good paw skeleton under experimental conditions only develops from limb buds of 11-day-old (40 to 45 somites) or older mouse embryos

allows the exclusion of the variables introduced into the system by the p o o r l y - d e f i n e d serum. A t the same time the differentiation achieved in the culture was at least as g o o d - often better - than that obtained in the presence of serum. T h e technique developed m a d e possible a systematic study of the r e q u i r e m e n t s (nutrients etc.) of the developing e m b r y o n i c tissues. T h e n we c h a n g e d to culturing limb buds in a suspension culture using a roller device and "penicillin-flasks" as culture vessels. This technique offered the great advantage that nutrients and drugs - in contrast to the Trowell-type setup - freely r e a c h e d the explant and also, if desired, could be easily r e m o v e d f r o m the explants. O n l y this technique allows a systematic study of drug effects for limited and defined periods of time during the culture period. With the suspension culture technique the explants also develop m o r e "naturally" (extended, round, with long fingers and "long b o n e s " and with few bends), in contrast to the Trowell-technique which leads to flat explants which are greatly distorted. With the two types of culture technique - the Trowell-technique and suspension culture - the extent of differentiation reached in culture critically d e p e n d e d on the d e v e l o p m e n t a l stage the organ culture is initiated with (cf. Fig. 9). T h e full d e v e l o p m e n t of the limbs - including cartilaginous b o n e anlagen of the h a n d skeleton - is only achieved if the culture is set up with limb buds f r o m l l - d a y - o l d m o u s e e m b r y o s or later stages. W h e n the culture is initiated with explants f r o m 10-day-old e m b r y o s , only the anlagen of "long b o n e s " develop in culture and none or very little of the hand skeleton. T h e r e a s o n for the comparatively p o o r differentiation when started with limb buds

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Fig. 10. Attempts to cultivate a "naked" mouse embryo in vitro. The culture was initiated with a ll-day-old mouse embryo removed from the embryonic membranes. Culture medium as for organ cultures of limb buds. After a three-day cultivation period in a roller device the embryo has grown and the cartilaginous bone anlagen of the upper extremity have differentiated. In addition, cartilaginous bone anlagen of other systems can be recognized (spinal cord, skull, pelvis, etc.). But the extent of development of the limbs is not better than that obtained in organ culture of the isolated limb buds

from 10-day-old (or younger) mouse embryos is not known to us. It may be that the culture medium is deficient in a component essential for differentiation when started on day 10 of gestation, but no longer required when the culture is initiated later. A n o t h e r possibility is that the interaction of the limb bud with some other tissue is a requirement for the early differentiation steps which is no longer present when the explant is removed from the embryo and put into culture. To test the latter hypothesis we tried to culture whole, naked embryos in the same medium. Here the interaction with other adjacent tissues should still be possible. Although we succeeded in obtaining differentiation of the limb when started with l l - d a y - o l d mouse embryos under these conditions (cf. Fig. 10) no better result was achieved with the whole embryo than with isolated limb buds when the culture was started with 10-day-old embryos. When using whole l 1-day-old embryos or older ones it is quite possible to observe the differentiation of other cartilaginous anlagen, such as spine, ribs, pelvis or skull. But culturing is made difficult by the amount of lactate produced by the large amount of tissue and it is hard to grow several embryos and to control the pH. Thus, we have routinely performed our studies with limb buds from 11- or 12-day-old mouse embryos ( 4 0 - 4 5 and 5 0 - 5 5 somites respectively) and we have cultured more than 50,000 explants during the last few years. A n example of development in culture is given in Figs. 9 and 11. The method can be considered a routine procedure by now in our laboratory and many variables of normal and pathological development have been measured. Some of the typical biochemical aspects have recently been summarized elsewhere (Barrach et al. 1980).

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Fig. 11. Differentiation of mouse limb buds (12-day-old embryos) in organ culture. This series shows the extent of differentiation obtained at various durations of culturing (day l to day 6 of the culture period)

Fig. 12. Reproducibihty of the differentiation of limb buds in organ culture. An example of a typical experiment is given. All limb buds (day 12 of gestation) have been selected from embryos with a constant number (52 + 53) of somites. A high degree of reproducibility can be seen. A similar extent of reproducibility is also to be seen in most series under pathological conditions

O n e of t h e a d v a n t a g e s of the o r g a n culture system is the high d e g r e e of r e p r o d u c i b i l i t y achieved if the system is s t a n d a r d i z e d by using explants f r o m d e f i n e d s o m i t e stages only (Fig. 12). T h u s , n o r m a l a n d a b n o r m a l d e v e l o p m e n t of the c a r t i l a g i n o u s b o n e a n l a g e n can be studied with m u c h less v a r i a n c e t h a n is possible in vivo.

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Table 2. Experimental setup to be used with the organ culture system Limb buds from

Normal embryos

Culture conditions Medium

Period

normal

total total

+ teratogen limited Embryos drug-pretreated in utero

normal

total total

+ teratogen limited total

+ antagonist limited Embryos pretreated with "inducers"

normal

total

+ potential

total

teratogen

limited

5. Pathological Development of Mammalian Limb Buds in Organ Culture A f t e r having established optimal experimental conditions for normal m o r p h o genetic differentiation of limb buds in organ culture, it was possible to study pathological development and the induction of "skeletal m a l f o r m a t i o n s " entirely in vitro. Already in earlier studies it had been observed that the system responds to "teratogenic agents" in a typical way and that an abnormal d e v e l o p m e n t closely resembling that found with the same agent in vivo can be observed in culture (Neubert et al. 1974b). For an assessment and a m o r e detailed study of abnormal d e v e l o p m e n t the organ culture system we developed can be used in a variety of modifications (Table 2). Firstly, it is possible - and we have m a d e ample use of it - to add a drug to the culture m e d i u m for a limited period of time only, culturing the limbs in the preceeding and the following periods in the presence of a normal medium. In this way, it is possible to study phase specificity in vitro - especially if the culture is initiated in addition with explants of varying developmental stages (Fig. 13). Typical examples of experimental series p e r f o r m e d , e.g., with inhibitors of R N A or protein synthesis are shown in Figs. 14 and 15. The first and sometimes also the second day of the culture period has been found to be most susceptible

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flask No.

day o f 1

2

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4

period 5

6

1.2.3. 4.5. 6.7. 8.9. 10. 11. 12. 13. 14.15.

Fig. 13. Experimental set-up to be used for studying a "phase-specific" effect of a teratogenic agent in organ culture. The agent to be tested is present in the culture medium for a limited period only. The culture can, in addition, be initiated with limb buds from embryos at different stages of development (day 10 to 13 of gestation)

, J with drug culture . . . . . . without drug

Fig. 14. Effect of a-amanitin added to the culture medium at various intervals of the culture period. The strongest effect of the inhibitor of RNA-synthesis is produced when added at the first day of culturing. Much less effect is seen if the inhibitor is present at the second half of the 6-day culture period

to the action of a number of teratogenic agents. This can be seen in the studies performed with a-amanitine, a typical inhibitor of transcription (Fig. 14). While a drastic interference with limb development is ~chieved when the inhibitor is present in the medium on the first day of the culture period, this effect is much less pronounced when a-amanitine is added to the medium on day 2 of culturing only. Little, if any effect is observed if the inhibitor is present on the 3rd day or later, although development is not completed at three days in culture (cf. Fig. 11). Although the highest susceptibility towards the action of cycloheximide, a typical inhibitor of protein synthesis at the 80S-level, also seems to be present at the first two days of culturing (Fig. 15) - the second day apparently

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Fig. 15. Effect of cycloheximide added to the culture medium at various intervals of the culture period. Here again the explants are susceptible to the inhibitor during the first days of cultivation, but development is also impaired, if the inhibitor of protein synthesis is present in the culture medium during the second half of the 6-day culture period

being m o r e susceptible than the first one - clear-cut interference with d e v e l o p m e n t can also be obtained when the inhibitor is added to the m e d i u m on the 4th to 6th day of the culture period, thus distinguishing the effect clearly f r o m that obtained with a-amanitine. W e have invested considerable time and effort to substantiate an effect of thalidomide in the organ culture system. Such an attempt is made difficult by two facts: 1) the extremely low water solubility of thalidomide and the finding that some solubilizing agents themselves interfere with limb d e v e l o p m e n t in culture (like D M S O which does so at concentrations exceeding 0.03%); and 2) the species routinely used as donors for our explants - rats and mice - do not typically respond to thalidomide with teratogenic effects in vivo. So far, we have not - in dozens of experiments - been successful in inducing abnormal development in vitro when adding thalidomide to the culture medium. But an impaired and abnormal development of the cartilaginous bone anlagen of the extremities could be observed when high concentrations ( 1 0 0 - 3 0 0 ~tg/ml) of some hydrolysis products of thalidomide (Fig. 16) were present in the culture m e d i u m (Neubert et al. 1978b). The significance of these findings is not clear at present, and unspecific effects can hardly be excluded at such high concentrations. But since most of these hydrolysis products are highly polar (di- or even tricarbonic acids), they may not penetrate into the explants at a high rate and extreme-appearing concentrations m a y be required in the m e d i u m to obtain at least some intracellular accumulation. A second experimental setup (Table 2) may be advantageous if the result of a teratogenic lesion set in vivo is to be followed by culturing the embryonic tissue exposed to the drug in utero. In this way maternal effects m a y be excluded and

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Fig. 16

H.-J. Barrach and D. Neubert

Fig. 17

Fig. 16. Effect of some hydrolysis products of thalidomide on differentiation of limb buds in organ culture, The very poorly water-soluble thalidomide hydrolyzes spontaneously to a variety of water-soluble products, Some of these products were added to the culture medium at a concentration of 300 ~g/ml. The cultures were initiated with limb buds of mouse embryos (NMRI) of different stages of development (day 10 or 11 of gestation). The picture shows the effect obtained after a 6-day culture period. The following hydrolysis products of thalidomide were used: I = N-phthalyl-L-glutamine; IV= N-phthalyl-D-glutamate; V = N-[O-carboxybenzoyl-D,L-glutamine; VI = N-[O-carboxybenzoyl]-D,L-isoglutamine. Abnormal development was induced by both the L- and the D-forms of the active derivatives Fig. 17. Result of an combined in vivo/in vitro study. NMRI-mice received 500mg/kg 5'-bromodesoxyuridine (BUdr) or 250 mg/kg hydroxyurea (HU) s.c. on day 11 of pregnancy. The embryos were dissected 15 h later, and the limb buds cultured for 6 days in a normal culture medium. A clear-cut pathological development can be followed in vitro and grossly malformed extremities develop in organ culture. (These experiments are part of a series performed in collaboration with Prof. Dr. R. Skalko, Johnson City, Tenn., USA)

morphological observations may easily be supplemented with biochemical s t u d i e s . T h e o r g a n a n l a g e n o f t h e e m b r y o d r u g - p r e t r e a t e d in u t e r o m a y b e c u l t u r e d in a n o r m a l m e d i u m o r t h e e f f e c t o f a s e c o n d t e r a t o g e n o r t h a t o f a n antagonist may be studied. By varying the interval between the injection of the compound to the pregnant animal and the start of the culturing period, the e x t e n t o f t h e l e s i o n m a y b e m o d i f i e d a n d its r e v e r s i b i l i t y can b e s t u d i e d . T h i s a p p r o a c h h a s b e e n u s e d b y us w i t h s e v e r a l a g e n t s a n d it has b e e n f o u n d t o b e a u s e f u l e x p e r i m e n t a l d e s i g n . A s a n e x a m p l e , Fig. 17 s h o w s d e f e c t s o b s e r v e d in

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limb buds cultivated in a normal medium after injection of 5'-bromodesoxyuridine or of hydroxyurea into the pregnant animals on day 11 of pregnancy, 15 h prior to the cultivation of the limb buds of the 12-day-old embryos. It is interesting that deformities can be induced in a way that is highly reproducible, which are confined to the "long bones" with little effect on the paw development. With other teratogenic agents it is possible to interfere selectively with the differentiation of the paw, using the same experimental set-up. This points to a high degree of specificity of the lesions produced. A third approach (Table 2) developed in our laboratory makes use of the possibility to "induce" certain enzyme systems - like the arylhydrocarbon hydroxylase ( A H H ) which activates many xenobiotics, which themselves are largely non-toxic, to the toxic metabolites. We have found that A H H can already be induced at the organogenesis stage in mouse and rat embryos by agents like benzo(a)pyrene, polychlorinated biphenyls or tetrachlorodibenzo-dioxin (Neubert and Tapken 1978) and it therefore is possible to use limb buds with such induced enzyme systems for in vitro cultivation studies. An important problem arising from toxicological studies using organ culture systems is the quantification of abnormal development produced in vitro. Three approaches appear feasible, and we have tested their applicabilities: 1) quantification with morphometric methods; 2) quantification with biochemical methods; 3) quantification according to the degree of interference with morphogenetic differentiation. Morphometric methods represent the simplest means of attempting quantification of differentiation processes. They also are the least reliable ones since it is hard or almost impossible to control the thickness of the explants, and the intensity of the staining (of specific and "unspecific" structures). This approach may give some meaningful results in the case of the Trowell-type culture, where the explants develop in a flat form (Neubert et al. 1974b). Much better results may be expected if a typical component of a developing tissue is monitored. With the limb buds used, this may be collagen or some glycoprotein component. The reliability of the biochemical method is much better than that of morphometric methods, but the explant is lost after the biochemical analysis. In our experience the correlation between morphometric and biochemical data is poor, pointing to the inadequacy of the first method. Both of the methods mentioned suffer from the inability to quantify alterations of morphogenetic differentiation and they are only successful if a pronounced interference with differentiation at the cellular level (cf. section 3) has occurred. We have already found in earlier studies (Neubert et al. 1974b) that drastic alterations of the morphogenetic differentiation may be produced without a clear-cut decrease in, e.g., the total amount of cartilage of the limb. The cartilage is just arranged in a pathological manner! The amount of cartilage formed, therefore, is only one parameter of the degree of differentiation achieved in vitro and it may not be the most important one. We have developed - and tested in a large number of experimental series a scoring system for the evaluation of the degree of differentiation achieved in

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culture (Neubert et al. 1978a). It is possible with the system to study quantitative relationships and to establish dose-response curves (Neubert et al. 1977). For teratological evaluations this technique has considerable advantages over the methods discussed before. But again, with this scoring system alone the type of abnormality produced is only poorly discriminated from other abnormalities. For a close analysis the different bone anlagen therefore have to be evaluated separately. W e have found that a discrimination into 1) paw, and 2) limb without paw is sufficient for m a n y purposes and we therefore use this system routinely (cf. N e u b e r t et al. 1977; Welsch et al. 1978).

6. Possibility of Supplementing Organ Culture Systems with Drug-metabolizing Capacities One of the disadvantages of an in vitro system utilizing embryonic or fetal tissues - especially f r o m rodents - lies in the inability of such tissues to activate a variety of substances to the reactive, teratogenic molecule. Therefore, it is of interest to attempt to supplement organ cultures or whole-embryo cultures with drug-metabolizing systems. A n u m b e r of possibilities exist for overcoming this difficulty of the active teratogenic metabolite not being formed in vitro (cf. Neubert and Barrach 1977b). Some of the most important approaches are listed in Table 3. Within the last years we have tested all the experimental approaches listed and - except for possibility 3a and b - have found them to be feasible and useful when applied and adapted to a given situation. Six situations possibly exist for an agent to be tested when drug metabolism is considered (Table 4): a) The agent as such is "active" and therefore capable of inducing abnormalities in vitro. b) The agent as such is "inactive" as a teratogen, but in vivo it is converted within the maternal c o m p a r t m e n t to a stable metabolite which can penetrate the placenta and act teratogenic.

TaMe 3. Possibility of supplementing an organ culture system with drug-metabolizing capacities 1. Serum of treated animal (or human) added to culture medium 2. Chemical preincubated with microsomes and cofactors; supernatant added to culture medium 3. Culture medium supplemented with a) intact (liver) cells b) microsomes c) solubilized microsomal enzymes d) other drug-metabolizing enzymes or complexes 4. Embryonic tissue pretreated with a) agents "inducing" drug-metabolizing enzymes in vivo; b) agents "inducing" drug-metabolizing enzymes in vitro

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Fig. 18. Effect of an alkylating metabolite of cyclophosphamide on differentiation of limb buds in organ culture. 4-hydroperoxy-cyclophosphamide (4-HPO-cpa) - which spontaneously converts to the labile 4-hydroxy-cyclophosphamide which is in equilibrium with aldophosphamide (cf. Brock and Hohorst 1977) - was added to the culture medium in which limb buds from normal, 12-day-old mouse embryos (NMRI-strain; 51-53 somites) differentiated. While 1 gg 4-HPO-cpa/ml only interfered very little with development (some alteration of radius development can be seen), drastic abnormalities are produced with 10 p~g 4-HPO-cpa/ml (ectrodactyly and almost complete absence of ulna and radius). Almost no differentiation takes place in the presence of 100 ~g 4-HPO-cpa/ml

c) T h e " i n a c t i v e " a g e n t can r e a c h t h e fetal tissue a n d is c o n v e r t e d within t h e fetal cells to an " a c t i v e " t e r a t o g e n i c substance. d) T h e " i n a c t i v e " a g e n t r e a c h e s the fetal tissue, b u t fetal r o d e n t tissue - in c o n t r a s t to t h a t o f p r i m a t e s - is not c a p a b l e of t r a n s f o r m i n g this c h e m i c a l to an " a c t i v e " t e r a t o g e n i c agent. e) F e t a l tissue c a n n o t c o n v e r t the agent to an " a c t i v e " , short-living t e r a t o g e n under normal conditions. Under pathological conditions (e.g., "induction" or c h a n g e in a m a j o r m e t a b o l i c r o u t e ) such a c o n v e r s i o n can t a k e place. f) T h e a g e n t in p r i n c i p l e is t e r a t o g e n i c o r can be c o n v e r t e d into a t e r a t o g e n , b u t it d o e s n o t r e a c h t h e c o n c e p t u s at a high e n o u g h c o n c e n t r a t i o n in vivo at c e r t a i n s t a g e s o f d e v e l o p m e n t (e.g., in certain species). T h e c o n s e q u e n c e s to be e x p e c t e d with the 6 situations listed in an in vitro s y s t e m a r e s h o w n in T a b l e 4. W h e n a t t e m p t i n g s e v e r a l a p p r o a c h e s to a d d a c y t o c h r o m e P450-type system in the o r g a n o r w h o l e - e m b r y o c u l t u r e ( p o i n t 3 o f T a b l e 3), w e h a v e f o u n d t h a t the a d d i t i o n o f a m i c r o s o m a l cell fraction g r e a t l y i n t e r f e r e s with t h e d i f f e r e n t i a t i o n s in vitro. W e f a v o u r the a p p r o a c h of a d d i n g a r e c o n s t i t u t e d c o m p l e x o f i s o l a t e d c y t o c h r o m e P450 to the culture m e d i u m ( K i t t e l et al. 1978). Such a c o m p l e x d o e s n o t i n t e r f e r e with the d e v e l o p m e n t in c u l t u r e a n d b y i s o l a t i n g the c y t o c h r o m e P450-type f r o m v a r i o u s sources a n d after the p r e t r e a t m e n t with s u i t a b l e " i n d u c i n g " agents, t h e m e t a b o l i z i n g activity can be m o d i f i e d c o n v e n i e n t l y a c c o r d i n g to the r e q u i r e m e n t s . U s i n g c y c l o p h o s p h a m i d e as a m o d e l - a l b e i t an especially s i m p l e o n e - we h a v e s u c c e e d e d in p r o d u c i n g typical a b n o r m a l i t i e s in vitro b y all of the following e x p e r i m e n t a l a p p r o a c h e s ( N e u b e r t et al. 1980): 1) a d d i t i o n o f t h e active m e t a b o l i t e to t h e culture (cf. B a r r a c h et al. 1978); 2) p r e i n c u b a t i o n o f c y c l o p h o s p h a m i d e with liver m i c r o s o m e s a n d a d d i t i o n o f t h e 100,000 g s u p e r n a t a n t to the c u l t u r e m e d i u m ;

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3) addition of serum of cyclophosphamide-pretreated mice to the culture medium; 4) addition of cyclophosphamide and of a reconstituted system of isolated cytochrome P450 to the culture medium. In each of the approaches mentioned very similar and typical "malformations" resulted during the cultivation (Fig. 18). Manson and Simons (1979) have recently published results of an approach in which limb buds were co-cultivated with hamster embryo cells. A clear-cut activation of the cyclophosphamide was achieved under these conditions. But the malformations induced were not too convincing. Although the studies performed so far have shown that it is possible to supplement the in vitro culture of embryonic tissues with a drug-metabolizing capacity, all these approaches must be considered preliminary and much more work is needed to derive at reliable and versatile systems that can be used rountinely with a high degree of reproducibility.

7. Applicability of Organ Culture Techniques for a "Screening" of Teratogenic Effects Attempts to establish in vitro systems replacing in vivo tests in the routine mass screening of chemicals (those used for medication, or even more those present as environmental substances) for potential embryotoxic or even for teratogenic capacities in our opinion represent an effort leading in the wrong direction. From a toxicological point of view such in vitro systems separately used cannot be expected to provide sufficient information on possible teratogenic hazards with relevance to the situation possibly existing in humans. The inefficiency of in vitro systems for a screening of a possible embryotoxic potential of a given chemical results from a number of considerations (Table 5): 1) From the embryotoxic effects mentioned in paragraph II it is basically not possible to predict with an in vitro test system an embryolethal potential, a potential to produce retardations or a potential to induce functional anomalies. Only a "teratogenic" potential - that is, a potential for inducing gross structural abnormalities - may be detected to a certain extent. 2) From the possible teratogenic effects only the "universal" teratogenic actions which cause interference with differentiation processes in general (cf. Neubert 1980; Neubert et al. 1980; Neubert and Barrach 1980) are likely to be detected with an in vitro system. A "specific" teratogenic potential - that is, the ability to induce a single, circumscript interference with embryonic differentiation at a defined stage of development - in all likelihood will not be detected with a single given in vitro system, and not even by a combination of several organ culture tests. 3) In each of the in vitro system known today, "false positive" effects can be produced by unspecific means, e . g . , by modification or interference with components of the culture medium. 4) Until now, drug-metabolism and other pharmacokinetic variables can only

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Table 5. Significance of in vitro techniques for the detection of an embryotoxic potential

Prenatal toxicity

Will be detected with in vitro techniques

Embryo-(feto-)toxic effect

embryo- (or feto-)lethal action growth retardation postnatal manifestations "teratogenic" potential

no

no no

possibly

Teratogenic potential

"universal" teratogenic potential "specific" teratogenic potential

probably probably not

"Universal" teratogenic potential

in all species if it needs metabolic "activation" if a special situation exists in humans with respect to drug metabolism or susceptibility

probably not necessarily

no

partly be controlled or mimicked in the in vitro tests - especially if the kind of drug-metabolism the chemical may undergo is not completely known. 5) certain components of the medium which are kept at an excess (ascorbate, cystein, etc.) m a y inactivate the chemical added to the culture medium, thus producing a "false negative" effect. If it is felt that it is sufficient to monitor for "universal" teratogens (cf. point 2), then simple embryonic systems, such as tests with chicken embryos (Jelinek 1976), systems monitoring differentiations at the cellular level (Pennypacker et al. 1978), or even tests using non-embryonic tissues, may suffice. The d e v e l o p m e n t of sophisticated mammalian organ culture techniques would not necessarily be required. But we feel that it is n o t adequate to screen for "universal" teratogenic effects exclusively. A simple consideration will clarify the situation with which we are faced and will lead to the following conclusions: a) A " p o s i t i v e " outcome of a simple in vitro test will not provide us with enough information to p e r f o r m a risk evaluation for the chemical in question with respect to embryotoxicity or teratogenicity with relevance to humans. Further animal experiments will be inevitable. b) A " n e g a t i v e " outcome of a simple in vitro system will not provide us with sufficient information to be reasonably sure that an embryotoxic or teratogenic risk with relevance to humans does not exist with the chemical or agent in question. Further animal experiments will again be inevitable. This shows that, with the situation as it exists now, in vitro "screening tests" for possible teratogenicity can only exceptionally be considered to be useful (perhaps if a given class of compounds is studied with well-known teratogenic

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capacities, like, e.g., thalidomide derivatives). In the majority of the cases they must yet be considered a waste of time, money and effort today, also if intended as "precreening" of agents which in addition will have to be tested in vivo in animal experiments (cf. Neubert and Barrach 1980).

8. Glossary of Terms Used in Prenatal Toxicology 5 embryo I fetus perinatal period embryotoxic or fetotoxic effect, embryotoxicity 1 abnormality, anomaly teratogenic effect teratogenicity 2

developmental stages from the fertilized egg up to the end of organogenesis phase developmental stages from the end of organogenesis up to birth period around birth, including late fetal and early postnatal life all possible types of toxic effects interfering with prenatal development every structural or functional deviation from normal development embryotoxic effect leading to structural abnormalities

malformation

structural (gross morphological) abnormality or defect

embryolethal or fetolethal effect embryo- or fetomortality growth retardation

embryotoxic effect incompatible with life

transplacental carcinogenesis day 0 of pregnancy 3 (~ 1st day of pregnancy) or of gestation 4

embryotoxic effect resulting in a too-small for-age-fetus; either immature or small but mature fetotoxic (or embryotoxic) effect leading to the development of a tumor in postnatal life caused by an agent applied prenatally first 24-hour period following conception or mating)

1 the term "embryo" is often misused to denominate all stages of prenatal development. In this case an "embryotoxic effect" might also be induced late in gestation 2 the term "teratogenic" is often incorrectly used to denominate an "embryotoxic"effect 3 with regard to mother 4 with regard to embryo 5 (According to Neubert et al. 1978)

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Experimental Conditions For the results presented in this paper, NMRI-mice (Han: NMRI, Zentratinstitut ftir Versuchstiere, Hannover/FRG) or Wistar-rats (Wistar TNO W/74, Versuchstierfarm Winkelmann, Paderborn/FRG) were used. The animals were kept under spf-conditions at a defined dark-light cycle (light from 9.00-21.00 h) at 24-25 ~ C and 50% humidity. Altromin | 1324 and tap water were given ad libitum. The animals were mated for 2 - 3 h at the end of the dark period. If sperm or vaginal plugs were detected, the 24-hour period after mating was designated as day 0 of pregnancy (e.g., day 11 = 264-288 h after mating). The females were sacrificed at the pregnancy stage indicated, and the embryos removed from the uterus and prepared for cultivation.

Whole-Embryo Culture. The whole embryo culture was performed with rat embryos at day 9.5 or 10.5 of gestation, according to the method of New (1966) as described by Cockroft (1977), using rat serum (100%) as culture medium. More recently we have used human serum (supplemented with glucose to give a 150 rag-% concentration) with very good success as a much more convenient culture medium. Limb Bud Culture. The limb buds (we mostly used forelimbs) of 10.5- to 12-day-old mouse embryos were excised under a dissection microscope. For the suspension culture technique used for obtaining the data presented in this paper, the limb buds were cultivated in 50 ml "penicillin"-flasks (Neubert and Barrach 1977b) in a roller device, turning with 25 rpm. The bottles contained 6 mt culture medium (with 15 limb buds) and they were closed airtight with a rubber stopper and a metal seal. They were flushed with an appropriate gas mixture (mostly 40% 02; 5% CO2; 55% N2). This technique abolishes the necessity of using a CO2-incubator which is costly, often unreliable and does not permit the study of the influence of varied gas phases - including that of toxic gases - within one experimental series. Once a day the bottles were gently shaken to counteract an aggregation of the limbs. The culture medium consisted of: 6400 mg/l NaC1; 425 mg/1 KC1; 330 rag/1 MgSO 4 9 7 H20; 2800 mg/1 NaHCO3; 113 mg/1 KHEPO4; 4400rag/1 glucose monohydrate; 225 mg/1 e-lysine hydrochloride; 150rag/1 L-histidine hydrochloride; 70 rag/1 L-argenine hydrochloride; 60 mg/z-tyrosine; 50 rag/1 z-valine; 40 mg/l L-leucine; 23 mg/1 z-isoleucine; 40 mg/l L-methionine; 40 rag/1 L-phenylalaline; 30 mg/1 L-tryptophan; 150 mg/1 L-glutamine; 140 mg/l L-cysteine hydrochloride; 600 rag/1 Ca lactate-5 H20; 150 rag/1 ascorbic acid; 6000 rag/1 bovine serum albumin; 15 mg/1 nicotinamide; 3 mg/1 thiamine HC1; 0.15 mg/l Ca pantothenate; 0.15 rag/1 riboflavine; 0.15 mg/1 pyridoxal hydrochloride; 0.15mg/l folic acid; 0.15mg/1 D(+)biotin; 0.75mg/l Dt-a-tocopherol; 38 mg/l choline chloride; 0.15 mg/1 inositol; 0.03 mg/1 cyanocobalamin; 12.5 mg/l streptomycin; 7.5 mg/1 penicillin; 16 mg/1 phenolred. This medium roughly corresponds to 75% Bigger's medium. Routinely the explants were cultured for 6 days and the medium was changed after 3 days. In some series, a

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d r u g - c o n t a i n i n g m e d i u m w a s u s e d f o r a l i m i t e d p e r i o d o n l y (cf. Fig. 13) a n d t h e e x p l a n t s d e v e l o p e d b e f o r e a n d a f t e r this p e r i o d in f r e s h m e d i u m ( w i t h o u t drug). In order to evaluate the degree of differentiation of the cartilaginous skeletal a n l a g e n , t h e e x p l a n t s w e r e f i x e d in B o u i n ' s s o l u t i o n , s t a i n e d w i t h A l c i a n B l u e , c l e a r e d w i t h 2 5 % a c e t i c a c i d , d r i e d in 7 0 % a n d 9 6 % e t h a n o l a n d k e p t in c e d a r oil. F o r q u a n t i f i c a t i o n , t h e s c o r i n g m e t h o d o f N e u b e r t e t al. (1978a) w a s u s e d . Photographs were taken using a Tessovar, Zeiss, FRG.

Acknowledgements. The original work presented in this paper was supported by grants by the Deutsche Forschungsgemeinschaft awarded to Sonderforschungsbereich 29 (Embryonale Entwicklung und Differenzierung - Embryopharmakologie) at the Freie Universit~it Berlin. We are indebted to Ruth Kreft and Susanne Barrach for their help in preparing the manuscript and to Irmela Baumann-Wilschke and Ursula Schwikowski for taking the photographs.

References Aydelotte MB, Kochhar DM (1972) Development of mouse limb buds in organ culture: Chondrogenesis in the presence of a proline analog, L-azetidine-2-carboxylic acid. Dev Biol 29 : 191-201 Barrach H-J, Rautenberg M, Tapken S, Neubert D (1975) Some biochemical characteristics of mouse limb buds differentiating in organ culture. In: Neubert D, Merker H-J (eds) New approaches to the evaluation of abnormal embryonic development. Thieme, Stuttgart, p 114 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. In: Neubert D, Merker H-J, Nau H, Langman J (eds) Role of Pharmacokinetics in Prenatal and Perinatal Toxicology. Thieme, Stuttgart, p 323 Barrach H-J, Grundmann K, Kinel J, Neubert D (1980) Some biochemical and toxicological aspects of mammalian limb development, Conference on Teratology, Carcinogenicity, and Mutagenicity, Sept. 17-19, 1979, Hunt Valley, Md., USA (in press) Brock N, Hohorst H-J (1977) The problem of specificity and selectivity of alkylating cytostatics: studies on N-2-chlorethylamido-oxazaphosphorines. Z. Krebsforsch. 88:185-215 Cockcroft DL (1977) Post implantation embryo culture. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, p 231 Eibs H-G, Spielmann H (1977) Preimplantation embryos, part II: Culture and transplantation. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, p 221 Jelinek R (1976) The view of an academic scientist on existing requirements for testing drugs and other agents on prenatal toxicity. In: van Julsingha EB, Tesh JM, Fara GM (eds) Advances in the detection of congenital malformations. European Teratological Society M. Roblin Printers, Chelmsford, Essex, p 153 Karkinen-J~i~iskel~iinen M, Sax6n L (1976) Advantages of organ culture techniques in teratology. In: Ebert JD, Marois M (eds) Tests of Teratogenicity in vitro. North Holland P, Amsterdam, p 275 Kittel J, Neubert D, Nau H (1978) Studies on isolated and reconstituted monooxygenases for incorporation into culture systems of embryonic tissue. In: Neubert D, Merker H-J, Nau H, Langman J (eds) Role of pharmacokinetics in prenatal and perinatal Toxicology. Thieme, Stuttgart, p 383 Lessm611mann U, Neubert D, Merker H-J (1975) Mammalian limb buds differentiating in vitro as a test system for the evaluation of embryotoxic effects. In: Neubert D, Merker H-J (eds) New approaches to the evaluation o f abnormal embryonic development. Thieme, Stuttgart, p 99

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Lessm611mann 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 Manson JM, Simons R (1979) In vitro metabolism of cyclophosphamide in limb bud culture. Teratology 19 : 149-158 Neubert D (1980) Prospektive in-vitro-Modelle als Ersatz ffir Langzeituntersuchungen. In: Zur Problematik von chronischen Toxizit~itsprtifungen, AMI-Berichte 1/1980. Dietrich Reimer Verlag, Berlin, p 277 Neubert D, Barrach H-J (1977a) Significance of in vitro techniques for the evaluation of embryotoxic effects. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, p 202 Neubert D, Barrach H-J (1977b) Techniques applicable to study morphogenetic differentiation of limb buds in organ culture. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, p 241 Neubert D, Barrach H-J (1980) Effect of environmental agents on embryonic development and the applicability of in vitro techniques for teratological testing, Toxicity testing of environmental agents: Current and future requirements, NATO Advanced Study Institute, Monte Carlo, 23-29, September 1979 (In press) Neubert D, Tapken S (1978) Some data on the induction of monooxygenases in fetal and neonatal mouse tissues. In: Neubert D, Merker H-J, Nau H, Langman J (eds) Role of pharmacokinetics in prenatal and perinatal toxicology. Thieme, Stuttgart, p 69 Neubert D, Merker H-J, Tapken S (1974a) Comparative studies on the prenatal development of mouse extremities in vitro and in organ culture. Naunyn-Schmiedeberg's Arch Pharmacol 286 : 251-270 Neubert D, Tapken S, Merker H-J (1974b) Induction of skeletal malformations in organ cultures of mammalian embryonic tissues, Naunyn-Schmiedeberg's Arch Pharmacol 286:271-282 Neubert D, Merker H-J, Barrach H-J, Lessm611mann U (1976) Biochemical and teratological aspects of mammalian limb bud development in vitro. In: Ebert JD, Marois M (eds) Tests of teratogenicity in vitro. North-Holland Publ Comp, Amsterdam, p 335 Neubert D, Lessm611mann U, Hinz N, DiUmann I, Fuchs G (1977) Interference of 6-mercaptopurine riboside, 6-methylmercaptopurine riboside and azathioprine in the morphogenetic differentiation of mouse extremities in vivo and in organ culture. Naunyn-Schmiedeberg's Arch Pharmacol 298 : 93-105 Neubert D, Hinz N, Baumann I, Barrach H-J (1978a) Attempt upon a quantitative evaluation of the degree of differentiation of or the degree of interference with development in organ culture. In: Neubert D, Merker H-J, Nau H, Langman J (eds) Role of pharmacokinetics in prenatal and perinatal toxicology. Thieme, Stuttgart, p 337 Neubert D, Tapken S, Baumann I (1978b) Influence of potential thalidomide metabolites and hydrolysis products on limb development in organ culture and on the activity of proline hydroxylase. - Further data on our hypothesis on the thalidomide embryopathy. In: Neubert D, Merker H-J, Nau H, Langman J (eds) Role of pharmacokinetics in prenatal and perinatal toxicology. Thieme, Stuttgart, p 355 Neubert D, Barrach H-J, Merker H-J (1980) Drug-induced damage to the embryo or fetus (Molecular and multilateral approach to prenatal toxicology). In: Grundmann E (ed) Current topics in pathology, Vol. "Drug-Induced Pathology". Springer, Heidelberg (in press) New DAT (1966) Development of rat embryos cultured in blood sera. J Reprod Fertil 12 : 509-524 Pennypacker JP, Wilk AL, Martin GR (1978) In vitro differentiation of mesenchyme cells into chondrocytes: Effect of teratogens in the presence or absence of drug-metabolizing liver preparations. In: Neubert D, Merker H-J, Nau H, Langman J (eds) Role of Pharmacokinetics in prenatal and perinatal toxicology. Thieme, Stuttgart, p 411 Spielmann H, Eibs H-G (1977) Preimplantation embryos. Part I: Laboratory equipment, preparation of media, sampling and handling of the embryos. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, p 210 Thesleff I (1977) In vitro development of oral tissues. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, p 252

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Trowell O A (1954) A modified technique for organ culture in vitro. Exp Cell Res 6 : 246- 248 Trowell O A (1959) The culture of mature organs in a synthetic medium. Exp Cell Res 16:118-147 Welsch F, Baumann I, Neubert D (1978) Effects of methyl-parathion and methyl-paraoxon on morphogenetic differentiation of mouse limb buds in organ culture. In: Neubert D, Merker H-J, Nau H, Langmann J (eds) Role of pharmacokinetics in prenatal a perinatal toxicology. Thieme, Stuttgart, p 351 Received May 5, 1980