Embryonic and fetal development: Fundamental reseatch

Reproducnve Toxwology. Vol 7, pp 155-164, 1993

0890-6238/93 $6 00 + 00 Copynght © 1993PergamonPress Ltd

Pnnted m the U S A All rights reserved

• Developmental Toxicity

EMBRYONIC A N D FETAL DEVELOPMENT: FUNDAMENTAL RESEARCH B. P. SCHMID,* P. H O N E G G E R , t a n d P. KUCERA'~ *Zyma SA, PrecllnlCal Development and Drug Safety, Nyon, Switzerland, tUmverslty of Lausanne, Institute of Physiology, Lausanne, Switzerland Abstract m Much progress has been made over the past decades in the development of in vitro techniques for the assessment of chemically induced effects in embryonic and fetal development. In vitro assays have originally been developed to provide information on the mechanism of action of normal development, and have hence more adequately been used in fundamental research. These assays had to undergo extensive modification to be used in developmental toxicity testing. The present paper focuses on the rat whole embryo culture system, but also reviews modifications that were undertaken for the in vitro chick embryo system and the aggregate cultures of fetal rat brain cells. Today these tests cannot replace the existing in vivo developmental toxicity tests. They can, however, be used to screen chemicals for further development or further testing. In addition, these in vitro tests provide valuable information on the mechanisms of developmental toxicity and help to understand the relevancy of findings for humans. In vitro systems, combined with selected in vivo testing and pharmacokinetic investigations in animals and humans, can thus provide essential information for human risk assessment. Key Words In vitro (techmques), chick e m b r y o cultures, rat e m b r y o cultures, fetal brain cell aggregation, alternatives, reproductive toxicity testmg, reproductive hazard, testing batteries

developmental toxicity, further modifications have been indispensable. The adaptation of original techniques to toxicologic investigations involves the testing of a certain number of adequately selected chemicals. On the other hand, further mechanistic studies are also important to be able to define an optimal protocol for the ultimate testing procedure. Irrespective of the nature of the test, such applications to toxicologic investigations can involve a variety of different steps, such as those presented in Figure 1. It is clear, of course, that each of the individual steps may involve more or less effort to satisfy the final needs. As indicated, it is also important that mechanistic studies have to be followed or paralleled by validation studies that can be carned out on small and/or large scales. These validation studies should, then again, be accompanied by mechanistic studies, making it possible to modify and adapt a given method in its broadest sense, e.g., making it applicable for the assessment of a wide range of chemicals. It is also important that those classes of compounds that provide different re-

INTRODUCTION Some twenty years ago, Moscona investigated and discovered the unique ability of dissociated fetal cells to reaggregate in culture, and to continue as spheroids the differentiation and growth process to a similar extent as the original tissues (1). The maintenance of whole rat embryo cultured in vitro during the major phase of development became reproduclbly possible, about 15 years ago, by the use of roller bottle techniques, particularly adapted gassing conditions, and the application of rat serum as the medium (2). The value of the chick embryo for the study of fundamental processes in developmental biology was discovered almost 40 years ago, and led subsequently to the whole chick embryo culture system similar to the one used for rat embryos (3). These three techniques were firstly developed for investigating mechanisms underlying physiologic developmental processes. For their application to A d d r e s s c o r r e s p o n d e n c e to Dr B. P Schmld, D e p a r t m e n t of P r e c h m c a l D e v e l o p m e n t & Drug Safety, CH-1260 N y o n , Switzerland 155

156

Reproductive Toxicology

Volume 7, Supplement 1, 1993

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Fig. 1. Schematic presentation of different phases of adaptauons and vahdatlon of m vitro techniques for reproductive toxicity testing The actlwt]es on the two discs presented here are complementary and can occur m parallel throughout the adaptation and vahdatlon process

sponses to the ones obtained from m VlVO systems are identified early, so that the system can be adapted accordingly, or in cases where this might not be possible, other systems can be developed (test battery approach) The present paper informs on and reviews some of our efforts undertaken to combine mechamstic studies for m vitro refinements with validation trmls using the rat whole embryo culture, the fetal rat brain cell cultures, and the in vitro whole chick embryo technique. TECHNICAL ASPECTS ON THE THREE IN VITRO SYSTEMS

The posttmplantation Rat Embryo Culture The experimental protocol that has been developed by New and his collaborators [for review, (2)] uses whole rat embryos explanted at day 9.5 of gestation into wals containing heat-inactwated male rat serum saturated with an O2-CO2 mixture. The vials are continuously rotated up to 48 hours at an incubation temperature of 37 °C. The medium is normally not renewed, but the oxygen concentration of the gas mixture is progressively changed during the incubation from 5 to 20%. At the end of the culture period, the size and vascularisation of the yolk sac, the crown-rump length, number of somltes, and morphologic abnormahtles are evaluated under a binocular microscope. A schematic outline for the culture technique is presented in Figure 2.

The Chtck Embryo In Vttro This method was modified from original technologies used in studies on the physiology and early chick embryogenesls (3-5). Today, the whole chick embryos are preincubated for 20 h (stage of the late gastrula), and then explanted into transparent silicone chambers where they develop under defined conditions for additional 3 days. The medium consists of a modified Tyrode solution mixed with hquid egg albumen (1:1 mixture ratio). The test compounds are directly added to the medium, and the chick embryos exposed to them up to 44 hours. The procedure is also schematically presented In Figure 3. As the early embryo is almost transparent, the survival, growth and morphogenesis of the skeletomotor, cardiovascular and nervous systems, and of the extraembryonlc membranes, can be observed directly under a binocular microscope Overall, 7 quantitative and 17 qualitative criteria are systemically used to score the development of each embryo (6). As for rat embryos the ontogenetlc stages of the chick embryos during the test correspond to the postconceptional weeks 2 to 5 in the human embryo, which is regarded as the most critical period in teratogenesls. In a previous intralaboratory validation, 8 compounds were tested in this system and found to produce, within 2 days, specific patterns of growth perturbatlons and anomalies. These effects were concentration-dependent, and repeated testing with

Embryomc and fetal cultures • B. P SCHMIDET AL

STRAIN: Han Wistar Rat

157

CULTURE CONDITIONS: According to Newts method Medium/Male Ret~

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d 9.5 - 11.5 post coitum; 2 Embryos/5ml Medium

ENDPOINT MEASUREMENTS: Morphology : 15 Features Differentiation : Morph. Scoring System. Somite Number Growth : Crown- Rump Length, Head Length Fig 2. Experimental scheme for the in vitro teratogenloty testing with the postlmplantatlon rat embryo culture (32, with permission).

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Fig. 3. Schematic presentation of the m vitro culture of chick embryos, and the relative endpomts that can be assessed in this system for investigations on normal or abnormal embryomc development.

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Volume 7, Supplement 1, 1993

nique, and have increased both the yield and the reproducibility of the cultures. Previous work has shown the usefulness of these cultures for studies in developmental toxicology (10,11). A schematic presentation of the test protocol is given in Figure 4 Generally, the cultures are treated continuously between day 5 and day 14. On day 14, cellular growth and maturation are monitored using several biochemical parameters, such as the activities of the neuron-specific enzymes choline acetyltransferase (CHAT), acetylchollne esterase (ACHE), glutamic acid decarboxylase (GAD); and the activity of the gha-speclfiC enzymes glutamine synthetase (GS) and 2' ,3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Furthermore, the release of intracellular lactate dehydrogenase, and DNA and protein content are used as an indicator for general cytotoxicity

the same chemicals, and evaluation by different persons and with eggs from different hens, yielded practically identical results (6). The experimental protocol described here was also used to present the investigations on a quantitative comparison of 12 compounds, between the chick and the rat embryos

The Aggregatmg Bram Cell Culture This technique is based on the fact that cells dissociated from embryonic tissues are able to reaggregate spontaneously, forming three-dimensional cell structures. Such cultures are able to mimic a series of morphogenetlc events occurring in VlVO, and to attain a high degree of cellular organization and differentiation (for review, 1,7,8). Several methodologic improvements and the use of a defined culture medium (9) have greatly simplified this tech-

275 fetal rat forebrains

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Embryomc and fetal cultures • B P SCHMIDET AL

Other than P450 metabolizing-dependent steps were also investigated. Bechter et al. (18), for example, were able to show that etretinate, an aromatic retmold, exerts its teratogemc activity mainly after metabolism by the carboxylic-ester hydrolase. Procarbazine, on the other hand, induced dysmorphogenesis only after the embryos were exposed to serum of procarbazine treated antmals; the S9-mlx alone did not mduce specific malformations (Figure 5). It is therefore recommended that hydrazmes are tested only after treatment of the whole animal. It could be demonstrated m m vitro investigations that the yolk sac has intrinsic metabohc capacities (19). In view of the special anatomic and physiologic role of the yolk sac in rodents, results obtained with the WEC system can thus also help to explain "rodent specific" responses (the yolk sac is absent In the majority of other in wtro techniques). As trypan blue has been shown to exert specific embryomc malformations via the yolk sac (20), azo dyes should therefore be subjected to the WEC system when being tested in vitro. The duration of the culture and treatment period was found to be an important issue during the process of modification of the WEC for screening purposes. Results showed that a 48-hour culture and exposure period is not only more sensitive, but also more relevant to the in VlVO ammal situation. Cadmium chloride, for example, did not induce malformations when mouse embryos were exposed to it for 24 h, but high incidences of non-closed neural tubes were observed after a 48 h period (Table 1). Also, in an interlaboratory validation study, we reported that 6-aminonlcotmamlde reduced specific

The first trial studies showed that neurotoxlc compounds elicited a distinct, concentration-dependent response pattern (10,1 I). In the present paper, an identical protocol was used to demonstrate the selective response of this system to treatment with ketoconazole and 6-aminonicotinamlde.

REFINEMENTS, VALIDATION, AND MECHANISTIC STUDIES Of the three described systems, the whole embryo culture (WEC) was the first system to undergo industrial applications. Initial studies were mostly based on existing data derived from in VlVO tests. They were, for example, undertaken to elucidate whether compounds (e.g., valproic acid) exert direct effects on the developing rodent embryo, and whether the types of malformations were comparable to those observed in vivo (12). Other in wtro investigations were targeted to identify whether effects m humans could possibly be "simulated" in wtro [e.g., growth retardation in connection with the fetal alcohol syndrome (13)]. Linked to this, different metabolites were tested in vitro to identify possible causatwe metabohtes. Many studies have successfully investigated the posslbihty of incorporating metabohzlng systems into the WEC. Techniques included mlcrosomal fractions and cofactors (14,15), adherence layers of hepatocytes (16), and hepatocyte suspension in coculture with embryos (17) Of the compounds known to require metabolic activation by P450 systems, all were also identified to cause reproducibly abnormal development m vitro.

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Reproductive Toxicology

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Volume 7, Supplement l, 1993

Table 1 Effects of cadmium chloride on mouse n e u r a l t u b e c l o s u r e m vitro Cadmmm chloride (× l0 6 M)

No of embryos treated

Embryos showing open neural tubes

Mean morphologlc score

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Mouse embryos were treated with different concentrations of cadmium chloride for 24 h (a) and 48 h (b) The numbers in parenthesis indicate the percentage of embryos with open neural tubes (35)

malformations after 48 h but not after 24 h of culture (17). These individual studies on the rat embryo m vitro contributed considerably to the understanding of the use of the WEC system for general screening purposes. The optimal culture medium should consist of pure (rat) serum, and the treatment and culture period should be 48 h. S9-mlx or other P450 metabolizing sources should be added if metabohzlng capacmes of a given compound are unknown. To reduce the number of embryos, we propose testing only for one subtoxlc concentration w~th a metabolizing source, and to compare these data with data from a given concentration containing the maternal compound only, to see whether there are modifications of the evaluated parameters. To assess the WEC's general use for screening purposes, its predictive value had then to be further evaluated. Individual scoring of control embryos across different laboratories (21), and the testing of specific classes of compounds (22) as well as chemicals of different origin were carried out (23,24). In these studies we were able to show that, despite the fact that the actual culture conditions differ between Individual laboratories, the assessment of quantitative and morphologlc parameters is reproducible (21). Classes of compounds with similar chemical structure (e.g., retmolds) gave similar types of malformations and resulted m rankmgs comparable to the in VlVO investigations (22). Compounds with different chemical structure or similar indication areas (e.g., antifungal), yielded different yet compound-specific types of malformations (25). The outcome (malformations/no-malformations) was generally in good accordance with what has been reported in the literature from In VlVO systems (22-24). Overall, the screening with the different chemi-

cals revealed that the WEC has a slight tendency to classify chemicals as so-called "false-positives," but shows a high sensitiwty for the identification of teratogenlc compounds The selection criteria for the poSltlVe and/or the negative responses were the concentration-dependent induction of specific malformations in the absence of an effect on overall growth (embryonic/yolk sac) or differentiation (somite number/yolk sac vascularazation) The window covered by the rat WEC is relatively small (2 days) in comparison to the dam's overall pregnancy stage (21 days), and the system still reqmres pregnant animals It would have been desirable to develop a system that does not rely on pregnant dams and that allows extended treatment periods. Unfortunately there is at present no in vitro system available that would combine these two advantages, and the fetal brain cell aggregation and the chick embryo culture in vitro had to be used in combination. First, a study was initiated, where 6 coded pairs of compounds were subjected to testing in the rat WEC and the chicken embryo in vitro. Figure 6 summarizes these data and shows that there was a good correlation concerning the ranking of compounds between the chicken and the rat embryos (27). Also, the types of malformations observed in the two species were comparable. These results suggest that the chicken embryo, cultured m vitro, represents a potentml alternative to the rat WEC, excluding the use of pregnant animals The chick embryo test in vitro may therefore obtain better acceptance than the chick in ovo embryo test, which was regarded as being too sensitive (28). The fetal brain cell aggregate cultures complementary results to the chick and the rat embryo. It could clearly distinguish between compounds affecting ghal cells or neurons (Figure 7). For example, a selective reduction of the neuron-speofic enzymes

Embryomc and fetal cultures • B P SCHMID ET AL

161

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C H I C K E M B R Y O IC5o log (tool/l) Fig 6. Comparison of the sensitivity and discrimination m the whole embryo culture and the in vitro chick embryo culture The IC50values for teratogenlclty were derived from Kucera et al. (27) The two test systems were able to detect two families of compounds according to the concentration-dependency of their effects (10 6 and 105-10 3 M), and to discriminate between the members of the same chemical class. As judged from the regression lines, the chick embryo was slightly more sensitive than the rat embryo Overall, however, concentrations to induce speofic malformations in the two systems are comparable Mol methoxyethanol, Mac: methoxyacetate, Caf caffeine, The. theophylline, Sad: sulfanilamide, Sdz: sulfadiazme, 4hp 4-hydroxypyndlne, Ana 6-amlnonlcotlnamide, Mnz metromdazole, Kzn: ketoconazole, Ro 1:Ro-1-5488 (all-trans retmolc acid), Ro 13: Ro-13-6307.

GAD and ChAT was found at relatively low concentrations of ketoconazole. In contrast, 6-aminonicotinamide proved to affect predominantly the glial parameters GS and CNP, and to a somewhat lesser extent also the GABAerglc marker GAD. Also, the results indicate that more specific parameters can be concentration-dependently affected in the absence of overall effects on total DNA or protein content. This system not only covers a different window of development, but also allows assessment of biochemical changes of fetal neuronal and glial differentiation processes. No comparable in vivo data are yet available in this domain. Amongst the in vitro cell culture systems used [for review see ECETOC Monograph (29)], assays with estabhshed cell lines have also been modified and investigated for screening of reproductive toxicants. The results on their validation indicate, however, that they are not highly predictive. This is not

unexpected as these tests take into consideration only a single endpolnt. In teratology, for example, many causes can directly or mdirectly lead to dysmorphogenests. The assumption that a battery of single tests on cell lines could have a better predlctweness could not be confirmed (30). The techmques presented in this paper make it possible to detect lethality, growth retardation, and Interferences with differentiation. They thus provide the most comprehensive assessment of embryotoxicity and teratogenicity. This does not mean that other in vitro tests could not be predictive of embryotoxic effects, but such tests may need to undergo further refinements for further testing of chemicals and more extensive validation, as, for example, proposed by Balls et al. (31). In the future, it would be desirable that in vitro tests, as presented here, are used In the development of new compounds in parallel with selected in vivo testing,

162

Reproductive Toxicology

A

Volume 7, Supplement 1, 1993

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(~tM) Ftg. 7 Examples of selective effects m aggregating rat brain cell cultures Aggregate cultures of the fetal rat telencephalon were treated from day 5 to day 14 with e~ther 6-ammomcotmamlde (A) or ketoconazole (B) Cell type-specific enzyme actwmes, total protein, and DNA were determined at day 14 The values are expressed as percentages of the total actwmes per culture flask measured m control cultures treated with the solvent only (e g , DMSO, final concentratmn of 0 5%) GS. glutamme synthetase, CNP 2',3'-cychc nucleoude 3'-phosphohydrolase, ChAT chohne acetyltransferase, ACHE. acetylchohnesterase, GAD. glutam~c acid decarboxylase, Prot. total protein content, DNA total DNA content

to obtain m o r e insight m the predictive value of such tests. T o d a y we can state that fundamental research in d e v e l o p m e n t a l biology has greatly contributed to d e v e l o p m e n t a l toxlctty testing. N o t only has it provided the basis for the accessibility for m vitro

screening tests but ~t has also helped to gain better understanding in the process of risk a s s e s s m e n t for humans (Figure 8) Acknowledgment- This work was supported by grants 401711078 from the Swiss National Research Foundation and 15-G29/ 88 from Foundation Fmanz-Pool 3R

Embryonic and fetal cultures

•

B P SCHMID ET AL

163

BIOkOGICAL

In Vivo

'I

t

ANIMALS

aml PhamHee-

HUMANS

F~g. 8. S c h e m a t i c flow-chart for t h e use o f in vitro t e s t s in t h e p r o c e d u r e for risk a s s e s s m e n t o f t e r a t o g e m c i t y (34, w~th permission).

REFERENCES 1 Moscona AA Patterns and mechanisms ofttssue reconstructlon from dlssoctated cells In Rudnlck, ed Developing cell systems and their control New York Ronald Press, 1960 45-70. 2 New DAT Whole embryo culture and the study of mammahan embryos dunng organogenesls B]ol Rev 1978, 53"81-122 3 Kucera P, Monnet-Tschudl F Early functional dlfferentlatmn m the chick embryonic d~sc interaction between me-

chanlcal actwlty and extraceUular matrix J Cell Scl 1987,$8 415-27 4 Kucera P Physiological approach to the early embryogenes~s In Marthy H-J, ed Expenmental embryology m aquatlc plant and ammal orgamsms NATO ASI series, vol A 195 New York. Plenum, 1990"377-88 5 Kucera P, Raddatz E Environmental pollution and embryomc development relevance of standardized toxteologlcal tests In. Marthy H-J, ed Expenmental embryology m aquatic plant and ammal organisms. NATO ASI senes, vol A 195 New York" Plenum, 1990.389-94

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Reproductive Toxacology

6 KuceraP, BurnandM-B Routme teratogemcgy test that uses chick embryos in v,tro Teratogenesls Carclnog Mutagen 1987,7 427-47 7 HoneggerP, RachelsonE Blochemlcaldffferentlatlonofmechanlcally dissociated mammalian brain ,n aggregating cell culture Brain Res 1976,109 335-54 8 Honegger P Biochemical differentiation in serum-free aggregating brain cell cultures In Bottenstem JE, Sato G, eds Cell culture m the neurosclences New York Plenum 1985 223 -43 9 Honegger P, Lenolr D, Favrod P Growth and differentiation of aggregating fetal brain cells m a serum-free defined medram Nature 1979,282 305-8 10 Honegger P, Werffeh P Use of aggregating cell cultures for toxicological studies Expenentia 1988,44 817-23 11 Honegger P, Schdter B Serum-free aggregate cultures of fetal rat brain and liver cells methodology and some practical apphcatlons m neurotoxlcology In Zblnden G, ed The brain m bits and pieces In vitro techniques in neuroblology, neuropharmacology and neurotoxicology Zolhkon, Switzerland MTC Verlag 1992 51-79 12 Kao J, Brown NA, Gouldlng EH, Fabro S Teratogemcity of valprolc acid in vivo and in vitro investigations Teratogenesis Carclnog Mutagen 1981,1 367-82 13 Brown NA, Gouldlng EH, Fabro S Ethanol embryotoxlclty direct effects on mammalian embryos in vitro Science 1979,206 573-5 14 Fantel AG, Greenaway JC, Juchau MR, Shepard TH Teratogemc bloactwatlon of cyclophospham,de in vitro Life Sciences 1979,25 67-72 15 Kltchm KT, Sanyal MK, Schmld BP A coupled mlcrosomalactivating/embryo culture system, toxicity of reduced b-mcotlnamlde adenine dlnucleotlde phosphate (NADPH) Blochem Pharmacol 1981,30 985-92 16 Oglesby LA, Ebron MT, Beyer BE, Carver BD, Kavlock RJ Cocultures of rat embryos and hepatocytes m vitro detection of a proteratogen Teratogenesis Carcmog Mutagen 1986,6 129-38 17 Plersma AH, Attenon J, Bechter R, et al Rodent postlmplantatxon embryo culture lnterlaboratory vahdation as a screenmg test for teratogemc compounds [Abstract] Reprod Toxlcol 1991,5 267 18 Bechter R, Terlouw GDC, Tsuchlya M, Tsuch~ya T, Klstler A Teratogenlcgy of arotmoids (retmolds) in the whole rat embryo culture Arch TOXlCOl 1992,66 193-7 19 Terlouw GDC, Bechter R Comparison of the metabolic activity of yolk sac tissue m the whole embryo and isolated yolk sac culture Reprod Toxicol 1992,6 85-92 20 Lloyd JB, Beck F Teratogenesls In Dingle JT, Fedd HB,

Volume 7, Supplement 1, 1993

21 22 23 24 25 26 27

28 29

30

31 32 33 34 35

eds Lysosomes m biology and pathology Amsterdam North-Holland, 1969 333-449 Van Maele-Fabry G, P~card JJ, Attenon P, et al Interlaboratory evaluation of three culture media for postimplantation rodent embryos Reprod Toxlcol 1991,5 417-26 Clcurel L, Schmld BP Post,mplantation embryo culture for the assessment of the teratogemc potential and potency of compounds Expenentia 1988,44 833-40 Schmld BP Teratogemclty testing of new drugs with the postlmplantatlon embryo culture system Concepts Toxicol 1985,3 46-57 Schmid BP Xenoblotlc influences on embryonic differentiation, growth and morphology in vitro Xenoblotlca 1985, 15 719-26 Bechter R, Schm,d B, Mayer FK Teratogemc potenUal of antlmycotlc drugs evaluated m the whole embryo culture system Food Chem Toxlcol 1986,24 641-2 C,curel L, Schmid BP Post,mplantatlon embryo culture validation with selected compounds for teratogenlclty testing XenobtoUca 1988,6 617-24 Kucera P, Cano E, Honegger P, Schdter B, Z01stra JA, Schmid B Vahdation ofwhole chick embryo cultures, whole rat embryo cultures and aggregating embryonic cell cultures using six pairs of coded compounds Toxicol In Vitro [In press] World Health Orgamzat~on Prmciplesforthetestmgofdrugs for teratogemcRy Technical Report Series, 364 WHO, 1967 European Chemical Industry Toxicology Center (ECETOC) Alternat,ve approaches for the assessment of reproductive toxacaty (with emphasis on embryotoxicRy/teratogemcaty) Monograph No 12 Brussels ECETOC, 1989 NTP (US National Toxicity Program) Evaluation of two in vatro teratology test systems Final report National Toxicology Program National Institute of Environmental Health Scaences NTP, Research Triangle Park, NC 1986 86-372 Balls M, Blaauboer B, Brustck D, et al Report and recommendations of the CAAT/ERGATT workshop on the vahdataon of toxacaty test procedures ATLA 1990,18 313-37 Schmld BP Action sates of known in wvo teratogens m extracorporeally exposed rat embryos Concepts Toxicol 1985, 3 74-85 Schmad BP, Tnppmacher A, Blanchl A Teratogemclty reduced m cultured rat embryos by the serum of procarbazme treated rats Toxicology 1982,25 53-60 Zljlstra J Reproductive toxicology In Balls M, Bridges J, Southee J, eds Animals and alternatave in toxicology New York Macmillan Scientific & Medical, 1992 191-5 Schmad BP, Kao J, Gouldmg E Evidence for reopening of the cranial neural tube in mouse embryos treated with cadmium chloride Expenentia 1983.41 271-2