Human Reproduction vol.6 no.l pp. 144-147, 1991
REVIEW
Whole embryo culture and teratogenesis
Boots Pharmaceuticals Research Department, Nottingham, UK
Whole embryo culture (WEC) is under evaluation in numerous laboratories to determine the role it should play in teratogen testing. There is general agreement that its role is to complement the Segment II Teratology studies required by regulatory authorities. Currently it is used as a 'screen' in order to minimize the number of compounds used in animal tests and also to determine the mechanism(s) of teratogenesis. This brief review focuses on the study of retinoid teratogenicity in WEC in order to illustrate these points. Key words: teratogenesis/embryo culture/retinoids Ethical and legal considerations effectively preclude the use of human postimplantation embryos in the study of teratogenesis. Direct evidence of the adverse effects of a pharmaceutical agent on the human fetus therefore comes only from inadvertent exposure to known teratogens, such as retinoids, or because the drug is thought to be safe in this respect, for example thalidomide. Epidemiological studies also provide useful information, as in the case of the Fetal Alcohol Syndrome, but cannot always establish a causal relationship. It is not surprising therefore that the list of proven human teratogens contains only ~20 compounds (see Table I in Smith et al., 1983) and that these account for only 2—3% of developmental defects in man. Although 65—70% of the remaining defects have no known cause, it would not be surprising if existing (and future) pharmaceuticals constituted (or will constitute) some of this group. In order to determine whether new chemical entities are teratogenic, pharmaceutical companies are required by various regulatory agencies worldwide to use animal models. In practice, teratogenicity tests are performed by dosing pregnant rats and rabbits during mid-gestation followed by examination of the fetuses at caesarean section (this is known as the Segment II or Teratology Study). Recently these in-vivo studies have been complemented by a wide range of in-vitro studies (Freeman and Brown, 1987; Flint, 1987) either as a 'screen' to prioritize a number of compounds before in-vivo studies, or to determine the mechanism of a positive result in an in-vivo teratogenicity study. The culture of whole rat embryos (WEC) is now widely used in this respect as well as for empirical research in teratogenesis in many academic laboratories (Cockroft and Steele. 1987). In common with other in-vitro techniques, it has ethical, financial and scientific advantages over in-vivo tests. Early teratological studies using WEC adopted the simple protocol of adding the (potential) teratogen directly to the culture 144
medium. A positive result was thought to indicate that it was the parent compound, and not a metabolite(s), which was teratogenic and that it was a direct effect, i.e. not mediated via the mother. There have been far too many of these studies to review comprehensively. Since most of them have used proven teratogens it is difficult to assess the value of WEC as a screen since, ideally, it should result in no (or only a few) 'false positive' results with non-teratogens. This situation is being rectified following the publication of a list of candidate compounds for in-vitro teratogenesis test evaluation (Smith et al., 1983). Such work is essential for the validation of WEC, a complicated process which has only just begun. The questions which must be answered before its role in screening can be finally determined are summarized in Table I. With regard to the ability of the embryo (and/or its membranes) to metabolize xenobiotics, it was generally assumed that compounds added directly to the culture medium without a metabolizing system did not undergo biotransformation. However, recent studies have shown that rat embryonic tissue is capable of xenobiotic metabolism (Juchau, 1987; Flint and Brown, 1987). The significance of these findings for teratology studies in WEC has yet to be firmly established. Nevertheless, it is possible to take into account the effects of metabolism, and
Table I. Questions to be answered during the validation of an in-vitro teratogenicity test (verbatim from Kimmel el al.. 1982) 1
What can be considered an acceptable measure of teratogenic potential, i.e. quantifiable endpoint. for in-vitro test systems? How closely should these endpoints be related to embryogenic events in the 'intact mammalian system"? If a test with an apparent 'developmentally irrelevant' endpoint proves to respond well to validation criteria, should it be considered for inclusion in a test battery for regulatory purposes?
2
To be accepted as part of a screening process, in-vitro systems will have to be validated for their ability to predict the teratogenic potential of an agent. What are the necessary criteria for validation9 How will the choice of compounds used in the validation be made, by structure, biologic activity, environmental use. etc ? How many compounds should be used for validation and what percentage of false-negatives and false-positives will be acceptable'7 On what basis will dosing be defined and how will it relate to the in-vivo situation?
3
How could in-vitro systems be applied to screening previously untested individual compounds or mixtures of compounds with unknown teratogenic potential? Consideration should be given to the specific experimental design, to the choice and administration of doses, and to the distinction of general toxicity from specific endpoints of developmental toxicity Could these systems be used to prioritize the further testing of unknown compounds9
4
Is there a requirement that in-vitro tests mimic the metabolic capacity of the in-vivo system? © Oxford University Press
Downloaded from https://academic.oup.com/humrep/article-abstract/6/1/144/875886 by University of West London user on 17 October 2018
C.E.Steele
Whole embryo culture and teratogenesis
- V '•
VA
' /
Fig. 1. Rat embryo explanted at 9 '/i days of gestation and cultured for 48 h in control serum. Bar = 1 mm. o, otocyst; m, first (mandibular) pharyngeal arch.
—m
i.
i/ -
•
&
.
-
•
Fig. 2. Rat embryos explanted at 9'/2 days of gestation and cultured for 48 h in serum containing 0.5 ^g/ml retinol (left) or retinoic acid (right). The position of the otocyst, level with the mandibular arch, is abnormal. At the points marked by an arrow the neural tube has failed to close from that point forward. Magnification and abbreviations as for Figure 1.
145
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**V
pharmacokinetics in general, either by the addition of microsomes and co-factors to the culture vessel (Fantel et al., 1979) or by using serum from a rat or human dosed with the chemical under investigation as the culture medium (Klein et al., 1980; Steele etal., 1974). Finally, less commonly used protocols include embryonic exposure in utero followed by explantation and culture (Steele et al., 1983), or removal and farther culture of an organ rudiment in order to prolong development in vitro (Kochhar, 1981). It may also be possible to inject materials directly into the viteUine vessels (Beck etal., 1987). The great majority of studies involve explantation of rat embryos at the headfold stage (9!/2 days gestation) and culture for 48 h by which time the embryo should have 25 — 30 somites. This is a period of rapid growth and differentiation and is often most sensitive to teratogenic insult. Relatively small differences in developmental stage at explantation could result in fairly large differences by the end of the culture period. It is therefore important to describe clearly the explanted embryos and perhaps give some indication of what proportion of the embryos are discarded. In one laboratory it is standard practice to reject — 15% of explanted embryos in order that control and treated groups all have embryos of the same very specific developmental stage. This is probably unneccessarily extravagant for most teratological studies. There are some studies which do not use the 9 Vi — 11 Vi day period. For example, in a study of the effects of localized administration of tunicamycin on the induction of cleft lip, Eto
C.E.Steele
Table II. Comparison of acidic p/fa values of ten retinoids with lowest toxic concentration in embryo culture Retinoid 13-c/.y-retinoic acid all-/ra«5-retinoic acid etretin
concentration (/ig/ml)
pKa 3.S ) 4 5.2 10 15 15 22 22
A'-butylretinamide etretinate
22 i
* ' 50
> 100
et al. (1981) cultured fetuses explanted late on day 11 to the 45 somite stage. Precise application of the drug was facilitated by means of a sterile human eyelash. At the other end of the scale, IVi day 'egg-cylinders' had to be used to study the role of vitamin E in the prevention of gestation-resorption since this is the latest stage at which vitamin E can be administered and development still proceed normally. These experiments showed that it is probably the antioxidant properties of vitamin E which are important in preventing embryonic death (Steele et al., 1974). Because abnormal development is often the result of an interaction between environmental and genetic factors, attempts have been made to culture mouse embryos, more being known of the genetics of this species than that of the rat. Unfortunately, this has met with limited, though not unpromising, success (Sadler and New, 1981) and the rat therefore remains the species of choice for teratogenicity studies in WEC. Whatever age of embryo or species is used it is important to have appropriate endpoints as a measure of toxicity, whether this be teratogenicity and/or embryotoxicity. In this respect a morphological scoring system (Brown and Fabro, 1981) may be appropriate as well as detailed examination of the embryos (Cockcroft and New, 1978). WEC enables precise control of factors such as developmental stage, duration and timing of exposure to parent compound (and/or metabolites) and the ability to monitor continuously the formation of an abnormality or the condition of an embryo, e.g. its heartrate (Robkin et al., 1974). This can be used to determine the aetiology of dysmorphogenesis where two or more factors are implicated, as in the case of maternal diabetes (Sadler and Horton, 1983) or the Fetal Alcohol Syndrome (Becker al., 1984), as well as to establish quantitative structure/activity relationships of a series of related compounds, e.g. carboxylic acids (Brown, 1987) and retinoids (see below). In addition, these can be complemented by distribution studies of the medium and embryonic/placental tissues (Bechter and Taccard, 1987). It is with the retinoids, natural and synthetic analogues of vitamin A, that there is a direct link between WEC and human reproduction since not only are these the latest examples of therapeutic agents (for the treatment of a wide range of dermatoses) proven to be teratogenic in man (Rosa et al., 1986) but they have also been extensively used in WEC and in rodent teratogenicity studies in-vivo. Briefly, the embryo culture experiments have shown the following facts. 146
Most retinoids have a direct, teratogenic effect on embryonic development in vitro (Figures 1 and 2) at relatively low concentrations (Morriss and Steele, 1977). An important exception to this is etretinate (marketed as 'Tigason') which must be metabolised to etretin to become teratogenic. This retinoid was inactive when added directly to the culture medium without a metabolising system (Steele et al., 1987). (ii) pA"a value (Table II) is clearly an important property of the retinoid molecule in respect of its teratogenic activity (Steele et al., 1987). In contrast, there have been conflicting results concerning cisltrans isomerization. Initial studies in mice (Goulding and Pratt, 1986) and rats (Steele et al., 1987) suggested that there is little difference between cis and trans isomers in their teratogenic potency. A further study in rats (Klug et al., 1989), however, showed all-rra/w retinoic acid to be more potent than 13-c/s retinoic acid. Although there is no obvious explanation for these different results, it is possible, as Klug et al. (1989) have proposed, that the effects of \3-cis retinoic acid added to the culture medium are the result of isomerization to a\\-trans retinoic acid, (iii) Retinoids can be detected in human serum using WEC (Steele et al., 1982). (iv) There is good correlation between rat embryo studies, in vitro and in vivo (Steele et al., 1983), and clinical malformations (Turton et al., 1989). In conclusion, the growing importance of WEC led to an international symposium devoted entirely to this technique, held at the Catholic University of Louvain, Belgium in April 1990. It became apparent that there was no clear consensus as to the precise role of WEC in teratogen testing. The most sensible approach would probably be to remain flexible and pragmatic in deciding when to use WEC and not try to establish rules and terminology prematurely. It should be remembered that in-vitro teratogenicity tests are not required, though they may be accepted, by governmental or regulatory authorities (such as the Food and Drug Administration in the USA and the Committee on the Safety of Medicines in the United Kingdom) and that they do not replace, but rather complement, the definitive Segment II teratology studies in vivo. It may, or may not, be significant that WEC has recently been used to replace preliminary reprotoxicity studies required to establish the doses for the Segment II studies (J.Tesh, personal communication). There is, however, no doubt concerning the value of WEC in mechanistic studies in teratogenesis.
References Bechter.R. and Taccard,G. (1987) Medium and tissue levels of Acyclovir and Etretinate in the rat whole embryo culture system. In Nau.H. and Scott,W.J. Jr, (eds), Pharmacokinetics in Teratogenesis Vol. II. CRC Press, Boca Raton, p. 197. Beck,F., Huxham.I.M. and Gulamhasein.A.P. (1984) In Porter,R.. O'Connor,M. and Whelan,J. (eds), Mechanisms of Alcohol Damage In Utero. Pitman. London, p.218 (Ciba Foundation Symposium 105). Beck,F., Mensah-Brown.E. and Pratten,M.K. (1987) Development of a method for assessing the acute toxicity of chemicals on early postimplantation embryos. Arch. Toxicol.. Suppl. 11. 155-158. Brown,N.A. (1987) Teratogenicity of carboxylic acids: distribution
Downloaded from https://academic.oup.com/humrep/article-abstract/6/1/144/875886 by University of West London user on 17 October 2018
JV-(4-hydroxyphenyl)retinamide A/-tetrazol-5-ylretinamide /V-(2-hydroxyethyl)retinamide /V-ethylretinamide 13-m-yV-ethylretinamide
1 )
(i)
Whole embryo culture and teratogenesis compounds for in vitro teratogenesis test evaluation. Teratogen. Carcinogen. Mutagen., 3, 461—480. Steele,C.E., Jeffery,E.H. and Diplock,A.T. (1974) The effect of vitamin E and synthetic antioxidants on the growth in vitro of explanted rat embryos. J. Reprod. Fertil., 38, 115-123. Steele,C.E., Plenefisch,J.D. and Klein,N.W. (1982) Abnormal development of culture rat embryos in rat and human sera prepared after vitamin A ingestion. Experientia, 38, 1237—1239. Steele,C.E., Marlow,R., TurtonJ.A. and Hicks.R.M. (1987) In-vitro teratogenicity of retinoids. Br. J. Exp. Pathol., 68, 215—223. Steele,C.E., Trasler,D.G. and New,D.A.T. (1983) An in vivo/in vitro evaluation of the teratogenic action of excess vitamin A. Teratology, 28, 209-214. Turton,J.A., Steele,C.E., Haselden,J.N. and Hicks,R.M. (1989) Clinical and experimental teratogenesis of retinoids. In Shroot.B. and Schaefer,H. (eds), Pharmacology and the Skin. Karger,Basel, p. 120. Received on July 2, 1990; accepted on September 18, 1990
147
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studies in whole embryo culture. In Nau,H. and Scott,W.J. Jr, (eds), Pharmacokinetics in Teratogenesis Vol. 11, CRC Press, Boca Raton, p.153. Brown,N.A. and Fabro,S. (1981) Quantitation of rat embryonic development in vitro: a morphological scoring system. Teratology, 24, 65-78. Cockroft,D.L. and New,D.A.T. (1978) Abnormalities induced in cultured rat embryos by hyperthermia. Teratology, 17, 277—284. Cockroft.D.L. and Steele,C.E. (1987) Postimplantation embryo culture and its application to problems in teratology. In Atterwill,C.K. and Steele,C.E. (eds), In Vitro Methods in Toxicology. Cambridge University Press, Cambridge, p.365. Eto.K.. Figueroa.A., Tamura,G. and Pratt,R.M. (1981) Induction of cleft lip in cultured rat embryos by localized administration of tunicamycin. J. Embryol. Exp. Morphol., 64, 1—9. Fantel,A.G., Greenaway,J.C, Juchau,M.R. and Shepard,T.H. (1979) Teratogenic bioactivation of cyclophosphamide in vitro. Life Sci., 25, 67-72. Flint,O.P. (1987) An in vitro test for teratogens using cultures of rat embryo cells. In Atterwill.C.K. and Steele,C.E. (eds), In Vitro Methods in Toxicology. Cambridge University Press, Cambridge, p.339. Flint,O.P. and Brown,L.P. (1987) Metabolism of teratogens by mammalian embryos: an in vitro investigation. In Nau.H. and Scott,W.J., Jr (eds), Pharmacokinetics in Teratogenesis Vol II. CRC Press, Boca Raton, FL, p. 133. Freeman,S.J. and Brown,N.A. (1987) Sub-mammalian and subvertebrate models in teratogenicity screening. In Atterwill,C.K. and Steele,C.E. (eds), In Vitro Methods in Toxicology. Cambridge University Press, Cambridge, p. 391. Goulding,E.H. and Pratt.R.M. (1986) Isotretinoin teratogenicity in mouse whole-embryo culture. J. Craniofac. Genet. Dev. BioL, 6, 99—112. Juchau,M.R. (1987) Whole embryo culture: preinduction in vivo and metabolising activity in vitro. In Nau,H. and Scott,W.J., Jr (eds), Pharmacokinetics in Teratogenesis Vol II, CRC Press, Boca Raton, p. 121. Kimmel,G.L., Smith,K., Kochhar,D.M. and Pratt,R.M. (1982) Overview of in vitro teratogenicity testing: Aspects of validation and application to screening. Teratogen. Carcinogen. Mutagen., 2. 221-229. Klein,N.W., Vogler,M.A., Chatot.C.L. and Pierro.L.J. (1980) The use of cultured rat embryos to evaluate the teratogenic activity of serum: cadmium and cyclophosphamide. Teratology, 21, 199—208. Klug,S., Creech Kraft,J., Wildi,E., Merker,H.-J., Persaud,T.V.N., Nau.H. and Neubert,D. (1989) Influence of 13-cw and a\\-trans retinoic acid on rat embryonic development in vitro : correlation with isomerisation and drug transfer to the embryo. Arch. Toxicol., 63, 185-192. Kochhar,D.M. (1981) Embryo explants and organ cultures in screening of chemicals for teratogenic effects. In Kimmel,C.A. and BuelkeSam,J. (eds), Developmental Toxicology. Raven Press, New York, p.3O3. Morriss.G.M. and Steele,C.E. (1977) Comparison of the effects of retinol and retinoic acid on post-implantation rat embryos in vitro. Teratology, 15, 109-120. Robkin.M., Shephard,T.H. and Baum,D. (1974) Autonomic drug effects on the heart rate of early rat embryos. Teratology, 9, 35-44. Rosa.F.W., Wilk,A.L. and Kelsey,F.O. (1986) Teratogen update: vitamin A congeners. Teratology, 33, 355-364. Sadler.T.W. and Horton,W.E. (1983) Effects of maternal diabetes on early embryogenesis. The role of insulin and insulin therapy. Diabetes, 32, 1070-1074. Sadler,T.W. and New,D.A.T. (1981) Culture of mouse embryos during neurulation. J. Embryol. Exp. Morphol., 66, 109-116. Smith, M.K., Kimmel,G.L., Kochhar.D.M., Shephard.T.H., Spielberg,S.P. and Wilson,J.G. (1983) A selection of candidate
REVIEW
Whole embryo culture and teratogenesis
Boots Pharmaceuticals Research Department, Nottingham, UK
Whole embryo culture (WEC) is under evaluation in numerous laboratories to determine the role it should play in teratogen testing. There is general agreement that its role is to complement the Segment II Teratology studies required by regulatory authorities. Currently it is used as a 'screen' in order to minimize the number of compounds used in animal tests and also to determine the mechanism(s) of teratogenesis. This brief review focuses on the study of retinoid teratogenicity in WEC in order to illustrate these points. Key words: teratogenesis/embryo culture/retinoids Ethical and legal considerations effectively preclude the use of human postimplantation embryos in the study of teratogenesis. Direct evidence of the adverse effects of a pharmaceutical agent on the human fetus therefore comes only from inadvertent exposure to known teratogens, such as retinoids, or because the drug is thought to be safe in this respect, for example thalidomide. Epidemiological studies also provide useful information, as in the case of the Fetal Alcohol Syndrome, but cannot always establish a causal relationship. It is not surprising therefore that the list of proven human teratogens contains only ~20 compounds (see Table I in Smith et al., 1983) and that these account for only 2—3% of developmental defects in man. Although 65—70% of the remaining defects have no known cause, it would not be surprising if existing (and future) pharmaceuticals constituted (or will constitute) some of this group. In order to determine whether new chemical entities are teratogenic, pharmaceutical companies are required by various regulatory agencies worldwide to use animal models. In practice, teratogenicity tests are performed by dosing pregnant rats and rabbits during mid-gestation followed by examination of the fetuses at caesarean section (this is known as the Segment II or Teratology Study). Recently these in-vivo studies have been complemented by a wide range of in-vitro studies (Freeman and Brown, 1987; Flint, 1987) either as a 'screen' to prioritize a number of compounds before in-vivo studies, or to determine the mechanism of a positive result in an in-vivo teratogenicity study. The culture of whole rat embryos (WEC) is now widely used in this respect as well as for empirical research in teratogenesis in many academic laboratories (Cockroft and Steele. 1987). In common with other in-vitro techniques, it has ethical, financial and scientific advantages over in-vivo tests. Early teratological studies using WEC adopted the simple protocol of adding the (potential) teratogen directly to the culture 144
medium. A positive result was thought to indicate that it was the parent compound, and not a metabolite(s), which was teratogenic and that it was a direct effect, i.e. not mediated via the mother. There have been far too many of these studies to review comprehensively. Since most of them have used proven teratogens it is difficult to assess the value of WEC as a screen since, ideally, it should result in no (or only a few) 'false positive' results with non-teratogens. This situation is being rectified following the publication of a list of candidate compounds for in-vitro teratogenesis test evaluation (Smith et al., 1983). Such work is essential for the validation of WEC, a complicated process which has only just begun. The questions which must be answered before its role in screening can be finally determined are summarized in Table I. With regard to the ability of the embryo (and/or its membranes) to metabolize xenobiotics, it was generally assumed that compounds added directly to the culture medium without a metabolizing system did not undergo biotransformation. However, recent studies have shown that rat embryonic tissue is capable of xenobiotic metabolism (Juchau, 1987; Flint and Brown, 1987). The significance of these findings for teratology studies in WEC has yet to be firmly established. Nevertheless, it is possible to take into account the effects of metabolism, and
Table I. Questions to be answered during the validation of an in-vitro teratogenicity test (verbatim from Kimmel el al.. 1982) 1
What can be considered an acceptable measure of teratogenic potential, i.e. quantifiable endpoint. for in-vitro test systems? How closely should these endpoints be related to embryogenic events in the 'intact mammalian system"? If a test with an apparent 'developmentally irrelevant' endpoint proves to respond well to validation criteria, should it be considered for inclusion in a test battery for regulatory purposes?
2
To be accepted as part of a screening process, in-vitro systems will have to be validated for their ability to predict the teratogenic potential of an agent. What are the necessary criteria for validation9 How will the choice of compounds used in the validation be made, by structure, biologic activity, environmental use. etc ? How many compounds should be used for validation and what percentage of false-negatives and false-positives will be acceptable'7 On what basis will dosing be defined and how will it relate to the in-vivo situation?
3
How could in-vitro systems be applied to screening previously untested individual compounds or mixtures of compounds with unknown teratogenic potential? Consideration should be given to the specific experimental design, to the choice and administration of doses, and to the distinction of general toxicity from specific endpoints of developmental toxicity Could these systems be used to prioritize the further testing of unknown compounds9
4
Is there a requirement that in-vitro tests mimic the metabolic capacity of the in-vivo system? © Oxford University Press
Downloaded from https://academic.oup.com/humrep/article-abstract/6/1/144/875886 by University of West London user on 17 October 2018
C.E.Steele
Whole embryo culture and teratogenesis
- V '•
VA
' /
Fig. 1. Rat embryo explanted at 9 '/i days of gestation and cultured for 48 h in control serum. Bar = 1 mm. o, otocyst; m, first (mandibular) pharyngeal arch.
—m
i.
i/ -
•
&
.
-
•
Fig. 2. Rat embryos explanted at 9'/2 days of gestation and cultured for 48 h in serum containing 0.5 ^g/ml retinol (left) or retinoic acid (right). The position of the otocyst, level with the mandibular arch, is abnormal. At the points marked by an arrow the neural tube has failed to close from that point forward. Magnification and abbreviations as for Figure 1.
145
Downloaded from https://academic.oup.com/humrep/article-abstract/6/1/144/875886 by University of West London user on 17 October 2018
**V
pharmacokinetics in general, either by the addition of microsomes and co-factors to the culture vessel (Fantel et al., 1979) or by using serum from a rat or human dosed with the chemical under investigation as the culture medium (Klein et al., 1980; Steele etal., 1974). Finally, less commonly used protocols include embryonic exposure in utero followed by explantation and culture (Steele et al., 1983), or removal and farther culture of an organ rudiment in order to prolong development in vitro (Kochhar, 1981). It may also be possible to inject materials directly into the viteUine vessels (Beck etal., 1987). The great majority of studies involve explantation of rat embryos at the headfold stage (9!/2 days gestation) and culture for 48 h by which time the embryo should have 25 — 30 somites. This is a period of rapid growth and differentiation and is often most sensitive to teratogenic insult. Relatively small differences in developmental stage at explantation could result in fairly large differences by the end of the culture period. It is therefore important to describe clearly the explanted embryos and perhaps give some indication of what proportion of the embryos are discarded. In one laboratory it is standard practice to reject — 15% of explanted embryos in order that control and treated groups all have embryos of the same very specific developmental stage. This is probably unneccessarily extravagant for most teratological studies. There are some studies which do not use the 9 Vi — 11 Vi day period. For example, in a study of the effects of localized administration of tunicamycin on the induction of cleft lip, Eto
C.E.Steele
Table II. Comparison of acidic p/fa values of ten retinoids with lowest toxic concentration in embryo culture Retinoid 13-c/.y-retinoic acid all-/ra«5-retinoic acid etretin
concentration (/ig/ml)
pKa 3.S ) 4 5.2 10 15 15 22 22
A'-butylretinamide etretinate
22 i
* ' 50
> 100
et al. (1981) cultured fetuses explanted late on day 11 to the 45 somite stage. Precise application of the drug was facilitated by means of a sterile human eyelash. At the other end of the scale, IVi day 'egg-cylinders' had to be used to study the role of vitamin E in the prevention of gestation-resorption since this is the latest stage at which vitamin E can be administered and development still proceed normally. These experiments showed that it is probably the antioxidant properties of vitamin E which are important in preventing embryonic death (Steele et al., 1974). Because abnormal development is often the result of an interaction between environmental and genetic factors, attempts have been made to culture mouse embryos, more being known of the genetics of this species than that of the rat. Unfortunately, this has met with limited, though not unpromising, success (Sadler and New, 1981) and the rat therefore remains the species of choice for teratogenicity studies in WEC. Whatever age of embryo or species is used it is important to have appropriate endpoints as a measure of toxicity, whether this be teratogenicity and/or embryotoxicity. In this respect a morphological scoring system (Brown and Fabro, 1981) may be appropriate as well as detailed examination of the embryos (Cockcroft and New, 1978). WEC enables precise control of factors such as developmental stage, duration and timing of exposure to parent compound (and/or metabolites) and the ability to monitor continuously the formation of an abnormality or the condition of an embryo, e.g. its heartrate (Robkin et al., 1974). This can be used to determine the aetiology of dysmorphogenesis where two or more factors are implicated, as in the case of maternal diabetes (Sadler and Horton, 1983) or the Fetal Alcohol Syndrome (Becker al., 1984), as well as to establish quantitative structure/activity relationships of a series of related compounds, e.g. carboxylic acids (Brown, 1987) and retinoids (see below). In addition, these can be complemented by distribution studies of the medium and embryonic/placental tissues (Bechter and Taccard, 1987). It is with the retinoids, natural and synthetic analogues of vitamin A, that there is a direct link between WEC and human reproduction since not only are these the latest examples of therapeutic agents (for the treatment of a wide range of dermatoses) proven to be teratogenic in man (Rosa et al., 1986) but they have also been extensively used in WEC and in rodent teratogenicity studies in-vivo. Briefly, the embryo culture experiments have shown the following facts. 146
Most retinoids have a direct, teratogenic effect on embryonic development in vitro (Figures 1 and 2) at relatively low concentrations (Morriss and Steele, 1977). An important exception to this is etretinate (marketed as 'Tigason') which must be metabolised to etretin to become teratogenic. This retinoid was inactive when added directly to the culture medium without a metabolising system (Steele et al., 1987). (ii) pA"a value (Table II) is clearly an important property of the retinoid molecule in respect of its teratogenic activity (Steele et al., 1987). In contrast, there have been conflicting results concerning cisltrans isomerization. Initial studies in mice (Goulding and Pratt, 1986) and rats (Steele et al., 1987) suggested that there is little difference between cis and trans isomers in their teratogenic potency. A further study in rats (Klug et al., 1989), however, showed all-rra/w retinoic acid to be more potent than 13-c/s retinoic acid. Although there is no obvious explanation for these different results, it is possible, as Klug et al. (1989) have proposed, that the effects of \3-cis retinoic acid added to the culture medium are the result of isomerization to a\\-trans retinoic acid, (iii) Retinoids can be detected in human serum using WEC (Steele et al., 1982). (iv) There is good correlation between rat embryo studies, in vitro and in vivo (Steele et al., 1983), and clinical malformations (Turton et al., 1989). In conclusion, the growing importance of WEC led to an international symposium devoted entirely to this technique, held at the Catholic University of Louvain, Belgium in April 1990. It became apparent that there was no clear consensus as to the precise role of WEC in teratogen testing. The most sensible approach would probably be to remain flexible and pragmatic in deciding when to use WEC and not try to establish rules and terminology prematurely. It should be remembered that in-vitro teratogenicity tests are not required, though they may be accepted, by governmental or regulatory authorities (such as the Food and Drug Administration in the USA and the Committee on the Safety of Medicines in the United Kingdom) and that they do not replace, but rather complement, the definitive Segment II teratology studies in vivo. It may, or may not, be significant that WEC has recently been used to replace preliminary reprotoxicity studies required to establish the doses for the Segment II studies (J.Tesh, personal communication). There is, however, no doubt concerning the value of WEC in mechanistic studies in teratogenesis.
References Bechter.R. and Taccard,G. (1987) Medium and tissue levels of Acyclovir and Etretinate in the rat whole embryo culture system. In Nau.H. and Scott,W.J. Jr, (eds), Pharmacokinetics in Teratogenesis Vol. II. CRC Press, Boca Raton, p. 197. Beck,F., Huxham.I.M. and Gulamhasein.A.P. (1984) In Porter,R.. O'Connor,M. and Whelan,J. (eds), Mechanisms of Alcohol Damage In Utero. Pitman. London, p.218 (Ciba Foundation Symposium 105). Beck,F., Mensah-Brown.E. and Pratten,M.K. (1987) Development of a method for assessing the acute toxicity of chemicals on early postimplantation embryos. Arch. Toxicol.. Suppl. 11. 155-158. Brown,N.A. (1987) Teratogenicity of carboxylic acids: distribution
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JV-(4-hydroxyphenyl)retinamide A/-tetrazol-5-ylretinamide /V-(2-hydroxyethyl)retinamide /V-ethylretinamide 13-m-yV-ethylretinamide
1 )
(i)
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