The use of a retinoid receptor antagonist in a new model to study vitamin A-dependent developmental events.

Multiple fetal anomalies occur in vitamin A deficient animals as well as in retinoic acid receptor gene 'knockout' mice, indicating that retinoic acid (an active metabolite of vitamin A) performs some essential functions in normal development. Additional approaches are needed to probe directly the stages and sites in the embryo where a presence of endogenous retinoic acid is indispensable. We have employed a new strategy for this purpose which involved an intervention in retinoic acid receptor (RAR)-dependent functions at specific developmental stages by means of a highly effective RAR antagonist, AGN 193109. We report that in an in vitro cell differentiation bioassay, AGN 193109 completely reversed the inhibitory action of a potent RAR agonist, AGN 190121. In pregnant mice, a single oral 1 mg/kg dose of the antagonist given on 8 day post coitum (dpc) produced a severe craniofacial anomaly (median cleft face or frontonasal dysplasia) and eye malformations in virtually all exposed fetuses. On the other hand, treatment on 11 dpc, a time in development when RARs are strategically expressed in the limb bud primordium, no limb anomalies could be induced by the antagonist. Even after a high dose of 100 mg/kg, limb development progressed normally in spite of the fact that measurable concentrations of the antagonist were present. Because retinoids are long known to influence skin morphology, we next monitored the effects of the antagonist on skin development. When given late in gestation, on 14 dpc, we found that the antagonist delayed differentiation and maturation of the fetal skin and hair follicles. We conclude that this model provides a convenient and pertinent system which enables us to seek and clarify true functions of retinoic acid and its cognate receptors in embryogenesis and in adult animals.

Developmental toxicity of purified fumonisin B1 in pregnant Syrian hamsters.

The purpose of this study was to characterize the developmental toxicity of fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme, on fetal Syrian hamsters. Fusarium moniliforme has been associated with a variety of diseases in animals and esophageal cancer in humans. Purified FB1 causes leukoencephalomalacia in horses and is hepatocarcinogenic in rats. Fumonisin B1 has been associated with fetal toxicity in rats and mice and has been suggested to be involved in reproductive failure in pregnant sows. Results from a preliminary developmental toxicity study using an aqueous extract of F. moniliforme corn-culture material in hamsters suggested that FB1 was a developmental toxicant. These results were verified using purified FB1. Six groups of ten time-mated female Syrian hamsters were dosed with 0.0-18 mg kg(-1) day(-1) of FB1 by gavage on days 8-12 of gestation and euthanized on day 15. Live fetuses were weighed and examined for gross external and internal abnormalities and skeletal anomalies. Purified fumonisin B1 was shown to cause dose-dependent fetal death and delayed fetal development without causing fetal abnormalities.

A novel Mycoplasma sp. associated with proliferative tracheitis and pneumonia in a Burmese python (Python molurus bivittatus).

Proliferative lymphocytic tracheitis and pneumonia were observed histologically in the respiratory tract of a captive Burmese python (Python molurus bivittatus). A mycoplasma species was isolated from the respiratory tissue. Polymerase chain reaction analysis of the 16S rRNA gene sequence of the isolate showed 0.90 similarity to Mycoplasma agassizii, an organism previously shown to cause respiratory disease in reptiles. Based on these findings, a novel Mycoplasma species was suspected to be the causative agent of respiratory disease in this snake.

Differential teratogenic response of mouse embryos to receptor selective analogs of retinoic acid.

Early events that initiate teratogenesis by Accutane or other retinoids in mammalian embryos remain unknown. It would be helpful for mechanistic considerations to know whether or not retinoids act through retinoid receptor-dependent pathways, and if they do, which of the two families of receptors (retinoic acid receptors - RARalpha, beta, gamma or retinoid X receptors - RXRalpha, beta, gamma) are more likely involved. We previously used an in vitro bioassay to demonstrate that those retinoid analogs with binding affinity and transactivational activity limited only to the RXRs have a low potential as teratogens. Here, we have extended the study to examine teratogenicity, in pregnant mice, of a number of synthetic retinoids with varying degrees of receptor selectivity. The ability of each compound to induce fetal limb and craniofacial defects after a single exposure on day 11 of gestation was assessed and compared to that of all-trans retinoic acid (RA). The highest dose selected was 100 mg/kg maternal body weight since such a regimen of all-trans RA affects virtually every exposed embryo without any indication of maternal toxicity. We found that although all RAR agonists were strong teratogens, their potencies varied over a wide magnitude. The teratogenic potencies and receptor transactivation profiles of RAR agonists were not directly correlated since compounds with similar receptor activities presented major differences in potencies. Three compounds were exclusively RXR agonists, and these were not teratogenic under our experimental conditions. Two additional compounds which turned out to be non-teratogenic were distinguished by the fact that they activated neither RARs nor RXRs. These data indicate that although RAR-dependent mechanisms are likely involved in retinoid-induced teratogenesis, there are additional factors which determine teratogenic potency. The absence of teratogenic response in the case of RXR agonists suggests that risk-benefit analyses of such receptor-selective compounds may be fruitful in further studies.

Diminished teratogenicity of retinoid X receptor-selective synthetic retinoids.

One feature that contraindicates the wide therapeutic use of retinoids is their teratogenicity. Synthetic retinoids are distinguishable from each other on the basis of their partial or exclusive preference in binding and activation of all-trans retinoic acid receptors (RARs) or retinoid X receptors (RXRs). Using mouse embryo limb bud cells in micromass cultures as a bioassay, we examined the inhibitory activities of a number of standard and novel retinoids on chondrogenic cell differentiation. Transient cotransfection of HeLa cells was used to measure the ability of each retinoid to induce transcription of a reporter gene by activating RAR alpha, RAR beta, RAR gamma, or RXR alpha chimeric constructs. All retinoids in this study that activated RARs to any degree in the cotransfection assay also inhibited chondrogenesis in vitro, whereas retinoids that were either specific for RXR or inactive in the cotransfection assay did not. The activity of RAR-selective agonists and the inactivity of RXR-specific agonists in the cotransfection assay correlated well with the relative teratogenicity of six of the representative retinoids studied when orally administered at day 11 to pregnant ICR mice.

Modulation of limb bud chondrogenesis by retinoic acid and retinoic acid receptors.

An excess of retinoic acid (RA) in the mouse embryo in utero produces hypochondrogenesis and severe limb bone deformities. Since one of the RA receptors--RAR-beta 2, is specifically induced in the limb bud cells upon treatment of embryos with teratogenic doses of RA, we investigated if this receptor played a role in teratogenesis by regulating the process of chondrogenesis. In micromass cultures of mouse limb bud mesenchymal cells, we found that a downregulation of RAR-beta 2 as well as several other RAR isoforms by supplementation of the culture medium with specific oligodeoxynucleotides stimulated chondrogenesis: cartilage nodule number, sulfated proteoglycans, and synthesis of collagen type IIB were all enhanced in a dose-dependent manner. However, only the antisense RAR-beta 2 probe efficiently prevented the strong inhibitory effects of exogenous RA on chondrogenesis in these cells. The data suggest that the RAR-RA complexes play a role in position-dependent patterning of the limb skeleton in normal development and that, in particular, RAR-beta 2 serves to prevent the mesenchymal cells from expressing their chondrogenic bias. Our results further strengthen the argument that RA-dependent elevation in RAR-beta 2 levels plays a unique role in RA-induced teratogenesis.

Placental transfer and developmental effects of 9-cis retinoic acid in mice.

9-cis retinoic acid (RA) is a naturally occurring isomer of all-trans RA. While both isomers can bind with high affinity and activate RA receptors, only 9-cis RA is the specific ligand for the retinoid X receptors. 9-cis RA has also been shown to be much more potent than all-trans RA in inducing digit duplication in the chick embryo wing bud. To gain further insight into its mechanisms, here we investigated the teratogenic activity in pregnant mice of 9-cis RA and compared it with those of all-trans RA and 13-cis RA. Using frequency and severity of limb reduction defects as well as palatal clefts in the resultant fetuses as indicators, we found that orally administered 9-cis RA was one-half as potent a teratogen as all-trans RA. That 9-cis RA was intrinsically less active than all-trans RA was deduced by comparing the inhibitory activities of the two retinoids in the limb bud mesenchymal cell micromass cultures using chondrogenesis as an end-point. Since placental transfer of cis isomers of RA is generally poor, we monitored the identities and amounts of retinoids in the embryo after administration of 9-cis RA to the mother. We found that 9-cis RA undergoes extensive metabolism and isomerization during absorption resulting in a number of metabolites in the maternal circulation within 30 min after administration. Although some of these metabolites remain to be identified, the most abundant RA isomers in the plasma coeluted with 13-cis RA.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of methods to predict minimal weight in high school wrestlers.

The purpose of this study was to compare the accuracy of body fat determinations and subsequent calculation of minimal weight (MW) by dual energy x-ray absorptiometry (DEXA), bioelectrical impedance (BIA), near-infrared photospectometry (NIR), and anthropometry (LOHMAN). Necessitated by mandatory state minimal weight testing, the methods were cross-validated on 95 Wisconsin high school wrestlers (mean +/- SD; age: 15.1 +/- 1.2 yr, height: 170.4 +/- 7.1 cm, weight: 63.4 +/- 9.8 kg). MW, defined as fat-free body/0.93, determined by hydrostatic weighing (HW) and residual volume via O2 dilution, served as the criterion. The validity of the four selected MW predictions were evaluated against HW by examining mean differences (MD), standard deviation differences (SDD), correlations (r), standard error of estimate (SEE), and total errors (TE). Statistically significant differences were shown between the methods and the criterion by t-tests; however, these were clinically small in Lohman (0.6 kg) and BIA (0.9 kg). TE ranged from 2.25 kg (Lohman) to 6.03 kg (NIR). The results indicated that Lohman skinfold equation provided the most accurate prediction of MW, demonstrating the highest correlation (0.972), lowest MD (0.6 kg), lowest SEE (2.12 kg), and lowest TE (2.25 kg) of the methods evaluated.

Analysis of high dysmorphogenic activity of Ro 13-6307, a tetramethylated tetralin analog of retinoic acid.

Certain synthetic retinoids differ widely from retinoic acid (RA) in teratogenic potency, being much more or much less effective than RA. It is assumed that the potency of a retinoid may depend on the nature of its interaction with cellular binding components (nuclear retinoic acid receptors or cytoplasmic binding proteins) and, as in the case of retinoids that are mammalian teratogens, on factors that determine its accessibility to the embryo. To investigate some of the factors that contribute to potency, we used a new synthetic retinoid Ro 13-6307 that differs in structure from RA in having an aromatic ring inserted in its side chain along with gem dimethyl modification of the natural cyclohexenyl ring. Pregnant ICR mice were given a single oral dose (0, 1, or 10 mg/kg) on day 11 of gestation, and the resultant teratogenic outcome was monitored on day 17. Direct effects on cell differentiation were obtained by exposing high density cultures of limb bud mesenchymal cells to a range of concentrations (0.3 ng/ml-3 micrograms/ml) of Ro 13-6307 and scoring for chondrogenic suppression. Concentrations reaching the embryo after maternal administration of Ro 13-6307 were measured by HPLC to quantify the analog for a period of 4 h after administration of the oral dose. We found that this retinoid was 40-fold as active as RA in both inducing teratogenesis and suppressing chondrogenesis, yet its concentration in the affected embryo was only a fraction of that achieved after an equivalent dose of RA was employed in a similar protocol.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinamides: hydrolytic conversion of retinoylglycine to retinoic acid in pregnant mice contributes to teratogenicity.

Retinamides are prominent among synthetic vitamin A derivatives (retinoids) which can prevent or reduce the incidence of certain carcinogen-induced neoplasms in animals. They also possess lower toxicity toward adult and developmental systems than natural retinoids, presumably because of the presence of an amide endgroup which resists ready hydrolysis. In this investigation, we compared the developmental toxicities in mice of N-(4-hydroxyphenyl)retinamide(4-HPR), N-ethylretinamide (ER) and two retinoylamino acids, N-(all-trans-retinoyl)glycine (RG) and N-(all-trans-retinoyl)-DL-leucine (RL), which are formed from retinoic acid and the alpha-amino acids; RG and RL were shown in a previous study to differ from each other and from retinoic acid in certain toxicity bioassays. We found that while 4-HPR, ER, and RL were only minimally embryotoxic, RG was uniquely active as a teratogen with potency equivalent to that of retinol, the precursor of retinoic acid. Since binding to cytoplasmic proteins and nuclear receptors is a function of the presence of an acidic endgroup in the retinoid molecule, we investigated if RG given to pregnant mice was converted to retinoic acid (RA) and if teratologically significant amounts were detectable in the embryo. A single 100 mg/kg dose of RG in oil vehicle was given orally to ICR mice on day 11 of gestation (plug day = day 0). Extraction and quantification by HPLC of the retinoids in the maternal plasma and in whole embryos were performed at hourly intervals for the first 10 h after dosing and at 26 h. RG was absorbed rapidly reaching peak levels in the maternal plasma at 1 h after the dose and maintained a level of 15 micrograms/mL for up to 4 h, before starting a decline. RG also transferred to the embryo reaching peak levels greater than 0.75 micrograms/g wet weight between 2 and 4 h after the dose. All-trans RA was detected in the maternal plasma and the embryo at 1 h after the dose, reaching peak levels at 2 h in both compartments (0.43 micrograms/mL or g), before starting a decline. Small quantities of 13-cis RG (a contaminant in the original solution comprising 2-3% by weight) and 13-cis RA were also detected in both compartments, but their amounts in the embryo were considered insufficient to contribute to teratogenicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Biotransformation of etretinate and developmental toxicity of etretin and other aromatic retinoids in teratogenesis bioassays.

Developmental toxicity of the anti-psoriatic drug etretinate (Tegison) and some features of its metabolic conversion to etretin and isoetretin were investigated in in vivo and in vitro teratogenesis bioassays. We found that a single dose of etretinate administered orally to pregnant mice on day 11 of gestation was a potent teratogen (ED50 = 26 mg/kg). Etretin (acitretin, Neotigason), given as a single dose, was about 8-fold less active as a teratogen than etretinate. A ring substituted congener of etretinate, Ro 11-4768, was essentially inactive under similar conditions. Although the mechanisms which operate to make Ro 11-4768 inactive in teratogenesis are unknown and intriguing, it is suggested that the differences between etretinate and etretin may be dependent on individual pharmacokinetic characteristics. The in vitro chondrogenesis bioassay confirmed previous reports that the presence of an acidic endgroup was necessary for suppression of chondrogenesis, and that on that basis etretin was an active inhibitor of chondrogenesis, whereas etretinate was not. Introduction of esterase into the culture medium resulted in complete hydrolysis of etretinate and a quantitative conversion to acid congeners sufficient to account for an appropriate suppression in chondrogenesis. Although limb bud cells were virtually incapable of converting etretinate to etretin in the absence of exogenous esterase, they did influence the metabolism so that in the presence of esterase, isoetretin rather than etretin was the major endproduct of etretinate hydrolysis. Since etretinate therapy endangers the conceptus for a prolonged period of time even after cessation of therapy, further studies are necessary to determine the nature and the extent of hazard posed by the storage and/or metabolism of etretinate.

Pharmacokinetic assessment of teratologically effective concentrations of an endogenous retinoic acid metabolite.

Retinoic acid is a natural vitamin A derivative that undergoes oxidative metabolism in the body to yield several metabolites, which apparently represent the products of a detoxification pathway. To assess if such metabolic conversions diminished teratogenic potency, one of the major metabolites (4-oxo-all-trans-retinoic acid) was tested for its teratogenic activity in pregnant ICR mice and further investigated for its pharmacokinetic features to determine if it accumulated in the embryo in concentrations sufficient to elicit a teratogenic response. Administration of single oral doses (10, 25, 50, or 100 mg/kg) of the compound to ICR mice on day 11 of gestation (plug day = day 0) produced dose-dependent frequencies of serious fetal anomalies of the type usually associated with the use of retinoic acid and other retinoids. The metabolite was equivalent in teratogenic potency to retinoic acid, and, in the instance of cleft palate frequency, it was even more active. Concentrations of 4-oxo-all-trans-retinoic acid and its 13-cis isomer were measured in the maternal plasma and whole embryos at 30 min to 10 hr after administration of the lowest (10 mg/kg) and the highest (100 mg/kg) teratogenic dose of 4-oxo-all-trans-retinoic acid by means of high-performance liquid chromatography methodology. Distribution of the compound in the maternal system and transfer to the embryo occurred rapidly with either dose. Peak concentration in the maternal plasma and the embryo persisted for 3-4 hr after the higher dose but not with the lower dose; however, elimination kinetics for the two dose levels were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Derivation of retinoic acid and metabolites from a teratogenic dose of retinol (vitamin A) in mice.

Megadose supplements of vitamin A are under suspicion as hazards to the developing embryo after the discovery that two vitamin A-related drugs, Accutane and Tigason, are human teratogens. Retinoic acid (all-trans-RA) is a natural metabolite of vitamin A which participates in many of the known functions of vitamin A and may be the active agent in teratogenesis. In this investigation we gave a single, high oral dose of retinol (vitamin A) to pregnant mice to assess its transplacental pharmacokinetics as well as to measure the formation and distribution of its metabolites in the embryo. Retinol was estimated to be 4-fold less active than retinoic acid in the whole animal teratogenesis and 20-fold less active in the in vitro bioassay. A fully teratogenic dose, 200 mg/kg, yielded considerable quantities of retinoic acid which were transferred to the embryo with kinetics similar to that of retinol. During the first 8 hr after administration of retinol, the metabolites (including all-trans-RA, 13-cis-RA, and 4-oxo-RA) constituted almost 50% of the quantity of all retinol derivatives found in the embryo. A comparison of combined peak concentrations of the metabolites (or their AUC values) with the extent of teratogenesis associated with them individually provided sufficient evidence to implicate the metabolites themselves as mediators of retinol-induced teratogenesis. However, since both retinol and retinoic acid were present in sufficient concentrations in the embryo to act as teratogens we cannot at present rule out the possibility that they may act independently. Further experimentation will be necessary to address whether retinoic acid detected in the embryo is the product of the embryo's own metabolic capability or is transferred from the maternal circulation.

Angiotensin II receptors in the lumbar spinal cord of the rat.

Locations of cells responsive to microiontophoretically applied angiotensin II (AII) were compared to distributions of AII receptor binding sites identified by autoradiography in the lumbar enlargement region of the rat spinal cord. Angiotensin II receptor binding sites were densely concentrated in the superficial layers of the dorsal horn. Considerably lower densities of binding sites were present in the remaining gray matter. Effects of microiontophoretically applied AII on lumbar spinal cord cells did not vary with location within the gray matter. AII facilitated firing of most cells in the lumbar cord whether the cells were in superficial or deeper laminae of the dorsal horn or in the ventral horn. The distribution of AII binding sites and the distribution of cells that were responsive to AII suggest that AII may play a role in modulating both sensory and motor functions of the spinal cord.

A comparative study of the lesions in cultured fibroblasts of humans and four species of animals with Chediak-Higashi syndrome.

The Chediak-Higashi syndrome (CHS) is an autosomal recessive genetic disease of humans, and clinically similar diseases occur in cats, mink, cattle, mice, killer whales, blue foxes, and silver foxes. It is characterized by incomplete albinism, increased susceptibility to infection, and the most distinctive hallmark, the presence of enlarged cytoplasmic granules in many cell types. The acid phosphatase-positive granules, lysosomes, of fibroblasts from control and CHS humans, cats, mink, cattle, and mice were examined. These studies represent the initial characterization of the lesions in fibroblasts of CHS cats, mink, and cattle. Fibroblasts from each species and genotype were stained histochemically for acid phosphatase, and morphometric analysis of the distribution of acid phosphatase-positive granules was performed. The lysosomes in the CHS fibroblasts tended to be restricted to the perinuclear area of the cytoplasm, whereas the lysosomes in the normal fibroblasts were generally more widely distributed in the cytoplasm. The lysosomes in the CHS fibroblasts of all species examined were also more enlarged and heterogeneous than those in the control fibroblasts.

Interspecific genetic complementation analysis with fibroblasts from humans and four species of animals with Chediak-Higashi syndrome.

Although the autosomal recessive disease Chediak-Higashi syndrome (CHS) has been described in humans, cats, mink, cattle, mice, killer whales, blue foxes, and silver foxes, and these conditions appear quite similar, no direct evidence of the homology of this disease in the various species has been presented. To determine if CHS in humans, cats, mink, cattle, and mice is due to a mutant gene at the homologous genetic locus in each species, or alternatively, if these are merely similar syndromes, genetic complementation analysis after interspecific somatic cell (fibroblast) hybridization was performed. "Paracrystal" formation was the criterion used for the determination of complementation. The initial studies in this report were designed to characterize paracrystal formation in control and CHS fibroblasts of these five species. Most of the control fibroblasts from each species (91-96.6%) formed paracrystals upon incubation with 25 micrograms/ml of the microtubule depolymerizing agent vinblastine sulfate. A significantly (P less than 0.05) smaller percentage of the CHS fibroblasts formed paracrystals after the same incubation (except CHS mice, with 90.2% paracrystals). It was found that 52% of the human CHS fibroblasts, 60% of cat CHS fibroblasts, 47% of mink CHS fibroblasts, and 53.8% of cow CHS fibroblasts formed paracrystals. For genetic complementation analysis, human CHS fibroblasts were fused to cat, mink, cow, or mouse CHS fibroblasts with polyethylene glycol. Control fusions were human CHS fibroblasts fused with human, cat, mink, cow, and mouse normal fibroblasts. The results of complementation analysis after the fusion of human CHS with cow CHS and human CHS with mouse CHS fibroblasts were inconclusive. A lack of complementation of human CHS with cat CHS and human CHS with mink CHS fibroblasts indicates that the disease is homologous in these species.

Developmental effects of isotretinoin and 4-oxo-isotretinoin: the role of metabolism in teratogenicity.

Previous observations have indicated that isotretinoin (IT), a drug in common use for therapy of cystic acne, is teratogenic in humans but possesses low embryotoxicity in pregnant mice, probably because of its shorter half-life and limited placental transfer in rodents. In human volunteers and patients, one major blood metabolite of IT is 4-oxo-isotretinoin (4-oxo-IT) which undergoes slower elimination than IT and may itself be a participant in teratogenesis. To investigate the problem of species differences displayed by IT and the role of its metabolism, embryotoxic effects of 4-oxo-IT were examined after its single or repeated intubations into pregnant ICR mice and compared with the effects of a similar regimen of IT. The two compounds were also tested for their relative ability to suppress chrondrogenesis in the in vitro cell and organ culture assays. We found that a single dose of 4-oxo-IT, 100 mg/kg, given on day 11 of gestation (plug day = day 0 of gestation) produced a moderate incidence of limb reduction defects and cleft palate (39% and 27% of surviving fetuses, respectively), while a dose of 150 mg/kg affected virtually every fetus. IT, on the other hand, produced no defects in fetuses exposed to similar dose levels. Repeated intubations with IT, however, resulted in increasing the frequencies of limb reduction defects and cleft palate to levels obtained after 4-oxo-IT administration. We found that a 3-hour interval between IT intubations was more effective in this regard than an 8-hour interval. Repeated IT intubations also uncovered sharper stage-dependency of limb and palatal defects than obtained otherwise.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic acid enhances the displacement of newly synthesized hyaluronate from cell layer to culture medium during early phases of chondrogenesis.

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid (1 microgram/ml or 3.3 X 10(-6) M). Since in control medium overt chondrogenesis begins on day 3, retinoic acid was introduced on day 2 so that the relationship between initial biosynthetic changes and inhibition of chondrogenesis could be studied. During the first 24 h of exposure the treated cells remained viable but suffered 10% inhibition in growth and synthesized [3H]glucosamine-labeled glycosaminoglycan at a level 24% below untreated cells. The amount of labeled hyaluronic acid released into the culture medium by the treated cells was, however, 2-fold greater, on a per cell basis, than that in the untreated cultures. It is suggested that the displacement of hyaluronate may play a role in the disruption of mesenchymal cell differentiation and of limb morphogenesis as observed in other systems.

Comparative teratogenic activities of two retinoids: effects on palate and limb development.

Two closely related retinoids, all-trans and 13-cis retinoic acids, were assessed for their relative activities as teratogens in ICR mice by monitoring the frequency with which either isomer produced discrete dysmorphogenesis of the embryonic limb and the secondary palate. A single oral dose of all-trans retinoic acid at 100 mg/kg on either day 11.5 or 12.0 of gestation (plug day = day one) was maximally effective; more than 90% of the treated embryos developed reduction defects of the limb bones and an equally high percentage also had cleft palate. The limb development was most sensitive on day 11.5 of gestation while the peak susceptibility for palatal clefts began on day 12.0. Under identical experimental conditions, treatment with 100 mg/kg 13-cis retinoic acid produced no apparent teratogenic effects. By assessing the relative incidence of readily identifiable malformations of the limb and palate associated with various doses of the two isomers, we found that 13-cis retinoic acid was four to eight times less embryopathic than all-trans retinoic acid. Since the mechanism of teratogenic action of retinoids is still far from clear, it is suggested that further studies on causative factors will be greatly assisted by the use of these two closely related retinoids, which substantially differ from each other in their teratogenic potency.