The synthetic retinoid AGN 193109 but not retinoic acid elevates CYP1A1 levels in mouse embryos and Hepa-1c1c7 cells.

The synthetic retinoid AGN 193109 is a potent pan retinoic acid receptor (RAR) antagonist. Treatment of pregnant mice with a single oral 1 mg/kg dose of this antagonist on day 8 postcoitum results in severe craniofacial (median cleft face or frontonasal deficiency) and eye malformations in virtually all exposed fetuses. Using differential display analysis, we have determined that CYP1A1 mRNA levels are elevated in mouse embryos 6 h following treatment with AGN 193109. Similarly, an elevation in CYP1A1 mRNA levels, protein levels, and aryl hydrocarbon hydoxylase activity occurs in Hepa-1c1c7 cells, with the maximal elevation observed when the cells were treated with 10(-5) M AGN 193109 for 4 to 8 h. Elevation in CYP1A1 mRNA levels in mouse embryos and Hepa-1c1c7 cells does not occur upon treatment with the natural retinoid, all-trans-retinoic acid. Finally, elevation in CYP1A1 mRNA levels was not observed when mutant Hepa-1c1c7 cells, which are defective in either the aryl hydrocarbon receptor (AhR) or aryl hydrocarbon receptor nuclear translocator (ARNT), were treated with AGN 193109. This suggests that the AhR/ARNT pathway and not the RAR/RXR pathway is mediating the elevation of CYP1A1 mRNA levels by AGN 193109, at least in the Hepa-1c1c7 cells. This is the first example of a retinoid that displays the abililty to regulate both the RAR/RXR and AhR/ARNT transcriptional regulatory pathways.

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.

Tretinoin: a review of the nonclinical developmental toxicology experience.

Tretinoin has been thoroughly evaluated for its potential as an embryofetal developmental toxicant. Oral tretinoin produces developmental anomalies in animal models; the minimal teratogenic dose is consistently 2.5 to 10 mg/kg. In contrast, topical application does not induce developmental malformations in laboratory animals. A structurally related compound, isotretinoin, is a potent toxicant in humans and animals; the lowest systemic dose that induces fetal anomalies varies more than 100-fold depending on the model. Oral isotretinoin is a more potent developmental toxicant than oral tretinoin in monkeys. Between-drug differences in the metabolism and transplacental transfer of the two retinoids account for the differences in toxicant potency. Pharmacokinetic studies reveal that absorption of tretinoin from the skin is poor and yields maternal plasma concentrations below the developmentally toxic threshold established after oral administration. Analysis of outcomes of developmental toxicology and pharmacokinetic studies suggests that the human risk of fetal anomalies is negligible after therapeutic application of topical tretinoin.

Murine toxicology and pharmacology of UAB-8, a conformationally constrained analog of retinoic acid.

(2E, 4E, 6E)-8-[3'-Ethyl-2'-(1-methylethyl)-2'-cyclohexen-1'-ylidene] -3, 7-dimethyl-2,4,6-octatrienoic acid (UAB-8) has potent activity in preventing papillomas on the skin of mice similar to that determined in a previous study for the homolog containing one less carbon atom. To evaluate the toxicological profile for UAB-8, relative to all-trans-retinoic acid (RA), female mice were dosed by oral gavage for 29 days with amounts of 0.05, 0.1, or 0.2 mmol/kg/day. For the two compounds, the effects on body weights were similar. Mice dosed with UAB-8, however, had a lower incidence of clinical signs of toxicity (alopecia, scaly skin, and limping). At necropsy, bone fractures, skin abnormalities, and splenomegaly were observed in some mice dosed with RA but not in any dosed with UAB-8. Lymph node hyperplasia was noted in some mice dosed with either dose of RA but only in those dosed with the highest dose of UAB-8. All dose levels of RA produced microscopic lesions in the bones of mice; only the highest dose of UAB-8 had this effect. RA and UAB-8 had similar effects on chondrogenesis in cultures of cells from mouse limb buds, an indication of comparable teratogenic effects. For mice dosed i.v. (10 mg/kg), there was a saturated phase of elimination of RA from plasma (Km = 0.61 microgram/ml and Vmax = 2572 micrograms/hr); no such phase was noted when UAB-8 was administered. UAB-8 had values for t1/2 alpha and t1/2 beta of 0.47 and 17.1 hr, respectively. Relative to RA, UAB-8 has a favorable toxicological profile and different pharmacokinetics.

Synthesis and structure-activity relationships of retinoid X receptor selective diaryl sulfide analogs of retinoic acid.

Retinoids exert their biological effects by binding to and activating nuclear receptors that interact with responsive elements on DNA to promote gene transcription. There are two families of retinoid receptors, the retinoic acid receptor (RAR) family and the retinoid X receptor (RXR) family, which are each further divided into three subclasses: RAR alpha, beta, gamma and RXR alpha, beta, gamma. Herein we describe the synthesis and structure-activity relationships of a new series of diaryl sulfide retinoid analogs that specifically bind and transactivate the RXRs. Furthermore, the sulfoxide and sulfone derivatives of these analogs are partial agonists which activate the RXRs only at high concentrations. Thus, these compounds possess a potential site of metabolic deactivation and may have less prolonged systemic effects than other compounds with arotinoid-like structures. We show also that these compounds have activity in nontransfected cells as demonstrated by their ability to induce TGase activity in HL-60 cells. Finally, we corroborate our earlier report that RXR-specific agonists may possess reduced teratogenic toxicity compared to RAR-specific agonists since these compounds are much less potent inhibitors of chondrogenesis than RAR-specific agonists such as TTNPB.

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)

Teratogenesis by retinoic acid analogs positively correlates with elevation of retinoic acid receptor-beta 2 mRNA levels in treated embryos.

Retinoic acid (RA) plays an important role during normal embryogenesis, however high doses of RA are teratogenic. Retinoic acid receptor-beta 2 (RAR-beta 2) mRNA and protein levels were previously demonstrated to undergo rapid elevation in susceptible tissues after treatment with teratogenic doses of RA. In this report we compared the effects of a number of retinoids, which represent a wide variety of chemical structures and which differ in their teratogenic potencies, on RAR-beta 2 mRNA levels in mouse embryos 6 hr after treatment. Retinoid treatments which result in a high incidence of limb defects elevated RAR-beta 2 mRNA levels similarly (10-14 fold in the limb buds, 4-8 fold in the head, and 2-4 fold in the remainder of the body). On the other hand, retinoid treatments which cause a low or no incidence of limb defects resulted in minor changes in RAR-beta 2 mRNA levels in each embryonic region. Therefore, a strong positive correlation was found between the elevation of RAR-beta 2 mRNA levels and the retinoids which produce limb defects. This provides further evidence that an elevation of RAR-beta 2 mRNA levels, and subsequently protein levels, is an important event involved in mediating the effects of RA during dysmorphogenesis.

A sustained elevation in retinoic acid receptor-beta 2 mRNA and protein occurs during retinoic acid-induced fetal dysmorphogenesis.

We have previously shown that oral treatment of pregnant mice with all-trans retinoic acid (RA) at doses which cause 100% fetal dysmorphogenesis results in a rapid elevation in the mRNA of one specific isoform of the RA receptor-beta, RAR-beta 2, in susceptible embryonic regions. To further investigate the involvement of RAR-beta 2 mRNA in teratogenesis, we have examined its expression in mouse embryos exposed to marginal/nonteratogenic and teratogenic dosing regimens of both 13-cis RA and all-trans RA. We have found that the mere elevation in embryonic RAR-beta 2 mRNA levels and free retinoid levels is not sufficient to result in dysmorphogenesis. Rather, retinoid-induced dysmorphogenesis of embryos appears to occur only when RAR-beta 2 mRNA and unbound retinoid levels remain elevated for at least 6-9 h following retinoid treatment resulting in a significant and prolonged elevation in RAR-beta protein levels.

Induction of RAR-beta 2 gene expression in embryos and RAR-beta 2 transactivation by the synthetic retinoid Ro 13-6307 correlates with its high teratogenic potency.

Vitamin A (retinol), its metabolite all-trans retinoic acid (RA), and many synthetic analogs (retinoids) express variable potencies as teratogens. Although biological activities of retinoids are mediated by nuclear RA receptors (RARs) and retinoid X receptors (RXRs), it is not known if any of these receptors mediate teratogenicity, and if the potency also depends on the nature of the ligand-receptor interactions. Previous evidence has implicated that one specific isoform, RAR-beta 2, does play a role in mediating retinoid teratogenicity. Here, we employed an aromatic retinoid with a triene side chain, Ro 13-6307, to study its interactions with RAR-beta 2 since its teratogenicity is much higher and its accessibility to the embryo is much lower than RA. A fully teratogenic dose of Ro 13-6307 (10 mg-kg) given to pregnant mice preferentially elevated the level of RAR-beta 2 mRNA in susceptible embryonic regions (maximal induction, 10- to 12-fold above control in limb buds) in a manner comparable to a fully teratogenic dose of all-trans RA (100 mg-kg). Using the RAR-beta 2 promoter linked to a reporter gene in cotransfection experiments, the efficacy of Ro 13-6307 and RAR-beta 2 in transcription transactivation was found to be 30-40 times greater than all-trans RA. Since the teratogenic potency of Ro 13-6307 is estimated from a previous study to be 44-fold greater than all-trans RA, we suggest that the teratogenicity of this synthetic retinoid is generally proportional to its ability to enhance receptor function.

Correlations of RAR isoforms and cellular retinoid-binding proteins mRNA levels with retinoid-induced teratogenesis.

Retinoic acid (RA) plays an important role in normal embryogenesis; however, excessive doses are teratogenic. At present, the molecular mechanisms responsible for these effects of RA are not well understood. The action of retinoids are believed to be mediated by two classes of proteins, nuclear receptors (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) and small cellular retinol-binding and retinoic acid-binding proteins (CRBP-I, CRBP-II, CRABP-I and CRABP-II). Teratogenic doses of RA increase the level of RAR-beta 2 mRNA, RAR-alpha 2 mRNA, CRBP-I mRNA and CRABP-II mRNA in mouse conceptuses and embryos. The elevation in the level of only RAR-beta 2 mRNA correlates with the target tissues, as well as developmental stages that are sensitive to the teratogenic effects of RA. In addition, we have screened a few other natural and synthetic retinoids with similar results. These results are consistent with the possibility that RAR-beta 2 may mediate at least some of the effects of retinoids during abnormal development.

Evidence that retinoic acid-induced apoptosis in the mouse limb bud core mesenchymal cells is gene-mediated.

The evidence presented here strengthens the argument that RA-induced truncation defects of the embryonic limb, and probably other teratogenic effects, are mediated by the nuclear retinoid receptors, particularly the RAR-beta 2 isoform. Although apoptotic cell death and an increased transglutaminase (tTG) activity accompany teratogenesis, it should be emphasized that the increased levels of RAR-beta 2 may influence additional events in limb development, e.g., modulation of connective tissue differentiation and an inhibition of chondrogenesis. Further work entails screening the effects of RA on genes targeted by the receptors.

Induction of tissue transglutaminase and apoptosis by retinoic acid in the limb bud.

Mesenchymal cells in the limb buds of midgestation mouse embryos suffer prominent cell death upon exposure to retinoic acid (RA), an event likely associated with the micromelic and phocomelic anomalies of the resultant fetuses. It has been suggested, but not yet shown, that cells die by an active process termed apoptosis rather than by necrotic cytolysis. In certain cell types, investigators have previously observed a specific and early effect of RA on transcriptional activation of the gene for tissue transglutaminase (tTG), an enzyme suspected to play a role in apoptosis. We report here a distinct but transient increase in tTG activity which accompanied the initiation of cell death in the mesenchymal cells located in the central core of RA-treated limb buds. We also ascertained microscopically that the cytological appearance of the affected cells was consistent with a characterization of the process of cell death as apoptosis.

Retinoic acid receptor beta 2 mRNA is elevated by retinoic acid in vivo in susceptible regions of mid-gestation mouse embryos.

Many of the biological effects of retinoic acid are mediated by its nuclear receptors (RAR-alpha, RAR-beta, and RAR-gamma), and each of these three receptors exist in multiple isoforms. As a first step to identify if any of the receptor isoforms are involved in dysmorphogenesis which is induced in mouse embryos after treatment with retinoic acid (RA), we examined the levels of mRNA of several isoforms of each RAR in the limb buds and other embryonic regions of normal and RA-treated embryos. Within 3 to 6 hr after treatment of mice on day 11 of gestation with RA, RAR-beta 2 mRNA levels in the whole embryo increased 7-fold while both RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated only 2-fold. Since RA treatment of day 11 embryos especially produces limb defects in virtually every embryo, we next examined individual embryonic regions separately. Limb buds showed the highest elevations in RAR-beta 2 mRNA levels (12-fold) compared to a moderate elevation in the head/craniofacial region (8-fold) and a small elevation in the remainder of the body (4-fold). In contrast, RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated in all these tissues to a similar extent, which amounted to only about a 2-fold increase. Retinol, the precursor of RA in the embryo, was also capable of elevating RAR-beta 2 mRNA levels in the limb bud, but the increase was delayed, apparently indicating that metabolic conversion of retinol to RA preceded the effect on mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Developmental changes in endogenous retinoids during pregnancy and embryogenesis in the mouse.

Vitamin A and its analogs (retinoids) have acquired particular significance in embryonic development since the discovery that retinoic acid (RA) possesses properties of an endogenous morphogen and that embryonic tissues contain specific nuclear receptors for RA. Since the mammalian embryo does not synthesize RA de novo but rather must acquire it directly or in a precursor form from the maternal circulation, we sought to establish the relationship between levels of RA, retinol, and retinyl esters in the maternal system and their acquisition by the embryo, particularly during organogenesis in the mouse. Results indicate profound changes in maternal vitamin A levels during pregnancy in the mouse. These changes were characterized by a large, transient decrease in plasma retinol levels coincident with the period of organogenesis (e.g. gestational Days 9-14), and an apparent increase in mobilization from hepatic stores to the conceptus. During organogenesis, the embryo exhibited a steady increase in retinol levels with little increase in retinyl esters and virtually no change in RA. Analysis of retinoid accumulation patterns in the embryonic liver indicate that functional onset of vitamin A storage occurs by mid-organogenesis. In contrast, placental levels of these retinoids remained unchanged throughout organogenesis. Analysis of the conceptus as a developmental unit revealed that during early organogenesis the majority of retinoids are contained in the placenta (8-fold more than in the embryo). However, by mid-organogenesis the retinoid content of the embryo exceeds that of the placenta. Together, these results provide evidence that pregnancy in the mouse is accompanied by pronounced alterations in maternal retinoid homeostasis that occur coincident with the period of high embryonic sensitivity to exogenous retinoids.

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)