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Hemimelic extra toes (Hx) arose spontaneously as a dominant mutation in B10.D2/nSnJ mice in 1967. It specifically affects the appendicular skeleton, causing variable foreshortening of the tibia (radius) and preaxial polydactylism. Early anatomical studies revealed anterior overgrowth of the autopod, with decreased apoptosis and increased mitosis in the anterior apical ectodermal ridge and underlying mesenchyme; overextension of apoptosis in the central zeugopod accounted for hemimelia. The Hx mutant phenotype was coarsely mapped to mouse chromosome (Chr) 5 and closely linked to engrailed-2 (En2) and Sonic hedgehog (Shh). This region is syntenic to human Chr 7q36 that harbors several dominant mutations affecting the hand. High-resolution genome mapping identified the Hx mutation as a G --> A base pair transition within Intron 5 of the murine Lmbr1 locus. The critical effect is on a multifunctional conserved regulatory element that acts as a limb-specific, long-distance cis-acting enhancer of Shh expression. As such, the Hx mutant phenotype results from ectopic Shh signals at the anterior margin of the limb bud that directly or indirectly alter FGF4 signaling from the apical ectodermal ridge. Given significant advances in understanding of embryonic development in general and limb development in particular, this review article reveals how research that once attracted interest of teratologists has advanced across the decades to pinpoint a critical molecular lesion and reveal a potential mechanism of a specific malformation that is found commonly in experimental teratology., ((c) 2010 Wiley-Liss, Inc.)

The question of how alterations in cell behavior produced by retinoic acid (RA) influenced the development of skeletogenic mesenchyme of the limb bud was examined in this study. Our established model was employed, which involves treatment of pregnant mice with a teratogenic dose of RA (100 mg/kg) on 11 days postcoitum (dpc) resulting in a severe truncation of all long bones of the forelimbs in virtually every exposed fetus. It is shown that RA, administered at a stage to induce phocomelia in virtually all exposed embryos, resulted in immediate appearance of enhanced cell death within the mesenchyme in the central core of the limb bud, an area destined for chondrogenesis. The central core mesenchyme, which in the untreated limb buds experiences a sharp decline in cell proliferation heralding the onset of chondrogenesis, demonstrated a reversal of the process; this mesenchyme maintained a higher rate of cell proliferation upon RA exposure. These events resulted in a truncation and disorganization of the chondrogenic anlage, more pronounced in zeugopodal mesenchyme than in the autopod. We conclude that an inhibition of chondrogenesis was secondary to a disruption in cellular behavior caused by RA, a likely consequence of misregulation in the growth factor signaling cascade.

The developing limb has been studied extensively and is a useful model to study morphogenesis. During embryogenesis, limb formation is initiated as a budding off from the embryonic lateral body wall. Limb pattern is specified by a series of epithelial-mesenchymal interactions, directing proximodistal, dorsoventral and anteroposterior axes. Vitamin A metabolites, especially retinoic acid, are known to play an important role in limb development, and the effects of retinoic acid may be mediated through the retinoid receptor signaling pathways. Accumulated evidence has shown that inadequate levels (excess or deficiency) of retinoic acid cause a wide range of limb malformations. Some species have the capacity to regenerate amputated limbs, and retinoids certainly affect this process, but there is debate regarding the extent that regeneration recapitulates development. In this review, phenotypic features, pathogenesis and the molecular basis of retinoid-induced limb malformations are discussed with a description of normal limb development and endogenous retinoid pathways.

Background: Eye malformations induced by vitamin A deficiency (VAD) during pregnancy is a major part of the VAD syndrome. But the signaling role of retinoic acid (RA) in ocular tissues is poorly understood. The goal of this study was to determine the role of retinoic acid receptor (RAR) in the development of eye and the possible signaling pathway., Methods: Time-pregnant mice were treated with 1 mg/kg dose of RAR antagonist AGN193109 (AGN) on 8 days postcoitum (dpc). Newborn mice and 18-dpc embryos were used for phenotype studies. Embryonic eyes of 18 dpc were sectioned for histological study. With immunohistochemistry and TUNEL method, we monitored the alternation of AP-2 expression and apoptotic cells in sections of 12- to 18-dpc embryos., Results: Treatment with AGN resulted in severe craniofacial and eye malformations in virtually all exposed fetuses. The ocular abnormalities included severe defects in anterior segments such as focal corneal thickening and eversion, absence of corneal endothelium and anterior chamber, differentiation defects of lens, as well as defects in posterior segment such as persistent hyperplastic primary vitreous and retinal eversions. The percentage of AP-2-positive cells in ocular tissues on 12, 14, and 18 dpc was significantly (P < 0.05) reduced in AGN-treated eyes compared to control ones. Additionally, the number of apoptotic cell was significantly (P < 0.05) increased in AGN-treated eyes., Conclusions: The blocking of RAR function can lead to ocular abnormalities that depict partial phenocopies of vitamin A-deficiency syndrome. Both an inhibition of expression of AP-2 and an enhancement of cell death contribute to AGN-induced ocular defects.

Background: Phocomelia, which is primarily due to a disruption in the proximodistal axis, is found in virtually all mouse embryos exposed to high doses of retinoic acid (RA) on 11 days post coitum (dpc)., Methods: To identify genes that potentially mediate the effects of retinoic acid (RA) on limb development, we have examined the expression of 9,000 clones from the IMAGE consortium by microarray analysis of RNA isolated from 11 dpc mouse forelimbs exposed to RA or vehicle for 6 hr. Eight genes that demonstrated altered expression were chosen for further study of their mRNA levels using RT-PCR. Protein levels were determined by Western blot analysis., Results: Of the 9,000 genes examined in the microarray, approximately 111 demonstrated altered expression (33 known genes and 78 ESTs). Of the eight known genes chosen for further study using RT-PCR, four mRNAs (PBX1a, PBX1b, IGF-Ia, and IGF-Ib) demonstrated consistent elevation ( approximately 3-fold) in their levels after RA treatment in both the forelimbs and hindlimbs as early as 3 hr after RA treatment. In addition to the two PBX1 isoforms, the mRNA level of the other two subtypes (PBX2 and PBX3) and the level of PBX1/2/3 protein were also found to be elevated in limb buds after RA treatment. Finally, we examined the expression of MEIS1, MEIS2, and MEIS3 because these proteins are necessary for PBX nuclear localization. The mRNA level of all three subtypes of MEIS were elevated approximately three- to four-fold in both the forelimbs and hindlimbs after RA treatment., Conclusions: Because both PBX and MEIS (and their orthologs) are believed to be involved in the control of proximodistal axis formation in mouse and fly limbs and IGFs in the development of limbs, we suggest that increases in PBX, MEIS and IGF-1 mRNA levels may contribute to proximodistal limb reduction defects caused by teratogenic doses of RA., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2001 Academic Press.)

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 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.

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.

(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.

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.

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.

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.

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)

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.

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.

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.

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.

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.

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)

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)

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 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)

It has been reported that fractionated doses of 13-cis-retinoic acid are disproportionately more embryotoxic in pregnant mice than is the same dose given in a single bolus. Here, we examined limited pharmacokinetic profiles of a single (100 mg/kg dose given to NMRI mice on day 11 of gestation) versus multiple (3 x 100 mg/kg, 4 h apart) doses in an effort to assess the relative contribution to teratogenicity made by the drug and/or its metabolites. The major plasma metabolite of 13-cis-retinoic acid in the mouse was 13-cis-retinoyl-beta-glucuronide, followed by the 4-oxo metabolites and all-trans-retinoic acid. Transfer to the mouse embryo was very efficient for all-trans-retinoic acid, whereas, it was tenfold less efficient for 13-cis-retinoic acid and 100-fold less efficient for 13-cis-retinoyl-beta-glucuronide. The isomer all-trans-retinoic acid was found in the placenta at concentrations two- to three-fold higher than in the plasma, suggesting placental accumulation as well as placental cis/trans isomerization. Since 13-cis-retinoyl-beta-glucuronide and 13-cis- and all-trans-retinoic acid were detected in the embryo after this multiple dosing schedule, any of the three or their combinations may have been involved in the induction of malformations, but all-trans-retinoic acid, a well-known potent teratogen detected at concentrations of between 590 and 80 ng/g for 10 critical hours during gestation, could have been the major component.

The potential usefulness of the retinoids, a large group of synthetic compounds chemically and structurally related to vitamin A, in the treatment of severe dermatologic diseases and in the prophylaxis and therapy against cancer is severely limited because of their potential teratogenicity. CASE analysis of published retinoid data from the hamster teratogenicity assay and the limb bud "spot" culture system has targeted the hydrophobic region of the retinoids as having the greatest effect on the range of potencies studied. In addition, log p's (as calculated by the CASE program) below a certain value appear to unilaterally result in nonteratogenic retinoids in the in vivo hamster assay system.

Maternal administration of a single dose of retinoic acid (vitamin A acid, 100 mg/kg) on either the 11th, 11 1/2, 12th, 12 1/2, 13th or 13 1/2 day of gestation produced phocomelia or partial phocomelia in ICR/DUB fetuses. The results depended upon the time of treatment and two gradients of effect were produced: 1) cranio-caudal gradient, since forelimb defects resulted from between days 11 and 13, while similar hindlimb abnormalities were produced by administration of retinoic acid 12 to 24 hours later: 2) proximo-distal gradient, due to the heterogenous sensitivity among individual bones of the limb. In the forelimb, early treatment (11th day) produced humero-unlar defects and later treatment (12th day) ulnoradial defects. A similar proximo-distal gradient was observed in the hindlimb. The use of teratological studies as a tool to assist morphogenetic investigation is discussed.

The Consensus Workshop on In Vitro Teratogenesis Testing recommended that test validation be facilitated by a listing of agents with defined teratogenicity; subsequently, a panel was convened to review and select such agents. This communication established a list of 47 compounds or conditions which demonstrate a wide range of teratogenicity in vivo. The agents were chosen primarily on the strength of the literature base denoting their in vivo effects. The tables note a number of general biological and toxicological characteristics for each agent, and the details of representative in vivo teratology studies are summarized and referenced. This list is intended to serve as a base for in vitro teratogenesis test validation and should prove useful in developing and identifying those systems which will contribute to a more effective testing program.

The capabilities of investigators in the fields of teratology and toxicology are greatly enhanced by the use of tissue culture procedures in unraveling the mechanisms of drug action. Techniques currently available for the culture of postimplantation mammalian embryos permit evaluation of their metabolic responses to potential teratogens even when the length of time embryos survive and develop in culture is too short to allow a conventional teratologic survey of malformations. A simple procedure for culturing mouse embryos during early organogenetic stages is described in this report that will be of value to teratologists; it avoids the requirements of special glassware and equipment by using ordinary capped test tubes which are rotated tomaintain and efficient nutritional and gaseous evnironment. Some studies concucted with this procedure to monitor the metabolism of embryo during the first 24 h of culture are summarized. Another aspect of tissue culture, organ culture, provides further manipulative capability by which embryonic organs can be maintained for long periods of time during which they develop and differentiate to an extent that their morphological and biochemical responses to a teratogen can usually be made. Comparative effects of several teratogenic agents and the relative concentration of each that produces a similar degree of response are summarized. It is concluded that organs are more sensitive to teratogens in culture than they are in vivo, and that different teratogens possess enough specificity to isolate their simple growth-retarding effect from the role they play in distrubing other specific developmental events.

The cmd mutation is associated with a systemic defect in all parts of the cartilaginous skeleton. Since limb buds isolated from the embryos and cultured in vitro develop the defect, the gene expression is independent of the humoral factors in the maternal/placental/embryonic environment. The limb development in the mutant progresses normally through early events in morphogenesis including the formation of mesenchymal cell condensations preparatory to chondrogenesis. The deviation first becomes apparent as soon as the chondrogenic cells begin the process of accumulation of extracellular matrix; cmd/cmd chondrogenic cells remain close to each other and lack the abundant extracellular matrix which accumulates between normal cells. Quantitatively normal levels of chondroitin sulfate proteoglycans are synthesized by the mutant limbs during precartilaginous stages. Subsequently, however, the mutant fails to attain the normally high levels of chondroitin sulfate synthesis. Its growth rate also slows down, as judged by the lowered protein synthesis in the mutant cultured limb buds. The lack of at least one species of protein molecules, ie, proteoglycan core-protein, is already known from previous studies on this mutation; an abnormal or a deficient core-protein was shown to lead to a virtually complete shut off of the biosynthesis of cartilage-specific proteoglycans in another mutation in the chick embryo [Goetinck, 1982]. It may be important to note that the cmd mutation does not seem to interfere with the process of determination of cartilage even though it interrupts virtually completely one important biosynthetic aspect of the chondrogenic cell differentiation pathway. The mutant chondrocytes, embedded as they are in an abnormal and proteoglycan-deficient matrix, begin to degenerate prematurely without first undergoing hypertrophy. Also, the process of ossification begins precociously in the shortened cartilage models of the mutant, hence resulting in overall shortening of the limbs. As assessed from the HA:s-GAG ratios during early embryonic limb development, some of the phenotypically normal embryos could be distinguished as recessive carriers of the mutation. Even though these carriers have an intermediate level of chondroitin sulfate proteoglycan synthesis, this does not interfere with their normal development during prenatal stages. It will be of practical importance to follow these carriers through subsequent postnatal stages and adult life to assess any long-term effects.

A dominant mutation in the mouse, Hemimelia-extra toe (Hmx), induces congenital limb malformations in heterozygotes. Typical expression includes axial shortening of the radius, tibia and talus ('hemimelia'), with supernumerary metacarpals, metatarsals, and digits ('polydactyly'). Pathogenesis was investigated during developmental stages 16 through 22 (11th through 15th days of gestation). Full expression was apparent during stage 20 when the limb pattern was comprised of pre-cartilaginous anlagen. Formation of a pre-axial protrusion on the autopod during stage 17 or 18 was the earliest gross abnormality, and foreshadowed the development of supernumerary digits. Microscopically, there was an alteration in the pattern of physiologic cellular degeneration (PCD) programmed to occur within the zeugopod and autopod. The 'opaque patch' (mesodermal necrotic zone normally occurring between tibial and fibular anlagen) was overextended pre-axially causing resorption of the tibial precartilage. Additionally, PCD normally occurring within the basal cell layer of the apical ectodermal ridge (AER) and the 'foyer primaire préaxial' was not expressed in the mutant autopod. This occurred in association with outgrowth of the protrusion. The pre-axial portion of the AER remained in an abnormally thickened, viable, proliferative state, and did not undergo scheduled degression. This may have been the basis for prolonged induction of pre-axial outgrowth. Paucity of mesenchymal cell filopodial processes extended along the basal lamina, as well as a rarefaction of the filamentous material normally associated with the mesodermal face of the basal lamina, was detected at the pre-axial AER-mesenchymal interface on stage 18. A potential involvement of epithelial-mesenchymal interactions in the induction of epithelial PCD is discussed.

The induction of polydactyly in mouse embryos heterozygous for Hemimelia-extra toe (Hmx) is associated with aberrant outgrowth of the developing autopod on day 12 of gestation. We have quantitated the rate of DNA synthesis and the activity of cAMP-phosphodiesterase (PDE) that is characteristic of the prospective polydactylous region. Mid-stage 18 hind-limb buds were labeled with [3H]dThd either in situ using whole embryo culture, or as isolated preaxial autopod fragments cultured on a membrane substratum. The mean specific activities of incorporation were compared for normal (+/+) and mutant (Hmx/+) genotypes. A significant (P less than or equal to 0.01) 19% increase, peculiar to the prospective polydactylous region, was measured after 4 hours in embryo culture. The same increment was detected after 4 hours in organ culture, but was amplified linearly to 55% when incubation was extended to 20 hours. During this period, continuous exposure to 1.0 mM IBMX (3-isobutyl-1-methyl xanthine), an inhibitor of cAMP-PDE activity, "slowed down" the rate of DNA synthesis to untreated +/+ proportions. When cAMP-PDE activity was assayed in uncultured autopods, a significant (P less than or equal to 0.01) 18% increase was detected within the prospective polydactylous region specifically on stage 18 of gestation. This is the developmental phase during which polydactylous outgrowth is induced in situ. Thus, uncontrolled cAMP-PDE activity may, in part, provoke the enhanced rate of cell proliferation.

The need for efficient methods to screen new chemicals, drugs, and environmental pollutants for their teratogenic activity is obvious. The method currently available, ie, pregnant-animal testing, is of considerable value but there are certain drawbacks which prevent reliance on this method alone as the predictive device. Nutritional state of the dam, variability in the developmental age of embryos from litter to litter and even within the same litter, metabolic differences between species, placental function, and a host of other factors must be taken into account before data obtained from animal testing can be logically extrapolated to human situation. Many of these variables are either eliminated altogether or at least can be controlled by the use of tissue culture techniques. After surveying a variety of in vitro systems, it is our opinion that organ culture, whole embryo culture, and a combination of the two offer at present the best potential for screening of suspected teratogens. These culture techniques provide a much better simulation of in vivo situations than isolated cells grown as monolayers. Among other advantages, these procedures allow one to exercise control over the effective concentration of the suspected teratogen to which an embryo is exposed and also the duration of this exposure. Since maternal metabolism or modification of the drug is routinely eliminated in these experiments, there is a need for exploring the use of drug-metabolizing preparations as additives to the culture medium. The choice of limb bud in the screening system is promising since during its development, the limb progresses through a succession of embryonic processes that are generally relevant to other organ systems as well. Hence, such a screening system may not only predict teratogenicity but also provide insight into the mechanisms by which a test chemical is teratogenic.

Various morphological and biochemical parameters were used to study the mode of interference by cytosine arabinoside (Ara-C) in critical phases of embryonic limb development. Inhibition of embryonic DNA synthesis occurred immediately after injection of Ara-C into the mother. The inhibition was dose-dependent and was substantial even after the nonteratogenic dose (2 mg/kg) of Arc-C. The pattern of limb bone deficiencies in Ara-C treated fetuses was specific for each developmental stage at which the treatment was given; the site of affect moved distalwards along the limb as the development advanced. The teratogenic dose was cytotoxic to mesenchymal cells with a high proliferation rate but did not affect others such as the future cartilage cells in which the rate of proliferation was lower. The existence of this differential susceptibility at each stage of development, together with information about the pattern of bone defects at the same stage, permitted us not only to define with some precision the cellular basis of origin of limb defects but also to infer the relative level of cell differentiation pertaining to each successive stage. Deoxycytidine, if injected simultaneously with and at doses eight times larger than Ara-C, afforded virtually complete protection against teratogenic effects. Deoxycytidine also prevented cell death in the limbs of Ara-C treated embryos. However, a dramatic increase in the frequency of polydactyly was found in the protected fetuses. The fact that the frequency of ectrodactyly in the protected fetuses decreased in inverse proportion to the frequency of polydactyly strengthened the notion that there may be a common cellular basis underlying these two types of digital defects. Striking changes were found in the structure of AER at 24 hours after Ara-C treatment; it was abnormally thickened into a gland-like structure and its inner edge facing the mesenchyme thickened into a gland-like structure and its inner edge facing the mesenchyme was thrown into several folds. This may constitute a response to impairment in the underlying mesenchyme with which AFR has long been considered to have an interdependent relationship.

The transplacental pharmacokinetics of single teratogenic doses of etretinate and motretinide were compared with particular emphasis on distribution and concentrations in the exposed embryos of the free acid metabolite, etretin. The three aromatic retinoids were also tested for their direct inhibitory effect on chondrogenesis in the limb bud mesenchymal cell "micromass" culture assay. After a standard dose of 100 mg/kg administered on day 11 of gestation in NMRI mice, all three compounds were teratogenic, but they differed from each other in potency. Etretinate was most active as a teratogen, equalling the potency of our standard all-trans-retinoic acid; every exposed fetus was deformed with severe shortening of all limb bones as well as cleft palate. Etretin was less potent than etretinate, and motretinide was considerably less active as a teratogen than the other two. In the in vitro assay, only etretin suppressed chondrogenesis and this activity was equivalent to that of all-trans-retinoic acid (IC50 of 12 ng/ml). Both etretinate and motretinide (which contain an ethyl ester and ethylamide terminal group, respectively) were essentially inactive in vitro, demonstrating the fact that a free carboxylic group may be a requirement for the in vitro suppression of chondrogenesis. These differences between the results obtained in vivo and in vitro could be resolved by pharmacokinetic investigations using HPLC methods. Both etretinate and motretinide were metabolized in vivo to etretin, their likely common teratogenic metabolite. The high teratogenic potency of etretinate was probably the result of high concentrations as well as AUC values of its metabolite etretin in the embryo. On the other hand, the comparatively low teratogenicity of motretinide could be related to approximately 5 x lower embryonic peak levels as well as AUC values of etretin. A comparison of these results with those previously obtained for all-trans- and 13-cis-retinoic acids confirms the correlation between embryonic exposure and teratogenic potency in the mouse. Our results indicate that pharmacokinetic studies are essential for the interpretation of relative teratogenic potencies of retinoids as well as apparent differences between in vivo and in vitro teratogenesis. A free carboxyl group at the terminal end of the tetraene chain was necessary for high activity of the retinoids studied.

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.

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)

The glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), has been shown to inhibit biosynthesis of purines and glycosaminoglycans, presumably by blocking the glutamine-dependent steps in the biosynthetic pathways. The teratogenic potential of DON on the developing mouse limb-bud in vivo and in vitro was studies in an attempt to discriminate whether DON is exerting its teratogenic effect by interfering with glycosaminoglycan or purine metabolism. A single intramuscular injection of DON (0-5 mg/kg) to ICR/DUB mice on day 10 of gestation resulted in 76% resorption, while fetuses surviving to day 17 exhibited growth retardation, median cleft lip, and limb malformation. Concurrent administration of L-glutamine (250 mg/kg) provided no protection against resorption of malformations, while 5-aminoimidazolecarboxamide (AIC, 250 mg/kg) decreased the resorption rate to 34% without significantly altering the incidence of malformations. Injection of DON alone on day 11 resulted in 87% of fetuses exhibiting limb formations, with only 2% resorption. Concurrent injection of AIC decreased the frequency of limb malformations to 32% L-Glutamine, D-glucosamine, or inosinic acid were without any protective effect in vivo. DON (5mug/ml medium) added in vitro to organ cultures of day 11 mouse limb-buds caused all limbs to evidence cartilage abnormalities. In this system, either L-glutamine ro D-glucosamine (0-5 mg/ml medium) provided protection against DON effects while AIC (0-5 mg/ml medium) offered no protection in vitro. These data suggest that DON exerts its effects in vivo by interfering with purine metabolism while in vitro its teratogenic action may be interruption of glycosaminoglycan biosynthesis. This may reflect upon the relative importance of growth and differentiation to limb development in vivo and in vitro. These data infer that limb development in vitro relies more on the differentiative process (differentiation of cartilage) than on growth, whereas limb development in vivo is dependent, at this stage, to a greater extent on growth for normal phenotypic expression.

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.

The inhibitory effect of 5-bromodeoxyuridine (BudR) - an analog of thymidine - on embryonic mouse limb-buds was studied in vitro employing an organ-culture system. The effect was found to be dose-related and also depended on the developmental stage of the donor embryos. Limbs at an early stage development (early 11 th-day embryos, somite stage 26-29) were extremely sensitive to the analog. Treatment with low levels (2-4 mug/ml) and for a relatively short period of time in cluture (2-3 days) completely and irreversibly suppressed chondrogenesis in these explants. Limbs from older embryos (somite stage 40 and up) were found to be much less sensitive to the inhibitory effect of the drug; a prolonged exposure to a much higher dose (100-150 mug/ml) resulted in an incomplete suppression of chondrogesesis. Only a 20% inhibition was observed in the cultures of limbs from mid-13th-day mouse embryos. After continuous growth in vitro, the limbs became progressively resistent to the analog and towards the end of the culture period had become refractory to the drug. The time of complete insensitivity appeared earlier in the cures of the limbs taken from older embryos than in the explants of youngerlimbs. These studies show that as limbs continue to differentiate in vivo or in vitro, they become increasingly resistent to the inhibitory effect of BudR in at least as far as the effect on the process of chondrogenesis is concerned. It is suggested that the relative sensitivity or insensitivity to the inhibitory effect of BudR may prove to be a useful parameter in evaluating the developmental stage of an organ.

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid in a dose-dependent fashion. Cells prelabeled with (3H) arachidonic acid were treated with 0.3 microgram/ml retinoic acid. Treatment with retinoic acid increased the (3H) fatty acid in the triglyceride fraction. Furthermore, treatment with retinoic acid enhanced the release of (3H) fatty acid upon stimulation of these cells with the divalent ionophore A23187. These data permit the suggestion that there may be a correlation between altered lipid metabolism and retinoic acid's ability to disrupt chondrogenic differentiation.

Because of the automatic restrictions and warning requirements imposed on substances identified by the state as "known to cause developmental toxicity," the Expert Committee recommends the use of criteria that emphasize human relevancy, biological plausibility, and evidence in support of a selective, adverse developmental effect at non-maternally-toxic doses. In many instances, data for substances of public concern will be insufficient at present to meet these criteria. The fact that a substance is not listed as "known to cause developmental toxicity" does not create a presumption that the substance is safe. The Expert Committee, therefore, urges that these substances be recommended for further testing and that high priority be given to conducting the necessary tests. The Expert Committee reiterates its concern that substances listed by the SAP be identified according to the toxic endpoints (cancer, male reproductive toxicity, female reproductive toxicity, and/or developmental toxicity) that led to listing. Further, the Expert Committee recommends that the state Health and Welfare Agency institute education programs emphasizing appropriate courses of action for citizens informed of exposures to substances known to the state to cause cancer, birth defects, or reproductive toxicity.

13-cis-Retinoic acid (isotretinoin) is teratogenic in man at therapeutic doses (0.5-1.5 mg/kg body wt), but only marginally teratogenic in the mouse at exceedingly high doses (greater than 100 mg/kg). On the other hand, the isomer all-trans-retinoic acid (tretinoin) is teratogenic in the mouse at dose levels which are 10 times lower than those for the 13-cis isomer. We have therefore studied whether the greatly different teratogenic potencies of these two compounds in the mouse are the result of differences in their transplacental kinetics. Following a single oral dose of 100 mg all-trans- or 13-cis-retinoic acid per kg body wt, concentrations of the parent drugs, of the C-13 isomerization products, as well as of their 4-oxo metabolites were determined in maternal plasma and embryo at two sensitive stages of organogenesis, i.e., Days 9 or 11 of gestation. All-trans-retinoic acid and its 4-oxo metabolite were transferred to the embryo to a much greater extent (embryo/maternal plasma concentration ratios, approximately 0.4) than the 13-cis-retinoic acid and its 4-oxo metabolite (embryo/maternal plasma concentration ratios, approximately 0.02). Embryo concentrations of all-trans-retinoic acid on Day 9 of gestation exceeded those on Day 11, whereas the embryo levels of 13-cis-retinoic acid were minimal at both gestational stages. The concentration of the 4-oxo metabolite of all-trans-retinoic acid was generally lower than that of the parent drug, whereas the level of the 4-oxo metabolite of the 13-cis-retinoic acid was comparable with or even higher than that of the parent compound. Concentrations of the C-13 isomerization products in maternal plasma were less than 20% of corresponding parent drug levels. However, due to the different extent of transfer of the two isomers, the concentration of all-trans-retinoic acid in the embryo exceeded that of the cis isomer even after administration of 13-cis-retinoic acid. Our results indicate that the low teratogenicity of 13-cis-retinoic acid in the mouse is the result of minimal placental transfer of this compound and of its 4-oxo metabolite, which contrast sharply with extensive placental transfer and high teratogenicity of the corresponding isomers with the all-trans configuration.