Devendra M. Kochhar

Embryotoxic, teratogenic, and metabolic effects of ribavirin in mice☆

Abstract To gain comparative perspective on the teratogenic mechanisms of action of drugs that are precursor analogs or antimetabolites of nucleic acids, we employed a new antiviral agent—ribavirin—which is known in other systems to inhibit the biosynthesis of guanine nucleotides. Pregnant ICR mice were injected at 10th–13th days of gestation (stages 14–20) with a single ip dose of ribavirin in the range of 10–200 mg/kg. All dosages in excess of 25 mg/kg were teratogenic. The optimal teratogenic dose varied with the stage of development, being higher at advanced stages of development. Depending on the dose and stage of treatment, virtually all parts of the skeleton including the craniofacial and limb bones were susceptible to ribavirin. Both the frequency and multiplicity of skeletal defects increased as the dose was raised. The stage dependency of defects in the orofacial bones was markedly apparent. Treatment on Day 10.5 resulted in shortened maxilla in all survivors, while treatment on either Day 11 or 11.5 resulted in a high frequency of reduction in the length of both upper and lowerjaws. Treatment on the 12th day resulted in a very low incidence of effect on the maxilla (4%) but a high frequency (88–100%) of reduction and deformation of the mandible. This enhanced susceptibility of individual facial bones at different stages of development with virtually no overlap provides an experimental model to study cellular phenomena underlying normal and abnormal facial development. Ribavirin, both in vivo and in vitro , inhibited embryonic DNA synthesis. The inhibition was transitory and did not seem to be directly related to the embryolethal activity of the drug. Although the role of metabolic inhibition in precipitating teratogenesis is not clear, cytotoxic action of ribavirin against proliferating limb bud mesenchymal cells is directly associated with the origin of limb deformities.

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

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

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.

A sustained elevation in retinoic acid receptor-β2 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.

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.

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

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

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.

A history of the Teratology Society.

BACKGROUND The 39-year history of the Teratology Society is reviewed. An abbreviated history is outlined in table form, along with listings of the Warkany Lectures, the postgraduate courses, and officers of the Society. METHODS A year-by-year description of the events, including the scientific and social content of the annual meetings and changes in the business of the Society, is given, in many cases using comments from the past presidents. The valuable and unique diversity of the members is discussed and illustrated, presenting the disciplines and main research area of the presidents. The number of submitted abstracts and the various categories are tabulated, averaging the number and type over four periods. Within the past 10 years, a significant increase in the number of abstracts dealing with epidemiology and developmental biology is evident. The Societys development is compared with that of a human, and the question is asked: Have we reached the maturational stage of old age or senescence, or is the Society still maturing gracefully? This question needs further discussion by all the members. RESULTS During the past 40 years, we have developed the scientific basis to prevent birth defects caused by rubella, alcoholism, and folate deficiency, as well as many other prenatal exposures. CONCLUSIONS We must now engage in the political battles to obtain the resources needed to conduct further research and to implement the prevention programs, as well as to provide care and rehabilitation for persons with birth defects.

The Hemimelic extra toes mouse mutant: Historical perspective on unraveling mechanisms of dysmorphogenesis

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.

Retinoids and Retinoid Receptors in Teratogenesis

There is overwhelming evidence that vitamin A (retinol), presumably through its metabolite retinoic acid, participates in organogenesis at several stages and sites during normal development. Besides the important role of retinol and retinoic acid (RA) as micronutrients in growth and development, these retinoids and their synthetic analogs are now viewed as drugs for treatment of oncologic and dermatologic diseases. An excess of vitamin A, RA, or several other synthetic analogs are teratogenic. Mechanisms involved in teratogenesis remain unsolved but are under active investigation in many laboratories. The attention has recently focused on a series of endogenous proteins which serve as nuclear receptors for natural retinoids as means to discover how retinoids intervene in diverse cellular functions and which of their cellular and molecular targets are crucial to the developing embryo. There are two classes of receptors, termed retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This brief review summarizes the results of our recent studies which suggest that: 1) the teratologic effects of retinoids are mediated by the nuclear receptors; 2) the heterodimer RXR/RAR pathway is the major mechanism for the induction of teratogenesis; 3) RXR‐selective synthetic retinoids have diminished teratogenicity; and 4) an overexpression of specific RARs in response to RA disrupt skeletal morphogenesis resulting in limb reduction defects.