Assessment of environmental stressors potentially responsible for malformations in North American anuran amphibians.

Several species of anuran amphibians from different regions across North America have recently exhibited an increased occurrence of malformations, predominantly of the hindlimb. Research concerning the potential causes of these malformations has focused extensively on three stressors: chemical contaminants, ultraviolet (UV) radiation, and parasitic trematodes. In this overview of recent work with each of these stressors, we assess their plausibility as contributors to the malformations observed in field-collected amphibians. There is as yet little evidence that chemical contaminants are responsible for the limb malformations. This includes chemicals, such as the pesticide methoprene, that could affect retinoid-signaling pathways that are critical to limb development. Exposure to UV radiation also seems to be an unlikely explanation for hindlimb malformations in amphibians. Although solar UV can cause hindlimb deficiencies in amphibians, a probabilistic assessment based on empirical dose-response and exposure data indicates that UV exposures sufficient to induce limb defects would be uncommon in most wetlands. Results of controlled studies conducted with some affected species and field-monitoring work suggest infection by digenetic trematodes as a promising explanation for the malformations observed in anurans collected from many field sites. Controlled experimentation with additional species and monitoring across a broader range of affected sites are required to assess fully the role of trematodes in relation to other stressors in causing limb malformations. If trematode infestations are indeed related to the recent increases in malformed amphibians, then the question remains as to what alterations in the environment might be causing changes in the distribution and abundance of the parasites.

Xenoendocrine disrupters-tiered screening and testing: filling key data gaps.

The US Environmental Protection Agency (EPA) is developing a screening and testing program for endocrine disrupting chemicals (EDCs) to detect alterations of hypothalamic-pituitary-gonadal (HPG) function, estrogen (ER), androgen (AR) and thyroid hormone synthesis and AR and ER receptor-mediated effects in mammals and other animals. High priority chemicals would be evaluated in the Tier 1 Screening (T1S) battery and chemicals positive in T1S would then be tested (Tier 2). T1S includes in vitro ER and AR receptor binding and/or gene expression, an assessment of steroidogenesis and mammalian (rat) and nonmammalian in vivo assays (Table 1). In vivo, the uterotropic assay detects estrogens and antiestrogens, while steroidogenesis, antithyroid activity, (anti)estrogenicity and HPG function are assessed in a 'Pubertal Female Assay'. (Anti-) androgens are detected in the Hershberger Assay (weight of AR-dependent tissues in castrate-immature-male rats). Fish and amphibian assays also are being developed. The fathead minnow assay can identify EDCs displaying several mechanisms of concern, including AR and ER receptor agonists and antagonists and inhibitors of steroid hormone synthesis. An amphibian metamorphosis assay is being developed to detect thyroid-active substances. Several alternative mammalian in vivo assays have been proposed. Of these, a short-term pubertal male rat assay appears most promising. An in utero-lactational screening protocol also is being evaluated. For Tier 2, the numbers of endocrine sensitive endpoints and offspring (F1) examined in multigenerational tests need to be expanded for EDCs. Consideration should be given to tailoring T2, based on the results of T1S. Tier 1 and 2 also should examine relevant mixtures of EDCs. Toxicants that induce malformations in AR-dependent tissues produce cumulative effects even when two chemicals act via different mechanisms of action.

Ambient solar UV radiation causes mortality in larvae of three species of Rana under controlled exposure conditions.

Recent reports concerning the lethal effects of solar ultraviolet-B (UV-B) (290-320 nm) radiation on amphibians suggest that this stressor has the potential to impact some amphibian populations. In this study embryos and larvae of three anuran species, Rana pipiens, Rana clamitans and Rana septentrionalis, were exposed to full-spectrum solar radiation and solar radiation filtered to attenuate UV-B radiation or UV-B and ultraviolet-A (UV-A) (290-380 nm) radiation to determine the effects of each wavelength range on embryo and larval survival. Ambient levels of solar radiation were found to be lethal to all three species under exposure conditions that eliminated shade and refuge. Lethality was ameliorated by filtration of UV-B radiation alone, demonstrating that ambient UV-B radiation is sufficient to cause mortality. Although several studies have qualitatively demonstrated the lethality of UV-B to early life stage amphibians this study demonstrates that the larval life stages of the three species tested are more sensitive than the embryonic stages. This suggests that previous reports that have not included the larval life stage may underestimate the risk posed to some anuran populations by increasing UV-B exposure. Furthermore, this study reports quantitative UV-B dosimetry data, collected in conjunction with the exposures, which can be used to begin the assessment of the impact of environmental changes which increase UV-B exposure of these anurans.

Stage- and species-specific developmental toxicity of all-trans retinoic acid in four native North American ranids and Xenopus laevis.

Within the last decade, there have been increasing reports of malformed amphibians across North America. Recently, it has been suggested that hind-limb malformations are a consequence of xenobiotic disruption of developmental pathways regulated by retinoids. To assess the validity of this hypothesis, the developmental toxicity of all-trans retinoic acid (RA) was examined in Xenopus laevis and four North American anurans, at several life stages. To determine the effects of RA on embryogenesis, mid-blastula stage embryos were exposed to 0, 6.25, 12.5, 25, or 50 ng RA/ml for 24 h. To evaluate the effects of RA on hind-limb development, early- and mid-limb bud stage tadpoles were exposed to RA concentrations of 0, 250, 500, 750, 1000, or 1250 ng RA/ml for 24 h. Mid-blastula RA exposure resulted in a concentration-dependent increase in dysmorphogenesis and mortality in the three species examined (R. clamitans, R. septentrionalis and X. laevis). RA exposure at stage 51 in X. laevis and stage 28 in R. sylvatica resulted in concentration-dependent increases in reductions and deletions of the hind limb. However, RA was ineffective at inducing hind-limb abnormalities in stages 26 and 28 of R. pipiens, stage 28 in R. clamitans, or stage 48 in X. laevis tadpoles. These results indicate that mid-blastula stage embryos are more sensitive to RA-induced dysmorphogenesis and mortality than limb-bud stage tadpoles. The significance of these findings is discussed in the context of the possible occurrence of retinoid mimics in the environment.

Role of TGF-beta in RA-induced cleft palate in CD-1 mice.

Retinoic acid (RA) plays an important role in embryogenesis, by regulating morphogenesis, cell proliferation, differentiation, and extracellular matrix production. RA exposure on gestational day (GD) 12 in CD-1 mice results in delayed palatal shelf elevation and subsequent clefts in the secondary palate. Given the dynamic and complex nature of palate development, it is not surprising that this system is susceptible to changes in retinoid levels. There is evidence that experimental manipulation of retinoid status during development alters normal transforming growth factor-beta (TGF-beta) status. To study the role of perturbation in TGF-beta levels in RA-induced cleft palate, gravid CD-1 mice were treated with 70 mg/kg RA on GD 12. We examined changes in TGF-beta proteins and the steady-state level of TGF-beta mRNA within the first 24 hr after exposure. The interactions between RA and TGF-beta s were very complex. RA differentially regulated the mRNA and protein levels of TGF-beta 1. Changes in mRNA steady-state levels were rapid and transient in nature, indicating a direct mediation by RA. Differential regulation was evident, because RA treatment resulted in an increase in TGF-beta 1 mRNA steady levels followed by a decrease in the intracellular and extracellular forms of TGF-beta 1 protein. Moreover, the patterns of localization and levels of TGF-beta 2 and TGF-beta 3 proteins were not dramatically affected, although there was an increase in TGF-beta 3 mRNA steady-state levels. The increases in mRNA steady-state levels for TGF-beta 2 and TGF-beta 3, as for TGF-beta 1, were rapid and transient in nature, again arguing for direct mediation by RA. These data provide evidence for interactions between RA and TGF-beta s, and indicate that RA is capable of differentially regulating TGF-beta isoforms through processes involving different stages of TGF-beta synthesis and secretion. Further, changes in TGF-beta isoforms were observed prior to changes in mesenchyme morphology and must be considered as mediators of RA's effects on mesenchyme development.

Mesenchymal changes associated with retinoic acid induced cleft palate in CD-1 mice.

Retinoic acid (RA) is teratogenic in many species and is an effective inducer of cleft palate in mice. The pathogenesis of cleft formation varies with the timing of exposure. It has been demonstrated, before formation of the palatal shelves, that RA exposure results in insufficient mesenchymal tissue, and palatal shelves fail to make contact. However, at the palatal shelf outgrowth stage, RA exposure affects shelf elevation and growth in rats, and possibly medial edge epithelium (MEE) differentiation in mice. The objective of this study was to examine the morphologic and functional changes associated with cleft formation in mice following exposure during shelf outgrowth. Particular emphasis was placed on evaluating the timing of palatal shelf elevation in RA exposed embryos and on identifying differentiation events occurring concurrently in the epithelium. On gestational day (GD) 12 (8:00 AM), gravid CD-1 mice were gavaged with 70 mg/kg RA or vehicle. This protocol produced a 100% incidence of cleft palate at term, allowing us to correlate the morphological and/or biochemical changes observed at pre-fusion time points. Embryos were collected at 12 hr intervals through GD 15, beginning 4 hr after exposure. Serial sections of embryos were either stained with H&E, with a battery of lectins [Sambucus nigra (SNA), Arachis hypogaea (PNA), Ricinus communis (RCA-1), Glycine max (SBA), Succinylated Wheat Germ (S-WGA)], or with a probe to hyaluronan. Throughout the period of normal palate development, the shelf mesenchyme showed increasing regional organization and progressive hydration and these changes were correlated with increase Hyaluronan (HA) deposition. RA treatment resulted in lose of regional organization and delayed mesenchyme hydration. In association with these changes there were reductions in HA deposition and extracellular matrix glycoconjugates recognized by PNA in the palate mesenchyme. Further there was a considerable delay in palatal shelf elevation and palate shelf did not make contact at the midline. Our data indicates, in embryos exposed on GD 12 to levels of RA sufficient to induce a 100% incidence of clefting, that cleft formation is a result of palatal shelves failing to make contact. Alterations in mesenchyme development and the subsequent delay in palate shelve elevation are central to RA-induced cleft formation following exposure at the palate shelf out growth stage.

Maternal and developmental toxicity of polychlorinated diphenyl ethers (PCDEs) in Swiss-Webster mice and Sprague-Dawley rats.

Polychlorinated diphenyl ethers (PCDEs) are industrial byproducts found in many ecosystems at low levels. PCDEs are not markedly toxic to adult rodents, but their developmental toxicity has not previously been examined. We evaluated the maternal and perinatal toxicity of nine PCDE congeners to outbred mice when compounds were administered from gestation day (GD) 6 through GD 15. 2,2',4,4',5,6'-hexaCDE and 2,3',4',6-tetraCDE decreased the number of pups born per female mated and the number of pups surviving per litter born. 2,2',4,4',5,5'-hexaCDE and 2,2',4,5,6'-pentaCDE decreased the number of litters born per female mated, without decreasing postnatal survival. The other PCDEs did not decrease survival either pre- or postnatally. None of the PCDEs caused absence of Harderian glands in surviving offspring at the doses administered. Neither induction of cytochromes P450 nor tissue residues of individual congeners correlated well with developmental toxicity. Three PCDEs were also evaluated in outbred (Sprague-Dawley) rats: 2,2',4,5,6'-pentaCDE and 2,3',4',6-tetraCDE, because of their toxicity to mice; 2,2',4,4',5,5'-hexaCDE, because it should exhibit PCB-like toxicity. Each congener was administered at three dose levels from GD 6 through GD 15. 2,2',4,5,6'-pentaCDE decreased the number of litters born at 100 mg/kg/day, and the survival of pups in litters carried to term, at both 50 and 100 mg/kg per day. Postnatal weight gain was also reduced. In contrast to its action in mice, 2,3',4',6-tetraCDE decreased neither the numbers of litters born nor postnatal survival of rat offspring, but did suppress postnatal weight gain at least through PD 5. As in mice, induction of cytochromes P450 was not well correlated with the developmental toxicity of individual congeners.