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Trenbolone is an androgen agonist used in cattle production and has been measured in aquatic systems associated with concentrated animal-feeding operations. In this study, the authors characterized the effects of aqueous exposure to 17β-trenbolone during larval Xenopus tropicalis development. Trenbolone exposure resulted in increased mortality of post-Nieuwkoop-Faber stage 58 tadpoles at concentrations ≥100 ng/L. Morphological observations and the timing of this mortality are consistent with hypertrophy of the larynx. Development of nuptial pads, a male secondary sex characteristic, was induced in tadpoles of both sexes at 100 ng/L. Effects on time to complete metamorphosis or body sizes were not observed; however, grow-outs placed in clean media for six weeks were significantly smaller in body size at 78 ng/L. Effects on sex ratios were equivocal, with the first experiment showing a significant shift in sex ratio toward males at 78 ng/L. In the second experiment, no significant effects were observed up to 100 ng/L, although overall sex ratios were similar. Histological assessment of gonads at metamorphosis showed half with normal male phenotypes and half that possessed a mixed-sex phenotype at 100 ng/L. Hypertrophy of the Wolffian ducts was also observed at this concentration. These results indicate that larval 17β-trenbolone exposure results in effects down to 78 ng/L, illustrating potential effects from exposure to androgenic compounds in anurans. Environ. Toxicol. Chem. 2012; 31: 2391-2398. © 2012 SETAC [ABSTRACT FROM AUTHOR]

In response to the initial Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. This research highlighted key limitations associated with relying on tail resorption as a measure of anti/thyroid activity. The most critical limitation being that tail tissues of tadpoles at metamorphic climax are insensitive to perturbation by thyroid axis agonists/antagonists. To improve upon the initial proposal, we have conducted experiments comparing the sensitivity of pre-metamorphic (stage 51) and pro-metamorphic (stage 54) larvae to the model thyroid axis disruptors methimazole (control, 6.25, 12.5, 25, 50, 100 mg/l), 6-propylthiouracil (PTU) (control, 1.25, 2.5, 5, 10, and 20 mg/l), and thyroxine (T4) (0.25, 0.5, 1, 2, 4 μg/l). Exposures were conducted using two different experimental designs. For experimental design 1, tadpoles were exposed to methimazole or PTU starting at either NF stage 51 or NF 54 for 14 days. For experimental design 2, tadpoles were exposed to PTU or T4 starting at NF stage 51 or NF 54 for 14 and 21 days, respectively. Methimazole and PTU, which are thyroid hormone synthesis inhibitors, both caused a concentration dependent delay in larval development. As determined from this endpoint, there were only minor differences in sensitivity observed among the two stages examined. Further, both compounds caused concentration dependent changes in thyroid gland morphology. These changes were characterized as reduced colloid, glandular hypertrophy, and cellular hyperplasia and hypertrophy. Treatment failed to negatively affect growth, even in tadpoles that experienced significant metamorphic inhibition. T4 treatment resulted in a concentration dependent increase in developmental rate, as would be expected. Similar to studies with methimazole, there were no differences in sensitivity among the two developmental stages examined. These results indicate that tadpoles in the early stages of metamorphosis are sensitive to thyroid axis disruption and that development of a short-term, diagnostic amphibian-based thyroid screening assay shows considerable promise. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Abstract: Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzyme''s activity by xenobiotic exposure has been shown to adversely affect gonad differentiation in a number of diverse species. We hypothesized that exposure to the aromatase inhibitor, fadrozole, during the larval development of the tropical clawed frog, Xenopus tropicalis, would result in masculinization of the developing female gonad. Tadpoles were exposed to fadrozole at nominal concentrations from 1 to 64μg/L in a flow-through system from <24h post-fertilization (Nieuwkoop Faber (NF) stage 15–20) to metamorphosis (NF stage 66). At metamorphosis, morphologically examined gonads indicated complete masculinization of all tadpoles at concentrations of 16μg/L and above and a significant bias in sex ratio towards males at concentrations of 1μg/L and above. No effects on time to metamorphosis, body mass, or body length were observed. A random subsample of frogs was raised to reproductive maturity (39 weeks post-fertilization) in control water. All frogs exposed as tadpoles to 16μg/L fadrozole or greater possessed testes at sexual maturity. Intersexed gonads characterized by the presence of both testicular and ovarian tissue were observed in 12% of frogs in the 4μg/L treatment. No differences in estradiol, testosterone, or vitellogenin plasma concentrations were observed in exposed males or females compared to controls. Females in the 4μg/L treatment possessed a significantly greater percentage of pre-vitellogenic oocytes than controls and were significantly smaller in body mass. No differences in sperm counts were observed in exposed males compared to controls. Results from this study demonstrate that larval exposure to an aromatase inhibitor can result in the complete masculinization of female gonads. These masculinized females are phenotypically indistinguishable from normal males at adulthood. Lower levels of aromatase inhibition resulted in intersexed gonads and possible female reproductive impairment at adulthood. These results indicate that exposure of amphibians to xenobiotics capable of inhibiting aromatase would result in adverse reproductive consequences. [Copyright &y& Elsevier]

Fathead minnows (Pimephales promelas) are a widely-used small fish model for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). Quantitative real-time PCR assays for measuring fathead minnow gonadotropin (GtH) beta subunit transcripts were developed and "baseline" transcript levels in pituitary tissue were examined over a range of age classes and spawning states. Among females, GtHbeta transcripts did not vary significantly with gonadal-somatic index or gonad stage. However, in males, follicle-stimulating hormone beta subunit transcripts decreased significantly with increasing gonad stage, while mean luteinizing hormone beta subunit expression trended in the opposite direction. GtHbeta transcript levels measured in pituitaries from fish that had spawned within the preceding 24 h were not significantly different from those from fish that were 2-3 days post-spawn. Exposure to the fungicide ketoconazole, a known steroidogenesis inhibitor, for 21 days significantly affected the abundance of GtHbeta transcripts in pituitary tissue in males, but not females. This study provides critical data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression. Results of such experiments should facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.