Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole.

Determining the effects of chemicals on the thyroid system is an important aspect of evaluating chemical safety from an endocrine disrupter perspective. Since there are numerous chemicals to test and limited resources, prioritizing chemicals for subsequent in vivo testing is critical. 2-Mercaptobenzothiazole (MBT), a high production volume chemical, was tested and shown to inhibit thyroid peroxidase (TPO) enzyme activity in vitro, a key enzyme necessary for the synthesis of thyroid hormone. To determine the thyroid disrupting activity of MBT in vivo, Xenopus laevis larvae were exposed using 7- and 21-day protocols. The 7-day protocol used 18-357 µg/L MBT concentrations and evaluated: metamorphic development, thyroid histology, circulating T4, circulating thyroid stimulating hormone, thyroidal sodium-iodide symporter gene expression, and thyroidal T4, T3, and related iodo-amino acids. The 21-day protocol used 23-435 µg/L MBT concentrations and evaluated metamorphic development and thyroid histology. Both protocols demonstrated that MBT is a thyroid disrupting chemical at the lowest concentrations tested. These studies complement the in vitro study used to identify MBT as a high priority for in vivo testing, supporting the utility/predictive potential of a tiered approach to testing chemicals for TPO activity inhibition. The 7-day study, with more comprehensive, sensitive, and diagnostic endpoints, provides information at intermediate biological levels that enables linking various endpoints in a robust and integrated pathway for thyroid hormone disruption associated with TPO inhibition.

Trenbolone causes mortality and altered sexual differentiation in Xenopus tropicalis during larval development.

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 17ß-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 17ß-trenbolone exposure results in effects down to 78 ng/L, illustrating potential effects from exposure to androgenic compounds in anurans.

Effects of 4-tert-octylphenol on Xenopus tropicalis in a long term exposure.

Endocrine disrupting chemicals that activate the estrogen receptor are routinely detected in the environment and are a concern for the health of both exposed humans and indigenous wildlife. We exposed the western clawed frog (Xenopus tropicalis) to the weak estrogen octylphenol from Nieuwkoop-Faber (NF) stage 46 tadpoles through adulthood in order to document the effects of a weak estrogen on the life history of an amphibian species. Frogs were exposed to 1, 3.3, 11 and 36 µg/L octylphenol in a continuous flow-through water system. Just prior to completion of metamorphosis (NF 65), a random subsample of froglets was collected and assessed, while the remaining frogs received continued exposure through 31 weeks of exposure when the remaining animals were sampled. Significant induction of the female egg yolk protein precursor vitellogenin was observed in the high treatment at the larval subsampling for both males and females, but not at the final sampling for either sex. No significant deviation from the control sex ratio was observed for either sampling period, suggesting minimal to no effect of octylphenol exposure on gonad differentiation. No effects in the adult frogs were observed for mortality, body mass and size, liver somatic index, estradiol and testosterone serum levels, sperm counts, or oocyte counts. The development and growth of oviducts, a female-specific secondary sex characteristic, was observed in males exposed to octylphenol. These results indicate that octylphenol exposure can induce vitellogenin in immature froglets and the development of oviducts in male adult frogs. The lack of effect observed on the developing gonads suggests that in amphibians, secondary sex characteristics are more susceptible to impact from estrogenic compounds than the developing gonads.

A method for the determination of genetic sex in the fathead minnow, Pimephales promelas, to support testing of endocrine-active chemicals.

Certain endocrine-active toxicants have been reported to completely sex reverse both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species, resulting in a phenotype indistinguishable from unaffected individuals. Detection of low-level sex reversal often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with predominantly genetic sex determination and cryptic/homomorphic sex chromosomes. Here we describe a method for determining the genetic sex in the commonly used ecotoxicological model, the fathead minnow (Pimephales promelas). Analysis of amplified fragment length polymorphisms (AFLP) in a spawn of minnows resulted in detection of 10 sex-linked AFLPs, which were isolated and sequenced. No recombination events were observed with any sex-linked AFLP in the animals examined (n=112). A polymerase chain reaction (PCR) method was then developed that determined the presence of one of these sex-linked polymorphisms for utilization in routine toxicological testing. Analyses of additional spawns from our in-house culture indicate that fathead minnows utilize a XY sex determination strategy and confirm that these markers can be used to genotype sex; however, this method is currently limited to use in laboratory studies in which breeders possess a defined genetic makeup. The genotyping method described herein can be incorporated into endocrine toxicity assays that examine the effects of chemicals on gonad differentiation.

Genotyping sex in the amphibian, Xenopus (Silurana) tropicalis, for endocrine disruptor bioassays.

Endocrine disrupting compounds have been shown to alter gonad differentiation in both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species. In some cases, these affected individuals are completely sex reversed and are morphologically indistinguishable from normal individuals of the opposite sex. Detecting shifts in sex ratios following chemical exposure often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with genetic sex determination and homomorphic sex chromosomes (such as amphibians and many fish). The ability to assess the genetic sex of individuals would allow for detection of sex reversal (genotype-phenotype mismatches) that have greater statistical power compared to examining changes in sex ratios. Utilizing amplified fragment length polymorphisms (AFLPs), we developed a method for genotyping sex in the amphibian, Xenopus (Silurana) tropicalis, that can be incorporated into endocrine disruptor screening assays that examine the effects of chemicals on gonad differentiation. AFLPs from 512 primer pairs were assessed in one spawn of X. tropicalis. Each primer pair yielded, on average, 100 fragments. In total 17 sex-linked AFLPs were identified, isolated, and sequenced. A recombination map of these AFLPs was generated using over 300 individuals with four AFLPs having a recombination rate of 0% with regard to sex. A BLASTn search of the X. tropicalis genome using these AFLP sequences resulted in identification of sex-linked scaffolds. Areas of these scaffolds were searched for additional polymorphisms that could be utilized for genotyping sex. Retrospective and prospective strategies for incorporating genotyping sex in endocrine disruptor bioassays with X. tropicalis were developed. A Monte Carlo simulation comparing analyzing data as sex ratio shifts versus assessment of sex reversal using genotyping demonstrates the increase in statistical power that can be obtained by genotyping sex in studies dealing with altered gonad differentiation. This approach to identifying sex-linked markers and developing sex genotyping methods is applicable to other species with genetic sex determination.