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.

Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis.

Thyroid axis disruption is an important consideration when evaluating risks associated with chemicals. Bioassay methods that include thyroid-related endpoints have been developed in a variety of species, including amphibians, whose metamorphic development is thyroid hormone (TH)-dependent. Inhibition of TH synthesis in these species leads to developmental delay, and assays designed to capture these effects take several weeks to complete. In an effort to develop a shorter term approach, the early responses of various endpoints were evaluated in Xenopus laevis throughout 8d of exposure to three TH synthesis inhibitors: methimazole (100mg/L), 6-propylthiouracil (6-PTU) (20mg/L), and perchlorate (4 mg/L). Endpoints included thyroid gland histology and cell numbers, circulating TH concentrations, and thyroidal TH and associated iodo-compounds. Thyroidal 3,5-diodo-L-tyrosine (DIT) and thyroxine (T4) were significantly reduced from day 2 onward by all three chemicals, while 3-monoiodo-L-tyrosine (MIT) was significantly reduced by methimazole and perchlorate, but not by 6-PTU. These reductions were the earliest indicators of TH synthesis inhibition. Histological effects were apparent on day 4 and became more exaggerated through day 8. However, reductions in circulating T4 and increases in thyroid gland cell numbers were not apparent until day 6. Reductions of thyroidal MIT, DIT, and T4 and circulating T4 are indicative of inhibitory effects of the chemicals on TH synthesis. Changes in thyroid histology and cell number represent compensatory effects modulated by circulating TSH. These observations establish a basis for the development of short term amphibian-based methods to evaluate thyroid axis effects using a suite of diagnostic endpoints.

Sensitivity of fetal rat testicular steroidogenesis to maternal prochloraz exposure and the underlying mechanism of inhibition.

The fungicide prochloraz (PCZ) induces malformations in androgen-dependent tissues in male rats when administered during sex differentiation. The sensitivity of fetal testicular steroidogenesis to PCZ was investigated to test the hypothesis that the reported morphological effects from maternal exposure were associated with reduced testosterone synthesis. Pregnant Sprague-Dawley rats were dosed by gavage with 0, 7.8, 15.6, 31.3, 62.5, and 125 mg PCZ/kg/day (n = 8) from gestational day (GD) 14 to 18. On GD 18, the effects of PCZ on fetal steroidogenesis were assessed by measuring hormone production from ex vivo fetal testes after a 3-h incubation. Lastly, PCZ levels in amniotic fluid and maternal serum were measured using liquid chromatography/mass spectroscopy and correlated to the inhibition of steroidogenesis. Fetal progesterone and 17alpha-hydroxyprogesterone production levels were increased significantly at every PCZ dose, whereas testosterone levels were significantly decreased only at the two high doses. These results suggest that PCZ inhibits the conversion of progesterone to testosterone through the inhibition of CYP17. To test this hypothesis, PCZ effects on CYP17 gene expression and in vitro CYP17 hydroxylase activity were evaluated. PCZ had no effect on testicular CYP17 mRNA levels as measured by quantitative real-time polymersase chain reaction. However, microsomal CYP17 hydroxylase activity was significantly inhibited by the fungicide (K(i) = 865nM). Amniotic fluid PCZ concentrations ranged from 78 to 1512 ppb (207-4014nM) and testosterone production was reduced when PCZ reached approximately 500 ppb, which compares favorably with the determined CYP17 hydroxylase K(i) (326 ppb). These results demonstrate that PCZ lowers testicular testosterone synthesis by inhibiting CYP17 activity which likely contributes to the induced malformations in androgen-dependent tissues of male offspring.

Evaluation of the methoxytriazine herbicide prometon using a short-term fathead minnow reproduction test and a suite of in vitro bioassays.

Prometon is one of the most consistently detected herbicides in the U.S. environment. However, no previous assessment of the potential for prometon or related methoxytriazine herbicides to act as endocrine-disrupting chemicals has been conducted. This study used an array of in vitro bioassays to assess whether prometon, atraton, terbumeton, or secbumeton might act as potent (ant)agonists of the aryl hydrocarbon, estrogen, androgen, or glucocorticoid receptors or as aromatase inhibitors or inducers in vitro. Potential effects of prometon were also evaluated using a 21-d fathead minnow reproduction assay. Concentrations of methoxytriazines, as great as 1 mg/L (4.4 microM), did not induce significant dioxin-like responses in H4IIE-luc cells, estrogenic responses in MVLN cells, or androgen or glucocorticoid receptor-mediated responses in MDA-kb2 cells, nor did the methoxytriazines significantly affect aromatase activity in vitro. In the fathead minnow assay, exposure to 20, 200, or 1,000 microg prometon/L significantly reduced the weight of the male fat pad (an androgen-responsive tissue) relative to body weight. Exposure to 20 microg prometon/L significantly increased female plasma testosterone concentrations, but the effect was not observed at greater concentrations. Overall, prometon did not significantly reduce fecundity over the 21-d exposure, nor were other endpoints, including plasma vitellogenin and estradiol concentrations, brain and ovary aromatase activity, and male tubercle index, significantly affected. Evidence from our work suggests that prometon may cause subtle endocrine and/or reproductive effects in fathead minnows, but no clear mechanism of action was observed. The relevance of these effects to hazard assessment for the pesticide is uncertain.

Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas).

Many chemicals that adversely affect reproduction and/or development do so through multiple pathways within the reproductive tract and hypothalamic-pituitary-gonadal axis. Notable in this regard are fungicides, such as prochloraz or fenarimol, which in mammals have the potential to impact endocrine function through inhibition of CYP enzymes involved in steroid metabolism, as well as through antagonism of the androgen receptor(s). The objective of our studies was to assess the effects of prochloraz and fenarimol on reproductive endocrine function in a model small fish species, the fathead minnow (Pimephales promelas), using both in vitro and in vivo assays. The two fungicides inhibited in vitro CYP19 aromatase activity in brain and ovarian homogenates from the fish, with prochloraz exhibiting a greater potency than fenarimol. Prochloraz and fenarimol also bound competitively to the cloned fathead minnow androgen receptor expressed in COS-1 cells. The two fungicides significantly reduced fecundity of the fish in a 21-day reproduction assay at water concentrations of 0.1 (prochloraz) and 1.0 (fenarimol) mg/l. The in vivo effects of prochloraz on plasma steroid (17beta-estradiol, testosterone, 11-ketotestosterone) and vitellogenin (an estrogen-responsive protein) concentrations, as well as on gonadal histopathology, were consistent with inhibition of steroidogenesis. Fenarimol also affected several aspects of endocrine function in vivo; however, the suite of observed effects did not reflect either aromatase inhibition or androgen receptor antagonism. These studies contribute to a better mechanistic understanding of the extrapolation of effects of endocrine-disrupting chemicals across vertebrate classes.

Partial life-cycle toxicity and bioconcentration modeling of perfluorooctanesulfonate in the northern leopard frog (Rana pipiens).

A number of recent monitoring studies have demonstrated elevated concentrations of perfluorooctanesulfonate (PFOS) in humans and wildlife throughout the world. Although no longer manufactured in the United States, the global distribution and relative persistence of PFOS indicates a need to understand its potential ecological effects. Presently, little is known concerning toxicity of PFOS in chronic exposures with aquatic species. Therefore, we evaluated the effects of PFOS on survival and development of the northern leopard frog (Rana pipiens) from early embryogenesis through complete metamorphosis. Exposures were conducted via water at measured PFOS concentrations ranging from 0.03 to 10 mg/L. Animals exposed to 10 mg/L began dying within approximately two weeks of test initiation. Survival was not affected by PFOS at lower concentrations; however, time to metamorphosis was delayed and growth reduced in the 3-mg/L treatment group. Tadpoles readily accumulated PFOS directly from water. Using a one-compartment bioaccumulation model, growth was shown to have a modest impact on steady-state PFOS concentrations. Variability in observed growth rates and the possible contribution of a size-dependent decrease in PFOS elimination rate contributed uncertainty to modeling efforts. Nevertheless, fitted uptake and elimination rate constants were comparable to those determined in earlier studies with juvenile rainbow trout. Overall, our studies suggest that R. pipiens is not exceptionally sensitive to PFOS in terms of either direct toxicity or bioconcentration potential of the chemical.

Characterization of responses to the antiandrogen flutamide in a short-term reproduction assay with the fathead minnow.

A short-term reproduction assay with the fathead minnow (Pimephales promelas) has been developed to detect chemicals with the potential to disrupt reproductive endocrine function controlled by estrogen- and androgen-mediated pathways. The objective of this study was to use the assay to characterize responses of fathead minnow reproductive endocrinology and physiology to the mammalian antiandrogen, flutamide. Male and female fish were exposed to nominal (target) concentrations of 50 and 500 microg flutamide/l for 21-days, following which plasma steroid and vitellogenin concentrations were determined and gonadal morphology assessed. Fecundity of the fish was significantly reduced by exposure to a measured test concentration of 651 microg flutamide/l. In addition, embryo hatch was significantly reduced at this concentration. Qualitative histological assessment of ovaries from females exposed to flutamide indicated a decrease in mature oocytes and an increase in atretic follicles. Testes of males exposed to flutamide exhibited spermatocyte degeneration and necrosis. Concentration-dependent increases in plasma testosterone and vitellogenin concentrations were observed in the females. Flutamide also altered reproductive endocrinology of male fathead minnows. Males exposed to 651 microg flutamide/l exhibited elevated concentrations of beta-estradiol and vitellogenin. In summary, the results of this study with the fathead minnow demonstrate that flutamide affects reproductive endocrine function in fish and that the type of hormonal pattern and histopathology effects observed are consistent with an antiandrogenic mode-of-action. Consequently, our findings suggest that the 21-day reproduction assay utilizing fathead minnows is a sensitive short-term screening method for the detection of endocrine-disrupting chemicals, including antiandrogens.

Evaluation of androstenedione as an androgenic component of river water downstream of a pulp and paper mill effluent.

This study evaluates a recent report indicating that androstenedione (4-androsten-3, 17-dione) contributes to the androgenicity of water downstream of a pulp and paper mill discharge on the Fenholloway River (FL, USA). Extraction and concentration of Fenholloway water with C18 solid-phase extraction columns followed by reverse-phase high-pressure liquid chromatography resulted in clearly defined fractions with in vitro androgenic activity in CV-1 cells that had been transiently cotransfected with human androgen receptor and reporter gene constructs. However, we were unable to detect androstenedione in the active fractions by gas chromatography/mass spectrometry. Mass spectrometry analyses of deionized and Fenholloway River water samples that had been spiked with androstenedione, then extracted and fractionated, revealed that the androgen was found only in inactive fractions. We conclude that, although androstenedione was present at easily detectable concentrations in the river water (> 100 ng/L), this compound is not associated with androgenic activity of water from the site.