Rainbow trout (Oncorhynchus mykiss) confined to respirometer-metabolism chambers were dosed with perfluorooctanoate (PFOA) by intra-arterial (i.a.) injection and sampled to obtain concentration time-course data for plasma, urine, and expired water. The data were then analyzed by compartmental modeling to estimate rates of renal and branchial clearance. Averaged across all animals, the renal clearance rate (1.35mL/h/kg) was more than ten times greater than the branchial clearance rate (0.12mL/h/kg). The average whole-body elimination half-life was 12.6d, which is somewhat longer than values obtained in previous studies with smaller trout. The tissue distribution of PFOA was assessed by collecting tissues at the end of chambered exposures and in a separate tissue time-course experiment. From the time-course study it appeared that an internal steady-state was established within 24h of i.a. injection. Consistent with previous studies, the rank order of PFOA concentration in tissues at steady state was: plasma>liver>kidney>muscle. In a second set of chambered experiments, fish were exposed to PFOA in water to determine the rate of branchial uptake. Branchial uptake rates were too low to assess directly by measuring PFOA concentrations in inspired and expired water. Uptake rate constants (mean 0.19L/d/kg; 0.1% uptake efficiency) were therefore estimated by compartmental modeling using plasma concentration time-course data and model parameters derived from the elimination experiments. It is clear from this effort that elimination of PFOA by trout occurs primarily via the renal route. This finding is consistent with numerous studies of mammals and suggests that trout possess membrane transporters that facilitate the movement of PFOA from plasma to urine.
Thyroid gland explant cultures from prometamorphic Xenopus laevis tadpoles were evaluated for their utility in assessing chemicals for thyroid hormone (TH) synthesis disruption. The response of cultured thyroid glands to bovine thyroid stimulating hormone (bTSH) and the TH synthesis inhibitors methimazole, 6-propylthiouracil, and perchlorate was determined. Thyroid glands continuously exposed for 12 days to graded concentrations of bTSH released thyroxine (T4) in a dose-dependent manner. Over time, the glands appeared to reach a constant daily rate of T4 release. This suggested that the T4 stores in the glands were initially depleted but continuous release was maintained by synthesis of new hormone. The potency of methimazole, 6-propylthiouracil, and perchlorate for inhibiting T4 release was determined using glands cotreated with a single maximally effective bTSH concentration and graded concentrations of chemical. Inhibition of T4 release was dose dependent for all three chemicals. Perchlorate was the most potent inhibitor of T4 release. Methimazole and 6-propylthiouracil exhibited lower potency than perchlorate but similar potency to each other. The IC(50) (mean ± SD) for inhibition of T4 release by the thyroid glands was 1.2 ± 0.55, 8.6 ± 1.3, and 13 ± 4.0 ?M for perchlorate, 6-propylthiouracil, and methimazole, respectively. This model system shows promise as a tool to evaluate the potency of chemicals that inhibit T4 release from thyroid glands and may be predictive of in vivo T4 synthesis inhibition in prometamorphic tadpoles.
As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression patterns in the brain of the amphibian model (Xenopus laevis) following in vivo exposure to a suite of T4 synthesis inhibitors. We specifically address the application of Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE), Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and LC-MS/MS to assess changes in relative protein expression levels. 2D PAGE and iTRAQ proved to be effective complementary techniques for distinguishing protein changes in the developing amphibian brain in response to T4 synthesis inhibition. This information served to evaluate the use of distinctive protein profiles as a potential mechanism to screen chemicals for endocrine activity in anurans. Regulatory pathways associated with proteins expressed as a result of chemical effect are reported. To our knowledge, this is also the first account of the anuran larvae brain proteome characterization using proteomic technologies. Correlation of protein changes to other cellular and organism-level responses will aid in the development of a more rapid and cost-effective, non-mammalian screening assay for thyroid axis-disrupting chemicals.
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.
Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzymes 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 64 microg/L in a flow-through system from < 24 h 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 16 microg/L and above and a significant bias in sex ratio towards males at concentrations of 1 microg/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 16 microg/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 4 microg/L treatment. No differences in estradiol, testosterone, or vitellogenin plasma concentrations were observed in exposed males or females compared to controls. Females in the 4 microg/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.
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 ?g/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 ?g/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 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 17?-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 17?-trenbolone exposure results in effects down to 78?ng/L, illustrating potential effects from exposure to androgenic compounds in anurans.
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