Increased Endocrine Activity of Xenobiotic Chemicals as Mediated by Metabolic Activation.

The US Environmental Protection Agency (USEPA) is faced with long lists of chemicals that require hazard assessment. The present study is part of a larger effort to develop in vitro assays and quantitative structure-activity relationships applicable to untested chemicals on USEPA inventories through study of estrogen receptor (ER) binding and estrogen-mediated gene expression in fish. The present effort investigates metabolic activation of chemicals resulting in increased estrogenicity.

Estrogenic Activity of Perfluoro Carboxylic and Sulfonic Acids in Rainbow Trout Estrogen Receptor Binding and Liver Slice Vtg mRNA Expression Assays.

Perfluoroalkylated substances (PFAS) such as carboxylic acids, and sulfonic acids were manufactured in high quantities and are ubiquitous environmental contaminants. These chemicals persist in the environment and tend to bioaccumulate. In the current study, the estrogenic potential of a series of perfluoro carboxylic acids and select perfluoro sulfonic acids were assessed in an rainbow trout estrogen receptor (rtER) binding assay and an rtER dependent vitellogenin (Vtg) expression rainbow trout liver slice assay.

Characterization of the Mechanistic Linkages Between Iodothyronine Deiodinase Inhibition and Impaired Thyroid-Mediated Growth and Development in Xenopus laevis Using Iopanoic Acid.

Iodothyronine deiodinases (DIO) are key enzymes that influence tissue-specific thyroid hormone levels during thyroid-mediated amphibian metamorphosis. Within the larger context of evaluating chemicals for thyroid system disrupting potential, chemical activity toward DIOs is being evaluated using high-throughput in vitro screening assays as part of U.S.

Xenopus laevis and human type 3 iodothyronine deiodinase enzyme cross-species sensitivity to inhibition by ToxCast chemicals.

Deiodinase enzymes are critical for tissue-specific and temporal control of activation or inactivation of thyroid hormones during vertebrate development, including amphibian metamorphosis. We previously screened ToxCast chemicals for inhibitory activity toward human recombinant Type 3 iodothyronine deiodinase enzyme (hDIO3) and subsequently produced Xenopus laevis recombinant dio3 enzyme (Xldio3) with the goals to identify specific chemical inhibitors of Xldio3, to evaluate cross-species sensitivity and explore whether the human assay results are predictive of the amphibian. We identified a subset of 356 chemicals screened against hDIO3 to test against Xldio3, initially at a single concentration (200 μM), and further tested 79 in concentration-response mode.

Conversion of Estrone to 17β-Estradiol: A Potential Confounding Factor in Assessing Risks of Environmental Estrogens to Fish.

Feminization of male fish and the role of endocrine-active chemicals in this phenomenon has been an area of intense study for many years. Estrone (E1), a natural steroid, is found in aquatic environments sometimes at high concentrations relative to the estrogenic steroids 17β-estradiol (E2) and 17α-ethynylestradiol. However, E1 has been less thoroughly studied than E2 or 17α-ethynylestradiol due in part to a relatively lower potency in metabolically limited estrogen receptor (ER) binding/activation assays.

In vitro screening for chemical inhibition of the iodide recycling enzyme, iodotyrosine deiodinase.

The iodide recycling enzyme, iodotyrosine deiodinase (IYD), is a largely unstudied molecular mechanism through which environmental chemicals can potentially cause thyroid disruption. This highly conserved enzyme plays an essential role in maintaining adequate levels of free iodide for thyroid hormone synthesis. Thyroid disruption following in vivo IYD inhibition has been documented in mammalian and amphibian models; however, few chemicals have been tested for IYD inhibition in either in vivo or in vitro assays.

Targeted Pathway-based In Vivo Testing Using Thyroperoxidase Inhibition to Evaluate Plasma Thyroxine as a Surrogate Metric of Metamorphic Success in Model Amphibian Xenopus laevis.

Chemical safety evaluation is in the midst of a transition from traditional whole-animal toxicity testing to molecular pathway-based in vitro assays and in silico modeling. However, to facilitate the shift in reliance on apical effects for risk assessment to predictive surrogate metrics having characterized linkages to chemical mechanisms of action, targeted in vivo testing is necessary to establish these predictive relationships. In this study, we demonstrate a means to predict thyroid-related metamorphic success in the model amphibian Xenopus laevis using relevant biochemical measurements during early prometamorphosis.

Metabolism of cyclic phenones in rainbow trout assays.

1. Cyclic phenones are chemicals of interest to the USEPA due to their potential for endocrine disruption to aquatic and terrestrial species.2.

Phenone, Hydroxybenzophenone, and Branched Phenone ER Binding and Vitellogenin Agonism in Rainbow Trout Models.

Phenones and hydroxy benzophenones are widely used as UV radiation filters, and in the manufacturing of insecticides and pharmaceuticals. Understanding the estrogenic potential these chemicals is of interest to the US Environmental Protection Agency and other international environmental organizations. The current study sequentially combined complementary rainbow trout estrogen receptor (rtER) binding and liver slice vitellogenin (Vtg) mRNA induction assays in the context of a defined ER-mediated adverse outcome pathway (AOP).

Screening the ToxCast Phase 1, Phase 2, and e1k Chemical Libraries for Inhibitors of Iodothyronine Deiodinases.

Deiodinase enzymes play an essential role in converting thyroid hormones between active and inactive forms by deiodinating the pro-hormone thyroxine (T4) to the active hormone triiodothyronine (T3) and modifying T4 and T3 to inactive forms. Chemical inhibition of deiodinase activity has been identified as an important endpoint to include in screening chemicals for thyroid hormone disruption. To address the lack of data regarding chemicals that inhibit the deiodinase enzymes, we developed robust in vitro assays that utilized human deiodinase types 1, 2, and 3 and screened over 1800 unique chemicals from the U.

Estrogenic activity of multicyclic aromatic hydrocarbons in rainbow trout (Oncorhynchus mykiss) in vitro assays.

A representative group of multicyclic aromatic hydrocarbons (MAHC) which can be further classified as bridged-ring (bridged-MAHC) or fused-ring (fused-MAHC) were examined for their ability to interact with the estrogen receptor of rainbow trout (rtER) in a hepatic cytosolic estrogen receptor competitive binding assay (cyto rtERαβ) and the vitellogenin (Vtg) mRNA gene activation liver slice assay. All five fused-MAHCs; naphthalene (NAFT), fluorene (FE), Fluoranthene (FAT), pyrene (PY), and 9,10-dihydroanthracene (DAC) had no estrogenic activity in the in vitro assays used. Five of the eight bridged-MAHCs; triphenylethylene (3PE), o-terphenyl (OTP), triphenylmethane (TPM), 1,1-diphenylethylene (DPE), and cis-stilbene (CSB) were positive in the rtER-binding assay.

Screening the ToxCast Phase 1 Chemical Library for Inhibition of Deiodinase Type 1 Activity.

Thyroid hormone (TH) homeostasis is dependent upon coordination of multiple key events including iodide uptake, hormone synthesis, metabolism, and elimination, to maintain proper TH signaling. Deiodinase enzymes catalyze iodide release from THs to interconvert THs between active and inactive forms, and are integral to hormone metabolism. The activity of deiodinases has been identified as an important endpoint to include in the context of screening chemicals for TH disruption.

Avoiding False Positives and Optimizing Identification of True Negatives in Estrogen Receptor Binding and Agonist/Antagonist Assays.

The potential for chemicals to affect endocrine signaling is commonly evaluated via receptor binding and gene activation, but these assays, especially antagonism assays, have potential artifacts that must be addressed for accurate interpretation. Results are presented from screening 94 chemicals from 54 chemical groups for estrogen receptor (ER) activation in a competitive rainbow trout ER (rtER) binding assay and a trout liver slice vitellogenin mRNA expression assay. Results from true competitive agonists and antagonists, and inactive chemicals with little or no indication of ER binding or gene activation were easily interpreted.

Temporal variation in the estrogenicity of a sewage treatment plant effluent and its biological significance.

Daily variation in the estrogenic activity of effluent released by a modern sewage treatment plant (STP) was measured and its effects on the physiology, behavior, and reproductive success of male fish were evaluated. As measured by an estrogen receptor binding assay, the daily estrogenic activity of this effluent was both high and extremely variable (42 +/- 25.4 [mean +/- SD] ng 17beta-estradiol (E2) equivalents/L; n = 18).

Comparison of relative binding affinities of endocrine active compounds to fathead minnow and rainbow trout estrogen receptors.

Twelve chemicals were tested for binding affinity to rainbow trout liver estrogen receptor (rbtER) and fathead minnow liver ER (fhmER). The chemicals included estradiol (E2), diethylstilbestrol (DES), ethinylestradiol (EE2), estrone (El), estriol, tamoxifen (TAM), genistein (GEN), p-nonylphenol (PNP), p-tert-octylphenol (PTOP), methoxychlor (MXC), testosterone, and methyltestosterone (MT). Relative binding affinity (RBA) was calculated for each chemical as a function of E2 binding to the receptor.

Use of trout liver slices to enhance mechanistic interpretation of estrogen receptor binding for cost-effective prioritization of chemicals within large inventories.

The cost of testing chemicals as reproductive toxicants precludes the possibility of evaluating large chemical inventories without a robust strategyfor prioritizing chemicals to test. The use of quantitative structure-activity relationships in early hazard identification is a cost-effective prioritization tool, but in the absence of systematic collection of interpretable test data upon which models are formulated, these techniques fall short of their intended use. An approach is presented for narrowing the focus of candidate ED chemicals using two in vitro assays: one optimized to measure the potential of chemicals to bind rainbow trout estrogen receptors (rtER), and a second to enhance interpretation of receptor binding data in a relevant biological system (i.

Mechanistic basis for estrogenic effects in fathead minnow (Pimephales promelas) following exposure to the androgen 17alpha-methyltestosterone: conversion of 17alpha-methyltestosterone to 17alpha-methylestradiol.

Exposure of adult fathead minnows (Pimephales promelas) to the androgen 17alpha-methyltestosterone (MT) produces both androgenic and estrogenic effects, manifested as nuptial tubercle formation in females, and vitellogenin production in males and females, respectively. The present study was conducted to determine if the unanticipated estrogenic effects are produced by conversion of MT via aromatase activity to 17alpha-methylestradiol (ME2). Aromatase activity at the end of a 7-day waterborne MT exposure (20, 200microg/l) was significantly decreased in ovarian microsomes and brain homogenates from exposed fish, to about 30-50% of control activity.

Effects of the androgenic growth promoter 17-beta-trenbolone on fecundity and reproductive endocrinology of the fathead minnow.

Trenbolone acetate is a synthetic steroid that is extensively used in the United States as a growth promoter in beef cattle. The acetate is administered to livestock via slow-release implants; some is converted by the animal to 17-beta-trenbolone, a relatively potent androgen receptor agonist in mammalian systems. Recent studies indicate that excreted 17-beta-trenbolone is comparatively stable in animal waste, suggesting the potential for exposure to aquatic animals via direct discharge, runoff, or both.