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. Phenolphthalin (PLIN) was shown not to bind rainbow trout (Oncorhynchus mykiss) ER (rtER) in a competitive binding assay, but vitellogenin (Vtg) expression was induced in trout liver slices exposed to 10-4 and 10-3.7 M PLIN. Phenolphthalein (PLEIN), a metabolite of PLIN, was subsequently determined to be formed when slices were exposed to PLIN. It binds rtER with a relative binding affinity to 17ß-estradiol of 0.020%. Slices exposed to PLEIN expressed Vtg messenger RNA (mRNA) at 10-4.3 , 10-4 , and 10-3.7 M, with no detectable PLIN present. Thus, Vtg expression noted in PLIN slice exposures was explained by metabolism to PLEIN in trout liver slices. A second model chemical, 4,4'-methylenedianiline (MDA), was not shown to bind rtER but did induce Vtg mRNA production in tissue slices at 10-4.3 , 10-4 , and 10-3.7 M in amounts nearly equal to reference estradiol induction, thus indicating metabolic activation of MDA. A series of experiments were performed to identify a potential metabolite responsible for the observed increase in activity. Potential metabolites hydroxylamine-MDA, nitroso-MDA, azo-MDA, and azoxy-MDA were not observed. However, acetylated MDA was observed and tested in both ER-binding and tissue slice Vtg induction assays. Environ Toxicol Chem 2023;42:2747-2757. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

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. EPA's ToxCast program. However, existing data gaps preclude any inferences between in vitro chemical inhibition of DIOs and in vivo outcomes relevant to ecological risk assessment. This study aimed to generate targeted data in a laboratory model species (Xenopus laevis) using a model DIO inhibitor, iopanoic acid (IOP), to characterize linkages between in vitro potency, in vivo biochemical responses, and adverse organismal outcomes. In vitro potency of IOP toward DIOs was evaluated using previously developed in vitro screening assays, which showed concentration-dependent inhibition of human DIO1 (IC50: 97 µM) and DIO2 (IC50: 231 µM) but did not inhibit human or X. laevis DIO3 under the assay conditions. In vivo exposure of larval X. laevis to 0, 2.6, 5.3, and 10.5 µM IOP caused thyroid-related biochemical profiles in the thyroid gland and plasma consistent with hyperthyroxinemia but resulted in delayed metamorphosis and significantly reduced growth in the highest 2 exposure concentrations. Independent evaluations of dio gene expression ontogeny, together with existing literature, supported interpretation of IOP-mediated effects resulting in a proposed adverse outcome pathway for DIO2 inhibition leading to altered amphibian metamorphosis. This study highlights the types of mechanistic data needed to move toward predicting in vivo outcomes of regulatory concern from in vitro bioactivity data.

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. Most chemicals had IC50 values lower for hDIO3 than for Xldio3 and many had steep Hill slopes (a potential indication of non-specific inhibition). However, eight of the most potent chemicals are likely specific inhibitors, with IC50 values of 14 µM or less, Hill slopes near -1 and curves not significantly different between species likely due to conservation of catalytically active amino acids. Controlling for assay conditions, human in vitro screening results can be predictive of activity in the amphibian assay. This study lays the groundwork for future studies using recombinant non-mammalian proteins to test cross-species sensitivity to chemicals. DISCLAIMER: The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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. Presented here are the development and application of a screening assay to assess susceptibility of IYD to chemical inhibition. With recombinant human IYD enzyme, a 96-well plate in vitro assay was developed and then used to screen over 1800 unique substances from the U.S. EPA ToxCast screening library. Through a tiered screening approach, 194 IYD inhibitors were identified (inhibited IYD enzyme activity by 20% or greater at target concentration of 200 µM). 154 chemicals were further tested in concentration-response (0.032-200 µM) to determine IC50 and rank-order potency. This work broadens the coverage of thyroid-relevant molecular targets for chemical screening, provides the largest set of chemicals tested for IYD inhibition, and aids in prioritizing chemicals for targeted in vivo testing to evaluate thyroid-related adverse outcomes.

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. The adverse outcome pathway for thyroperoxidase inhibition leading to altered amphibian metamorphosis was used to inform a pathway-based in vivo study design that generated response-response relationships. These causal relationships were used to develop Bayesian probabilistic network models that mathematically determine conditional dependencies between biochemical nodes and support the predictive capability of the biochemical profiles. Plasma thyroxine concentrations were the most predictive of metamorphic success with improved predictivity when thyroid gland sodium-iodide symporter gene expression levels (a compensatory response) were used in conjunction with plasma thyroxine as an additional regressor. Although thyroid-mediated amphibian metamorphosis has been studied for decades, this is the first time a predictive relationship has been characterized between plasma thyroxine and metamorphic success. Linking these types of biochemical surrogate metrics to apical outcomes is vital to facilitate the transition to the new paradigm of chemical safety assessments.

Conversion of Estrone to 17ß-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 17ß-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. Recent evidence suggests that in vivo biotransformation of E1 to E2 may occur in fathead minnows (Pimephales promelas) residing in environments with high concentrations of E1, such as near wastewater treatment plants. The enzymes likely responsible for this biotransformation, 17ß-hydroxysteroid dehydrogenases (17ßHSDs), have been well characterized in mammals but to a lesser extent in fish species. In the present study, a novel systematic analysis of amino acid sequence data from the National Center for Biotechnology Information database demonstrated that multiple 17ßHSD isoforms are conserved across different fish species. Experimentally, we showed that metabolically active hepatic cytosolic preparations from 2 commercially important salmonid species, rainbow trout and lake trout, biotransformed E1 to E2 to a degree sufficient to alter results of competitive ER binding assays. These results from in silico and in vitro analyses indicate that E1 and biotransformation may play a significant role in adverse effects on development and reproduction of a variety of fish species in contaminated aquatic environments. Environ Toxicol Chem 2020;39:2028-2040. Published 2020. This article is a US Government work and is in the public domain in the USA.

Metabolism of cyclic phenones in rainbow trout in vitro assays.

1. Cyclic phenones are chemicals of interest to the USEPA due to their potential for endocrine disruption to aquatic and terrestrial species.2. Prior to this report, there was very limited information addressing metabolism of cyclic phenones by fish species and the potential for estrogen receptor (ER) binding and vitellogenin (Vtg) gene activation by their metabolites.3. The main objectives of the current research were to characterize rainbow trout (rt) liver slice-mediated in vitro metabolism of model parent cyclic phenones exhibiting disparity between ER binding and ER-mediated Vtg gene induction, and to assess the metabolic competency of fish liver in vitro tests to help determine the chemical form (parent and/or metabolite) associated with the observed biological response.4. GC-MS, HPLC and LC-MS/MS technologies were applied to investigate the in vitro biotransformation of cyclobutyl phenyl ketone (CBP), benzophenone (DPK), cyclohexyl phenyl ketone (CPK) mostly in the absence of standards for metabolite characterization.5. It was concluded that estrogenic effects of the studied cyclic phenones are mediated by the parent chemical structure for DPK, but by active metabolites for CPK. A definitive interpretation was not possible for CBP and CBPOH (alcohol), although a contribution of both structures to gene induction is suspected.

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 rtERαß) 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. The additional three bridged-MAHC's; trans-stilbene (TSB), tetraphenylethylene (4PE), and 4,4-di-tertbutylphenyl (DtBB) were determined to be non-binders due to isomeric configuration, solubility limitation, and possible steric hinderance. It is possible that the bridged-MAHCs bind to the rtER through a proposed aromatic-aromatic stacking (π-π interaction) facilitated by perpendicular ring orientation achieved through free rotation of the bridged rings. The fused-ring structures are locked in a planar configuration which doesn't allow for rotation of rings perpendicular to one another. This first report of the rtER-binding of bridged-MAHCs in fish demonstrates binding for a class of chemicals normally not thought of as having an affinity for the estrogen receptor and further supports the versatility or promiscuity of ER ligand selectivity.

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.S. EPA's ToxCast phase 1_v2, phase 2, and e1k libraries. Initial testing at a single concentration identified 411 putative deiodinase inhibitors that produced inhibition of 20% or greater in at least 1 of the 3 deiodinase assays, including chemicals that have not previously been shown to inhibit deiodinases. Of these, 228 chemicals produced enzyme inhibition of 50% or greater; these chemicals were further tested in concentration-response to determine relative potency. Comparisons across these deiodinase assays identified 81 chemicals that produced selective inhibition, with 50% inhibition or greater of only 1 of the deiodinases. This set of 3 deiodinase inhibition assays provides a significant contribution toward expanding the limited number of in vitro assays used to identify chemicals with the potential to interfere with thyroid hormone homeostasis. In addition, these results set the groundwork for development and evaluation of structure-activity relationships for deiodinase inhibition, and inform targeted selection of chemicals for further testing to identify adverse outcomes of deiodinase inhibition.

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. To begin to address the potential for chemicals to inhibit these enzymes an adenovirus expression system was used to produce human deiodinase type 1 (DIO1) enzyme, established robust assay parameters for nonradioactive determination of iodide release by the Sandell-Kolthoff method, and employed a 96-well plate format for screening chemical libraries. An initial set of 18 chemicals was used to establish the assay, along with the known DIO1 inhibitor 6-propylthiouracil as a positive control. An additional 292 unique chemicals from the EPA's ToxCast phase 1_v2 chemical library were screened. Chemicals were initially screened at a single high concentration of 200 µM to identify potential DIO1 inhibitors. There were 50 chemicals, or 17% of the TCp1_v2 chemicals tested, that produced >20% inhibition of DIO1 activity. Eighteen of these inhibited DIO1 activity >50% and were further tested in concentration-response mode to determine IC50s. This work presents an initial effort toward identifying chemicals with potential for affecting THs via inhibition of deiodinases and sets the foundation for further testing of large chemical libraries against DIO1 and the other deiodinase enzymes involved in TH function.

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 in vitro 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. However, results for numerous industrial chemicals were more challenging to interpret, including chemicals with: (1) apparent competitive binding curves but no gene activation, (2) apparent binding and gene inhibition with evidence of either cytotoxicity or changes in assay media pH, (3) apparent binding but non-competitive gene inhibition of unknown cause, or (4) no rtER binding and gene inhibition not due to competitive ER interaction but due to toxicity, pH change, or some unknown cause. The use of endpoints such as toxicity, pH, precipitate formation, and determination of inhibitor dissociation constants (Ki) for interpreting the results of antagonism and binding assays for diverse chemicals is presented. Of the 94 chemicals tested for antagonism only two, tamoxifen and ICI-182780, were found to be true competitive antagonists. This report highlights the use of two different concentrations of estradiol tested in combination with graded concentrations of test chemical to provide the confirmatory evidence to distinguish true competitive antagonism from apparent antagonism.

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). Liver VTG mRNA expression in male fathead minnows (FHM) covaried with the binding assay estimates, suggesting that these fluctuations are biologically relevant. Tests which exposed male FHMs to either fluctuating levels of E2, a constant concentration of E2 (time-weighted to reflect average concentrations), or control (no E2) demonstrated that while the estrogenic activity of this effluent was detrimental to male spawning success, the fact that its concentration varied in a daily manner was without additional influence. The variability of the effluent's estrogenicity suggests that studies concerned with the effects of STP effluents should collect multiple daily samples and then test them on an appropriate time-weighted basis.

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. The estrogens DES, EE2, and E1 bound with high affinity to both receptors, with respective RBAs of 583, 166, and 28% (fathead minnow) and 179, 89, and 5% (rainbow trout). Relative binding affinity of E3, TAM, and GEN for both fhmER and rbtER were moderate, with values between 0.3 and 5%. The alkylphenols had weak affinity for the ERs with RBAs for the fhmER of 0.1 and 0.01 for PNP and PTOP, respectively. Corresponding values for the rbtER are 0.027 and 0.009. Estradiol ([3H]E2) only partially was displaced from both the fhmER and the rbtER by MXC, T, and MT. Comparison of RBAs of the chemicals tested for fhmER and rbtER indicates that the rank order of RBAs essentially are the same for both species.

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.e., fish liver tissue). Results of rtER competitive binding assays for 16 chemicals yielded calculable relative binding affinities (RBA) from 179 to 0.0006% for 13 chemicals and partial or no binding for an additional 3 chemicals. Eleven lower to no affinity chemicals (RBA < 0.1%) were further tested in trout liver slices to measure induction of rtER-dependent vitellogenin (VTG) mRNA in the presence of chemical passive partitioning (from media to multiple hepatocyte layers in the slice) and liver xenobiotic metabolism. VTG induction in slices was observed in a concentration-dependent manner for eight chemicals tested that had produced complete displacement curves in binding assays, including the lowest affinity binder with an RBA of 0.0006%. Two chemicals with only partial binding curves up to their solubility limit did not induce VTG. The monohydroxy metabolite of methoxychlor was the only chemical tested that apparently bound rtER but did not induce VTG mRNA. Data are presented illustrating the utility of the two assays in combination for interpreting the role of metabolism in VTG induction, as well as the sensitivity of the assays for measuring enantiomer selective binding and ER-mediated induction. The combined approach appears particularly useful in interpreting the potential relevance of extremely low affinity chemical binding to fish receptors (RBA = 0.01-0.0001%) within a defined toxicity pathway as a basis for prioritizing within large chemical inventories of environmental concern.

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. Although aromatase activity was decreased by 7 days, it is possible that the conversion of MT to ME2 occurred soon after initial exposure. In support of this, ME2 was detected in plasma samples of the fish following the 7-day exposure, confirming their ability convert the androgen MT to the estrogen ME2. The concentration of ME2 in plasma was within the range of plasma 17ss-estradiol (E2) found in control female fathead minnows (4-5ng/ml). These results, in conjunction with competitive binding assays that indicate ME2 binds to the fathead minnow estrogen receptor with a relative binding affinity of 68.3% of E2, support the hypothesis that aromatization of MT to ME2 contributes to the estrogenic effects in fathead minnows following exposure to this androgen.

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. However, little is known concerning the toxicity of trenbolone to fish. Our goal was to assess the effects of 17-beta-trenbolone on reproductive endocrinology of the fathead minnow (Pimephales promelas). An in vitro competitive binding study with the fathead minnow androgen receptor demonstrated that 17-beta-trenbolone had a higher affinity for the receptor than that of the endogenous ligand, testosterone. Male and female fish were exposed for 21 d to nominal (target) concentrations of 17-beta-trenbolone ranging from 0.005 to 50 microg/L. Fecundity of the fish was significantly reduced by exposure to measured test concentrations > or = 0.027 microg/ L. The 17-beta-trenbolone was clearly androgenic in vivo at these concentrations, as evidenced by the de novo production in females of dorsal (nuptial) tubercles, structures normally present only on the heads of mature males. Plasma steroid (testosterone and beta-estradiol) and vitellogenin concentrations in the females all were significantly reduced by exposure to 17-beta-trenbolone. The 17-beta-trenbolone also altered reproductive physiology of male fathead minnows, albeit at concentrations much higher than those producing effects in females. Males exposed to 17-beta-trenbolone at 41 microg/L (measured) exhibited decreased plasma concentrations of 11-ketotestosterone and increased concentrations of beta-estradiol and vitellogenin. Overall, our studies indicate that 17-beta-trenbolone is a potent androgen and reproductive toxicant in fish. Given the widespread use of trenbolone acetate as a growth promoter, and relative stability of its metabolites in animal wastes, further studies are warranted to assess potential ecological risk.