In vitro metabolism assessment of thiacloprid in rainbow trout and rat by LC-UV and high resolution-mass spectrometry.

Thiacloprid (THI) is a widely used neonicotinoid insecticide where concerns have been raised regarding low absorption by crops, substantial distribution in surrounding areas, and potential adverse effects to terrestrial and aquatic organisms.Prior to this study, there was very limited information addressing the ex vivo (precision-cut liver slices) metabolism of THI by fish species and the metabolic pathways regulating its potential for adverse effects.The in vitro and ex vivo biotransformation pathway of THI is defined by the formation of three primary metabolites (TM1, TM2 and TM3) via separate paths differentiated by reductive decyanation, reductive dechlorination with hydration and dealkylation processes, respectively.Kinetic rates were calculated for the rat microsomal decyanation of THI into TM1 (Km = 299.2 µM and Vmax = 5.3 pmol/min/mg), and for the dealkylation of THI into TM3 (Km = 368.9 µM and Vmax = 3.95 pmol/min/mg).Formation confirmation and identity inference of THI metabolites in absence of standards were achieved by LC-UV and High Resolution-MS strategies.The in vitro and ex vivo metabolic products of THI are conserved both across species (rat and Rainbow trout) and levels of biological organization (microsomes and liver slices), as previously reported for the neonicotinoid insecticides Imidacloprid and Acetamiprid.

In vitro metabolism of imidacloprid and acetamiprid in rainbow trout and rat.

Providing an alternative to pyrethroids, organophosphates, and carbamates, the neonicotinoids are now the most widely used insecticides in the world. They are water soluble and relatively stable in soil and water which allows for run-off through surface waters and thus potentially impacting aquatic species and environments.While the mammalian metabolism of neonicotinoids has been studied extensively, there is a lack of understanding of their metabolism in fish species. The current study constitutes the first report of the metabolism of imidacloprid (IMI) and acetamiprid (AC) in rainbow trout.Formation of respective metabolites 5-hydroxy-imidacloprid and N-desmethyl-acetamiprid was conserved across orders of biological organization in both microsomal and liver slice assays.Michaelis-Menten kinetics were determined for the microsomal conversion of IMI to 5-hydroxy-imidacloprid in rainbow trout (Km = 79.2 µM; Vmax = 0.75 pmole/min/mg) and rat (Km = 158.7 µM; Vmax = 38.4 pmole/min/mg). Kinetics for the microsomal demethylation of AC to N-desmethyl-acetamiprid were determined in the rat (Km = 70.9 µM; Vmax = 10 pmoles/min/mg). N-desmethyl-acetamiprid was found in detectable but below quantifiable levels across the range of test concentrations which precluded a calculation of kinetic rate constants in rainbow trout (RBT).Ultimately, the formation of the metabolites 5-hydroxy-imidacloprid and N-desmethyl-acetamiprid was conserved across RBT and rat species.

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.

A comparison of fish pesticide metabolic pathways with those of the rat and goat.

Ecological risk assessments are often limited in their ability to consider metabolic transformations for fish species due to a lack of data. When these types of evaluations are attempted they are often based on parent chemical only, or by assuming similarity to available mammalian metabolic pathways. The metabolism maps for five pesticides (fluazinam, halauxifen-methyl, kresoxim-methyl, mandestrobin, and tolclofos-methyl) were compared across three species. A rapid and transparent process, utilizing a database of systematically collected information for rat, goat, and fish (bluegill or rainbow trout), and using data evaluation tools in the previously described metabolism pathway software system MetaPath, is presented. The approach demonstrates how comparisons of metabolic maps across species are aided by considering the sample matrix in which metabolites were quantified for each species, differences in analytical methods used to identify metabolites in each study, and the relative amounts of metabolites quantified. By incorporating these considerations, more extensive rat and goat metabolism maps were found to be useful predictors of the more limited metabolism of the five pesticides in fish.

PFAS Biotransformation Pathways: A Species Comparison Study.

Limited availability of fish metabolic pathways for PFAS may lead to risk assessments with inherent uncertainties based only upon the parent chemical or the assumption that the biodegradation or mammalian metabolism map data will serve as an adequate surrogate. A rapid and transparent process, utilizing a recently created database of systematically collected information for fish, mammals, poultry, plant, earthworm, sediment, sludge, bacteria, and fungus using data evaluation tools in the previously described metabolism pathway software system MetaPath, is presented. The fish metabolism maps for 10 PFAS, heptadecafluorooctyl(tridecafluorohexyl)phosphinic acid (C6/C8 PFPiA), bis(perfluorooctyl)phosphinic acid (C8/C8 PFPiA), 2-[(6-chloro-1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexyl)oxy]-1,1,2,2-tetrafluoroethanesulfonic acid (6:2 Cl-PFESA), N-Ethylperfluorooctane-1-sulfonamide (Sulfuramid; N-EtFOSA), N-Ethyl Perfluorooctane Sulfonamido Ethanol phosphate diester (SAmPAP), Perfluorooctanesulfonamide (FOSA), 8:2 Fluorotelomer phosphate diester (8:2 diPAP), 8:2 fluorotelomer alcohol (8:2 FTOH), 10:2 fluorotelomer alcohol (10:2 FTOH), and 6:2 fluorotelomer sulfonamide alkylbetaine (6:2 FTAB), were compared across multiple species and systems. The approach demonstrates how comparisons of metabolic maps across species are aided by considering the sample matrix in which metabolites were quantified for each species, differences in analytical methods used to identify metabolites in each study, and the relative amounts of metabolites quantified. Overall, the pathways appear to be well conserved across species and systems. For PFAS lacking a fish metabolism study, a composite map consisting of all available maps would serve as the best basis for metabolite prediction. This emphasizes the importance and utility of collating metabolism into a searchable database such as that created in this effort.

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 in vitro rainbow trout estrogen receptor (rtER) binding assay and an ex vivo rtER dependent vitellogenin (Vtg) expression rainbow trout liver slice assay. Perfluoro carboxylic acids with perfluoroalkyl chain lengths of four to six did not significantly bind to the rtER or induce Vtg expression in liver slices. Perfluoro carboxylic acids with chain lengths of seven to ten, and sulfonic acids with seven and eight carbon chains bound to the rtER, but with low relative binding affinities. While affinity for the rtER increased with increasing chain length the highest affinity measured was only 0.0025% relative to the endogenous hormone 17ß-estradiol at 100%. Both the eight-carbon carboxylic acid and eight-carbon sulfonic acid induced Vtg expression in ex vivo liver slices. However, toxicity did not allow expression to achieve maximum efficacy relative to estradiol.

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.

MetaPath: an electronic knowledge base for collating, exchanging and analyzing case studies of xenobiotic metabolism.

The MetaPath knowledge base was developed for the purpose of archiving, sharing and analyzing experimental data on metabolism, metabolic pathways and crucial supporting metadata. The MetaPath system grew out of the need to compile and organize the results of metabolism studies into a systematic database to facilitate data comparisons and evaluations. Specialized MetaPath data evaluation tools facilitate the review of pesticide metabolism data submitted for regulatory risk assessments as well as exchange of results of complex analyses used in regulation and research. Customized screen editors called Composers were developed to automate data entry into MetaPath while also streamlining the production of agency specific study summaries such as the Data Evaluation Records (DER) used by the US EPA Office of Pesticide Programs. Efforts are underway through an Organization for Economic Co-operation and Development (OECD) work group to extend the use of DER Composers as harmonized templates for rat metabolism, livestock residue, plant residue and environmental degradation studies.

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.

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.

Characterization and analysis of estrogenic cyclic phenone metabolites produced in vitro by rainbow trout liver slices using GC-MS, LC-MS and LC-TOF-MS.

Cyclic phenones are chemicals of interest to the USEPA and international organizations due to their potential for endocrine disruption to aquatic and terrestrial species. The metabolic conversion of cyclic phenones by liver hepatocytes and the structure of main metabolites yielded have not been assessed in fish species. As part of a larger project, in this study we investigated the structure of metabolites produced in vitro by rainbow trout (rt) liver slices after exposure to the model cyclic phenones benzophenone (DPK), cyclobutyl phenyl ketone (CBP) and cyclohexyl phenyl ketone (CPK). While only one distinct metabolite was detected for DPK and CBP (benzhydrol and CBPOH, respectively), CPK yielded nine positional isomers (M1-M9) as products. In absence of standards, improved inference of CPK metabolites tentative structures was achieved by combining GC-MS with and without derivatization, LC with tandem MS, LC with high resolution time of flight (TOF) MS and LC fractionation data with CPK phase II conjugative metabolism information. Data supported that CPK is metabolized by phase I oxidation of the cyclohexyl ring and not the phenyl group as predicted by metabolism simulators. CPK metabolites M1 and M2 (MW 186), were proposed to be cyclohexenyl-derivatives. Also, M6-M9 were proposed to be hydroxylated metabolites (MW 204), with the potential for undergoing phase II conjugative metabolism to glucuronides and sulfates. Finally, M3, M4 and M5 were proposed as cyclohexanone-derivatives of CPK (MW 202), resulting from the limited redox-interconversion of their hydroxylated pairs M8, M6 and M7, respectively. Assessment of metabolite role in biological responses associated with endocrine disruption will advance the development of methods for species extrapolation and the understanding of differential sensitivity of species to chemical exposure.

Metabolism of Diazinon in Rainbow Trout Liver Slices.

INTRODUCTION: Understanding biotransformation pathways in aquatic species is an integral part of ecological risk assessment with respect to the potential bioactivation of chemicals to more toxic metabolites. The long-range goal is to gain sufficient understanding of fish metabolic transformation reactions to be able to accurately predict fish xenobiotic metabolism. While some metabolism data exist, there are few fish in vivo exposure studies where metabolites have been identified and the metabolic pathways proposed. Previous biotransformation work has focused on in vitro studies which have the advantage of high throughput but may have limited metabolic capabilities, and in vivo studies which have full metabolic capacity but are low throughput. An aquatic model system with full metabolic capacity in which a large number of chemicals could be tested would be a valuable tool. MATERIALS AND METHODS: The current study evaluated the ex vivo rainbow trout liver slice model, which has the advantages of high throughput as found in vitro models and non-dedifferentiated cells and cell to cell communication found in in vivo systems. The pesticide diazinon, which has been previously tested both in vitro and in vivo in a number of mammalian and aquatic species including rainbow trout, was used to evaluate the ex vivo slice model as a tool to study biotransformation pathways. RESULTS/DISCUSSION: While somewhat limited by the analytical chemistry method employed, results of the liver slice model, mainly that hydroxypyrimidine was the major diazinon metabolite, are in line with the results of previous rainbow trout in vivo studies. CONCLUSION: Therefore, the rainbow trout liver slice model is a useful tool for the study of metabolism in aquatic species.

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.

Rate and capacity of hepatic microsomal ring-hydroxylation of phenol to hydroquinone and catechol in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol at 11 and 25 degrees C by directly measuring the production of two potentially toxic metabolites, hydroquinone (HQ) and catechol (CAT). An HPLC method with integrated ultraviolet and electrochemical detection was used for metabolite identification and quantification at low (pmol) formation rates found in fish. The Michaelis-Menten saturation kinetics for the production of HQ and CAT over a range of phenol concentrations were determined at trout physiological pH. The apparent Km's for the production of HQ and CAT at 11 degrees C were 14+/-1 and 10+/-1 mM, respectively, with Vmax's of 552+/-71 and 161+/-15 pmol/min per mg protein. The kinetic parameters for HQ and CAT at 25 degrees C were 22+/-1 and 32+/-3 mM (Km) and 1752+/-175 and 940+/-73 pmol/min per mg protein (Vmax), respectively. The calculated increase in metabolic rate per 10 degrees C temperature rise (Q(10)) was 2.28 for HQ and 3.53 for CAT production. These experiments assess the potential for metabolic bioactivation in fish through direct quantification of putative reactive metabolites at the low, but toxicologically significant, chemical concentrations found in aquatic organisms. This work initiates a series of studies to compare activation pathway, rate, and capacity across fish species, providing a basis for development of biologically-based dose response models in diverse species.

An in vivo microdialysis method for the qualitative analysis of hepatic phase I metabolites of phenol in rainbow trout (Oncorhynchus mykiss).

Development of reliable and accurate methodologies for determination of xenobiotic hepatic biotransformation rate and capacity parameters is important to the derivation of precise physiologically-based toxicokinetic (PB-TK) models. Biotransformation data incorporated into PB-TK models has, for the most part, depended on in vitro techniques designed to mimic the in vivo environment; however, data from direct in vitro/in vivo comparisons is limited. In this investigation we describe for the first time a method using in vivo microdialysis (MD) to qualitatively assess hepatic xenobiotic biotransformation of phenol in an unanesthetized fish. MD probes were surgically implanted into the livers of adult rainbow trout which were subsequently confined to respirometer-metabolism chambers. Phenol (1-300 mM) was delivered directly to the liver via the MD probe at a perfusion rate of 1 microl min(-1) which consistently resulted in a relative delivery of 77-85% of the phenol in the perfusate to the tissue over a 3 day experimental time frame. Location of the probe within the liver was also shown to have no effect on the delivery of phenol or on the type or quantity of phase I metabolites formed. Production of hydroquinone (HQ) and catechol (CAT), the primary phase I metabolites of phenol, was monitored through direct sampling of the hepatic extracellular fluid space via the MD probe. HQ and CAT production increased with increasing time of perfusion and with increasing concentration of phenol delivered to the liver. In the future, data obtained through in vivo MD will be useful in resolving uncertainties in biotransformation rate and capacity parameters, which are central to fish PB-TK modeling of chemical disposition.

Effects of anesthesia (tricaine methanesulfonate, MS222) on liver biotransformation in rainbow trout (Oncorhynchus mykiss).

The effect of tricaine methanesulfonate (MS222) on rainbow trout liver biotransformation rates was investigated with a microsomal model; an in vitro preparation that can be employed with or without the use of an anaesthetic. Two experimental sets of rainbow trout microsomes were tested; one representing in vivo or surgical tricaine exposures and the other representing in vitro tissue/organ collection tricaine exposures. Microsomal incubations were performed on these two experimental groups with phenol as substrate to assess the effects of tricaine on Phase I (ring-hydroxylation) and II (glucuronidation) liver biotransformation by monitoring production of hydroquinone (HQ), catechol (CAT), and phenylglucuronide (PG). The use of a 2-h 100 mg/l exposure of tricaine for surgical anesthesia with or without 24-h recovery did not significantly (P< or =0.05) affect rates of phenol (Phase I and II) biotransformation rates; nor, did the 5-min 300 mg/l tricaine exposure for isolated organ/tissue collection significantly (P< or =0.05) affect phenol (Phase I and II) biotransformation rates. There were also no significant statistical differences (P< or =0.05) in P450 protein levels, or 7-ethoxyresorufin-O-deethylase (EROD) activity in these microsomal assays between any of the tricaine treated rainbow trout and controls.

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.