RESUMO
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.
Assuntos
Benzofenonas/metabolismo , Disruptores Endócrinos/metabolismo , Oncorhynchus mykiss/metabolismo , Animais , Cromatografia Líquida , Estrogênios , Espectrometria de Massas em Tandem , VitelogeninasRESUMO
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.
Assuntos
Cabras/metabolismo , Oncorhynchus mykiss/metabolismo , Perciformes/metabolismo , Praguicidas/farmacocinética , Aminopiridinas/farmacocinética , Animais , Feminino , Masculino , Redes e Vias Metabólicas , Compostos Organotiofosforados/farmacocinética , Ratos , Especificidade da Espécie , Estrobilurinas/farmacocinéticaRESUMO
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.
Assuntos
Bases de Dados Factuais , Bases de Conhecimento , Xenobióticos/farmacocinética , Animais , Poluentes Ambientais/farmacocinética , Humanos , Inativação Metabólica , Medição de Risco , SoftwareRESUMO
Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.
Assuntos
Ecotoxicologia , Medição de Risco , Animais , Dermatite Fototóxica , Humanos , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Pesquisa , Estupor/induzido quimicamente , Biologia de Sistemas , Vitelogênese/efeitos dos fármacosRESUMO
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.
Assuntos
Bioensaio , Estrogênios/farmacologia , Compostos Heterocíclicos/farmacologia , Hidrocarbonetos Aromáticos/farmacologia , Oncorhynchus mykiss/metabolismo , Animais , Ligação Competitiva , Citosol/efeitos dos fármacos , Citosol/metabolismo , Compostos Heterocíclicos/química , Hidrocarbonetos Aromáticos/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vitelogeninas/genética , Vitelogeninas/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
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.
Assuntos
Cromatografia Líquida/métodos , Disruptores Endócrinos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Fígado , Oncorhynchus mykiss/metabolismo , Animais , Benzofenonas/análise , Benzofenonas/metabolismo , Cicloexanos/análise , Cicloexanos/metabolismo , Disruptores Endócrinos/análise , Disruptores Endócrinos/metabolismo , Fígado/química , Fígado/metabolismoRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Cyprinidae/metabolismo , Hormônios/metabolismo , Oncorhynchus mykiss/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Endocrinologia , Feminino , Ligantes , Masculino , TermodinâmicaRESUMO
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.
Assuntos
Catecóis/metabolismo , Hidroquinonas/metabolismo , Microssomos Hepáticos/metabolismo , Oncorhynchus mykiss/fisiologia , Fenol/metabolismo , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Hidroxilação , CinéticaRESUMO
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.
Assuntos
Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Oncorhynchus mykiss/metabolismo , Análise de Variância , Animais , Biodegradação Ambiental/efeitos dos fármacos , Catecóis/metabolismo , Relação Dose-Resposta a Droga , Glucuronatos/metabolismo , Hidroquinonas/metabolismo , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Fenol/metabolismoRESUMO
The potential for temperature to influence estrogen-mediated responses in poikilothermic animals suggested that temperature may be an important variable to consider when using an estrogen-responsive reporter gene in a rainbow trout cell line to test chemicals for estrogenic activity. Rainbow trout hepatoma cells (RTH 149) incubated at 11 or 18 degrees C were cotransfected with an estrogen-responsive luciferase reporter plasmid and a plasmid containing a constitutively expressed rainbow trout estrogen receptor. The RTH-149 cells were then exposed to estradiol, with samples collected at 24-h intervals. The 72-h effective concentration for 50% maximal response (EC50) for estrogen-responsive luciferase activity at 11 degrees C was 3.8 x 10(-9) M and 7.4 x 10(-10) M at 18 degrees C. The efficacy of estradiol was lower at 11 degrees C. The maximal response to estradiol in cells at 11 degrees C was generally two- to threefold greater than controls (mean = 2.6-fold), whereas the maximal response at 18 degrees C was three- to fourfold greater than controls (mean = 3.2-fold). Ethinylestradiol, a strong estrogen receptor agonist, was similar to estradiol in potency (relative potency = 0.8) and efficacy at the two temperatures. The EC50 of the weak estrogen receptor agonist 4-tert-pentylphenol was 7.6 x 10(-7) M at 11 degrees C and 6.9 x 10(-7) M at 18 degrees C; its potency relative to 17beta-estradiol was not significantly different at the two temperatures, 0.00036 and 0.00054 at 11 degrees C and 18 degrees C, respectively. The estrogen-responsive reporter gene activity produced by 10(-8) M estradiol was completely inhibited by the two estrogen hormone receptor antagonists, ZM 189,154 and ICI 182,780, at 10(-6) M concentration of either antagonist. Although there may be slight differences in responses between the two temperatures tested here, this assay can be used to effectively determine the relative estrogenic activity of chemicals within the physiological temperature range of rainbow trout.
Assuntos
Carcinoma Hepatocelular/genética , Estradiol/análogos & derivados , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Estrogênios/metabolismo , Etinilestradiol/farmacologia , Fulvestranto , Oncorhynchus mykiss , Receptores de Estrogênio/metabolismo , Temperatura , Tetra-Hidronaftalenos/farmacologiaRESUMO
The demonstrated ability of a variety of structurally diverse chemicals to bind to the estrogen receptor has raised the concern that chemicals in the environment may be causing adverse effects through interference with nuclear receptor pathways. Many structure-activity relationship models have been developed to predict chemical binding to the estrogen receptor as an indication of potential estrogenicity. Models based on either two-dimensional or three-dimensional molecular descriptions that have been used to predict potential for binding to the estrogen receptor are the subject of the current review. The utility of such approaches to predict binding potential of diverse chemical structures in large chemical inventories, with potential application in a tiered risk assessment scheme, is discussed.
Assuntos
Poluentes Ambientais/toxicidade , Modelos Teóricos , Relação Quantitativa Estrutura-Atividade , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/fisiologia , Animais , Previsões , Humanos , Medição de RiscoRESUMO
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.
Assuntos
Estrogênios/metabolismo , Esgotos , Eliminação de Resíduos Líquidos/métodos , Purificação da Água/métodos , Animais , Cyprinidae , Peixes , Resíduos Industriais , Masculino , Ligação Proteica , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Tempo , Vitelogeninas/química , Poluentes Químicos da Água/análiseRESUMO
The molecular modeling is traditionally based on analysis of minimum energy conformers. Such simplifying assumptions could doom to failure the modeling studies given the significant variation of the geometric and electronic characteristics across the multitude of energetically reasonable conformers representing the molecules. Moreover, it has been found that the lowest energy conformers of chemicals are not necessarily the active ones with respect to various endpoints. Hence, the selection of active conformers appears to be as important as the selection of molecular descriptors in the modeling process. In this respect, we have developed effective tools for conformational analysis based on a genetic algorithm (GA), published in J. Chem. Inf. Comput. Sci. (1994, 34, 234; 1999, 39 (6), 997) and J. Chem. Inf. Model. (2005, 45 (2), 283). This paper presents a further improvement of the evolutionary algorithm for conformer generation minimizing the sensitivity of conformer distributions from the effect of smoothing parameter and improving the reproducibility of conformer distributions given the nondeterministic character of the genetic algorithm (GA). The ultimate goal of the saturation is to represent the conformational space of chemicals with an optimal number of conformers providing a stable conformational distribution which cannot be further perturbed by the addition of new conformers. The generation of stable conformational distributions of chemicals by a limited number of conformers will improve the adequacy of the subsequent molecular modeling analysis. The impact of the saturation procedure on conformer distributions in a specific structural space is illustrated by selected examples. The effect of the procedure on similarity assessment between chemicals is discussed.
RESUMO
Mathematical chemistry has afforded a variety of research areas with important tools to understand and predict the behavior of chemicals without having to consider the complexities of three-dimensional conformations of molecules. Predictive toxicology, an area of increasing importance to toxicity assessments critical to molecular design and risk management, must be based on more explicit descriptions of structure, however. Minimum energy conformations are often used for convenience due, in part, to the difficulty of computing a representative population of conformers in all but rigid structures. Such simplifying assumptions fail to reveal the variance of the stereoelectronic nature of molecules as well as the misclassification of chemicals which initiate receptor-based toxicity pathways. Because these errors impact both the success in discovering new lead and the identification of possible hazards, it is important that mathematical chemistry develop additional tools for conformational analysis. This paper presents a new system for automated 2D-3D migration of chemicals in large databases with conformer multiplication. The main advantages of this system are its straightforward performance, reasonable execution time, simplicity and applicability to building large 3D chemical inventories. The module for conformer multiplication within the 2D-3D migration system is based on a new formulation of the genetic algorithm for computing populations of possible conformers. The performance of the automated 2D-3D migration system in building a centralized 3D database for all chemicals in commerce worldwide is discussed. The applicability of the 3D database in assessing the impact of molecular flexibility on identifying active conformers in QSAR analysis and assessing similarity between chemicals is illustrated.
Assuntos
Algoritmos , Modelos Químicos , Automação , Bases de Dados Factuais , Desenho de Fármacos , Conformação Molecular , Relação Quantitativa Estrutura-Atividade , Esteroides/químicaRESUMO
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.