RESUMO
There is a pressing need for more-holistic approaches to fisheries assessments along with growing demand to reduce the health impacts of sample collections. Metabolomic tools enable the use of sample matrices that can be collected with minimal impact on the organism (e.g., blood, urine, and mucus) and provide high-throughput, untargeted biochemical information without the requirement of a sequenced genome. These qualities make metabolomics ideal for monitoring a wide range of fish species, particularly those under protected status. In the current study, we surveyed the relative abundances of 120 endogenous metabolites in epidermal mucus across eight freshwater fish species belonging to seven phylogenetic orders. Principal component analysis was used to provide an overview of the data set, revealing strong interspecies relationships in the epidermal mucous metabolome. Normalized relative abundances of individual endogenous metabolites were then used to identify commonalities across multiple species, as well as those metabolites that showed notable species specificity. For example, taurine was measured in high relative abundance in the epidermal mucus of common carp (Cyprinus carpio), northern pike (Esox lucius), golden shiner (Notemigonus crysoleucas), rainbow trout (Oncorhynchus mykiss), and rainbow smelt (Osmerus mordax), whereas γ-amino butyric acid (GABA) exhibited a uniquely high relative abundance in flathead catfish (Pylodictis olivaris). Finally, hierarchical cluster analysis was used to evaluate species relatedness as characterized by both the epidermal mucous metabolome (phenotype) and genetic phylogeny (genotype). This comparison revealed species for which relatedness in the epidermal mucous metabolome composition closely aligns with phylogenetic relatedness (e.g., N. crysoleucas and C. carpio), as well as species for which these two measures are not well aligned (e.g., P. olivaris and Polyodon spathula). These, and other findings reported here, highlight novel areas for future research with fish, including development of epidermal mucous-based markers for non-invasive health monitoring, sex determination, and hypoxia tolerance.
Assuntos
Carpas , Cyprinidae , Ictaluridae , Oncorhynchus mykiss , Osmeriformes , Animais , Filogenia , Metaboloma , Esocidae , Muco , Água Doce , Oncorhynchus mykiss/metabolismoRESUMO
Greater knowledge of biotransformation rates for ionizable organic compounds (IOCs) in fish is required to properly assess the bioaccumulation potential of many environmentally relevant contaminants. In this study, we measured in vitro hepatic clearance rates for 50 IOCs using a pooled batch of liver S9 fractions isolated from rainbow trout (Oncorhynchus mykiss). The IOCs included four types of strongly ionized acids (carboxylates, phenolates, sulfonates, and sulfates), three types of strongly ionized bases (primary, secondary, tertiary amines), and a pair of quaternary ammonium compounds (QACs). Included in this test set were several surfactants and a series of beta-blockers. For linear alkyl chain IOC analogues, biotransformation enzymes appeared to act directly on the charged terminal group, with the highest clearance rates for tertiary amines and sulfates and no clearance of QACs. Clearance rates for C12-IOCs were higher than those for C8-IOC analogues. Several analogue series with multiple alkyl chains, branched alkyl chains, aromatic rings, and nonaromatic rings were evaluated. The likelihood of multiple reaction pathways made it difficult to relate all differences in clearance to specific molecular features the tested IOCs. Future analysis of primary metabolites in the S9 assay is recommended to further elucidate biotransformation pathways for IOCs in fish.
Assuntos
Fígado/metabolismo , Oncorhynchus mykiss/metabolismo , Animais , Biotransformação , Extratos Hepáticos/metabolismo , Compostos Orgânicos/químicaRESUMO
Measured rates of intrinsic clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro intrinsic clearance of six reference compounds (benzo[a]pyrene, 4-nonylphenol, di-tert-butyl phenol, fenthion, methoxychlor and o-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. O-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro intrinsic clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro clearance values exhibited a reduced level of interlaboratory variability (5.3-38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro intrinsic clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment.
Assuntos
Bioensaio/métodos , Criopreservação , Hepatócitos/metabolismo , Laboratórios , Oncorhynchus mykiss/metabolismo , Animais , Separação Celular , Sobrevivência Celular , Masculino , Reprodutibilidade dos Testes , Xenobióticos/metabolismoRESUMO
Biotransformation rates measured using cryopreserved trout hepatocytes can be extrapolated to the whole animal to predict metabolism impacts on chemical bioaccumulation. Future use of these methods within a regulatory context requires, however, that they be optimized and standardized. Specifically, questions exist concerning gender differences in metabolism, cryopreservability of cells, and the accuracy of in vitro-in vivo scaling factors. 2. In this study, we evaluated hepatocytes from juvenile male and female trout. No gender differences in cell size, protein abundance, cytochrome P450 content, ethoxyresorufin-O-deethylase activity, uridine diphosphate glucuronosyltransferase activity or intrinsic clearance of pyrene were observed for freshly isolated hepatocytes. There was a small difference in measured glutathione-S-transferase activity (<25%; males > females). 3. Cells were cryopreserved by two methods: direct placement into liquid N2 vapor and controlled, slow-rate freezing. Comparable live recovery and enzymatic activity were observed regardless of freezing method or gender. Cells cryopreserved in liquid N2 vapor exhibited activity levels similar to those of freshly isolated cells, although there were small but significant differences in pyrene clearance and glutathione-S-transferase activity (frozen < fresh). Hepatocellularity values did not differ by sex. 4. These results suggest that hepatocytes from male and female juvenile trout may be used interchangeably for in vitro-in vivo metabolism extrapolations.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Animais , Biotransformação , Sobrevivência Celular , Criopreservação , Sistema Enzimático do Citocromo P-450/química , Feminino , Masculino , Nitrogênio/química , Oncorhynchus mykiss , Medição de RiscoRESUMO
In vitro and in silico methods that can reduce the need for animal testing are being used with increasing frequency to assess chemical risks to human health and the environment. The rate of hepatic biotransformation is an important species-specific parameter for determining bioaccumulation potential and extrapolating in vitro bioactivity to in vivo effects. One approach to estimating hepatic biotransformation is to employ in vitro systems derived from liver tissue to measure chemical (substrate) depletion over time which can then be translated to a rate of intrinsic clearance (CLint). In the present study, cryopreserved hepatocytes from humans, rats, and rainbow trout were used to measure CLint values for 54 industrial and pesticidal chemicals at starting test concentrations of 0.1 and 1 µM. A data evaluation framework that emphasizes the behavior of Heat-Treated Controls (HTC) was developed to identify datasets suitable for rate reporting. Measured or estimated ("greater than" or "less than") CLint values were determined for 124 of 226 (55 %) species-chemical-substrate concentration datasets with acceptable analytical chemistry. A large percentage of tested chemicals exhibited low HTC recovery values, indicating a substantial abiotic loss of test chemical over time. An evaluation of KOW values for individual chemicals suggested that in vitro test performance declined with increasing chemical hydrophobicity, although differences in testing devices for mammals and fish also likely played a role. The current findings emphasize the value of negative controls as part of a rigorous approach to data quality assessment for in vitro substrate depletion studies. Changes in current testing protocols can be expected to result in the collection of higher quality data. However, poorly soluble chemicals are likely to remain a challenge for CLint determination.
Assuntos
Criopreservação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Adulto , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Criopreservação/métodos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Masculino , Oncorhynchus mykiss , Praguicidas/metabolismo , Praguicidas/toxicidade , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
There is significant concern regarding potential impairment of fish reproduction associated with endocrine disrupting chemicals. Aromatase (CYP19) is a steroidogenic enzyme involved in the conversion of androgens to estrogens. Inhibition of aromatase by chemicals can result in reduced concentrations of estrogens leading to adverse reproductive effects. These effects have been extensively investigated in a small number of laboratory model fishes, such as fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio). But, differences in sensitivity among species are largely unknown. Therefore, this study took a first step toward understanding potential differences in sensitivity to aromatase inhibitors among fishes. Specifically, a standard in vitro aromatase inhibition assay using subcellular fractions of whole tissue homogenates was used to evaluate the potential sensitivity of 18 phylogenetically diverse species of freshwater fish to the nonsteroidal aromatase inhibitor fadrozole. Sensitivity to fadrozole ranged by more than 52-fold among these species. Five species were further investigated for sensitivity to up to 4 additional nonsteroidal aromatase inhibitors, letrozole, imazalil, prochloraz, and propiconazole. Potencies of each of these chemicals relative to fadrozole ranged by up to 2 orders of magnitude among the 5 species. Fathead minnow, Japanese medaka, and zebrafish were among the least sensitive to all the investigated chemicals; therefore, ecological risks of aromatase inhibitors derived from these species might not be adequately protective of more sensitive native fishes. This information could guide more objective ecological risk assessments of native fishes to chemicals that inhibit aromatase.
Assuntos
Inibidores da Aromatase/farmacologia , Reprodução/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Fadrozol/farmacologia , Feminino , Peixes , Água Doce , Especificidade da EspécieRESUMO
Chemical monitoring data were collected in surface waters from 57 Great Lakes tributaries from 2010 to 13 to identify chemicals of potential biological relevance and sites at which these chemicals occur. Traditional water-quality benchmarks for aquatic life based on in vivo toxicity data were available for 34 of 67 evaluated chemicals. To expand evaluation of potential biological effects, measured chemical concentrations were compared to chemical-specific biological activities determined in high-throughput (ToxCast) in vitro assays. Resulting exposure-activity ratios (EARs) were used to prioritize the chemicals of greatest potential concern: 4nonylphenol, bisphenol A, metolachlor, atrazine, DEET, caffeine, tris(2butoxyethyl) phosphate, tributyl phosphate, triphenyl phosphate, benzo(a)pyrene, fluoranthene, and benzophenone. Water-quality benchmarks were unavailable for five of these chemicals, but for the remaining seven, EAR-based prioritization was consistent with that based on toxicity quotients calculated from benchmarks. Water-quality benchmarks identified three additional PAHs (anthracene, phenanthrene, and pyrene) not prioritized using EARs. Through this analysis, an EAR of 10-3 was identified as a reasonable threshold above which a chemical might be of potential concern. To better understand apical hazards potentially associated with biological activities captured in ToxCast assays, in vitro bioactivity data were matched with available adverse outcome pathway (AOP) information. The 49 ToxCast assays prioritized via EAR analysis aligned with 23 potentially-relevant AOPs present in the AOP-Wiki. Mixture effects at monitored sites were estimated by summation of EAR values for multiple chemicals by individual assay or individual AOP. Commonly predicted adverse outcomes included impacts on reproduction and mitochondrial function. The EAR approach provided a screening-level assessment for evidence-based prioritization of chemicals and sites with potential for adverse biological effects. The approach aids prioritization of future monitoring activities and provides testable hypotheses to help focus those efforts. This also expands the fraction of detected chemicals for which biologically-based benchmark concentrations are available to help contextualize chemical monitoring results.
Assuntos
Rotas de Resultados Adversos , Monitoramento Ambiental/métodos , Ensaios de Triagem em Larga Escala/métodos , Poluentes Químicos da Água/análise , Great Lakes Region , Rios , Qualidade da ÁguaRESUMO
Biotransformation may substantially impact the toxicity and accumulation of xenobiotic chemicals in fish. However, this activity can vary substantially within and among species. In this study, liver S9 fractions from rainbow trout (4-400â¯g) were used to evaluate relationships between fish body mass and the activities of phase I and phase II metabolic enzymes. An analysis of log-transformed data, expressed per gram of liver (gâ¯liver-1), showed that total cytochrome P450 (CYP) concentration, UDP-glucuronosyltransferase (UGT) activity, and glutathione S-transferase (GST) activity exhibited small but significant inverse relationships with fish body weight. In contrast, in vitro intrinsic clearance rates (CLIN VITRO,INT) for three polycyclic aromatic hydrocarbons (PAHs) increased with increasing body weight. Weight normalized liver mass also decreased inversely with body weight, suggesting a need to express hepatic metabolism data per gram of body weight (gâ¯BW-1) in order to reflect the metabolic capabilities of the whole animal. When the data were recalculated in this manner, negative allometric relationships for CYP concentration, UGT activity, and GST activity became more pronounced, while CLIN VITRO,INT rates for the three PAHs showed no significant differences across fish sizes. Ethoxyresorufin O-deethylase (EROD) activity normalized to tissue weight (gâ¯liver-1) or body weight (gâ¯BW-1) exhibited a non-monotonic pattern with respect to body weight. The results of this study may have important implications for chemical modeling efforts with fish.
Assuntos
Microssomos Hepáticos/enzimologia , Modelos Biológicos , Oncorhynchus mykiss/fisiologia , Xenobióticos/toxicidade , Algoritmos , Animais , Tamanho Corporal , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Proteínas de Peixes/metabolismo , Glucuronosiltransferase/metabolismo , Glutationa Transferase/metabolismo , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Desintoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , Microssomos Hepáticos/metabolismo , Oncorhynchus mykiss/sangue , Oncorhynchus mykiss/crescimento & desenvolvimento , Tamanho do Órgão , Hidrocarbonetos Policíclicos Aromáticos/sangue , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Reprodutibilidade dos Testes , Toxicocinética , Xenobióticos/sangue , Xenobióticos/metabolismoRESUMO
The U.S. Environmental Protection Agency's ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with potential apical effects relevant to risk assessors. Thus, efforts are underway to develop AOPs relevant to pathway-specific perturbations detected in ToxCast assays. Previous work identified a "cytotoxic burst" (CTB) phenomenon wherein large numbers of the ToxCast assays begin to respond at or near test chemical concentrations that elicit cytotoxicity, and a statistical approach to defining the bounds of the CTB was developed. To focus AOP development on the molecular targets corresponding to ToxCast assays indicating pathway-specific effects, we conducted a meta-analysis to identify which assays most frequently respond at concentrations below the CTB. A preliminary list of potentially important, target-specific assays was determined by ranking assays by the fraction of chemical hits below the CTB compared with the number of chemicals tested. Additional priority assays were identified using a diagnostic-odds-ratio approach which gives greater ranking to assays with high specificity but low responsivity. Combined, the two prioritization methods identified several novel targets (e.g., peripheral benzodiazepine and progesterone receptors) to prioritize for AOP development, and affirmed the importance of a number of existing AOPs aligned with ToxCast targets (e.g., thyroperoxidase, estrogen receptor, aromatase). The prioritization approaches did not appear to be influenced by inter-assay differences in chemical bioavailability. Furthermore, the outcomes were robust based on a variety of different parameters used to define the CTB.
Assuntos
Rotas de Resultados Adversos , Substâncias Perigosas/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Testes de Toxicidade/métodos , Toxicologia/métodos , Animais , Disponibilidade Biológica , Sobrevivência Celular/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Substâncias Perigosas/metabolismo , Humanos , Valor Preditivo dos TestesRESUMO
Isolated hepatocytes and liver S9 fractions have been used to collect in vitro biotransformation data for fish as a means of improving modeled estimates of chemical bioaccumulation. To date, however, there have been few direct comparisons of these 2 methods. In the present study, cryopreserved trout hepatocytes were used to measure in vitro intrinsic clearance rates for 6 polycyclic aromatic hydrocarbons (PAHs). These rates were extrapolated to estimates of in vivo intrinsic clearance and used as inputs to a well stirred liver model to predict hepatic clearance. Predicted rates of hepatic clearance were then evaluated by comparison with measured rates determined previously using isolated perfused livers. Hepatic clearance rates predicted using hepatocytes were in good agreement with measured values (<2.1-fold difference for 5 of 6 compounds) under 2 competing binding assumptions. These findings, which may be attributed in part to high rates of PAH metabolism, are similar to those obtained previously using data from liver S9 fractions. For 1 compound (benzo[a]pyrene), the in vivo intrinsic clearance rate calculated using S9 data was 10-fold higher than that determined using hepatocytes, possibly due to a diffusion limitation on cellular uptake. Generally, however, there was good agreement between calculated in vivo intrinsic clearance rates obtained using either in vitro test system. These results suggest that both systems can be used to improve bioaccumulation assessments for fish, particularly when vitro rates of activity are relatively high, although additional work is needed to determine if the chemical domain of applicability for each system differs. Environ Toxicol Chem 2017;36:463-471. Published 2016 SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
Assuntos
Hepatócitos/metabolismo , Extratos Hepáticos/metabolismo , Fígado/metabolismo , Modelos Biológicos , Oncorhynchus mykiss/metabolismo , Animais , Benzo(a)pireno/farmacocinética , Biotransformação , Células Cultivadas , Cinética , Taxa de Depuração Metabólica , Hidrocarbonetos Policíclicos Aromáticos/farmacocinética , Cultura Primária de Células , Poluentes Químicos da Água/farmacocinéticaRESUMO
Adverse outcome pathways (AOPs) describe toxicant effects as a sequential chain of causally linked events beginning with a molecular perturbation and culminating in an adverse outcome at an individual or population level. Strategies for developing AOPs are still evolving and depend largely on the intended use or motivation for development and data availability. The present review describes 4 ecotoxicological AOP case studies, developed for different purposes. In each situation, creation of the AOP began in a manner determined by the initial motivation for its creation and expanded either to include additional components of the pathway or to address the domains of applicability in terms of chemical initiators, susceptible species, life stages, and so forth. Some general strategies can be gleaned from these case studies, which a developer may find to be useful for supporting an existing AOP or creating a new one. Several web-based tools that can aid in AOP assembly and evaluation of weight of evidence for scientific robustness of AOP components are highlighted. Environ Toxicol Chem 2017;36:1429-1449. © 2017 SETAC.
Assuntos
Ecotoxicologia/métodos , Animais , Fluoxetina/metabolismo , Fluoxetina/toxicidade , Substâncias Perigosas/toxicidade , Humanos , Inseticidas/toxicidade , Receptores de Esteroides/agonistas , Receptores de Esteroides/metabolismo , Medição de Risco , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Poluentes Químicos da Água/químicaRESUMO
Inflation of the posterior and/or anterior swim bladder is a process previously demonstrated to be regulated by thyroid hormones. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP). First, fathead minnow embryos were exposed to 0.6, 1.9, or 6.0 mg/L or control water until 6 d postfertilization (dpf), at which time posterior swim bladder inflation was assessed. To examine anterior swim bladder inflation, a second study was conducted with 6-dpf larvae exposed to the same IOP concentrations until 21 dpf. Fish from both studies were sampled for T4/T3 measurements and gene transcription analyses. Incidence and length of inflated posterior swim bladders were significantly reduced in the 6.0 mg/L treatment at 6 dpf. Incidence of inflation and length of anterior swim bladder were significantly reduced in all IOP treatments at 14 dpf, but inflation recovered by 18 dpf. Throughout the larval study, whole-body T4 concentrations increased and T3 concentrations decreased in all IOP treatments. Consistent with hypothesized compensatory responses, deiodinase-2 messenger ribonucleic acid (mRNA) was up-regulated in the larval study, and thyroperoxidase mRNA was down-regulated in all IOP treatments in both studies. These results support the hypothesized adverse outcome pathways linking inhibition of deiodinase activity to impaired swim bladder inflation. Environ Toxicol Chem 2017;36:2942-2952. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
Assuntos
Sacos Aéreos/efeitos dos fármacos , Cyprinidae/crescimento & desenvolvimento , Iodeto Peroxidase/metabolismo , Ácido Iopanoico/toxicidade , Poluentes Químicos da Água/toxicidade , Sacos Aéreos/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Cyprinidae/metabolismo , Regulação para Baixo/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Iodeto Peroxidase/antagonistas & inibidores , Iodeto Peroxidase/genética , Larva/efeitos dos fármacos , Larva/metabolismo , RNA Mensageiro/metabolismo , Espectrometria de Massas em Tandem , Tiroxina/análise , Tri-Iodotironina/análise , Poluentes Químicos da Água/químicaRESUMO
Trout provide a relatively easy source of hepatocytes that can be cryopreserved and used for a range of applications including toxicity testing and determination of intrinsic clearance. Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess metabolic stability of xenobiotics in fish by means of a substrate depletion approach. Variations on these methods, troubleshooting tips, and directions for use of extrapolation factors to express results in terms of in vivo intrinsic clearance are included. These protocols have been developed for rainbow trout, but can be adapted to other fish species with appropriate considerations.