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.

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.

Kinetics and effects of dichloroacetic acid in rainbow trout.

Halogenated acetic acids (HAAs) produced by chlorine disinfection of municipal drinking water represent a potentially important class of environmental contaminants. Little is known, however, about their potential to adversely impact fish and other aquatic life. In this study we examined the kinetics and effects of dichloroacetic acid (DCA) in rainbow trout. Branchial uptake was measured in fish confined to respirometer-metabolism chambers. Branchial uptake efficiency was <5%, suggesting passive diffusion through aqueous channels in the gill epithelium. DCA concentrations in tissues following prolonged (72, 168, or 336 h) waterborne exposures were expressed as tissue:plasma concentration ratios. Concentration ratios for the kidney and muscle at 168 and 336 h were consistent with the suggestion that DCA distributes primarily to tissue water. Reduced concentration ratios for the liver, particularly at 72 h, indicated that DCA was highly metabolized by this tissue. Routes and rates of elimination were characterized by injecting chambered animals with a high (5.0mg/kg) or low (50 microg/kg) bolus dose. DCA was rapidly cleared by naïve animals resulting in elimination half-lives (t(1/2)) of less than 4h. Waterborne pre-treatment of fish with DCA increased the persistence of a subsequently injected dose. In high dose animals, pre-treatment caused a 4-fold decrease in whole-body clearance (CL(B)) and corresponding increases in the area under the plasma concentration-time curve (extrapolated to infinity; AUC(0-->infinity)) and t(1/2). Qualitatively similar results were obtained in low dose fish, although the magnitude of the pre-treatment effect ( approximately 2.5-fold) was reduced. Renal and branchial clearance contributed little (combined, <3% of CL(B)) to the elimination of DCA. Biliary elimination of DCA was also negligible. The steady-state volume of distribution (V(SS)) did not vary among treatment groups and was consistent with results of the tissue distribution study. DCA had no apparent effects on respiratory physiology or acid-base balance; however, the concentration of blood lactate declined progressively during continuous waterborne exposures. A transient effect on blood lactate was also observed in bolus injection experiments. The results of this study suggest that clearance of DCA is due almost entirely to metabolism. The pathway responsible for this activity exhibits characteristics in common with those of mammalian glutathione S-transferase zeta (GSTzeta), including non-linear kinetics and apparent suicide inactivation by DCA. Observed effects on blood lactate are probably due to the inhibition of pyruvate dehydrogenase kinase in aerobic tissues and may require the participation of a monocarboxylase transport protein to move DCA across cell membranes.

Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas).

There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead minnows representing three different ages and stages of reproductive activity, and from fathead minnows exposed to the aromatase inhibitor fadrozole for 7d. The goal was to determine whether measures of a single aromatase endpoint in either brain or ovary tissue would be sufficient to understand and predict system-wide effects of endocrine disrupting chemicals on aromatase activity and transcript levels. Aromatase activity in the ovary, but not brain, varied significantly with age/reproductive category, with adults held in non-reproductive conditions showing significantly lower activity than juveniles and reproductively-active adults. Significant correlations between isoform-specific transcript levels and aromatase activity were observed for ovary tissue, but those relationships were not robust for all age/reproductive categories, nor were they sustained in fadrozole-treated fish. In vitro, fadrozole inhibited the aromatase activity of brain and ovary post-mitochondrial supernatants with similar potency (IC50s = 8.82 +/- 1.58 and 6.93 +/- 0.80 microM for brain and ovary, respectively), despite large differences in the magnitude of activity. In vivo, fadrozole altered aromatase activity and isoform-specific transcript levels in both brain and ovary tissue, but concentration-response relationships were different for each tissue. Aromatase activity and P450aromB mRNA expression in brain showed a dose-dependent decrease at concentrations greater than 5.55 microg/L. In contrast, ovary activity showed an inverted U-shaped concentration-response consistent with the interplay between increased P450aromA transcript levels in ovary and competitive inhibition of the aromatase enzyme. As a whole, results of this study did not reveal any robust correlations between brain and ovary aromatase activity and/or isoform-specific mRNA expression. However, they were consistent with the current body of evidence related to teleost aromatase regulation, suggesting that increased understanding of the biology of aromatase may facilitate system-wide understanding of effects on aromatase based on relatively few measured endpoints.

A comparison of the lethal and sublethal toxicity of organic chemical mixtures to the fathead minnow (Pimephales promelas).

The joint toxic effects of known binary and multiple organic chemical mixtures to the fathead minnow (Pimephales promelas) were defined at both the 96-h 50% lethal effect concentration (LC50) and sublethal (32-d growth) response levels for toxicants with a narcosis I, narcosis II, or uncoupler of oxidative phosphoralation mode of toxic action. Experiments were designed to define the degree of additive joint toxicity for mixtures of specific xenobiotics that are believed to act through a similar or different primary mode of toxic action. Our results support the general conclusion that concentration addition is expected for the joint toxicity of similarly acting toxicants. When chemicals were thought to act by a dissimilar mechanism, the combined effects we observed at both of the response levels tested were less than predicted by concentration addition, but usually more toxic than that predicted by the independent action/response addition model. It was demonstrated in multichemical mixtures that several toxicants can act together in a nearly additive fashion to produce effects even when they are present at concentrations below their individual no-observed-effect concentration. Concentration-response relationships for test chemicals at both the lethal and sublethal responses were defined for each of the three modes of toxic action studied. When normalized for potency, it was observed that one relationship could be defined to predict lethality to juvenile fathead minnows when exposed to individual chemicals with either a narcosis I, narcosis II, or uncoupler mode of toxic action. These sublethal relationships were similar for the narcosis I and narcosis II test chemicals, but a steeper response was observed for tests conducted with uncouplers.