In Vitro-In Vivo Extrapolation of Hepatic Biotransformation Data for Fish. III. An In-depth Case Study with Pyrene.

Computational models that predict chemical bioaccumulation in fish generally account for biotransformation using an apparent first-order whole-body rate constant (kB ; d-1 ). The use of such models requires, therefore, that methods exist for estimating kB , ideally without the need to expose live animals. One promising approach for estimating kB involves the extrapolation of measured in vitro intrinsic clearance (CLIN VITRO,INT ) to the whole animal (in vitro-in vivo extrapolation, [IVIVE]). To date, however, the accuracy of such predictions has been difficult to assess due to uncertainties associated with one or more extrapolation factors and/or a mismatch between fish used to generate in vitro data and those used to conduct in vivo exposures. In the present study we employed a combined in vitro and in vivo experimental approach to evaluate the IVIVE procedure using pyrene (PYR) as a model chemical. To the extent possible, measured rates of CLIN VITRO,INT were extrapolated to estimates of kB using extrapolation factors based on measured values. In vitro material (liver S9 fraction) was obtained from fish exposed to PYR in a controlled bioconcentration study protocol. Fish from the same study were then used to estimate in vivo kB values from an analysis of chemical depuration data. Averaged across four study groups, kB values estimated by IVIVE underestimated those determined from in vivo data by 2.6-fold. This difference corresponds to a 4.1-fold underestimation of true in vivo intrinsic clearance, assuming the liver is the only site of biotransformation. These findings are consistent with previous work performed using mammals and have important implications for use of measured CLIN VITRO,INT values in bioaccumulation assessments with fish. Environ Toxicol Chem 2023;42:1501-1515. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Biotransformation Potential of Cationic Surfactants in Fish Assessed with Rainbow Trout Liver S9 Fractions.

Biotransformation may substantially reduce the extent to which organic environmental contaminants accumulate in fish. Presently, however, relatively little is known regarding the biotransformation of ionized chemicals, including cationic surfactants, in aquatic organisms. To address this deficiency, a rainbow trout liver S9 substrate depletion assay (RT-S9) was used to measure in vitro intrinsic clearance rates (CLint ; ml min-1 g liver-1 ) for 22 cationic surfactants that differ with respect to alkyl chain length and degree of methylation on the charged nitrogen atom. None of the quaternary N,N,N-trimethylalkylammonium compounds exhibited significant clearance. Rapid clearance was observed for N,N-dimethylalkylamines, and slower rates of clearance were measured for N-methylalkylamine analogs. Clearance rates for primary alkylamines were generally close to or below detectable levels. For the N-methylalkylamines and N,N-dimethylalkylamines, the highest CLint values were measured for C10 -C12 homologs; substantially lower clearance rates were observed for homologs containing shorter or longer carbon chains. Based on its cofactor dependency, biotransformation of C12 -N,N-dimethylamine appears to involve one or more cytochrome P450-dependent reaction pathways, and sulfonation. On a molar basis, N-demethylation metabolites accounted for up to 25% of the N,N-dimethylalkylamines removed during the 2-h assay, and up to 55% of the removed N-methylalkylamines. These N-demethylation products possess greater metabolic stability in the RT-S9 assay than the parent structures from which they derive and may contribute to the overall risk of ionizable alkylamines. The results of these studies provide a set of consistently determined CLint values that may be extrapolated to whole trout to inform in silico bioaccumulation assessments. Environ Toxicol Chem 2021;40:3123-3136. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates.

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 2-h incubations relative to those obtained in the absence of PMSF and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish. Environ Toxicol Chem 2021;40:148-161. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

In vitro-in vivo extrapolation of hepatic and gastrointestinal biotransformation rates of hydrophobic chemicals in rainbow trout.

Hepatic in vitro biotransformation assays, in combination with in vitro-in vivo extrapolation (IVIVE) and bioaccumulation modeling, can be used to support regulatory bioaccumulation assessments. In most applications, however, these methods ignore the possibility of extrahepatic metabolism. Here we evaluated intestinal biotransformation in rainbow trout using S9 fractions prepared from the upper intestinal (GIT) epithelium. Measured levels of activity determined using standard substrates for phase I and phase II biotransformation enzymes were within 2-fold of activities measured in hepatic S9 fractions. In vitro intrinsic clearance rates for 2-ethylhexyl-4-methoxycinnamate (EHMC; an organic sunscreen agent) and two polycyclic aromatic hydrocarbons (pyrene [PYR] and benzo(a)pyrene [BAP]) were significantly higher in liver S9 fractions than in GIT S9 fractions. For octocrylene (OCT; a second sunscreen agent), however, in vitro intrinsic clearance rates were higher in GIT S9 fractions compared to liver S9 fractions. An existing 'liver only' IVIVE model was expanded to consider biotransformation in both the liver and GIT. Relevant IVIVE scaling factors were developed by morphological, histological, and biochemical evaluation of trout intestines. For chemicals biotransformed at higher rates by hepatic S9 fractions (i.e., BAP, PYR, EHMC), the 'liver & GIT' model yielded whole-body biotransformation rate constants (kMET) that were within 1.2 to 1.4-fold of those estimated using the 'liver only' model. In contrast to these findings, the mean kMET for OCT obtained using the 'liver & GIT' model was 3.3 times higher than the mean kMET derived using the 'liver only' model and was in good agreement with empirical kMET estimates determined previously for trout (<20 % difference). The results of this study suggest that current 'liver only' IVIVE approaches may underestimate in vivo biotransformation rates for chemicals that undergo substantial biotransformation in the GIT.

Biotransformation of Polycyclic Aromatic Hydrocarbons by Trout Liver S9 Fractions: Evaluation of Competitive Inhibition Using a Substrate Depletion Approach.

Environmental contaminants frequently occur as part of a chemical mixture, potentially resulting in competitive inhibition among multiple substrates metabolized by the same enzyme. Trout liver S9 fractions were used to evaluate the biotransformation of 3 polycyclic aromatic hydrocarbons (PAHs): phenanthrene, pyrene, and benzo[a]pyrene, tested as binary mixtures. Initial rates of biotransformation were determined using a substrate-depletion approach. The resulting data were then fitted by simultaneous nonlinear regression to a competitive inhibition model. In each case, the PAH possessing the lower Michaelis-Menten affinity constant (KM ) competitively inhibited biotransformation of the other compound. Inhibition constants determined for the lower-KM compound were generally close to previously determined KM values, consistent with the suggestion that phase I biotransformation of PAHs is largely catalyzed by one or a small number of cytochrome P450 enzymes. The use of a substrate-depletion approach to perform enzyme-inhibition studies imposes practical limitations on experimental design and complicates the interpretation of derived kinetic constants. Nevertheless, the resulting information may have utility for chemical hazard assessments as well as the design and interpretation of controlled laboratory studies. Depletion experiments informed by measured chemical concentrations in tissues may also provide a means of determining whether enzyme inhibition occurs under relevant environmental conditions. Environ Toxicol Chem 2019;38:2729-2739. Published 2019 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.

Allometric scaling of hepatic biotransformation in rainbow trout.

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 (4-400 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 (g 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 (g 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 (g liver-1) or body weight (g 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.

Toxicokinetics of the neonicotinoid insecticide imidacloprid in rainbow trout (Oncorhynchus mykiss).

Studies were conducted to determine the distribution and elimination of imidacloprid (IMI) in rainbow trout. Animals were injected with a low (47.6 µg/kg), medium (117.5 µg/kg) or high (232.7 µg/kg) dose directly into the bloodstream and allowed to depurate. The fish were then sampled to characterize the loss of IMI from plasma and its appearance in expired water (all dose groups) and urine (medium dose only). In vitro biotransformation of IMI was evaluated using trout liver S9 fractions. Mean total clearance (CLT) values determined by non-compartmental analysis of plasma time-course data were 21.8, 27.0 and 19.5 mL/h/kg for the low, medium and high dose groups, respectively. Estimated half-lives for the same groups were 67.0, 68.4 and 68.1 h, while fitted values for the steady-state volume of distribution (VSS) were 1.72, 2.23 and 1.81 L/kg. Branchial elimination rates were much lower than expected, suggesting that IMI is highly bound in blood. Renal clearance rates were greater than measured rates of branchial clearance (60% of CLT in the medium dose group), possibly indicating a role for renal membrane transporters. There was no evidence for hepatic biotransformation of IMI. Collectively, these findings suggest that IMI would accumulate in trout in continuous waterborne exposures.

Measurement of kinetic parameters for biotransformation of polycyclic aromatic hydrocarbons by trout liver S9 fractions: Implications for bioaccumulation assessment.

In vitro substrate depletion methods developed by the pharmaceutical industry are being used with increasing frequency to support chemical bioaccumulation assessments for fish. However, the application of these methods to high log K ow chemicals poses special challenges. Biotransformation of three polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions. Measured activity declined with incubation time and was reduced by acetone (used as a spiking solvent) at concentrations greater than 0.5%. Addition of alamethicin, a pore-forming peptide used to support UDP-glucuronosyltransferase activity, also reduced activity in a concentration-dependent manner. The substrate concentration dependence of activity was evaluated to estimate K M and V max values for each compound. Derived kinetic constants suggested that all three PAHs are transformed by the same reaction pathway and indicated an inverse correlation between K M and chemical log K ow. Binding effects on activity were evaluated by measuring unbound chemical concentrations across a range of S9 protein levels. Reaction rates were proportional to the unbound concentration except when these concentrations approached saturating levels, providing a direct demonstration of the free chemical hypothesis. These findings suggest that previous in vitro work with high log K ow compounds was conducted at inappropriately high substrate concentrations resulting in underestimation of true in vivo activity. Preliminary calculations also indicate that PAH metabolism in fish may approach saturation during standardized in vivo testing efforts, potentially resulting in concentration-dependent accumulation and/or steady-state levels of accumulation greater than those which occur in a natural setting.

Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish.

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.

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide.

1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency. 2. Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. 3. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. 4. These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.

Toxicokinetics of perfluorooctane sulfonate in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) confined to respirometer-metabolism chambers were dosed with perfluorooctane sulfonate (PFOS) by intra-arterial injection and sampled to obtain concentration time-course data for plasma and either urine or expired water. The data were then analyzed using a 2-compartment clearance-volume model. Renal and branchial clearance rates (mL/d/kg) determined for all experiments averaged 19% and 81% of total clearance, respectively. Expressed as mean values for all experiments, the steady-state volume of distribution was 277 mL/kg and the terminal half-life was 86.8 d. Additional animals were exposed to PFOS in water, resulting in an average calculated branchial uptake efficiency of 0.36%. The renal clearance rate determined in the present study is approximately 75 times lower than that determined in earlier studies with perfluorooctanoate (PFOA). Previously, it was suggested that PFOA is a substrate for membrane transporters in the trout kidney. The present study suggests that glomerular filtration may be sufficient to explain the observed renal clearance rate for PFOS, although a role for membrane transporters cannot be ruled out. These findings demonstrate that models developed to predict the bioaccumulation of perfluoroalkyl acids by fish must account for differences in renal clearance of individual compounds.

Toxicokinetics of perfluorooctanoate (PFOA) in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) confined to respirometer-metabolism chambers were dosed with perfluorooctanoate (PFOA) by intra-arterial (i.a.) injection and sampled to obtain concentration time-course data for plasma, urine, and expired water. The data were then analyzed by compartmental modeling to estimate rates of renal and branchial clearance. Averaged across all animals, the renal clearance rate (1.35mL/h/kg) was more than ten times greater than the branchial clearance rate (0.12mL/h/kg). The average whole-body elimination half-life was 12.6d, which is somewhat longer than values obtained in previous studies with smaller trout. The tissue distribution of PFOA was assessed by collecting tissues at the end of chambered exposures and in a separate tissue time-course experiment. From the time-course study it appeared that an internal steady-state was established within 24h of i.a. injection. Consistent with previous studies, the rank order of PFOA concentration in tissues at steady state was: plasma>liver>kidney>muscle. In a second set of chambered experiments, fish were exposed to PFOA in water to determine the rate of branchial uptake. Branchial uptake rates were too low to assess directly by measuring PFOA concentrations in inspired and expired water. Uptake rate constants (mean 0.19L/d/kg; 0.1% uptake efficiency) were therefore estimated by compartmental modeling using plasma concentration time-course data and model parameters derived from the elimination experiments. It is clear from this effort that elimination of PFOA by trout occurs primarily via the renal route. This finding is consistent with numerous studies of mammals and suggests that trout possess membrane transporters that facilitate the movement of PFOA from plasma to urine.

Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish.

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.

Hepatic clearance of 6 polycyclic aromatic hydrocarbons by isolated perfused trout livers: prediction from in vitro clearance by liver S9 fractions.

Isolated perfused trout livers were used to evaluate in vitro-in vivo metabolism extrapolation procedures for fish. In vitro depletion rates for 6 polycyclic aromatic hydrocarbons (PAHs) were measured using liver S9 fractions and extrapolated to the intact tissue. Predicted hepatic clearance (CLH) values were then compared with values exhibited by intact livers. Binding in liver perfusates was manipulated using bovine serum albumin (BSA) and was characterized by solid-phase microextraction. Additional studies were conducted to develop binding terms (f U; calculated as the ratio of unbound fractions in liver perfusate [f U,PERF] and the S9 system [f U,S9]) used as inputs to a well-stirred liver model. Hepatic clearance values for pyrene and benzo[a]pyrene, predicted by extrapolating in vitro data to the intact tissue, were in good agreement with measured values (< 2-fold difference). This can be partly attributed to the rapid rate at which both compounds were metabolized by S9 fractions, resulting in perfusion-limited clearance. Predicted levels of CLH for the other PAHs underestimated observed values although these differences were generally small (< 3-fold, except for naphthalene). Setting f U = 1.0 improved clearance predictions at the highest tested BSA concentration (10mg/ml), suggesting that trout S9 fractions exhibit lower levels of intrinsic activity than the intact tissue or that the full binding assumption (ie, f U = f U,PERF/f U,S9) underestimates the availability of hydrophobic substrates to hepatic metabolizing enzymes. These findings provide qualified support for procedures currently being used to predict metabolism impacts on chemical accumulation by fish based on measured rates of in vitro activity.

Enantiomer-specific in vitro biotransformation of select pharmaceuticals in rainbow trout (Oncorhynchus mykiss).

The occurrence of pharmaceuticals in the environment represents a challenge of emerging concern. Many pharmaceuticals are chiral compounds; however, few studies have examined the relative toxicity of pharmaceutical enantiomers to wildlife. Further, our understanding of stereospecific pharmacokinetics remains largely informed by research on humans and a few well-studied laboratory test animals, and not by studies conducted with environmentally relevant species, including fish. The objective of this study was to investigate whether rainbow trout display stereospecific in vitro metabolism of three common chiral pharmaceuticals. Metabolism by trout liver S9 fractions was evaluated using a substrate depletion approach, which provides an estimate of intrinsic hepatic clearance (CL(IN VITRO,INT)). No biotransformation was observed for rac-, R-, or S-fluoxetine. Ibuprofen, including both enantiomers and the racemic mixture, appeared to undergo slow metabolism, but the resulting substrate depletion curves did not differ significantly from those of inactive controls. Contrary to relative clearance rates in humans, S(-)-propranolol was more rapidly cleared than the R(+)-enantiomer. This work demonstrates that relative clearance rates and the effects of racemic mixtures in trout could not have been predicted based on human data. Additional research describing species differences and exploring tools for species extrapolation in biomedical and environmental studies is needed.

Comparative pharmaceutical metabolism by rainbow trout (Oncorhynchus mykiss) liver S9 fractions.

The occurrence of pharmaceuticals in the environment presents a challenge of growing concern. In contrast to many industrial compounds, pharmaceuticals undergo extensive testing prior to their introduction to the environment. In principle, therefore, it may be possible to employ existing pharmacological safety data using biological "read-across" methods to support screening-level bioaccumulation environmental risk assessment. However, few approaches and robust empirical data sets exist, particularly for comparative pharmacokinetic applications. For many pharmaceuticals, the primary cytochrome P450 (CYP) enzymes responsible for their metabolism have been identified in humans. The purpose of the present study was to employ a comparative approach to determine whether rainbow trout biotransform pharmaceuticals known to be substrates for specific human CYPs. Seven compounds were selected based on their primary metabolism in humans by CYP3A4, CYP2D6, or CYP2C9. Five additional test compounds are known to be substrates for multiple CYPs. Metabolism by rainbow trout liver S9 fractions was evaluated using a substrate-depletion approach, which provided an estimate of intrinsic hepatic clearance (CLIN VITRO,INT ). An isotope dilution liquid chromatography-tandem mass spectrometry method was employed for quantitation of parent chemical concentrations. Only 2 general CYP substrates demonstrated measurable levels of substrate depletion. No significant biotransformation was observed for known substrates of human CYP2D6, CYP2C9, or CYP3A4. The results of this study provide novel information for therapeutics that fish models are likely to metabolize based on existing mammalian data. Further, these results suggest that pharmaceuticals may possess a greater tendency to bioaccumulate in fish than previously anticipated.

Toward improved models for predicting bioconcentration of well-metabolized compounds by rainbow trout using measured rates of in vitro intrinsic clearance.

Models were developed to predict the bioconcentration of well-metabolized chemicals by rainbow trout. The models employ intrinsic clearance data from in vitro studies with liver S9 fractions or isolated hepatocytes to estimate a liver clearance rate, which is extrapolated to a whole-body biotransformation rate constant (kMET ). Estimated kMET values are then used as inputs to a mass-balance bioconcentration prediction model. An updated algorithm based on measured binding values in trout is used to predict unbound chemical fractions in blood, while other model parameters are designed to be representative of small fish typically used in whole-animal bioconcentration testing efforts. Overall model behavior was shown to be strongly dependent on the relative hydrophobicity of the test compound and assumed rate of in vitro activity. The results of a restricted sensitivity analysis highlight critical research needs and provide guidance on the use of in vitro biotransformation data in a tiered approach to bioaccumulation assessment.

Optimization of an isolated perfused rainbow trout liver model: Clearance studies with 7-ethoxycoumarin.

To date, research with isolated perfused fish livers has been limited by the relatively short time period during which stable performance can be achieved. In the present study, modifications to existing methods were employed with the goal of extending the usable life of an isolated perfused trout liver preparation. Liver performance was evaluated by measuring O(2) consumption (VO(2)), vascular resistance, K(+) leakage, glucose flux, lactate flux, and clearance of a model metabolic substrate, 7-ethoxycoumarin (CL(H,7-EC)). Livers perfused with solutions containing 15, 38, or 150microM bovine serum albumin (BSA) exhibited relatively stable physiological performance for up to 10h. CL(H,7-EC) decreased rapidly between 1 and 2h in all livers tested, possibly due in part to accumulation of 7-EC within the tissue. CL(H,7-EC) declined slowly thereafter, decreasing by 30-40% between 2 and 10h. A linear equation was subsequently developed to correct measured levels of clearance for this decrease in metabolic activity over time. To illustrate the value of this preparation, experiments were conducted to examine the effects of protein binding on 7-EC clearance. Clearance rates corrected for declining activity (CL(H,7-EC,CORR)) changed in nearly direct proportion to changes in the free concentration of 7-EC efferent to the liver, as predicted by theoretical models of liver function. Additional studies were performed to characterize the concentration-dependence of 7-EC clearance. The rate of substrate disappearance from the perfusate increased in proportion to the total concentration of 7-EC afferent to the liver resulting in constant levels of CL(H,7-EC,CORR). CL(H,7-EC,CORR) values for four livers averaged 12.1+/-2.5mL/h/g-liver (mean+/-SD, n=57 individual determinations) and were in good agreement with an estimate of hepatic clearance obtained by extrapolating published in vitro data from isolated trout hepatocytes. The extended viability of isolated trout livers achieved in this study creates new opportunities for research on hepatic function in fish.

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.

Use of online microdialysis sampling to determine the in vivo rate of phenol glucuronidation in rainbow trout.

A quantitative microdialysis (MD) sampling method was used to study phenol (PH) glucuronidation in vivo in rainbow trout. The method employs internal calibrators to account for changes in MD probe performance (in vitro-to-in vivo and sample-to-sample) and yields data of high temporal resolution that are well suited for developing kinetic models. Initially, trout were dosed with phenyl glucuronide (PG) by intravascular infusion for 24 h and then depurated for 48 h. Measured concentrations of PG in blood were well described by a one-compartment clearance-volume model. Massbalance calculations showed that 93% of infused PG was eliminated in urine during the depuration period. Peak concentrations of PG in urine averaged 3.4 times higher than those in blood, and the fitted PG clearance constant (15.7 ml/kg/h) was about 2.6 times higher than the reported glomerular filtration rate for trout. These findings confirm earlier work suggesting that PG is actively secreted by the trout kidney. In a second set of experiments, trout were exposed continuously to PH in water. In vivo rate constants for PH glucuronidation were estimated using a pair of linked (PH and PG) one-compartment clearance-volume models. Expressed on a whole-fish basis, the glucuronidation rate averaged 0.049/h, which was about 7% of the total rate of PH elimination. This study demonstrates the utility of quantitative MD sampling for kinetic studies of xenobiotic metabolism in fish.