Patrick N. Fitzsimmons

Branchial elimination of superhydrophobic organic compounds by rainbow trout (Oncorhynchus mykiss)

The branchial elimination of pentachloroethane and four congeneric polychlorinated biphenyls by rainbow trout was measured using a fish respirometer-metabolism chamber and an adsorption resin column. Branchial elimination was characterized by calculating a set of apparent in vivo blood:water partition coefficients (P(BW)). Linear regression was performed on the logarithms of P(BW) estimates and the log K(OW) value for each compound to give the fitted equation: log P(BW)=0.76 x log K(OW)-1.0 (r(2)=0.98). The linear nature of this relationship provides support for existing models of chemical flux at fish gills and suggests that a near equilibrium condition was established between chemical in venous blood entering the gills, including dissolved and bound forms, and dissolved chemical in expired branchial water. In vivo P(BW) estimates were combined with P(BW) values determined in vitro for a set of lower log K(OW) compounds (Bertelson et al., Environ. Toxicol. Chem. 17 (1998) 1447-1455) to give the fitted relationship: log P(BW)=0.73 x log K(OW)-0.88 (r(2)=0.98). The slope of this equation is consistent with the suggestion that chemical binding to non-lipid organic material contributes substantially to blood:water chemical partitioning. An equation based on the composition of trout blood (water content and the total amount of organic material) was then derived to predict blood:water partitioning for compounds with log K(OW) values ranging from 0 to 8: log P(BW)=log[(10(0.73 log K(ow)) x 0.16)+0.84].

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.

In vitro‐in vivo extrapolation of quantitative hepatic biotransformation data for fish. II. Modeled effects on chemical bioaccumulation

Hypothetical in vitro biotransformation rate and affinity values for fish were extrapolated to a set of in vivo whole-body metabolism rate constants. A one-compartment model was then used to investigate potential effects of metabolism on chemical bioaccumulation as a function of octanol/water partitioning (Kow). In a second model-based effort, in vitro data were incorporated into a physiologically based toxicokinetic (PBTK) model for fish. The two models predict similar effects on bioaccumulation when calculated in vivo intrinsic clearance values (CL(IN VIVO,INT) are less than 50% of estimated liver blood flow (Q(LIVER). When CL(IN VIVO,INT) approaches Q(LIVER), the PBTK model predicts a greater effect on bioaccumulation than the one-compartment model. This result is attributed to the structure of the PBTK model, which provides for first-pass clearance of chemicals taken up from food. Uncertainties inherent to in vitro-in vivo extrapolations of hepatic metabolism data include the effects of protein binding, inaccurate estimation of in vivo metabolism by in vitro assays, and failure to account for metabolism in other tissues. Model-based predictions of bioaccumulation within a natural setting also must account for possible metabolism at multiple trophic levels. The models described in this study can be used to perform in vitro-in vivo metabolism comparisons with fish, estimate in vitro biotransformation parameters on the basis of measured chemical residues in field-collected animals, and calculate the level of in vitro metabolic activity required to limit bioaccumulation of all compounds to a specified value.

Assessment of Metabolic Stability Using the Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species‐specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold‐water species. Protocols for other species (e.g., carp, a warm‐water species) can be developed based on these procedures as long as the specific considerations are taken into account. Curr. Protoc. Toxicol. 53:14.10.1‐14.10.28.

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

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

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.

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.

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.

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.

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

Abstract 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.