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.

No abstract

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.

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.

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.