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Hepatic clearance of 6 polyaromatic aromatic hydrocarbons by isolated trout livers: Prediction from in vitro clearance by liver S9 fractions
Citation:
Nichols, J., A. Hoffman, T. Ter Laak, AND P. Fitzsimmons. Hepatic clearance of 6 polyaromatic aromatic hydrocarbons by isolated trout livers: Prediction from in vitro clearance by liver S9 fractions. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 136(2):359-372, (2013).
Impact/Purpose:
Mathematical models based on log Kow relationships accurately predict the accumulation of many compounds by fish and other aquatic biota, provided that they do not undergo substantial biotransformation (metabolism). Metabolism reduces the extent to with chemicals accumulate in fish. Unfortunately, metabolism rates do not correlate simply with log Kow. For this reason, metabolism is the greatest uncertainty in many bioaccumulation assessments for fish. The goal of this effort was to critically evaluate methods currently being used to extrapolate measured in vitro rates of metabolism to the intact animal. In this effort we concentrated on the first part of this extrapolation procedure, which translates in vitro metabolism to an estimate of hepatic clearance. The metabolism of six polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions. Measured metabolism rates were extrapolated to the intact liver using extrapolation factors and chemical binding terms developed in our laboratory. Predicted hepatic clearance rates were then compared to measured values obtained using an isolated perfused trout liver preparation. Good agreement between measured and predicted rates of clearance was observed for all six PAHs. Based on these observations it was suggested that most or all of the chemical provided to the liver in blood plasma is available to be acted on by metabolizing enzymes. Models of liver function that calculate hepatic clearance as a function of the free chemical fraction in plasma may therefore underestimate actual levels of clearance. The use of in vitro assays to predict metabolism impacts on chemical bioaccumulation in fish has been proposed as a part of a tiered assessment process. This study provides strong support for continued development of these procedures.
Description:
An isolated perfused trout liver preparation was used to evaluate in vitro-to-in vivo metabolism extrapolation procedures for fish. Hepatic clearance (CLH) studies were conducted with six polyaromatic hydrocarbons (PAH) using an experimental design wherein each liver acted as its own control. Binding in liver perfusates was manipulated by changing the amount of bovine serum albumin (BSA) in solution, and was characterized by solid-phase microextraction (SPME). Additional studies were conducted to develop a set of chemical-specific binding terms (fU; calculated as the ration of unbound fractions in liver perfusate (fU,PERF) and the S9 system (fU,S9) used to measure intrinsic metabolic clearance) used as inputs to a well-stirred liver model. Predicted levels of CLH for pyrene and benzo[a]pyrene were within a factor of two of measured values. This outcome can be attributed to the rapid rate at which both compounds are metabolized by liver S9 fractions, resulting in perfusion-limited clearance. Predicted levels of CLH for the other PAHs tended to underestimate observed values, although these differences were generally small (less than a factor of 3 for all compounds except naphthalene). Setting fU = 1.0 (assuming that chemical concentrations available for metabolism in vitro and in the intact tissue are effectively the same) tended to improve predictions at the highest tested BSA concentration (10 g/L), suggesting that the full binding assumption (i.e., fU = fU,PERF/fU,S9) underestimates the availability of neutral hydrophobic substrates to metabolizing enzymes of the liver. 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.
