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Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish
Citation:
Fay, K., P. Fitzsimmons, A. Hoffman, AND J. Nichols. Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, Pensacola, FL, 36(2):463-471, (2017).
Impact/Purpose:
The tendency for chemicals to accumulate in fish and other aquatic biota is a critical component of chemical risk assessments performed by U.S. EPA under Toxic Substances Control Act (TSCA), as amended by the Frank R. Lautenberg Chemical Safety for the 21st Century Act. Chemical accumulation in fish can be measured directly in whole-animal exposures, but these methods are expensive, time consuming, and require many test animals. For this reason, most bioaccumulation assessments are performed using predictive computational models. The primary uncertainty in these assessments is the tendency for exposed animals to transform chemicals, thereby limiting their accumulation. In vitro (“test tube”) methods are needed that can be used to measure chemical biotransformation to improve these modeled bioaccumulation predictions. Cryopreserved hepatocytes isolated from rainbow trout were tested as an in vitro system for measuring biotransformation, and by extension hepatic clearance for 6 polycyclic aromatic hydrocarbons, which are components of petrochemical pollution. Clearance estimates generated using hepatocytes were compared to those obtained previously using trout liver homogenates (S9 fraction) to determine if one in vitro test system performed better than the other. The results of this study indicate that trout hepatocytes performed as well as liver S9 fractions for the chemicals studied. Furthermore, hepatic clearance predictions obtained using either system were in good agreement with measured hepatic clearance values. These results suggest that both in vitro systems can be used to improve bioaccumulation assessments for fish.
Description:
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 two 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 to 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 two 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 one 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.
