Citation:

Nichols, J., M. Ladd, AND P. Fitzsimmons. Measurement of kinetic parameters for biotransformation of polycyclic aromatic hydrocarbons by trout liver S9 fractions: Implications for bioaccumulation assessment. Applied In Vitro Toxicology. Mary Ann Liebert, Inc., Larchmont, NY, 4(4):365-378, (2018). https://doi.org/10.1089/aivt.2017.0005

Impact/Purpose:

In vitro biotransformation assays that employ biological material derived from liver tissue (S9 or microsomal fractions, hepatocytes) are being used with increasing frequency to inform bioaccumulation assessments for fish. The assays are used to estimate intrinsic hepatic clearance which is extrapolated to the intact animal (in vitro-in vivo extrapolation) and used as an input to established computational models. Modeled levels of chemical accumulation obtained in this manner are much closer to measured values than predictions obtained assuming no metabolism. To date, however, there has been a trend toward under-prediction of true in vivo biotransformation rates which results in systematic overestimation of measured accumulation. The goal of this study was to critically evaluate a widely used assay (trout S9 fraction) and determine whether issues associated with the assay itself are responsible for under-prediction of true in vivo activity. The results show that assay performance is dependent on incubation time, substrate concentration, and S9 protein concentration. Unbound chemical concentrations in the assay were used to evaluate these findings on a free or unbound chemical basis. These findings suggest that existing discrepancies between measured and modeled levels of accumulation for high log Kow compounds in fish can be largely explained by past use of inappropriately high starting substrate concentrations. Based on these findings, we make several recommendations regarding the use of these assays in bioaccumulation assessment for fish, including the need for targeted preliminary testing, limits on the working lifetime of the preparation, and appropriate use of spiking solvents. These recommendations will be incorporated into OECD Test Guidelines for these methods, which are currently in development. When followed, these recommendations will provide improved estimates of chemical accumulation in fish, increasing current confidence in modeled predictions for high log Kow chemicals that undergo biotransformation.

Description:

Substrate depletion methods were used to measure in vitro biotransformation of three polycyclic aromatic hydrocarbons (PAHs) by trout liver S9 fractions. Measured levels of activity declined with incubation time and were reduced by acetone at spiking concentrations greater than 0.5%. Addition of alamethicin, a pore-forming peptide used to support UDP-glucuronosyltransferase activity, also reduced activity in a dose-dependent manner. The concentration-dependence of activity was evaluated to estimate the Michaelis-Menten kinetic constants Km and Vmax for each compound. Derived kinetic constants suggest that all three PAHs are transformed by the same reaction pathway and illustrated a positive correlation between Km and chemical log Kow. 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 chemical concentration except when these concentrations approached saturating levels, providing a direct demonstration of the free chemical hypothesis. These findings provide critical guidance for optimal use of the S9 assay and suggest that previous in vitro work with fish has been conducted at inappropriately high starting substrate concentrations resulting in underestimation of in vivo activity that occurs in most natural settings. The relationship between tested starting substrate concentrations and those achieved in standardized in vivo bioaccumulation testing efforts is less clear, and these is some chance that PAH metabolism could saturate at high aqueous exposure levels resulting in concentration-dependence of measured accumulation.

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