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Addition of phenylmethylsulfonyl fluoride (PMSF) substantially increases the working lifetime of the trout liver S9 substrate depletion assay resulting in improved detection of low intrinsic clearance rates
Citation:
Nichols, J., A. Hoffman, J. Swintek, S. Droge, AND P. Fitzsimmons. Addition of phenylmethylsulfonyl fluoride (PMSF) substantially increases the working lifetime of the trout liver S9 substrate depletion assay resulting in improved detection of low intrinsic clearance rates. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, Pensacola, FL, 40(1):148-161, (2021). https://doi.org/10.1002/etc.4901
Impact/Purpose:
In vitro substrate depletion assays are being used with increasing frequency to obtain biotransformation rate data needed to run predictive bioaccumulation models for fish. One such assay, the trout liver S9 depletion assay, is preferred because biological material required to run the assay (trout liver S9 fraction) is easy to prepare and use. However, a current limitation of the assay is that initial rates of enzyme activity tend to decline over time due to proteolytic degradation of biotransformation enzymes. The purpose of this study was to determine whether protease inhibitors could be employed to extent the working lifetime of the S9 assay, thereby enhancing its utility for fish bioaccumulation assessments. Of the different treatment approaches attempted, that which provided the greatest benefit was addition of 10 mM phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, directly to S9 reaction mixtures. Addition of 10 mM PMSF to the assay resulted in a 6-fold increase in the working lifetime of the preparation. A subsequent power analysis showed that this increase in assay performance results in substantially improved detection of low, but environmentally relevant rates of in vitro intrinsic clearance. The results of this work greatly enhance the utility of the trout S9 assay and have broad implications for use of this and similar assays in screening-level chemical assessments.
Description:
The performance of a trout liver S9 substrate depletion assay was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or the S9 reaction mixture. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on initial clearance of phenanthrene (PHEN), a model CYP substrate, but resulted in significant improvements in retention of initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects of PMSF on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in higher rates of PHEN clearance in long-term (2 h) incubations relative to those obtained in the absence of PMSF, and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish.
