The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general‐purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short‐term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this 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 on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 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. Environ Toxicol Chem 2021;40:148–161. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

中文翻译：

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添加苯甲基磺酰氟可延长鳟鱼肝脏 S9 底物消耗测定的工作寿命，从而改进低固有清除率的检测

鳟鱼肝脏 S9 底物消耗测定的活性已显示随着时间的推移而下降，这可能是由于生物转化酶的蛋白水解降解。为了解决这个问题，在向肝脏匀浆缓冲液和/或 S9 反应混合物中添加苯甲基磺酰氟 (PMSF) 或通用蛋白酶抑制剂混合物后，评估了检测性能。在短期（25 或 30 分钟）耗竭实验中，将 PMSF 添加到肝脏匀浆缓冲液和/或 S9 反应混合物中对菲（一种模型细胞色素 P450 底物）的清除影响很小或没有影响，但显着改善了这种保留随着时间的推移的初始活动。当添加到匀浆缓冲液或反应混合物中时，蛋白酶抑制剂混合物强烈抑制初始活性。考虑到对肝脏羧酸酯酶的潜在影响，确定最佳的处理方法是在 S9 反应混合物中添加 10 µM PMSF。与没有 PMSF 的情况相比，在混合物中添加 10 µM PMSF 可显着提高 2 小时孵育中菲的清除率，并使制剂的工作寿命延长 6 倍。统计功效分析的结果表明，与目前的做法相比，通过增加检测的工作寿命，向反应混合物中添加 PMSF 可以显着提高对低体外清除率的检测。环境毒物化学2021；40：148–161。© 2020 SETAC。本文由美国政府雇员贡献，他们的工作在美国属于公共领域。