A comparative analysis of select P450 enzymes in uninduced and PB/BNF-induced hamster and rat liver S9

Abstract

The assessment of potentially carcinogenic N-nitrosamine impurities in drugs has become crucial for the pharmaceutical industry to ensure public safety. The in vitro Ames test, which uses rat or hamster liver S9 for metabolic activation, is an important component of regulatory test batteries for assessing mutagenicity and has been the traditional method for assessing the potential mutagenicity of chemicals, including N-nitrosamines. This test, however, has shown inconsistencies with some N-nitrosamines, raising concerns about the liver S9's ability to activate N-nitrosamines to their proximate mutagens. Assays from Vivid® CYP450 Screening Kits and the 7-benzyloxyquinoline assay were used to measure substrate activities of P450 enzymes involved in N-nitrosamine metabolism in rat and hamster liver S9. Both uninduced and induced rat and hamster liver S9 preparations were used. The results provide a comparative assessment of the metabolic competency of the rodent S9s to metabolize N-nitrosamines to their mutagenic forms. Hamster S9 consistently showed increased CYP activity compared to rat S9 under the same conditions. Induced rat S9 also displayed relatively high conversion levels, with the greatest increase in 7-benzyloxyquinoline conversion (CYP3A-like activity) over uninduced (15.7-fold). The highest increase observed with induced hamster S9 was for CYP2A6-like activity which was induced over 7.8-fold and was ∼60-fold higher in induced hamster S9 compared to induced rat S9. These results demonstrate that both rat and hamster S9 contain relevant P450 enzyme activities for N-nitrosamine bioactivation, but hamster S9 is recommended for nitrosamine in vitro tests due to its overall higher P450 activity levels.