CYP450 quantification and metabolic activation capacity of rat and hamster liver S9 fractions: implications for nitrosamine mutagenicity testing

N-nitrosamine impurities are a major concern in the pharmaceutical industry and public safety. The assessment of this chemically diverse group using conventional regulatory test batteries, including the Ames test (OECD TG 471), remains challenging. Consequently, refined strategies such as the Enhanced Ames Test (EAT) have been developed and recommended by regulatory authorities, requiring 30% induced rat and hamster S9. Recently, we showed that hamster S9 metabolizes short-chain N-nitrosamines more efficiently than rat S9, likely due to differences in the induction pattern of metabolizing enzymes. Metabolic activation by cytochrome P450 isoenzymes (CYP) is a critical step in the bioactivation of N-nitrosamines. Building on our previous publication, the present work focusses on key CYPs and sets the proteome data into perspective with in vitro mutagenicity data for selected N-nitrosamines. Targeted proteomics quantified CYP1A2, 2A6, 2C8/9/19, 2D6, 2E1, and 3A4 of rat and hamster orthologs in uninduced, Aroclor 1254-, and PB/NF-induced rat and hamster liver S9. In rat S9, induction markedly increased CYP1A2 and CYP3A1 (> 40-fold). For the selected set of CYPs, most protein levels in hamster S9 decreased or increased only slightly (< 6-fold) following induction, although baseline levels were higher than those in uninduced rat S9. Nevertheless, induced hamster S9 generated the strongest Ames responses for NDBA and 1-CPNP, indicating that induction enhances CYP activity beyond protein quantity alone. N-nitroso-atenolol was negative under all rat S9 conditions but positive in TA1535 only with induced hamster S9, indicating that larger NDSRIs require elevated metabolic activity for detection. These findings provide a mechanistic basis for species- and induction-specific differences in mutagenicity detection and contribute to a better understanding of the metabolic activation capacity of S9.