Mutagenicity of N-nitrosodiethylamine and its mechanism of action in liver and bone marrow of Big Blue rats evaluated using in vivo mutation assays and gene expression analysis

Both the cII mutation assay and DS revealed significantly higher mutation frequencies in the liver compared to bone marrow, consistent with the liver’s greater metabolic activation capacity. In contrast, the Pig-a assay in peripheral blood failed to consistently detect mutagenic effects from NDEA exposure. Mutation spectrum analysis by DS revealed a predominance of T > A transversions (35%-38%) and T > C transitions (32%-35%) in the liver, whereas mutations in bone marrow were more evenly distributed.

RNA sequencing provided supporting context, revealing significant differences in cytochrome P450 genes in the liver as key contributors to tissue-specific responses. NDEA exposure induced activation of proliferative and pro-tumorigenic pathways such as MYBL2, E2F3, and PRKAR2B signaling in liver. While bone marrow exhibited suppression of translation and immune-related pathways, including EIF2 signaling, following NDEA treatment.

These transcriptomic differences likely reflect tissue-specific susceptibility and mode of action. Overall, our study highlights the importance of integrating multiple mutation detection methods to assess tissue-specific genotoxicity. The findings underscore the critical role of metabolic activation and the regulation of proliferative, pro-tumorigenic, translational, and immune pathways in driving NDEA-induced mutagenesis, and support the use of sensitive next-generation sequencing-based approaches to enhance nitrosamine risk assessment.