Comparative DNA damage induced by eight nitrosamines in primary human and macaque hepatocytes

N-nitrosamines have been increasingly detected in human drugs, raising serious safety concerns due to their potential mutagenicity and carcinogenicity. In order to expand upon the human data available on these drug impurities, we previously used metabolically competent HepaRG human hepatoma cells to evaluate the genotoxicity of eight small-molecule nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. In this study, we used the comet assay to further investigate the DNA damage induced by the eight nitrosamines in primary human hepatocytes (PHHs) from three individual donors and primary macaque hepatocytes (PMHs) from freshly isolated livers of six rhesus macaques. In addition, expression of genes encoding Phase I and II metabolic enzymes and the activities of the enzymes were compared in PHHs and PMHs, and Western blot was used to analyze protein biomarkers of DNA damage and apoptosis in PMHs. All eight nitrosamines induced significant DNA damage in both PHHs and PMHs; with the exception of NDMA, higher fold increases in % tail DNA were detected in PMHs compared to PHHs. Greater interindividual variability in CYP gene expression, enzyme activities, and DNA damage responses was observed in PHHs compared to PMHs. Benchmark concentration (BMC) modeling analysis showed that PHHs had more conservative BMC₅₀ values than PMHs for most nitrosamines tested. Nonetheless, correlation analysis demonstrated that DNA damage data generated by PMHs and 3D HepaRG spheroids were comparable to those of PHHs. Western blot analysis suggested a potential role for the ethyl group in regulating protein expression in the DNA damage and apoptosis pathways for nitrosamines. Overall, this study provides human-relevant DNA damage responses for the eight nitrosamines and indicates that differences in genotoxic potency between PHHs and PMHs are likely related to CYP enzyme activity.