Investigating the discriminatory potential of urinary DNA adductomics in smokeless tobacco-treated rats and head-neck cancer patients

Abstract

The genotoxicity of smokeless tobacco (ST) remains an essential public health issue. We conducted a comprehensive study of rats exposed to ST and head and neck cancer patients to elucidate the DNA adduct profiles. ST exposure assessment used alkaloids and tobacco-specific nitrosamines in rat urine. High ST doses led to significantly elevated nicotine, cotinine, N-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) levels, indicating substantial exposure. DNA adductomics identified 17 significant DNA adducts using a neutral loss scan in liquid chromatography-mass spectrometry in rat urine samples upon ST exposure, highlighting the intricate landscape of DNA modifications associated with ST using univariate and multivariate statistical analyses. LC-ESI-QTOF-MS/MS analysis and accurate mass measurements of DNA adducts unveiled specific adducts upregulated in response to ST exposure, particularly those associated with methylation and NNK release. Intriguing correlations emerged between DNA adducts and alkaloids-TSNA in ST-exposed rat urine. Nicotine correlated positively with N2-paraldol-dG and O2/O4-[4-(3-Pyridyl)-4-oxobut-1-yl] thymidine in high-dose rats. NNK correlated with 6O-methylgaunine, while NNN correlated with 6-hydroxy-1-N2-propanodeoxyguanosine and 7-methylguanine. With this aim, we also applied analysis to human samples, where we observed specific DNA adducts for ST users, controls, and subjects with HNC. Two-way ANOVA showed that ST users and cancer patients had higher DNA adduct levels in human urine, which aids cancer risk assessment, early detection, and environmental exposure evaluation. This comprehensive study explores DNA adducts caused by ST exposure across rats and head and neck cancer patients, illuminating genotoxic mechanisms and health risks and providing valuable insights for future preventive and therapeutic strategies.