Evaluation of cytotoxic and genotoxic effects of nicotine, tobacco-specific nitrosamines, and dried tobacco leaf extract on HepG2

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During the tobacco crop sorting process, workers come in contact with various substances found in dried leaves, including tobacco-specific nitrosamines: N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and nicotine (NIC). The objective of this study was to assess the cytotoxic and genotoxic effects initiated by exposure to aqueous extract of dry tobacco leaves (TLE) and by compounds NNN, NNK, and NIC, both alone and in combination, using the human hepatocarcinoma cell line (HepG2) as a model. The relationship of exposure with mechanisms of oxidative damage was also assessed. To determine cytotoxicity, the MTT assay was employed, and to assess genotoxicity, the alkaline comet assay and its modified version with the enzyme FPG were utilized, along with the measurement of telomere length. Further systems biology tools were employed to generate the protein network for the combination of NIC, NNN, and NNK. No marked cytotoxicity was observed through the MTT assay. Genotoxicity was detected at the highest concentrations of TLE (1.25 and 5 mg/ml), for NIC, NNN, NNK, and in combinations of these samples. The modified comet assay with formamidopyrimidine DNA glycosylase (FPG) enzyme found that genotoxicity was related to oxidative damage. No significant difference in telomere length was observed. Through in silico analysis, data demonstrated that the predominant proteins associated with the combined agents NNN, NNK, and NIC exhibited connections with signaling pathways related to oxidative damage mechanisms and carcinogenic processes.