The DNA Repair Protein MGMT Protects against the Genotoxicity of <i>N</i>-Nitrosodimethylamine, but Not <i>N</i>-Nitrosodiethanolamine and <i>N</i>-Nitrosomethylaniline, in Human HepG2 Liver Cells with CYP2E1 Expression

<i>N</i>-nitrosamines are genotoxic contaminants that occur in the diet, consumer products, and the environment. More recently, <i>N</i>-nitrosamines were also detected as drug impurities. After uptake, <i>N</i>-nitrosamines undergo metabolic activation by cytochrome P450 monooxygenases (CYPs), resulting in DNA damage and tumor formation. In this study, the genotoxicity and cytotoxicity of three <i>N</i>-nitrosamines with structurally distinct substituents, <i>N</i>-nitrosodimethylamine (NDMA), <i>N</i>-nitrosodiethanolamine (NDELA) and <i>N</i>-nitrosomethylaniline (NMA), were analyzed in human HepG2 liver cell models proficient or deficient in CYP2E1 biotransformation. Furthermore, the impact of the DNA repair protein <i>O</i>⁶-methylguanine-DNA methyltransferase (MGMT) was investigated. The novel genetically engineered HepG2-CYP2E1 cell line strongly expressed CYP2E1, which was not detectable in wildtype (WT) HepG2 cells. We then confirmed that the CYP2E1 substrate NDMA caused <i>O</i>⁶-methyldesoxyguanosine adducts and DNA strand breaks in a CYP2E1-dependent manner, leading to cytotoxicity. By the same approach, we demonstrated that NDELA induced DNA strand breaks in HepG2-CYP2E1 cells, whereas no effect was observed for NMA. However, NMA was revealed to cause DNA cross-links. Furthermore, both NDELA and NMA were cytotoxic in HepG2-CYP2E1 cells, but not in WT cells. Subsequently, the pharmacological MGMT inhibitor <i>O</i>⁶-benzylguanine was used to deplete MGMT in both HepG2 cell models. MGMT inhibition clearly increased DNA strand break levels due to NDMA exposure, whereas DNA strand break formation by NDELA and NMA were not affected by inhibiting MGMT. In line with these findings, the clastogenic effects of NDMA were potentiated in the absence of MGMT. In contrast to that, NDELA- and NMA-induced clastogenicity was not influenced by MGMT inhibition. Taken together, our study revealed that all three structurally diverse <i>N</i>-nitrosamines are cytotoxic and clastogenic in a CYP2E1-dependent manner, while only NDMA and NDELA caused DNA strand breaks. Furthermore, we demonstrated for the first time that DNA repair by MGMT does not confer protection against NDELA and NMA-triggered DNA strand break induction and clastogenicity.