| 3-methylindole (3MI) is a preferential pneumotoxicant known to be present in cigarette smoke at concentrations previously demonstrated to cause pulmonary damage in experimental animals. Human cytochrome P450 (CYP450) enzymes, including 1A1, 2A13, and 2F1 catalyze the dehydrogenation of 3MI to the reactive intermediate 3-methyleneindolenine (3MEIN). This metabolite alkylates DNA. Alkyl DNA adducts have previously been demonstrated to play a role in the induction of xenobiotic-mediated apoptosis, mutagenesis, and carcinogenesis. In addition, 3MI induces the expression of cytochrome P450 enzymes in experimental animals. However, the ability of 3MI to induce these effects in human lung has not been elucidated to date. The hypothesis for this work is that cytochrome P450-mediated metabolism of 3MI causes DNA damage, cytochrome P450 induction, and mutagenesis in human lung.Low micromolar concentrations of 3MI produced extensive CYP450-dependent DNA damage in cultured primary normal human epithelial (NHBE) cells, as measured by the comet assay. The observed DNA damage was primarily due to single strand breaks. Exposure to subtoxic concentrations of 3MI resulted in the phosphorylation and nuclear localization of p53, whereas only at high micromolar concentrations of 3MI was apoptosis observed, indicating that there is a window of exposure in which 3MI can cause extensive DNA damage without inducing cell death.Quantitative real-time PCR and western blot analysis indicated that exposure to low concentrations of 3MI induced both transcription and translation of 1A1 and 2F1 in NHBE cells. Ames assay analysis of 3MI-mediated mutagenesis demonstrated that 3MI is a more potent mutagen than the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK) when either human lung microsomes or the recombinant lung enzymes 2F3 and 2A13 were used as the activating system. Taken together, these results demonstrate for the first time the highly genotoxic and mutagenic properties of 3MI in human lung and indicate that 3MI may potentially play a role in human lung carcinogenesis. |
