N-Nitrosodimethylamine-mediated cytotoxicity in a cell line expressing P450 2E1: evidence for apoptotic cell death.

N-Nitrosodimethylamine (NDMA) is an acute hepatotoxin and potent carcinogen. The metabolic activation of NDMA to reactive metabolites is a critical step for the expression of its toxic and carcinogenic potential. We have previously demonstrated a strong correlation between methylation of cellular macromolecules and NDMA-mediated cytotoxicity, and we have demonstrated that reactive oxygen species may partially contribute to the toxic effects in P450 2E1-expressing cells. The mode of cell death in NDMA-treated monolayer cultures exhibited the following characteristics: (i) condensation of nuclear chromatin as demonstrated by using Hoechst 33258 staining, (ii) DNA fragmentation as detected by combining pulsed field and conventional agarose gel electrophoresis, and (iii) DNA double strand breaks determined by using the in situ terminal deoxynucleotidyl transferase assay and flow cytometric analysis. These results indicate that reactive metabolites of NDMA trigger activation of the signal pathway for apoptotic cell death in these P450-expressing cells. The NDMA-mediated cell death was partially prevented by the endonuclease inhibitor, aurintricarboxylic acid, as well as the caspase inhibitors, acetyl-Asp-Glu-Val-Asp-CHO and acetyl-Tyr-Val-Ala-Asp-CHO. The cell cycle distribution was altered in NDMA-treated cells resulting in an increase in the G2/M phase and a decrease in the G1 phase. Our results suggest that DNA degradation, the inability to complete DNA repair, the biochemical events associated with G2/M arrest, and the process of apoptotic death all result from P450 2E1-catalyzed metabolism of NDMA.