An evaluation of the roles of metabolic denitrosation and alpha-hydroxylation in the hepatotoxicity of N-Nitrosodimethylamine.

N-Nitrosodimethylamine (NDMA) is a potent hepatotoxic agent in rats. NDMA causes cell death that does not correlate with known mechanisms of toxicity such as the production of oxidative stress or covalent binding to proteins. The following studies were designed to determine whether NDMA cytotoxicity is the result of metabolic denitrosation or alpha-hydroxylation of the nitrosamine. We determined the toxicity of various metabolites of NDMA in monolayer cultures of primary rat hepatocytes. NDMA was toxic at 0.1 mM in our cultures, but the metabolites formaldehyde, methanol, and methylamine were not toxic at this concentration. We used diazeniumdiolates that spontaneously release nitric oxide (NO) in aqueous media to deliver NO to hepatocytes in culture. The results show that, while NO released from diazeniumdiolates causes death in hepatocytes, the levels of NO produced during NDMA metabolism are insufficient to account for the toxicity of the nitrosamine. NDMA-d6, the fully deuteriated form of NDMA that undergoes approximately twice as much denitrosation in vivo as NDMA, was significantly less cytotoxic than NDMA. In contrast, N-nitroso(acetoxymethyl)methylamine (AcO-NDMA), a stable precursor of the methanediazonium ion, was found to cause toxicity equivalent of NDMA on a molar basis. Altogether, our results with methylamine, formaldehyde, methanol, the diazeniumdiolates, and NDMA-d6 indicate that NDMA toxicity is not the result of metabolic denitrosation, while the toxicity of AcO-NDMA provides strong strong evidence that the formation of the methanediazonium ion via alpha-hydroxylation leads to cell death.