The relationship between N-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

Abstract

The metabolism of N-nitrosodimethylamine (NDMA) and its methylation of DNA were simultaneously determined in hepatocytes isolated from untreated and saline- and pyrazole-treated male Sprague-Dawley rats. Metabolism of NDMA was directly measured by monitoring its disappearance via gas chromatography coupled with a sensitive and specific detector for N-nitrosamines. DNA methylation was determined in the same cells employed in the metabolism studies using a monoclonal antibody-based competitive ELISA procedure specific for O6-methyldeoxyguanosine (6-Me-dG). The apparent K(m) and V(max) for NDMA metabolism are 61 μM and 56 pmol/min/106 cells respectively for hepatocytes isolated from untreated rats. It was found that the addition of pyrazole to the in vitro hepatocyte incubations caused a dose-dependent inhibition of both metabolism and DNA methylation. However, when DNA methylation is expressed as a function of NDMA metabolized, there is no significant difference between hepatocyte incubations without or with pyrazole, with an average value of 79 nmol 6-Me-dG/mol dG/nmol NDMA metabolized. Based on the pyrazole inhibition studies, cytochrome P450IIE1 is responsible for at least 60% of the DNA methylation in rat hepatocytes. In pyrazole-pretreated rats there was an inconsistent increase in NDMA metabolism, but when metabolism was elevated so was DNA methylation. In contrast, microsomes isolated from pyrazole-pretreated rats consistently showed elevated metabolism of NDMA. Based on the simultaneous determination of adduct levels and metabolism, there is ~ 1 6-Me-dG adduct formed/133,000 NDMA molecules metabolized in the uninduced hepatocytes.