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

The metabolism of N-nitrosodimethylamine (NDMA) and its methylationof DNA were simultaneously determined in hepatocytes isolatedfrom untreated and saline- and pyrazole-treated male Sprague-Dawleyrats. Metabolism of NDMA was directly measured by monitoringits disappearance via gas chromatography coupled with a sensitiveand specific detector for N-nitrosamines. DNA methylation wasdetermined in the same cells employed in the metabolism studiesusing a monoclonal antibody-based competitive ELISA procedurespecific for O⁶-methyldeoxyguanosine (6-Me-dG). The apparentK_m and V_max, for NDMA metabolism are 61 µM and 56 pmol/min/10⁶cells respectively for hepatocytes isolated from untreated rats.It was found that the addition of pyrazole to the in vitro hepatocyteincubations caused a dose-dependent inhibition of both metabolismand DNA methylation. However, when DNA methylation is expressedas a function of NDMA metabolized, there is no significant differencebetween hepatocyte incubations without or with pyrazole, withan average value of 79 nmol 6-Me-dG/mol dG/nmol NDMA metabolized.Based on the pyrazole inhibition studies, cyto-chrome P450IIE1is responsible for at least 60% of the DNA methylation in rathepatocytes. In pyrazole-pretreated rats there was an inconsistentincrease in NDMA metabolism, but when metabolism was elevatedso was DNA methylation. In contrast, microsomes isolated frompyra zole-pretreated rats consistently showed elevated metabolismof NDMA. Based on the simultaneous determination of adduct levelsand metabolism, there is 1 6-Me-dG adduct formed/133 000 NDMAmolecules metabolized in the uninduced hepatocytes.