The relationship between N-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes.
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 Km and Vmax for NDMA metabolism are 61 microM and 56 pmol/min/10(6) 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 approximately 1 6-Me-dG adduct formed/133 000 NDMA molecules metabolized in the uninduced hepatocytes.
Encell
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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Foiles
P G
PG
Gold
B
B
D000818:Animals; D002273:Carcinogens / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004128:Dimethylnitrosamine / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D008745:Methylation; D051381:Rats; D017207:Rats, Sprague-Dawley
D002273:Carcinogens; D004247:DNA; C015558:O(6)-methyl-2'-deoxyguanosine; D003849:Deoxyguanosine; D004128:Dimethylnitrosamine