Journal Article

The relationship between <i>N</i>-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

Lance Encell, Peter G. Foiles and Barry Gold

in Carcinogenesis

Volume 17, issue 5, pages 1127-1134
Published in print May 1996 | ISSN: 0143-3334
e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/17.5.1127
The relationship between N-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

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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 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, cyto-chrome 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 pyra zole-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.

Journal Article.  0 words. 

Subjects: Clinical Cytogenetics and Molecular Genetics

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