Encell L, Foiles P G, Gold B
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Carcinogenesis. 1996 May;17(5):1127-34. doi: 10.1093/carcin/17.5.1127.
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.
在从未经处理、经生理盐水处理和经吡唑处理的雄性Sprague-Dawley大鼠分离出的肝细胞中，同时测定了N-亚硝基二甲胺（NDMA）的代谢及其DNA甲基化情况。通过气相色谱结合一种对N-亚硝胺敏感且特异的检测器监测NDMA的消失情况，直接测定其代谢。使用基于单克隆抗体的针对O6-甲基脱氧鸟苷（6-Me-dG）的竞争性ELISA程序，在代谢研究中使用的相同细胞中测定DNA甲基化。从未经处理的大鼠分离出的肝细胞中，NDMA代谢的表观Km和Vmax分别为61微摩尔和56皮摩尔/分钟/10⁶个细胞。研究发现，在体外肝细胞培养中添加吡唑会导致代谢和DNA甲基化的剂量依赖性抑制。然而，当将DNA甲基化表示为代谢的NDMA的函数时，未添加或添加吡唑的肝细胞培养之间没有显著差异，代谢的NDMA每摩尔dG产生6-Me-dG的平均值为79纳摩尔。基于吡唑抑制研究，细胞色素P450IIE1至少负责大鼠肝细胞中60%的DNA甲基化。在经吡唑预处理的大鼠中，NDMA代谢有不一致的增加，但当代谢升高时，DNA甲基化也升高。相比之下，从经吡唑预处理的大鼠分离出的微粒体始终显示NDMA代谢升高。基于加合物水平和代谢的同时测定，在未诱导的肝细胞中，每代谢133000个NDMA分子大约形成1个6-Me-dG加合物。
