Fadlallah S, Lachapelle M, Krzystyniak K, Cooper S, Denizeau F, Guertin F, Fournier M
Département des Sciences Biologiques and TOXEN, Université du Québec à Montréal, Canada.
Int J Immunopharmacol. 1994 Jul;16(7):583-91. doi: 10.1016/0192-0561(94)90109-0.
A non-invasive approach in immunopathological risk assessment was applied for analysis of the in vivo formation of DNA adducts. DNA methylation was studied in peripheral blood lymphocytes (PBLs) collected from Sprague-Dawley rats exposed to a single dose (75 mg/kg b.w.) of N-nitrosodimethylamine (NDMA). Three different techniques were applied for characterization and quantification of DNA adducts: (i) colloidal gold ultraimmunocytochemical localization of O6-methylguanosine (O6-meG)-DNA adducts, using affinity-purified, polyclonal antibody directed against O6-meG, (ii) quantitative assay using enzyme-linked immunosorbent assay (ELISA), amplified by the avidin-biotin (AB) system, and (iii) high-performance liquid chromatography (HPLC). The O6-meG-immunoreactive sites in PBLs seem to be concentrated in the nucleus. However, significant immunolabelling was also noted in the cytoplasm of the in vivo NDMA-exposed PBLs. Control preparations showed no specific gold immunolabelling. The O6-meG-DNA adduct formation in PBLs and hepatocytes, at 2-24 h following the exposure to NDMA, was analogous for both types of cells. The data showed high correlation for the ELISA and HPLC analytical methods. The data suggest an efficient O6-metG-DNA repair mechanism in lymphocytes, possibly analogous to the enzymatic repair of DNA adducts in liver cells.
一种免疫病理学风险评估的非侵入性方法被用于分析DNA加合物的体内形成。对从接受单剂量（75毫克/千克体重）N-亚硝基二甲胺（NDMA）的Sprague-Dawley大鼠采集的外周血淋巴细胞（PBL）进行DNA甲基化研究。应用三种不同技术对DNA加合物进行表征和定量：（i）使用针对O6-甲基鸟苷（O6-meG）的亲和纯化多克隆抗体，对O6-甲基鸟苷（O6-meG）-DNA加合物进行胶体金超免疫细胞化学定位；（ii）使用酶联免疫吸附测定（ELISA）并通过抗生物素蛋白-生物素（AB）系统进行扩增的定量测定；以及（iii）高效液相色谱（HPLC）。PBL中的O6-meG免疫反应位点似乎集中在细胞核中。然而，在体内暴露于NDMA的PBL的细胞质中也观察到明显的免疫标记。对照制剂未显示特异性金免疫标记。在暴露于NDMA后2至24小时，PBL和肝细胞中O6-meG-DNA加合物的形成在两种细胞类型中相似。数据显示ELISA和HPLC分析方法具有高度相关性。数据表明淋巴细胞中存在有效的O6-甲基鸟嘌呤-DNA修复机制，可能类似于肝细胞中DNA加合物的酶促修复。
