Godschalk R W, Maas L M, Van Zandwijk N, van 't Veer L J, Breedijk A, Borm P J, Verhaert J, Kleinjans J C, van Schooten F J
Department of Health Risk Analysis and Toxicology, Maastricht University, The Netherlands.
Carcinogenesis. 1998 May;19(5):819-25. doi: 10.1093/carcin/19.5.819.
The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.
用于DNA加合物定量的³²P后标记分析方法为检测内源性DNA反应性化合物暴露提供了契机。大多数人体生物监测研究采用白细胞（WBC）或通过支气管肺泡灌洗（BAL）获得的细胞作为DNA来源，但目前仍不清楚哪种细胞类型是接触香烟烟雾相关基因毒素的最可靠指标。首先，我们在体外将分离的WBC亚群与10微摩尔苯并[a]芘（B[a]P）孵育18小时后，通过核酸酶P1（NP1）富集的³²P后标记法检测DNA加合物水平。单核细胞中的DNA加合物水平最高（10.7±2.9个加合物/10⁸个核苷酸，n = 8），其次是淋巴细胞（5.9±1.7，n = 8）和粒细胞（0.5±0.2，n = 8）。其次，测定了（非）吸烟志愿者的BAL细胞和WBC亚群中的芳香族DNA加合物水平。在吸烟个体中，通过NP1富集法，加合物水平的排序为：BAL细胞（3.7±1.0，n = 5）＞单核细胞（2.0±0.5，n = 8）≥淋巴细胞（1.6±0.4，n = 8）＞粒细胞（0.8±0.2，n = 8）；通过丁醇富集的³²P后标记法，单核细胞（9.0±3.2，n = 5）≥淋巴细胞（8.0±2.1，n = 6）＞粒细胞（2.1±0.3，n = 7）。与非吸烟者相比，吸烟者WBC亚群中的芳香族DNA加合物水平显著更高，但使用丁醇富集法时粒细胞中的DNA加合物除外。第三，在一大组吸烟个体（n = 78）的单核白细胞（MNC，即单核细胞加淋巴细胞）和BAL细胞中研究了剂量反应关系。MNC中的加合物水平与每日香烟焦油暴露量相关（r = 0.31，P＜0.01）。BAL细胞中的加合物水平似乎与吸烟包年数相关，但校正年龄后这种关系消失。丁醇提取使MNC中的DNA加合物水平提高了5 - 6倍，而对同一批个体的BAL - DNA进行丁醇提取时，加合物水平仅提高了2倍。³²P后标记的两种富集方法在BAL细胞中具有相关性（r = 0.86，P＜0.001，n = 12）。我们得出结论，特别是MNC是检测吸烟相关DNA加合物的良好替代指标。
