Venkatachalam S, Denissenko M, Wani A A
Department of Radiology, Ohio State University, Columbus 43210, USA.
Carcinogenesis. 1995 Sep;16(9):2029-36. doi: 10.1093/carcin/16.9.2029.
Mutagenicity and carcinogenicity of the ubiquitous environmental pollutant benzo[a]pyrene is mediated via its reactive diol epoxide metabolite, anti-BPDE, with the predominant formation of N(2)-deoxyguanine adducts in genomic DNA. Polyclonal and monoclonal antibodies specific for (+/-)-anti-BPDE DNA adducts were used for the quantitative detection of genotoxic damage in DNA treated in vitro and in vivo with ( +/- )-anti-BPDE. In non-competitive enzyme-linked immunosorbent assay the polyclonal antiserum (BP1) exhibited higher affinity, avidity and sensitivity than the monoclonal antibody (5D2). A linear antibody binding response was observed over a wide carcinogen dose range with a detection limit of < 0.1 fmol adducts in immobilized DNA. Non-competitive immuno-slot blot assay could detect 0.2 adducts/10(6) nucleotides induced by < 1 nM ( +/- )-anti-BPDE. The high sensitivity and mono-adduct specificity of non-competitive immunoassays allowed the detailed study of ( +/- )-anti-BPDE-DNA adduct processing human cells exposed to very low levels of the genotoxin. Analysis of polyclonal antiserum binding sites in DNA from repair-proficient human fibroblasts revealed adduct removal rates directly proportional to the initial genotoxic insult. Despite efficient repair, substantial damage persisted in repair-proficient cells exposed to high doses of carcinogen. At low levels of initial damage (0.882 and 3.44 +/- 0.17 adducts/ 10(6) nucleotides) approximately 50% repair was observed after 4 and 8 h respectively. Cells removed approximately 40% of the lesions in 8 h at an intermediate level of damage (20.7 +/- 1.5 adducts/10(6) nucleotides). At higher DNA damage levels (105 +/- 8 and 177 +/- 1 adduct/10(6) nucleotides) 33 and 19% of the lesions respectively were repaired in 24 h. Repair-deficient xeroderma pigmentosum group A fibroblast cells did not show any significant loss of antibody binding sites at high or low initial modification levels. These data suggest that the level of initial DNA damage has a significant impact on the overall efficiency of cellular repair, with potential implications for the biological consequences of deleterious DNA lesions in mammalian cells.
普遍存在的环境污染物苯并[a]芘的致突变性和致癌性是通过其活性二醇环氧化物代谢物反式-苯并[a]芘二醇环氧化物（anti-BPDE）介导的，在基因组DNA中主要形成N(2)-脱氧鸟苷加合物。使用针对(+/-)-anti-BPDE DNA加合物的多克隆和单克隆抗体，对体外和体内用(+/-)-anti-BPDE处理的DNA中的遗传毒性损伤进行定量检测。在非竞争性酶联免疫吸附测定中，多克隆抗血清（BP1）比单克隆抗体（5D2）表现出更高的亲和力、亲合力和灵敏度。在较宽的致癌物剂量范围内观察到线性抗体结合反应，固定化DNA中加合物的检测限<0.1 fmol。非竞争性免疫斑点印迹测定可以检测到<1 nM(+/-)-anti-BPDE诱导的0.2个加合物/10(6)个核苷酸。非竞争性免疫测定的高灵敏度和单加合物特异性使得能够详细研究暴露于极低水平基因毒素的人细胞中(+/-)-anti-BPDE-DNA加合物的处理情况。对来自修复能力正常的人成纤维细胞的DNA中多克隆抗血清结合位点的分析表明，加合物的去除率与初始遗传毒性损伤直接相关。尽管修复效率较高，但暴露于高剂量致癌物的修复能力正常的细胞中仍存在大量损伤。在初始损伤水平较低（0.882和3.44 +/- 0.17个加合物/10(6)个核苷酸）时，分别在4小时和8小时后观察到约50%的修复。在中等损伤水平（20.7 +/- 1.5个加合物/10(6)个核苷酸）下，细胞在8小时内去除了约40%的损伤。在较高的DNA损伤水平（105 +/- 8和177 +/- 1个加合物/10(6)个核苷酸）下，24小时内分别修复了33%和19%的损伤。修复缺陷的A型着色性干皮病成纤维细胞在高或低初始修饰水平下均未显示出抗体结合位点的任何显著损失。这些数据表明，初始DNA损伤水平对细胞修复的整体效率有显著影响，这对哺乳动物细胞中有害DNA损伤的生物学后果具有潜在意义。
