Quan T, States J C
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan 48201, USA.
Mol Carcinog. 1996 May;16(1):32-43. doi: 10.1002/(SICI)1098-2744(199605)16:1<32::AID-MC5>3.0.CO;2-Q.
Gene-specific DNA damage levels were determined by quantitative polymerase chain reaction (QPCR) after treating cytochrome P450 (CYP) 1A1-expressing xeroderma pigmentosum fibroblasts with [3H]benzo[a]pyrene-trans-7,8-dihydrodiol ([3H]BPD) or [3H]benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide ([3H]BPDE). DNA damage in the p53 gene (which is transcriptionally active) and the beta-globin gene (which is transcriptionally inactive) was measured in cells treated with 3H-anti-BPDE, 3H-BPD, and 3H-BPD. DNA adduct formation in the genome overall was determined by measuring the incorporation of 3H into DNA. DNA damage in a p53 gene fragment (exons 8-9, 445 bp) was readily detected by QPCR. DNA damage was either not detected or much reduced in a similarly sized target in the beta-globin gene (exons 1-2, 551 bp). At equivalent levels of genomic DNA adducts, BPD treatment induced more damage in the p53 gene than BPDE treatment did. The lesion frequencies in the p53 and beta-globin genes in purified DNA treated with BPDE in vitro were the same, indicating that there was no sequence-specific basis for preferential lesion formation in the p53 gene in treated cells. DNA damage in both the p53 and beta-globin genes showed a dose response to 3H-BPD. The frequency of BPD-induced lesions in the p53 gene was sixfold to sevenfold greater than in the beta-globin gene and 200- to 300-fold greater than in bulk DNA. The BPD-induced lesion frequency in the beta-globin gene was 30- to 50-fold greater than in bulk DNA. The data indicate that the distribution of BPDE-induced DNA lesions is dramatically nonrandom and suggest that the nonrandomness is governed by DNA sequence composition, chromatin structure, and dose rate.
在用[3H]苯并[a]芘-反式-7,8-二氢二醇（[3H]BPD）或[3H]苯并[a]芘-反式-7,8-二氢二醇-9,10-环氧化物（[3H]BPDE）处理表达细胞色素P450（CYP）1A1的着色性干皮病成纤维细胞后，通过定量聚合酶链反应（QPCR）测定基因特异性DNA损伤水平。在用3H-反式-BPDE、3H-BPD和3H-BPD处理的细胞中，测量了p53基因（转录活跃）和β-珠蛋白基因（转录不活跃）中的DNA损伤。通过测量3H掺入DNA来确定基因组总体中的DNA加合物形成。通过QPCR很容易检测到p53基因片段（外显子8-9，445 bp）中的DNA损伤。在β-珠蛋白基因（外显子1-2，551 bp）中大小相似的靶标中，要么未检测到DNA损伤，要么损伤大大减少。在基因组DNA加合物水平相当的情况下，BPD处理比BPDE处理在p53基因中诱导的损伤更多。体外经BPDE处理的纯化DNA中p53和β-珠蛋白基因的损伤频率相同，这表明在处理的细胞中，p53基因中优先形成损伤没有序列特异性基础。p53和β-珠蛋白基因中的DNA损伤均显示出对3H-BPD的剂量反应。BPD诱导的p53基因损伤频率比β-珠蛋白基因高6至7倍，比总体DNA高200至300倍。BPD诱导的β-珠蛋白基因损伤频率比总体DNA高30至50倍。数据表明，BPDE诱导的DNA损伤分布极不随机，并表明这种非随机性受DNA序列组成、染色质结构和剂量率的支配。
