Yang L L, Maher V M, McCormick J J
Proc Natl Acad Sci U S A. 1980 Oct;77(10):5933-7. doi: 10.1073/pnas.77.10.5933.
The ability of normal diploid human fibroblasts and excision repair-deficient xeroderma pigmentosum cells (XP12BE, complementation group A) to excise potentially cytotoxic or mutagenic lesions induced in DNA by (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BzaP-diol epoxide) was determined. Large populations of cells were prevented from replicating by being grown to confluence; after 3 days they were exposed to tritiated BzaP-diol epoxide for 2 hr. One set of cultures was immediately released and assayed for the number of residues covalently bound to DNA, percent survival of colony-forming ability, and frequency of induced mutations. After various periods of time in confluence, other sets were similarly released and assayed. The normal cells exhibited a gradual increase in survival with time held in confluence (recovery from potentially cytotoxic lesions) which was directly correlated with a gradual loss of radioactivity from their DNA and a gradual decrease in the frequency of induced mutations. In contrast, no loss of radioactively labeled carcinogen from the DNA of the XP12BE cells could be detected during a 6-day period and their percent survival and frequency of induced mutations did not change. DNA from normal cells harvested immediately after treatment or after 2, 4, or 8 days in confluence was enzymatically hydrolyzed and analyzed by high-pressure liquid chromatograhy. Only a single peak was detected that cochromatographed with a standard prepared from deoxyguanosine treated with BzaP-diol epoxide. The kinetics of decrease of tritium label in this specific peak corresponded to the decrease in radioactivity of the total DNA with time and with the kinetics of recovery of the cells from the potentially cytotoxic and mutagenic effects of BzaP-diol epoxide. These results suggest that the N2-deoxyguanosine adduct is responsible for these biological effects and indicate that excision repair of this lesion by the normal human cells is "error free."
测定了正常二倍体人成纤维细胞和切除修复缺陷型着色性干皮病细胞（XP12BE，互补组A）对由（±）-7β，8α-二羟基-9α，10α-环氧-7,8,9,10-四氢苯并[a]芘（苯并[a]芘二醇环氧化物，BzaP-diol epoxide）诱导的DNA中潜在细胞毒性或诱变性损伤的切除能力。大量细胞通过生长至汇合而被阻止复制；3天后，将它们暴露于氚标记的BzaP-diol epoxide中2小时。一组培养物立即被释放，并测定与DNA共价结合的残基数量、集落形成能力的存活率以及诱导突变的频率。在汇合状态下经过不同时间段后，其他组也同样被释放并进行测定。正常细胞在汇合状态下随着时间推移存活率逐渐增加（从潜在细胞毒性损伤中恢复），这与它们DNA中放射性的逐渐丧失以及诱导突变频率的逐渐降低直接相关。相比之下，在6天时间内未检测到XP12BE细胞DNA中放射性标记致癌物的丧失，并且它们的存活率百分比和诱导突变频率没有变化。处理后立即收获或在汇合状态下2天、4天或8天后收获的正常细胞的DNA经酶促水解，并通过高压液相色谱法进行分析。仅检测到一个与用BzaP-diol epoxide处理的脱氧鸟苷制备的标准品共色谱的单一峰。该特定峰中氚标记减少的动力学与总DNA放射性随时间的降低以及细胞从BzaP-diol epoxide的潜在细胞毒性和诱变作用中恢复的动力学相对应。这些结果表明N2-脱氧鸟苷加合物是这些生物学效应的原因，并表明正常人细胞对该损伤的切除修复是“无差错的”。
