Wei S J, Chang R L, Bhachech N, Cui X X, Merkler K A, Wong C Q, Hennig E, Yagi H, Jerina D M, Conney A H
Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, State University of New Jersey, Piscataway 08855.
Cancer Res. 1993 Jul 15;53(14):3294-301.
Chinese hamster V-79 cells were exposed to a high dose (0.30-0.48 microM; 32% cell survival), an intermediate dose (0.04-0.10 microM; 100% cell survival) or a low dose (0.01-0.02 microM; 97% cell survival) of (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+)-BPDE] which is the ultimate carcinogenic metabolite of benzo(a)pyrene. The mutation frequency for cells treated with dimethyl sulfoxide vehicle or with low, intermediate or high dose of (+)-BPDE were 1, 10, 52 or 514 8-azaguanine-resistant colonies/10(5) survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene was amplified by the polymerase chain reaction and sequenced. Altogether, 368 (+)-BPDE-induced mutant clones were examined. At all doses, base substitutions were the most prevalent mutations observed (about 72% of the mutant clones), followed by exon deletions (about 26% of the mutant clones) and frame-shift mutations (about 6% of the mutant clones). At the high cytotoxic dose, 7 of 120 base substitutions occurred at AT base pairs (6%) and 113 at GC base pairs (94%). At the intermediate noncytotoxic dose, 20 of 82 base substitutions occurred at AT base pairs (24%) and 62 at GC base pairs (76%). At the low noncytotoxic dose, 27 of 76 base substitutions were at AT base pairs (36%) and 49 were at GC base pairs (64%). The results indicated that decreasing the dose of (+)-BPDE decreased the proportion of mutations at GC base pairs and increased the proportion of mutations at AT base pairs. At the dose of (+)-BPDE was decreased, there was a dose-dependent decrease in the proportion of GC-->TA transversions (from 69% to 42% of the base substitutions) and a dose-dependent increase in the proportion of AT-->CG transversions (from 1% to 25% of the base substitutions). The data also indicated dose-dependent differences in (+)-BPDE-induced exon deletions and hot spots for base substitutions at GC and AT base pairs. Although more than 99% of the (+)-BPDE-induced mutations at guanine occurred on the nontranscribed strand of DNA, (+)-BPDE-induced mutations at adenine occurred on both the transcribed and nontranscribed strands. The ratio of mutations at adenine on the transcribed strand to mutations at adenine on the nontranscribed strand was 35:19 in (+)-BPDE-treated V-79 cells.(ABSTRACT TRUNCATED AT 400 WORDS)
将中国仓鼠V - 79细胞暴露于高剂量（0.30 - 0.48微摩尔；32%细胞存活率）、中等剂量（0.04 - 0.10微摩尔；100%细胞存活率）或低剂量（0.01 - 0.02微摩尔；97%细胞存活率）的(+)-7R,8S - 二羟基 - 9S,10R - 环氧 - 7,8,9,10 - 四氢苯并(a)芘[(+)-BPDE]，其为苯并(a)芘的最终致癌代谢物。用二甲基亚砜载体处理的细胞，以及用低、中、高剂量(+)-BPDE处理的细胞，其突变频率分别为1、10、52或514个8 - 氮杂鸟嘌呤抗性集落/10⁵个存活细胞。分离出独立的8 - 氮杂鸟嘌呤抗性克隆，并通过逆转录制备互补DNA。次黄嘌呤（鸟嘌呤）磷酸核糖基转移酶（HPRT）基因的编码区通过聚合酶链反应扩增并测序。总共检测了368个(+)-BPDE诱导的突变克隆。在所有剂量下，碱基替换是观察到的最普遍的突变（约占突变克隆的72%），其次是外显子缺失（约占突变克隆的26%）和移码突变（约占突变克隆的6%）。在高细胞毒性剂量下，120个碱基替换中有7个发生在AT碱基对（6%），113个发生在GC碱基对（94%）。在中等非细胞毒性剂量下，82个碱基替换中有20个发生在AT碱基对（24%），62个发生在GC碱基对（76%）。在低非细胞毒性剂量下，76个碱基替换中有27个在AT碱基对（36%），49个在GC碱基对（64%）。结果表明，降低(+)-BPDE剂量会降低GC碱基对处突变的比例，并增加AT碱基对处突变的比例。随着(+)-BPDE剂量降低，GC→TA颠换的比例呈剂量依赖性降低（从碱基替换的69%降至42%），而AT→CG颠换的比例呈剂量依赖性增加（从碱基替换的1%增至25%）。数据还表明，(+)-BPDE诱导的外显子缺失以及GC和AT碱基对处碱基替换的热点存在剂量依赖性差异。虽然超过99%的(+)-BPDE诱导的鸟嘌呤突变发生在DNA的非转录链上，但(+)-BPDE诱导的腺嘌呤突变发生在转录链和非转录链上。在(+)-BPDE处理的V - 79细胞中，转录链上腺嘌呤的突变与非转录链上腺嘌呤的突变之比为35:19。（摘要截于400字）
