Aflatoxin B-1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines
Aflatoxin B-1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines
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DOI：
10.1093/carcin/18.7.1291
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发表时间：
1997-07-01
期刊：
影响因子：
4.7
通讯作者：
Pfeifer, AMA
中科院分区：
文献类型：
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作者：
Mace, K;Aguilar, F;Pfeifer, AMA
流行病学证据支持饮食中黄曲霉毒素B-1（AF B（1））暴露、人类原发性肝细胞癌（HCC）发生和p53肿瘤抑制基因突变之间的关系。然而，所观察到的p53突变、AFB（1）DNA加合物及其激活途径之间的相关性尚未阐明。考虑到物种和组织特异性，开发相关的细胞体外模型可以显著有助于了解化学前致癌物（如AFB（1））在人体中的细胞毒性和遗传毒性机制。为此，将保留大部分II相酶但I相活性显著降低的非致瘤性SV 40永生化人肝上皮细胞系（THLE细胞）用于稳定表达人CYP 1A 2、CYP 2A 6、CYP 2B 6和CYP 3A 4 cDNA。与人肝细胞相比，四种基因工程细胞系（T5- 1A 2、T5 - 2A 6、T5- 2B 6和T5- 3A 4）产生高水平的特异性CYP 450蛋白，并显示出与CYP 450表达相关的相当或更高的催化活性。T5- 1A 2、T5- 2A 6、T5- 2B 6和T5- 3A 4细胞系对AFB（1）的细胞毒作用表现出非常高的敏感性，分别比用不含CYP 450 cDNA的表达载体转染的对照T5-neo细胞敏感约125、2、2和15倍。在CYP 450表达细胞中，纳摩尔剂量的AFB（1）诱导DNA加合物形成，包括AFB（1）-N-7-鸟嘌呤、-嘧啶和-二醇加合物。此外，T5- 1A 2细胞显示AFM（1）-DNA加合物。在相似的总DNA加合物水平下，T5- 1A 2和T5- 3A 4细胞均显示p53基因密码子249处AGG至AGT的颠换，相对频率为15 × 10 - 6。相比之下，只有T5- 3A 4细胞在密码子250处显示高频率的CCC至ACC颠换，而在T5- 1A 2细胞中发现的第二最频繁的突变是密码子250的第一和第二位置处的C至T转换。在T5- 2A 6细胞中未检测到明显的AFB（1）诱导的p53突变。因此，人肝细胞中特异性CYP 450基因的差异表达可以调节AFB产生的细胞毒性、DNA加合物水平和p53突变频率（1）。
Epidemiological evidence has been supporting a relationship between dietary aflatoxin B-1 (AFB(1)) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB(1) DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB(1), in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB(1) and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB(1)-induced DNA, adduct formation including AFB(1)-N-7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM(1)-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB(1)-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB(1).