Creton S, Zhu H, Gooderham N J
Molecular Toxicology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK.
Toxicol Sci. 2005 Apr;84(2):335-43. doi: 10.1093/toxsci/kfi075. Epub 2005 Jan 5.
The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of meat, induces tumors of the prostate, colon, and mammary gland when fed to rats. PhIP is readily absorbed and efficiently metabolized to a genotoxic derivative by CYP1 enzymes. Although metabolism and mutational potential of PhIP have previously been well characterized, the intervening cellular and genomic responses to the chemical are not fully understood. We have examined the cellular response to PhIP exposure in human mammary epithelial MCF10A cells, which retain characteristics of normal breast epithelial cells. Because these cells fail to activate PhIP, they were cocultured with a human lymphoblastoid cell line MCL-5, which constitutively expresses CYP1A1, and have been transfected to express human CYPs1A2, 2A6, 3A4, and 2E1. The MCL-5 cells were irradiated (2,000 rads) prior to coculture, rendering them unable to replicate yet still retaining metabolic competency. MCF10A cells were treated (in the presence of MCL-5 cells) with PhIP (1-100 microM) and harvested at various time-points. Compared to DMSO control, treatment (24 or 48 h) with PhIP resulted in a significant dose-dependent fall in cell number. Cells treated for 48 h then cultured in the absence of PhIP (and MCL-5 cells) for a further 6 days showed a much greater dose-dependent reduction in cell number. Flow cytometric analysis indicated that PhIP treatment (48 h) resulted in a dose-dependent accumulation of cells in the G1 population. Western blotting revealed elevated expression of p53 and the cyclin dependent kinase inhibitor p21WAF1/CIP1 after PhIP treatment. Levels of MDM2, a negative regulator of p53, and the hypophosphorylated form of RB were also elevated, consistent with the triggering of G1 cell cycle checkpoint. These cell cycle effects are critical, as they enable cells to effect genome repair, accept mutation, or eliminate excessively damaged cells.
杂环胺2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶（PhIP）在肉类烹饪过程中形成，给大鼠喂食时会诱发前列腺、结肠和乳腺肿瘤。PhIP易于吸收，并被CYP1酶高效代谢为具有遗传毒性的衍生物。尽管PhIP的代谢和致突变潜力此前已得到充分表征，但对该化学物质的中间细胞和基因组反应尚未完全了解。我们研究了人乳腺上皮MCF10A细胞对PhIP暴露的细胞反应，该细胞保留了正常乳腺上皮细胞的特征。由于这些细胞无法激活PhIP，因此将它们与人淋巴母细胞系MCL-5共培养，MCL-5组成性表达CYP1A1，并已被转染以表达人CYP1A2、2A6、3A4和2E1。MCL-5细胞在共培养前接受照射（2000拉德），使其无法复制，但仍保留代谢能力。MCF10A细胞（在MCL-5细胞存在下）用PhIP（1-100微摩尔）处理，并在不同时间点收获。与二甲基亚砜对照相比，用PhIP处理（24或48小时）导致细胞数量显著的剂量依赖性下降。处理48小时后再在无PhIP（和MCL-5细胞）的情况下培养6天的细胞，其细胞数量显示出更大的剂量依赖性减少。流式细胞术分析表明，PhIP处理（48小时）导致细胞在G1期群体中剂量依赖性积累。蛋白质免疫印迹显示，PhIP处理后p53和细胞周期蛋白依赖性激酶抑制剂p21WAF1/CIP1的表达升高。p53的负调节因子MDM2和RB的低磷酸化形式水平也升高，这与G1细胞周期检查点的触发一致。这些细胞周期效应至关重要，因为它们使细胞能够进行基因组修复、接受突变或清除受损过度的细胞。
