Moorthy Bhagavatula, Muthiah Kathirvel, Fazili Inayat S, Kondraganti Sudha R, Wang Lihua, Couroucli Xanthi I, Jiang Weiwu
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.
Biochem Biophys Res Commun. 2007 Mar 23;354(4):1071-7. doi: 10.1016/j.bbrc.2007.01.103. Epub 2007 Jan 26.
Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 micro g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 degrees C for 2h, giving rise to 9 adducts, as determined by (32)P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16h, followed by MC (1 micro M) treatment for 24h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.
细胞色素CYP1A（CYP1A）酶催化3-甲基胆蒽（MC）生物活化为具有基因毒性的代谢产物。在此，我们验证了如下假说：CYP1A2催化MC-DNA加合物的形成，这些加合物优先在CYP1A1启动子区域形成，从而导致CYP1A1基因表达受到调控。当与野生型（WT）肝微粒体（2mg）及NADPH在37℃孵育2小时时，MC与含人CYP1A1启动子的质粒DNA（50μg，pGL3-1A1）共价结合，通过32P后标记法测定，产生了9种加合物。80%的加合物位于启动子区域。将加合的质粒瞬时转染大鼠肝癌（H4IIE）细胞16小时，随后用MC（1μM）处理24小时，与未加合的对照相比，报告基因（荧光素酶）表达受到75%的抑制。我们的结果表明，CYP1A2在CYP1A1启动子区域序列特异性MC-DNA加合物形成中起关键作用，导致CYP1A1基因表达减弱。
