Shimada T, Gillam E M, Sandhu P, Guo Z, Tukey R H, Guengerich F P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.
Carcinogenesis. 1994 Nov;15(11):2523-9. doi: 10.1093/carcin/15.11.2523.
Bacterial assays were used to examine the activation of 14 known procarcinogens by cytochrome P450 (P450) enzymes. Human P450s 1A1, 1A2 and 3A4 were expressed in Escherichia coli with slight modification of their N-terminal sequences. Genotoxicity was measured by the induction of the SOS response in Salmonella typhimurium NM2009 (TA1535/pSK1002/pNM12), which contains a umuC regulatory sequence attached to the lacZ reporter gene. Conditions for analysis were examined using E. coli membranes and purified enzymes. Membrane fractions, fortified with NADPH-P450 reductase, were found to be useful preparations for measuring activation of the procarcinogens. Conditions of linearity were established for these assays and the systems were applied to several particular problems related to bioactivation of procarcinogens by P450s. The patterns of activation of the 14 individual chemicals were consistent with the literature developed using human liver microsomes, purified liver P450s and other approaches. The P450s expressed in bacterial membranes could be inhibited by antibodies. 7,8-Benzoflavone inhibited P450s 1A1 and 1A2 and stimulated P450 3A4 in the membranes. The contributions of P450s 1A1 and 1A2 were distinguished with some of the arylamines and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Recombinant P450 3A4 was found to be more active than P450 1A2 in the activation of aflatoxin B1 at all substrate concentrations examined.
采用细菌试验检测细胞色素P450（P450）酶对14种已知前致癌物的激活作用。人P450 1A1、1A2和3A4在大肠杆菌中表达，其N端序列略有修饰。通过检测鼠伤寒沙门氏菌NM2009（TA1535/pSK1002/pNM12）中SOS应答的诱导来测定遗传毒性，该菌株含有与lacZ报告基因相连的umuC调控序列。使用大肠杆菌膜和纯化酶检测分析条件。发现用NADPH - P450还原酶强化的膜部分是用于测量前致癌物激活作用的有用制剂。为这些试验建立了线性条件，并将该系统应用于与P450对前致癌物的生物激活相关的几个特定问题。14种单独化学物质的激活模式与使用人肝微粒体、纯化的肝P450和其他方法所得到的文献报道一致。细菌膜中表达的P450可被抗体抑制。7,8 - 苯并黄酮抑制膜中的P450 1A1和1A2，并刺激P450 3A4。通过一些芳胺和7,8 - 二羟基 - 7,8 - 二氢苯并[a]芘区分了P450 1A1和1A2的作用。发现在所有检测的底物浓度下，重组P450 3A4在黄曲霉毒素B1的激活中比P450 1A2更具活性。
