Activation of procarcinogens by human cytochrome P450 enzymes expressed in Escherichia coli. Simplified bacterial systems for genotoxicity assays.
Activation of procarcinogens by human cytochrome P450 enzymes expressed in Escherichia coli. Simplified bacterial systems for genotoxicity assays.
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DOI：
10.1093/carcin/15.11.2523
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发表时间：
1994-11
期刊：
影响因子：
4.7
通讯作者：
T. Shimada;E. Gillam;P. Sandhu;Zuyu Guo;R. Tukey;F. Guengerich
中科院分区：
文献类型：
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作者：
T. Shimada;E. Gillam;P. Sandhu;Zuyu Guo;R. Tukey;F. Guengerich
细菌试验用于检查细胞色素P450（P450）酶对14种已知前致癌物的激活作用。人P450 1A 1、1A 2和3A 4在大肠杆菌中表达，其N-末端序列略有修饰。通过诱导鼠伤寒沙门氏菌NM 2009（TA 1535/pSK 1002/pNM 12）中的SOS反应测定遗传毒性，该菌株含有与lacZ报告基因连接的umuC调控序列。用E.大肠杆菌膜和纯化的酶。膜馏分，强化与NADPH-P450还原酶，被发现是有用的制剂，用于测量激活的原致癌物。建立了这些测定的线性条件，并将该系统应用于与P450生物活化前致癌物有关的几个特定问题。14种化学物质的激活模式与使用人肝微粒体、纯化肝P450和其他方法开发的文献一致。P450在细菌细胞膜上的表达可被抗体所抑制。7，8-苯丙酮对P450 1A 1和1A 2有抑制作用，对P450 3A 4有刺激作用。P450 1A 1和1A 2的贡献与一些芳胺和7，8-二羟基-7，8-二氢苯并[a]芘不同。重组P450 3A 4被认为是更积极的比P450 1A 2在激活黄曲霉毒素B1在所有底物浓度检查。
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.