Ueng Y F, Shimada T, Yamazaki H, Guengerich F P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Chem Res Toxicol. 1995 Mar;8(2):218-25. doi: 10.1021/tx00044a006.
Human cytochromes P450 (P450) 1A2 and P450 3A4 were expressed in Escherichia coli, purified, and used in reconstituted oxidation systems. The optimal system for P450 3A4 included a mixture of phospholipids, sodium cholate, cytochrome b5, GSH, and MgCl2. Relatively high catalytic activities were obtained with such a system for aflatoxin (AF) B1 3 alpha-hydroxylation and 8,9-epoxidation. P450 3A4 was more active than P450 1A2 in forming genotoxic AFB1 oxidation products. Analysis of the AFB1 products indicated that P450 3A4 formed AFQ1 and the exo-8,9-epoxide; P450 1A2 formed AFM1, a small amount of AFQ1, and both the exo- and endo-8,9-epoxides. The endo epoxide is essentially nongenotoxic in the umu test, as found previously in bacterial mutagenicity assays [Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) J. Am. Chem. Soc. 116, 1603-1609]. 7,8-Benzoflavone (alpha-naphthoflavone, alpha NF) stimulated AFB1 (exo) 8,9-epoxidation and inhibited 3 alpha-hydroxylation in human liver microsomes and a reconstituted P450 3A4 system but was a potent inhibitor of all reactions catalyzed by P450 1A2. Plots of AFB1 3 alpha-hydroxylation and 8,9-epoxidation vs AFB1 concentration were sigmoidal in both human liver microsomes and the reconstituted P450 3A4 system. The results are consistent with the view that P450 3A4 is a major human liver P450 enzyme involved in AFB1 activation, although the in vivo situation may be more complex due to the presence of the enzyme in the gastrointestinal tract.
人细胞色素P450（P450）1A2和P450 3A4在大肠杆菌中表达、纯化，并用于重组氧化系统。P450 3A4的最佳系统包括磷脂、胆酸钠、细胞色素b5、谷胱甘肽和氯化镁的混合物。用这样的系统对黄曲霉毒素（AF）B1进行3α-羟基化和8,9-环氧化可获得相对较高的催化活性。在形成具有遗传毒性的AFB1氧化产物方面，P450 3A4比P450 1A2更具活性。对AFB1产物的分析表明，P450 3A4形成AFQ1和外消旋8,9-环氧化物；P450 1A2形成AFM1、少量AFQ1以及外消旋和内消旋8,9-环氧化物。如先前在细菌诱变性试验中所发现的，内消旋环氧化物在umu试验中基本无遗传毒性[Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) J. Am. Chem. Soc. 116, 1603 - 1609]。7,8-苯并黄酮（α-萘黄酮，α NF）在人肝微粒体和重组P450 3A4系统中刺激AFB1（外消旋）8,9-环氧化并抑制3α-羟基化，但它是P450 1A2催化的所有反应的强效抑制剂。在人肝微粒体和重组P450 3A4系统中，AFB1 3α-羟基化和8,9-环氧化与AFB1浓度的关系图均为S形。结果与以下观点一致：P450 3A4是参与AFB1活化的主要人肝P450酶，尽管由于该酶在胃肠道中的存在，体内情况可能更复杂。
