Shimada T, Gillam E M, Oda Y, Tsumura F, Sutter T R, Guengerich F P, Inoue K
Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan.
Chem Res Toxicol. 1999 Jul;12(7):623-9. doi: 10.1021/tx990028s.
Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High-performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of approximately 3 nmol min(-)(1) nmol of P450(-)(1) only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min(-)(1) nmol of P450(-)(1); the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]pyrene carcinogenesis.
用细胞色素P450（P450）和NADPH - P450还原酶cDNA转化的大肠杆菌所获得的膜中表达的重组人酶，用于鉴定在体外催化苯并[a]芘氧化转化最具活性的人P450酶。发现通过在反应混合物中加入纯化的环氧化物水解酶（从大鼠或人肝脏中分离），P450 1A1和P450 1B1酶将苯并[a]芘激活为导致鼠伤寒沙门氏菌NM2009中umu基因表达诱导的遗传毒性产物的能力增强。高效液相色谱分析表明，仅当存在环氧化物水解酶时，P450 1B1催化苯并[a]芘生成反式 - 7,8 - 二羟基 - 7,8 - 二氢苯并[a]芘的水平约为3 nmol min⁻¹ nmol P450⁻¹，而P450 1A1（与水解酶一起）催化苯并[a]芘的活性是P450 1B1催化活性的十分之一。动力学分析表明，在该测定系统中，生成反式 - 7,8 - 二羟基 - 7,8 - 二氢苯并[a]芘的Vmax与Km之比在CYP1B1中比在CYP1A1中高3.2倍。发现其他人类P450（包括P450 1A2、2E1和3A4）对生成反式 - 7,8 - 二羟基 - 7,8 - 二氢苯并[a]芘的活性非常低或无法检测到。发现含有纯化的P450 1B1、兔肝NADPH - P450还原酶和人肝环氧化物水解酶的重组系统以0.86 nmol min⁻¹ nmol P450⁻¹的速率催化苯并[a]芘生成反式 - 7,8 - 二羟基 - 7,8 - 二氢苯并[a]芘；发现活性很大程度上取决于系统中胆酸钠的存在。这些结果表明，P450 1B1是催化苯并[a]芘氧化生成反式 - 7,8 - 二羟基 - 7,8 - 二氢苯并[a]芘的主要酶，并且P450 1B
