Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by recombinant human cytochrome P4501B1 and purified liver epoxide hydrolase
Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by recombinant human cytochrome P4501B1 and purified liver epoxide hydrolase
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DOI：
10.1021/tx990028s
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发表时间：
1999-07-01
影响因子：
4.1
通讯作者：
Inoue, K
中科院分区：
文献类型：
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作者：
Shimada, T;Gillam, EMJ;Inoue, K
利用细胞色素P450 （P450）和NADPH-P450还原酶cdna转化大肠杆菌膜中表达的重组人酶，鉴定了在体外催化苯并[a]芘氧化转化中最活跃的人P450酶。P450 1A1和P450 1B1酶能增强苯并[a]芘对鼠伤寒沙门菌NM2009中umu基因表达的基因毒性产物的激活作用，这种基因毒性产物能诱导鼠伤寒沙门菌NM2009中umu基因的表达。高效液相色谱分析表明，P450 1B1催化苯并[a]芘生成反式-7,8-二羟基-7,8-二氢苯并[a]芘的量与P450（-1）的3 nmol / min(-1) nmol / min相近，P450 1A1（含该水解酶）催化苯并[a]芘的活性仅为P450 1B1催化活性的十分之一。动力学分析表明，CYP1B1中生成反式-7,8-二羟基-7,8-二氢苯并[a]芘的V-max与K-m比值比CYP1A1高3.2倍。其他人类p450（包括p450 1A2, 2E1和3A4）对反式7,8-二羟基-7,8-二氢苯并[a]芘的形成具有非常低或检测不到的活性。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体系能以0.86 nmol min(-1) nmol的P450-1催化苯并[A]芘生成反式7,8-二羟基-7,8-二氢苯并[A]芘；这些活性在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]芘氧化为反式7,8-二羟基7,8-二氢苯并[a]芘的主要酶，P450 1B1的催化功能可能决定了个体对苯并[a]芘致癌的易感性。
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 similar to 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[la]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.