Hatanaka N, Yamazaki H, Oda Y, Guengerich F P, Nakajima M, Yokoi T
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, 920-0934, Kanazawa, Japan.
Mutat Res. 2001 Oct 18;497(1-2):223-33. doi: 10.1016/s1383-5718(01)00254-6.
Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.
研究了在大肠杆菌膜中与人细胞色素P450（P450）1家族酶和NADPH-细胞色素P450还原酶（NPR）共表达时1-硝基芘（1-NP）的代谢活化情况。在没有任何P450系统的情况下，1-NP可诱导鼠伤寒沙门氏菌TA1535/pSK1002中的umu基因表达，但其活性受细菌O-乙酰转移酶（OAT）和硝基还原酶水平的影响。观察到人类P450 1B1/NPR膜对1-NP的代谢活化，且其受测试菌株中OAT水平的影响。在OAT过表达菌株NM2009中，P450 1B1对1-NP（0.3微摩尔）的代谢活化活性为750 umu单位/分钟/纳摩尔P450 1B1。使用TA1535/pSK1002或OAT缺陷菌株NM2000时，1-NP（3 - 30微摩尔）的代谢活化情况相似（约300 umu单位/分钟/纳摩尔P450 1B1）。在OAT过表达菌株NM2009中，P450 1B1在P450 1家族酶中对1-氨基芘（1-AP）活化的催化活性最高，表明通过N-羟基化/O-乙酰化形成氮鎓离子。高效液相色谱（HPLC）分析显示，P450 1B1催化1-NP（10微摩尔）生成了1-硝基芘-6-醇，以及1-硝基芘-3-醇、1-硝基芘-8-醇和反式-4,5-二羟基-4,5-二醇-1-硝基芘。这些结果表明，1-NP在低底物浓度下可通过硝基还原/O-乙酰化被人类P450 1B1活化为基因毒性剂，在高浓度下可能通过环氧化（与OAT无关）被活化。
