Stiborová M, Frei E, Breuer A, Wiessler M, Schmeiser H H
Faculty of Science, Department of Biochemistry, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic.
Mutat Res. 2001 Jun 27;493(1-2):149-60. doi: 10.1016/s1383-5718(01)00171-1.
Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the (32)P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (alpha-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.
马兜铃酸（AA）是一种天然存在的肾毒素和致癌物，与一种独特类型的肾纤维化有关，这种肾纤维化被称为中草药肾病（CHN），可发展为尿路上皮癌。了解哪些酶参与AA的激活和/或解毒对于评估个体对这种天然致癌物的易感性很重要。我们使用核酸酶P1和（32）P后标记分析的1-丁醇萃取富集程序，研究了前列腺素H合酶（PHS）将AA激活为形成DNA加合物的代谢物的能力。PHS是肾脏和尿路上皮组织中的一种重要酶。含有高水平PHS的公羊精囊（RSV）微粒体产生的AA-DNA加合物模式与CHN患者肾组织中发现的模式一致。7-（脱氧腺苷-N（6）-基）马兜铃内酰胺I、7-（脱氧鸟苷-N（2）-基）马兜铃内酰胺I和7-（脱氧腺苷-N（6）-基）马兜铃内酰胺II被鉴定为AAI形成的AA-DNA加合物。两种加合物，7-（脱氧鸟苷-N（2）-基）马兜铃内酰胺II和7-（脱氧腺苷-N（6）-基）马兜铃内酰胺II，由AAII产生。根据鉴定出的DNA加合物的结构，硝基还原是AA代谢激活的关键途径。通过不同的辅因子和抑制剂证明了PHS作为RSV微粒体中激活酶的身份。只有吲哚美辛，一种PHS的选择性抑制剂，显著降低了RSV微粒体形成的加合物的量。NADPH：CYP还原酶的抑制剂（α-硫辛酸）和一些细胞色素P450（CYP）的选择性抑制剂无效。同样，只有PHS的辅因子花生四烯酸和过氧化氢支持AAI和AAII的DNA加合物形成，而NADPH和NADH无效。这些结果证明了PHS在RSV微粒体系统中AAI和AAII激活途径中的关键作用，并且用纯化的酶，即绵羊PHS-1得到了证实。这里呈现的结果是首次报道证明PHS-1对硝基芳香化合物的还原激活作用。
