Stiborova Marie, Mares Jaroslav, Levova Katerina, Pavlickova Jana, Barta Frantisek, Hodek Petr, Frei Eva, Schmeiser Heinz H
Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
Neuro Endocrinol Lett. 2011;32 Suppl 1:121-30.
The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model.
DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC.
Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed.
Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.
已证实马兜铃属植物中的草药成分马兜铃酸（AA）可导致马兜铃酸肾病（AAN）、巴尔干地方性肾病（BEN）及其尿路上皮恶性肿瘤。AAN和BEN的一个共同特征是，并非所有接触AA的个体都会患肾病和肿瘤。这些不同反应的一个原因可能是催化AA生物转化的酶活性存在个体差异。因此，确定主要参与AA主要毒性成分AAI代谢的酶，并详细了解其催化特异性至关重要。因此，本研究旨在评估大鼠模型中细胞色素P450（CYP）介导的AAI氧化解毒和还原激活作用。
采用32P后标记法的核酸酶P1版本研究DNA加合物的形成。通过高效液相色谱法测定大鼠肝微粒体中CYP介导的AAI解毒代谢产物8-羟基马兜铃酸I（AAIa）的体外形成。
大鼠肝脏CYP既能通过将AAI氧化为AAIa使其解毒，又能在体外将这种致癌物还原激活为环状N-酰基亚硝鎓离子，从而形成AAI-DNA加合物。为了确定肝脏CYP在AAI去甲基化和激活中的作用，研究了CYP诱导剂和选择性CYP抑制剂对AAIa和AAI-DNA加合物形成的调节作用。基于这些研究，我们认为CYP1A1和1A2在通过将AAI去甲基化为AAIa使其解毒过程中起主要作用，并且在缺氧条件下也在将AAI激活为形成DNA加合物的物质过程中起主要作用。使用含有重组表达大鼠CYP、NADPH:CYP还原酶和/或细胞色素b5的杆状病毒转染昆虫细胞微粒体（Supersomes™），证实了CYP1A1和1A2在体外这两个反应中均起主要作用。
基于本研究及以往研究结果，我们提出大鼠体内AAI的激活和解毒主要取决于AAI与CYP1A1/2或NADPH（P）H:醌氧化还原酶的结合亲和力、它们的周转率以及CYP1A对AAI氧化和还原之间的平衡。
