Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
Environ Toxicol Pharmacol. 1997 Sep;3(4):245-50. doi: 10.1016/s1382-6689(97)00019-7.
The reduction of the naphthoquinone derivative, lapachol, which is responsible for its bioactivation was examined using microsomal preparations and NADPH-cytochrome P450 reductase (P450 reductase). Phenobarbital (PB) pretreatment resulted in an induction of enzyme activities for cytochrome c reduction (1.54 times) and lapachol reduction (1.20 times) by hepatic microsomal preparation of rats. The specific activity of lapachol reduction by purified P450 reductase showed 56-fold higher than that by untreated liver microsomes. Addition of antibody against P450 reductase (2 mg of IgG/mg of protein) to the microsomal incubation mixture caused an immunoinhibition of cytochrome c (32%) and lapachol (19%) reduction activities, suggesting that P450 reductase catalyzes lapachol reduction. Generation of superoxide anion radical (1321 nmol/mg per min) in approximately equivalent amounts of with NADPH consumption (941 nmol/mg per min) was detected during metabolism of lapachol by P450 reductase. Electron spin resonance (ESR) experiments confirmed generation of superoxide anion radical and hydroxyl radical as these 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adducts. Incubation of lapachol with P450 reductase caused a cleavage of DNA which was reduced in the presence of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase(1), and hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) and thiourea. Taken together, these results indicate that lapachol is bioactivated by P450 reductase to reactive species, which promote DNA scission through the redox cycling based generation of superoxide anion radical.
醌衍生物拉帕醇的还原反应，负责其生物激活，使用微粒体制剂和 NADPH-细胞色素 P450 还原酶（P450 还原酶）进行了检查。苯巴比妥（PB）预处理导致大鼠肝微粒体制剂中细胞色素 c 还原（1.54 倍）和拉帕醇还原（1.20 倍）的酶活性诱导。与未处理的肝微粒体相比，纯化的 P450 还原酶的拉帕醇还原比活性高 56 倍。向微粒体孵育混合物中添加针对 P450 还原酶的抗体（2mg IgG/mg 蛋白）导致细胞色素 c（32%）和拉帕醇（19%）还原活性的免疫抑制，表明 P450 还原酶催化拉帕醇还原。在 P450 还原酶代谢拉帕醇时，检测到与 NADPH 消耗（941 nmol/mg per min）相当数量的超氧阴离子自由基（1321 nmol/mg per min）的产生。电子自旋共振（ESR）实验证实了超氧阴离子自由基和羟基自由基的产生，因为这些是 5,5'-二甲基-1-吡咯啉 N-氧化物（DMPO）加合物。拉帕醇与 P450 还原酶孵育会导致 DNA 断裂，而在 Cu、Zn-超氧化物歧化酶（Cu、Zn-SOD）、过氧化氢酶（1）和羟基自由基清除剂如二甲基亚砜（DMSO）和硫脲存在的情况下，DNA 断裂会减少。总之，这些结果表明，拉帕醇被 P450 还原酶生物激活为活性物质，通过基于氧化还原循环的超氧阴离子自由基的产生促进 DNA 断裂。
