Shimada H, Hirai K, Simamura E, Pan J
Department of Anatomy, Kanazawa Medical University, Ishikawa, Japan.
Arch Biochem Biophys. 1998 Mar 1;351(1):75-81. doi: 10.1006/abbi.1997.0557.
We investigated the existence of an NADH-dependent paraquat (PQ) reduction system in rat liver mitochondria (Mt) in respect to the cytotoxic mechanisms of PQ. The outer membrane fractions, free from the contamination of inner membranes but with a few microsomes, catalyzed rotenone-insensitive NADH, but not NADPH, oxidation by menadione or PQ. Anti-NADH-cytochrome b5 reductase antibody and its inhibitor p-hydroxymercuribenzonate did not inhibit the NADH-PQ reduction activity. Therefore, the respiratory systems of the inner membranes and microsomal cytochrome P450 systems could not have been responsible for the reaction. Dicoumarol, an inhibitor of NAD(P)H-quinone oxidoreductase (NQO), dose dependently suppressed the NADH oxidation in the outer membrane via PQ as well as menadione, with I50 values of 190 (for menadione) and 150 microM (for PQ). Because of a lower sensitivity to NADPH and the higher doses of dicoumarol required for its inhibition, the activity in the outer membrane may be an "NADH-quinone oxidoreductase" which partly differs from the NQO previously reported. This outer membrane enzyme produced superoxide anions in the presence of both NADH and PQ and was too tightly membrane-bound to be extracted by Triton X-100 and deoxycholate. From these results, we concluded that the free radical-producing mitochondrial NADH-quinone oxidoreductase is a novel oxidation-reduction system participating in PQ toxicity. This is in good agreement with our previous results showing that PQ selectively damaged Mt in vivo and in vitro, resulting in cell death (K.-I. Hirai et al., 1992, Toxicology 72, 1-16).
我们针对百草枯（PQ）的细胞毒性机制，研究了大鼠肝线粒体（Mt）中是否存在依赖烟酰胺腺嘌呤二核苷酸（NADH）的百草枯还原系统。外膜组分未受内膜污染，但含有少量微粒体，可催化鱼藤酮不敏感的NADH（而非NADPH）被甲萘醌或PQ氧化。抗NADH - 细胞色素b5还原酶抗体及其抑制剂对羟基汞苯甲酸并不抑制NADH - PQ还原活性。因此，内膜的呼吸系统和微粒体细胞色素P450系统不可能是该反应的原因。双香豆素是烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H） - 醌氧化还原酶（NQO）的抑制剂，它通过PQ以及甲萘醌剂量依赖性地抑制外膜中的NADH氧化，对甲萘醌的半数抑制浓度（I50）值为190μM，对PQ为150μM。由于对外源NADPH敏感性较低且抑制其活性所需双香豆素剂量较高，外膜中的活性可能是一种“NADH - 醌氧化还原酶”，与先前报道的NQO有所不同。这种外膜酶在同时存在NADH和PQ时会产生超氧阴离子，并且与膜结合紧密，无法被 Triton X - 100和脱氧胆酸盐提取。基于这些结果我们得出结论，产生自由基的线粒体NADH - 醌氧化还原酶是参与PQ毒性的一种新型氧化还原系统。这与我们之前的结果高度一致，即PQ在体内和体外均能选择性地损伤线粒体，导致细胞死亡（平井启一等人，1992年，《毒理学》72卷，第1 - 16页）。
