Yamamoto H, Hirose K, Hayasaki Y, Masuda M, Kazusaka A, Fujita S
Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Arch Toxicol. 1999 Nov;73(8-9):457-64. doi: 10.1007/s002040050635.
The Long-Evans Cinnamon (LEC) rat is a mutant strain of rats that accumulate copper (Cu) in the liver in much the same way as individuals who suffer from Wilson's disease (WD) and has been suggested as a model for this disease. Lipid peroxidation (LPO) is considered to be involved in the toxic action of Cu in the livers of LEC rats. We investigated the mechanism of LPO in the livers of LEC rats showing apparent signs of hepatitis. Several-fold higher LPO levels were observed in post-mitochondrial supernatant (S-9) fraction of livers from hepatitic LEC rats than in those from Wistar rats. To mimic living cells, we introduced NADPH-generating system (NADPH-gs) into the S-9 incubation system. Thus was ensured a constant supply of NADPH to vital enzymes that may be directly or indirectly involved in the generation and/or elimination of reactive oxygen species (ROSs), such as glutathione reductase (GSSG-R), which require NADPH for their reactions. The levels of LPO in liver S-9 from hepatitic LEC rats were further increased by incubating liver S-9 at 37 degrees C in the presence of NADPH-gs. This increase was inhibited by EDTA, butylated hydroxytoluene (BHT), and catalase (CAT), suggesting that some metal, most likely the accumulated Cu, and ROSs derived from hydrogen peroxide (H2O2) are involved in the increased levels of LPO in the livers of hepatitic LEC rats. The requirement of NADPH-gs for enhanced LPO in the livers of hepatitic LEC rats indicates the consumption of NADPH during reactions leading to LPO. It is known that H2O2, and consequently hydroxyl radical are generated during Cu-catalyzed glutathione (GSH) oxidation. The cyclic regeneration of GSH from GSSG by NADPH-dependent GSSG-R in the presence of NADPH-gs may cause sustained generation of hydroxyl radical in the presence of excess free Cu. The generation of H2O2 in S-9 fraction of livers from hepatitic LEC rats was observed to be significantly higher than that in S-9 fraction of livers from non-hepatitic LEC rats and Wistar rats. Moreover, in addition to the reported decrease in glutathione peroxidase (GPX) activity, we found that CAT activity was markedly decreased in LEC rats with hepatitis. The increased generation of H2O2 with reduced activities of GPX and CAT may result in cellular accumulation of H2O2 in the liver of hepatitic LEC rats. Taken altogether, it is suggested that the accumulated H2O2 undergoes the Fenton-type reaction with also accumulated free Cu, thus generating hydroxyl radical in the livers of hepatitic LEC rats and increasing LPO levels in these animals.
长 Evans 肉桂色（LEC）大鼠是一种大鼠突变品系，其肝脏中铜（Cu）的蓄积方式与患有威尔逊病（WD）的个体非常相似，并且已被提议作为这种疾病的模型。脂质过氧化（LPO）被认为与 Cu 在 LEC 大鼠肝脏中的毒性作用有关。我们研究了出现明显肝炎迹象的 LEC 大鼠肝脏中 LPO 的机制。在患肝炎的 LEC 大鼠肝脏的线粒体后上清液（S - 9）部分中观察到的 LPO 水平比 Wistar 大鼠肝脏中的高几倍。为了模拟活细胞，我们将 NADPH 生成系统（NADPH - gs）引入 S - 9 孵育系统。这样就确保了向可能直接或间接参与活性氧（ROS）生成和/或消除的关键酶持续供应 NADPH，例如谷胱甘肽还原酶（GSSG - R），其反应需要 NADPH。通过在 37℃下在 NADPH - gs 存在的情况下孵育肝脏 S - 9，患肝炎的 LEC 大鼠肝脏 S - 9 中的 LPO 水平进一步升高。这种升高受到乙二胺四乙酸（EDTA）、丁基羟基甲苯（BHT）和过氧化氢酶（CAT）的抑制，表明某些金属，很可能是蓄积的 Cu，以及源自过氧化氢（H₂O₂）的 ROS 参与了患肝炎的 LEC 大鼠肝脏中 LPO 水平的升高。患肝炎的 LEC 大鼠肝脏中增强 LPO 需要 NADPH - gs，这表明在导致 LPO 的反应过程中 NADPH 被消耗。已知在 Cu 催化的谷胱甘肽（GSH）氧化过程中会生成 H₂O₂，进而生成羟基自由基。在 NADPH - gs 存在的情况下，通过 NADPH 依赖性 GSSG - R 从 GSSG 循环再生 GSH 可能会在存在过量游离 Cu 的情况下导致羟基自由基的持续生成。观察到患肝炎的 LEC 大鼠肝脏 S - 9 部分中 H₂O₂的生成明显高于非患肝炎的 LEC 大鼠和 Wistar 大鼠肝脏 S - 9 部分中的生成。此外，除了报道的谷胱甘肽过氧化物酶（GPX）活性降低外，我们发现患肝炎的 LEC 大鼠中 CAT 活性明显降低。GPX 和 CAT 活性降低导致 H₂O₂生成增加，可能会导致患肝炎的 LEC 大鼠肝脏中 H₂O₂的细胞内蓄积。综上所述，提示蓄积的 H₂O₂与同样蓄积的游离 Cu 发生芬顿型反应，从而在患肝炎的 LEC 大鼠肝脏中生成羟基自由基并增加这些动物体内的 LPO 水平。
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