Quantitative Evaluation and Prediction of Drug-Toxicity Due to Lipid Peroxidation

体外和体内研究了药物诱导的脂质过氧化作用。1.建立了谷胱甘肽的高效液相色谱检测方法。用邻苯二甲醛标记洗脱液中的谷胱甘肽进行测定。用硫代巴比妥酸反应物质(TBA-RS)、荧光物质和高分子量蛋白质聚集体评价脂质过氧化反应。通过测量荧光寿命对大鼠肝脏微粒体中形成的荧光物质进行了表征，发现荧光物质至少有三种不同的荧光寿命。这在微粒体蛋白质和脂质中也是如此。微粒体酶在脂质过氧化过程中活性降低，膜流动性降低。给大鼠灌胃对乙酰氨基酚。血浆谷草转氨酶(GOT)升高，肝匀浆TBA-RS轻度升高。扑热息痛给药前，大鼠腹腔注射马来酸二乙酯，肝微粒体内可见荧光物质和高分子量蛋白质聚集体。这表明谷氨酰胺在对乙酰氨基酚诱导的脂质过氧化中起重要作用。四氯化碳和阿霉素中也观察到了同样的情况。这些评价脂质过氧化反应的指标可用于体内脂质过氧化反应的检测。研究了醋氨酚对体外培养肝细胞的影响。肝细胞与对乙酰氨基酚孵育。当产生TBA-RS时，细胞存活率下降。同时，培养液中GOT含量增加，细胞内谷胱甘肽含量降低。在较高浓度的扑热息痛作用下，TBA-RS的形成受到抑制，细胞活力没有下降，这可能与扑热息痛本身的抗氧化作用有关。因此，本研究表明脂质过氧化在扑热息痛肝毒性中起重要作用。

Drug-induced lipid peroxidation was investigated in vitro and in vivo. 1. The method to determine glutathione was set up using HPLC which was supplied by this grant. The determination was carried out by labeling eluted glutathione with o-phthalaldehyde.2. Lipid peroxidation was evaluated by thiobarbituric acid reactive substances (TBA-RS), fluorescent substances and high molecular weight protein aggregates. Fluorescent substances formed in peroxidized microsomes of rat liver were characterized by measuring fluorescence lifetime.Fluorescent substances were found to have at least three different fluorescence lifetimes. This was the same in microsomal proteins and lipids. Microsomal enzymes decreased their activities during lipid peroxidation and membrane fluidity also decreased.3. Acetaminophen was orally administered to rats. Plasma glutamic-oxaloacetic transaminase (GOT) increased and TBA-RS in liver homogenates slightly increased. When diethylmaleate was injected intraperitoneally to rats before acetaminophen administration, fluorescent substances and high molecular weight protein aggregates were found in liver microsomes. This indicates that glutatione plays an important role in acetaminophen-induced lipid peroxidation. The same was observed in carbon tetrachloride and adriamycin. These indices to evaluate lipid peroxidation were found to be useful to detect in vivo lipid peroxidation.4. Effects of acetaminophen on isolated hepatocytes were investigated. Hepatocytes were incubated with acetaminophen. When TBA-RS was produced, cell viability decreased. Simultaneously, GOT in the medium increased and intracellular glutathione decreased. At higher concentration of acetaminophen, TBA-RS formation was depressed and cell viability was not decreased,which was probably due to the antioxidant effect of acetaminophen itself. Thus, this study showed that lipid peroxidation played an important role in acetaminophen-induced hepatotoxicity.