Tang W, Borel A G, Abbott F S
Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Drug Metab Dispos. 1996 Apr;24(4):436-46.
The hepatotoxic metabolite of the anticonvulsant drug valproic acid (VPA), namely (E)-2-propyl-2,4-pentadienoic acid (E)-2,4-diene VPA), is known to react with glutathione (GSH) in vivo. Although glutathione-S-transferase (GST) was suspected of being the catalyst for this conjugation reaction, this was yet to be confirmed. In this study, GST activities were detected in the hepatic cytosolic and sonic-disrupted mitoplast fractions isolated from male Sprague-Dawley rats by using 1-chloro-2,4-dinitrobenzene as a substrate. An elevation of GST activities by 45 to 100% was observed after pretreatment of rats with phenobarbital (PB). Subsequently, these apparent GST activities were examined for their effects on the in vivo conjugation of GSH with N-acetyl-S-((E)-2-propyl-2,4-pentadienoyl)cysteamine (2,4-diene VPA-NACA), a structural mimic of (E)-2,4-diene VPA coenzyme A thioester. Reaction products were identified and quantitated by combined liquid chromatography-tandem mass spectrometry. The GST-mediated conjugation of GSH with 2,4-diene VPA-NACA produced two structural isomers via either 5,6- or 1,6-addition of GSH. Only the 1,6- addition product was found for the spontaneous conjugation reaction (control). Quantitatively, GSH conjugates formed in the cytosolic fraction were 23-fold that of control. An additional 1.5-fold enhancement was observed in the cytosolic fraction from PB-treated rats. The production of the GSH conjugates was increased by 2-fold for reactions involving the sonic-disrupted mitoplasts, either from untreated or PB-treated rats. Partially purified GST was found to catalyze the conjugation reactions in a fashion similar to that of the isolated subcellular fractions. No reaction with GSH could be detected for the free acid form of (E)-2,4-diene VPA. As was the case with the in vitro data, two structural isomers of GSH conjugates were detected in the bile of rats that received (E)-2,4-diene VPA. These results indicate that in vivo production of the GSH conjugates of (E)-2,4-diene VPA is most likely catalyzed by GST enzymes, with the esterified diene being essential for the conjugation reaction. In a separate experiment, 2,4-diene VPA-NACA was observed to alkylate reduced oxytocin through one or both cysteine residues. Thus, the toxicity of (E)-2,4-diene VPA might be produced via either GST-promoted depletion of cellular GSH, or a direct modification of key proteins, or both.
抗惊厥药物丙戊酸（VPA）的肝毒性代谢产物，即（E）-2-丙基-2,4-戊二烯酸（（E）-2,4-二烯VPA），已知在体内会与谷胱甘肽（GSH）发生反应。尽管怀疑谷胱甘肽-S-转移酶（GST）是这种结合反应的催化剂，但尚未得到证实。在本研究中，以1-氯-2,4-二硝基苯为底物，检测了从雄性Sprague-Dawley大鼠分离的肝细胞质和超声破碎的线粒体部分中的GST活性。用苯巴比妥（PB）预处理大鼠后，观察到GST活性升高了45%至100%。随后，检查了这些表观GST活性对GSH与N-乙酰-S-（（E）-2-丙基-2,4-戊二烯酰）半胱胺（2,4-二烯VPA-NACA）（（E）-2,4-二烯VPA辅酶A硫酯的结构模拟物）体内结合的影响。通过液相色谱-串联质谱联用对反应产物进行鉴定和定量。GST介导的GSH与2,4-二烯VPA-NACA的结合通过GSH的5,6-或1,6-加成产生了两种结构异构体。自发结合反应（对照）仅发现了1,6-加成产物。定量分析，细胞质部分形成的GSH结合物是对照的23倍。在PB处理大鼠的细胞质部分中观察到额外的1.5倍增强。对于涉及未处理或PB处理大鼠的超声破碎线粒体的反应，GSH结合物的产生增加了2倍。发现部分纯化的GST以与分离的亚细胞部分相似的方式催化结合反应。未检测到（E）-2,4-二烯VPA的游离酸形式与GSH的反应。与体外数据一样，在接受（E）-2,4-二烯VPA的大鼠胆汁中检测到了GSH结合物的两种结构异构体。这些结果表明，（E）-2,4-二烯VPA的GSH结合物在体内的产生很可能由GST酶催化，酯化二烯对于结合反应至关重要。在另一个实验中，观察到2,4-二烯VPA-NACA通过一个或两个半胱氨酸残基使还原型催产素烷基化。因此，（E）-2,4-二烯VPA的毒性可能通过GST促进的细胞内GSH消耗或关键蛋白质的直接修饰，或两者兼而有之产生。
