Lock E A, Schnellmann R G
Biochemical Toxicology Section, ICI Central Toxicology Laboratory, Cheshire, United Kingdom.
Toxicol Appl Pharmacol. 1990 Jun 1;104(1):180-90. doi: 10.1016/0041-008x(90)90293-4.
An early event in the nephrotoxicity of haloalkene cysteine conjugates is their metabolism by cysteine conjugate beta-lyase to generate a reactive "thiol moiety" which binds to protein. This reactive metabolite(s) has been reported to cause mitochondrial dysfunction. We have examined the effect of three haloalkene cysteine conjugates on the activity of rat renal cortical cytosolic glutathione reductase and mitochondrial lipoyl dehydrogenase, two enzymes which have been reported to be inhibited by S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in the liver. N-Acetyl-S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L- cysteine (N-acetyl PCBC) produced a time- and concentration-dependent inhibition of glutathione reductase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 215 microM. The enzyme activity from male rat kidney was more sensitive to N-acetyl PCBC than that from female rat kidney. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, and bis-p-nitrophenyl phosphate, an amidase inhibitor, blocked the effect of N-acetyl PCBC on glutathione reductase, indicating that metabolism by the cytosol is required to produce enzyme inhibition. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC) and DCVC are also noncompetitive inhibitors of glutathione reductase but are less active than N-acetyl PCBC with Ki's of 2.6 and 6.2 mM for DCVC and TFEC, respectively, DCVC produced a time- and concentration-dependent inhibition of lipoyl dehydrogenase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 762 microM. TFEC and PCBC also inhibit lipoyl dehydrogenase. Aminooxyacetic acid blocked the effect of DCVC, TFEC, and PCBC on lipoyl dehydrogenase, indicating that metabolism by the mitochondrial fraction is required to produce enzyme inhibition. Glutathione reductase activity in the renal cortex of male rats treated with 200 mg/kg hexachloro-1,3-butadiene (HCBD) was inhibited as early as 1 hour after dosing, before signs of marked morphological damage. The activity of lipoyl dehydrogenase was also reduced but was only statistically significant 8 hr after dosing when there was marked renal dysfunction. These findings indicate that the reactive thiol moiety formed by cysteine conjugate beta-lyase cleavage of PCBC can inhibit both glutathione reductase and lipoyl dehydrogenase activities in vivo following HCBD administration. We suggest that such inhibition is a general phenomenon, occurring with diverse and as yet unidentified renal proteins. The critical nature of mitochondrial function and the generation of reactive metabolites within this compartment make this organelle a prime target.
卤代烯烃半胱氨酸共轭物肾毒性的早期事件是它们被半胱氨酸共轭β-裂解酶代谢，生成一种与蛋白质结合的反应性“硫醇部分”。据报道，这种反应性代谢物会导致线粒体功能障碍。我们研究了三种卤代烯烃半胱氨酸共轭物对大鼠肾皮质胞质谷胱甘肽还原酶和线粒体硫辛酰胺脱氢酶活性的影响，这两种酶在肝脏中已被报道会被S-(1,2-二氯乙烯基)-L-半胱氨酸（DCVC）抑制。N-乙酰-S-(1,2,3,4,4-五氯-1,3-丁二烯基)-L-半胱氨酸（N-乙酰PCBC）对谷胱甘肽还原酶产生了时间和浓度依赖性抑制，动力学研究表明这种抑制是非竞争性的，Ki为215μM。雄性大鼠肾脏中的酶活性比雌性大鼠肾脏中的酶活性对N-乙酰PCBC更敏感。半胱氨酸共轭β-裂解酶抑制剂氨基氧乙酸和酰胺酶抑制剂双对硝基苯基磷酸阻断了N-乙酰PCBC对谷胱甘肽还原酶的作用，表明需要胞质代谢才能产生酶抑制作用。S-(1,1,2,2-四氟乙基)-L-半胱氨酸（TFEC）和DCVC也是谷胱甘肽还原酶的非竞争性抑制剂，但活性低于N-乙酰PCBC，DCVC和TFEC 的Ki分别为2.6和6.2 mM。DCVC对硫辛酰胺脱氢酶产生了时间和浓度依赖性抑制，动力学研究表明这种抑制是非竞争性的，Ki为762μM。TFEC和PCBC也抑制硫辛酰胺脱氢酶。氨基氧乙酸阻断了DCVC、TFEC和PCBC对硫辛酰胺脱氢酶的作用，表明需要线粒体部分的代谢才能产生酶抑制作用。用200 mg/kg六氯-1,3-丁二烯（HCBD）处理的雄性大鼠肾皮质中的谷胱甘肽还原酶活性在给药后1小时就受到抑制，此时还没有明显的形态学损伤迹象。硫辛酰胺脱氢酶的活性也降低了，但只有在给药8小时后出现明显肾功能障碍时才具有统计学意义。这些发现表明，PCBC经半胱氨酸共轭β-裂解酶裂解形成的反应性硫醇部分在给予HCBD后可在体内抑制谷胱甘肽还原酶和硫辛酰胺脱氢酶的活性。我们认为这种抑制是一种普遍现象，发生在多种尚未鉴定的肾蛋白上。线粒体功能的关键性质以及该细胞器内反应性代谢物的产生使这个细胞器成为主要靶点。
