Zhang G H, Stevens J L
W. Alton Jones Cells Science Center, Lake Placid, New York 12946.
Toxicol Appl Pharmacol. 1989 Aug;100(1):51-61. doi: 10.1016/0041-008x(89)90091-4.
An important step in understanding the mechanism underlying the tubular specificity of the nephrotoxicity of toxic cysteine conjugates is to identify the rate-limiting steps in their activation. The rate-limiting steps in the activation of toxic cysteine conjugates were characterized using isolated proximal tubules from the rat and 35S-labeled S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAC-DCVC) as model compounds. The accumulation by tubules of 35S radiolabel from both DCVC and NAC-DCVC was time and temperature dependent and was mediated by both Na+-dependent and independent processes. Kinetic studies with DCVC in the presence of sodium revealed the presence of two components with apparent Km and Vmax values of (1) 46 microM and 0.21 nmol/mg min and (2) 2080 microM and 7.3 nmol/mg.min. NAC-DVVC uptake was via a single system with apparent Km and Vmax values of 157 microM and 0.65 nmol/mg.min, respectively. Probenecid, an inhibitor of the renal organic anion transport system, inhibited accumulation of radiolabel from NAC-DCVC, but not from DCVC. The covalent binding of 35S label to cellular macromolecules was much greater from [35S]DCVC than from NAC-[35S]DCVC. Analysis of metabolites showed that a substantial amount of the cellular NAC-[35S]DCVC was unmetabolized while [35S]DCVC was rapidly metabolized to bound 35S-labeled material and unidentified products. The data suggest that DCVC is rapidly metabolized following transport, but that activation of NAC-DCVC depends on a slower rate of deacetylation. The results are discussed with regard to the segment specificity of cysteine conjugate toxicity and the role of disposition in vivo in the nephrotoxicity of glutathione conjugates.
了解毒性半胱氨酸共轭物肾毒性的肾小管特异性潜在机制的一个重要步骤是确定其激活过程中的限速步骤。使用来自大鼠的分离近端小管以及35S标记的S-(1,2-二氯乙烯基)-L-半胱氨酸（DCVC）和N-乙酰基-S-(1,2-二氯乙烯基)-L-半胱氨酸（NAC-DCVC）作为模型化合物，对毒性半胱氨酸共轭物激活过程中的限速步骤进行了表征。DCVC和NAC-DCVC的35S放射性标记在小管中的积累是时间和温度依赖性的，并且由钠依赖性和非依赖性过程介导。在有钠存在的情况下用DCVC进行的动力学研究表明存在两个组分，其表观Km和Vmax值分别为(1) 46 microM和0.21 nmol/mg·min以及(2) 2080 microM和7.3 nmol/mg·min。NAC-DVVC的摄取通过单一系统，其表观Km和Vmax值分别为157 microM和0.65 nmol/mg·min。丙磺舒是肾有机阴离子转运系统的抑制剂，它抑制了NAC-DCVC的放射性标记积累，但不抑制DCVC的。35S标记与细胞大分子的共价结合在[35S]DCVC中比在NAC-[35S]DCVC中要多得多。代谢物分析表明，细胞内大量的NAC-[35S]DCVC未被代谢，而[35S]DCVC迅速代谢为结合的35S标记物质和未鉴定产物。数据表明，DCVC在转运后迅速代谢，但NAC-DCVC的激活取决于较慢的脱乙酰化速率。就半胱氨酸共轭物毒性的节段特异性以及体内处置在谷胱甘肽共轭物肾毒性中的作用对结果进行了讨论。
