Eyre R J, Stevens D K, Parker J C, Bull R J
Pharmacology/Toxicology Graduate Program, Washington State University, Pullman, USA.
J Toxicol Environ Health. 1995 Dec;46(4):443-64. doi: 10.1080/15287399509532048.
Covalent binding of radiolabel to tissue proteins following [14C]trichloroethene (TRI) exposure has been used as a measure of TRI activation. Gross binding of 14C label does not differentiate between alternate routes of metabolism and can be confounded when there is significant metabolic incorporation of radiolabel. We examined the covalent association of 14C label to hepatic and renal proteins in male F344 rats and B6C3F1 mice following oral treatment with [14C]TRI and three metabolites of TRI: [14C]trichloroacetate (TCA), [14C]dichloroacetate (DCA), and [14C]dichlorovinylcysteine (DCVC) in vivo. Association of radiolabel from [14C]TRI with hepatic proteins reached a maximum at 2 and 4 h in mouse and rat hepatic proteins, respectively. Association of radiolabel with renal proteins reached a maximum at 8 h in both species. An approach was developed based upon formation of protein adducts that release acetate and monochloroacetate (MCA) on acid hydrolysis. These adducts were found to be specifically associated with the activation of DCVC to reactive intermediates. Acetate and MCA were identified by using two different conditions of high-performance liquid chromatography (HPLC) separation with differing selectivity. Diethylmaleate and aminooxyacetic acid pretreatment inhibited the formation of these adducts from TRI, consistent with requirements for glutathione and beta-lyase. No evidence of these adducts was detected following [14C]TCA and [14C]DCA treatment. Renal acid-labile adduct formation from 25 mg/kg DCVC was approximately 12-fold greater in male B6C3F1 mice than in male F344 rats. They accounted for 7.8 and 4.6% of the total adducts to renal protein in rats and mice, respectively. Acid-labile adducts formed from 1000 mg/kg TRI were approximately two times greater in mice than rats. In this case, they accounted for 1.4 and 3.3% of the total adduct formed in renal proteins from TRI (corrected for metabolic incorporation), respectively. This greater dilution of adducts associated with DCVC in renal proteins of the rat suggests that covalent binding of TRI has less specificity for the DCVC pathway in rats than in mice.
[14C]三氯乙烯（TRI）暴露后放射性标记与组织蛋白的共价结合已被用作TRI活化的一种衡量指标。14C标记的总结合无法区分代谢的替代途径，并且当放射性标记存在显著的代谢掺入时可能会产生混淆。我们在雄性F344大鼠和B6C3F1小鼠经口给予[14C]TRI及其三种代谢物：[14C]三氯乙酸（TCA）、[14C]二氯乙酸（DCA）和[14C]二氯乙烯基半胱氨酸（DCVC）后，检测了14C标记与肝脏和肾脏蛋白的共价结合情况。[14C]TRI的放射性标记与小鼠和大鼠肝脏蛋白的结合分别在2小时和4小时达到最大值。放射性标记与两种动物肾脏蛋白的结合在8小时达到最大值。基于蛋白质加合物的形成开发了一种方法，该加合物在酸水解时会释放乙酸盐和一氯乙酸盐（MCA）。发现这些加合物与DCVC活化为反应性中间体有特异性关联。通过使用两种具有不同选择性的高效液相色谱（HPLC）分离条件来鉴定乙酸盐和MCA。马来酸二乙酯和氨基氧基乙酸预处理抑制了TRI形成这些加合物，这与谷胱甘肽和β-裂解酶的需求一致。在[14C]TCA和[14C]DCA处理后未检测到这些加合物的证据。雄性B6C3F1小鼠中由25mg/kg DCVC形成的肾脏酸不稳定加合物比雄性F344大鼠大约高12倍。它们分别占大鼠和小鼠肾脏蛋白总加合物的7.8%和4.6%。由1000mg/kg TRI形成的酸不稳定加合物在小鼠中比在大鼠中大约高两倍。在这种情况下，它们分别占TRI在肾脏蛋白中形成的总加合物（校正代谢掺入）的1.4%和3.3%。大鼠肾脏蛋白中与DCVC相关的加合物稀释度更高，这表明TRI的共价结合对大鼠中DCVC途径的特异性低于小鼠。
