Tullis K, Olsen H, Bombick D W, Matsumura F, Jankun J
Department of Environmental Toxicology, University of California, Davis 95616.
J Biochem Toxicol. 1992 Summer;7(2):107-16. doi: 10.1002/jbt.2570070208.
A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21ras protein, or indirectly measured by the specific binding of 3H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P less than 0.05, Student t test) levels of hepatic p21ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the difference became statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21ras protein which did not become statistically significant even at 24 hr host-dosing. TCDD evoked increases in hepatic p21ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a 3H-GTP binding protein with MW of approximately 21 Kd. 3H-GTP binding in total hepatic membranes was also elevated (P less than 0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of approximately 21 Kd.
进行了一系列体内和体外实验，以确定给予2,3,7,8-四氯二苯并-对-二恶英（TCDD）对c-ras表达的影响。通过对p21ras蛋白进行免疫测定，或通过3H-GTP与肝细胞膜制剂的特异性结合进行间接测量，来确定对照组和TCDD处理组之间c-ras表达的差异。腹腔注射亚致死剂量的TCDD可显著提高（P<0.05，Student t检验）Sprague-Dawley大鼠和TCDD敏感的C57BL/6J小鼠肝脏中p21ras蛋白的水平。在C57BL/6J小鼠中毒的早期阶段就出现了这种增加。最早在给药后6小时可检测到增加，给药后12小时和24小时差异具有统计学意义。相比之下，TCDD耐受的DBA/2J小鼠肝脏中p21ras蛋白仅有轻微增加，即使在给药后24小时也没有达到统计学显著水平。TCDD引起的C57BL/6J小鼠肝脏中p21ras蛋白增加伴随着GTP与肝细胞膜特异性结合的增加。在sephadex G-50上对溶解的大鼠肝膜蛋白进行柱色谱分析表明，给予TCDD会增加一种分子量约为21 Kd的3H-GTP结合蛋白的水平。给C57BL/6J小鼠给药后6小时和24小时，总肝细胞膜中的3H-GTP结合也升高了（P<0.05，Fisher PLSD多重比较检验），但正如预期的那样，TCDD的作用不如在细胞膜中那么明显。TCDD处理增加了从豚鼠肝脏纯化的RNA体外翻译产物中发现的一种21 Kd蛋白的水平。基于其结合GTP的能力、被针对rasHa和Ki蛋白的多克隆抗体沉淀以及随后的SDS-PAGE显示出一条约21 Kd的单一蛋白带，该蛋白被鉴定为c-ras蛋白。
