Van Pelt F N, Kenna J G
Department of Pharmacology and Toxicology, St Mary's Hospital Medical School (Imperial College of Science, Technology and Medicine, London, U.K.
Biochem Pharmacol. 1994 Aug 3;48(3):461-71. doi: 10.1016/0006-2952(94)90275-5.
Immune responses to novel, halothane metabolite-modified protein antigens (tri-fluoroacetylated proteins; TFA-proteins) have been implicated in the pathogenesis of halothane hepatitis. The aim of the present study was to investigate and characterize expression of TFA-proteins in cultures of rat hepatocytes which were exposed to halothane in vitro. Following exposure to halothane, the hepatocytes were harvested, then subcellular fractions were prepared and were analysed by immunoblotting for expression of antigens recognized by a rabbit anti-TFA antiserum, and by antibodies in sera from two patients with halothane hepatitis. Hepatocytes exposed to halothane in vitro were shown to express novel microsomal protein antigens, which exhibited molecular masses that were identical to the molecular masses of the major TFA-protein antigens expressed in vivo, in livers of halothane-treated rats (100, 80 and 60 kDa). Experiments in which hepatocytes were exposed to halothane in the presence of SKF-525A, or were exposed to deuterated halothane in place of halothane, confirmed that these novel antigens were TFA-modified proteins whose generation required cytochrome P450-mediated metabolism of halothane. The maximal levels of TFA-antigens expressed in vitro were about 30% of the levels expressed in halothane-treated rats in vivo. Maximal expression of the TFA-antigens in vitro occurred when hepatocytes were exposed to halothane at doses which yielded concentrations of the drug in culture medium of about 13 microM. Expression of the antigens in vitro occurred slowly, with an apparent half-time of about 8 hr. Overall, these results demonstrate that the properties of the TFA-antigens expressed in cultured hepatocytes in vitro closely resemble the properties exhibited by the antigens expressed in vivo, in livers of halothane-treated rats.
对新型氟烷代谢物修饰的蛋白质抗原（三氟乙酰化蛋白质；TFA-蛋白质）的免疫反应被认为与氟烷性肝炎的发病机制有关。本研究的目的是调查和表征体外暴露于氟烷的大鼠肝细胞培养物中TFA-蛋白质的表达。暴露于氟烷后，收获肝细胞，然后制备亚细胞组分，并通过免疫印迹分析兔抗TFA抗血清以及两名氟烷性肝炎患者血清中的抗体所识别的抗原的表达。体外暴露于氟烷的肝细胞显示表达新型微粒体蛋白质抗原，其分子量与氟烷处理大鼠肝脏中体内表达的主要TFA-蛋白质抗原的分子量相同（100、80和60 kDa）。在肝细胞存在SKF-525A的情况下暴露于氟烷，或用氘代氟烷代替氟烷进行暴露的实验证实，这些新型抗原是TFA修饰的蛋白质，其产生需要细胞色素P450介导的氟烷代谢。体外表达的TFA抗原的最大水平约为体内氟烷处理大鼠表达水平的30%。当肝细胞暴露于氟烷的剂量使培养基中药物浓度约为13 microM时，体外TFA抗原的表达达到最大值。抗原在体外的表达缓慢，表观半衰期约为8小时。总体而言，这些结果表明，体外培养的肝细胞中表达的TFA抗原的特性与体内氟烷处理大鼠肝脏中表达的抗原所表现出的特性非常相似。
