FORMATION OF TRIFLUOROACETYLATED PROTEIN ANTIGENS IN CULTURED RAT HEPATOCYTES EXPOSED TO HALOTHANE IN-VITRO

Immune responses to novel, halothane metabolite-modified protein antigens (trifluoroacetylated 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 mu M. 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.