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

Previous investigations have implicated an immune response to trifluor oacetylated proteins (TFA-proteins) in the pathogenesis of halothane h epatitis. The objective of this study was to establish conditions for generation of TFA-proteins in hepatocytes exposed to halothane in vitr o. Monolayer cultures of rat hepatocytes were incubated in sealed flas ks with or without added halothane, then subcellular fractions were pr epared by differential centrifugation and analysed by immunoblotting f or reactivity with anti-TFA antiserum. The specificity of the antiseru m was verified by hapten inhibition with N-epsilon-TFA-L-lysine. TFA-p roteins were generated when hepatocytes were cultured with halothane, but not when hepatocytes were cultured without halothane, and were con centrated in the microsomal fraction. Generation of TFA-proteins was g reater when hepatocytes were exposed to an initial halothane concentra tion of about 0.17 mM-halothane than when hepatocytes were exposed to higher concentrations (0.6 mM and 1.4 mM). The molecular masses -of th e major TFA-proteins produced in vitro (100, 80 and 60 kDa) were very similar, if not identical, to the molecular masses of the major TFA-pr oteins produced in livers of rats treated ip with halothane in vivo, a s were the kinetics of TFA-protein formation and turnover.