GENERATION AND DETECTION OF NEOANTIGENS IN GUINEA-PIG LIVER SLICES INCUBATED WITH HALOTHANE

The volatile anesthetic halothane can be biotransformed by the hepatic cytochrome P-450 system to produce a reactive intermediate, trifluoroacetyl chloride, capable of covalently binding to liver proteins. The product of this reaction, the trifluoroacetyl lysinyl moiety, can act as an epitope to alter protein antigenicity. An in vitro system has been developed to produce halothane induced neoantigens and to study conditions for formation in the liver. Liver slices, capable of halothane biotransformation, provide a viable means for mechanistic studies. Liver slices (1 cm diameter, 300-mu-m thick) from male Hartley guinea pigs (600-800 g) were exposed to either 1.0 or 1.7 mM halothane (media concentration) in 95% O2/5% CO2 for 12 h. Covalent binding was determined using C-14-halothane. Neoantigens were detected by Western immunoblot analysis using rabbit anti-trifluoroacetylated albumin antiserum. Covalent binding was detected by 1 h of incubation and increased linearly through 12 h (20.7-48.5 nmole equiv/mg protein). Covalent binding preceded and correlated with the appearance of neoantigens. By 12 h of incubation, five neoantigens were seen with molecular weights ranging from 51 to 97 kD. These neoantigens have molecular weights similar to those seen in vivo. Liver slices exposed to deuterated halothane, which is oxidatively metabolized to a lower extent, did not develop neoantigens. This in vitro model system can be used to examine the mechanism for covalent binding and neoantigen production in the hepatocyte.