GLUTATHIONE-S-TRANSFERASE IS A TARGET FOR COVALENT MODIFICATION BY A HALOTHANE REACTIVE INTERMEDIATE IN THE GUINEA-PIG LIVER

The anesthetic halothane is bioactivated by the liver cytochrome P450 system to the reactive intermediate, trifluoroacetyl chloride, which c an acylate liver protein. Cytosolic glutathione-S-transferase (GST) wa s identified as a major target for protein adduct formation in guinea pig liver slices exposed to halothane. To determine if GST is also a t arget in vivo, male Hartley guinea pigs were exposed to 1% halothane i n 40% O-2 for 4 h. At 10 h post exposure, livers were removed and micr osomal and cytosolic fractions prepared. Past studies have shown these conditions resulted in maximal covalent binding of halothane intermed iates to hepatic protein. Protein was isolated by ethanol precipitatio n and washed with trichloroacetic acid to remove unbound metabolites. Cytosolic GST was isolated by gel filtration and S-hexylglutathione af finity chromatography to electrophoretic purity. Protein adducts were quantified using a covalently bound fluorine assay. Covalent binding o f a halothane intermediate to cytosolic and microsomal protein was det ermined as 2.0 +/- 0.4 and 13.2 +/- 2.3 nmol F/mg protein, respectivel y. Liver glutathione depletion by buthionine sulfoximine pretreatment produced an increase in covalent binding only to cytosolic proteins (3 .3 +/- 0.4 nmol F/mg protein). Adduct formation to cytosolic GST was d etermined to be 4.7 +/- 1.6 nmol F/mg protein. Glutathione-S-transfera se is a target for covalent modification in the liver following an inh alation exposure to halothane.