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 can acylate liver protein. Cytosolic glutathione-S-transferase (GST) was identified as a major target for protein adduct formation in guinea pig liver slices exposed to halothane. To determine if GST is also a target in vivo, male Hartley guinea pigs were exposed to 1% halothane in 40% O2 for 4 h. At 10 h post exposure, livers were removed and microsomal and cytosolic fractions prepared. Past studies have shown these conditions resulted in maximal covalent binding of halothane intermediates to hepatic protein. Protein was isolated by ethanol precipitation and washed with trichloroacetic acid to remove unbound metabolites. Cytosolic GST was isolated by gel filtration and S-hexyl-glutathione affinity chromatography to electrophoretic purity. Protein adducts were quantified using a covalently bound fluorine assay. Covalent binding of a halothane intermediate to cytosolic and microsomal protein was determined as 2.0 +/- 0.4 and 13.2 +/- 2.3 nmol F/mg protein, respectively. 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 determined to be 4.7 +/- 1.6 nmol F/mg protein. Glutathione-S-transferase is a target for covalent modification in the liver following an inhalation exposure to halothane.
Brown
A P
AP
Department of Anesthesiology, College of Medicine, University of Arizona, Tucson 85724.
Gandolfi
A J
AJ
D000280:Administration, Inhalation; D000818:Animals; D001711:Biotransformation; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D005978:Glutathione / Q000502:physiology; D005982:Glutathione Transferase / Q000187:drug effects / Q000378:metabolism*; D006168:Guinea Pigs; D006221:Halothane / Q000378:metabolism* / Q000633:toxicity; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D011485:Protein Binding
D005982:Glutathione Transferase; D005978:Glutathione; D006221:Halothane