Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothane.
The anesthetic halothane can be bioactivated to the reactive intermediate, trifluoroacetyl chloride, which can covalently bind to liver protein. The product of this reaction is trifluoroacetyl-N-epsilon-lysine which can act as a foreign epitope in altering both protein immunogenicity and antigenicity. An in vitro liver slice system was used to study the formation of protein adducts following exposure to halothane. Liver slices (30-35 mg wet weight, 250-300 microns thick) from adult male Hartley guinea pigs (600-800 g) were exposed to [14C]halothane (0.6-0.9 microCi, 1.0-1.7 mM) in 95% O2/5% CO2 for 1, 6 and 12 h. The slices were homogenized and subcellular fractions prepared. Proteins were resolved by electrophoresis and bound radioactivity was detected by scintillation counting and autoradiography. Greater than 80% of detectable radioactivity to whole liver cell protein was localized in the 20-30-kDa range and increased in a linear fashion over the 12-h incubation period. Covalent binding was localized to two proteins of 27 kDa and 26 kDa present in the cytosolic compartment. Purification followed by N-terminal amino acid sequence analysis of the 27-kDa protein has identified it to be homologous with glutathione-S-transferase b. This cytosolic protein appears to be the major target for trifluoroacetylation in liver slices exposed to halothane.
Brown
A P
AP
Department of Anesthesiology, University of Arizona, Tucson 85724.
Hastings
K L
KL
Gandolfi
A J
AJ
Liebler
D C
DC
Brendel
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D046508:Culture Techniques; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006168:Guinea Pigs; D006221:Halothane / Q000494:pharmacology*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D011506:Proteins / Q000302:isolation & purification*
D011506:Proteins; D005982:Glutathione Transferase; D006221:Halothane