Metabolic activation of halothane to neoantigens in C57Bl/10 mice: immunochemical studies.
C57Bl/10 mice were given halothane (10 mmol/kg, intraperitoneally) and microsomal proteins were analysed for the presence of trifluoroacetylated (TFA) neoantigens by SDS-gel electrophoresis followed by immunoblotting using a polyclonal anti-TFA antibody. In microsomal preparations from liver, lung and olfactory tissues, a 54 kDa TFA adduct was detectable 1 h after dosing. After 3-48 h, multiple bands were detected in liver (45-100 kDa) and in the lung (26-57 kDa) and in one experiment in which [14C]halothane was given, several immunoreactive bands from liver microsomes were shown to contain a covalently bound metabolite of the drug. In olfactory tissue, initially (1 h), a major band of 54 kDa and a less prominent component of about 50 kDa were seen. The number of bands increased at later times but the additional bands were far fewer than in liver. The rate of decay of the 54 kDa adduct was also measured in both liver and olfactory microsomes and found to be compatible with the reported turnover of total liver cytochrome P-450. 24 h after treating mice with halothane (10 mmol/kg), no TFA neoantigens could be detected on the outer cell surface of isolated viable hepatocytes when analysed by fluorescence activated flow cytometry. In contrast, non-viable cells, or those fixed in acetone were all positive. Using immunohistochemistry, TFA neoantigens were demonstrated in the centrilobular area of the liver, the non-ciliated bronchiolar epithelial (Clara) cells of the lung, proximal tubular cells of the kidney and the respiratory and olfactory epithelium of nasal tissues.
Heijink
E
E
MRC Toxicology Unit, Carshalton, Surrey, UK.
De Matteis
F
F
Gibbs
A H
AH
Davies
A
A
White
I N
IN
D000818:Animals; D000941:Antigens / Q000032:analysis / Q000378:metabolism; D001711:Biotransformation; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005453:Fluorescence; D006221:Halothane / Q000276:immunology / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000166:cytology; D008168:Lung / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000276:immunology / Q000378:metabolism; D008862:Microsomes, Liver / Q000276:immunology / Q000378:metabolism; D009831:Olfactory Mucosa / Q000378:metabolism
D000941:Antigens; D006221:Halothane