Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection.
Potentially serious idiosyncratic reactions associated with sulfamethoxazole (SMX) include systemic hypersensitivity reactions and hepatotoxicity. Covalent binding of SMX to proteins subsequent to its N-hydroxylation to form N4-hydroxysulfamethoxazole (SMX-HA) is thought to be involved in the pathogenesis of these reactions. A polyclonal antibody was elicited in rabbits against a SMX--keyhole limpet hemocyanin conjugate that recognized covalent protein adducts of SMX in microsomal protein and was used to characterize the covalent binding of SMX and its putative reactive metabolites to hepatic protein in vivo and in vitro. In vitro covalent binding of SMX to rat and human liver microsomal protein was NADPH-dependent, while binding of SMX-HA was not dependent on NADPH. SMX and SMX-HA produced similar patterns of covalent binding, with major protein targets in the region of 150, 100 (two bands), 70 (two bands), and 45-55 kDa. The pattern of covalent binding to human and rat liver microsomal protein was similar. Binding of SMX-HA was completely eliminated by GSH or by addition of cytosolic fractions and acetylcoenzyme A. The acetoxy metabolite of SMX also led to covalent binding, but it was primarily attributable to the formation of SMX-HA from acetoxySMX. In vivo exposure of rats to SMX did not result in detectable covalent binding by the methods employed. When rat liver slices were incubated with 2 mM SMX or 500 microM SMX-HA, no toxicity was observed and yet covalent binding of SMX-HA to 130, 100, 70, and 55 kDa proteins could be detected. These results confirm that covalent binding of SMX occurs via the formation of SMX-HA and that covalent binding of SMX-HA in vitro results from its conversion to the more reactive nitroso metabolite. Acetylation of SMX-HA protected against its covalent binding. Further studies are required to determine how this in vitro covalent binding relates to in vivo covalent binding in humans and to either direct or immune-mediated cytotoxicity in SMX idiosyncratic drug reactions.
Cribb
A E
AE
Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Nuss
C E
CE
Alberts
D W
DW
Lamphere
D B
DB
Grant
D M
DM
Grossman
S J
SJ
Spielberg
S P
SP
D000818:Animals; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D009249:NADP / Q000502:physiology; D011485:Protein Binding / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013420:Sulfamethoxazole / Q000031:analogs & derivatives / Q000378:metabolism*
D009249:NADP; D013420:Sulfamethoxazole