Covalent Binding of Sulfamethoxazole Reactive Metabolites to Human and Rat Liver Subcellular Fractions Assessed by Immunochemical Detection

Abstract

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.

Pharmacogenetics of the arylamine N-acetyltransferases

“…In slow acetylators, a higher proportion of the drug is Nhydroxylated and consequently, these individuals are at a greater risk of sulphonamide-induced toxicity. [114][115][116] However, as pointed out by Spielberg, the incidence of severe adverse side effects to sulphonamides is much less than the incidence of the slow acetylator phenotype suggesting that other factors predispose individuals to idiosyncratic adverse reactions. 113 Risk of developing side effects, such as neurotoxicity or haemolytic anemia, to dapsone therapy is very similar to that described for the sulphonamides.…”

Antigenicity and immunogenicity of sulphamethoxazole: demonstration of metabolism‐dependent haptenation and T‐cell proliferation in vivo

“…Previous in vivo studies to characterize hapten formation in the liver were unsuccessful (Cribb et al, 1996). This may have been due to the very ecient detoxication mechanisms that are present in the liver, which would have precluded covalent binding (Gill et al, 1997;Cribb et al, 1995).…”

“…Further (auto)oxidation yields a nitroso compound (Cribb et al, 1991;Naisbitt et al, 1996) that can haptenate proteins, including the surface of viable lymphocytes (Naisbitt et al, 1999). These observations and identi®cation of (a) sulphamethoxazole-substituted hepatic proteins in vitro (Cribb et al, 1996), (b) sulphamethoxazole-substituted serum proteins and anti-SMX antibodies in patient sera (Meekins et al, 1994;Daftarian et al, 1995;Gruchalla et al, 1998), and (c) CD8 + dermal T-cells that proliferate in response to microsome-generated sulphamethoxazole metabolites (Hertl et al, 1995) are consistent with the involvement of both drug metabolism and the immune system in the pathogenesis of drug allergy.…”

Bioactivation, protein haptenation, and toxicity of sulfamethoxazole and dapsone in normal human dermal fibroblasts☆

“…Reactions to these drugs may range from a mild morbilliform reaction to more severe cutaneous involvement, such as Stevens Johnson Syndrome or toxic epidermal necrolysis (Cribb et al, 1996a;Svensson et al, 2001). Several of the sulfonamides associated with CDRs have been found to undergo bioactivation in the liver to reactive arylhydroxylamine metabolites (Cucinelli et al, 1972;Shear and Spielberg, 1985;Cribb and Spielberg, 1992;Cribb et al, 1995;Cribb et al, 1996b). Formation of these arylhydroxylamines may lead to covalent adduction with cellular proteins and cause cytotoxicity (Cribb et al, 1996b;Manchanda et al, 2002;Naisbitt et al, 2002).…”