title: Novel non-labile covalent binding of sulfamethoxazole reactive metabolites to cultured human lymphoid cells.
abstract: Sulfamethoxazole (SMX) causes rare hypersensitivity syndrome reactions characterized by fever and multi-organ toxicity. Covalent binding of SMX reactive metabolites to cellular proteins has been demonstrated but the link between cytotoxicity and targets of covalent binding has not been explored. We therefore investigated the relationship between covalent binding of the reactive SMX-hydroxylamine (SMX-HA) metabolite, and its cytotoxicity to a hystiocytic lymphoma (U937) cell line. Incubation of U937 cells with 0-1 mM SMX-HA for 3 h resulted in dose-dependent cytotoxicity, as assessed by tetrazolium dye conversion at 24 h. SMX-HA caused dose-dependent covalent binding to cellular proteins as assessed by immunoblotting with SMX antisera at 3 and 24 h. Covalent binding was predominantly to proteins of approximately 45, 59 and 75 kDa, but other targets were also observed. The relative extent of binding to proteins was significantly different from the relative cytotoxicity at 24 h. Further, cells surviving at 24 h also had extensive covalent binding. Covalent binding was observed under reducing (beta-mercaptoethanol) and non-reducing conditions to plasma membrane and microsomal but not cytosolic proteins. This non-labile covalent binding has not been previously reported. These observations suggest that extensive covalent binding does not necessarily lead to cell death, allowing the accumulation of potentially immunogenic drug-protein conjugates. These observations in whole cells may be relevant to the immunopathogenesis of SMX hypersensitivity syndrome reactions.
authors.affiliation: Laboratory of Comparative Pharmacogenetics, Atlantic Veterinary College, PEI Health Research Institute, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, Canada, C1A 4P3.
authors.forename: Mukesh
authors.initials: M
authors.lastname: Summan
authors.forename: Alastair E
authors.initials: AE
authors.lastname: Cribb
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000276:immunology / Q000493:pharmacokinetics* / Q000633:toxicity; D000906:Antibodies / Q000378:metabolism; D001711:Biotransformation; D015153:Blotting, Western; D003600:Cytosol / Q000276:immunology / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013420:Sulfamethoxazole / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism* / Q000493:pharmacokinetics* / Q000633:toxicity; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D020298:U937 Cells
chemical_list: D000890:Anti-Infective Agents; D000906:Antibodies; D013778:Tetrazolium Salts; D013844:Thiazoles; C059873:sulfamethoxazole hydroxylamine; C022616:thiazolyl blue; D013420:Sulfamethoxazole