Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity

Abstract

1 Bioactivation of sulphamethoxazole (SMX) to chemically-reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro. 2 Flow cytometry revealed binding of N-hydroxy (SMX-NHOH) and nitroso (SMX-NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX-NO was reduced by exogenous glutathione (GSH). 3 SMX-NHOH and SMX-NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism. 4 Fluorescence HPLC showed that SMX-NHOH and SMX-NO depleted CYS and GSH in bu er, and to a lesser extent, in cells and plasma. 5 Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX-NHOH and SMX-NO than those inducing loss of membrane viability, with SMX having no e ect. Lymphocytes were signi®cantly (P50.05) more sensitive to the direct cytotoxic e ects of SMX-NO than neutrophils. 6 Partitioning of SMX-NHOH into red blood cells was signi®cantly (P50.05) lower than with the hydroxylamine of dapsone. 7 Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity.

Sulfamethoxazole and Its Metabolite Nitroso Sulfamethoxazole Stimulate Dendritic Cell Costimulatory Signaling

“…4C) and not observable immediately after incubation of SMX with cells. By contrast, SMX-NO bound rapidly to both intra-and extracellular proteins, consistent with previous work from several laboratories including ours (4,5,9,(13)(14)(15).…”

“…1) (3,12). SMX-NO can bind covalently to cysteine residues on cellular protein (4,(13)(14)(15)(16) and this binding, above a threshold, causes direct toxicity (5,16) but also provides an antigenic signal to SMX-NO-specific T cells (9,17). Furthermore, Ag-specific T cells have been isolated and cloned from hypersensitive patients and have been characterized in terms of their functionality and phenotype (6 -8, 18, 19).…”

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

“…Sulphamethoxazole hydroxylamine circulates in blood and tissues and is excreted in human (Gill et al, 1996;Mitra et al, 1996) and rat urine (Gill et al, 1997). 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.…”

“…Antigen formation on cell surfaces was determined by¯ow cytometry using a speci®c anti-sulphamethoxazole IgG antibody (1 : 500; 40 ml) and a FITC-conjugated anti-IgG secondary antibody (Naisbitt et al, 1999). Forward and side scatter were measured simultaneously and the FITC¯uores-cence of cells was acquired on¯uorescence channel FL1.…”

“…To measure proliferation, [ Cell surface antigen formation, within the proliferation assay, was also determined by¯ow cytometry using a speci®c antisulphamethoxazole antibody (Naisbitt et al, 1999).…”