Toxicity and metabolism of sulphamethoxazole and its metabolites in keratinocytes

Chloée Nadeau, M. Rieder MD Ph.D, Dave Freeman Ph.D. Depts. of Paediatrics and Physiology and Pharmacology, University of Western Ontario, and Robarts Research Institute, London, ON, Canada.

Cutaneous drug reactions (CDRs) are common adverse drug reactions (ADRs), occurring in up to 30% of all documented cases. 4% of patients receiving sulfamethoxazole (SMX) have CDRs,, which rises in HIV positive patients (40% to 60%) (Naldi et al. 1999; Svensson et al. 2001; Masters et al. 2003) . SMX is responsible for most of the CDRs and its effect in keratinocytes (Kcs) is poorly understood. The aims of this study are to determine the toxicity of SMX and its metabolite sulphamethoxazole-hydroxylamine (SMX-HA) in HaCaT cells, an adult keratinocyte cell line, and to quantitate the amount of SMX metabolite produced in HaCaT cells and to verify protein-hapten formation with SMX, SMX-HA and sulphamethoxazole-nitroso (SMX-NO).

HaCaT cells were incubated with increased concentration of SMX (0-3200µM) and SMX-HA (0-800µM) over increasing incubation time. After incubation, cellular viability was determined by using a MTT cytotoxicity assay. Also, HaCaT cells were incubated for 0-24 hours with SMX (0-1500 µM). Then, SMX metabolite production, acetyl-sulphamethoxazole (AC-SMX), was measured by using HPLC analysis. Haptenation assay was performed by incubating HaCaT cells with 100 µM of SMX, SMX-HA or SMX-NO for 6 hours. A second haptenation assay was performed by incubating SMX 800 µM, with or without pre-incubation with DEM, for 24. Cells were harvested and lysed and SMX-protein haptenation was determined using western blot analysis.

The HaCaT cells cytotoxicity assays demonstrate concentration-dependent and time-dependent decline in cell viability with SMX-HA. At 800 µM, the maximum cell viability is 30.02% ± 3.05%. With SMX, there is a very small decline of cell viability at high SMX concentration. HPLC analysis demonstrates that HaCaT cells produce AC-SMX (1%). Western blot analysis showed significant haptenation of proteins with SMX-HA and SMX-NO (bands between 30 to 125kDa), but not with SMX. Also, HaCaT cells pre-treated with DEM and then treated with SMX 800uM, demonstrate a specific hapten-protein formation band at 35kDa.

In conclusion, these data suggest that keratinocytes are sensitive to reactive-metabolite mediatedcytotoxicity. This may be important in the pathogenesis of CDRs. This data also suggests that keratinocytes metabolize SMX to AC-SMX metabolites under pharmacological concentration. Also, the production of AC-SMX in this adult keratinocyte cell line appears to be 1000 time higher than previous work published using neonatal keratinocytes (Reilly et al. 2000). Western blot analysis demonstrates total GSH reduction can produce hapten-protein formation with SMX in HaCaT cells. The involvement of SMX-HA and SMX-NO protein haptenation in CDRs remains to be clarified.

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Svensson et al. (2001). Pharmacol Rev 53(3): 357-79.