Reactive oxygen species are increased in cells incubated with reactive sulphamethoxazole derivatives

Kim Chau2,3, M. Jane Tucker1,3, Michael Rieder1,2,3,4. Departments Paediatrics1, Physiology & Pharmacology2, University of Western Ontario3 and John P. Robarts Research Institute4.

Adverse drug reactions (ADRs) are an important cause of morbidity and mortality. ADRs occur frequently during therapy with sulphamethoxazole (SMX) (2-5% of the general population, 40-60% of HIV infected individuals) (Gordin et al., 1984) . Metabolism of sulphamethoxazole is believed to be a critical determinant in the pathogenesis of serious ADRs, including severe hypersensitivity reactions. The molecular mechanism(s) for these reactions remains unclear (Rieder et al., 1988).

To study the mechanism of cytotoxicity induced by SMX metabolites and to determine the contribution of apoptosis in reactive-metabolite mediated cell death.

Jurkat E6.1 cells were incubated with 200 µM SMX or 0 - 800 µM sulphamethoxazole hydroxylamine (SMX-HA) for 0 - 2 hours. After incubation, cell viability was determined using MTT assay (Bellamy, 1992) and reactive oxygen species (ROS) formation determined using 2’,7’- dichlorfluorescein-diactate (DCFH-DA) (LeBel et al., 1992) . To study apoptosis, a nnexin V and 7-Amino Actinomycin D (7-AAD) were measured using FACS analysis (van Engeland et al., 1998) . Data are expressed as percentage of control cells. Significant differences were determined by one-way ANOVA with repeated measures and Dunnett’s multiple comparisons test.

A concentration- and time- dependent toxicity was demonstrated with SMX-HA at all time points, e.g., 47.8 ± 4.5% viability at 400 µM and 9.5 ± 3.5% at 800 µM at 2 hours (n=4, p<0.01). T he exposure of Jurkat cells to increasing concentrations of SMX-HA produced a similar formation of ROS, with a 450 ± 65- fold increase at 50 µM SMX-HA and 600 ± 83- fold at 800 µM SMX-HA compared to controls at 2 hours (n=5, p<0.05). Furthermore, Jurkat cells treated with SMX-HA show a similar increase in the percentage of cells staining positive for annexin V and 7AAD.

Jurkat E6.1 cells undergo SMX-HA-induced, but not SMX-induced cell death. This is consistent with the suggested role of reactive drug metabolites in hypersensitivity ADRs . The molecular pathway(s) of this effect is currently under study in our laboratory.

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Gordin, F. M., et al. (1984) Ann Intern Med 100, 495-499.
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Rieder, M. J., et al. (1988). J Pharmacol Exp Ther 244, 724-728.
van Engeland, M., et al. (1998). Cytometry 31, 1-9.