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Sulfamethoxazole (SMX) is an effective drug for the management of opportunistic infections, but its use is limited by hypersensitivity reactions, particularly in HIV-infected patients. The oxidative metabolite SMX-nitroso (SMX-NO), is thought to be a proximate mediator of SMX hypersensitivity, and can be reduced in vitro by ascorbate or glutathione. Leukocytes from patients with SMX hypersensitivity show enhanced cytotoxicity from SMX metabolites in vitro; this finding has been attributed to a possible "detoxification defect" in some individuals. The purpose of this study was to determine whether variability in endogenous ascorbate or glutathione could be associated with individual differences in SMX-NO cytotoxicity. Thirty HIV-positive patients and 23 healthy control subjects were studied. Both antioxidants were significantly correlated with the reduction of SMX-NO to its hydroxylamine, SMX-HA, by mononuclear leukocytes, and both were linearly depleted during reduction. Controlled ascorbate supplementation in three healthy subjects increased leukocyte ascorbate with no change in glutathione, and significantly enhanced SMX-NO reduction. Ascorbate supplementation also decreased SMX-NO cytotoxicity compared to pre-supplementation values. Rapid reduction of SMX-NO to SMX-HA was associated with enhanced direct cytotoxicity from SMX-NO. When forward oxidation of SMX-HA back to SMX-NO was driven by the superoxide dismutase mimetic, Tempol, SMX-NO cytotoxicity was increased, without enhancement of adduct formation. This suggests that SMX-NO cytotoxicity may be mediated, at least in part, by redox cycling between SMX-HA and SMX-NO. Overall, these data indicate that endogenous ascorbate and glutathione are important for the intracellular reduction of SMX-NO, a proposed mediator of SMX hypersensitivity, and that redox cycling of SMX-HA to SMX-NO may contribute to the cytotoxicity of these metabolites in vitro.

Sulphonamides, such as sulphamethoxazole (SMX) and the related sulphone dapsone (DDS), show a higher incidence of cutaneous drug reactions (CDRs) in patients with the acquired immunodeficiency syndrome (AIDS) compared with human immunodeficiency virus (HIV) negative patients. During HIV infection, pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are increased. We hypothesized that this increase in pro-inflammatory cytokines may increase the toxicity of the arylhydroxylamine metabolites of SMX (S-NOH) and DDS (D-NOH) in keratinocytes through a reduction in glutathione (GSH) content. We evaluated the effect of TNF-alpha on GSH levels in normal human epidermal keratinocytes (NHEK) and found a significant decrease in GSH after 24h. Pre-treatment with TNF-alpha also resulted in an increase in the recovery of D-NOH, but failed to alter drug-protein covalent adduct formation in NHEK. We also evaluated the effect of TNF-alpha, IL-1 beta, interferon-gamma (IFN-gamma), lipopolysaccharide (LPS) and conditioned media (obtained from monocytes stimulated with LPS) on the cytotoxicity of pre-formed arylhydroxylamine metabolites in NHEK. Priming cells with cytokines did not significantly alter the cytotoxicity of the metabolites. The effect of pre-treatment with TNF-alpha on reactive oxygen species (ROS) generation in NHEK was also determined. While ROS formation in NHEK was increased in the presence of D-NOH, TNF-alpha did not alter the level of ROS generation. Our data suggest that the level of GSH reduction induced by pro-inflammatory cytokines does not predispose NHEK to cellular toxicity from either S-NOH or D-NOH.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated in humans with hypersensitivity reactions, to include fever, skin eruptions, hepatotoxicity, and blood dyscrasias. These reactions also occur in dogs, the only non-human species known to develop a similar spectrum of sulfonamide hypersensitivity. Sulfonamide hypersensitivity is not well understood, but has been hypothesized to be due to the generation of the reactive oxidative metabolite, nitroso sulfamethoxazole (SMX-NO). SMX-NO, unlike the parent sulfonamide, is cytotoxic in vitro, haptenizes tissue proteins, and is immunogenic in rodents. The purpose of this pilot study was to determine whether SMX-NO, when administered to dogs, would lead to drug-tissue adducts, anti-drug antibodies, antioxidant depletion, or clinical evidence of drug hypersensitivity. Four dogs were randomized to one of four treatments: SMX-NO 1 mg/kg; SMX-NO 3 mg/kg; SMX-NO 10 mg/kg; or vehicle control. Dosing was by the intraperitoneal route, once daily for four consecutive days per week, for 2 weeks total, followed by a third week of observation. Following this, all dogs were challenged with trimethoprim-sulfamethoxazole, 25 mg/kg for 12 h for 2 weeks. No dog developed clinical or biochemical evidence of drug hypersensitivity. Plasma cysteine and leukocyte reduced glutathione were not depleted during dosing; however, ascorbate was significantly depleted by week 2 following SMX-NO at 10 mg/kg. Anti-SMX antibodies (IgG or IgM by ELISA) were not detected in any dogs at any time points. SMX-hemoglobin adducts were detected in the spleen in SMX-NO dosed dogs; however, these adducts were not accompanied by an immunologic or systemic response. The results of this pilot study indicate that SMX-NO dosing in dogs, using a dosing protocol shown to be immunogenic in other species, produces modest ascorbate depletion and hemoglobin adduct formation, but is insufficient to produce an immunologic response or a clinical syndrome of sulfonamide hypersensitivity in this susceptible species.

Sulfamethoxazole hypersensitivity may be caused by production of the protein-reactive metabolite nitroso sulfamethoxazole (SMX-NO) and interaction of SMX-NO with T-cells. We have characterised the nature of the immune response induced by administration of sulfamethoxazole, sulfamethoxazole metabolites and nitrosobenzene to BALB/c mice. Drugs were administered over a 13-day period to induce polarised cytokine secretion profiles. Proliferation was measured by [(3)H] thymidine incorporation. Cytokine secretion was monitored by ELISA. Results were compared with those provoked by exposure to type 1 and type 2 chemical allergens, 2,4-dinitrochlorobenzene (DNCB) and trimellitic anhydride (TMA). CD4(+) or CD8(+) T-cells were depleted ex vivo to identify the primary source of cytokines. Lymph node activation was observed following treatment with DNCB, TMA, nitrosobenzene and SMX-NO, but not with sulfamethoxazole or sulfamethoxazole hydroxylamine (SMX-NHOH). DNCB and TMA induced type 1 and type 2 cytokine profiles, respectively. SMX-NO treatment stimulated the production of high levels of IL-5, variable amounts of IFN-gamma, and relatively low levels of IL-10 and IL-4. Nitrosobenzene-activated lymph node cells secreted only low levels of IFN-gamma and IL-5. Depletion of CD4(+) or CD8(+) T-cells from SMX-NO stimulated lymph node cells revealed that CD4(+) T-cells were the major source of IL-5. In conclusion, the data presented indicates that subcutaneous administration to mice of SMX-NO, but not the parent drug, stimulated the secretion of high levels of IL-5 from activated CD4(+) T-cells, which is consistent with the clinical profile of the drug.

The popliteal lymph node (PLN) assay has been proposed as a tool to predict drugs and chemicals with the potential to induce systemic autoimmune reactions in man. In this assay, weight and cellularity indices typically are the measured endpoints. The present study was conducted to test whether incorporation of tritiated thymidine could improve sensitivity of the PLN assay. Male and female Balb/c mice were injected with 20 microCi of [3H]-methyl-thymidine intravenously 7 days after receiving 0.5, 1 or 2 mg of diphenylhydantoin, streptozotocin, sulfamethoxazole, ofloxacin, phenobarbital, or metformin intradermally. Results obtained with incorporation of tritiated thymidine were compared to weight indices. No consistent or marked differences in these endpoints were noted whatever the compound used. This study shows that incorporation of tritiated thymidine does not improve sensitivity of the PLN assay.

It has been suggested, on the basis of work in cell-free systems, that sulfonamide hydroxy-lamines are metabolized to nitroso metabolites which may be the proximate toxins mediating sulfonamide hypersensitivity reactions. We performed time-course experiments investigating the toxicity of the hydroxylamine and nitroso derivatives of sulfamethoxazole to investigate this hypothesis. The nitroso derivative of sulfamethoxazole was significantly more toxic than the hydroxylamine derivative (P < 0.05). When the LC50 was compared over time, there was a significant decrease in the LC50 of the hydroxylamine of sulfamethoxazole over time, while there was no change in the LC50 of the nitroso derivative. There was an equivalent reduction in toxicity demonstrated when the hydroxylamine or nitroso derivatives were co-incubated with glutathione. This supports the role of the nitroso as a proximate toxin mediating sulfonamide hypersensitivity reactions and suggests an explanation for the high rate of adverse reactions to sulfonamides among patients with AIDS.