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(The FASEB Journal. 1999;13:1688-1698.)
© 1999 FASEB

Cytotoxicity of sulfonamide reactive metabolites: apoptosis and selective toxicity of CD8+ cells by the hydroxylamine of sulfamethoxazole

DAVID A. HESS*, MARGARET E. SISSON, HAMZA SURIA{dagger}, JOHN WIJSMAN, RAM PUVANESASINGHAM, JOAQUÍN MADRENAS{dagger} and MICHAEL J. RIEDER§1,2

* Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada;
{dagger} Departments of Medicine, Microbiology and Immunology, University of Western Ontario, Transplantation and Immunobiology Group, John P. Robarts Research Institute, London, Ontario, Canada; and
§ Departments of Pediatrics, Pharmacology and Toxicology, Children's Hospital of Western Ontario, University of Western Ontario, Gene Therapy and Molecular Virology Group, John P. Robarts Research Institute, London, Ontario, Canada

1Correspondence: Departments of Pediatrics and Pharmacology and Toxicology, Children's Hospital of Western Ontario, University of Western Ontario, 800 Commissioners Rd. E. London, Ontario, Canada, N6J 1Y5. E-mail: mrieder{at}julian.uwo.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment with sulfonamide antibiotics in HIV-infected patients is associated with a high incidence (> 40%) of adverse drug events, including severe hypersensitivity reactions. Sulfonamide reactive metabolites have been implicated in the pathogenesis of these adverse reactions. Sulfamethoxazole hydroxylamine (SMX-HA) induces lymphocyte toxicity and suppression of proliferation in vitro; the mechanism(s) of these immunomodulatory effects remain unknown. We investigated the cytotoxicity of SMX-HA via apoptosis on human peripheral blood mononuclear cells and purified cell subpopulations in vitro. CD19+, CD4+, and CD8+ cells were isolated from human peripheral blood by positive selection of cell surface molecules by magnetic bead separation. SMX-HA induced significant CD8+ cell death (67 ± 7%) at 100 µM SMX-HA, with only minimal CD4+ cell death (8 ± 4%). No significant subpopulation toxicity was shown when incubated with parent drug (SMX). Flow cytometry measuring phosphatidylserine externalization 24 h after treatment with 100 µM and 400 µM SMX-HA revealed 14.1 ± 0.7% and 25.6 ± 4.2% annexin-positive cells, respectively, compared to 3.7 ± 1.2% in control PBMCs treated with 400 µM SMX. Internucleosomal DNA fragmentation was observed in quiescent and stimulated PBMCs 48 h after incubation with SMX-HA. Our data show that CD8+ cells are highly susceptible to the toxic effects of SMX-HA through enhanced cell death by apoptosis.—Hess, D. A., Sisson, M. E., Suria, H., Wijsman, J., Puvanesasingham, R., Madrenas, J., Rieder, M. J. Cytotoxicity of sulfonamide reactive metabolites: apoptosis and selective toxicity of CD8+ cells by the hydroxylamine of sulfamethoxazole.


Key Words: cytochrome P450 • MACS • apoptosis • cell viability


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SULFONAMIDE ANTIBIOTICS ARE commonly used for the therapy of infections in transplantation and for AIDS-related complications (1 , 2) . Sulfonamide use for treatment of opportunistic infections in patients with AIDS is associated with increased rates of adverse reactions (> 40%), which has provoked interest in the pathogenesis of these potentially life-threatening reactions (1 , 2) Severe adverse reactions to sulfonamides appear to involve the initial production of sulfonamide reactive metabolites, which arise in vivo via the cytochrome P450 (CYP)3 mono-oxygenase system, primarily CYP 2C9 (Fig. 1 ) (3 , 4) . The pathophysiology of these events is complex, with genetic and metabolic factors contributing to the development of adverse drug reactions. Factors leading to a relative increase in the amount of reactive metabolite produced include oxidative P450 enzyme induction (4 5 6 7) , oxidative metabolism of drug by monocytes and neutrophils (6) , and N-acetyl transferase slow acetylation phenotype leading to reduced excretion of parent compound (7) . Factors decreasing cells ability to detoxify the metabolite include disease or metabolite-induced cellular glutathione deficiency and genetic glutathione synthase deficiency (8 9 10) . Disease factors in patients with AIDS such as viral load, concurrent medications, and disease progression may also increase cell sensitivity to reactive metabolites (11 , 12) .



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  		Figure 1. Proposed mechanism of sulfonamide adverse reactions. The majority of an SMX dose is conjugated by N-acetyltransferase and subsequently excreted in the urine. Under oxidative conditions, SMX is bioactivated to the chemically active hydroxylamine metabolite (SMX-HA), primarily by cytochrome P450 2C9. Further oxidation of SMX-HA to the nitroso (SMX-NO) derivative can occur through spontaneous oxidation or by cytochrome P450 oxidation. The hydroxylamine and nitroso derivatives of SMX have been implicated in sulfonamide adverse drug reactions and induce immune cell cytotoxicity, impaired proliferation, and function.

Subsequent steps in the pathogenesis of these adverse drug reactions after bioactivation appear to be mediated by the immune system (1 , 13) . Our laboratory previously isolated and synthesized the hydroxylamine metabolite of sulfamethoxazole (SMX-HA) (11) . SMX-HA has been demonstrated to be an important component in sulfonamide hypersensitivity reactions and has been implicated in sulfonamide-mediated immunomodulatory effects (11 , 14) . SMX-HA produces concentration-dependent toxicity when incubated with peripheral blood mononuclear cells (PBMCs) (10) and decreases T cell proliferation induced by phytohemagglutinin (PHA) and phorbol myristate acetate (PMA) (15 , 16) . This inhibition was significant at concentrations of hydroxylamine that were not associated with a decrease in cell viability. SMX-HA has also been shown to suppress pokeweed mitogen-driven human immunoglobulin M and immunoglobulin G production in vitro (17) . The mechanism of cytotoxicity and immune suppression for both cellular and humoral responses remains unknown.

Evaluation of idiosyncratic drug reactions in predisposed individuals is limited by ethical concerns arising from rechallenge with the suspected offending agent. Early studies investigated the toxic effect of aromatic amine metabolites on PBMCs using microsomal drug metabolizing systems in vitro (18) . Chemical synthesis of hydroxylamine and nitroso derivatives of sulfamethoxazole (Fig. 1) led to the development of in vitro `rechallenge' assays to diagnose sulfonamide hypersensitivity on the basis of increased lymphocyte sensitivity to reactive metabolites (11 , 19) . Increased sensitivity to SMX-HA was also observed in PBMCs and cell lines infected with HIV (12) . Time course toxicity studies of the hydroxylamine and nitroso derivatives of sulfamethoxazole have shown that the nitroso metabolite was significantly more toxic than the hydroxylamine derivative immediately after metabolite exposure, whereas the hydroxylamine metabolite exposure increased cell death over a 48 h period (20) . These findings suggested that nitroso metabolites induced cell death primarily by necrosis occurring within 4 h of exposure. Cell deletion via necrotic and/or apoptotic mechanism(s) cannot be ruled out by the time course toxicity for the hydroxylamine metabolite of sulfamethoxazole (20) . The aim of this study was to further characterize the cytotoxicity produced by sulfonamide hydroxylamine metabolites, specifically with respect to cellular subpopulation targets, and the mechanism(s) of cell death via apoptosis and/or necrosis.

Cell death can occur by one of two distinct mechanisms: necrosis or apoptosis. Necrosis occurs when cells are exposed to extreme variances from physiological conditions (e.g., hypothermia, hypoxia, ischemia, etc.), which result in damage to the plasma membrane (21) . Serious physical or chemical insult may result in an impairment of the cell's ability to maintain homeostasis, and an influx of water and extracellular ions leads to cell/organelle swelling and complete lysis (22) . This process requires no energy investment by the cell and is often associated with the release of lysosomal enzymes and random DNA digestion. In vivo, necrotic cell death is often associated with extensive damage to contiguous cells, resulting in an intense inflammatory response (23) . Apoptosis represents an ATP-dependent, tightly regulated mode of cell death characterized by chromatin segregation, cell shrinkage, cytoplasmic condensation, membrane blebbing, and formation of membrane bound apoptotic bodies (24 , 25) . Biochemical changes characteristic of apoptosis include externalization of membrane phosphatidylserine (PS), the activation of intracellular cysteine proteases (caspases), and alterations in mitochondrial membrane permeability resulting in the release of cytochrome c and other protein factors into the cytoplasm (25 26 27 28 29) . The biochemical hallmark of apoptosis is the internucleosomal fragmentation of the genomic DNA, an irreversible event that commits the cell to die and occurs before changes in plasma membrane permeability (25 , 30 , 31) . Physiological stimuli (CD95/Fas ligand system for CTL-mediated killing) (32 , 33) , many exogenous compounds (dexamethazone, methotrexate) (34 , 35) , and biological agents (chemokines, cytokines) (21 , 22) can induce or inhibit apoptosis in T lymphocytes in vitro. Apoptosis in vivo is normally not associated with an inflammatory response since compartmentalized cell constituents are immediately engulfed by phagocytes and macrophages (36) . Lymphocytes exposed to an increase in the concentration of cytotoxic agents in vitro may change the mode of cell death from apoptosis to necrosis (37) . In addition, a decrease in intracellular levels of glutathione markedly enhances the cytotoxicity of alkylating agents, with the mode of cell death switching from apoptosis to necrosis (23) . Little is known about the concentration- or GSH-dependent induction of apoptosis vs. necrosis in human lymphocytes by reactive drug metabolites produced by sulfonamide metabolism in vivo.

PS is a negatively charged phospholipid located predominantly in the inner leaflet of the viable cell plasma membrane (38) . In 1992, Fadok and co-workers (39) discovered that cells undergoing apoptosis expose PS in the outer membrane leaflet while retaining membrane integrity. During necrosis, cell membrane integrity is lost in the absence of PS switching. Thus, PS externalization is considered an early detection marker for apoptosis (40) . Annexin-V binds preferentially to phospholipid species such as PS and shows minimal binding to other membrane constituents, such as phosphatidylcholine and sphingomyelin (31 , 41) . Measurement of annexin-V binding in conjunction with a dye exclusion test to establish cell membrane integrity was used as a sensitive probe for PS exposure during the initial stages of apoptosis (31 , 40 , 42) .

To further elucidate the immune effects of sulfonamide reactive metabolites we have characterized the cytotoxic effects of SMX-HA on purified subpopulations of human PBMCs, using a magnetic-activated cell sorter (MACS) for rapid and accurate purification of lymphocyte subpopulations (43 , 44) . Cells were purified by positive selection for the subtype specific surface receptors: CD3 (T lymphocytes), CD4, CD8, or CD19 (B-lymphocytes) (45 46 47) . Separating PBMCs into subtypes and elucidation of a cytotoxic mechanism will determine the specific cellular target(s) of sulfonamide reactive metabolites and provide insight into the role of injured or expiring cells in the propagation of hypersensitivity reactions in response to initial oxidative insult.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
PBMC preparation
Venous blood from healthy volunteers was diluted with an equal volume of HEPES buffered saline, layered on Hypaque-Ficoll (specific gravity 1.077; Sigma Chemical Co., St. Louis, Mo.), and centrifuged for 30 min at 400 x g. The interface containing PBMCs was washed three times in HEPES buffer. PBMCs were resuspended in RPMI 1640 media (Gibco BRL, Burlington, Ontario, Canada) supplemented with 10% fetal calf serum (Whittaker, Bethesda, Md.), penicillin-streptomycin (100 IU/ml and 100 µg/ml, respectively; Gibco BRL), and 50 µM ß-mercaptoethanol (Sigma Chemical Co.). PBMCs were incubated at a density of 107 cells/ml in 150 mm diameter Falcon polystyrene petri dishes for 2 h at 37°C to remove plastic adherent cells. The remaining cells were then washed and resuspended in ice-cold HEPES buffer for subpopulation purification or treatment with SMX-HA, SMX, or dimethyl sulfoxide (DMSO) (Sigma Chemical Co.) vehicle controls, as described previously (10) .

Cell subpopulation isolation
PBMCs were subdivided into subpopulations using the characteristic cell surface receptors CD19, CD3, CD4, and CD8. The magnetic-activated cell separation system (MiniMACS) (Miltenyi Biotec Inc., Sunnyvale, Calif.) used in this investigation has been described in detail by Miltenyi et al. (46) . Briefly, cells were isolated through positive selection by antibody/magnetic bead binding to subtype-specific surface receptors. Passing the labeled cells through a coated stainless steel pellet-filled separation column under a magnetic field isolated the subpopulation of interest. Elution of the column in the absence of the magnetic field recovered a pure cell sample defined by the antibody used in the selection. PBMC subpopulations were checked for purity by fluorescence-activated cell sorter (FACS) analysis (> 98% CD3+; > 90% CD19+; > 95% CD4+; > 95% CD8+). Samples of CD8+ and CD4+ cell populations isolated by MiniMACS separation were also incubated with a monoclonal antibody recognizing CD3 (Immunotech, Marseille, France). FACS revealed that the fraction of cells CD4+/CD3+ or CD8+/CD3+ was ~88% or 80%, respectively. These data indicated that the majority of cells in the CD4 or CD8 isolated subpopulations were T helper lymphocytes or cytotoxic/suppressor T lymphocytes, respectively.

Cell viability assay
Cell samples from the PBMCs, CD3+, CD19+, CD4+, and CD8+ cell populations were incubated at 100,000 cells/well in triplicate with increasing concentrations (0–400 µM) of SMX or SMX-HA (Dalton Chemicals, Missassagua, Ontario, Canada) in flat-bottom, 96-well microtiter plates for 2 h at 37°C. SMX and SMX-HA were dissolved in < 1% DMSO final well concentration. DMSO control (< 1%) for all experiments was not associated with any cytotoxic effects. After incubation, the cells were washed three times by centrifugation in HEPES buffer. The samples were incubated for 18 h in RPMI 1640 at 37°C to allow for SMX-HA-mediated cell death to occur. Cell viability was quantified by 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy-fluorescein (Molecular Probes Inc., Eugene, Oreg.) staining of viable cells. Cell viability for each cell subpopulation was quantified by concentration fluorescence analysis on a Baxter fluorometer (Idexx Laboratories Inc., Westbrook, Maine) at 410 nm wavelength. Percent cell viability was calculated using the following formula:

Induction of apoptosis
Apoptosis was induced by exposing PBMCs to 15,000 µJ ionizing UV irradiation in a Stratagene (Stratagene, LaJolla, Calif.) (29) UV Stratalinker 1800 (annexin-V experiments only) or by PBMC incubation in the presence of 400 µM 5-azacytidine (Sigma Chemical Co.) (48 , 49) . Azacytidine is a poisonous nucleoside analog and inhibitor of DNA methylation that induces intranucleosomal DNA fragmentation and cell death via apoptosis (48 , 49) . The process is protein synthesis dependent and requires the up-regulation of p53. Experiments were performed in Falcon 6-well, flat-bottom plates with RPMI 1640 containing 10% fetal calf serum (Gibco BRL) for 24 or 48 h after drug treatment.

Cell staining and flow cytometry
We used bivariate flow cytometry and cell staining with fluorescein isothiocyanate- (FITC) labeled annexin-V (green fluorescence), simultaneously with propidium iodide (PI) stain (red fluorescence), to discriminate intact cells (annexin-/PI-) from apoptotic cells (annexin+/PI-) and necrotic cells (annexin+/PI+) after treatment with SMX-HA (40 , 41) . Annexin staining of apoptotic cells was performed using FITC-labeled annexin-V (Roche Diagnostics, Laval, Quebec, Canada). PBMCs (2 x 106) were washed (twice) using iced phosphate-buffered saline and incubated for 30 min in binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2), 1 µg/ml PI (Molecular Probes), and 1 µg/ml FITC-labeled annexin-V (42) . FACS analysis for annexin-V and PI staining was performed on a Becton Dickson FACScan (Becton Dickson, Mountainview, Calif.), with a minimum of 10,000 cells/sample analyzed. Data analysis was performed with CellQuest software (Becton Dickson) on a Power Macintosh 7600 (Apple Computer Inc., Cupertino, Calif.). Negative controls included unstained viable PBMCs, viable PBMCs stained with PI only, viable cells stained with annexin-V only, and viable cells stained with annexin-V and PI. Untreated cells displayed < 5% annexin+/PI- after 24 h and 6 h incubation.

DNA fragmentation assay
We used DNA fragment gel electrophoresis as a marker of end-stage apoptosis in order to confirm the induction and progression of apoptosis/necrosis by SMX-HA on human PBMCs, as described by Walker et al. (49) . Briefly, PBMCs (2 x 106) were treated with SMX (negative control), azacytidine (positive control), or SMX-HA (test samples) for 2 h, washed by centrifugation in HEPES buffer, and incubated (37°C at 5% CO2) for 24 or 48 h in RPMI 1640 with 10% fetal calf serum, with or without PHA (5 µg/ml) stimulation. Harvested cells were centrifuged, resuspended in 200 µl lysis buffer (1% sodium dodecyl sulfate, 100 mM NaCl 1 mM EDTA, and 1 M Tris-HCl, pH 7.5), and incubated for 10 min at room temperature. Each lysate was treated with 100 µg proteinase K (Bioshop Canada, Burlington, Ontario, Canada) for 2 h at 55°C, followed by treatment with 100 µg RNaseA (Bioshop Canada) for 2 h at 37°C. Genomic DNA was subsequently precipitated by adding 1 ml of 100% ice-cold ethanol and removed by swirling with a glass pipette. The remaining solution was centrifuged at 16,000 x g for 10 min at 4°C to pellet low molecular weight DNA fragments. Both genomic and fragmented DNA were resuspended in 20 µl of 10 mM Tris-HCl pH 8.5. DNA samples were mixed with 4 µl of 6x loading dye (MBI Fermentas, Flamborough, Ontario, Canada) and analyzed using a 1% agarose gel (Roche Diagnostics) prestained with 1 mM ethidium bromide (Sigma Chemical Co.). Each gel electrophoresis included a 1 kb DNA ladder (MBI Fermentas).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SMX-HA cytotoxicity of purified PBMC subgroups
Comparing the viability of the selected purified cellular subpopulations with the viability of PBMCs at the indicated concentrations (0–400 µM) (Fig. 2 A, B) allowed us to dissect the effect(s) of SMX-HA treatment on cellular toxicity. The parent drug, SMX, did not reduce the viability of any purified cell group or PBMCs; all cell types remained > 90% viable at concentrations of SMX up to 400 µM (Fig. 2A ). The reactive metabolite, SMX-HA, induced a strong, concentration-dependent decrease in cell viability for the PBMCs, CD3+, and CD8+ cellular subpopulations (~50–70% cell death at 100–400 µM SMX-HA) (Fig. 2B ). Comparatively, CD4+ cells displayed only a minimal loss in viability when incubated with SMX-HA (~15–20% cell death at 100–400 µM SMX-HA) (Fig. 2B ). The CD19+ B-cell subgroup showed an intermediate level of cytotoxicity with SMX-HA (~30–40% cell death at 100–400 µM SMX-HA) (Fig. 2B ). Therefore, the CD4+ cells appeared to be resistant to the toxic effects of SMX-HA in comparison to the other cell subpopulations studied. Cytotoxicity curves for the PBMCs, CD3+ and CD8+ subpopulations (Fig. 2B ), when treated with SMX-HA for 2 h showed no significant difference in cell viability between groups for the range of concentrations investigated. However, the CD4+ subpopulation exhibited a significant increase in viability when compared with PBMCs, CD3+, or CD8+ cells at concentrations of SMX-HA > 100 µM (P<0.05). It should be noted that SMX and SMX-HA were solubilized in DMSO, and DMSO treatment of cell populations showed no significant decrease in cell viability over untreated cell populations (data not shown).



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  		Figure 2. Reduced viability of PBMCs and purified cell subpopulations after exposure to SMX-HA. PBMCs ({blacksquare}), CD3+ cells ({diamondsuit}), CD19+ cells (•), CD4+ cells ({blacktriangleup}), and CD8+ cells (*) were incubated with 0–400 µM SMX (A) or 0–400 µM SMX-HA (B), and cell viability was measured 18 h after treatment. DMSO controls had no effect on the viability of any cell populations. Data represent the mean ± standard error of experiments performed in triplicate. Purified lymphocyte subpopulations were derived from the PBMCs of five healthy volunteers.

SMX-HA-induced membrane externalization of PS
Annexin-V-FITC staining of SMX-HA-treated human PBMCs revealed significantly increased detection of PS in the outer leaflet of the cellular plasma membrane. Figure 3 and Fig. 4illustrate characteristic samples of PS detection in the membrane of quiescent lymphocytes by FACS analysis, 6 and 24 h after SMX-HA exposure. Similar results were obtained from identical experiments performed on the PBMCs isolated from five control individuals. Vehicle (DMSO) and parent drug (SMX) did not induce increased levels of PS externalization or PI access to the cell interior when compared to untreated annexin-V-FITC- and PI-stained cells (Figs. 3 and 4) . Treatment of PBMCs with 15,000 µJ UV irradiation induced a significant increase in annexin+/PI- (lower right quadrant, representing intact apoptotic cells) and annexin+/PI+ (upper right quadrant, representing breached apoptotic or necrotic cells) populations at both 6 and 24 h (Figs. 3 and 4) . Quadrant statistics over the 24 h time course demonstrated increasing levels of cell death and PS externalization with increased incubation time. SMX treatment (400 µM) induced 4.3% and 4.9% apoptotic cells (annexin+/PI-) at 6 and 24 h, respectively. Untreated PBMCs from the same experiment showed background PS detection at 3.9% and 4.6% at 6 and 24 h, respectively (data not shown). These data suggested that both DMSO and SMX were unable to induce PS externalization above background levels. In contrast, SMX-HA treatment (100 µM) of PBMCs for 2 h induced a significant increase in the frequency of annexin+ cells, with 13.7% of cells annexin+/PI- and 4.5% of cells annexin+/PI+ at 6 h, and 15.5% of cells annexin+/PI- and 12.7% of cells annexin+/PI+ at 24 h (Figs. 3 and 4) . Figure 5 illustrates a statistically significant increase in the levels of PS externalization for 400 µM and 100 µM SMX-HA when compared with untreated, DMSO, or parent drug (SMX) controls (P<0.01).



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  		Figure 3. PS externalization in PBMCs exposed to SMX-HA after 6 h PBMCs incubated with 0–400 µM SMX-HA were stained with 1 µg/ml annexin-V-FITC and 1 µg/ml PI 6 h after treatment. FACS revealed annexin-V-labeled PS in the external leaflet of the plasma membrane (annexin+) or PI-labeled DNA in disrupted cells (PI+). Quadrant statistics for labeled cells are displayed for A) UV, B) DMSO, C) 400 µM SMX, D) 25 µM SMX-HA, E) 100 µM SMX-HA, F) 400 µM SMX-HA-treated cells. Increased annexin+/PI- cells were detected as SMX-HA concentration increased. SMX had no effect on PS externalization compared to untreated controls. Similar results were obtained from identical experiments performed on PBMCs from five individuals.



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  		Figure 4. PS externalization in PBMCs exposed to SMX-HA after 24 h PBMCs incubated with 0–400 µM SMX-HA were stained with 1 µg/ml annexin-V-FITC and 1 µg/ml PI 24 h after treatment. FACS revealed annexin-V-labeled PS in the external leaflet of the plasma membrane (annexin+) or PI-labeled DNA in disrupted cells (PI+). Quadrant statistics for labeled cells are displayed for A) UV, B) DMSO, C) 400 µM SMX, D) 25 µM SMX-HA, E) 100 µM SMX-HA, F) 400 µM SMX-Ha-treated cells. Increased annexin+ cells were detected as SMX-HA concentration increased. SMX had no effect on PS externalization compared to untreated controls. Similar results were obtained from identical experiments performed on the PBMCs from five individuals.



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  		Figure 5. Frequency of annexin+/PI- cells in PBMCs treated with SMX-HA. PBMCs were incubated with 0–400 µM SMX-HA and stained with 1 µg/ml annexin-V-FITC and 1 µg/ml PI 24 h after treatment. FACS analysis revealed annexin-V-labeled PS in the external leaflet of the plasma membrane (annexin+) or PI-labeled DNA in disrupted cells (PI+). Data represents the mean ± standard error of the frequency of annexin+/PI- cells (lower right quadrant) displayed on the PBMCs of five control volunteers. A significant increase in annexin+/PI- cells was observed at 100 µM and 400 µM SMX-HA compared to untreated PBMCs (P<0.01).

SMX-HA-induced DNA fragmentation
The biochemical hallmark of apoptosis is internucleosomal cleavage of the genomic DNA into 180–200 bp fragments, resulting in the characteristic DNA ladder formation by DNA gel electrophoresis (27 , 29 , 50) . DNA fragmentation analysis was performed on the DNA isolated from human PBMCs, both quiescent and PHA-stimulated, at 24 and 48 h after treatment with SMX-HA. SMX-HA did not induce observable levels of DNA fragmentation at 24 h in either unstimulated or PHA-stimulated cell populations (data not shown). At 48 h, DNA fragmentation banding patterns were observed at 400 µM SMX-HA for quiescent PBMCs and PHA-stimulated PBMCs (Fig. 6 ). Lower concentrations of SMX-HA (100 µM or 25 µM) did not induce DNA fragmentation in PBMCs at 48 h post-treatment. The data displayed in Fig. 6 are representative of similar experiments performed on the PBMCs of three healthy volunteers. Parent drug SMX and lower concentrations of SMX-HA (100 µM and 25 µM) did not induce observable DNA fragmentation.



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  		Figure 6. Internucleosomal DNA fragmentation induced by SMX-HA. SMX-HA-treated PBMCs were stimulated with 5 µg/ml PHA and incubated for 48 h Cells were lysed, treated with 100 µg proteinase K and 100 µg RNase A, and the genomic DNA was precipitated with pure ethanol. Low molecular weight DNA fragments were separated by agarose gel electrophoresis. Lane 1, DNA ladder; lane 2, vehicle control (DMSO); lane 3, parent drug (SMX); lanes 4, 5-azacytidine; lane 5, 400 µM SMX-HA. DNA fragmentation was observed in 5-azacytidine-treated PBMCs (lane 4) and in 400 µM SMX-HA (lane 5) -treated PBMCs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The use of drug therapy has been of benefit in reducing death and disability, but substantial increases in the incidence and severity of adverse drug reactions have resulted. Adverse reactions complicate the course of 3–5% of hospitalized patients treated with a drug (51 , 52) , increasing patient recovery time and the economic cost of therapy (53) . Sulfonamide hypersensitivity reactions include severe adverse events such as erythema multiforme or Stevens-Johnson syndrome, usually after 10 to 14 days of therapy (54 55 56) . The rates of adverse drug events to sulfonamide antibiotics are ~5% in the general population, and increase to a reported rate of > 40% in patients with AIDS (57 58 59 60) . These adverse events do not appear to be related to the antifolate activity of sulfonamides, which is responsible for the antibacterial activity of sulfonamides (10 , 55) .

Sulfonamides are aromatic amines and are metabolized primarily, but not exclusively, by N-acetyltransferase (9 , 61 , 62) . A fraction of the parent drug is available for oxidative metabolism and is readily converted in vivo to a reactive intermediate such as the hydroxylamine (Fig. 1) (4 , 5 , 10 , 63) . We have previously demonstrated that the initial events in sulfonamide hypersensitivity reactions appear to involve bioactivation of the drug (10 , 11) , and altered sulfonamide metabolism is implicated in the increased rate of adverse reactions to sulfonamides demonstrated among patients with AIDS (2 , 12 , 57 , 64) . The ongoing pathogenesis of sulfonamide hypersensitivity reactions after initial bioactivation appears to involve propagation by the immune system (15 16 17 , 65 , 66) , but the mechanistic details of how this occurs remain unclear.

Sulfonamide reactive metabolites are both cytotoxic and immunomodulatory (15 , 16 , 67) . Understanding the effect(s) of reactive sulfonamide metabolites with respect to specific cellular subtypes is important in understanding how immunity contributes in determining which patients tolerate therapy and which patients are at risk for adverse drug reactions. The studies described above have investigated the role of sulfonamide reactive metabolites as cytotoxic agents on purified PBMC subpopulations in vitro in order to better understand the role of specific cellular subtypes in drug metabolite-induced immune suppression and cytotoxicity. We also investigated the mechanism of cell death by SMX-HA with respect to the possible role of apoptosis as a primary mode of cell deletion in response to cell damage by sulfonamide reactive metabolites.

We have demonstrated that significant toxicity among B cells and T cells occurs at micromolar concentrations of SMX-HA. The loss of viability is most evident among CD8+ cells, whereas purified CD4+ cells seem to be more resistant to SMX-HA-induced cell death (Fig. 2B ) in vitro. The MiniMACS isolation procedure did not contribute additional cell death to SMX-HA-treated, magnetic bead-labeled cell populations, as evidenced by similar levels of cell death observed between unlabeled PBMCs and antibody-labeled CD3+ or CD8+ populations (44 , 46) . PBMC and CD3+ lymphocyte populations displayed nearly identical viability curves when compared with SMX-HA susceptible CD8+ cells (Fig. 2B ). This observation suggests that SMX-HA-resistant characteristics of CD4+ cells may not occur in mixed cell populations (for example, CD3+ or PBMCs). In fact, it appears that cell death may be enhanced in any population where CD8+ cells are present. It is possible that cell injury and toxicity to SMX-HA susceptible cells could cause damage to neighboring cells by the release of harmful cellular constituents. However, it is difficult to segregate SMX-HA-induced toxic events from cell-mediated toxic events in mixed cell populations, especially since CD4+ SMX-HA-resistant cells comprise ~30% of the PBMC population.

Subpopulation cytotoxicity in vitro presents at concentrations that would be predicted to occur in vivo under the conditions of high-dose therapy, particularly in regions of localized oxidative metabolism (12 , 20) . This suggests that at clinically relevant concentrations of reactive metabolites in genetically predisposed individuals, CD4+ cells remain viable and therefore would be available to alter normal immune responses, and thus propagate adverse reactions to sulfonamides in these individuals. Even at sublethal concentrations, SMX-HA is able to alter normal immune cell function, inhibiting PBMC proliferation in vitro (1 , 15) , reducing antibody production (17) , and interfering with T cell signaling and cytokine elaboration (65 , 68) . Abnormal lymphocyte function and cell-mediated toxicity appear to be implicated in the clinical representation of severe hypersensitivity reactions to sulfonamide therapy via immune cell targeting of SMX-HA haptenated proteins on the surface of cells (1 , 54 , 69) .

Differential toxicity of CD4+ vs. CD8+ subsets may have direct relevance to the increased incidence of adverse reactions to sulfonamides among patients with AIDS (1 , 12 , 60 , 70 , 71) . The depletion of CD4+ cells during the course of HIV infection may leave a greater proportion of circulating T cells as the more susceptible CD8+ subset. The mechanism(s) and clinical implications of differential lymphocyte subset toxicity by reactive drug metabolites in HIV-infected patients remains unclear; however, HIV-infected MOLT-3 cells and PBMCs isolated from patients with HIV infection are significantly more sensitive to the toxic effects of SMX-HA in vitro (12) .

CD4+ cells may also be better equipped to resist oxidative stress than CD8+ cells. We have previously demonstrated that thiols exert protective effects with respect to reactive sulfonamide metabolites by conjugation of the hydroxylamine metabolite and by preventing its further oxidation to the more toxic nitroso derivative (20) . It has been demonstrated that there are subset specific variations in cell surface thiol and intracellular glutathione expression, most notably in patients with AIDS (2 , 72) . The concentration of cell surface thiols is increased among CD4+ and CD19+ cells but not CD8+ cells in the setting of AIDS, suggesting that cellular thiol content and increased CD8+ susceptibility to reactive metabolites may be involved specifically in the increased rate of adverse drug reactions seen among patients with AIDS (72) .

The mechanism of cell death associated with reactive sulfonamide metabolites is unknown, but it has been speculated that necrosis is a primary mechanism of cell death, with apoptosis making a minor contribution (20) . To address this, we studied the development of apoptosis using two methods: annexin-FITC staining and DNA fragmentation. At 6 and 24 h after incubation, membrane changes representative of apoptosis were demonstrated in cells incubated with SMX-HA at concentrations of 100 µmol and greater (Figs. 4 and 5) . At 24 h, the number of annexin+/PI- cells was increased; there is also an observed increase in the number of annexin+/PI+ cells. These data suggest that SMX-HA-treated cells (as well as UV-treated cells, Figs. 3 and 4 ) are progressing through the active process of apoptosis with PS externalization of intact membranes, acting as an early indicator of the apoptotic process (31 , 40 , 41) . Higher frequencies of double-positive cells at the later time period suggest a progression of the early apoptotic cells culminating in the late morphological changes of apoptosis such as membrane blebbing and DNA fragmentation. There was no evidence of apoptosis demonstrated by DNA fragmentation at 24 h; however, DNA fragmentation was observed at 48 h after SMX-HA exposure (Fig. 6) . This indicated that the SMX-HA-induced apoptotic process occurred over 24–48 h in the majority of dying cells. No cell death or evidence of apoptosis was seen at 25 µmol or less SMX-HA, which is considered a sublethal concentration of SMX-HA, inducing less than 10% cell death in human PBMCs (10 , 16) . SMX-HA at these concentrations has been associated with immunosuppressive effects of mitogen-stimulated T cell proliferation (16) , illustrating the importance of separating the cytotoxic vs. immune modulating concentrations of SMX-HA in vivo.

We previously speculated that necrosis was the primary mechanism of cellular injury associated with sulfonamide reactive metabolites (20) , especially with the immediate form of cell death induced within 4 h by the nitroso metabolite. Our data suggest that the mechanism of cell death induced by SMX-HA may involve two distinctly different types of cellular injury, with apoptosis being more important as a mechanism of cellular death than was previously appreciated (20) . Low levels of cell death are seen after 4 h that may be due to necrosis, but it appears that the predominant mechanism of cell death after exposure to SMX-HA metabolites is apoptosis. With respect to the induction and propagation of adverse drug reactions to sulfonamide therapy, we speculate that the apoptotic (as opposed to the necrotic) mechanism of cell death may act as a protective mechanism causing the deletion of functionally modified, reactive metabolite damaged lymphocytes in a noninflammatory fashion. Future experimentation will focus on the mechanism of cell death induced in cells isolated from 1) patients with a history of ADRs to sulfonamides, and 2) patients with HIV infection in order to address whether the presence or absence of apoptosis correlates to the onset or severity of ADRs (73) .

In this study we have illustrated that hydroxylamine metabolite of sulfamethoxazole can induce apoptosis in human PBMCs in vitro. This is the first report of a reactive metabolite of an antimicrobial agent inducing apoptosis in human PBMCs. We have also demonstrated that CD8+ cells are far more susceptible to SMX-HA-induced cell toxicity in comparison to CD4+ cells. The detailed mechanism of hydroxylamine-mediated cellular toxicity and the molecular target(s) for reactive sulfonamide metabolites remain undetermined. There are a number of possible targets for reactive sulfonamide metabolites, which may include critical proteins on the cell surface or in the intracellular environment. Association of a reactive metabolite with macromolecules can produce adducts that may interfere with cell function through membrane damage, DNA damage, or alterations in key signal transduction pathways, culminating in apoptosis. We are currently investigating possible surface and intracellular target(s) for these metabolites. The potential impact of reactive metabolite haptenation in the setting of HIV infection also remains to be defined, and we are currently addressing this question.


   ACKNOWLEDGMENTS
 
The authors would like to acknowledge the contributions of Dr. M. Jane Tucker in the experimental design and review of this manuscript. This work was supported by a grant from the Medical Research Council of Canada and the National Health Research and Development Program-Health Canada.


   FOOTNOTES
 
2 Dr. M. J. Rieder holds a career award from the Medical Research Council of Canada.

3 Abbreviations: CYP, cytochrome P450; DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; LC50, lethal concentration in 50% of cells; MACS, magnetic-activated cell separation; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutanin; PI, propidium iodide; PMA, phorbol myristate acetate; PS, phosphatidylserine; SMX, sulfamethoxazole; SMX-HA, sulfamethoxazole hydroxylamine.

Received for publication January 29, 1999. Revised for publication April 19, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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