Introduction

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One of the major risks of pharmacotherapy is adverse drug reactions (Weinshilboum, 1987; Rieder, 1993). Adverse drug reactions are a significant cause of morbidity and mortality, and hinder effective therapy (Goldstein et al., 1984). Approximately 5% of patients develop adverse reactions during therapy, and 5 to 10% of patients develop adverse events during hospitalization (Bates et al., 1997; Rieder, 1997; Pirmohamed and Park, 1999). In the US, adverse drug reactions are believed to be the fourth most common cause of death (Pirmohamed and Park, 1999).

Predictable adverse drug reactions account for more than 80% of all reactions (Pirmohammed and Park, 1999). They are dose-dependent, can be anticipated from the drug's pharmacology, and resolve when dose is reduced. Unpredictable or idiosyncratic adverse drug reactions are less common but account for some of the most serious adverse events. These reactions are not related to the known pharmacology of the drug, do not show any simple dose-response relation, resolve only when treatment is discontinued, and even then can sometimes progress (Park et al., 1992). Clinical manifestations are remarkably diverse and include fever, skin rash, and multiorgan involvement (Park et al., 1987; Rieder, 1993, 1994; Pirmohamed et al., 1996). Although uncommon, many of these reactions are life threatening.

It has recently been appreciated that drug metabolism may be a key factor in the pathogenesis of many unpredictable drug hypersensitivities. Drug metabolism is usually thought of as generating polar, inactive metabolites that are readily excreted from the body. However, phase I enzymes, particularly isozymes of cytochrome P450, may bioactivate drugs to chemically reactive or toxic metabolites. The initial pathogenesis of idiosyncratic hypersensitivity reactions seems to involve bioactivation of parent drugs to reactive metabolites (Spielberg et al., 1981; Shear et al., 1985). The further propagation of these reactions seems to be mediated by the immune system (Hess and Rieder, 1997).

The clinical manifestations of an adverse event seem to be largely determined by the immune response generated (Hess and Rieder, 1997; Coleman, 1998). Most drugs and metabolites are less than 1000 Da and are conventionally not considered immunogenic. A hapten is a small-molecular-weight species that is immunogenic when conjugated to protein but not in free form. Our current belief is that drug hypersensitivity is largely based on hapten formation, in that drug-protein conjugates are recognized as an immunogen rather than the small drug molecules themselves (Coleman, 1998). Immune responses may be directed against the drug, part of the carrier molecule, or both. Conjugation can lead to formation of two distinct types of antigenic determinants, the hapten itself or a structurally modified carrier molecule. The nature of the interaction between antigen and immune system will determine the type of tissue injury observed (Park and Kitteringham, 1990; Bolzacchini et al., 1998).

A key characteristic for an agent to function as a hapten is the ability to form stable bonds with nucleophilic groups on proteins under aqueous conditions (Park et al., 1987). For most drugs, it is assumed that bioactivation of the drug to a chemically reactive metabolite, such as an epoxide, quinone, nitroso derivative, or acyl halide, acts as a hapten and is processed by antigen presenting cells as the ultimate immunogen (Park et al., 1995; Coleman, 1998; Coleman and Blanca, 1998). The specific immune response mounted is dependent on antigen processing and presentation. Presentation of a haptenated peptide can lead to an immune response in which the hapten is recognized as the major antigenic determinant or in which the peptide itself is the major determinant (Martin and Weltzien, 1994; Kalish, 1995; Weltzien et al., 1996).

The sulfonamides are antimicrobial agents associated with serious hypersensitivity adverse reactions (Shear et al., 1986; Rieder et al., 1989). Most of a sulfonamide dose ( 95%) is metabolized by N-acetyltransferase to a nontoxic acetylated conjugate (Cribb et al., 1995). A small fraction of a sulfonamide dose, however, is also metabolized to a reactive hydroxylamine metabolite, primarily by CYP2C9 (Cribb et al., 1995). Evolution of these reactions seems to be caused by the immune system (Hess and Rieder, 1997). Naisbitt et al. (1999) have demonstrated that reactive sulfonamide derivatives can bind to the surface of white blood cells. The subsequent fate of these bound derivatives and the role of haptens in the immune component of sulfonamide hypersensitivity reactions are poorly understood; this research was conducted to define hapten formation in response to sulfonamides and their reactive metabolites (Naisbitt et al., 1999).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

MOLT-3 Cell Line. MOLT-3 cells are a human, peripheral blood, T lymphoblast cell line maintained in suspension in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, 100 U/ml penicillin and 50 µM 2--mercaptoethanol at 37°C in a humidified atmosphere containing 5% CO₂.

HEPA 1C1C7 Cell Line. HEPA 1C1C7 cells are a mouse liver hepatoma cell line. Cultures were maintained in Dulbecco's modified Eagle's medium with high glucose (Invitrogen) supplemented with 10% fetal calf serum at 37°C in a humidified atmosphere containing 5% CO₂.

Purification of SMX-Keyhole Limpet Hemocyanin Immunization Rabbit Serum. Post SMX-keyhole limpet hemocyanin immunization rabbit serum samples were previously collected and frozen at 20°C. Rabbit serum samples were purified using a Protein G Sepharose 4 Fast Flow column (Pharmacia LKB, Uppsala, Sweden) to select only IgG and its subclasses from the crude serum. Rabbit serum (10 ml) was dialyzed overnight in 2 liters of PBS, pH 7.2, using Spectra/Por 6 and 7 Molecularporous dialysis membrane (1.8 cm/ml) (Spectrum, Dallas, TX). The protein G column was equilibrated by filling with binding buffer (0.2 M sodium phosphate, pH 7.0) and allowing column to drain. After immunization, rabbit serum was diluted 1:1 with binding buffer to ensure proper ionic strength and pH for optimal binding and was then placed in 5-ml aliquots over the equilibrated column, allowing the serum to absorb into the gel. Binding buffer (~30 ml) was passed through the column to wash away any unbound materials. Elution buffer (1.0 M glycine-HCl, pH 2.7) was then added to the column to elute the bound IgG. The antibody fractions were collected in 1-ml aliquots in Eppendorf tubes containing 100 µl of neutralization buffer (1.0 M Tris-HCl, pH 9.0) which allows for immediate renaturing of the purified sample. The absorbance of each of the 1-ml collection fractions was measured using Bio-Rad (Hercules, CA) protein estimation assay at a wavelength of 650 nm. Antibody collections of (10 mg/ml) were collected, dialyzed overnight in PBS, and stored at 20°C until required.

Protein Estimation. Protein concentrations were obtained using a Bio-Rad DC protein assay applying the following colorimetric microplate assay protocol. To obtain a standard curve dilutions of bovine serum albumin (BSA) protein standard were prepared ranging from 3.125 to 1000 µg/ml. Five microliters of standards and samples were pipetted into a clean, dry, microtiter plate followed by 25 µl of reagent A (alkaline copper tartrate solution) and 200 µl of reagent B (dilute Folin phenol reagent). The plate was gently agitated to mix the reagents and, after 15 min, absorbances were read at 650 nm using the plate-reader (Kinetic microplate reader; Molecular Devices, Sunnyvale, CA).

Analysis of Sulfa-Specific Cell Surface Protein Binding Using Flow Cytometry. MOLT-3 cells (2 × 10⁶) or HEPA 1C1C7 cells (70% confluent; 60-mm dish) were incubated with concentrations of SMX (Sigma-Aldrich, St. Louis, MO), SMX-HA (Rieder et al., 1988), or SMX-NO (Rieder et al., 1995) from 0 to 800 µM in HEPES for time points from 0 to 30 min at 37°C. After incubation, cells were thoroughly washed three times with ice-cold PBS to remove any unbound drug. All subsequent incubations were carried out at 4°C. Cells were then incubated with blocking solution (5% goat serum in PBS) for 15 min followed by rabbit anti-SMX IgG antibody (1:500 in blocking solution) for 1 h. After the incubation, cells were washed three times with cold PBS and incubated with human absorbed biotin-conjugated goat anti-rabbit antiserum (1:500 in PBS) for 1 h followed by washing twice in cold PBS and incubation with streptavidin-FITC (1:1000 in PBS) for 15 min. The MOLT-3 cells were washed once with cold PBS and resuspended in 400 µl of PBS before being transferred to Falcon flow cytometry tubes (Falcon Plastics, Oxnard, CA) containing 100 µl of 8% formaldehyde. Hepa 1C1C7 cells were further incubated in 400 µl of 5 mM EDTA in PBS for 10 min to remove cells from the plate, transferred to Falcon flow cytometry tubes containing 100 µl of 8% formaldehyde and analyzed on the FACscan for cell surface immunofluorescence. For each flow cytometry analysis, the control cells were 1) untreated, unlabeled; 2) untreated, fully labeled; 3) treated, unlabeled; 4) treated, 2° antibody and label only. Sulfa-specific binding was defined by the percentage increase in immunofluorescence compared with the untreated, fully labeled control. A control gate for viable cells using propidium iodide was used for each experiment. Each sample was analyzed in duplicate.

Analysis of Sulfa-Specific Intracellular and Cell Surface Protein Binding Using Fluorescent Microscopy. HEPA 1C1C7 cells were plated directly on glass slides (tissue culture chamber/slides; Lab-Tek, Naperville, IL), allowed to grow to 50 to 70% confluence, and then incubated with 25 µM SMX, SMX-HA, or SMX-NO in HEPES for 1 h at 37°C. Cells were washed with cold PBS and fixed with a methanol/acetone (1:1, v/v) solution for 5 min at 20°C. After fixation, cells were washed with TBS (PBS + 0.05% Tween 20) and permeabilized with 0.05% Triton X-100 in PBS. Cells were then incubated in blocking solution (1% BSA, 5% goat serum, 0.05% Triton X-100 in PBS) for 30 min followed by liver acetone powder absorbed rabbit anti-SMX IgG antibody (1:500) for 1 h at room temperature. After the incubation, cells were washed in TBS and incubated with FITC-goat anti-rabbit antibody (1:200) (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 30 min at room temperature. Cells were then washed with TBS and PBS before mounting with coverslips for microscopy. Fluorescence was visualized using a fluorescent microscope (IX50; Olympus, Tokyo, Japan) with 40× lens and Endow GFP (Chroma Technology Corp., Brattleboro, VT) filter set.

Analysis of Sulfa-Specific Intracellular and Cell Surface Protein Binding Using Confocal Microscopy. Hepa ICIC7 cells were cultured on coverslips in Petri dishes (60 mm) to 30% confluence and incubated with various concentrations of SMX, SMX-HA, or SMX-NO in HEPES for various time points (0-30 min) at 37°C. Cells were washed with cold PBS and fixed with a methanol/acetone (1:1, v/v) solution for 5 min at 20°C. To visualize cell surface binding alone, cells were washed three times with PBS and incubated in blocking solution (1% BSA, 5% goat serum in PBS) for 30 min followed by liver acetone powder-absorbed rabbit anti-SMX IgG antibody (1:500) for 1 h at room temperature. After the incubation, cells were washed three times in PBS and incubated with FITC-goat anti-rabbit antibody (1:200) for 30 min at room temperature. Cells were then washed with PBS before removing coverslips and mounting on slides for confocal microscopy. Staining for intracellular as well as cell surface binding was as described previously.

Identification of Haptenated Cellular Proteins Using Western Blot Analysis. MOLT-3 cells (5 × 10⁵) or HEPA 1C1C7 cells (70% confluent, 60-mm dish) were incubated with varying concentrations of SMX, SMX-HA, or SMX-NO (0-100 µM) in HEPES for varying time points at 37°C. Cells were thoroughly washed three times in cold PBS and resuspended with lysis buffer (10% Triton X-100, 3 M NaCl, 1 M Tris, pH 7.5, 0.5 M EDTA, Complete mini protease cocktail inhibitor tablet) for 30 min on ice. The HEPA cells were harvested from the dish using 5 mM EDTA in PBS. Lysed cells were then microcentrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was collected and protein concentration was determined using the Bio-Rad protein estimation assay. Protein concentration was standardized to (400 µg/ml) and samples were diluted in 4× sample buffer (8% SDS, 8% 2-mercaptoethanol, 0.5 M Tris, pH 6.8, 40% glycerol, and 0.02% bromphenol blue) and boiled for 5 min to denature the proteins. Proteins were separated according to molecular weight using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a Bio-Rad miniprotein II gel apparatus. Hapten inhibition experiments were performed to confirm the immunochemical recognition of the drug ligand (data not shown). Sample (8-10 µg) was loaded onto a 15-cm 10% discontinuous polyacrylamide separating gel (H₂O, 30% bis-acrylamide mix, 1.5 M Tris, pH 8.8, 10% SDS, 10% ammonium persulfate, N,N,N',N'-tetramethylethylenediamine) with a 5% stacking gel (H₂O, 30% bis-acrylamide mix, 0.5 M Tris, pH 6.8, 10% SDS, 10% ammonium persulfate, N, N, N', N'-tetramethylethylenediamine) according to the procedure of Laemmli (1970). Gels were run in a chamber containing 1× running buffer (glycine, Tris, SDS) at 150 V for approximately 1 h or until the samples reached the bottom of the gel. The gel was then transferred onto a polyvinylidene difluoride membrane (Immobilion-P; Millipore Corporation, Bedford, MA) using the semidry electrophoretic transfer apparatus (Trans-Blot SD; Bio-Rad) for 43 min at 16 V. After protein transfer, the polyvinylidene difluoride membrane was blocked overnight at room temperature with 5% Carnation skim milk powder in TBST (3 M NaCl, 1 M Tris pH 8.0, 0.1% Tween 20). The membrane was then incubated with the rabbit anti-SMX IgG antibody (1:500 in TBST containing 0.5% skim milk powder) for 2 h followed by washing three times with TBST and incubation with the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:20,000 in TBST) (Jackson Immunoresearch Laboratories) for 1 h at room temperature. The membrane was then washed three times in TBST and the sulfa-specific haptenated proteins were visualized using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) and developed on autoradiographic film (BioMax; Eastman Kodak, Rochester, NY) at suitable exposure times.

Analysis of the Effects of Antioxidants on Sulfa-Specific Haptenation Using Flow Cytometry and Western Blot Analysis. MOLT-3 cells (5 × 10⁶) were incubated with varying concentrations of glutathione (Calbiochem, San Diego, CA), ascorbic acid (BDH Inc., Poole, Dorset, UK) and N-acetylcysteine (Roche Applied Science, Indianapolis, IN) in combination with 100 µM of either SMX-HA or SMX-NO for 1 h at 37°C. After incubation cells were stained and analyzed on the flow cytometer as described above or lysed and analyzed by Western blot analysis as described above.

Analysis of Sulfa-Specific Bound Protein Internalization. The internalization of sulfa-specific bound proteins over time was assessed by flow cytometry. Hepa 1C1C7 cells were cultured to 70% confluence in 60-mm dishes and incubated with either 50 µM SMX-HA or 25 µM SMX-NO in HEPES for 10 min at 37°C. The cells were then placed on ice, washed two times with ice-cold HEPES, and incubated for an additional 15 min in a blocking solution consisting of 5% goat serum in cold HEPES. The cell sulfa-specific haptenated proteins were specifically labeled on ice with a rabbit anti-SMX IgG antibody diluted 1:500 in blocking solution for 1 h and any unbound antibody was subsequently washed away by washing twice in cold-HEPES. The cells were warmed up to 37°C by washing twice in prewarmed HEPES and were incubated in HEPES at 37°C for the time periods indicated in the figures. The cells were immediately placed on ice and washed twice with cold HEPES. Any of the immunolabeled cell surface proteins remaining on the cell surface were then labeled with human absorbed biotin-conjugated goat anti-rabbit (1:1000) followed by streptavidin-FITC (1:1000) and analyzed by FACscan. The sulfa-specific protein internalization was defined as the percentage loss of immunolabeled surface proteins compared with cells kept at 4°C.

Analysis of Sulfa-Specific Bound Protein Internalization in the Presence of Inhibitors. The sulfa-specific protein internalization was assessed in the presence of 0.45 M sucrose, which is an inhibitor of clathrin-mediated internalization, and 50 µg/ml nystatin (ICN Biomedicals Inc., Costa Mesa, CA), which is an inhibitor of caveolae- mediated internalization and inhibits all membrane internalization. Hepa 1C1C7 cells were pretreated with either 0.45 M sucrose for 30 min or 50 µg/l nystatin for 3 h at 37°C. Cells were then exposed to 50 µM SMX-HA or 25 µM SMX-NO and analyzed for percentage internalization above.

Statistical Analysis. Dose-response and time course data were analyzed using Prism (GraphPad Software, San Diego, CA). Statistical significance was determined using paired two-tailed t test with p < 0.05. In the case of multiple determinations, analysis was performed by analysis of variance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Analysis of Haptenation by SMX, SMX-HA, and SMX-NO. To determine whether SMX or its reactive metabolites bind to cellular proteins, flow cytometry, fluorescent microscopy, and confocal microscopy techniques were employed. SMX, SMX-HA, or SMX-NO was incubated with MOLT-3 cells and cells were subsequently examined for binding to membrane proteins. Results (Fig. 1A) demonstrated that the parent compound SMX did not bind to membrane proteins to any significant degree and, even at high concentrations (800 µM), mean binding was less than 9.9 ± 4.6%. SMX-HA bound to 46 ± 4.7% of membrane proteins at 25 µM and increased to a maximum binding of 78.7 ± 3.0% at 800 µM concentration (Fig. 1B). SMX-NO however, showed 74.7 ± 2.6% binding at 25 µM with maximum binding of 80.7 ± 4.7% at 50 µM (Fig. 1C). At higher concentrations (> 200 µM), binding was actually shown to decrease.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Dose response of extracellular protein binding by SMX, SMX-HA, and SMX-NO in MOLT-3 cells using flow cytometry. MOLT-3 cells were treated with various concentrations of SMX (A), SMX -HA (B), or SMX-NO (C) and analyzed for cell surface binding using flow cytometry. Controls were fully treated and unstained (C1) or fully treated and stained only with the secondary antibody (C2) to account for nonspecific binding. Binding was calculated as a percentage increase in the mean fluorescence of the immunolabeled, fully treated cells compared with immuno-labeled untreated control cells. There is a significant increase in binding associated with SMX-HA and SMX-NO compared with SMX (p > 0.05). Data show the mean ± S.E.M. of three independent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Time course of extracellular protein binding by SMX, SMX-HA, and SMX-NO in HEPA 1C1C7 cells using flow cytometry. HEPA 1C1C7 cells were treated with 25, 50, and 100 µM concentrations of SMX-HA (A) or SMX-NO (B) for 5, 10, 15, and 30 min at 37°C. Cells were analyzed for cell surface binding using flow cytometry as described under Materials and Methods. C, summary table of the maximum percentage binding at each concentration. Binding was calculated as a percentage increase in the mean fluorescence of the treated cells compared with immunolabeled untreated control cells. There is a significant time-related increase in binding up to time 10 min (p > 0.05). Data show the mean ± S.E.M. of three independent experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of temperature on extracellular protein binding by SMX, SMX-HA, and SMX-NO in HEPA 1C1C7 cells using flow cytometry. HEPA 1C1C7 cells were incubated at 37°C and 4C with either 50 µM SMX-HA (A) or 25 µM SMX-NO (B) for 5, 10, 15, and 30 min, respectively. Cells were analyzed for cell surface binding using flow cytometry. C, summary table of the maximum percentage binding at each concentration. Binding was calculated as percentage increase in the mean fluorescence of the treated cells compared with immunolabeled untreated control cells. There is significantly more binding at 37°C versus 4°C for times under 10 min (p > 0.05). Data show the mean ± S.E.M. of three independent experiments.

Identification of Haptenated Cellular Proteins. Flow cytometry and microscopy experiments described previously have demonstrated that sulfa-specific binding to cellular proteins occurs with the reactive metabolites SMX-HA and SMX-NO but not the parent compound SMX. To establish the specific proteins were being haptenated by the sulfa metabolites, MOLT-3 cells were incubated with DMSO, SMX, SMX-HA, or SMX-NO and their cell lysates were run on a 10% SDS-PAGE; Western blot analysis was performed using a sulfa-specific polyclonal antibody. A protein band at approximately 64 kDa was detected in all lanes, including the DMSO control, indicating that it is a nonspecific protein band (Fig. 4). Incubation of cells with SMX-HA and SMX-NO revealed more than 20 protein bands (Fig. 4); however, the intensity and the total number of protein bands detected was greater with SMX-NO. Particularly strong bands can be seen at ~55, 80, and 150 kDa in both SMX-HA and SMX-NO lanes when the other protein bands are of approximately equal intensity.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of protein binding by SMX, SMX-HA, and SMX-NO in MOLT-3 cells. MOLT-3 cells were incubated with 100 µM SMX, SMX-HA, or SMX-NO for 60 min at 37°C. "No drug" represents incubation with vehicle DMSO only. Whole-cell lysates (10 µg) were run on 10% SDS-PAGE and examined for sulfa-specific binding using Western blot analysis.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Dose response of protein binding by SMX, SMX-HA, and SMX-NO in MOLT-3 cells using Western blot analysis. MOLT-3 cells were treated with either 12.5, 25, 50, 100, 200, and 400 µM of either SMX-HA (A) or SMX-NO (B) for 60 min at 37°C. "No drug" represents incubation with vehicle DMSO only. Whole-cell lysates (10 µg) were run on 10% SDS-PAGE and examined for sulfa-specific binding using Western blot analysis.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of protein binding by SMX, SMX-HA, and SMX-NO in HEPA 1C1C7 cells. HEPA 1C1C7 cells were treated with 100 µM SMX, SMX-HA, or SMX-NO for 60 min at 37°C. "No drug" represents incubation with vehicle DMSO only. Whole-cell lysates (10 µg) were run on 10% SDS-PAGE and examined for sulfa-specific binding using Western blot analysis.

View larger version (68K): [in this window] [in a new window]   Fig. 7.   Protein binding by SMX-HA and SMX-NO in HEPA 1C1C7 cells at different time points using Western blot analysis. HEPA 1C1C7 cells were treated with 50 µM of SMX-HA or SMX-NO at 37°C at for 5 min (A) or 60 min (B). Whole-cell lysates (10 µg) were run on 10% SDS-PAGE and examined for sulfa-specific binding using Western blot analysis.

Confocal Studies of Cellular Protein Binding. Flow cytometry experiments (Figs. 1-3) illustrated sulfa-specific binding patterns to cell membrane proteins but did not give any information about what was occurring inside the cell. The very faint amounts of green seen associated with SMX (Fig. 8B) are the same as the DMSO control (Fig. 8A), suggesting background fluorescence only and not SMX-specific binding. However, in the case of the cells incubated with SMX-HA (Fig. 8C) and SMX-NO (Fig. 8D), there is a great deal of fluorescence, indicating sulfa-specific binding. Interestingly, binding seems to be localized to specific punctata and is not simply diffused throughout the cell.

View larger version (50K): [in this window] [in a new window]   Fig. 8.   Analysis of HEPA1C1C7 cellular protein binding by SMX, SMX-HA, and SMX-NO using fluorescent microscopy. HEPA 1C1C7 cells were incubated vehicle DMSO alone (A) or 25 µM SMX (B), SMX-HA (C), or SMX-NO (D) for 60 min at 37C. Cells were fixed, permeabilized, stained for sulfa-specific binding, and analyzed on the fluorescence microscope as described under Materials and Methods.

View larger version (50K): [in this window] [in a new window]   Fig. 9.   Analysis of HEPA1C1C7 cell membrane protein binding by SMX, SMX-HA, and SMX-NO using confocal microscopy. HEPA 1C1C7 cells were incubated vehicle DMSO alone for 60 min (A), 50 µM SMX for 60 min (B), 50 µM SMX-HA for 5 and 60 min (C), or 50 µM SMX-NO for 5 and 60 min (D). Cells were immunostained for sulfa-specific binding, fixed, and analyzed on the confocal microscope as described under Materials and Methods. Each depicts FITC staining alone, phase contrast of cells alone, and an overlay of FITC staining with the phase contrast.

View larger version (89K): [in this window] [in a new window]   Fig. 10.   Analysis of HEPA1C1C7 intracellular and extracellular protein binding by SMX, SMX-HA, and SMX-NO using confocal microscopy. HEPA 1C1C7 cells were incubated at 37°C with 50 µM SMX-HA for 5 and 60 min (A) or 25 µM SMX-NO for 5 and 60 min (B). DMSO and SMX control pictures were identical to those seen in Fig. 9, A and B. Cells were fixed, permeabilized, immunostained, and analyzed on the confocal microscope for sulfa-specific binding as described under Materials and Methods. Each depicts FITC staining alone, phase contrast of cells alone, and an overlay of FITC staining with the phase contrast.

View larger version (59K): [in this window] [in a new window]   Fig. 11.   Analysis of HEPA1C1C7 intracellular and extracellular protein binding by SMX, SMX-HA and SMX-NO at 4C using confocal microscopy. HEPA 1C1C7 cells were incubated at 4°C with 50 µM SMX-HA (A) or 25 µM SMX-NO (B). Cells were fixed, permeabilized, immunostained, and analyzed on the confocal microscope for sulfa-specific binding as described under Materials and Methods. Each depicts FITC staining alone, phase contrast of cells alone, and an overlay of FITC staining with the phase contrast.

Analysis of the Effects of Antioxidants on Sulfa-Specific Binding. To examine the effects of antioxidants on sulfa-specific binding, MOLT-3 cells were coincubated with either glutathione, N-acetylcysteine, or ascorbic acid, along with SMX-HA or SMX-NO. Results (Fig. 12) indicate that 1.0 mM glutathione was able to decrease SMX-HA-specific binding by 71.7 ± 8.2% and SMX-NO-specific binding by 45.8 ± 5.4% (Fig. 12A). As well, 1.0 mM N-Acetylcysteine decreased SMX-HA and SMX-NO binding by 70.4 ± 1.9 and 29.5 ± 3.1%, respectively (Fig. 12B). The greatest decrease in binding was seen by 1.0 mM ascorbic acid, which decreased SMX-HA binding by 80.5 ± 1.0% and SMX-NO binding by 41.9 ± 5.4% (Fig. 12C). The results of these studies as well as the effects of these antioxidants at lower concentrations are summarized in Fig. 12D. Western blot analysis was then carried out to determine whether the above antioxidants decreased binding of specific cellular proteins. The results of Western blot analysis revealed a decrease in protein binding with the antioxidants corresponding to the flow cytometry data with both the SMX-HA and the SMX-NO (Fig. 12E). The Western blot shown in Fig. 13E is that of glutathione; however, the blots for N-acetylcysteine and ascorbic acid were identical. There were no specific proteins that were inhibited by the antioxidants; rather, a generalized decrease in all sulfa-bound proteins was seen.

View larger version (26K): [in this window] [in a new window]   Fig. 12.   Effect of antioxidants on extracellular protein binding by SMX, SMX-HA, and SMX-NO in MOLT-3 cells. A to D, MOLT-3 cells were incubated for 60 min at 37°C with 100 µM SMX-HA or SMX-NO in the absence or presence of GSH (A), N-acetyl-cysteine (NAC) (B), or ascorbic acid (AA) (C) at 0.5 and 1.0 mM concentrations. Cells were analyzed for cell surface binding in the absence and presence of these antioxidants using flow cytometry. Results are displayed as a percentage decrease in mean fluorescence in the presence and absence of antioxidants compared with cells treated with the metabolites alone. There was a significant decrease in binding associated with increasing antioxidant concentrations for SMX-HA for all three antioxidants (p < 0.05). D, summary table of the percentage decreases in binding at each condition (% decrease in binding ± S.E.). Data shows the mean ± S.E.M. of three independent experiments. E, MOLT-3 cells were incubated for 60 min at 37°C with 100 µM SMX, SMX-HA, or SMX-NO in the absence or presence of 1.0 mM GSH. Whole-cell lysates were run on 10% SDS-PAGE and examined for sulfa-specific binding using Western blot analysis. Similar results were obtained when MOLT-3 cells were incubated in the presence of NAC or AA (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 13.   Time course of disappearance of extracellular proteins haptenated by SMX, SMX-HA, and SMX-NO from the cell surface using flow cytometry. HEPA 1C1C7 cells were incubated at 10 min at 37°C with either 50 µM SMX-HA (A) or 25 µM SMX-NO (B) and internalization of proteins bound by these metabolites was determined at 0, 2.5, 5, 10, 15, and 30 min using flow cytometry as described under Materials and Methods. Internalization was defined as the percentage loss of antibody labeled cellular proteins (mean fluorescence) over the various time periods. There is a significant time-related disappearance up to 15 min (p > 0.05). C, summary table of the maximum internalization for each experimental condition. Data represent the mean ± S.E.M. of three independent experiments.

Analysis of Haptenated Cellular Protein Internalization. Confocal analysis of sulfa-specific protein haptenation illustrated that binding was occurring on the cell membrane as well as inside the cell. Therefore, we used flow cytometry to examine internalization of cellular proteins from the cell membrane into the cell. Results (Fig. 13) indicate that 60.12 ± 6.05% of cell membrane proteins bound by SMX-HA are internalized within 30 min. Correspondingly, 53.99 ± 4.30% of cell membrane proteins bound by SMX-NO are internalized within 30 min.

View larger version (19K): [in this window] [in a new window]   Fig. 14.   Effect of inhibitors of disappearance of extracellular proteins haptenated by SMX-HA and SMX-NO from the cell membrane using flow cytometry. HEPA 1C1C7 cells were preincubated with 0.45 M sucrose or 50 mg/ml Nystatin for 30 min and 3 h, respectively, at 37°C. Cells were then incubated at 10 min at 37°C with either 50 µM SMX-HA (A) or 25 µM SMX (B) and internalization of proteins bound by these metabolites was determined at 0, 2.5, 5, 10, 15, and 30 min using flow cytometry as described under Materials and Methods. Cells were also maintained at 4°C under all treatment conditions and analyzed for internalization. Internalization was defined as the percentage loss of antibody-labeled cellular proteins (mean fluorescence) over the various time periods. There is no significant difference in the percentage disappearance associated with incubation with sucrose; in contrast, there is a significant decrease in disappearance associated with incubation with nystatin up to 15 min (p > 0.05). C, summary table of the maximum internalization for each experimental condition. Data represent the mean ± S.E.M. of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Sulfonamides are common antimicrobial agents. Unfortunately, sulfa therapy is often associated with adverse drug reactions (Rieder et al., 1989; Pirmohamed and Park, 1999). The most severe adverse events are hypersensitivity reactions. The mechanisms by which sulfonamides produce hypersensitivity adverse reactions are unclear; however, the clinical manifestations suggest involvement of the immune system (Hess and Rieder, 1997; Naisbitt et al., 1999). Bioactivation of sulfonamides to reactive metabolites seems to play a major role in the initial steps leading to these reactions (Shear et al., 1986; Rieder et al., 1989). Studies have shown that in vivo and in vitro metabolic production of electrophilic reactive metabolites may lead to binding to essential cellular proteins, hapten formation, and activation of immune responses (Meekins et al., 1994; Cribb et al., 1995, 1996). Anti-SMX antibodies, as well as specific T cell responses, have been detected in patients experiencing hypersensitivity reactions to sulfonamides but also in patients who tolerate therapy (Daftarian et al., 1995; Mauri-Hellweg et al., 1995; Gill et al., 1997). There is very little knowledge of the immune response to sulfa haptenated proteins; understanding this response would provide insights into the biology of xenobiotic-immune system interactions leading to idiosyncratic adverse drug reactions.

Sulfa-Specific Haptenation to Cellular Proteins. The reactive metabolites SMX-HA and SMX-NO, but not SMX, bound to cellular proteins at concentrations of 25 µM and above. Maximum plasma concentrations of SMX can reach 1.5 to 2 mM in patients on high-dose cotrimoxazole; concentrations of SMX-HA can be 10 to 20% of the dose and SMX-NO is less than 2% (Cribb et al., 1995). The concentrations at which haptenation occurs in vitro are within the therapeutic range achieved in vivo. This binding occurred within 5 min of incubation. Because the nitroso is inherently more reactive (Cribb et al., 1995), it is not surprising that maximum binding of the nitroso occurs at lower concentrations and more quickly than with SMX-HA.

Identification of Sulfa-Specific Haptenated Cellular Proteins. Specific proteins haptenated by SMX-HA and SMX-NO in vitro were detected using Western blot analyses. Sulfa-specific haptenation revealed a binding pattern of more than 20 bands with both the SMX-HA and SMX-NO metabolites in both cell lines. The intensity and the total number of haptenated protein bands detected was greater with SMX-NO, probably because of its greater reactivity.

Effect of Antioxidants on Sulfa-Specific Protein Haptenation. Addition of antioxidants to cells incubated with reactive metabolites of SMX attenuated sulfa-specific binding with both the hydroxylamine and nitroso metabolites, although attenuation was greater with SMX-HA. Antioxidants such as GSH may be responsible for reducing reactive metabolites to inactive compounds and higher levels in the liver may explain why SMX-mediated hepatotoxicity is rarely observed clinically (Cribb et al., 1996).

Internalization of Haptenated Cellular Proteins. Sulfa-specific haptenation can be visualized on the cell membrane as well as intracellularly. Binding pattern occurred at specific sites on the membrane within 5 min of incubation. The confocal images show specific and not indiscriminate areas at which binding is localized on the cell membrane. Looking intracellularly within 5 min of exposure to metabolite, haptenated cellular proteins seem to be in vesicles near the plasma membrane, but later distribute throughout the cell (excluding the nucleus). This suggests a mechanism of internalization of haptenated proteins from the cell membrane into the cell.

View larger version (25K): [in this window] [in a new window]   Fig. 15.   Proposed pathogenesis of hypersensitivity reactions to sulfamethoxazole. Sulfamethoxazole can be acetylated but also is metabolized to its reactive metabolites SMX-HA and SMX-NO. These reactive metabolites can covalently bind to cellular proteins, forming immunogenic hapten-protein complexes. These can then be internalized, processed, and presented for recognition by cells of the immune system, leading to drug hypersensitivity.