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Vol. 62, Issue 5, 1011-1026, November 2002
Section of Paediatric Clinical Pharmacology, Toxicology & Experimental Therapeutics, Departments of Paediatrics, Pharmacology & Toxicology, and Medicine, Faculty of Medicine & Dentistry, University of Western Ontario, Ontario, Canada (T.M., D.H., M.J.R.); and Child Health Research Institute - Robarts Research Institute Children's Hospital of Western Ontario London, Ontario, Canada (L.D., S.G.F., M.J.R.)
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Abstract |
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Adverse drug reactions are a major problem complicating medical therapy. The pathogenesis of many severe adverse drug reactions, notably hypersensitivity reactions, is poorly understood. The sulfonamides are associated with severe hypersensitivity reactions. The initial pathogenesis seems to be caused by bioactivation of the parent drug to a reactive intermediate and subsequent propagation by the immune system. The determinants of the immune response are not known. We explored the formation of sulfonamide haptens in Molt-3 and HEPA 1C1C7 cells after incubation with sulfamethoxazole (SMX), the hydroxylamine of sulfamethoxazole (SMX-HA), or the nitroso of sulfamethoxazole (SMX-NO). Haptenation was demonstrated with SMX-HA and SMX-NO but not SMX; this occurred at concentrations below that associated with toxicity (significant haptenation was seen at 25 to 50 µM). Thus, haptenation occurred presumably onto viable cells. Haptenation occurred rapidly; haptenation of cell surface proteins was demonstrated within 5 min. This did not occur indiscriminately; confocal microscopy demonstrated haptenation onto specific sites on the cell membrane. We found that haptenation was significantly inhibited by thiols and other antioxidants (p < 0.05). Sulfonamide-specific haptens were rapidly internalized by what seemed to be a caveolae-dependent process. It seems that sulfonamide reactive metabolites haptenated specific cell surface proteins that are rapidly internalized. Understanding the specific protein target(s) for haptenation and how these haptens are processed will be important in understanding the immune mediation of sulfonamide hypersensitivity adverse drug reactions.
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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
).
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Materials and Methods |
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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.
Cells of lymphoblastic origin, such as MOLT-3 cells, have been used as an in vitro model for a number of studies of drug toxicity (Shear et al., 1985Purification 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.
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Results |
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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.
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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.
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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.
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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.
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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.
To determine the mechanism of internalization, HEPA cells were preincubated with inhibitors of internalization before being exposed to SMX-HA and SMX-NO. The inhibitors used were incubation at 4°C (which inhibits overall cell membrane internalization), sucrose (which inhibits clathrin-mediated internalization), and nystatin (which is an inhibitor of caveolae-mediated internalization). Incubating cells at 4°C with the reactive sulfa metabolites decreased maximum internalization of sulfa-bound proteins from 60.12 ± 6.05 to 5.68 + 0.91% (p < 0.05) with SMX-HA and from 53.99 ± 4.30 to 6.04 + 1.46% (p < 0.05) with SMX-NO (Fig. 14). Sucrose decreased maximum internalization of sulfa-bound proteins to 45.52 ± 6.68% with SMX-HA and to 45.16 ± 3.35% (Fig. 14). The overall effect of sucrose was minimal, suggesting that clathrin-mediated internalization is not a major mechanism by which sulfa-bound proteins are internalized. Alternatively, nystatin was found to decrease the initial rate of binding up to 15 min; over the entire time-course, nystatin was found to display an overall significant inhibition in sulfa-bound protein internalization (Fig. 14) (p < 0.05). From these results, it seems that early internalization of haptenated cellular proteins (< 15 min) may occur through a caveolae-dependent mechanism.
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Discussion |
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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.
A number of particularly strong bands can be visualized in both cell types; however, only the 55-kDa band is clearly common. It is likely that there are other common protein bands, but better separation is needed for a clear correlation. It is not known whether these intense protein bands are specific high-affinity targets for protein haptenation or simply a reflection of relative abundance in the cell. Detection of haptenated proteins over time shows that selective proteins are haptenated to the same extent at 5 min as they are at 60 min of incubation with the metabolite. Alternately, other proteins show increased haptenation over time. This pattern indicates some degree of selectivity. Initial binding of reactive metabolites to cellular proteins may be a specific selective process. However, over time and with increased concentrations, binding to cellular proteins in vitro may become more general and nondiscriminate. It should be noted that there are a large number of candidate proteins in these bands (Lefkovitz et al., 2000Effect 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.
Virtually all eukaryotic cells continually ingest bits of plasma membrane as small pinocytic (endocytic) vesicles that later return to the cell surface. The endocytic part of the cycle usually begins at specialized regions of the plasma membrane called clathrin-coated pits. The lifetime of clathrin coated pits is short; within a minute of being formed, they are invaginated into the cell and pinched off to form clathrin-coated vesicles, which within seconds shed their coats and fuse with early endosomes. In most animal cells, clathrin-coated pits and vesicles provide an efficient pathway known as receptor-mediated endocytosis for taking up specific macromolecules bound to cell surface receptors or transmembrane proteins. The plasma membrane of most cells also has distinct invaginations called caveolae, thought to bud off to form calveolin-coated vesicles that provide another mechanism for endocytosis. Inhibiting clathrin-mediated endocytosis did not significantly decrease internalization of sulfa-haptenated proteins from the cell membrane. Inhibition of caveolae-mediated endocytosis decreased the time of internalization but not the overall amount or protein that was internalized. This indicates that caveolae-mediated endocytosis may be important in early internalization of haptenated proteins but does not rule out other mechanism of internalization. Two sets of endosomes can be readily distinguished in labeling experiments: early endosomes, just beneath the plasma membrane within 1 to 2 min of incubation, and late endosomes, which accumulate closer to the Golgi apparatus and nucleus within 15 min. The intracellular haptens seen at 5 min near the plasma membrane may be in the early endosomes, whereas by 60 min, they are in late endosomes. Endogenous antigens (such as drugs and metabolites) can be endocytosed by antigen-presenting cells, routed to endosomes, and associated with MHC class II to elicit a specific T-cell response. We have demonstrated that the reactive metabolites SMX-HA and SMX-NO bind covalently to cellular proteins at concentrations found in plasma during sulfonamide therapy. Sulfa-specific binding seems to be somewhat discriminate; specific proteins were detected early after incubation with metabolites. Antioxidants such as GSH, N-acetylcysteine, and ascorbic acid were able to attenuate sulfa-specific binding. This provides further support for their key role as a defense mechanism against sulfa toxicity and may account for interpatient variability in toxic response. Internalization of sulfa-bound proteins was also demonstrated, indicating specific mechanisms of internalization and processing of these haptenated structures. Understanding specific protein target(s) for sulfonamides will be important in understanding how induction of an immune response can be clinically manifested as an adverse drug reaction to sulfonamide therapy (Fig. 15). Further characterization of the mechanisms of haptenation, as well as understanding hapten processing, is essential to gain a better understanding of the pathobiology of sulfonamide hypersensitivity reactions.
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Footnotes |
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Received December 26, 2001; Accepted June 10, 2002
This research was supported by a grant from the Canadian Institutes of Health Research and a studentship from the Department of Pediatrics, University of Western Ontario
Address correspondence to: Dr. Michael J. Rieder, Section of Pediatric Clinical Pharmacology, Toxicology and Experimental Therapeutics, Department of Pediatrics, Children's Hospital of Western Ontario, 800 Commissioner's Road East London, Ontario, Canada N6C 2V5. E-mail: mrieder{at}julian.uwo.ca
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Abbreviations |
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SMX, sulfamethoxazole; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SMX-HA, hydroxylamine of sulfamethoxazole; SMX-NO, nitroso of sulfamethoxazole; FITC, fluorescein isothiocyanate; TBS, phosphate-buffered saline and 0.05% Tween 20; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; GSH, glutathione.
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