Enzyme-mediated protein haptenation of dapsone and sulfamethoxazole in human keratinocytes: I. Expression and role of cytochromes P450

Article abstract of DOI:10.1124/jpet.106.105858

Cutaneous drug reactions (CDRs) are among the most common adverse drug reactions and are responsible for numerous minor to life-threatening complications. Several arylamine drugs, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation, resulting in adduction to cellular proteins. These adducted proteins may initiate the immune response that ultimately results in a CDR. Recent studies have demonstrated that normal human epidermal keratinocytes (NHEKs) can bioactivate these drugs, resulting in protein haptenation. We sought to identify the enzyme(s) responsible for this bioactivation in NHEKs. Using immunofluorescence confocal microscopy and an adduct-specific enzyme-linked immunosorbent assay (ELISA), we found that N-acetylation of the primary amine of SMX and DDS markedly reduced the level of protein haptenation in NHEKs. Detection of mRNA and/or protein confirmed the presence of CYP3A4, CYP3A5, and CYP2E1 in NHEKs. In contrast, although a faint band suggestive of CYP2C9 protein was detected in one NHEK sample, a CYP2C9 message was not detectable. We also examined the ability of chemical inhibitors of cytochromes P450 (aminobenzotriazole and 1-dichloroethylene) and cyclooxygenase (indomethacin) to reduce protein haptenation when NHEKs were incubated with SMX or DDS by either confocal microscopy or ELISA. These inhibitors did not significantly attenuate protein adduction with either SMX or DDS, indicating that cytochromes P450 and cyclooxygenase do not play important roles in the bioactivation of these xenobiotics in NHEKs and thus suggesting the importance of other enzymes in these cells.

Full text of DOI:10.1124/jpet.106.105858

0022-3565/06/3191-488–496
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 319:488–496, 2006
Vol. 319, No. 1
105858/3142335
Printed in U.S.A.
Enzyme-Mediated Protein Haptenation of Dapsone and
Sulfamethoxazole in Human Keratinocytes: I. Expression and
Role of Cytochromes P450
Piyush M. Vyas, Sanjoy Roychowdhury, Farah D. Khan, Thomas E. Prisinzano,
Jatinder Lamba, Erin G. Schuetz, Joyce Blaisdell, Joyce A. Goldstein, Kimber L. Munson,
Ronald N. Hines, and Craig K. Svensson
Divisions of Pharmaceutics (P.M.V., S.R., F.D.K., C.K.S.) and Medicinal and Natural Products Chemistry (T.E.P.), College of
Pharmacy, University of Iowa, Iowa City, Iowa; Department of Pharmaceutical Sciences, St. Jude Children’s Hospital,
Memphis, Tennessee (J.L., E.G.S.); Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina (J.B., J.A.G.); and Department of Pediatrics and Pharmacology and
Toxicology, Medical College of Wisconsin and Children’s Research Institute, Children’s Hospital of Wisconsin, Milwaukee,
Wisconsin (K.L.M., R.N.H.)
Received April 7, 2006; accepted July 3, 2006
ABSTRACT
Cutaneous drug reactions (CDRs) are among the most common nation in NHEKs. Detection of mRNA and/or protein confirmed
adverse drug reactions and are responsible for numerous minor the presence of CYP3A4, CYP3A5, and CYP2E1 in NHEKs. In
to life-threatening complications. Several arylamine drugs, contrast, although a faint band suggestive of CYP2C9 protein
such as sulfamethoxazole (SMX) and dapsone (DDS), undergo was detected in one NHEK sample, a CYP2C9 message was
bioactivation, resulting in adduction to cellular proteins. These not detectable. We also examined the ability of chemical inhib-
adducted proteins may initiate the immune response that ulti- itors of cytochromes P450 (aminobenzotriazole and 1-dichlo-
mately results in a CDR. Recent studies have demonstrated roethylene) and cyclooxygenase (indomethacin) to reduce pro-
that normal human epidermal keratinocytes (NHEKs) can bio- tein haptenation when NHEKs were incubated with SMX or
activate these drugs, resulting in protein haptenation. We DDS by either confocal microscopy or ELISA. These inhibitors
sought to identify the enzyme(s) responsible for this bioactiva- did not significantly attenuate protein adduction with either
tion in NHEKs. Using immunofluorescence confocal micros- SMX or DDS, indicating that cytochromes P450 and cycloox-
copy and an adduct-specific enzyme-linked immunosorbent ygenase do not play important roles in the bioactivation of
assay (ELISA), we found that N-acetylation of the primary amine these xenobiotics in NHEKs and thus suggesting the impor-
of SMX and DDS markedly reduced the level of protein hapte- tance of other enzymes in these cells.
Sulfonamides are used in the treatment of numerous in- 75% of the patients suffering from this ailment (Hughes,
fectious diseases. Their effectiveness in the treatment of 1987; Goldie et al., 2002). However, adverse drug reactions,
Pneumocystis carinii pneumonia, especially in AIDS pa- especially those of a cutaneous nature, limit their use. These
tients, has increased their importance as therapeutic agents reactions most commonly occur 7 to 10 days after initiation of
in the modern antimicrobial era (Cribb et al., 1996a). Sulfa-
methoxazole (SMX) and the sulfone dapsone (DDS) are
widely used antimicrobials for the treatment of Pneumocystis
carinii pneumonia resulting in the recovery of approximately
therapy and are associated with fever and skin rash. Morbil-
liform or maculopapular nonurticarial types of skin rash are
most commonly observed with these agents. Some patients,
however, progress to Stevens-Johnson syndrome or toxic epi-
dermal necrolysis, which can have a mortality rate as high as
40 to 50%. A multiorgan syndrome, manifested as fever, rash,
eosinophilia, and hepatotoxicity, has also been reported in
patients receiving these drugs (Dujovne et al., 1967; Berg and
Daniel, 1987; Rieder et al., 1989; Svensson et al., 2001).
This work was supported in part by National Institutes of Health Grants
AI41395 and GM63821 (to C.K.S.) and GM60346 (to E.G.S.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.105858.
ABBREVIATIONS: SMX, sulfamethoxazole; DDS, dapsone; NHEK, normal human epidermal keratinocyte; CYP450, cytochrome(s) P450; COX,
cyclooxygenase; MADDS, monoacetyl dapsone; ABT, 1-aminobenzotriazole; INDO, indomethacin; 7-BQ, 7-benzyloxyquinoline; 7-HQ, 7-hy-
droxyquinoline; DCE, trans-dichloroethylene; ELISA, enzyme-linked immunosorbent assay; NASMX, N-acetyl sulfamethoxazole; PCR, polymerase
chain reaction; PBS, phosphate-buffered saline; ANOVA, analysis of variance; DMSO, dimethyl sulfoxide.
488

Protein Haptenation of Dapsone and Sulfamethoxazole
489
Several studies have demonstrated that SMX and DDS the bioactivation and subsequent protein adduction of SMX
undergo biotransformation to form reactive N-arylhydroxy- and DDS in NHEKs.
lamine metabolites (Fig. 1), which are believed to be respon-
sible for causing cutaneous drug reactions (Cribb et al.,
1996a; Reilly and Ju, 2002; Svensson, 2003). It has been
proposed that the bioactivation of these drugs at the site of
Materials and Methods
Materials. Monoacetyl dapsone (MADDS) was generously pro-
vided by Parke-Davis (now Pfizer, Ann Arbor, MI). DDS, SMX,
manifestation (i.e., skin) may be a critical element in the
4-dimethylaminopyridine, 1-aminobenzotriazole (ABT), troleando-
initiation of these reactions (Reilly et al., 2000). Indeed, we
mycin, disulfiram, and indomethacin (INDO) were obtained from
Sigma (St. Louis, MO). 7-Benzyloxyquinoline (7-BQ) and 7-hy-
droxyquinoline (7-HQ) were purchased from Gentest (Bedford, MA).
have previously demonstrated that normal human epidermal
keratinocytes (NHEKs) are able to bioactivate SMX and DDS
to their respective arylhydroxylamine metabolites (sulfame- DDS and SMX hydroxylamine metabolites were synthesized as de-
scribed previously (Vyas et al., 2005). Trans-dichloroethylene (DCE)
was obtained from TCI America (Portland, OR). Rabbit anti-sera
were raised against SMX- and DDS-keyhole limpet hemocyanin con-
jugates and specificity assessed as described previously (Reilly et al.,
2000). Rat tail collagen (type I) was obtained from Sigma. Normal
human epidermal keratinocytes (as first passage cells) and keratin-
ocyte culture media were obtained from CAMBREX (Walkersville,
MD). The HaCaT cell line was generated by Dr. N. Fusenig (DKFZ
Heidelberg, Heidelberg, Germany) and obtained from Dr. Michael
thoxazole hydroxylamine and dapsone hydroxylamine)
(Reilly et al., 2000) and that such bioactivation results in
protein haptenation (Roychowdhury et al., 2005).
Studies have demonstrated that skin cells express a vari-
ety of drug-metabolizing enzymes, such as cytochromes P450
(CYP450), cyclooxygenase (COX), flavin-containing monoox-
ygenases, and peroxidases, which may bioactivate numerous
chemical agents (Kanekura et al., 1998; Rys-Sikora et al.,
2000; Baron et al., 2001; Janmohamed et al., 2001; Saeki et Southhall (Johnson and Johnson, Skillman, NJ). Microtiter ELISA
plates (96-well) were obtained from Rainin Instruments (Woburn,
MA). Rabbit anti-CYP2C9 antibody was custom made by Covance
Research Products Inc. (Denver, PA). Donkey anti-rabbit IgG conju-
gated to horseradish peroxidase was obtained from Amersham Phar-
macia Biotech Inc. (Piscataway, NJ). SuperSignal West Pico Chemi-
luminescent Substrate was purchased from Pierce (Rockford, IL),
and a SynGene GeneGnome chemiluminescence detection system
was obtained from Synoptics (Cambridge, UK). K03 monoclonal anti-
CYP3A4 has been described previously (Beaune et al., 1985); rabbit
al., 2002). Hydroxylation of SMX and DDS by various
CYP450s has been reported (Cribb et al., 1995; Mitra et al.,
1995; Gill et al., 1999; Winter et al., 2000). Several arylamine
drugs, including procainamide, are oxidized to arylhydroxy-
lamine metabolites by COX-2 (Liu and Levy, 1998; Goebel et
al., 1999).
In the present investigation, we sought to identify the
enzyme(s) that catalyze the bioactivation of SMX and DDS,
leading to protein haptenation in NHEKs. Our studies dem- ␣-3A5 primary antibody was purchased from Gentest. Goat-anti-
rabbit IgG conjugated with Alexa fluor 488 and goat anti-rabbit
antibody conjugated with alkaline phosphatase and YoYo-1 were
purchased from Molecular Probes (Eugene, OR). Bradford assay
reagent was purchased from Pierce. Immunomount was obtained
from Vector Laboratories (Burlingame, CA). Trizol was purchased
from Invitrogen (Carlsbad, CA). All other chemicals and reagents
were purchased from Sigma or Fisher Scientific (Chicago, IL). Hu-
man liver microsomes were processed at St. Jude Children’s Re-
search Hospital (Nashville, TN), and tissue was provided by the
Liver Tissue Procurement and Distribution System (National Insti-
tutes of Health Contract N01-DK-9-2310 and by the Cooperative
Human Tissue Network.
onstrate that although COX-1/2, CYP3A4, and CYP2E1 are
able to catalyze the bioactivation of SMX and DDS in vitro,
inhibitors of these enzymes do not reduce adduct formation
when NHEKs are exposed to either SMX or DDS. These data
suggest that these enzymes do not play an important role in
Synthesis of N-Acetylsulfamethoxazole. SMX (250 mg) was
dissolved in 1 ml of acetic anhydride in the presence of 0.5 g of
4-dimethylaminopyridine and 10 ml of dichloromethane. The reac-
tion was carried out for 1 h at room temperature with continuous
stirring to obtain N-acetylsulfamethoxazole (NASMX). Excess of ace-
tic anhydride was removed by neutralizing the reaction mixture with
saturated sodium bicarbonate solution and NASMX obtained by
recrystallizing the product in an acetone and hexane (1:3) mixture.
NASMX was characterized by NMR, and the purity was determined
by high-performance liquid chromatography-UV. Product purity was
98%, whereas the observed yield was 79%.
Cell Culture. Adult NHEKs and immortalized HaCaT cells were
cultured as detailed previously (Reilly et al., 2000). In brief, cells
were propagated in 75-cm2 flasks using basal media (KBM-2) sup-
plemented with bovine pituitary extract (7.5 mg/ml), human epider-
mal growth factors (0.1 ng/ml), insulin (5 ␮g/ml), hydrocortisone, (0.5
␮g/ml), epinephrine, transferrin, gentamicin (50 ␮g/ml), and ampho-
tericin (50 ng/ml) at 37°C in an atmosphere containing 5% CO2.
Media were replaced every 2 to 3 days. When cell cultures reached
near confluency (70–90%), cells were disaggregated using 0.025%
trypsin/0.01% EDTA in HEPES followed by neutralization with 2
volumes of Trypsin neutralizing solution. Cell suspensions were then
centrifuged at 220g for 5 min followed by washing in basal media and
resuspension in KGM-2 (supplemented growth medium). Cells were
Fig. 1. Scheme for the bioactivation of SMX and DDS giving rise to
protein haptenation. MPO, myeloperoxidase; NAT, N-acetyltransferase.

490
Vyas et al.
then either subjected to subculturing or cryopreservation for further suspended in storage buffer (100 mM potassium phosphate, pH 7.4,
purposes. All experiments were performed using third to fourth
passage cells.
1.0 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 20 ␮M butylated
hydroxytoluene, and 2 mM phenylmethylsulfonyl fluoride), lysates
Determination of mRNA for CYP2C9 in NHEKs. Total RNA were generated by sonication, and protein concentrations were de-
was isolated from fourth passage NHEKs and seventh passage termined using the Bio-Rad protein assay. Lysate (30 ␮g) was sep-
HaCaT cells using Trizol (Invitrogen) following the manufacturer’s arated on 10% SDS-polyacrylamide gels and immunoblotted with
protocol. cDNA was generated using 500 ng of total RNA in a syn-
thesis reaction using SuperScript II RT with random hexamers ac-
monoclonal anti-CYP3A4 K03 or rabbit ␣-3A5 (Gentest), followed by
appropriate secondary antibodies coupled with peroxidase. The blot
cording to the supplier’s protocol. PCR was performed using the was developed with the ECL detection system (Amersham Bio-
CYP2C9-specific primers 5Ј-GATCTGCAATAATTTTTCTC-3Ј and sciences).
5Ј-TCTCAGGGTTGTGCTTGTC-3Ј with diluted amounts of cDNA
from the keratinocytes resulting in a 280-bp product. Plasmid NHEKs (106 cells) were incubated in KGM-2 overnight. After 24 h,
CYP2C9 cDNA in pCW was used as a positive control for amplifica- the media were discarded and replaced with fresh media. 7-BQ (50
Determination of Catalytic Activity for CYP3A4 in NHEKs.
tion. Actin was also amplified from the keratinocyte cDNA using the ␮M), a substrate for CYP3A4, was added to measure the catalytic
primers 5Ј-TCATGAAGTGTGACGTTGACATCCGT-3Ј and 5Ј- activity of CYP3A4 in these cells. The plate was incubated for 30 min
CCTAGAAGCATTTGCGGTGCACGATG-3Ј, resulting in a 285-bp at 37°C after the addition of 7-BQ followed by the fluorescence
fragment. Amplifications were performed in 20-␮l reactions using measurement every 30 min for 24 h using the CytoFluor multiwell
Amplitaq Gold (Applied Biosystems, Foster City, CA) according to fluorescent plate reader (excitation wavelength, 410 nm; emission
the manufacturer’s protocol with a final concentration of MgCl2 of 2 wavelength, 538 nm). The fluorescent metabolite of 7-BQ, 7-HQ, was
mM. Cycling conditions consisted of an initial denaturation at 94°C also added to NHEKs at varying concentrations (0, 5, 10, 25, and 50
for 5 min followed by 40 cycles of 94°C for 30 s, 53°C for 10 s, and
72°C for 10 s. Reactions were analyzed on 4% agarose gels stained
with ethidium bromide.
␮M) to determine the ability to detect this metabolite in the presence
of NHEKs.
Determination of CYP2E1 in NHEKs. CYP2E1 mRNA expres-
Immunoblot Analysis for CYP2C Proteins in NHEKs. Cell sion was quantified in NHEKs and HaCaT cells essentially as de-
lysates and yeast-expressed recombinant CYP2C proteins were frac- scribed by Haufroid et al. (2001). Briefly, total RNA was isolated
tionated by electrophoresis in SDS-10% (w/v) polyacrylamide gels from keratinocytes using Trizol reagent (Invitrogen) and quantified
and transferred to nitrocellulose membranes. Membranes were im- using a Quant-iT RiboGreen kit (Invitrogen) as per the manufactur-
munoblotted with a polyclonal rabbit anti-CYP2C9 antibody (1:500) er’s instructions. Using 2 ␮g of total RNA from each cell preparation,
raised to bacterially expressed human CYP2C9 and donkey anti- cDNA was prepared using random hexamer primers and ImProm-II
rabbit IgG conjugated to horseradish peroxidase. This anti-CYP2C9 reverse transcriptase following the procedures outlined in the Im-
polyclonal antibody to bacterially expressed recombinant CYP2C9 Prom-II Reverse Transcription System kit (Promega, Madison, WI).
was made for National Institute of Environmental Health Sciences RT-PCR reactions to quantify CYP2E1 mRNA concentrations were
by Covance using a standard National Institute of Environmental carried out in a total volume of 40 ␮l containing 5.5 mM MgCl2, 0.2
Health Sciences rabbit antibody production protocol. Yeast-ex- mM each deoxyribonucleotide, 0.3 ␮M of each PCR primer, 0.2 ␮M
pressed CYP2C proteins were used as standards on immunoblots fluorescent probe, 5 or 25 ng of keratinocyte cDNA, 0.01 U/␮l uracil
because there is no N-terminal modification for cDNA expression in N-glycosylase, and 0.025 U/␮l AmpliTaq Gold Polymerase (TaqMan
yeast. Therefore, the recombinant yeast-expressed CYP2C proteins PCR Core Reagents kit; Applied Biosystems) in an Opticon System
have mobilities identical to those of the human CYP proteins ex- (MJ Research, Watertown, MA). 18S rRNA concentrations were de-
pressed in vivo. Specific bands were visualized with SuperSignal termined using the TaqMan Ribosomal RNA Control Reagents (Ap-
West Pico Chemiluminescent Substrate and a SynGene GeneGnome plied Biosystems), which were designed to generate a 187-bp prod-
chemiluminescence detection system.
Determination of mRNA for CYP3A4 and CYP3A5 in
uct. Each 40-␮l PCR reaction contained 5.5 mM MgCl2, 0.2 mM each
deoxyribonucleotide, 0.05 ␮M of each PCR primer, 0.2 ␮M fluores-
NHEKs. Total RNA was isolated from NHEKs and HaCaT cells cent probe, 5 or 25 ng of keratinocyte cDNA, 0.01 U/␮l uracil N-
using Trizol Reagent (Invitrogen) and first strand cDNA was syn- glycosylase, and 0.025 U/␮l AmpliTaq Gold Polymerase (TaqMan
thesized from 3 ␮g of total RNA according to the manufacturer’s PCR Core Reagents kit; Applied Biosystems).
instructions (SuperScript First Strand cDNA Synthesis kit; Invitro-
Determination of the Cytotoxicity of Inhibitors in NHEKs.
gen). Amplification of CYP3A4 cDNA (GenBank accession no. To determine the maximal noncytotoxic concentrations of various
AF182273) from first-strand cDNA was performed using: forward, inhibitors in NHEKs for the inhibition of the respective enzymes, the
5Ј-CCCAGACTTGGCCATGGAAACC-3Ј; and reverse, 5Ј-GAG- cytotoxicity of concentrations ranging from 25 ␮M to 25 mM were
GTCTCTGGTGTTCTCAG-3Ј primers that annealed to nucleotides examined. Cytotoxicity was determined using an impermeable DNA
in exons 1 and 13. PCR was carried out in a total reaction volume of binding dye (YoYo-1), as we have described previously (Vyas et al.,
50 ␮l consisting of 5ϫ PCR buffer with 1.5 mM MgCl2, 1 ␮l, 5 pmol 2005).
of each primer, 0.2 mM dNTP (Life Technologies, Gaithersburg,
ELISA Analysis of Drug/Metabolite-Protein Adducts. For-
MD), and 2.5 U of TaqDNA Polymerase (Expand High Fidelity PCR mation of covalent adducts following SMX or DDS exposure, in the
system; Boehringer Mannheim, Mannheim, Germany). PCR con- presence or absence of the selective enzyme inhibitors, was deter-
sisted of at initial denaturation at 94°C for 3 min followed by 35 mined by cultivating NHEKs (1 ϫ 106 cells) for 24 h in 50-ml
cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, centrifuge tubes containing 10 ml of complete growth medium. The
synthesis at 72°C for 90 s, and a final extension at 72°C for 10 min. concentrations of the selective inhibitors used were the maximum
No CYP3A4 was detected, so an aliquot of the first round product noncytotoxic concentrations of those inhibitors in NHEKs. Cells were
was PCR amplified for a second round with the same primers and then incubated with selective enzyme inhibitors for 3 h followed by
conditions to detect whether very small amounts of mRNA were SMX (1 mM ascorbic acid was added before the addition of SMX) or
present. CYP3A5 was amplified using: P1-forward, 5Ј-AACAGC- DDS treatment for 3 h (concentrations specified under Results).
CCAGCAAACAGCAGC-3Ј (forward); and P2-reverse, 5Ј-TAAGC- Following total 6-h incubation, tubes were centrifuged at 220g for 5
CCATCTTTATTTCAAGGT-3Ј primers as described previously min to pellet the cells. Covalent adducts were determined as previ-
(Kuehl et al., 2001), and product was detected after the first round
amplification.
Determination of Protein for CYP3A4 and CYP3A5 in
ously described (Vyas et al., 2005).
Immunocytochemistry. Drug/metabolite-protein covalent ad-
duct formation was visualized using immunofluorescence confocal
NHEKs. NHEKs, HaCaT cells, and human liver samples were re- microscopy. Cells were grown on collagen-coated (0.1 mg/ml) cover-

Protein Haptenation of Dapsone and Sulfamethoxazole
491
slips placed in Petri dishes containing 2 ml of complete growth data that did not pass the normality test. For the comparison be-
medium. After 24 h, cultures were subjected to different selective tween more than two groups, ANOVA and the Holm-Sidak method
enzyme inhibitors for 3 h (maximum noncytotoxic concentrations for multiple pair-wise comparisons were used. p Ͻ 0.05 was consid-
were used), followed by SMX or DDS treatment for 3 h (concentra- ered to be significant.
tions specified under Results). After the 3-h incubation, cells were
washed (three times) with phosphate-buffered saline (PBS; 0.05 M
sodium phosphate, 0.15 M NaCl, pH 7.4) and fixed for 20 min with
4% paraformaldehyde in PBS. After fixation, cultures were washed
Covalent Adduct Formation with DDS and SMX and
three times with PBS followed by blocking for 60 min with Tris-
casein buffer containing 0.3% Triton X-100 and overnight incubation
with the anti-DDS or anti-SMX antisera (1:500 diluted in blocking
buffer) at 4°C. Coverslips were then washed with PBS, incubated for
3 h at 37°C with the fluorochrome-conjugated secondary antibody
Results
Their Acetylated Metabolites in NHEKs. Protein hapte-
nation of the parent arylamine drugs, DDS and SMX, was
readily detected by both confocal microscopy and ELISA
(Figs. 2 and 3). N-Acetylation of SMX or DDS resulted in
(Alexa Fluor-488-labeled goat-anti-rabbit IgG, 1:500 diluted in block- marked attenuation of protein haptenation. As we have
ing buffer), and mounted on glass slides using Immunomount con-
taining antifade reagent.
noted previously, the amount of adduct formed with DDS
appears to be greater than that seen with SMX, although this
may represent differences in antisera affinity for the respec-
tive adducts.
Expression of Various Cytochromes P450 in Keratin-
ocytes. NHEKs and an immortalized keratinocyte cell line
(HaCaT) were probed for the presence of various CYP450
known to catalyze the formation of the arylhydroxylamine
metabolites of SMX and DDS in vitro. As shown in Fig. 4,
Fluorescence images were acquired with a Zeiss Laser Scanning
Microscope (LSM 510, Zeiss Axiovert stand, Zeiss 63ϫ and 20ϫ
objective lenses; Carl Zeiss GmbH, Jena, Germany) using excitation
at 488 nm. Emission was set to a long-pass filter at 505 nm.
Image Analysis. For imaging with the confocal laser scanning
microscope, laser attenuation, pinhole diameter, photomultiplier
sensitivity, and offset were kept constant for every set of experi-
ments. Images were acquired from three different view fields of each
slide. The obtained images were quantitatively analyzed for changes CYP2C9 mRNA was not detected in cells from cultures of
in fluorescence intensities within regions of interests (boxes drawn
over cell somata) using the NIH Image J software (Bethesda, MD).
Fluorescence values from minimum of three view fields consisting of
15 to 20 NHEK cells in each field from three different slides of each
treatment were averaged and expressed as mean (S.D.) fluorescence
intensity.
Statistical Analysis. Data are presented as mean (S.D.). Data
were analyzed using SigmaStat (Systat Software, Inc., Point Rich-
mond, CA). Statistical comparisons between two groups were made
using either Student’s t test (parametric method) for normalized
primary keratinocytes (NHEKs) and HaCaT cells using mul-
tiple dilutions of cDNA. However, immunoblot analysis sug-
gested the possible presence of CYP2C9 (a diffuse polypep-
tide band with approximately the same mobility as
recombinant CYP2C9) in the first sample of NHEKs, but that
was not seen in the second sample of cells from the same
patient or in HaCaT cells. Moreover, CYP2C8, CYP2C18, or
CYP2C19 were not detected in either cell type (Fig. 5). The
recombinant CYP2C protein standards were expressed in a
data or Friedman’s rank sum test (nonparametric method) for the yeast cDNA expression system as described previously (Gold-
Fig. 2. Protein haptenation in NHEKs incubated with DDS or MADDS. A, NHEKs were incubated in the presence of vehicle (1% DMSO) or 800 ␮M
DDS or MADDS for 24 h. Cells were then imaged using confocal microscopy (objective lens, 20ϫ) as described under Materials and Methods.
Micrographs are representative images from each incubation condition. B, images for control, DDS, and MADDS in A were analyzed by NIH Image
J software and fluorescence intensity from a minimum of three view fields of three different slides of each treatment (with 15–20 cells/field) were
averaged and expressed as mean (S.D.) fluorescence intensity (arbitrary units). Results were analyzed using ANOVA with the Holm-Sidak method for
multiple pair-wise comparisons. ء, p Ͻ 0.05 compared with control; ءء, p Ͻ 0.05 compared with control or MADDS. C, determination of covalent
adducts of DDS and MADDS (800 ␮M, 24 h) in NHEKs by ELISA as described under Materials and Methods. Data presented represent the mean (S.D.)
optical density of three different experiments having three replicates in each experiment. Data were analyzed using ANOVA with the Holm-Sidak test
for multiple pair-wise comparisons. ء, p Ͻ 0.05 compared with NHEKs incubated with vehicle or MADDS.

492
Vyas et al.
Fig. 3. Protein haptenation in NHEKs incubated with SMX or NASMX. A, NHEKs were incubated in the presence of vehicle (1% DMSO) or 800 ␮M
SMX or NASMX for 24 h. Cells were then imaged using confocal microscopy (objective lens, 63ϫ) as described under Materials and Methods.
Micrographs are representative images from each incubation condition. B, images for control, SMX, and NASMX in A were analyzed by NIH Image
J software and fluorescence intensity from a minimum of three view fields of three different slides of each treatment (with 5–10 cells/field) were
averaged and expressed as mean (S.D.) fluorescence intensity (arbitrary units). Results were analyzed using ANOVA with the Holm-Sidak method for
multiple pair-wise comparisons. ء, p Ͻ 0.05 compared with control and NASMX. C, determination of covalent adducts of SMX and NASMX (800 ␮M,
24 h) in NHEKs by ELISA as described under Materials and Methods. Data presented represent the mean (S.D.) optical density of three different
experiments having three replicates in each experiment. Data were analyzed using ANOVA with the Holm-Sidak test for multiple pairwise
comparisons. ء, p Ͻ 0.05 compared with NHEKs incubated with vehicle or NASMX.
Fig. 5. Immunoblot analysis for CYP2C9 protein expression in NHEKs
Fig. 4. Blot gel analysis of PCR products for CYP2C9 mRNA expression
and HaCaT cells. Fourth passage NHEKs and HaCaT cells were fraction-
in NHEKs and HaCaT cells. Fourth passage NHEKs (4ˆNHEK) and
ated by electrophoresis on SDS-polyacrylamide gels as described under
HaCaT (7ˆHaCaT) cells were probed for mRNA expression using
Materials and Methods and probed for CYP2C proteins using a polyclonal
CYP2C9-specific primers, as described under Materials and Methods.
antibody to CYP2C9, which recognizes all of the human CYP2C proteins.
Various dilutions of the cDNA were evaluated as denoted. Note the
Replicate sets of cells were analyzed (designated as first and second
absence of a band in both cell types corresponding to CYP2C9.
shipments). YE, yeast expressed (recombinant proteins).
stein et al., 1994). Interestingly, although both CYP3A4 and mRNA was also found to be expressed in both NHEKs and
CYP3A5 mRNA were observed in NHEKs, only CYP3A5 HaCaT cells (Fig. 7), whereas CYP2E1 protein was barely
mRNA was observed in HaCaT cells (Fig. 6A). Immunoblot detectable (data not shown).
analysis using an antibody specific for CYP3A5 yielded pos-
CYP3A4 Activity in NHEKs. After the addition of 7-BQ,
itive results in both NHEKs and HaCaT cells, as did an no increase in fluorescence above background (NHEKs alone)
antibody that recognizes both CYP3A4 and CYP3A5 (Fig. was observed (data not shown). In contrast, a concentration-
6B). However, the levels of CYP3A4 and CYP3A5 proteins dependent increase in fluorescence was observed when the
were much lower compared with the human livers. CYP2E1 expected metabolite (7-HQ) was added to NHEKs (data not

Protein Haptenation of Dapsone and Sulfamethoxazole
493
ELISA (Fig. 9). Similar results were obtained when inhibi-
tors were added simultaneously with SMX or DDS and incu-
bated for 3 h (data not shown). We also found that trolean-
domycin (a potent inhibitor of CYP3A4) and disulfiram
(CYP2E1 inhibitor) did not attenuate the protein haptena-
tion with either DDS or SMX (data not shown).
Discussion
The biotransformation of xenobiotics to reactive metabo-
lites is believed to be responsible for a wide range of adverse
reactions. Sulfonamides such as SMX and the sulfone DDS
have been reported to be bioactivated to arylhydroxylamine
metabolites, which readily auto-oxidize to arylnitroso species
(Fig. 1). These metabolites are believed to initiate the cascade
of events that ultimately provoke a cutaneous drug reaction
(Cribb et al., 1996b; Svensson, 2003). We have proposed
previously that bioactivation in the skin may play an impor-
tant role in these reactions (Reilly et al., 2000). Indeed,
incubation of NHEKs with SMX or DDS results in protein
haptenation (Roychowdhury et al., 2005).
In vitro studies have shown that the bioactivation of these
parent arylamine drugs to their arylhydroxylamine metabo-
lites may be mediated by various oxidizing enzymes such as
CYP2C9, CYP2E1, CYP3A4, and MPO (Cribb et al., 1990,
1995; Uetrecht, 1990; Mitra et al., 1995; Gill et al., 1999;
Winter et al., 2000). Several arylamine drugs, including pro-
cainamide, are oxidized to arylhydroxylamine metabolites by
COX-2 in vitro (Liu and Levy, 1998; Goebel et al., 1999).
Which of these enzymes, if any, mediates the bioactivation of
SMX and DDS in NHEKs is unknown.
Fig. 6. A, RT-PCR analysis for CYP3A4 and CYP3A5 mRNA expression
in NHEKs and HaCaT cells. Fourth passage NHEKs and HaCaT cells
were probed for mRNA expression using CYP3A4- and CYP3A5-specific
primers and the products resolved on agarose gels as described under
Materials and Methods. Note the absence of CYP3A4 mRNA in HaCaT
cells. B, immunoblot analysis for CYP3A expression in NHEKs, HaCaT
cells, and human livers. Fourth passage NHEKs, HaCaT cells, and hu-
man livers were probed for CYP3A proteins using a monoclonal antibody
that recognizes both CYP3A4 and CYP3A5 or an anti-CYP3A5-specific
antibody. Human liver microsomes from a CYP3A5 expressor (right) and
a nonexpressor (left) were run as positive controls.
To assess the importance of arylamine oxidation, we eval-
uated the impact of SMX and DDS N-acetylation on protein
haptenation in NHEKs. We found that the N-acetyl metab-
olites gave rise to a lower level of protein haptenation as
measured by both ELISA and confocal microscopy (Figs. 2
and 3). It should be noted that it is possible that the apparent
reduction in adduct formation could be secondary to a re-
duced affinity of the antisera for the adduct in the presence of
an acetyl group on the drug, as opposed to an actual reduc-
tion in the amount of adduct formed. Differentiation of these
two potential explanations could only be accomplished by the
characterization of and development of chemical methods for
the quantification of the drug-protein adducts.
Fig. 7. Quantitative RT-PCR assessment of CYP2E1 mRNA in NHEKs
and HaCaT cells. CYP2E1 mRNA was quantified in NHEKs and HaCaT
cells as described under Materials and Methods. CYP2E1 mRNA was
normalized to 18S rRNA. NHEKs represent data from second passage
cells from one patient (subject 1) and third passage cells from a different
patient (subject 2).
Before evaluating the effect of various enzyme inhibitors
on protein haptenation with these drugs, we sought to con-
firm the presence of the most probable enzymes for bioacti-
vation in NHEKs. Because HaCaT cells are widely used as an
alternative to primary cultures of NHEKs, together with our
shown), indicating that the metabolite is readily detected previous observation of protein haptenation in these cells
down to 5 ␮M in NHEK incubations. when exposed to SMX or DDS (Roychowdhury et al., 2005),
Covalent Adduct Formation of DDS and SMX in the we also examined the presence of CYP450 enzymes in this
Presence and Absence of Inhibitors of Various En- cell line. CYP2C9 appears to be the most important enzyme
zymes. The effect of various enzyme inhibitors on protein for bioactivating SMX and DDS in the human liver (Cribb et
haptenation in NHEKs exposed to SMX or DDS was evalu- al., 1995; Gill et al., 1999; Winter et al., 2000). We did observe
ated by both confocal microscopy and ELISA. For each inhib- a polypeptide band with the approximate mobility of recom-
itor, we used the highest concentration that did not cause binant CYP2C9 in the one sample of NHEKs. This band was
cytotoxicity in NHEKs. As shown in Fig. 8, a broad inhibitor not seen in a second sample of NHEKs from the same patient
of CYP450 (ABT) that inhibits CYP3A4 and CYP2C9, a se- or in either shipment of HaCaT cells. In addition, there was
lective inhibitor of CYP2E1 (DCE), and an inhibitor of COX no evidence of other members of the CYP2C family of en-
(INDO) failed to reduce the protein haptenation of DDS in zymes in either NHEKs or HaCaT cells when probed by
NHEK cells. Similar results were observed with SMX by immunoblot. Moreover, we did not detect the presence of

494
Vyas et al.
Fig. 8. Protein haptenation of DDS in NHEKs in the presence of inhibitors of CYP450s and cyclooxygenase. NHEKs were incubated for 3 h in
the presence of vehicle (1% DMSO), 5 mM ABT, 5 mM DCE, or 100 ␮M INDO. The concentrations of inhibitors selected were the maximal
concentrations that did not increase cell death in NHEKs under the incubation conditions. After preincubation with inhibitors, cells were
incubated for an additional 3 h with 250 ␮M DDS. Covalent adducts were quantified using confocal microscopy (A) or ELISA (B), as described
under Materials and Methods. A, quantification of adducts was performed as described under Materials and Methods. Control, NHEKs incubated
with vehicle (1% DMSO) alone. Results were analyzed using ANOVA with Holm-Sidak method for multiple pair-wise comparisons. ء, p Ͻ 0.05
compared with NHEKs incubated with vehicle alone. B, covalent adducts were determined by the ELISA as described under Materials and
Methods. Data presented represent the mean (S.D.) optical density of three different experiments having three replicates in each experiment.
Data were analyzed statistically using ANOVA with the Holm-Sidak test for multiple pair-wise comparisons. ء, p Ͻ 0.05 compared with NHEKs
incubated with vehicle alone.
CYP2C9 message in either cell type (Fig. 4). Yengi et al.
(2003) have previously reported the expression of CYP2C9,
CYP2C18, and CYP2C19 transcripts in human skin samples.
Because these investigators probed full thickness skin, it is
not possible to determine which skin cells gave rise to these
transcripts. Saeki et al. (2002) have observed the presence of
CYP2C transcripts in human keratinocytes from several sub-
jects. However, their use of nonspecific primers does not
permit identification of the specific CYP2C genes that were
expressed in these cells. Moreover, transcripts can be de-
tected in the absence of detectable protein. Although our data
suggest the possible presence of a low level of CYP2C9 pro-
tein in NHEKs, further studies are needed to more carefully
determine the expression of this enzyme in human skin.
The results shown in Fig. 6 suggest that CYP3A5 is the
protein detected in both NHEKs and HaCaT cells because it
was detected by an antibody that is immunospecific for
CYP3A5. Moreover, CYP3A5 mRNA was readily amplified
from NHEKs and HaCaT cells, whereas a second round of
PCR amplification was required to detect CYP3A4 mRNA
transcript in NHEK cells (Fig. 6B). To our knowledge, this
Fig. 9. Protein haptenation of SMX in NHEKs in the presence of inhib-
differential expression of CYP3A enzymes has not been dem-
onstrated previously in keratinocytes. The differential ex-
itors of CYP450s and cyclooxygenase. NHEKs were incubated for 3 h in
the presence of vehicle (1% DMSO), 5 mM ABT, 5 mM DCE, or 100 ␮M
pression of CYP3A4/5 proteins, especially much lower ex-
pression in NHEKs and HaCaT cells as compared with liver,
might suggest the minor role of these enzymes for the bioac-
tivation of these parent drugs in skin. Interestingly, we were
unable to demonstrate the metabolism of a CYP3A4 sub-
strate (7-BQ) in NHEKs, although its metabolite (7-HQ) was
readily detected when added directly to NHEKs. This sug-
gests a very low level of CYP3A4-mediated catalytic activity
INDO. The concentrations of inhibitors selected were the maximal con-
centrations that did not increase cell death in NHEKs under the incuba-
tion conditions. After preincubation with inhibitors, cells were incubated
for an additional 3 h with 250 ␮M SMX. Covalent adducts were quantified
using ELISA as described under Materials and Methods. Data presented
represent the mean (S.D.) optical density of three different experiments
having three replicates in each experiment. Data were analyzed statisti-
cally using ANOVA with the Holm-Sidak test for multiple pair-wise
comparisons. ء, p Ͻ 0.05 compared with NHEKs incubated with vehicle
alone.

Protein Haptenation of Dapsone and Sulfamethoxazole
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in these cells. We were also able to demonstrate the presence
of CYP2E1 mRNA in both NHEKs and HaCaT cell types (Fig.
7). This is consistent with the reports of other investigators
who have demonstrated the presence of CYP2E1 transcripts
and protein in NHEKs (Baron et al., 2001; Saeki et al., 2002).
Based on the mRNA and protein data, we concluded that
CYP2C9 was not a likely mediator of the bioactivation of
these drugs in NHEKs. Indeed, preliminary studies demon-
strated that sulfaphenazole, an inhibitor of CYP2C9, did not
inhibit protein haptenation in NHEKs exposed to SMX or
DDS (Wurster et al., 2004). To evaluate the role of CYP3A4/5
and CYP2E1 in the generation of drug-protein adducts, we
evaluated the effect of inhibitors of these enzymes (at their
maximum noncytotoxic concentration) on protein haptena-
tion in NHEKs exposed to SMX or DDS. Our results indicate
that a general inhibitor of CYP450, ABT [which has been
shown to completely (ϳ90%) inhibit most of the major
CYP450, such as CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4]
(Balani et al., 2002), did not reduce the covalent adducts
formed after exposure of NHEKs to DDS and SMX (Figs. 8
and 9). The selective inhibitor of CYP2E1 (DCE) also failed to
decrease adduct formation. Other inhibitors of CYP3A4
(troleandomycin) and CYP2E1 (disulfiram) also did not re-
duce the protein haptenation of DDS and SMX in NHEKs
(data not shown). These results suggest that CYP450 does
not play a major role in the oxidative metabolism of these
drugs in NHEKs. In addition, although COX-2 has been
shown to mediate the oxidation of arylamines in vitro, an
inhibitor of cyclooxygenase (indomethacin) did not attenuate
the protein haptenation in NHEKs exposed to either DDS or
SMX (Figs. 8 and 9). This is consistent with our recent
observation that recombinant COX-2 does not mediate the
oxidation of these drugs (Vyas et al., 2006).
Hence, our studies confirm our previous observations that
NHEKs are able to bioactivate SMX and DDS, giving rise to
haptenated proteins. We found that CYP450 that are impor-
tant in the bioactivation of these drugs in the liver do not
appear to play a significant role in their bioactivation in
NHEKs. It should be noted, however, that recent data sug-
gests the level of CYP450 expression may differ as keratin-
ocytes differentiate (Du et al., 2006). Nevertheless, we ob-
served significant protein haptenation in NHEKs in the
absence of evidence for involvement of CYP450. These stud-
ies demonstrate that the role of various drug-metabolizing
enzymes in the bioactivation of drugs may vary from tissue to
tissue. Likewise, although COX-2 has been reported to oxi-
dize arylamines, neither COX-1 nor COX-2 appears to con-
tribute to the bioactivation of these drugs in NHEKs. In our
companion paper to this current investigation, we demon-
strate the role of flavin-containing monooxygenase and per-
oxidases in the bioactivation of these arylamine drugs in
keratinocytes.
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acterization of the formation and localization of sulfamethoxazole and dapsone-
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Acknowledgments
We acknowledge the assistance of William Wurster and Radita
Nemes in conducting preliminary studies in support of the project.
We also thank the staff of the Central Microscopy Research Facility
at The University of Iowa, which is supported by the Office of the
Vice President for Research, for technical assistance.
Svensson CK, Cowen EW, and Gaspari AA (2001) Cutaneous drug reactions. Phar-
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Address correspondence to: Dr. Craig K. Svensson, Dean, College of Phar-
macy, Nursing, and Health Sciences, Purdue University, West Lafayette, IN
47907. E-mail: svensson@purdue.edu