Enzyme-Mediated Protein Haptenation of Dapsone and
Sulfamethoxazole in Human Keratinocytes: II. Expression and
Role of Flavin-Containing Monooxygenases and Peroxidases
Piyush M. Vyas, Sanjoy Roychowdhury, Sevasti B. Koukouritaki, Ronald N. Hines,
Sharon K. Krueger, David E. Williams, William M. Nauseef, and Craig K. Svensson
Division of Pharmaceutics (P.M.V., S.R., C.K.S.), College of Pharmacy, and Inflammation Program, Department of Internal
Medicine, Carver College of Medicine (W.M.N.), University of Iowa and Veterans Administration Hospital, Iowa City, Iowa;
Department of Pediatrics and Pharmacology and Toxicology (S.B.K., R.N.H.), Medical College of Wisconsin and Children’s
Research Institute, Children’s Hospital and Health Systems, Milwaukee, Wisconsin; and Department of Environmental and
Molecular Toxicology and the Linus Pauling Institute (S.K.K., D.E.W.), Oregon State University, Corvallis, Oregon
Received April 7, 2006; accepted July 19, 2006
Arylamine compounds, such as sulfamethoxazole (SMX) and
dapsone (DDS), are metabolized in epidermal keratinocytes to
arylhydroxylamine metabolites that auto-oxidize to arylnitroso
derivatives, which in turn bind to cellular proteins and can act
as antigens/immunogens. Previous studies have demonstrated
that neither cytochromes P450 nor cyclooxygenases mediate
this bioactivation in normal human epidermal keratinocytes
(NHEKs). In this investigation, we demonstrated that methima-
zole (MMZ), a prototypical substrate of the flavin-containing
monooxygenases (FMOs), attenuated the protein haptenation
observed in NHEKs exposed to SMX or DDS. In addition,
recombinant FMO1 and FMO3 were able to bioactivate both
SMX and DDS, resulting in covalent adduct formation. Western
blot analysis confirmed the presence of FMO3 in NHEKs,
whereas FMO1 was not detectable. In addition to MMZ, 4-ami-
nobenzoic acid hydrazide (ABH) also attenuated SMX- and
DDS-dependent protein haptenation in NHEKs. ABH did not
alter the bioactivation of these drugs by recombinant FMO3,
suggesting its inhibitory effect in NHEKs was due to its known
ability to inhibit peroxidases. Studies confirmed the presence of
peroxidase activity in NHEKs; however, immunoblot analysis
and reverse transcription-polymerase chain reaction indicated
that myeloperoxidase, lactoperoxidase, and thyroid peroxidase
were absent. Thus, our results suggest an important role for
FMO3 and yet-to-be identified peroxidases in the bioactivation
of sulfonamides in NHEKs.
Biotransformation of nonreactive drugs or chemicals to
reactive intermediates or metabolites is believed to be an
important step in the provocation of numerous adverse drug
reactions, especially those that seem to be immune-mediated.
Although the liver is the major site for the generation of
reactive metabolites, organs other than the liver are often the
primary or secondary target for toxicity. Because the ability
of unstable metabolites to distribute from the liver to other
organs is questionable, it has been proposed that extrahe-
patic metabolism (e.g., in circulating cells) may be important
in mediating such toxic events (Uetrecht, 1992).
Skin eruptions after systemic drug administration repre-
sent one of the most commonly reported adverse drug reac-
tions. Many drugs associated with cutaneous drug reactions
(CDRs) are known to be metabolized to reactive metabolites
(Roychowdhury and Svensson, 2005). However, the survival
of such metabolites during transit from liver to the skin is
undocumented. We have suggested that bioactivation of
drugs in the skin may play an important role in the initiation
of CDRs (Reilly et al., 2000). Using sulfonamides as model
This work was supported in part by National Institutes of Health Grants
AI41395 and GM63821 (to C.K.S.), HL038650 (to D.E.W. and S.K.K.), and
CA53106 (to R.N.H.) and by the Department of the Veterans Administration
(Merit Review Grant to W.M.N.).
Article, publication date, and citation information can be found at
ABBREVIATIONS: CDR, cutaneous drug reaction; SMX, sulfamethoxazole; DDS, dapsone; S-NOH, sulfamethoxazole hydroxylamine; D-NOH,
dapsone hydroxylamine; NHEK, normal human epidermal keratinocyte; FMO, flavin-containing monooxygenase; PRX, peroxidase; KCZ, keto-
conazole; MMZ, methimazole; ABH, 4-aminobenzoicacid hydrazide; DAB, 3,3?-diaminobenzidine tetrahydrochloride; ELISA, enzyme-linked
immunosorbent assay; TPO, thyroid peroxidase; LPO, lactoperoxidase; MPO, myeloperoxidase; HRP, horseradish peroxidase; PBS, phosphate-
buffered saline; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcription; PCR, polymerase chain reaction; HEK, human embryonic
kidney; bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid; TNB, 5-thio-2-nitrobenzoic acid; TBS, Tris-buffered saline; ANOVA, analysis
of variance; DMSO, dimethyl sulfoxide.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 319:497–505, 2006
Vol. 319, No. 1
Printed in U.S.A.
compounds to test this hypothesis, we have shown the bioac-
tivation of sulfamethoxazole (SMX) and dapsone (DDS) to
their respective arylhydroxylamine metabolites (S-NOH, D-
NOH) in normal human epidermal keratinocytes (NHEKs)
(Reilly et al., 2000). Moreover, we have demonstrated that
incubation of such cells with the parent compounds gives rise
to protein haptenation (Roychowdhury et al., 2005).
Although the formation of the arylhydroxylamine metabo-
lites of these compounds in the liver is mediated primarily
via cytochromes P450 (Cribb et al., 1995; Mitra et al., 1995;
Winter et al., 2000), it is unclear what enzyme(s) bioactivate
these drugs in NHEKs. In the first of these two companion
articles, we demonstrate that cytochromes P450 do not play
a major role in the protein haptenation observed in NHEKs
exposed to SMX or DDS. In addition, although other inves-
tigators have reported that cyclooxygenase-2 is able to oxi-
dize arylamine compounds (Goebel et al., 1999), our results
indicate that neither cyclooxygenase-1 nor -2 is involved in
the protein haptenation observed in these cells (Vyas et al.,
2006). Hence, we probed the role of flavin-containing mono-
oxygenases (FMOs) and peroxidases (PRXs) in this phenom-
enon. Our results indicate that FMOs and PRXs are the
primary enzymes that bioactivate these drugs in NHEKs and
thereby generate haptenated proteins. Based on these find-
ings, extrapolation of observations in one organ of bioactiva-
tion to another (e.g., liver to skin) must be made cautiously.
Materials and Methods
Materials. DDS, SMX, ketoconazole (KCZ), methimazole (MMZ),
4-aminobenzoic acid hydrazide (ABH), magnesium chloride, NADP?,
glucose 6-phosphate, glucose-6-phosphate dehydrogenase, 3,3?-dia-
minobenzidine tetrahydrochloride (DAB), and rat tail collagen (type
I) were obtained from Sigma (St. Louis, MO). DDS and SMX hydrox-
ylamine metabolites were synthesized as described previously (Vyas
et al., 2005). Rabbit antisera were raised against SMX- and DDS-
keyhole limpet hemocyanin conjugates and specificity assessed as
described previously (Reilly et al., 2000). Normal human epidermal
keratinocytes and keratinocyte culture media were obtained from
CAMBREX (Walkersville, MD). Microtiter ELISA plates (96 wells)
were obtained from Rainin Instruments (Woburn, MA). Goat-anti-
rabbit IgG conjugated with Alexa fluor 488 and goat anti-rabbit
antibody conjugated with alkaline phosphatase, Amplex red reagent,
and YoYo-1 were purchased from Molecular Probes (Eugene, OR).
Mouse monoclonal antibody to human thyroid peroxidase (TPO) and
rabbit polyclonal anti-mouse IgG antibody conjugated to alkaline
phosphatase was purchased from Abcam (Cambridge, MA). Pure
TPO was obtained from Fitzgerald Industries International, Inc.
(Concord, MA). Human salivary gland mRNA, used as a positive
control for lactoperoxidase (LPO), was obtained from Clontech
(Mountain View, CA). A human promyelocytic cell line (PLB-985
cells) known to express myeloperoxidase (MPO) was obtained from
Dr. Tom Redo (Tucker et al., 1987) and used as a positive control for
MPO. Polyclonal antibodies raised against human FMO1 and FMO3
peptides and horseradish peroxidase-conjugated goat anti-rabbit IgG
were obtained from Gentest (Woburn, MA). Protein mol. wt. stan-
dards (bench mark prestained protein ladder) were from Invitrogen
(Carlsbad, CA). Donkey anti-rabbit-horseradish peroxidase (HRP)
antibody, nitrocellulose membranes, and enhanced chemilumines-
cence Western blotting kits were purchased from Amersham Phar-
macia Biotech (Arlington Heights, IL). The one-step nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate, micro-
bicinchoninic acid protein assay, and Bradford assay reagent were
purchased from Pierce Chemical Company (Rockford, IL). Immuno-
mount was obtained from Vector Laboratories (Burlingame, CA).
The pCEL1001 plasmid containing the Celera 19600414014330
cDNA representing full-length human FMO1, position ?61 to ?2172
inserted into pCMVSport 6 at the SpeI site, Trizol reagent, TOPO
cloning kits, and all reagents for baculovirus protein expression were
purchased from Invitrogen. RNeasy and One-Step RT-PCR kits were
obtained from Qiagen (Valencia, CA). Criterion precast acrylamide
gels and the Criterion Gel Electrophoresis system were purchased
from Bio-Rad (Hercules, CA). All other chemicals and reagents were
purchased from Sigma or Fisher Scientific (Chicago, IL).
Cell Culture. NHEK cells were cultured as detailed previously
(Reilly et al., 2000). In brief, cells were propagated in 75-cm2flasks
using basal media (KBM-2) supplemented with bovine pituitary
extract (7.5 mg/ml), human epidermal growth factor (0.1 ng/ml),
insulin (5 ?g/ml), hydrocortisone (0.5 ?g/ml), epinephrine, trans-
ferrin, gentamicin (50 ?g/ml), and amphotericin B (50 ng/ml)
(KGM-2) at 37°C in a humidified atmosphere containing 5% CO2.
Media was replaced every 2 to 3 days. When the cultures were nearly
confluent (70% to 90%), cells were disaggregated using 0.025% tryp-
sin/0.01% EDTA in HEPES buffer followed by neutralization with 2
volumes of trypsin neutralizing solution. Cell suspensions were then
centrifuged at 220g for 5 min followed by washing in KBM-2 and
resuspension in KGM-2. Cells were then either subcultured or cryo-
preserved for further purposes. All experiments were performed
using third to fourth passage cells.
Determination of KCZ, MMZ, and ABH Cytotoxicity in
NHEKs. To determine the maximal noncytotoxic concentrations of
KCZ, MMZ, and ABH in NHEKs to block the respective enzymes, the
cytotoxicity of concentrations ranging from 25 ?M to 25 mM was
examined. Cytotoxicity was determined using an impermeable DNA
binding dye (YoYo-1), as we have described previously (Vyas et al.,
ELISA Analysis of Drug/Metabolite-Protein Adducts. For-
mation of covalent adducts after SMX or DDS exposure, in the
presence or absence of KCZ, MMZ, or ABH, was determined by
cultivating NHEKs (1 ? 106cells) for 24 h in 50-ml centrifuge tubes
containing 10 ml of complete growth medium. The caps of the tubes
were slightly loosened to maintain the aerobic conditions. The con-
centrations of KCZ, MMZ, and ABH used were the maximum non-
cytotoxic concentrations determined in NHEKs. Cells were then
incubated with the compounds 3 h before or immediately before
addition of SMX (1 mM ascorbic acid was added before the addition
of SMX as we have previously reported that ascorbic acid increases
the protein haptenation in NHEKs; Roychowdhury et al., 2005) or
DDS treatment for 3 h (concentrations specified under Results).
Following the total 6- or 3-h incubations, tubes were centrifuged at
220g for 5 min to pellet the cells. Covalent adducts were determined
as described previously (Vyas et al., 2005).
Immunocytochemistry. Drug/metabolite-protein covalent ad-
duct formation was also visualized using immunofluorescence con-
focal microscopy. Cells were grown on collagen-coated (0.1 mg/ml)
coverslips placed in Petri dishes containing 2 ml of complete growth
medium. After 24 h, cultures were subjected to KCZ, MMZ, or ABH
for 3 h (maximum noncytotoxic concentrations were used), followed
by SMX or DDS treatment for 3 h (concentrations specified under
Results). After the total 6-h incubation, cells were washed (three
times) with phosphate-buffered saline (PBS; 0.05 M sodium phos-
phate, 0.15 M NaCl, pH 7.4) and fixed for 20 min with 4% parafor-
maldehyde in PBS. After fixation, cultures were washed 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 (Alexa
Fluor-488 labeled goat-anti-rabbit IgG, 1:500 diluted in blocking
buffer), and mounted on glass slides using Immunomount containing
Fluorescence images were acquired with a Zeiss Laser Scanning
Microscope (LSM 510, Zeiss Axiovert stand, Zeiss 63? and 20?
Vyas et al.
objective lens; Carl Zeiss GmbH, Jena, Germany) using excitation at
488 nm. Emission was set to a long-pass filter at 505 nm.
Image Analysis. For confocal laser scanning microscopy, laser
attenuation, pinhole diameter, photomultiplier sensitivity, and offset
were kept constant for every set of experiments. Images were ac-
quired from three different view fields of each slide. The obtained
images were quantitatively analyzed for changes in fluorescence
intensities within regions of interests (boxes drawn over cell somata)
using NIH Image J software (Bethesda, MD). Fluorescence values
from a 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.
Peroxidase Assay in NHEKs. PRX activity was determined
using the fluorescent dye Amplex red. In brief, 5 ? 106NHEK cells
were lysed in 0.5 ml of 10 mM sodium phosphate buffer, pH 7.4. The
cell lysate was centrifuged at 2040g for 2 min in a microcentrifuge to
obtain the supernatant fraction. Protein content of the supernatant
fraction was determined using Bradford assay. Various amount of
protein of the supernatant fraction (10–50 ?g) was mixed with 50
?M Amplex red reagent and 1 mM H2O2in a microtiter plate and
then incubated in the dark at room temperature for 1 h. Following
the incubation period of 1 h, the reactions were read in a fluorescence
plate reader (Cytofluor) at an excitation wavelength of 530 nm and
an emission wavelength of 580 nm. The PRX activity was determined
by measuring the observed fluorescence intensity.
The presence of PRXs was also determined by mixing 300 ?l of cell
lysate supernatant with 0.27 mM DAB and 1.4 mM H2O2in 100 mM
potassium acetate buffer, pH 5.0, in a microtiter plate. Following
incubation in the dark at room temperature for 24 h, absorbance at
450 nm was measured to assess the presence of PRXs in the NHEK
MPO Immunoblot Analysis. Lysates of NHEKs or PLB-985
cells and purified MPO were solubilized in SDS sample buffer and
separated by SDS-PAGE in 9% acrylamide gel. Immunoblots were
probed with rabbit polyclonal antibody to human MPO (Nauseef et
al., 1983) followed by secondary donkey anti-rabbit antibody conju-
gated to HRP (Amersham).
Reverse Transcription-Polymerase Chain Reaction (RT-
PCR) Amplification for MPO and LPO. Total RNA was isolated
from human salivary glands, NHEKs, PLB-985, and human embry-
onic kidney (HEK)-293 cells using a Qiagen RNeasy kit following the
manufacturer’s protocol. cDNA was generated using 1750 ng of total
RNA in a synthesis reaction using Qiagen One Step Reverse Tran-
scription (RT) with random hexamers according to the supplier’s
protocol. Polymerase chain reaction (PCR) amplification was per-
formed using the MPO-specific forward primer, 5?-AAC ATC ACC
ATC CGC AAC CAG AT-3?; and reverse primer, 5?-AAT GCA GGA
AGT GTA CTG CAG TT-3?, resulting in a 1.2-kb product, and the
LPO-specific forward primer, 5?-TCA TGC AG T GGG GTC AGA
TTG TGG A-3?; and reverse primer, 5?-CGG AAG GCG AAG GTG
AAG ACA TTG G-3?, resulting in a 744-bp product. PLB cells and
human salivary glands were used as a positive control for MPO and
LPO, respectively, and HEK cells were used as negative control for
both reactions. Glyceraldehyde-3-phosphate dehydrogenase was
used as housekeeping gene and was amplified from the keratinocyte
cDNA using the primers 5?-CCA CCA CCC TGT TGC TGT AGC-3?
and 5?-GGA TCC CCA CAG TCC ATG CCA-3? resulting in a 455-bp
fragment. Amplifications were performed in 50-?l reactions. Cycling
conditions for MPO and glyceraldehyde-3-phosphate dehydrogenase
consisted of an initial incubation at 50°C for 30 min followed by
denaturation at 95°C for 15 min, then 25 cycles of 94°C for 1 min,
53°C for 30 s, and 72°C for 2 min, and a final 72°C extension for 10
min. Cycling conditions for LPO consisted of an initial incubation at
50°C for 30 min followed by 37 cycles of 94°C for 2.5 min, 68°C for
30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min.
Reactions were analyzed on 0.7% agarose gels stained with ethidium
MPO Chlorination Assay. MPO-mediated hypochlorous acid
formation was measured using a previously reported method for
quantification of taurine chloramine (Kettle and Winterbourn, 1994).
In brief, 5 ? 107NHEK cells suspended in 10 ml of Hanks’ balanced
salt solution without Ca2?or Mg2?were incubated with 1 mM
diisopropylfluorophosphate to inhibit endogenous serine proteases.
Cells were washed, resuspended in 0.5 ml of relaxation buffer (10
mM PIPES, 3 mM NaCl, 3.5 mM MgCl2, 100 mM KCl, pH 7.3)
containing 1 mM ATP(Na)2and cavitated in 350 psi N2, as described
previously for neutrophils (Borregaard et al., 1983). The cavitate was
collected in relaxation buffer containing 1 mM EGTA and centri-
fuged at 200g for 10 min to remove unbroken cells and nuclei. The
postnuclear supernatant was centrifuged at 100,000g ? 20 min to
obtain cytosol and membrane for subsequent study. Reaction buffer
(Dulbecco’s PBS ? 20 mM taurine ? 1 mM glucose) was added to
cytosol and supernatant and 100 ?l of each were incubated at 37°C
for 90 min in the presence or absence of a cell-free H2O2-generating
system, composed of 10 mM acetaldehyde and 0.01 U of xanthine
oxidase in reaction buffer. The reaction was terminated by adding
200 U of catalase and the reaction mixture was cooled on ice for 10
min before the addition of 1 mM 5-thio-2-nitrobenzoic acid (TNB;
extinction coefficient ? 14,100 M/cm) and incubated for 5 min in the
dark at room temperature. The absorbance was measured at 412 nm
(A412) to assess the concentration of TNB in solution, and the hypo-
chlorous acid produced was calculated by determining the loss in
A412of the sample without acetaldehyde compared with the A412of
the sample with acetaldehyde and dividing by 28,200, the molar
extinction coefficient for 2 mol yellow TNB reacting with 1 mol
taurine chloramine to form 1 mol colorless 5,5?-dithiobis(2-nitroben-
TPO Immunoblot Analysis. NHEK cell lysate (75 ?g) and pu-
rified TPO were solubilized in SDS sample buffer and separated by
PAGE in 10% SDS gel. Immunoblots were probed with mouse mono-
clonal antibody to human TPO followed by secondary rabbit anti-
mouse-alkaline phosphatase-conjugated antibody. Nitro blue tetra-
zolium/5-bromo-4-chloro-3-indolyl phosphate was used as substrate
for the secondary antibody to determine the presence of TPO.
Expression of Human Recombinant FMO3 and FMO1. A
baculovirus/insect cell system was used for cloning and expression of
FMO3 and FMO1. FMO1 cDNA was amplified by PCR from
pCEL1001 using the forward primer, 5?-CAC CAT GGC CAA GCG
AGT TGC-3?, and the reverse primer, 5?-TTT TAC TTA TAG GAA
AAT CAG AAA AAT AG-3? (underlined sequences correspond to the
start and stop codon positions in the forward and reverse primers,
respectively). The resulting 1606-bp amplicon representing FMO1
sequences from ?98 to ?1703 (accession no. NM_002021) was cloned
into the pENTR/SD/D-TOPO plasmid using a TOPO cloning kit
The original full-length FMO3 cDNA was amplified from an adult
liver RNA sample using RT-PCR and the forward primer, 5?-TTG
GAC AGG ACG TAG ACA CA-3?, and reverse primer, 5?-TGG GTA
TTG TCA GTA ACT TTC A-3?. The resulting 1711-bp amplicon was
cloned into the pCR2.1 vector using AT cloning, resulting in
To move the FMO3 cDNA into the pENTR shuttle vector, PCR
amplification was performed using linearized pRNH696 as a tem-
plate and the forward primer, 5?-CAC CAT GGG GAA GAA AGT
G-3?, and reverse primer, 5?-GAT GAT TAG GTC AAC ACA AG-3?.
The resulting 1604-bp amplicon was then cloned into the pENTR/
SD/D-TOPO plasmid using the TOPO cloning kit. The resulting
cDNA clone, pRNH829, contains FMO3 sequences from position ?91
to ?1697 (accession no. NM_001002294). For both the FMO1 and
FMO3 shuttle vectors, the fidelity of the amplified products were
verified by complete DNA sequence analysis of the cDNA inserts.
Baculovirus and proteins were produced in ovary cells from Spo-
doptera frugiperda (Sf9) with the BaculoDirect system that uses
lambda phage integration sites for the recombination reaction with
BaculoDirect linear DNA. Once the primary virus was produced, it
FMOs and Peroxidases in Protein Haptenation in Keratinocytes
was amplified and proteins were expressed as described elsewhere
(Henderson et al., 2004; Krueger et al., 2005). FAD was added (10
?g/ml) to the cell culture media during protein production to ensure
that the level of this essential cofactor would not be limiting. Micro-
somes were prepared from cells harvested at approximately 96 h
postinfection. Protein concentration was determined by the Bradford
method, whereas the FMO content was determined by a high-per-
formance liquid chromatography-based method (Henderson et al.,
2004) that measures the FAD concentration. FAD concentration was
corrected for background content. Substrate-dependent NADPH ox-
idation by FMO1 and FMO3 was performed as described previously
(Henderson et al., 2004).
DDS- and SMX-Dependent Adduct Formation Catalyzed by
Human Recombinant FMO3 and FMO1. An incubation mixture
containing FMO3 or FMO1 (50 ?g of microsomal protein), MgCl2(3.3
mM), NADP?(0.065 mM), glucose 6-phosphate (3.3 mM), glucose-6-
phosphate dehydrogenase (0.1 U), and DDS or SMX (100 ?M) in
Tris-glycine buffer (50 mM, pH 9.5) was incubated for 1 h at 37°C.
Heat-inactivated FMO3 and FMO1 (90°C for 5 min) were used as
negative controls to account for nonspecific binding of the antisera in
this experiment. After 1 h of incubation, the reaction mixture was
left overnight at 4°C for complete adhesion of protein to the micro-
titer plate. After 24 h, covalent adducts were determined by an
adduct-specific ELISA as described previously (Reilly et al., 2000).
Determination of FMO3 and FMO1 Protein in NHEKs. SDS-
PAGE, using 10% resolving gels and a Tris-glycine running buffer,
was performed according to the method of Laemmli (1970). Fraction-
ation was carried out using the Criterion Precast Gel Electrophoresis
System (Bio-Rad), followed by electrophoretic transfer to a nitrocel-
lulose membrane using the Criterion Blotter (Bio-Rad) (Towbin et
al., 1979). Twenty micrograms of microsomal protein was used for
each sample of NHEKs. Nonspecific binding sites were blocked by
overnight incubation at 4°C with 5% nonfat dry milk in Tris-buffered
saline (TBS; 25 mM Tris, pH 7.5, 150 mM NaCl). The blots were then
incubated for 1 h at room temperature with FMO1 or FMO3 primary
antibody diluted 1:5000 in TBS containing 0.5% nonfat dry milk.
After extensive washing with several changes of TBS supplemented
with 0.1% Tween 20, the blots were incubated for 1 h at room
temperature with horseradish peroxidase-conjugated goat anti-rab-
bit IgG diluted 1:10,000 in TBS containing 0.5% nonfat dry milk.
Blots were then washed with several changes of TBS supplemented
with 0.1% Tween 20 and processed for detection by enhanced chemi-
luminescence according to the manufacturer’s instructions. The lu-
minescence produced was detected by exposure of Fuji Super RX film
(Fisher Scientific) and after digitizing the image using a Hewlett
Packard 6300C scanner (Boise, ID), the integrated OD of immuno-
reactive protein bands was determined using a Kodak DC120 digital
camera and Digital Science 1D version 3.0 software (New Haven,
CT). In all blots, bands corresponding to the protein of interest
(FMO1 or FMO3) were identified by reference to the baculovirus-
expressed FMO1 and FMO3 and mol. wt. standards.
D-NOH-Dependent Adduct Formation in the Presence of
Peroxidase Inhibitor and FMO Competitive Substrate. Protein
haptenation after D-NOH exposure to NHEK cells, in the presence or
absence of ABH or MMZ, was determined. In brief, NHEKs (1 ? 106
cells) were incubated for 24 h in 50-ml centrifuge tubes containing 10
ml of complete growth medium maintaining the sterile aerobic con-
ditions. Cells were then incubated with D-NOH (100 ?M) and im-
mediate addition of the PRX inhibitor (ABH, 5 mM) or the FMO
competitive substrate (MMZ, 5 mM). The samples were incubated for
3 h more followed by ELISA analysis for the covalent adducts as
described previously (Vyas et al., 2005).
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 the Student’s t test (parametric method) for normalized
data or Friedman’s rank sum test (nonparametric method) for the
data that did not pass the normality test. For comparisons between
more than two groups, ANOVA and the Holm-Sidak method for
multiple pair-wise comparisons were used. p ? 0.05 was accepted as
DDS- and SMX-Dependent Protein Haptenation in
the Presence and Absence of Peroxidase Inhibitors in
NHEKs. We have observed previously that DDS- and SMX-
dependent protein haptenation is dose-dependent (Roy-
chowdhury et al., 2005). Various oxidizing enzymes including
cytochromes P450, COX, FMOs, and PRXs might play an
important role in the bioactivation of these drugs in NHEKs.
In our previous studies, we found that cytochromes P450 and
COX do not play major roles in the bioactivation of these
drugs in NHEKs, which led to the focus in this study on the
role of PRXs and FMOs in mediating the bioactivation lead-
ing to protein haptenation. To determine the role of PRX-
mediated bioactivation of DDS and SMX, the effect of the
PRX inhibitors ABH and KCZ (Kettle et al., 1997; Cornejo et
al., 1998) on protein haptenation in NHEKs exposed to DDS
was evaluated by both confocal microscopy and ELISA. ABH
has been generally used for MPO inhibition, but as a hydra-
zide inhibitor, it also inhibits nonspecific PRXs (Ator et al.,
1987; Kettle et al., 1995). KCZ, although usually used for
CYP3A inhibition, has also been reported to inhibit PRXs
(Cornejo et al., 1998). For each inhibitor, we used the highest
inhibitor concentration that did not cause NHEK cytotoxic-
ity. As shown in Fig. 1, A and B, ABH and KCZ reduced
DDS-dependent protein haptenation in NHEK cells by 35 to
45% and 16 to 24%, respectively. ABH and MMZ caused
similar reductions in SMX-dependent protein haptenation
(data not shown). Similar results were obtained when inhib-
itors were added simultaneously with SMX or DDS and in-
cubated for 3 h (data not shown).
Presence of Peroxidases in NHEKs. Reduction of cova-
lent adduct formation by PRX inhibitors ABH and KCZ sug-
gested the presence of PRXs in NHEKs. To further explore
the presence of PRX activity in NHEKs, we used the Amplex
red reagent for measurement of PRX activity. In the presence
of H2O2, the Amplex red reagent reacts with PRXs to produce
the red fluorescent oxidation product, resorufin, which has
absorption and fluorescence emission maxima of approxi-
mately 571 and 585 nm, respectively. As shown in Fig. 2,
fluorescence increased with increasing amount of NHEK cell
lysate, confirming the presence of PRX activity in these cells.
HRP (100 ?U) was used as a positive control for activity and
the ability of ABH (5 mM) to inhibit HRP confirmed the
ability of ABH to inhibit PRXs (Fig. 2). It is noteworthy that
no measurable increase in fluorescence occurred in a cell-free
system in the absence of HRP (data not shown). The presence
of PRXs in NHEKs was also demonstrated by the DAB assay,
which gave rise to the brown precipitate expected from the
oxidation of DAB (data not shown).
MPO in NHEKs. The reduction in protein haptenation by
the PRX inhibitors, ABH and KCZ, together with the pres-
ence of PRX activity in NHEKs, suggested a role for PRXs in
DDS and SMX bioactivation. Because MPO has been shown
to bioactivate arylamines to their hydroxylamine metabolites
(Uetrecht et al., 1988), we probed for the presence of MPO in
NHEKs. Immunoblot analysis (Fig. 3) failed to detect the
presence of MPO. In contrast, MPO was readily detected in a
Vyas et al.
myeloid cell line known to express this PRX. The band in
NHEKs that comigrated with a band in pure MPO is believed
to be that of degradation products because of the heat treat-
ment of these samples. Thus, to probe further for the pres-
ence of MPO, we also used RT-PCR amplification and were
unable to observe MPO mRNA in NHEKs (data not shown).
In addition, we also failed to find evidence of MPO-like ac-
tivity in NHEKs using the chlorination assay (data not
LPO in NHEKs. The absence of MPO suggested other
PRXs may be responsible for DDS and SMX bioactivation.
Because LPO also has been shown to oxidize arylamines to
their nitroso metabolites (Gorlewska-Roberts et al., 2004), we
sought to determine the presence of LPO in NHEKs. LPO
mRNA was readily detected in human salivary glands by
RT-PCR amplification (positive control) but was nondetect-
able in NHEKs. The HEK cells also showed the absence of
LPO mRNA (negative control) (Fig. 4).
TPO in NHEKs. The absence of MPO and LPO led us to
assess whether or not TPO was present in NHEKs. TPO has
been shown to oxidize SMX to S-NOH (Gupta et al., 1992).
Thus, we probed the presence of TPO in NHEKs by immu-
noblot analysis. TPO was absent in NHEKs. Using purified
TPO as a positive control, the expected signal at 105 kDa was
observed, along with another less intense band at 35 kDa.
Neither protein was present in NHEK lysates from three
separate patient samples (data not shown).
DDS- and SMX-Dependent Protein Haptenation in
the Presence and Absence of FMO Inhibitors in
NHEKs. The effect of MMZ as a prototypical FMO substrate
was used to probe the potential role of this enzyme family on
SMX- and/or DDS-dependent protein haptenation in NHEKs.
Fig. 1. DDS-dependent protein haptenation in NHEKs in the presence of peroxidase inhibitors. NHEKs were incubated for 3 h in the presence of
vehicle (1% DMSO), 5 mM ABH, or 100 ?M KCZ. After preincubation with inhibitors, cells were incubated for an additional 3 h with 250 ?M DDS.
A, cells were immunostained followed by confocal imaging, and fluorescence intensity of cells exposed to vehicle, DDS, or DDS ? inhibitors was
determined by NIH Image J software. Control, NHEKs incubated with vehicle (1% DMSO) alone. Data presented represent the mean ? S.D.
fluorescence intensity of nine replicates. B, covalent adducts in cells exposed to vehicle, DDS, or DDS ? inhibitors as determined by ELISA. Data
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. ?, p ? 0.05 compared with NHEKs incubated with vehicle alone, ??, p ?
0.05 compared with NHEKs incubated with vehicle and DDS, ???, p ? 0.05 compared with NHEKs incubated with vehicle, DDS and DDS ? KCZ.
Fig. 2. Presence of peroxidases in NHEKs. Various amounts of NHEK
cell lysate supernatant fraction (10–50 ?g) were mixed with 50 ?M
Amplex red reagent and 1 mM H2O2in a microtiter plate. Following
incubation in the dark for 1 h at room temperature, fluorescence was
measured using an excitation wavelength of 530 nm and an emission
wavelength of 580 nm. Data represent mean ? S.D. of six replicates.
Fig. 3. Immunoblot analysis for MPO protein expression in NHEKs.
Fourth passage NHEK cells were probed for MPO proteins using specific
primary and secondary antibodies as described under Materials and
Methods. Lane 1, cultured myeloid cell line as a positive control showing
the presence of 90-kDa MPO precursor and 59-kDa MPO heavy subunit.
Lane 2, pure MPO showing the presence of the 59-kDa MPO heavy
subunit. Lanes 3 and 4, 105and 8 ? 105NHEK cells, respectively, in
which MPO is absent.
FMOs and Peroxidases in Protein Haptenation in Keratinocytes
The highest concentration of MMZ that did not cause cyto-
toxicity in NHEKs was used. As shown in Fig. 5, MMZ
reduced the DDS- and SMX-dependent protein haptenation
by 40 to 50% in NHEK cells when measured by ELISA.
Similar results were obtained when MMZ was added simul-
taneously with SMX or DDS and incubated for 3 h (data not
shown). Moreover, using confocal microscopy to detect hap-
tenated proteins (as opposed to ELISA) yielded similar re-
sults (data not shown).
DDS- and SMX-Dependent Adduct Formation Cata-
lyzed by Human Recombinant FMO3 and FMO1. To
further elucidate the possible role of FMO in metabolizing
these drugs, we studied the in vitro bioactivation of DDS and
SMX with recombinant FMO enzymes. Both recombinant
FMO3 and FMO1 were able to bioactivate DDS and SMX, as
shown by the formation of covalent adducts detected by
ELISA (Fig. 6). Incubation of DDS with FMO3 showed higher
adduct formation compared with FMO1. Heat inactivation of
recombinant FMO3 and FMO1 reduced adduct formation by
53 to 60% and 40 to 46%, respectively, compared with noni-
nactivated enzyme. Despite heat inactivation, significant ab-
sorbance was observed. This may be due to incomplete inac-
tivation of the enzymes or nonspecific binding of the antisera
to the recombinant proteins. Alternatively, there may be
some level of chemical-mediated oxidation of DDS that re-
sults in protein haptenation in the absence of enzyme activ-
ity. Preliminary studies have shown this latter effect occurs
to a low degree with bovine serum albumin (M. Farley, S.
Roychowdhury, P. M. Vyas, and C. K. Svensson, personal
communication). Significantly, no inhibition of FMO1 or
FMO3 catalyzed DDS-dependent protein haptenation was
observed when coincubated with ABH. The optical density
representing the protein haptenation of DDS by recombinant
FMO3 or FMO1 was 2.83 (?0.18) and 0.54 (?0.11), respec-
tively, whereas the same incubations in the presence of ABH
exhibited an optical density of 2.84 (?0.11) for FMO3 and
0.55 (?0.14) for FMO1.
Analysis of FMO in NHEKs. Immunoblot analysis was
used to determine the presence of FMO1 and FMO3 in
NHEKs. Other FMOs were not evaluated because FMO3 and
FMO1 are the major FMOs that play a role in the oxidation
of various xenobiotics (Chung et al., 2000). As shown in Fig.
7, FMO3 was readily detected, whereas FMO1 was undetect-
able in NHEK cells. Thus, it seems that only FMO3 is in-
volved in the observed bioactivation of DDS and SMX to their
respective hydroxylamine metabolites in human keratino-
D-NOH-Dependent Adduct Formation in Presence of
a PRX Inhibitor and an FMO Competitive Substrate.
Because the combination of MMZ and ABH were found to
inhibit protein haptenation in NHEKs to the same level as
that seen with either agent alone, we sought to determine
whether these compounds inhibited sequential steps in the
bioactivation leading to protein haptenation. For this reason,
we examined protein haptenation after D-NOH exposure in
NHEK cells, in the presence or absence of MMZ or ABH. As
shown in Fig. 8, ABH was able to reduce the protein hapte-
nation significantly when the cells were treated with D-NOH,
indicating a role for PRXs in the conversion of D-NOH to
dapsone nitroso. MMZ, however, did not reduce the protein
haptenation in NHEKs exposed to D-NOH.
Numerous xenobiotics have been reported to undergo bio-
activation to reactive metabolites that cause cellular toxicity
(Uetrecht, 1990; Svensson, 2003). Such metabolism to reac-
tive intermediates mostly is mediated through enzymatic or
Fig. 4. Northern blot analysis for LPO mRNA expression in NHEKs.
Fourth passage NHEK cells were probed for LPO mRNA expression as
described under Materials and Methods. Human salivary gland was used
as positive control. LPO mRNA was detected as a band at 744 bp. HEK
cells were used as negative control.
Fig. 5. SMX- and DDS-dependent pro-
tein haptenation in NHEKs in the pres-
ence of an FMO competitive substrate.
NHEKs were incubated for 3 h in the
presence of vehicle (1% DMSO) or 5 mM
MMZ. After preincubation with inhibitor,
cells were incubated for an additional 3 h
with 250 ?M DDS (A) or SMX (B). Cova-
ELISA as described under Materials and
Methods. Data represent the mean ?
S.D. optical density of three different ex-
periments with three replicates in each
experiment. Data were compared using
ANOVA with the Holm-Sidak test for
multiple pair-wise comparisons. ?, p ?
0.05 compared with NHEKs incubated
with vehicle alone. ??, p ? 0.05 compared
with NHEKs incubated with vehicle and
Vyas et al.
chemical oxidation to form the toxic intermediates capable of
binding cellular proteins to form covalent adducts (Lin et al.,
2006). Oxidation of SMX and DDS to their arylhydroxy-
lamine and then subsequently to arylnitroso metabolites is
believed to be a critical step in their ability to cause CDRs
(Naisbitt et al., 1999; Svensson et al., 2001). Various hepatic
enzymes, such as the cytochrome P450-dependent monooxy-
genases, cyclooxygenases, and PRXs, have been shown to be
active in SMX and DDS metabolism (Cribb et al., 1990; Mitra
et al., 1995; Goebel et al., 1999; Winter et al., 2000). Although
survival of reactive metabolites during transit from liver to
skin has not been demonstrated, evidence suggests the pres-
ence of the hydroxylamine metabolites of these drugs in
plasma and urine after therapeutic doses (Coleman et al.,
1992; Mitra et al., 1995; Winter et al., 2000). It is noteworthy
that the concentration of SMX in skin blisters of subjects
receiving this drug achieves 82% of those observed in plasma
(Krolicki, 2002). Hence, it has been demonstrated that the
parent compound readily penetrates to the epidermal layer of
We have proposed the bioactivation of such compounds in
the epidermal layer of the skin, primarily in keratinocytes
(Reilly et al., 2000). The ability of NHEKs to metabolize DDS
and SMX to their hydroxylamine metabolites with the sub-
sequent formation of cellular protein covalent adducts is
consistent with this hypothesis (Reilly et al., 2000; Roy-
chowdhury et al., 2005). Studies to identify the enzyme or
enzymes responsible for DDS and SMX bioactivation in
NHEKs indicated that neither the CYP450-dependent mono-
oxygenases nor cyclooxygenases are responsible for the ob-
served bioactivation (see companion article). Thus, we sought
to identify other enzymes that are known to oxidize
arylamines and might be involved in the bioactivation pro-
cess in keratinocytes.
PRXs have been shown to bioactivate arylamine drugs
resulting in the formation of arylhydroxylamine metabolites
(Uetrecht et al., 1988; Cribb et al., 1990). A potential role of
PRXs in the bioactivation of DDS and SMX in NHEKs was
demonstrated by the ability of the general PRX inhibitors,
ABH and KCZ (Kettle et al., 1997; Cornejo et al., 1998), to
significantly reduce DDS-dependent protein haptenation in
NHEKs (Fig. 1). Although both MPO and LPO have been
shown to specifically oxidize arylamine drugs to their
Gorlewska-Roberts et al., 2004), no data were found to sup-
port the presence of either enzyme in keratinocytes. Using
Fig. 6. Human recombinant FMO mediated DDS and
SMX adduct formation. Human recombinant FMO3 (A)
or FMO1 (B) was incubated with DDS or SMX (100 ?M)
in an NADPH-regenerating system for 1 h at 37°C as
mentioned under Materials and Methods. Heat-inacti-
vated FMO3 and FMO1 were used as negative controls
(gray bars) versus activated FMO3 and FMO1 (black
bars). Covalent adducts were determined by an adduct-
specific ELISA as described under Materials and Meth-
ods. Data represent mean ? S.D. of six replicates. ?, p ?
0.05 compared with the incubation containing heat in-
activated FMO3 or FMO1.
Fig. 7. FMO3 and FMO1 protein expression in NHEKs. Fourth passage
NHEK cells were probed for FMO1 and FMO3 proteins using specific
primary and secondary antibodies as described under Materials and
Methods. A, FMO1 expression: lanes 1 to 5, 25, 100, 250, 350, and 500
fmol of baculovirus-expressed human FMO1, respectively; lane 6, 40 ?g;
and lane 7, 60 ?g of NHEK proteins. B, FMO3 expression: lanes 1 to 5, 50,
75, 100, 250, and 500 fmol of baculovirus-expressed human FMO3, re-
spectively; lane 6, 40 ?g of NHEKs; and lane 7, 60 ?g of NHEK proteins.
Fig. 8. A, scheme representing the sequential bioactivation of DDS to
dapsone nitroso leading to protein haptenation. B, D-NOH-dependent
protein haptenation in NHEKs in the presence of a PRX inhibitor and an
FMO competitive substrate. NHEKs were incubated with 100 ?M D-
NOH for 3 h in the presence of 5 mM ABH or 5 mM MMZ. Covalent
adducts in cells exposed to vehicle (1% DMSO), D-NOH, or D-NOH ?
inhibitors were determined by adduct-specific ELISA as mentioned under
Materials and Methods. Data represent the mean ? S.D. optical density
of six replicates. Data were analyzed statistically using ANOVA with the
Holm-Sidak test for multiple pair-wise comparisons. ?, p ? 0.05 compared
with NHEKs incubated with vehicle alone, ??, p ? 0.05 compared with
NHEKs incubated with vehicle, D-NOH, and D-NOH ? MMZ.
FMOs and Peroxidases in Protein Haptenation in Keratinocytes
both immunoblotting and RT-PCR amplification, we failed to
find evidence for the expression of either MPO protein or
mRNA in NHEKs (Figs. 3 and 4), and MPO activity was not
observed using a chlorination assay. In addition, we found no
evidence for LPO mRNA in NHEKs.
The lack of evidence for MPO or LPO expression in
NHEKs, in contrast to the evidence for both general PRX
activity and SMX- and DDS-dependent protein haptenation,
suggests that other peroxidases may be involved in SMX and
DDS bioactivation in these cells. It is noteworthy that TPO
has been shown to oxidize SMX to its arylhydroxylamine
metabolite in vitro (Gupta et al., 1992). This observation,
together with evidence for the presence of hypothalamic-
pituitary-thyroid axis-related genes expressed in skin, sug-
gests TPO may be a good candidate for the PRX-dependent
bioactivation of these drugs in NHEKs (Slominski et al.,
2002). However, our results showing the absence of TPO in
NHEK cells suggest the presence of other oxidizing enzymes
that might be inhibited by ABH play a role in the metabolism
of these parent drugs.
Other bioactivating enzymes, such as FMOs, are important
for the oxidization of arylamine drugs (Cashman, 2000). Be-
cause previous studies have suggested the presence of FMOs
in keratinocytes (Janmohamed et al., 2001), we sought to
determine the role of these enzymes in the bioactivation of
SMX and DDS in NHEKs. Using an FMO substrate, MMZ
(Nace et al., 1997), we found a 40 to 50% reduction in the
protein haptenation for both DDS and SMX (Fig. 5). In vitro
studies demonstrated that recombinant FMO1 and FMO3
can oxidize SMX and DDS, although incubation with FMO3
resulted in a higher level of protein haptenation (Fig. 6).
Immunoblot analysis confirmed the presence of FMO3 in
these cells, whereas FMO1 was not detected (Fig. 7). It is
possible that the failure to detect this latter FMO is due to
the use of low-affinity antibodies. Although FMO3 (and pos-
sibly other unidentified FMOs) seems to be responsible for
this bioactivation, our results showing the inability of ABH to
inhibit the observed FMO3 activity (data not shown) and the
percent inhibition observed with the various inhibitors sug-
gested that unidentified PRXs or oxidizing enzymes are re-
sponsible for between 30 and 40% of SMX and DDS bioacti-
vation, whereas FMO3 is responsible for 50 to 60% of this
activity in keratinocytes.
Because we found that combined exposure of NHEKs to
MMZ and ABH was neither additive nor synergistic in its
ability to inhibit protein haptenation upon exposure to SMX
or DDS, we considered the potential that these compounds
attenuated sequential steps in the formation of covalent ad-
ducts in NHEKs. Upon formation, S-NOH and D-NOH can
undergo autooxidation leading to their respective nitroso spe-
cies. It is this latter species that is believed to be the penul-
timate metabolite for the haptenation of cellular proteins.
Our demonstration that ABH but not MMZ attenuates pro-
tein haptenation in NHEKs exposed to D-NOH suggests that
PRXs enhance the oxidation of the arylhydroxylamine to the
arylnitroso species (Fig. 8A).
The results reported herein, together with our companion
study, demonstrate that the relative role of various enzymes
in the bioactivation of SMX and DDS differ in keratinocytes
compared with liver. Although CYP2C9 and CYP3A4 are
important enzymes mediating the oxidation of these drugs in
liver, they do not play a significant role in keratinocytes.
Consistent with this conclusion, previous studies failed to
demonstrate a clear association between variant CYP2C9
alleles and the incidence of sulfonamide-induced CDRs (Pir-
mohamed et al., 2000).
Taken together, these data indicate that FMO3 plays an
important role in the oxidation of arylamines in NHEKs,
whereas PRXs play an important role in the subsequent
formation of the arylnitroso species. As functional variant
FMO3 alleles have been identified (Koukouritaki and Hines,
2005), it will be important to determine whether such vari-
ants influence the predisposition of individuals to sulfon-
amide-induced CDRs. Likewise, environmental and genetic
factors that alter the expression of these enzymes should be
probed for their potential role in altering the predisposition
to these reactions.
We acknowledge the technical assistance of Jamie Schlomann,
Sally McCormick, and Kevin Leidal in conducting studies for MPO
and LPO 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.
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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: firstname.lastname@example.org
FMOs and Peroxidases in Protein Haptenation in Keratinocytes