Reduction of sulfamethoxazole hydroxylamine (SMX-HA) by the mitochondrial amidoxime reducing component (mARC)

Article abstract of DOI:10.1021/tx500174u

Under high dose treatment with sulfamethoxazole (SMX)/trimethoprim (TMP), hypersensitivity reactions occur with a high incidence. The mechanism of this adverse drug reaction is not fully understood. Several steps in the toxification pathway of SMX were investigated. The aim of our study was to investigate the reduction of sulfamethoxazole hydroxylamine (SMX-HA) in this toxification pathway, which can possibly be catalyzed by the mARC-containing N-reductive enzyme system. Western blot analyses of subcellular fractions of porcine tissue were performed with antibodies against mARC-1, mARC-2, cytochrome b₅ type B, and NADH cytochrome b₅ reductase. Incubations of porcine and human subcellular tissue fractions and of the heterologously expressed human components of the N-reductive enzyme system were carried out with SMX-HA. mARC-1 and mARC-2 knockdown was performed in HEK-293 cells. Kinetic parameters of the heterologously expressed human protein variants V96L, A165T, M187 K, C246S, D247H, and M268I of mARC-1 and G244S and C245W of mARC-2 and N-reductive activity of 2SF, D14G, K16E, and T22A of cytochrome b₅ type B were analyzed. Western blot analyses were consistent with the hypothesis that the mARC-containing N-reductive enzyme system might be involved in the reduction of SMX-HA. In agreement with these results, highest reduction rates were found in mitochondrial subcellular fractions of porcine tissue and in the outer membrane vesicle (OMV) of human liver tissue. Knockdown studies in HEK-293 cells demonstrated that mARC-1 and mARC-2 were capable of reducing SMX-HA in cell metabolism. Investigations with the heterologously expressed human mARC-2 protein showed a higher catalytic efficiency toward SMX-HA than mARC-1, but none of the investigated human protein variants showed statistically significant differences of its N-reductive activity and was therefore likely to participate in the pathogenesis of hypersensitivity reaction under treatment with SMX. (Chemical Equation Presented).

Full text of DOI:10.1021/tx500174u

Article
pubs.acs.org/crt
Reduction of Sulfamethoxazole Hydroxylamine (SMX-HA) by the
Mitochondrial Amidoxime Reducing Component (mARC)
Gudrun Ott,† Birte Plitzko,† Carmen Krischkowski,† Debora Reichmann,‡ Florian Bittner,‡
Ralf R. Mendel,‡ Thomas Kunze,† Bernd Clement,*,† and Antje Havemeyer†
†Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel,
Gutenbergstrasse 76, D-24118 Kiel, Germany
‡Department of Plant Biology, Braunschweig University of Technology, Humboldtstrasse 1, D-38106 Braunschweig, Germany
ABSTRACT: Under high dose treatment with sulfamethoxazole
(SMX)/trimethoprim (TMP), hypersensitivity reactions occur with
a high incidence. The mechanism of this adverse drug reaction is
not fully understood. Several steps in the toxification pathway of
SMX were investigated. The aim of our study was to investigate the
reduction of sulfamethoxazole hydroxylamine (SMX-HA) in this
toxification pathway, which can possibly be catalyzed by the mARC-
containing N-reductive enzyme system. Western blot analyses of subcellular fractions of porcine tissue were performed with
antibodies against mARC-1, mARC-2, cytochrome b5 type B, and NADH cytochrome b5 reductase. Incubations of porcine and
human subcellular tissue fractions and of the heterologously expressed human components of the N-reductive enzyme system
were carried out with SMX-HA. mARC-1 and mARC-2 knockdown was performed in HEK-293 cells. Kinetic parameters of the
heterologously expressed human protein variants V96L, A165T, M187 K, C246S, D247H, and M268I of mARC-1 and G244S
and C245W of mARC-2 and N-reductive activity of 2SF, D14G, K16E, and T22A of cytochrome b5 type B were analyzed.
Western blot analyses were consistent with the hypothesis that the mARC-containing N-reductive enzyme system might be
involved in the reduction of SMX-HA. In agreement with these results, highest reduction rates were found in mitochondrial
subcellular fractions of porcine tissue and in the outer membrane vesicle (OMV) of human liver tissue. Knockdown studies in
HEK-293 cells demonstrated that mARC-1 and mARC-2 were capable of reducing SMX-HA in cell metabolism. Investigations
with the heterologously expressed human mARC-2 protein showed a higher catalytic efficiency toward SMX-HA than mARC-1,
but none of the investigated human protein variants showed statistically significant differences of its N-reductive activity and was
therefore likely to participate in the pathogenesis of hypersensitivity reaction under treatment with SMX.
Metabolites of approved drugs or the parent drugs
themselves that contain a primary aromatic amine structure
are often associated with a high incidence of idiosyncratic drug
reactions.12
For example, the 5-amino-2-nitrobenzotrifluoride metabolite
of the approved drug flutamide is a primary aromatic amine
metabolized to N-hydroxy-5-amino-2-nitrobenzotrifluoride,
which is considered to be involved in the hepatotoxicity of
flutamide.13 SMX is an example of an approved drug containing
a primary aromatic amine structure that is metabolized to SMX-
HA, a metabolite causing hypersensitivity reactions.
SMX is administered in a fixed combination with TMP for
the treatment of uncomplicated urinary tract infections and in
higher doses for the prophylaxis of Pneumocystis jirovecii
pneumonia. Patients receiving high-dose SMX/TMP show a
significantly higher incidence of adverse drug reactions;
especially HIV-infected patients show more frequent and
more severe reactions, for example, the potentially life
threatening Stevens-Johnsons syndrome (SJS).14,15 Actually,
SMX/TMP is also one alternative in the treatment of
INTRODUCTION
■
The mARC-containing N-reductive enzyme system consists of
one of the two mARC paralogues, mARC-1 (NP_073583.3) or
mARC-2 (NP_060368.2), cytochrome b5 type B
(NP_085056.2), and NADH cytochrome b5 reductase
(NP_015565.1).1 This enzyme system is able to catalyze the
reduction of N-hydroxylated substrates in the presence of
NADH.2 Several studies provided evidence that these structures
are reduced independent of the rest of their structural formula
like benzamidoxime, N-hydroxymelagatran, N-hydroxysulfona-
mides, N-hydroxy-valdecoxib, N-hydroxylated nucleobases and
nucleosides, upamostat (Mesupron), N-oxides, oximes, or N-
hydroxyamidinohydrazones.3−8 This variety of substrates
underlines the role of the mARC-containing enzyme system
in drug development and detoxification.7,9 A putatively
physiologic substrate of the mARC-containing N-reductive
enzyme system is the nitric oxide precursor Nω-hydroxy-L-
arginine (NOHA), which is involved in the complex regulation
of nitric oxide biosynthesis.10 All substrates are reduced under
aerobic conditions.2 Recently, the involvement of the mARC-
containing N-reductive enzyme system in the reduction of
nitrite to NO under anaerobic conditions was demonstrated.11
Received: May 9, 2014
Published: August 29, 2014
© 2014 American Chemical Society
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a
Scheme 1. Possible Participation of the mARC-Containing Enzyme System in the Metabolism of SMX
a
Metabolism and toxification pathways of SMX according to van der Ven et al.24 and Sanderson et al.48 Abbreviations: CYP, cytochrome P450; Cyt
b5 B, cytochrome b5 type B; Cyt b5 R, NADH cytochrome b5 reductase; NAT, N-acetyltransferase; UDPGT, uridine 5′-diphospho-
glucuronosyltransferase.
community acquired methicillin-resistant Staphylococcus aureus
(MRSA) skin and soft-tissue infections.16
NADH cytochrome b5 reductase, we aimed to add a more
detailed investigation of the reduction of SMX-HA with
particular focus on a possible contribution of the mARC-
containing enzyme system.1,34,35 Moreover, the influence of
protein variants encoded by nonsynonymous SNPs on this
reduction was likewise investigated in constitutive studies.
Much research is done to explain the mechanism of adverse
drug reactions under SMX, especially the hypersensitivity
reaction. Polymorphisms in several genes encoding enzymes in
the metabolic pathway of SMX were investigated, for example,
CYP2C9, GSTM1, GSTT1, GSTP1, and NAT2, the slow
acetylator genotype and phenotype, CYB5A, CYB5R3, and
NAT2, the NAT1 alleles *10 and *11, NAT2, and the
glutamate cysteine ligase catalytic subunit (GCLC).17−23 All
findings of these and many other studies enable a multifactorial
pathogenesis and not one explanatory cause.
MATERIALS AND METHODS
■
Chemicals. SMX-HA was purchased from Dalton Pharma Services
(Toronto, Kanada) and SMX from Sigma-Aldrich (Steinheim,
Germany).
Preparation of Subcellular Fractions. Subcellular fractions of
porcine liver and thyroid gland (i.e., mitochondria, microsomes,
cytosol, and homogenate and post nuclear supernatant (PNS),
respectively) were prepared by gradient centrifugation.7,36 Human
liver was obtained from the University of Kiel, Medical Department.
Prior consent of the local ethics committee and from the donors
before removal of the liver pieces was granted.33 Subcellular fractions
of human liver (i.e., outer membrane vesicle (OMV), mitochondria,
and homogenate) were prepared from pooled livers of cancer patients
in a similar manner to the method described by de Kroon et al.37
SDS PAGE and Western Blot Analysis. Protein (10, 12, or 15
μg) of each subcellular fraction of porcine thyroid gland and 25 μg of
protein of the total cellular protein of knockdown studies were
separated on a 12.5% SDS-PA gel under reducing conditions.38
Separated proteins were transferred to a PVDF membrane (Hybond-
P, Amersham GE Healthcare), blocked, and incubated with primary
antibodies against MOSC1 (=mARC-1, 1:10000 for the analysis of
subcellular fractions and 1:500 for the analysis of knockdown; Abgent,
San Diego, CA), MOSC2 (=mARC-2, 1:10000 for the analysis of
subcellular fractions and 1:500 for the analysis of knockdown, Sigma-
Aldrich, Taufkirchen, Germany), Cyt b5 B (1:10000; Sigma-Aldrich,
Taufkirchen, Germany), Cyt b5 R3 (1:1000; Acris antibodies, Herford,
Germany), VDAC2 (voltage dependent anion channel 2, 1:1000, Acris
antibodies, Herford, Germany), calnexin (1:10000; Acris antibodies,
Herford, Germany), and GAPDH (glycerinaldehyde-3-phosphate
dehydrogenase, 1:8000; Sigma-Aldrich, Taufkirchen). Incubation
with secondary horseradish peroxide conjugated anti-rabbit IgG or
anti-goat IgG antibodies (1:10000; Jackson immune Research
Laboratories, Suffolk United Kingdom) followed. Chemiluminescence
was detected by using the Amersham ECL Plus Western Blotting
Detection System (GE Healthcare, UNK) according to the
manufacturer’s protocol.
SMX is metabolically inactivated by cytochrome P450
monooxygenases, UDP-glucuronosyltransferases, and N-acetyl-
transferases 1 and 2.24 The latter represent the enzymes of the
main metabolic route of SMX. These enzymes acetylate the
drug to the inactive metabolite N-acetyl-sulfamethoxazole.25,26
In addition, a toxification pathway is known that consists of
several steps leading to a redox-cycle.27 SMX can be oxidized by
CYP2C9 to the toxic metabolite SMX-HA.25,28 In the following
step, SMX-HA is autoxidized to the nitroso intermediate, which
is highly reactive and able to bind to cellular proteins, finally
leading to an immune response.25,29−31 On the other hand,
nitroso-SMX can be reduced by glutathione to SMX-HA, which
can be detoxified to SMX by reduction.29 This step can possibly
be catalyzed by the mARC-containing N-reductive enzyme
(Scheme 1).
SMX-induced hypersensitivity is viewed as an idiosyncratic
adverse drug reaction.23 Polymorphisms in genes that encode
proteins involved in the reduction of SMX-HA to SMX might
be able to lead to increased toxicity. This reduction has been
investigated intensively regarding participation of the enzymes
cytochrome b5 type A and NADH cytochrome b5 reductase and
regarding the influence of some variant proteins.19,32 Ten years
ago, we already demonstrated that the reduction of SMX-HA is
catalyzed by porcine and human subcellular liver fractions.33
After discovering the mARC-containing enzyme system in our
laboratory, which is responsible for the reduction of N-
hydroxylated substrates, and based on the finding that it
consists of the components mARC, cytochrome b5 type B, and
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Expression, Purification and Determination of Protein,
Molybdenum, Heme and FAD Content of Recombinant
Proteins. Expression of mARC-1 and mARC-2 followed standard
protocol. Variants of MARC1, MARC2, and CYB5B were generated by
PCR mutagenesis using primers carrying the desired mutation.
Accuracy of PCR mutagenesis was confirmed by DNA sequencing
(GATC, Konstanz, Germany). mARC-1 and mARC-2 wild-type
proteins, variants V96L, A165T, M187 K, C246S, D247H, and
M268I of mARC-1, and variants G244S and C245W of mARC-2 were
expressed in Escherichia coli TP 1000 cells, an E. coli strain that is able
to express the eukaryotic form of the molybdenum cofactor (Moco).
Expression of cytochrome b5 type B wild-type protein and variants
2SF, D14G, K16E, and T22A was performed in E. coli DL41. Cells
were supplemented with 1 mM aminolevulinic acid to support heme
synthesis. Expression of NADH cytochrome b5 reductase was also
performed in E. coli DL41. Expressions in E. coli DL41 followed the
method described by Kurian et al.6,32,39
Protein concentrations were determined by BCA assay (Pierce BCA
Protein Assay Kits, Thermo Scientific, USA) after precipitating
proteins using Compat-Able protein assay preparation reagent set
(Thermo Scientific). Measurement of each duplicate was repeated
twice.
Molybdenum content of the mARC proteins was measured by
inductively coupled plasma mass spectrometry (ICP-MS). Measure-
ment of each duplicate was repeated three times.
defined concentrations of the synthetic reference substance (from 0.05
to 500 μM), which were incubated and worked up under the same
conditions as the experimental samples without adding cofactor and
using heat-denatured protein. The standard curves were linear in this
range with regression coefficients of 0.999 for SMX. Quantification
was performed on the basis of peak area. The obtained signals were
compared with those of the same amount of SMX dissolved in the
mobile phase. The recovery rate after incubation and sample work up
was 104 12%. The detection limit was about 0.5 μM. Characteristic
retention times were 10.3 0.5 min for SMX-HA and 12.9 0.6 min
for SMX.33
Cell Line and Reagents for Knockdown Experiments. HEK-
293 human embryonic kidney cells were purchased from Cell Lines
Service (Eppelheim, Germany). Opti-MEM, minimum essential
medium, sodium pyruvate solution, sodium bicarbonate, minimum
Eagles’s medium nonessential amino acids, FBS, trypsin, L-glutamine,
PBS, Lipofectamine RNAiMAX, Stealth Select RNAi siRNA targeting
human MOSC1 (MOSC1HSS127704), and Stealth Select RNAi
siRNA negative control were obtained from Invitrogen. ON-
TARGETplus SMARTpool siRNA targeting human MOSC2 was
purchased from Thermo Scientific. Complete protease inhibitor
cocktail was acquired from Roche Applied Science (Mannheim,
Germany).1
Cell Culture. HEK-293 cells were maintained in minimum essential
medium supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate, and 1.5 g/L sodium
bicarbonate. The cell line was incubated at 37 °C in 5% CO2.1
siRNA Transfection and Design of Knockdown Experiments.
HEK-293 cells were reverse transfected according to the manufac-
turer’s transfection protocol from Invitrogen. Briefly, siRNA oligos
were diluted in Opti-MEM in 24-well plates and incubated with
transfection reagent to form liposome−siRNA complexes. The cells
were trypsinized, counted, and suspended in culture medium. The cell
suspension was then added to the liposome−siRNA complexes. Effects
of the siRNA-mediated knockdown were examined on day 5 after
transfection. Negative controls included a nontargeting siRNA control
(scrambled siRNA) and transfection reagents without siRNA. Down-
regulation of protein expression was verified by Western blot analysis.1
Determination of the N-reduction of SMX-HA in HEK 293
cells. For N-reduction studies, culture medium was removed, and cells
were carefully washed and subsequently preincubated with substrate-
free incubation buffer (Hanks’ balanced salt solution containing 10
mM HEPES, pH 7.4) at 37 °C for 10 min. Then 250 μL of incubation
buffer containing 3 mM SMX-HA, 0.5% DMSO, and 1 mM ascorbic
acid was added to each well simultaneously, and cells were incubated
at 37 °C for 180 min. After the designated time, incubation medium
was carefully removed and centrifuged to eliminate cellular
contaminants and debris. The supernatant was analyzed by HPLC as
described above.1
Heme content was determined by measurement of difference
spectra according to Estrabrook and Werringloer, which is based on
the reduction of cytochrome b5 type B in the presence of NADH
cytochrome b5 reductase by NADH (Merck, Darmstadt, Germany).40
The assay was performed in duplicate.
FAD content of NADH cytochrome b5 reductase was determined
according to Whitby.41 The assay was performed in duplicate.
A detailed description of all methods was published.39
Determination of mARC-Catalyzed N-Reduction of SMX-HA.
Incubation mixtures with subcellular fractions contained 0.01−0.2 mg
of protein. Incubation mixtures with recombinant expressed proteins
were adjusted on the cofactors and consisted of mARC containing 120
pmol of molybdenum, cytochrome b5 type B containing 60 pmol of
heme, and NADH cytochrome b5 reductase containing 6 pmol of
FAD. Incubation mixtures contained different substrate concentrations
in 100 mM potassium phosphate buffer, pH 6.0 (subcellular fractions),
or 20 mM 4-morpholineethansulfonic acid (MES), pH 6.0
(recombinant proteins), 1 mM ascorbic acid, and 6.6% DMSO.
Incubations were performed under aerobic conditions at 37 °C. After 3
min of preincubation, the reaction was started by addition of 1 mM
NADH and stopped after 20 min by addition of ice-cold methanol.
Precipitated proteins were separated by centrifugation (5 min, 10000
rpm), and the amount of SMX in the supernatant was determined by
high-performance liquid chromatography (HPLC). Incubations were
carried out in duplicate, each of which was measured twice. Each
incubation was carried out in duplicate containing an external standard
with an amount of 5 μM SMX, each of which was measured twice.
Determination of kinetic parameters showed that the reduction
followed Michaelis−Menten kinetics. Initially, data was checked for
substrate inhibition by using Lineweaver−Burk plot. Kinetic
parameters Vmax and Km were determined through Michaelis−Menten
plot using SigmaPlot 11.0. In cases of substrate inhibition, kinetic
parameters were calculated from data in the linear range of the
Lineweaver−Burk plot.
HPLC Method for the Separation of SMX-HA and SMX. The
HPLC system consisted of a Waters 1525 HPLC pump, a Waters 717
autosampler, and a Waters 2487 dual absorbance detector combined
with Waters Breeze software, version 3.30. HPLC analysis was
performed on a Waters Symmetry C18 column (5 μm, 250 mm × 4.5
mm) and a Phenomenex Security Guard Cartridge System C18 (4 mm
× 3.0 mm). Elution was carried out isocratically with acetonitrile/
water/pure acetic acid/triethylamine (30:68.5:1:0.5, v/v/v/v). pH was
not adjusted. The flow rate was kept at 0.6 mL/min, and detection was
carried out at 254 nm. The recovery rate and quantification limit of the
metabolite SMX were determined using incubation mixtures with
Total Cellular Protein Extraction. To harvest cellular protein,
the medium was removed, and cells were washed with ice-cold PBS
and collected by centrifugation. After PBS was removed, cells were
resuspended in ice-cold lysis buffer (1% (v/v) Nonidet P-40, 150 mM
NaCl, 50 mM Tris, pH 8.0, protease inhibitor cocktail) and shaken for
60 min at 4 °C. Then cell lysates were cleared by centrifugation. The
supernatant contained the total cellular protein extract.1
Statistical Analysis. Data analysis of statistically significant
differences was performed with SigmaPlot 11.0 using t test or one
way ANOVA followed by a suitable post-hoc test with p < 0.05
considered significant.
RESULTS
■
HPLC Method for the Separation of SMX-HA and
SMX. The HPLC method for the separation and quantification
of SMX-HA and SMX is based on methods published earlier.33
Solubility of the SMX-HA was optimized according to Kurian
et al.32 The method is characterized by an accurate
quantification in the range of 0.05−500 μM SMX in the
presence of protein matrix and the cosubstrate NADH/NAD+.
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The separation of both compounds was achieved within 18
min. A representive chromatogram is shown in Figure 1.
Figure 1. HPLC analysis for the separation of SMX-HA and SMX.
Representive chromatogram of an incubation mixture containing
human recombinant expressed proteins.
N-Reductive Activity Is Enriched in Mitochondria. The
reduction of SMX-HA was investigated in subcellular fractions
of (i) porcine liver and thyroid gland and (ii) human liver.
Incubations with the purified subcellular fractions led to
formation of SMX, which was quantified by HPLC.
Mitochondrial fractions of both porcine tissues showed the
highest reduction rates (Figure 2A,B). This is in agreement
with the expression pattern of the components of the mARC-
containing N-reductive enzyme system (Figure 2B). For
positive control, incubations with our model substrate
benzamidoxime were carried out under the same conditions
as incubations with SMX-HA. In these control incubations, N-
reductive activity was solely enriched in mitochondrial fractions
as well. For both substrates, enrichment ratios were found in
the same range. Investigation of subcellular fractions of human
liver confirmed the result for porcine tissue. The highest
conversion rate of the reduction of SMX-HA was found in
OMV of human liver followed by conversion rate of the
mitochondrial fraction. In the homogenate, N-reduction of
SMX-HA was not detectable (Figure 3). Compared with the
model substrate benzamidoxime conversion rates were lower
for SMX-HA, but enrichment ratios were found in the same
range as well as for the model substrate (data not shown).
Reduction of SMX-HA Requires the Presence of All
Three Components of the Human N-Reductive Enzyme
System. Studying the reduction of SMX-HA with recombinant
expressed human enzymes revealed that the reaction was
dependent on NADH and that the highest conversion rates
were detectable with the complete enzyme system, that is,
mARC-1 or mARC-2, cytochrome b5 type B, and NADH
cytochrome b5 reductase. Lack of one component or the
replacement of cytochrome b5 type B for cytochrome b5 type A
resulted in a decreased or almost abrogated activity (Figure 4).
mARC-1 and mARC-2 Knockdown in HEK 293-cells.
siRNA-mediated mARC knockdown studies were carried out in
human cells to find out whether the mARC proteins were
involved in the reduction of SMX-HA in these cells. Specific
activities in HEK 293-cells were determined on day 5 after
transfection. Knockdown of mARC-1 affected the specific
activity of the reduction of SMX-HA significantly (p < 0.001)
compared with negative control (NC). Single knockdown of
mARC-2 did not have any influence on the reduction due to
the low expression of mARC-2 in HEK 293-cells. Double
knockdown of mARC-1 and mARC-2 affected the specific
activity of the reduction of SMX-HA significantly (p < 0.001)
compared with the single mARC-1 knockdown. The siRNA-
mediated down regulations of the proteins of interest were
verified by Western blot using anti-mARC-1, anti-mARC-2, and
anticalnexin antibodies (Figure 5).
Figure 2. Reduction of SMX-HA is enriched in mitochondrial
subfractions of porcine liver (A) and porcine thyroid gland (B).
Protein (0.01−0.2 mg) was incubated with 5 mM SMX-HA and 1 mM
NADH or NADPH in 100 mM potassium phosphate buffer, pH 6.0,
containing 1 mM ascorbic acid and 3.3% DMSO under aerobic
conditions. Incubation was carried out in duplicate, each measured
twice by HPLC. Western blot analyses of subcellular fractions of
porcine thyroid gland showed that the N-reductive activity was in
agreement with the presence of the components of the N-reductive
enzyme system. Abbreviations: MWM, molecular weight marker; Mt,
mitochondria; Ms, microsomes; Cyt, cytosol; PNS, postnuclear
supernatant; Hom, homogenate; VDAC, voltage dependent anion
channel; GAPDH, glycerinaldehyde-3-phosphate dehydrogenase.
human mARC wild-type (WT) proteins and variants,
cytochrome b5 type b and NADH cytochrome b5 reductase
and increasing amounts of substrate (0.1−10 mM) in the
presence of NADH. Arithmetic means of Vmax and Km and their
Kinetic Parameters of Human mARC WT and Variants.
Reduction of SMX-HA was carried out with recombinant
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Figure 3. Reduction of SMX-HA is enriched in the mitochondrial
subfraction and OMV of human liver. Protein (0.05 mg) was
incubated with 5 mM SMX-HA and 1 mM NADH in 100 mM
potassium phosphate buffer, pH 6.0, containing 1 mM ascorbic acid
and 6.6% DMSO under aerobic conditions. Incubation was carried out
in duplicate, each measured twice by HPLC. Abbreviations: OMV,
outer membrane vesicle; n.d., not detectable.
Figure 5. mARC-1 and mARC-2 knockdown in HEK-293 cells. HEK-
293 cells were transfected with 20 nM of scrambled (NC) siRNA or
mARC-1 or mARC-2 siRNA. For simultaneous knockdown, 10 nM
mARC-1 and 10 nM mARC-2 siRNA were transfected. Specific
activities in cells were determined as described in Materials and
Methods on day 5 after transfection. Results are presented as means
SD (n = 3): *p < 0.05; **p < 0.01; ***p < 0.001. The siRNA-
mediated down regulation of the proteins of interest was verified by
Western blot using anti-mARC-1, anti-mARC-2, or anticalnexin
(loading control) antibody.
Table 1. Vmax and Km Values of the Reduction of SMX-HA by
Human mARC WT and Variants
mARC
protein
number of
protein lots
Vmax (nmol SMX/(min mg
Km (mM
SMX-HA)
protein))
mARC-1
95 40
105 24
72 17
90 28
73 26
77 44
mARC-1
V96L
4
1
2
2
2
2
1
0.8 0.5
0.8 0.3
0.7 0.4
1.0 0.6
1.0 0.6
0.7 0.2
0.9 0.2
A165T
M187 K
C246S
D247H
M268I
Figure 4. Reduction of SMX-HA requires the presence of all three
components of the human N-reductive enzyme system. mARC
containing 120 pmol of molybdenum, cytochrome b5 containing 60
pmol of heme, and NADH cytochrome b5 reductase containing 6 pmol
of FAD were incubated with 3 mM SMX-HA and 1 mM NADH in 20
mM MES buffer, pH 6.0, containing 1 mM ascorbic acid and 6.6%
DMSO under aerobic conditions. Data are means of duplicates SD.
103
mARC-2
5
mARC-2
G244S
3
1
2
135 31
130 26
88 24
0.5 0.2
0.6 0.2
0.5 0.1
C245W
standard deviations are shown in Table 1. One way ANOVA
did not reveal any statistical differences in the kinetic
parameters of the variants compared with WT.
and C245W, and human cytochrome b5 type B variants 2SF,
D14G, K16E, and T22A were incubated with SMX-HA. Each
incubation contained a control with an incubation mixture of
the same mARC and cytochrome b5 type B WT proteins and
N-reductive activities obtained with protein variants were
related to these controls. By this, it became evident that none of
the cytochrome b5 type B variants affected the N-reductive
activity more than mARC variants alone (data not shown).
Catalytic efficiency was calculated as kcat/Km from kinetic
parameters. Arithmetic means of the values and their standard
deviations are shown in Figure 6. Catalytic efficiency of human
mARC-1 WT was 410
175 s−1 M−1. Statistically, catalytic
efficiency of human mARC-2 WT (840 260 s−1 M−1) was
significantly different compared with mARC-1 WT (p < 0.001, t
test, Sigma-Plot 11.0).
DISCUSSION
N-Reductive Activity of Incubation Mixtures Contain-
ing Human mARC and Cytochrome b5 Type B Variants.
All combinations of human mARC-1 variants V96L, A165T,
M187 K, C246S, D247H, and M268I, mARC-2 variants G244S
■
The mARC-containing N-reductive enzyme system is localized
in the outer mitochondrial membrane and able to catalyze the
reduction of N-hydroxylated substrates.2,34,42
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Reductions that take place in microsomal fractions usually
rely on NADPH as cosubstrate, whereas reductions that take
place in mitochondrial fractions usually require NADH as
cosubstrate as postulated in the hypothetical reaction cycle of
amidoxime reduction by mARC.2 Our results give evidence that
the reduction of SMX-HA is an NADH dependent reaction
(Figure 2).
The use of the porcine enzyme system in these initial
experiments successfully led to the hypothesis of an
involvement of the mARC-containing N-reductive enzyme
system in this reaction. Following studies were necessary to
verify this hypothesis for the human enzyme system.
In humans the main tissue involved in metabolism is the
liver. For the investigation of subcellular fractions, OMV,
mitochondria, and homogenate of human liver were available.
Results showed that highest reduction rates for SMX-HA were
detectable in OMV followed by mitochondria (Figure 3). This
result is in agreement with the results for our model substrate
benzamidoxime and with the results received from subcellular
porcine fraction using further substrates.3,7,10
Incubations with heterologously expressed human proteins
easily allow differentiation between the single components of
the N-reductive enzyme system and enable us to identify those
components that are essential for reduction in vitro. Results
showed that all three components of the N-reductive enzyme
system, that is, mARC-1 or mARC-2, cytochrome b5 type B,
and NADH cytochrome b5 reductase, had to be present for
exhibition of N-reductive activity. mARC-1 and mARC-2 were
indeed interchangeable, but the lack of one component led to
almost absent N-reductive activity (Figure 4).
In past years, several studies investigated the reduction of
SMX-HA to SMX with an enzyme system containing
cytochrome b5 type A and NADH cytochrome b5 reduc-
tase.32,45 The influence of protein variants encoded by SNPs in
the genes of these enzymes was ruled out, as well as the
influence of ascorbate and glutathione on the expression levels
of these enzymes.19,20,46,47 Our in vitro investigations likewise
showed that it was possible to substitute cytochrome b5 type B
for cytochrome b5 type A, albeit N-reductive activity was
decreased. In this respect, however, recent knock down studies
gave solid evidence that only cytochrome b5 type B is part of
the mARC-containing N-reductive enzyme system in vivo.1,35
The most important experiments supporting our hypothesis
analyzing the cell metabolism of SMX-HA in vivo were the
knockdown studies of mARC-1 and mARC-2 in HEK-293 cells.
Knockdown of mARC-1 in HEK-293 cells showed a significant
decrease in the reduction of SMX-HA, whereas the knockdown
of mARC-2 did not affect the reduction of SMX-HA in these
cells. This result was also observed for the model substrate
benzamidoxime and is attributed to the low expression of
mARC-2 in HEK-293 cells compared with the expression of
mARC-1.1 Interestingly, effects of a mARC-2 knockdown were
detectable when mARC-1 was also knocked down. The double
knockdown led to a further significant decrease in the reduction
rates of SMX-HA compared with the single knockdown of
mARC-1 (Figure 5).
Figure 6. Catalytic efficiency of human mARC WT and variants.
mARC containing 120 pmol of molybdenum, cytochrome b5
containing 60 pmol of heme, and NADH cytochrome b5 reductase
containing 6 pmol of FAD were incubated with 0.1−10 mM SMX-HA
and 1 mM NADH in 20 mM MES buffer, pH 6.0, containing 1 mM
ascorbic acid and 6.6% DMSO under aerobic conditions. Catalytic
efficiency was calculated from kinetic parameters. Data are means of
duplicates SD of the number of protein lots mentioned in Table 1.
WT proteins are presented as black bars and variant proteins as gray
bars.
A first assay for the proof of the participation of the mARC-
containing N-reductive enzyme system is the enrichment of
reductive activity in mitochondrial subcellular tissue fractions.
Therefore, our studies with the substrate SMX-HA started with
the investigations of subcellular tissue fractions of porcine liver
and thyroid gland. The liver plays a major role in metabolic and
detoxification pathways and represents the organ that is always
investigated first regarding its N-reductive activity.7 The thyroid
gland was chosen as an extrahepatic tissue showing noticeably
high N-reductive activity in subcellular mitochondrial frac-
tions.7 Furthermore, several publications report about hypo-
thyroidism under treatment with SMX/TMP.43 Probably
effects of sulfonamides on the thyroid gland depend on the
species to which the drug is administered. The thyroid gland of
rodents shows a greater sensitivity to derangement by drugs,
chemicals, and physiologic perturbations than the one of
human beings. Sensitive species like rat, dog, and mice more
frequently develop thyroid nodules after long-term treatment
with sulfonamides than insensitive species like monkey, guinea
pig, chicken and human beings.44 Our study is just able to give
a hint that the mARC-containing N-reductive enzyme system in
porcine thyroid is able to reduce SMX-HA. Further studies are
necessary to find whether it plays an important role. Our
investigations with the substrate SMX-HA showed that N-
reductive activity was only enriched in subcellular mitochon-
drial fractions of porcine liver and thyroid gland. Western blot
analyses of all components of the N-reductive enzyme system
showed that all of them were detectable in the subcellular
mitochondrial fraction. Control stains for mitochondria,
microsomes, and cytosol confirmed purity of the mitochondrial
and cytosolic fraction. Only the microsomal fraction showed
the slightest contamination with mitochondria and cytosol. N-
reductive activity was in agreement with the expression pattern
of the components of the mARC-containing N-reductive
enzyme system (Figure 2).
After ensuring that the mARC-containing N-reductive
enzyme system is capable of reducing SMX-HA to SMX in
cells, our studies continued with the in vitro investigation of
human heterologous expressed mARC-1, mARC-2, and
cytochrome b5 type B protein variants and their possible
influence on the N-reductive capacity. Six protein variants of
human mARC-1, two of human mARC-2, and four of human
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cytochrome b5 type B, whose effects on the reduction of our
model substrate benzamidoxime have already been analyzed,
were investigated for their reductive capacity toward SMX-
HA.1,39 The mARC-1 variants V96L, A165T, M187 K, C246S,
D247H, and M268I, which did not influence the reduction of
benzamidoxime, likewise showed unaltered activity when SMX-
HA was used as substrate.39 While the protein variants G244S
and C245W of mARC-2 showed a statistically significant
decrease in the catalytic efficiency of benzamidoxime reduction,
only a non-statistically significant decrease in the catalytic
efficiency of SMX-HA reduction was observed.39 Briefly
summarized, the results for the N-reductive activity of mARC
variants toward the substrate SMX-HA are in agreement with
the results concerning the model substrate benzamidoxime.
Furthermore, Km values for the reduction of SMX-HA were
determined in a range of 0.5 to 1 mM SMX-HA. Under high-
dose SMX/TMP treatment (3200 mg SMX per day) a
concentration of 0.02−0.023 mM SMX-HA occurs in the
blood volume for a typical adult (4.7−5 L), which fits in the
determined kinetic parameters.
The human cytochrome b5 type B variants S2F, D14G,
K16E, and T22A did not influence the reduction of
benzamidoxime and, in view of the results for the substrates
benzamidoxime and SMX-HA for the mARC variants, a
significant influence on the reduction of SMX-HA therefore
seems to be unlikely.1 Thus, N-reductive activity of the four
protein variants of cytochrome b5 type B were not only
investigated together with mARC wild-type proteins but also
with the respective mARC variants. The results showed that
protein variants of cytochrome b5 type B were not able to
influence N-reductive activity of mARC wild-type proteins
significantly. These results were expected and are consistent
with those published for the substrate benzamidoxime.
Additionally, it was not possible to induce a further decrease
of N-reductive activity when protein variants of cytochrome b5
type B were incubated together with protein variants of mARC.
Thus, the protein variants of cytochrome b5 type B are
incapable of inducing a relevant effect on the reduction of
SMX-HA as well.
Besides SMX-HA, only NOHA was reduced more efficiently by
mARC-2 compared with mARC-1.10 These results demand a
further explanation of the differences between mARC-1 and
mARC-2, which have to be a necessary part of further
investigations.
CONCLUSIONS
■
In this study, we demonstrated that the detoxification of SMX-
HA to SMX is catalyzed by the mARC-containing N-reductive
enzyme system. This hypothesis was proposed based on the
first results of the investigations of porcine subcellular tissue
fractions, further supported by investigations of subcellular
fractions of human liver, and finally mARC-1 and mARC-2
knockdown studies using HEK-293 cells gave in vivo evidence.
Thus, mARC plays an important role in the metabolism of
SMX and its metabolites. Recent studies were not able to detect
any effects of polymorphisms MARC1, MARC2, and CYB5B on
the N-reductive activity using the model substrate benzamidox-
ime. The encoded human protein variants had not been
investigated with the substrate SMX-HA so far. Therefore, the
ten heterologously expressed human protein variants encoded
by SNPs in MARC1, MARC2, and CYB5B were investigated in
this study, and a decrease in the reduction of SMX-HA was
excluded.
Our results allow the conclusion that the detoxification
pathway of SMX-HA to SMX is catalyzed by the mARC-
containing N-reductive enzyme system. In vitro, the hetero-
logously expressed human protein variants do not affect the
reduction of SMX-HA. It can be suspected that they might not
participate in the pathogenesis of hypersensitivity reactions
under treatment with SMX. But this hypothesis can only be
formulated very vaguely because further effects that might be
caused by influences on the expression levels of the proteins, by
anchoring of the protein in the outer mitochondrial membrane,
and by posttranslational modifications of the protein cannot be
excluded so far.
AUTHOR INFORMATION
■
All investigated protein variants were encoded by non-
synonymous SNPs in MARC1, MARC2, and CYB5B genes. A
previous study determined genotype frequencies of these
nonsynonymous SNPs in MARC1 and MARC2 in a Caucasian
cohort.39 Homozygous carriers of the variant genotype were
only detectable for one SNP in MARC1, which encodes the
protein variant A165T, with a frequency of 7.1%. Analyzing the
kinetic parameters of this important variant did not show a
statistically significant difference in the reduction of SMX-HA
compared with the wild-type protein. Therefore, this
investigation could not establish a connection between a
polymorphism in MARC1 and a decrease in reduction capacity
of SMX-HA that might possibly underlie the pathogenesis of
hypersensitivity reactions.
Corresponding Author
*Tel:+49-431-8801126. Fax: +49-431-8801352. E-mail:
bclement@pharmazie.uni-kiel.de.
Present Address
†Debora Reichmann: Universite
́
Grenoble-Alpes, iRTSV-
LCBM-Biocatalyse; CNRS, IRTSV-LCBM-Biocatalyse; CEA,
iRTSV-LCBM-Biocatalyse, 38000 Grenoble, France.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Financial support by the Deutsche Forschungsgemeinschaft
(DFG, Grant Number CL56/9-1, ME 1266/24-1) is greatly
appreciated.
The mARC proteins, mARC-1 and mARC-2, show over-
lapping substrate preferences.2 Results of incubations with
heterologously expressed proteins showed that most of the
investigated substrates, for example, N-hydroxylated nucleo-
bases and nucleosides, including the model substrate
benzamidoxime show a higher catalytic efficiency in the
reduction when incubations were performed with mARC-
1.6,7,39 The present investigation with SMX-HA is the second
that shows a statistically significant increase of catalytic
efficiency when incubations were performed with mARC-2.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Petra Koster and Sven Wichmann for their excellent
̈
technical assistance.
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Chemical Research in Toxicology
Article
trimethoprim-sulfamethoxazole in the ambulatory setting. Curr. Drug
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ABBREVIATIONS
■
Cyt b5 B/CYB5B, cytochrome b5 type B; Cyt b5 R/CYB5R3,
NADH cytochrome b5 type reductase; mARC/MARC,
mitochondrial amidoxime reducing component; Moco, molyb-
denum cofactor; SMX, sulfamethoxazole; SMX-HA, sulfame-
thoxazole hydroxylamine; SNP, single nucleotide polymor-
phism; TMP, trimethoprim; WT, wild-type
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