Chemical and Enzymatic Transformations of Nimesulide to GSH Conjugates through Reductive and Oxidative Mechanisms

Article abstract of DOI:10.1021/acs.chemrestox.5b00290

Nimesulide (NIM) is a nonsteroidal anti-inflammatory drug, and clinical treatment with NIM has been associated with severe hepatotoxicity. The bioactivation of nitro-reduced NIM (NIM-NH₂), a major NIM metabolite, has been thought to be responsible for the hepatotoxicity of NIM. However, we found that NIM-NH₂ did not induce toxic effects in primary rat hepatocytes. This study aimed to investigate other bioactivation pathways of NIM and evaluate their association with hepatotoxicity. After incubating NIM with NADPH- and GSH-supplemented human or rat liver microsomes, we identified two types of GSH conjugates: one was derived from the attachment of GSH to NIM-NH₂ (NIM-NH₂-GSH) and the other one was derived from a quinone-imine intermediate (NIM-OH-GSH). NIM-NH₂-GSH was generated not only by the oxidative activation of NIM-NH₂ but also from the reductive activation of NIM. Both NADPH and GSH could act as reducing agents. Moreover, aldehyde oxidase also participated in the reductive activation of NIM. NIM-OH-GSH was generated mainly from NIM via epoxidation with CYP1A2 as the main catalyzing enzyme. NIM was toxic to both primary human and rat hepatocytes, with IC₅₀ values of 213 and 40 μM, respectively. Inhibition of the oxidative and reductive activation of NIM by the nonspecific CYP inhibitor 1-aminobenzotriazole and selective aldehyde oxidase inhibitor estradiol did not protect the cells from NIM-mediated toxicity. Moreover, pretreating cells with l-buthionine-sulfoximine (a GSH depletor) did not affect the cytotoxicity of NIM. These results suggested that oxidative and reductive activation of NIM did not cause the hepatotoxicity and that the parent drug concentration was associated with the cytotoxicity.

Full text of DOI:10.1021/acs.chemrestox.5b00290

Article
pubs.acs.org/crt
Chemical and Enzymatic Transformations of Nimesulide to GSH
Conjugates through Reductive and Oxidative Mechanisms
Lei Zhou, Xiaoyan Pang, Cen Xie, Dafang Zhong, and Xiaoyan Chen*
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, P.R. China
S
* Supporting Information
ABSTRACT: Nimesulide (NIM) is a nonsteroidal anti-
inflammatory drug, and clinical treatment with NIM has
been associated with severe hepatotoxicity. The bioactivation
of nitro-reduced NIM (NIM-NH2), a major NIM metabolite,
has been thought to be responsible for the hepatotoxicity of
NIM. However, we found that NIM-NH2 did not induce toxic
effects in primary rat hepatocytes. This study aimed to
investigate other bioactivation pathways of NIM and evaluate
their association with hepatotoxicity. After incubating NIM
with NADPH- and GSH-supplemented human or rat liver
microsomes, we identified two types of GSH conjugates: one
was derived from the attachment of GSH to NIM-NH2 (NIM-
NH2-GSH) and the other one was derived from a quinone-imine intermediate (NIM-OH-GSH). NIM-NH2-GSH was generated
not only by the oxidative activation of NIM-NH2 but also from the reductive activation of NIM. Both NADPH and GSH could
act as reducing agents. Moreover, aldehyde oxidase also participated in the reductive activation of NIM. NIM-OH-GSH was
generated mainly from NIM via epoxidation with CYP1A2 as the main catalyzing enzyme. NIM was toxic to both primary human
and rat hepatocytes, with IC50 values of 213 and 40 μM, respectively. Inhibition of the oxidative and reductive activation of NIM
by the nonspecific CYP inhibitor 1-aminobenzotriazole and selective aldehyde oxidase inhibitor estradiol did not protect the cells
from NIM-mediated toxicity. Moreover, pretreating cells with L-buthionine-sulfoximine (a GSH depletor) did not affect the
cytotoxicity of NIM. These results suggested that oxidative and reductive activation of NIM did not cause the hepatotoxicity and
that the parent drug concentration was associated with the cytotoxicity.
other metabolic activation pathways for NIM. Mingatto et al.14
reported that a cytochrome P450 inhibitor, proadifen, could
INTRODUCTION
■
Nimesulide (NIM) is a nonsteroidal anti-inflammatory drug
stimulate NIM-induced cytotoxicity in isolated rat hepatocytes;
that selectively inhibits cyclooxygenase-2 (COX-2).1,2 Severe
thus, the parent drug by itself was presumed to be the main
and even fatal hepatotoxicity3−6 related to its use has been
factor responsible for the toxic effect. However, they did not
observed in clinical practice. Owing to the relatively significant
consider the possible effect of reductive activation on the
adverse reactions generated by it, NIM has been removed from
cytotoxicity of NIM. It is well-documented that reductive
the market in Finland and Spain. Bioactivation is one of the
activation of some nitroaromatic drugs is implicated in their
important mechanisms of drug-induced hepatotoxicity.7−11
toxicity.15−17 NIM has an aromatic-nitro group in its structure.
Studies have demonstrated that a major NIM metabolite,
Until now, there have been no literature reports on the
nitro-reduced NIM (NIM-NH2), was capable of forming an
reductive activation mechanism and the role of reductive
electrophilic diiminoquinone intermediate and reactive nitro-
activation in NIM-induced hepatotoxicity.
gen-based species, which were, respectively, mediated by
Thus, the current study aimed to evaluate the potential
P45012 and myeloperoxidase.13 The bioactivation of NIM-
bioactivation pathways of NIM and to conduct a preliminary
NH2 was presumed to be responsible for the hepatotoxicity of
investigation on the correlation between the formation of
NIM. However, our pilot experiment and a previous study14
reactive metabolites and the hepatotoxicity of NIM.
both demonstrated that NIM-NH2, even at an extremely high
concentration (1 mM), did not cause toxicity to primary rat
hepatocytes, although a large amount of GSH conjugates were
observed. This result suggested that bioactivation of NIM-NH2
was weakly correlated with the hepatotoxicity of NIM.
We preliminarily detected two types of GSH conjugates in
NADPH- and GSH-supplemented human liver microsomes
(HLM) incubated with NIM but only one GSH conjugate in
HLM incubated with NIM-NH2, indicating the presence of
MATERIALS AND METHODS
■
Materials. HLM, human liver cytosol (HLC), rat liver microsomes
(RLM), rat liver cytosol (RLC), and recombinant cytochrome P450
(CYP) enzymes CYP1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1,
Received: July 9, 2015
© XXXX American Chemical Society
A
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3A4, 3A5, and 4A11 were supplied by BD Gentest (Woburn, MA,
USA). NIM, microsomal epoxide hydrolase (MEH), GSH, NADPH,
ABT, 1-chloro-2,4-dinitrobenzene (CDNB), L-buthionine-sulfoximine
(BSO), estradiol, allopurinol, and ethacrynic acid were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Cryopreserved primary human
hepatocytes were obtained from Xeno-Tech (Lenexa, KS, USA). NIM-
NH2 and hydroxyl des-nitro NIM (NIM-OH) were synthesized
according to the methods of Li et al.12 and Macpherson et al.,
respectively;18 MS/MS data for the synthesized compounds were
identical to those reported in the literature.12,18 All other reagents and
solvents were either analytical or HPLC grade.
Instrumentation. Chromatographic separation and MS detection
were performed using an Acquity UPLC system (Waters Corp.,
Milford, MA, USA) and a TripleTOF 5600+ system (AB Sciex,
Concord, Ontario, Canada), respectively. Semipreparative HPLC
studies were performed using a Shimadzu LC-6AD pump equipped
with a UV detector. NMR experiments were conducted with a Bruker
Avance III 400 spectrometer.
Synthesis of the Nitroso Intermediate of NIM (NIM-NO). A
100 mg aliquot of NIM was dissolved in 25 mL of methanol; then, 5
mg of activated Pd/C (5% w/w) was added into the solution. The
mixture was degassed, refilled with hydrogen gas, and then stirred
under a hydrogen atmosphere for about 2 min, thereby generating a
solution containing NIM, NIM-NO, and NIM-NH2. The resulting
solution was filtered through double Whatman qualitative filter paper
coupled with bergmeal. NIM-NH2 was isolated from the mixed
solution using semipreparative liquid chromatography with a YMC
ODS-A C18 reversed-phase column (Shimadzu Corp., Kyoto, Japan);
methanol and water were used as the mobile phase (40:60 v/v).
However, the separation of NIM-NO and NIM failed, possibly owing
to their similar polarity.
Liver Microsome Incubations. Stock solutions of NIM, NIM-
NH2, and NIM-OH were dissolved in dimethyl sulfoxide (DMSO)
and then diluted to the desired concentration using phosphate-
buffered saline (PBS, 100 mM) containing MgCl2 (0.5 mM). The final
DMSO concentration was 0.1% (v/v).
NIM (5 μM), NIM-OH (50 nM or 250 nM), and NIM-NH2 (30
nM) were separately incubated with 1.0 mg/mL HLM or RLM in the
presence of NADPH (2.0 mM) and GSH (5.0 mM). The total
medium volume was 200 μL of PBS. GSH-free control samples were
also prepared. After 60 min of incubation at 37 °C in a water bath,
reactions were terminated by adding an equal volume of ice-cold
acetonitrile. Each incubation was performed in duplicate.
To investigate the formation pathway of NIM-NH2-GSH, we used
other incubation systems with either NIM (5 μM) or NIM-NH2 (30
nM) as the substrate: one system contained NADPH and GSH at
different concentration levels, whereas the other was an HLM
incubation system supplemented with NADPH, GSH, and a
nonspecific CYP inhibitor, ABT (1.0 mM). Moreover, to elucidate
the mechanism of NIM-OH-GSH formation, various concentrations of
MEH (1.0, 2.0, 5.0, and 10 mg/mL) were added into the HLM
incubations of NIM supplemented with NADPH and GSH.
Liver Cytosol Incubations. The incubation mixtures contained
NIM (5 μM), GSH (5.0 mM), HLC or RLC (1.0 mg/mL), and PBS
in a final volume of 200 μL. After 60 min of incubation in a water bath
at 37 °C, the reactions were terminated using an equal volume of ice-
cold acetonitrile. Cytosol-free control samples were also prepared. To
assess the involvement of aldehyde oxidase, its selective inhibitor,
estradiol (1 or 10 μM), was added into the cytosol incubations. Each
treatment was performed in duplicate.
metabolite in other incubations was then expressed as a percentage
relative to the highest level.
Cytotoxicity of NIM in Primary Human and Rat Hepatocytes.
Primary human hepatocytes were revived from cryopreservation and
seeded in a 48-well collagen I-coated plate (BD Gentest) at a density
of 6.0 × 105 cells/mL. The procedures involving hepatocyte isolation
from rats were performed in accordance with Guide for the Care and
Use of Laboratory Animals at Shanghai Institute of Materia Medica,
Chinese Academy of Sciences. Primary rat hepatocytes were prepared
according to a previously published two-step collagenase perfusion
technique19,20 with some modifications: the flow rates of the perfusion
and digestion buffers were set at 25 and 10 mL/min, respectively, and
collagenase type IV was used instead of type I. The fresh primary rat
hepatocytes were also seeded at a density of 6.0 × 105 cells/mL in a
48-well collagen I-coated plate. The primary human or rat hepatocytes
were then cultured for 4 h prior to the addition of NIM or NIM/
inhibitor mix. All cell incubations were performed in triplicate and
maintained at 37 °C in a humidified incubator containing 95% O2/5%
CO2.
After 24 h of incubation with various concentrations of NIM (10,
25, 50, 100, 200, and 500 μM), cell viability was determined using the
Cell Counting Kit-8 assay (Dojindo Molecular Technologies,
Gaithersburg, MD, USA) following the manufacturer’s protocol. In
addition, the absorbance data at 450 nm were collected using a 96-well
plate reader. The absorbance of the treated cells was compared with
that of the control, in which the cells were exposed only to the vehicle,
which were considered to be 100% viable.
Various inhibitors or the GSH depletor were added in the NIM
incubations to evaluate the relationship between bioactivation and
cytotoxicity. These reagents included nonspecific CYP inhibitor ABT
(1.0 mM), specific aldehyde oxidase inhibitor estradiol (10 μM), and
GSH depletor BSO (100 μM). Media from the triplicate incubations
were collected and pooled to improve the MS sensitivity. A 500 μL
aliquot of the pooled sample was precipitated with acetonitrile,
evaporated to dryness, and then reconstituted in 80 μL of a water/
methanol (90:10 v/v) solution. A 10 μL aliquot of the reconstituted
solution was then injected into the UPLC/TripleTOF 5600+ system
for analysis.
Sample Pretreatment. Unless otherwise noted, the sample was
pretreated by adding 300 μL of acetonitrile for every 300 μL of each
sample. The mixture was vortexed for 1 min and subsequently
centrifuged at 11 000g for 5 min. The supernatant was evaporated to
dryness at 40 °C in a nitrogen stream and then reconstituted in 80 μL
of a water/methanol (90:10 v/v) solution. A 10 μL aliquot of the
reconstituted solution was injected into the UPLC/TripleTOF 5600+
system for analysis.
UPLC/TripleTOF 5600+ MS Analysis. Chromatographic separa-
tion was conducted using Acquity UPLC HSS T3 column (100 mm ×
2.1 mm i.d., 1.8 μm; Waters) at 45 °C. The mobile phase was a
mixture of 5 mM ammonium acetate containing 0.05% formic acid (A)
and methanol (B) at a flow rate of 0.45 mL/min. The gradient elution
was started from 5% B and maintained for 1 min; the gradient was
increased linearly to 25% B in the next 7 min and then increased to
75% B for another 7 min. Afterward, the gradient was rapidly increased
to 99% B for 1 min, reduced to 5% B for 1 min, and, finally,
maintained at 5% B for 3 min to equilibrate the column.
MS detection was performed using a TripleTOF 5600+ mass
spectrometer (AB Sciex). The mass range was set at m/z 100−1000,
and the other parameters were set as follows: ion spray voltage, 5500
V; declustering potential, 80 V; ion source heater, 500 °C; curtain gas,
40 psi; ion source gas 1, 55 psi; and ion source gas 2, 55 psi. Collision
energies of 10 and 30 eV were used for TOF MS and product ion
scans, respectively, and a collision energy spread of 15 was used in the
MS/MS experiment. Information-dependent acquisition (IDA),
together with real-time multiple mass defect filter, was used to trigger
acquisition of MS/MS spectra. Unless otherwise noted, the
metabolites were detected in positive electrospray ionization mode
(ESI (+)).
Recombinant Human CYP Enzyme Incubations. NIM (5 μM)
and NIM-OH (5 μM) were separately incubated with recombinant
human CYP enzymes (CYP1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19,
2D6, 2E1, 3A4, 3A5, and 4A11; 50 pmol/mL) in the presence of
NADPH (2.0 mM) and GSH (5.0 mM). Incubation conditions were
similar to those for the microsomal incubations. The peak areas of
NIM-OH-GSH or NIM-OH were recorded to determine the relative
contribution of the selected CYP enzyme in the formation of these
metabolites. The highest area of each metabolite detected in the
incubation was normalized to 100%. The area of the specific
Statistical Analysis. Data are presented as the mean standard
deviation (SD). Data were compared using unpaired, two-tailed
B
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Figure 1. Extracted ion chromatograms of NIM-NH2-GSH in ESI (+) (left) and NIM-OH-GSH in ESI (−) (right) in NADPH- and GSH-
supplemented HLM with NIM (A), GSH-supplemented HLC with NIM (B), and NADPH- and GSH-supplemented HLM with NIM-NH2 (C).
Student’s t-test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 were
considered to be statistically significant. In the cytotoxicity experiment,
the media from the triplicate incubations were pooled; thus, the data
are presented as the mean of the triplicate incubations. The difference
in metabolite formation in the variously treated hepatocytes was
assessed by directly comparing the determined absolute values without
statistical significance tests.
RESULTS
■
Identification of GSH Conjugates in Liver Microsomes
and Liver Cytosol. The coincubation of NIM with NADPH-
and GSH-supplemented HLM revealed two GSH conjugates (I
and II). The representative UPLC/TripleTOF MS chromato-
grams of the GSH conjugates are illustrated in Figure 1A.
GSH conjugate I, with a retention time of 10.0 min, exhibited
a protonated molecule at m/z 584.149, which was 305.069 Da
larger than that of NIM-NH2 (m/z 279.080). This finding
suggested the attachment of one molecule of GSH to NIM-
NH2 and the removal of two hydrogen atoms. GSH conjugate I
was named NIM-NH2-GSH. Further fragmentation revealed
product ions at m/z 505.162, 376.118, 309.039, and 232.070
(Figure 2A). The fragment ions at m/z 505.162 and 309.039
Figure 2. Product ion scan spectra of NIM-NH2-GSH in ESI (+) (A)
and NIM-OH-GSH in ESI (−) (B). The inset is the tentative
fragmentation pattern.
were, respectively, formed from the removal of the methyl-
sulfonyl side chain and the C−S bond of the glutathionyl
moiety, whereas the product ion at m/z 376.118 was generated
from the cleavage of the pyroglutamic acid moiety and the
methylsulfonyl side chain. The ion at 232.070 was derived from
the cleavage of the methylsulfonyl side chain and the C−S
bond of the glutathionyl moiety.
The fragment ion at m/z 272.090 corresponded to γ-glutamyl-
dehydroalanyl-glycine. The MS signal of NIM-OH-GSH was
much higher in ESI (−) than in ESI (+); thus, we chose the
negative ionization mode to detect NIM-OH-GSH.
In the GSH-supplemented HLC incubation with NIM, NIM-
NH2-GSH was also detected, but no NIM-OH-GSH formed
(Figure 1B). Similar to the results in human liver subcellular
fraction incubations, NIM-NH2-GSH was found in incubations
in RLM and RLC, and NIM-OH-GSH was found in
incubations in RLM (Figure S1, Supporting Information). In
addition, when NIM-NH2 was used as substrate, only NIM-
NH2-GSH was detected in the NADPH- and GSH-
supplemented HLM incubation (Figure 1C). The formation
In addition, GSH conjugate II, with a retention time of 10.1
min, exhibited a deprotonated molecule at m/z 583.119, which
was 305.070 Da larger than that of NIM-OH (m/z 278.049).
This finding indicated the addition of one molecule of GSH to
NIM-OH and the removal of two hydrogen atoms. GSH
conjugate II was named NIM-OH-GSH. Figure 2B shows the
MS fragmentation patterns of NIM-OH-GSH. The fragment
ion at m/z 503.125 resulted from the loss of the methylsulfonyl
side chain, whereas the product ion at 310.022 was generated
via the cleavage of the C−S bond of the glutathionyl moiety.
C
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Figure 3. Extracted ion chromatograms of NIM-NH2-GSH (m/z 584.149) in various incubation systems with NIM (left) or NIM-NH2 (right) as the
substrate: nonenzymatic incubation containing NADPH and GSH only (A), NADPH- and GSH-supplemented HLM incubation (B), and HLM
incubation supplemented with NADPH, GSH, and ABT (C).
pathways of NIM-NH2-GSH and NIM-OH-GSH were further
characterized in the subsequent investigations.
Formation of NIM-NH2-GSH. To elucidate the formation
pathway of NIM-NH2-GSH, we incubated NIM or NIM-NH2
in various enzymatic and nonenzymatic systems. As shown in
Figure 3, when NIM (5 μM) was used as the substrate, NIM-
NH2-GSH was observed in the nonenzymatic incubation
containing NADPH and GSH and in the NADPH- and
GSH-supplemented HLM incubation with and without ABT;
the yield of every incubation was nearly the same. When NIM-
NH2 (30 nM) was used as the substrate, NIM-NH2-GSH was
generated only in the NADPH- and GSH-supplemented HLM
incubation, and its yield was nearly identical to that of the NIM
incubation. However, no GSH conjugate was detected in the
other two incubation systems. These results indicated that
NIM-NH2-GSH formation in incubations with NIM was
independent of P450s, whereas that in incubations with
NIM-NH2 was P450-mediated.
We further investigated the respective role of GSH and
NADPH on NIM-NH2-GSH formation in the nonenzymatic
incubation. When NIM was incubated with GSH at 37 °C for
60 min, only a trace amount of NIM-NH2 and NIM-NH2-GSH
was generated (Figure S2, Supporting Information). The yields
Figure 4. Effect of GSH concentration on the formation of NIM-NH2
and NIM-NH2-GSH in the incubation containing NIM and GSH (A).
of these metabolites were elevated with the increase of the GSH
concentration from 0.5 to 5.0 mM, and they did not change
with further increases in the GSH concentration (Figure 4A).
When NADPH was added into the NIM incubation containing
5.0 mM GSH, the yields of these metabolites were markedly
improved; the effect of NADPH on these metabolites’
formation was also concentration-dependent (Figure 4B).
Moreover, there was a good positive correlation between the
yield of NIM-NH2-GSH and that of NIM-NH2 in the
nonenzymatic incubations (R2 = 0.968).
Effect of NADPH concentration on the formation of NIM-NH2 and
NIM-NH2-GSH in the NIM incubation supplemented with 5.0 mM
GSH (B). The inset shows the correlation between NIM-NH2-GSH
and NIM-NH2 formation at NADPH concentrations of 0.2−2.0 mM.
The slope of the line represents the relative ratio of NIM-NH2-GSH to
NIM-NH2 formation at different NADPH concentrations.
13.9 min was detected (Figure 5A). This product displayed a
protonated molecule at m/z 293.060, which was 15.995 Da
smaller than the m/z 309.055 of NIM, indicating the removal of
O from NIM. Further fragmentation produced ions at m/z
215.082 and 185.084 (Figure S3, Supporting Information);
these ions were respectively derived from the loss of the
methylsulfonyl side chain and the further removal of the nitroso
Reactivity of NIM-NO with GSH. The solution produced
from the reaction that lasted for 2 min between NIM and
hydrogen was analyzed using UPLC/Triple TOF 5600+ MS.
Besides NIM and NIM-NH2, a product with a retention time of
D
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Figure 5. Extracted ion chromatograms of NIM-NO (left), NIM-NH2 (middle), and NIM-NH2-GSH (right) in different samples: NIM-NO solution
(A), a mixture of NIM-NO and GSH that was directly injected into the UPLC/TripleTOF 5600+ MS for analysis (B), and a mixture of NIM-NO
and GSH that was evaporated to dryness at 40 °C in a nitrogen stream before MS analysis (C). The NIM-NO used was a mixture of NIM-NO and
NIM in this experiment.
group. On the basis of the accurate molecular mass and MS
fragmentation patterns, we hypothesized that the product was
NIM-NO.
(Shimadzu Corp., Kyoto, Japan) with a mixture of acetonitrile
and water (20:80 v/v) as the mobile phase. The substitution
position of GSH on the sulfonaniline ring was characterized by
1
1H NMR analysis. Compared with the H NMR spectrum of
The reactivity of NIM-NO with GSH was evaluated by
adding GSH into the mixed solution containing NIM-NO and
NIM. After 5 min of incubation at 37 °C, an equal volume of
ice-cold acetonitrile was added. The resulting solution was
divided into two parts. One part was directly injected into the
UPLC/TripleTOF 5600+ MS for analysis; the other part was
evaporated to dryness at 40 °C in a nitrogen stream and then
reconstituted in an equal volume of an acetonitrile/water
(50:50 v/v) solution before MS analysis. In the presence of
GSH, NIM-NO disappeared, accompanied by the formation of
NIM-NH2 and NIM-NH2-GSH in both samples with different
pretreatments (Figure 5B,C). Differently, in addition to NIM-
NH2-GSH, another GSH conjugate with a retention time of
10.8 min was detected in the sample injected directly (Figure
5B). When a higher GSH concentration and a longer
incubation time were applied, the yield of the GSH conjugate
at 10.8 min declined and coincided with increased formation of
NIM-NH2 and NIM-NH2-GSH (data not shown).
MS fragmentation patterns of the GSH conjugate at 10.8 min
were examined. Product ions at m/z 376.121, 306.077, and
177.033 were observed (Figure S4, Supporting Information).
The product ion at m/z 376.121 was generated from the
removal of the pyroglutamic acid moiety and the methyl-
sulfonyl side chain. The fragment ions at m/z 306.077 and
177.033 were, respectively, derived from the cleavage of the N−
S bond and the further loss of the pyroglutamic acid moiety.
We presumed that GSH was attached to the N position rather
than the benzene ring because no characteristic product ions
formed by the cleavage of the cysteinyl C−S bond were
detected for this aromatic GSH conjugate.21
NIM-NH2, the proton signal at δ 6.40 ppm (dd, J = 8.4, 2.4 Hz,
1H) was missing in the 1H NMR spectrum of NIM-NH2-GSH
(Figure S5, Supporting Information), demonstrating that GSH
substitution occurred at position C5 of the sulfonaniline ring.
Involvement of Aldehyde Oxidase in Reductive
Activation. NIM-NH2-GSH was also detected in GSH-
supplemented HLC or RLC incubations, and the yield of the
GSH conjugate in the cytosol incubations was much higher
than that in the cytosol-free incubations, suggesting that a
cytosolic reductase might be involved in the reductive
activation. Aldehyde oxidase has been implicated in the
reduction of some nitro-containing drugs.22,23 Thus, we
evaluated the role of aldehyde oxidase in the reductive
activation of NIM. As shown in Figure 6, estradiol, a selective
aldehyde oxidase inhibitor,24 suppressed NIM-NH2 and NIM-
NH2-GSH formation in a concentration-dependent manner in
both HLC and RLC. Ten micromolar estradiol decreased the
yields of NIM-NH2 and NIM-NH2-GSH by 38 and 43%,
respectively, in HLC; it also reduced the formation of NIM-
NH2 and NIM-NH2-GSH by 44 and 53%, respectively, in RLC
at 10 μM.
Formation of NIM-OH-GSH. NIM-OH is one of the major
metabolites of NIM found in human urine.18 Owing to its
phenol group, NIM-OH can potentially form a quinone-imine
intermediate.25−29 Thus, we presumed that NIM-OH-GSH was
derived mainly from NIM-OH bioactivation. To verify our
hypothesis, we incubated NIM-OH in NADPH- and GSH-
supplemented HLM; the substrate concentration was low (50
nM), which was nearly identical to the NIM-OH yield formed
in the NADPH-supplemented HLM with NIM (5 μM).
Unexpectedly, almost no NIM-OH-GSH was discernible. At a
Moreover, NIM-NH2-GSH was isolated from the mixed
solution using a YMC ODS-AQ C18 reversed-phase column
E
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Table 1. Effect of MEH on the Formation of NIM-OH and
a
NIM-OH-GSH in HLM
metabolites (% of control SD)
MEH concentration (mg/mL)
NIM-OH
NIM-OH-GSH
0
100.0 4.1
88.8 6.4
100.0 3.9
82.9 5.3
1
2
81.4 3.0*
59.8 4.3**
37.5 1.2***
75.1 2.6*
55.7 2.6***
34.7 1.3***
5
10
a
Data are presented as the mean of two separate experiments. The
yield of each metabolite in the incubations without MEH (control
group) was normalized to 100%. The yields of the metabolites in other
incubations were expressed as a percentage relative to the control
group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs the control group.
Figure 6. Effect of estradiol (1 or 10 μM) on NIM-NH2 and NIM-
NH2-GSH formation in GSH-supplemented HLC (A) and RLC (B)
incubated with NIM. Data are reported as the mean of two separate
samples. *, P < 0.05; **, P < 0.01 vs the control group without any
inhibitor.
higher concentration (250 nM), the yield of NIM-OH-GSH in
the NIM-OH incubation was comparable with that in the NIM
incubation. These results indicated that NIM-OH-GSH was
formed mainly from NIM rather than from NIM-OH because
the yield of the GSH conjugate in the NIM-OH incubation was
much lower than that in the NIM incubation.
Figure 7. Formation of metabolites in incubations with recombinant
human CYP isozymes with NIM (A) and NIM-OH (B) as the
substrate. Data are displayed as the mean of two separate samples.
We predicted epoxidation30,31 to be the pathway of NIM-
OH-GSH formation from NIM. The epoxide of NIM possibly
exhibited a denitrated rearrangement32 to form a quinone-
imine intermediate, which could be trapped by GSH to form
NIM-OH-GSH or reduced to generate NIM-OH. To test this
possibility, various concentrations of MEH were added into the
HLM incubation of NIM in the presence of NADPH and GSH.
As shown in Table 1, MEH reduced the yields of NIM-OH and
NIM-OH-GSH in a concentration-dependent manner. At 10
mg/mL MEH concentration, the yields of NIM-OH and NIM-
OH-GSH were reduced by 62 and 65%, respectively. This
finding supported the hypothesis mentioned earlier.
NIM-OH-GSH was assessed. Multiple CYP isozymes, including
CYP2C19, CYP2D6, CYP1B1, CYP3A4, CYP2C9, CYP2B6,
and CYP1A2, were found to be involved in this bioactivation
pathway (Figure 7B).
Bioactivation and Cytotoxicity of NIM in Primary
Human and Rat Hepatocytes. Concentration-dependent
toxicity was observed in both primary human and rat
hepatocytes after incubation with NIM for 24 h (Figure 8).
The calculated IC50 values of NIM on human and rat cell
viability were approximately 213 and 40 μM, respectively
(calculated by GraphPad Prism 5.0).
To test the role of the oxidative and reductive activation of
NIM on its cytotoxicity, various inhibitors or a GSH depletor
were added into the primary rat hepatocytes’ culture medium to
examine changes in cytotoxicity. No obvious cytotoxicity was
observed in cells with the inhibitors or depletor alone.
Treatment with the nonspecific CYP inhibitor ABT reduced
cell viability by 28.3, 25.0, and 24.3% at NIM concentrations of
CYP Enzymes Involved in NIM-OH-GSH Formation.
NIM-OH-GSH was detected only in HLM compared with
HLC, indicating that P450 enzymes were involved in NIM-
OH-GSH formation. To identify the specific CYP enzyme, a
panel of recombinant human CYP isozymes was screened for
their activities. NIM-OH and NIM-OH-GSH were detected
mainly in the NIM incubation catalyzed by CYP1A2 and to a
lesser extent by CYP3A4 (Figure 7A). Furthermore, the
capability of CYP isozymes to oxidize NIM-OH to form
F
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Table 2. Formation of GSH Conjugates (%) in Primary Rat
a
Hepatocytes under Various Treatments
NIM concentration (μM)
GSH conjugates
NIM-NH2-GSH
treatment
hepatocyte
25
50
100
100
61.4
63.3
82.7
100
−
100
61.7
62.0
82.0
100
−
100
55.4
54.3
71.2
100
−
+ABT (1 mM)
+estradiol (10 μM)
+BSO (100 μM)
hepatocyte
NIM-OH-GSH
+ABT (1 mM)
+estradiol (10 μM)
+BSO (100 μM)
85.9
56.7
73.2
Figure 8. Toxic effects of NIM on the viability of primary human and
rat hepatocytes. Each column represents the mean of triplicate
samples. The cells that were exposed only to vehicle were considered
to be 100% viable. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs groups
with vehicle only.
79.3
87.3
85.2
a
The yields of NIM-NH2-GSH and NIM-OH-GSH in the incubations
without inhibitors or the GSH depletor (control group) were
considered to be 100%. The ratio of GSH conjugate formation in
other treatments was expressed as a percentage relative to the control
group.
25, 50, and 100 μM, respectively (Figure 9A). Analysis of the
culture media from the ABT-treated cells showed an increase in
incubations pretreated with the GSH depletor BSO (Figure
9A,B), although the yields of NIM-NH2-GSH and NIM-OH-
GSH were significantly attenuated (Table 2).
DISCUSSION
■
This study explored the potential metabolic activation pathways
of NIM, including reductive and oxidative activation, and
preliminarily evaluated the role of these metabolic activation
pathways in the hepatotoxicity of NIM.
Two types of GSH conjugates were identified in the HLM or
RLM incubation supplemented with NIM, NADPH, and GSH.
The high-resolution MS demonstrated that one conjugate was
formed by the attachment of GSH to NIM-NH2 (NIM-NH2-
GSH) and the other was derived from a quinone-imine
intermediate (NIM-OH-GSH). Theoretically, nucleophilic
addition of GSH may take place at the C3, C5, and C6
positions of the sulfonaniline ring of both GSH conjugates.
However, we detected only one peak for each type of GSH
conjugate. The C5 position was the least hindered position
compared with the other two positions;12 thus, these GSH
conjugates were presumed to be derived from the nucleophilic
attack of GSH at the C5 position. Other regioisomers were not
observed, possibly because their yields were too low to be
detected.
NIM-NH2-GSH was also generated in HLM incubations
with NIM-NH2 as substrate, and this process was P450-
dependent, consistent with the findings of a previous study.12
By contrast, NIM-NH2-GSH formation in HLM incubation
with NIM was apparently independent of P450s. A trace
amount of NIM-NH2 and NIM-NH2-GSH was observed in the
incubations containing only NIM and GSH, and the addition of
NADPH greatly accelerated the formation of these metabolites.
On the basis of the above-mentioned observations, we
presumed that NIM-NH2-GSH in the NIM incubations was
derived from reductive activation. Both GSH and NADPH
could act as a reductant, and the reduction ability of NADPH
was apparently higher than that of GSH.
Figure 9. Effects of the nonspecific CYP inhibitor ABT (1 mM),
aldehyde oxidase inhibitor estradiol (10 μM), and GSH depletor BSO
(100 μM) on the cytotoxicity (A) and remaining content of NIM (B)
in primary rat hepatocytes exposed to NIM (25, 50, and 100 μM). For
the cell viability, each column shows the mean of triplicate samples. *,
P < 0.05; **, P < 0.01 vs their respective incubations without
inhibitors or depletor. Remaining NIM content in various incubations
was expressed as a percentage relative to the initial content.
the remaining NIM content compared to that in the respective
incubation without ABT (Figure 9B). Additionally, ABT totally
inhibited NIM-OH-GSH formation, but it suppressed NIM-
NH2-GSH generation only by about 40% (Table 2). Treatment
with estradiol, a specific aldehyde oxidase inhibitor,24 reduced
NIM-NH2-GSH formation by about 40% in incubations with
NIM (Table 2). However, the cell viability was almost
unchanged in the incubations with estradiol compared with
those without estradiol (Figure 9A). In addition, a nearly
identical amount of NIM was observed in the incubations with
or without estradiol (Figure 9B). Furthermore, the cell viability
and the amount of NIM were almost unaltered in the
To investigate the mechanism of reductive activation, we
synthesized the reductive intermediate of NIM. In the reaction
system of NIM and hydrogen, NIM-NO and NIM-NH2 were
detected, but the hydroxylamine intermediate was not. Some
aromatic hydroxylamine compounds have been reported to
readily undergo disproportionation reactions to yield amide and
nitroso,33 which might explain the absence of the hydroxyl-
G
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Chemical Research in Toxicology
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Scheme 1. Proposed Metabolic Activation Pathways of NIM
amine intermediate of NIM. Much larger amounts of NIM-
NH2 and NIM-NH2-GSH were generated in the incubation
system containing NIM, NIM-NO, and GSH (Figure 5) than in
the incubation system containing NIM and GSH (Figure S2),
indicating the high reactivity of NIM-NO with GSH. In
addition to NIM-NH2 and NIM-NH2-GSH, another GSH
conjugate with a retention time of 10.8 min also formed.
However, the GSH conjugate was extremely unstable, with a
high propensity to transform into NIM-NH2 and NIM-NH2-
GSH. On the basis of its MS fragmentation patterns, the
attachment of GSH to the N position, forming sulphenamide,
was speculated. For NIM-NH2-GSH, the substitution of GSH
on the C5 position of the sulfonaniline ring was confirmed by
1H NMR analysis. Thus, we proposed a plausible mechanism
for the reductive activation of NIM. As displayed in Scheme 1,
NIM probably underwent reduction from nitro to nitroso, then
to hydroxylamine, and, finally, to amino. In the presence of
GSH, NIM-NO readily reacted with GSH, possibly leading to
the successive formation of a semimercaptal intermediate and a
sulphenamide intermediate.34 Some of the sulphenamide
intermediate might bind with GSH to give NIM-NH2, and
the rest might be rearranged to form NIM-NH2-GSH.
The formation of NIM-NH2 and NIM-NH2-GSH was also
observed in GSH-supplemented HLC or RLC incubations, and
the yields of these metabolites were significantly suppressed by
estradiol, a selective aldehyde oxidase inhibitor, indicating the
involvement of aldehyde oxidase in NIM reduction. It is worth
pointing out that estradiol did not chemically interfere with
NIM reduction because estradiol (1 and 10 μM) did not
change the yields of NIM-NH2 and NIM-NH2-GSH in the
NIM incubation supplemented with NADPH and GSH.
Xanthine oxidase, another important cytosolic enzyme, is
closely related to aldehyde oxidase.35 Allopurinol is a frequently
used specific inhibitor of xanthine oxidase.23 With the addition
of different concentrations of allopurinol (10 and 100 μM) in
the nonenzymatic incubations containing NIM, GSH, and
NADPH, the formation of NIM-NH2 and NIM-NH2-GSH
increased in a concentration-dependent manner (Figure S6,
Supporting Information). The reason for this may be that
allopurinol was also a good hydroxyl radical scavenger36,37 and
could prevent the conversion of the hydroxylamine back to
NIM. Thus, allopurinol was not a suitable inhibitor for the
evaluation of the role of xanthine oxidase in NIM reduction.
In addition to aldehyde oxidase and xanthine oxidase, we also
investigated the effect of glutathione S-transferase (GST) in
both HLC and RLC on NIM reductive activation. The yield of
NIM-NH2-GSH was not reduced in the cytosol incubations
with ethacrynic acid, a known inhibitor of some isoforms of
GST.38 Moreover, a positive correlation between the yield of
NIM-NH2-GSH and that of NIM-NH2 was found in the cytosol
incubations with different enzyme concentrations (data not
shown). These findings collectively demonstrated that GST did
not play a major role in the reductive activation of NIM.
Chang et al. reported that the quinoid metabolites of equine
estrogens were potent inhibitors of GST.39 The effect of
estradiol on the activity of GST was also evaluated in our
studies. The conjugation of 1-chloro-2,4-dinitrobenzene
(CDNB) with GSH was determined to measure the activity
of GST.39,40 It was found that the conjugation of CDNB with
GSH in HLC and RLC was not affected by estradiol even at
100 μM but that it was significantly reduced by ethacrynic acid
at 20 μM, indicating that estradiol was not an efficient inhibitor
of GST.
Moreover, we presumed that NIM-OH-GSH was derived
from the bioactivation of NIM-OH, one of the major
metabolites found in human urine;18 NIM-OH was anticipated
to form a quinone-imine intermediate owing to its phenol
group.25−29 However, our studies demonstrated that NIM-OH-
GSH was derived mainly from the NIM incubation rather than
from the NIM-OH incubation. Additionally, in the incubation
of NIM-NH2 with NADPH- and GSH-supplemented HLM, no
NIM-OH or NIM-OH-GSH was observed, excluding the
possibility that these metabolites were derived from the
oxidation of NIM-NH2. The presence of MEH reduced the
formation of both NIM-OH-GSH and NIM-OH in the NIM
incubations. Thus, a plausible mechanism of NIM bioactivation
H
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Chemical Research in Toxicology
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to form NIM-OH-GSH is depicted in Scheme 1. The first step
in this bioactivation possibly involved NIM epoxidation to yield
a nitro-epoxide, which was subsequently denitrated32 and
rearranged to form the quinone-imine. The resulting quinine-
imine could either react with GSH to generate NIM-OH-GSH
or be reduced to form NIM-OH.
NIM-OH-GSH formation was P450-dependent. CYP1A2
was the main enzyme catalyzing the generation of NIM-OH-
GSH and NIM-OH from NIM. However, NIM-OH-GSH
formation from NIM-OH was primarily CYP2C19-mediated.
These findings further indicated that NIM-OH-GSH in the
NIM incubation did not mainly originate from the oxidation of
NIM-OH. Otherwise, the collective catalysis of CYP1A2 and
2C19 was necessary for NIM-OH-GSH formation in the NIM
incubations.
Therefore, chronic administration of NIM should be avoided,
especially in patients who are susceptible to NIM accumulation.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.chemres-
tox.5b00290.
Extracted ion chromatograms of NIM-NH2-GSH and
NIM-OH-GSH in NADPH- and GSH-supplemented
RLM and GSH-supplemented RLC; extracted ion
chromatograms of NIM-NH2 and NIM-NH2-GSH in
incubations containing NIM and GSH; product ion scan
spectrum of NIM-NO; product ion scan spectrum of the
1
GSH conjugate with a retention time of 10.8 min; H
NIM was toxic to primary human and rat hepatocytes and
displayed a higher degree of toxicity in the latter, which was
probably due to species-specific differences in NIM-induced
hepatotoxicity. Furthermore, we evaluated the role of reductive
activation and oxidative activation in NIM-induced cytotoxicity.
Similar results were observed in primary human and rat
hepatocytes. Since rat hepatocytes were more susceptible to
NIM hepatotoxicity, we evaluated the relationship between
metabolic activation and cytotoxicity using rat hepatocytes.
Inhibition of the oxidative activation by the nonspecific CYP
inhibitor ABT increased the degree of cytotoxicity in primary
rat hepatocytes, which was consistent with the result reported
by Mingatto et al.14 In the incubations with ABT, we found an
increased level of nonmetabolized NIM, a total suppression of
NIM-OH-GSH generation, and a partial inhibition of NIM-
NH2-GSH formation; the remaining NIM-NH2-GSH was
presumed to be derived from reductive activation, which was
independent of P450s. Inhibition of the reductive activation by
selective aldehyde oxidase inhibitor estradiol did not protect the
cells from NIM-mediated toxicity. Estradiol reduced NIM-
NH2-GSH formation by about 40%. The incomplete inhibition
of the reductive activation by estradiol was presumably due to
the presence of biological reducing agents, such as NADPH and
GSH, and other nitroreductases from microsome and
mitochondria.22,41,42 Furthermore, cell treatment with the
GSH depletor BSO did not change the cytotoxicity in the
NIM incubations, although the formation of NIM-NH2-GSH
and NIM-OH-GSH was significantly suppressed. Together, all
of the evidence collectively showed that the reductive and
oxidative activation of NIM did not cause cytotoxicity in
primary rat hepatocytes. The sustained amount of non-
metabolized NIM correlated well with the cytotoxicity; thus,
the metabolic activation acted as the detoxifying pathways of
NIM.
Overall, we have characterized the reductive and oxidative
activation pathways of NIM. NIM-NH2-GSH was generated
not only by the oxidative activation of NIM-NH2 but also from
the reductive activation of NIM. Both NADPH and GSH could
act as reducing agents. Aldehyde oxidase was also involved in
the reductive activation of NIM. Moreover, NIM-OH-GSH was
generated mainly from NIM via epoxidation; CYP1A2 was the
major catalyzing enzyme. Inhibition of the bioactivation
pathways did not reduce the cytotoxicity of NIM. The
sustained amount of nonmetabolized NIM was closely
associated with its degree of cytotoxicity. Although NIM was
less toxic to primary human hepatocytes than to rat
hepatocytes, the NIM concentration may accumulate to high
amounts after chronic administration and lead to liver injury.
NMR spectra of NIM-NH2-GSH and NIM-NH2;
extracted ion chromatograms of NIM-NH2 and NIM-
NH2-GSH in the NADPH- and GSH-supplemented
nonenzymatic incubations with or without allopurinol
(PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel./Fax: +86-21-50800738. E-mail: xychen@simm.ac.cn.
Funding
This work was supported by the National Natural Science
Foundation of China (no. 81573500).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Jinghua Yu and Zitao Guo for their kind
assistance in the cytotoxicity experiments. We also thank Peng
Lei for synthesizing NIM-NO.
ABBREVIATIONS
■
NIM, nimesulide; NIM-NH2, nitro-reduced NIM; NIM-OH,
hydroxyl des-nitro NIM; NIM-NO, the nitroso intermediate of
NIM; NIM-NH2-GSH, a GSH conjugate from the attachment
of GSH to NIM-NH2; NIM-OH-GSH, a GSH conjugate from a
quinone-imine intermediate; GSH, glutathione; NADPH, β-
nicotinamide adenine dinucleotide 2′-phosphate reduced
tetrasodium salt; ABT, 1-aminobenzotriazole; BSO, L-buthio-
nine-sulfoximine; MEH, microsomal epoxide hydrolase;
CDNB, 1-chloro-2,4-dinitrobenzene; HLM, human liver micro-
somes; CYP, cytochrome P450; HLC, human liver cytosol;
RLM, rat liver microsomes; RLC, rat liver cytosol; ESI,
electrospray ionization; IDA, information-dependent acquis-
ition; UPLC/TripleTOF 5600+ MS, ultraperformance liquid
chromatograph−triple-time-of-flight 5600+ mass spectrometry
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