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ANTIOXIDANTS & REDOX SIGNALING
Volume 11, Number 1, 2009
© Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2008.2073
Original Research Communication
H
2
S Protects Against Methionine–Induced Oxidative
Stress in Brain Endothelial Cells
Neetu Tyagi, Karni S. Moshal, Utpal Sen, Thomas P. Vacek, Munish Kumar,
William M. Hughes Jr., Soumi Kundu, and Suresh C. Tyagi
Abstract
Homocysteine (Hcy) causes cerebrovascular dysfunction by inducing oxidative stress. However, to date, there
are no strategies to prevent Hcy-induced oxidative damage. Hcy is an H
2
S precursor formed from methionine
(Met) metabolism. We aimed to investigate whether H
2
S ameliorated Met-induced oxidative stress in mouse
brain endothelial cells (bEnd3). The bEnd3 cells were exposed to Met treatment in the presence or absence of
NaHS (donor of H
2
S). Met-induced cell toxicity increased the levels of free radicals in a concentration-depen-
dent manner. Met increased NADPH-oxidase-4 (NOX-4) expression and mitigated thioredxion-1(Trx-1) ex-
pression. Pretreatment of bEnd3 with NaHS (0.05 mM) attenuated the production of free radicals in the pres-
ence of Met and protected the cells from oxidative damage. Furthermore, NaHS enhanced inhibitory effects of
apocynin, N-acetyl-l-cysteine (NAC), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD),
N
-nitro-l-arginine methyl ester (L-NAME) on ROS production and redox enzymes levels induced by Met. In
conclusion, the administration of H
2
S protected the cells from oxidative stress induced by hyperhomocys-
teinemia (HHcy), which suggested that NaHS/H
2
S may have therapeutic potential against Met-induced ox-
idative stress. Antioxid. Redox Signal. 11, 25–33.
25
Introduction
M
ETHIONINE
(M
ET
)
IS AN ESSENTIAL AMINO ACID
and is me-
tabolized to homocysteine (Hcy), a sulfhydryl-contain-
ing nonprotein amino acid (32). It has been suggested that hy-
perhomocysteinemia (HHcy) is an independent risk factor for
neurodegenerative diseases such as dementia, Alzheimer’s
disease (AD) and stroke (5, 16, 22, 28, 29, 33). Previous stud-
ies have shown that Hcy is critically involved in the patho-
genesis of neurodegenerative disorders (11, 12). These unfa-
vorable vascular effects of Hcy are believed to be due to one
or both of the following: generation of reactive oxygen species
(ROS) [including hydrogen peroxide (H
2
O
2
) and superoxide
anion (O
2
-) (10, 20, 47)], and a decrease in endothelial nitric
oxide (NO) bioavailability (27, 31, 37) that play a critical role
in endothelial cell damage and dysfunction.
Although hydrogen sulfide (H
2
S) has been recognized as
a toxic gas, recent H
2
S research has been focused on its pro-
tective role in cardiovascular disease conditions. Like nitric
oxide (NO) and carbon monoxide (CO) (40, 41), which are
considered two gaseous transmitters, H
2
S has been shown
to be the third gaseous transmitter (39); moreover, H
2
S plays
important roles in several diseases. NO, CO, and H
2
S share
distinct properties which qualify them as gasotransmitters:
a) they are small molecules of gas; b) they are freely perme-
able across membrane, do not act via specific membrane re-
ceptors; c) they are produced enzymatically (17). H
2
S has
been demonstrated to stimulate heme oxygenase expression
and CO production and has bidirectional effects on the in-
ducible NO synthase (23). However, much less is known
about the physiological role of H
2
S.
H
2
S is endogenously generated in various mammalian tis-
sues from Met–Hcy–Cys metabolism through the action of
cystathione--synthetase (CBS) and/or cystathionine -lyase
(CSE) enzymes (21). H
2
S is a toxic gas and may act as a func-
tional regulator in the nervous and cardiovascular systems
Department of Physiology and Biophysics, School of Medicine, University of Louisville, Louisville, Kentucky.
(14). Although its neuromodulatory role has been demon-
strated, little is known about its protective role in oxidative
stress. Interestingly, recent studies have shown that H
2
S is
neuroprotective (4, 5, 18, 19). Physiological concentrations of
H
2
S in plasma have been reported to be between 45 Mand
300 M(48, 50). At physiological concentrations, H
2
S inhibits
smooth muscle cell proliferation via the mitogen-activated
protein kinase pathway and protects the following tis-
sues/cells against oxidative stress: neurons, cardiomyocytes,
pancreatic -cells, and vascular smooth muscle cells (19.3).
H
2
S induces apoptosis by activating ERK and pro-caspase-3
(44). It has also been established that H
2
S directly opens the
K
ATP
channel and causes reduction of vasorelaxation and
transient blood pressure (49). However, the role of H
2
S in
the regulation of oxidative stress in endothelial cells is still
unclear. In addition, H
2
S acts as an endogenous scavenger
for reactive oxygen species and reactive nitrogen species
(RNS) (8, 9, 42). Therefore, the purpose of the present study
was to determine a potential role of H
2
S in preventing Met-
induced oxidative damage in bEnd3 endothelial cells by
modulating the production of ROS/RNS.
Materials and Methods
Materials
DCFH-DA(2,7-dichlorodihydrofluoresceindiacetate),
DL-methionine (Met), Dulbecco’s modified Eagle’s medium
(DMEM), dimethyl sulfoxide (DMSO), N-acetyl-l-cysteine
(NAC), sodium hydrosulfide (NaHS), reduced glutathione
(GSH), catalase (CAT), apocynin, superoxide dismutase
(SOD), N
-nitro-l-arginine methyl ester (L-NAME), and DL-
propargylglycine (PAG) were purchased from Sigma Chem-
ical Company (St. Louis, MO). Specific antibodies against
NOX-4 and Trx-1 were purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA). Fetal bovine serum (FBS), phos-
phate-buffered saline (PBS), penicillin, and streptomycin were
obtained from Gibco (Grand Island, NY). 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), super-
oxide dismutase (SOD) assay kit were obtained from Cay-
man Chemical (Ann Arbor, MI).
Methods
Cell culture. bEnd3, an immortalized mouse brain endo-
thelial cell line originally generated in 1990 (26), now com-
mercially available at American Type Culture Collection,
Manassas (ATCC, VA), was grown according to the sup-
plier’s instructions in DMEM supplemented with 4.5 g/l glu-
cose, 3.7 g/l sodium bicarbonate, 4 mMglutamine, 10% FBS,
100 U/ml penicillin, and 100 g/ml streptomycin, pH 7.4.
Cells were maintained in a humid chamber at 37°C in an
atmosphere of 95% air and 5% CO
2
in 25 cm
2
tissue culture
flasks (38). Confluent 25 cm
2
flasks were trypsinized and
seeded at a density of 0.5–1.0 10
4
cells/cm
2
on to 6–12-
well cell culture plate, and allowed to grow to 70%–80%
confluence.
Cell viability assay. Cell viability was determined by an
MTT assay as originally described by Mosmann (27). In brief,
bEnd3 cells were plated at a density of 10
5
cells/well on
to 96-well tissue culture plates and incubated with Met
(0.114 –2.3 mM), NaHS (0.05 –0.5 mM), and 500 MPAG (in-
hibitor of CSE) in serum-free DMEM/F12 at 37°C for 24 h.
Then, 10 l of MTT reagent (5 mg/ml) was added to each
well, and the plates were incubated for another 4 h. The
medium was removed and wells were rinsed twice with PBS.
To each well, 100 l of crystal dissolving solution was added
at room temperature to dissolve the formazan crystals for 5
min. The absorbance was measured at 570 nm with a spectra-
max3000 plate reader (Molecular devices, Sunnyvale, CA).
Measurements of homocysteine. Homocysteine was mea-
sured by using high pressure liquid chromatography
(HPLC) as described earlier (33). HPLC analyses were per-
formed using Class-VP 5.0 chromatograph (Shimadzu,
Tokyo, Japan) containing a LC-10ADvp pump, a SIL-
10ADvp auto-injector, a CTO-10Avp column oven, and a
SPD-10Avp detector. The temperature inside the column was
maintained at 37°C during analyses.
Sample preparation. Culture supernatants were collected
and centrifuged to remove cell debris. To determine the Hcy
level in the supernatants, 200 l of supernatant was diluted
with 100 l of water and then 300 l of 9 Murea (pH 9.0)
was added. Fifty microliters of n-amyl alcohol was added to
the solution as an antifoaming agent. Reduction of disulfides
and cleavage of the protein-bound sulfur-containing amino
acids was performed by the addition of 50 l of NaBH
4
so-
lution (10%, wt/vol) in 0.1 NNaOH. To perform the reac-
tion, samples were incubated in a water bath at 50°C for 30
min. Samples were cooled at room temperature, and the re-
action was stopped by the addition of 500 l of 20%
trichloroacetic acid. The proteins were separated by cen-
trifugation for 4 min at 12,000 g, and supernatants were fil-
tered using a 0.45 m Millipore filter (32).
Measurement of H
2
S production. bEnd3 (10
5
cells/well)
were grown briefly in a 10 cm
2
dish. Cells became confluent
following 24 h of treatment with Met at different concentra-
tions. H
2
S concentration in bEnd3 cells was measured as de-
scribed previously (7, 48, 50).
Intracellular fluorescence measurement of reactive oxygen
species. In order to measure the oxidized DCF levels in cells,
we used the probe, 2-7-dichlorodihydrofluorescein diacetate
(H
2
DCF-DA), as described previously (36). This membrane-
permeable probe enters the cells and produces a fluorescent
signal after intracellular oxidation by ROS. bEnd3 (10
5
cells/well) were grown briefly in 96-well plates. Cells were
grown to confluence and treated for 24 h in serum-free
DMEM/F12 media with or without the following: Met, NaHS,
other various agents at different concentrations. The cells were
washed with PBS, loaded with probe, DCFH-DA (10 M), and
incubated in dark for 2 h at 37°C in PBS. Thereafter, the cells
were washed three times with PBS to remove the excess probe.
Oxidized DCF was quantified by monitoring the DCF fluo-
rescence intensity with excitation at 485 nm and emission at
530 nm with a spectra-max3000 plate reader (Molecular de-
vices). Values were expressed in arbitrary units.
In situ labeling of ROS. bEnd3 cells were grown on 8-well
cover glass plates and serum starved before the treatments
with the following: 1.14 mMMet, 0.05 mMNaHS, or 1.14
mMMet
0.05 mMNaHS for 24 h. Ros formation was vi-
sualized as described previously (36, 37).
TYAGI ET AL.26
Measurement of intracellular superoxide levels. The intra-
cellular superoxide anion radicals were detected using su-
peroxide dismutase (SOD) assay, using a kit from Cayman
Chemicals. Briefly, bEnd3 were plated and treated with dif-
ferent agents in serum free DMEM/F12 media at 37°C for 24
h. Then cells were scraped with a rubber policeman and son-
icated in cold 20 mMHEPES buffer, pH 7.2, containing 1 mM
EGTA, 210 mMmannitol, and 70 mMsucrose, according to
the manufacturer’s recommendation. SOD activity was de-
termined by spectrophotometry as the ability to inhibit the
reduction of nitroblue tetrazolium (NBT) induced by xan-
thine-xanthine oxidase (1).
Immunoblot analysis. Cells were lysed in ice-cold-modi-
fied RIPA lysis buffer (Tris-HCl, 50 mM, pH 7.4; NP-40, 1%;
0.25% Na-deoxycholate, 150 mMNaCl; 1 mMEDTA; 1 mM
PMSF; 1 g/ml each of aprotinin, leupeptin, pepstatin; 1 mM
Na
3
VO
4
; 1 mMNaF). Protein content of the lysate was de-
termined using BCA protein assay (Pierce, Rockford, IL) kit.
Protein samples were mixed with 1:1 vol/vol ratio with 2X
sample loading buffer [800 l glycerol, 1 ml 0.5 mMTris-
HCl (pH 6.8), 1.6 ml 10% (wt/vol) SDS, 400 l 2-mercap-
toethanol, 400 l 0.05% (wt/vol) Bromophenol blue], boiled
at 95–100°C for 5 min. Samples were cooled to room tem-
perature and centrifuged to precipitate cell debris. Equal
amounts of protein (20 g) for each group were resolved by
10–15% SDS-PAGE. Protein was then electrophoretically
transferred to a nitrocellulose membrane (BioRad, Hercules,
CA). Transferred protein was blocked with 5% nonfat dry
milk in TBS-T (50 mMTris-HCl, 150 mMNaCl, 0.1% Tween-
20, pH 7.4) for 1 h at room temperature. The blot was then
incubated with appropriate primary antibody in blocking so-
lution according to the supplier’s specific instructions. Next,
the blot was washed with TBS-T three times for 10 min each.
The blots were incubated with appropriate horseradish per-
oxidase-conjugated secondary antibody for 2 h at room tem-
perature. Four more 10 min washes were performed, and
ECL Plus substrate (Amersham Biosciences, Pittsburgh, PA)
was applied to the blot for 5 min. The blot was developed
using X-ray film (RPI Corp, Inc., Mount Prospect, IL) with a
Kodak 2000A developer (Eastman Kodak, Rochester,NY).
Image analysis was performed using UMAX PowerLock II
(Taiwan, Republic of China).
Data analysis and statistics. Results were expressed as
means SEM from at least seven independent experiments.
Both paired and unpaired Student’s ttests were used, where
appropriate, for comparing the mean values between con-
trol and tested groups. The difference between mean values
of multiple groups was analyzed by one-way analysis of
variance (ANOVA), followed by a Scheffe’s post-hoc analy-
sis. Statistical significance was considered at p0.05. The
arbitrary densitometry units (AU) were represented as per-
centage relative to control.
Results
Effect of NaHS on Met-induced cytotoxicity
To determine the protected role of hydrogen sulfide on
Met-induced cytotoxicity, bEnd3 were cultured with Met
with or without NaHS (donor of H
2
S) at different concen-
trations for 24 h. The cell death was significantly increased
in Met (0.114–2.3 mM) and NaHS (0.25–0.5 mM)-treated cells
after 24 h compared to untreated cells. This effect was not
observed when cells were treated with lower concentrations
of Met or NaHS (Fig. 1A and B). The increase in cell death
by high Met (1.14 mM) was attenuated by the pre-treatment
with NaHS (0.05 and 0.1 mM), respectively (Fig. 1C). But this
effect was reduced significantly by PAG (inhibitor of CSE)
pretreatment (Fig. 1D).
H
2
S INHIBITS METHIONINE–INDUCED OXIDATIVE STRESS 27
FIG. 1. Effect of hydrogen sulfide
on Met–induced cytotoxicity. (A)
Viability of bEnd3 cells treated with
different concentrations of Met for
24 h; n7; *p0.05; **p0.01. (B)
Cell viability measured after treat-
ment with different concentrations
of NaHS for 24 h; n7; *p0.05;
**p0.01. (Cand D) Cells were in-
cubated with Met (1.14 mM) for 24
h in the presence or absence of
NaHS or PAG; in (C) n7 in each
group; *p0.05; **p0.05; ***p
0.05. In (D), n7 in each group,
*p0.05;
#
p0.05;
$
p0.01;
p
0.01.
Role of NaHS in Hcy accumulation
Cell culture medium was collected after 24 h from control
(0.114 mMMet), high (1.14 mMMet), NaHS (0.05 mM), and
1.14 mMMet NaHS (0.05 mM) treated cells. The medium
was analyzed by HPLC. There was increased accumulation
of Hcy in the high Met (1.14 mMMet)-treated group com-
pared to that in control (Fig. 2A). Hcy levels were 3.6 0.5,
23.38 3.4, 5.1 0.6, and 11.3 1.2 Min control Met, high
Met, NaHS, and high Met NaHS treated cells, respectively.
These levels were similar and largely comparable with in vivo
studies (4). The increase in Hcy accumulation in the high Met
group was attenuated by NaHS treatment, which suggested
that H
2
S was a potent inhibitor of Hcy formation.
H
2
S levels
To determine the effect of endogenously generated H
2
S,
bEnd3 cells were treated with different concentrations (0.114–
2.3 mM) of Met for 24 h. H
2
S production rate was markedly
increased in a concentration-dependent manner (Fig. 2B).
NaHS attenuated Met-induced increases
in intracellular ROS
Because the cytotoxicity of Met is known to be mediated
mainly by oxidative stress, we investigated whether NaHS
affects ROS formation by high Met using DCFH-DA fluo-
rescence. Incubation of cells with different concentrations
(0.114–2.3 mM) of Met for 24 h resulted in significant in-
creases in ROS production in comparison with untreated
cells (Fig. 2C). When the cells were treated with Met (1.14
mM) for different time periods (6, 12, 24 h), the 24 h treat-
ment showed significant increases in ROS production (data
not shown). When cells were incubated with antioxidant
(NAC) or H
2
O
2
scavenger (CAT), there were marked de-
creases in ROS production induced by the treatment with
Met (1.14 mM, Fig. 2D). Furthermore, addition of NaHS (0.05
mM) significantly increased the inhibitory effects of NAC on
Met-induced ROS production, while the effect could not be
found in the CAT-treated group.
Interestingly, in-situ labeling (Fig. 3A–D) showed that the
levels of intracellular ROS were increased in cells treated
TYAGI ET AL.28
FIG. 2. Effect of hydrosulfide on homo-
cysteine accumulation. (A) Data from chro-
matography of homocysteine from cell cul-
ture medium. The effect of the addition of
Met (1.14 mM) and NaHS (0.05 mM) on Hcy
levels; *p0.05 compared to control (CT),
**p0.01compared to Met. (B) The mea-
surement of H
2
S in culture media after treat-
ment with different concentrations of Met
for 24 h. The cells were collected and ho-
mogenized to measure the H
2
S production
rate. N 7 for each group, *p0.05; **p
0.01. (C) ROS production, detected by 5-(6)-
chloromethyl-2,7-dichlorodihy-
droflurorescein diacetate (DCFH-DA) stain-
ing after incubation of cells with Met at
various concentrations for 24 h, *p0.05;
**p0.01. (D) Effect of hydrogen sulfide on
Met-induced reactive oxygen species (ROS)
production in bEnd3 cells, ROS production
was detected after treatment with Met (1.14
mM) in the presence or absence of NAC (50
M), CAT (5 /ml) with or without NaHS,
respectively; n7 for each group, *p
0.05; **p .01;
##
p0.01;
p0.01.
FIG. 3. In situ labeling of ROS. bEnd3 cells were grown on
glass 8-well chambers and exposed to different treatments for
24 h. ROS production was evaluated by staining the cells with
DCFH-DA. Images were acquired by laser confocal microscope
(FluoView 1000) at an excitation of 488 nm and emission of 525
nm representative micrograph. (A) Control; (B) 1.14 mMMet;
(C) 0.05 mMNaHS, (D) 1.14 mMMet 0.05 mMNaHS; mag-
nification 100. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of
this article at www.liebertonline.com/ars).
with Met (1.14 mM, Fig. 3B), indicated by the increases in
DCFH-DA fluorescence. However, when cells were treated
with both NaHS (0.05 mM) and Met (1.14 mM), DCFH-DA
fluorescence was decreased (Fig. 3D). This suggested that
Met-induced intracellular ROS accumulation was attenuated
by hydrogen sulfide. Cells treated with NaHS (0.05 mM)
alone showed weak DCFH-DA fluorescence (Fig. 3C), simi-
lar to that of untreated cells (Fig. 3A).
NaHS attenuated Met-induced peroxynitrite formation
The generation of ONOO
requires rapid interaction of
NO and O
2
.
. To determine the effects of the interaction of
Met and NaHS on ONOO
formation, cells were treated
with different concentrations (0.114–2.3 mM) of Met for 24 h
in the presence or absence of the NOS inhibitor, L-NAME
(100 M), NADPH oxidase inhibitor apocynin (100 M),
O
2
.
scavenger SOD (200 U/ml), with or without NaHS (0.05
mM). Figure 4A shows a concentration-dependent increase
in Met-induced ONOO
formation. However, when bEnd3
cells were co-treated with NaHS, Met (1.14 mM), apocynin,
or SOD, it ameliorated the inhibitory effect of apocynin, SOD,
and L-NAME, respectively (Fig. 4B).
NaHS attenuated Met-induced superoxide
anion production
To determine the protected role of hydrogen sulfide on
Met-induced superoxide anion, we examined the release of
superoxide anion by chemiluminescence assay. Figure 4C
H
2
S INHIBITS METHIONINE–INDUCED OXIDATIVE STRESS 29
FIG. 4. Effect of H
2
S on Met-induced
generation of peroxynitrite (ONOO
).
(A) The bEnd3 cells were treated with Met
1.14 mM, Met 1.14 mML-NAME (100
M) or Met (1.14 mM), L-NAME (100
M)NaHS (0.05 mM) for 24 h and ROS
were detected by using DCFH-DA probe;
**p0.05 compared to corresponding Met
treatment. p0.05 compared to Met
and L-NAME (100 M) treatment. (B) ROS
production was detected after bEnd3 cells
were incubated for 24 h with Met (1.14
mM) in the presence or absence of apoc-
ynin (100 M) or SOD (200 U/ml), with or
without NaHS (0.05 mM), respectively.
N7 in each group; *p0.05; **p0.01;
##
p0.01;
p0.05;
p0.05. (C) Met-
induced superoxide (O
2
.
) production and
effect of H
2
S in bEnd3: O
2
.production
was measured after bEnd3 cells were
treated with different concentrations of
Met for 24 h. (D) O
2
.
production was de-
termined in the treated cells after 24 h in-
cubation with NaHS at different concen-
trations. N7 for each group. *p 0.05
compared to control.
FIG. 5. Effect of hydrogen sulfide on Met-sup-
pressed thioredoxin (Trx) expression. (A) bEnd3
cells were cultured and treated with different con-
centrations (0.05–0.1 mM) of NaHS alone for 24h;
*p0.05 compared to control. (B) bEnd3 cells were
treated with Met (1.14 mM) in the presence or ab-
sence of the antioxidant NAC (50 M), GSH (1 mM),
with or without NaHS, respectively, for 24 h. Trx
protein was measured in cell lysates by Western
blot analysis, and membranes were stripped and
reprobed with -actin for equal loading. Bottom:
graphical presentation of Trx (fold change over con-
trol). N7 for each group. *p 0.05 vs. untreated
cells,
#
p0.05 or
##
p0.01 vs. cells treated with
Met in.
p0.05 vs. cells treated with Met NAC,
p0.05 vs. cells treated with Met GSH.
shows that 24 h of incubation with Met (1.14 mM) signifi-
cantly increased O
2
.
levels in a concentration-dependent
manner. The bEnd3 cells were incubated with different con-
centrations (0.05–0.25 mM) of NaHS for 24 h. The O
2
.
pro-
duction was significantly increased only in 0.25 mMNaHS-
treated cells, as compared to untreated cells (Fig. 4D).
Effect of NaHS on redox enzymes levels
To determine the protective role of hydrogen sulfide on
Met-induced imbalance between the redox enzymes, we ex-
amined the Trx-1 and NOX-4 protein levels by Western blot
analysis. Incubation of cells with 0.05–0.1 mMNaHS for 24
h resulted in a statistically significant increase in Trx-1 pro-
tein expression level (Fig. 5A) in comparison with untreated
cells. Met (1.14 mM) alone resulted in a significant decrease
in Trx-1 protein expression levels in comparison to the un-
treated control (Fig. 5B). Simultaneous incubation of Met
(1.14 mM) with either NAC (50 M) or GSH (1 mM) resulted
in a significant increase of the Met downregulated Trx-1 pro-
tein expression compared with Met alone. Furthermore,
when bEnd3 cells were treated with a combination [Met (1.14
mM); Met NAC; Met NAC NaHS; Met GSH; or
Met GSH NaHS], the treatment reduced the effect of
Met on Trx-1 protein expression as shown in Fig. 5B.
There was a concentration-dependent decrease in NOX-4
protein expression (Fig. 6A) after cells were incubated with
NaHS (0.05 or 0.1 mM) for 24 h. Met (1.14 mM) alone sig-
nificantly induced NOX-4 protein expression (Fig. 6A). Met-
induced NOX-4 protein expression was markedly decreased
in cells when they were pretreated with either NAC (50 M)
or GSH (1 mM). Furthermore, addition of NaHS (0.05 mM)
significantly increased the inhibitory effects of NAC and
GSH on Met-induced NOX-4 protein production in compar-
ison with that in the absence of NaHS (Fig. 6).
Discussion
Our present study attempted to examine a novel link be-
tween the protective role of H
2
S towards oxidative stress
caused by HHcy in brain endothelial cells. Methionine, an
essential amino acid, is converted to Hcy that then promotes
neurodegenerative diseases through endothelial dysfunction
(24, 25, 34). It is important to note that there are other re-
spondents to increases in ROS, such as HO-1. Homocysteine
generates ROS via the auto-oxidation of the thiol group (36,
44) or by decreasing the endothelial heme oxygenase-1 (HO-
1) activity (30). The transcriptional upregulation of the HO-
1 gene downregulated intracellular ROS (29). H
2
S is an en-
dogenous metabolic product of Met by the trans-sulfuration
pathway that is dependent on two important enzymes: CBS
and CSE (39). It has been demonstrated that H
2
S reacts with
at least four different ROS, superoxide radical anion, hy-
drogen peroxide, peroxynitrite, and hypochorite (8, 9, 41).
All these compounds are highly reactive and their interac-
tion with H
2
S resulted in the protection of proteins and lipids
from ROS/RNS-mediated damage (41, 42). Therefore, we hy-
pothesize that H
2
S may protect the cerebro-vasculature
against Met-induced endothelial damage.
Our present study results show that high Met (1.14 mM)
significantly decreased bEnd3 cells viability. Expectedly, the
addition of NaHS (0.05 or 0.1 mM) significantly increased
the cell viability as compared to the cells treated with high
Met. Although H
2
S (0.1 mM) reduced cell viability, this
was merely the effect of chemical cytotoxicity rather than
physiological effect. But this effect was reduced significantly
by PAG pretreatment (Fig. 1D), a result consistent with pre-
TYAGI ET AL.30
FIG. 6. Effect of hydrogen sulfide on Met-In-
duced NOX-4 expression. (A) Serum-starved
bEnd3 cells were treated for 24 h with different
concentration (0.05–0.1 mM) of NaHS alone for 24
h; *p 0.05 compared to control. (B) Serum-
starved bEnd3 cells were either left untreated or
were treated with Met (1.14 mM) in the presence
or absence of the antioxidant NAC (50 M), GSH
(1 mM) with or without NaHS, respectively, for
24 h. After treatment, cell lysates were analyzed
with 10–15% SDS-PAGE and subsequently by
Western blot analysis; membranes were stripped
and reprobed with -actin for equal loading. Bot-
tom: graphical presentation of NOX-4 (fold change
over control). N7 for each group, *p 0.05 vs.
untreated cells.
p0.05;
p0.05 vs. cells
treated with Met group.
#
p0.05 vs. cells treated
with Met NAC,
##
p0.01 vs. cells treated with
Met GSH.
FIG. 7. Schematic presentation of proposed mechanism
for the protective role of H
2
S towards Met-induced oxida-
tive stress in bEnd3 cells.
vious studies (32, 36, 43). PAG is an inhibitor of CSE, an en-
zyme responsible for endogenous H
2
S formation. We found
that, in the presence of PAG, cell viability was decreased in
the setting of high Met.
The rapid interaction of superoxide with nitric oxide gen-
erated peroxynitrite, a potent mediator of oxidant-induced
cellular injury (15, 34). In the present study, we demonstrated
that ROS production was increased in a concentration-de-
pendent manner after bEnd3 cells were treated with differ-
ent doses of Met for 24 h. Met-induced ROS production was
effectively blocked by NAC and H
2
O
2
scavenger, CAT, and
concentration-dependently inhibited by the NOS inhibitor,
L-NAME. This provides evidence that Met can induce not
only H
2
O
2,
but also ONOO
generation in mouse brain en-
dothelial cells (Fig. 2). This finding is supported by the ob-
served inhibitory effect of SOD and the effect of NADPH ox-
idase inhibitor, apocynin, on ROS formation (Fig. 3).
According to previous studies, H
2
S worked as a scavenger
of oxygen-derived free radicals (9), which could contribute
to the protective role of NaHS against the toxicity of H
2
O
2
in vitro and in vivo model (45). We observed that Met-induced
O
2
production was markedly reduced by the O
2
scav-
engers (SOD) as well as by NaHS (Fig. 4).
In addition, H
2
S enhanced NO production via ERK1/2 ac-
tivation, which suggested that H
2
S may cooperate with NO
in modulating their biological effects (13). Our results
showed that high levels of H
2
S can induce ROS and RNS for-
mation, but low levels of H
2
S can decrease H
2
O
2
, ONOO
,
and O
2
generation induced by Met in bEnd3 cells. Fur-
thermore, our results indicated that low concentrations of
H
2
S combined with certain agents, such as NAC, apocynin,
SOD, or L-NAME, may synergistically increase their antiox-
idant effects, scavenge ROS, and protect vascular endothe-
lium from Met-induced oxidant stress and cytotoxicity (Fig.
4). This is in contrast to functioning directly as an antioxi-
dant. This protective role of H
2
S may be similar to the pro-
tection of neurons from oxidative stress (18). The failure of
NaHS combined with CAT to reduce Met-induced ROS for-
mation might be explained by the proposed low efficiency
of CAT compared with NAC in removing H
2
O
2
at low con-
centrations (46). H
2
S protects the cell damage by decreasing
the ROS production by increasing SOD. The mechanism of
Met-induced oxidative stress is not yet well known. Some
studies suggested that Met-induced oxidative stress is pos-
sible through an NADPH oxidase-mediated pathway (36).
Our results showed that Met (1.14 mM) significantly in-
creased NOX-4 expression and, consequently, decreased Trx
expression. bEnd3 cells treated with a combination of high
Met, antioxidant, and NaHS may synergistically increase Trx
expression and decrease NOX-4 expression, in comparison
with the group treated with Met alone.
In this study, we employed different approaches to de-
termine the protective role of H
2
S on Met–induced oxidative
damage. We presented evidence that H
2
S is an important
modulator of cellular cytotoxicity via redox cell pathways in
the pathogenic conditions associated with HHcy. In conclu-
sion, the data presented here provides a new mechanism by
which H
2
S reduces oxidative damage induced by Met. Thus,
H
2
S protected bEnd3 cells from oxidative damage. When
considering previous studies along with our results with re-
spect to antioxidant activity of H
2
S and its effect on Met, we
suggest that administration of H
2
S might be an interesting
potentially preventive strategy for reducing cerebrovascular
complications in hyperhomocysteinemia (Fig. 7). However,
the molecular mechanisms of a putative protective role of
H
2
S in neurogenerative pathogenesis should be further in-
vestigated.
Limitations
In “mild” human hyperhomocysteinemia (which is asso-
ciated with an increased neurodegenerative diseases),
plasma Hcy levels range from 15 to 30 mol/l. However,
only a fraction of total plasma Hcy is in the reduced form in
vivo. The concentrations used in the present study represent
a 100-fold dose. Three ranges of hyperhomocysteinemia
are defined as follows: moderate (16–30 M), intermediate
(31–100 M), and severe (100 M) (2, 6). Extracellular thi-
ols are oxidized. Only a fraction of total plasma Hcy is in the
reduced form in vivo and in vitro. NaHS, a donor of H
2
S, was
used at physiologically relevant concentrations (41).
Abbreviations
bEnd3, mouse brain microvascular endothelial cells; CAT,
catalase; CBS, cystathione--synthetase; CSE, cystathionine
-lyase; GSH, reduced glutathione; Hcy, homocysteine;
HHcy, hyperhomocysteinemia; H
2
S, hydrogen sulfide; L-
NAME, N
-nitro-l-arginine methyl ester; Met, methionine;
NAC, N-acetyl-l-cysteine; NaHS, sodium hydrogen sulfide;
NOX4, NADPH-oxidase-4; PAG, DL-propargylglycine. ROS,
reactive oxygen species; SOD, superoxide dismutase; Trx-1
,thioredxion-1.
Acknowledgments
Supported in part by the National Institutes of Health
grants HL-75185, HL-71010, and NS-51568.
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Address reprint requests to:
Suresh C. Tyagi, PhD
Department of Physiology and Biophysics
School of Medicine
University of Louisville
Louisville, KY 40202
E-mail: suresh.tyagi@louisville.edu
Date of first submission to ARS Central, March 8, 2008; date
of final revised submission, July 16, 2008; date of acceptance,
July 16, 2008.
H
2
S INHIBITS METHIONINE–INDUCED OXIDATIVE STRESS 33
