Open Journal of Apoptosis, 2013, 2, 1-11 Published Online January 2013 (
Gsta4 Null Mouse Embryonic Fibroblasts Exhibit
Enhanced Sensitivity to Oxidants: Role of
4-Hydroxyn onen al in O xidant Toxicity*
Kevin E. McElhanon1,2, Chhanda Bose2,3, Rajendra Sharma1,2, Liping Wu1,2,
Yogesh C. Awasthi4, Sharda P. Singh1,2#
1Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, USA
2Central Arkansas Veterans Healthcare System, Little Rock, USA
3Department of Internal Medicine, Nephrology Division, University of Arkansas for Medical Sciences, Little Rock, USA
4Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, USA
Received November 28, 2012; revised December 29, 2012; accepted January 8, 2013
The alpha class glutathione s-transferase (GST) isozyme GSTA4-4 (EC2.5.1.18) exhibits high catalytic efficiency to-
wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of
cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated con-
centrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a
signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To ex-
amine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we dis-
rupted the Gsta4 gene that encodes GSTA4-4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts
(MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of
GSTA4-4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and
DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of
4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regu-
lated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of
caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, acti-
vation of stress-related kinases in 4-HNE(Ac)3 treated Gsta4 null MEF were inversely correlated with the catalytic effi-
ciency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4-4 plays a major role in protecting cells from
the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE.
Keywords: Glutathione Transferase; Protein Adducts; Lipid Peroxidation, Oxidants, Stress Kinases
1. Introduction
Lipid aldehydes generated duri ng li pid peroxidation (LPO)
are highly reactive species capable of electro philic attack
on DNA and proteins [1-4]. 4-HNE, one of the major end
products of LPO, causes cytotoxicity and genotoxicity by
inducing necrosis and pro-apoptotic signaling through
multiple pathways at supraphysiological concentrations
[5-7]. 4-HNE is also involved in regulation of gene ex-
pression and c ell cycle signal ing in a concentrati on depen-
dent manner [8-11]. We and others [12,13] have shown
that 4-HNE causes activation and phosphorylation of the
c-jun-N -te rminal k ina se (JN K ) an d p3 8 mito gen act i vate d
protein kinases (p38 MA PK) in lung endothelial cells and
contributes to apoptotic response in K562 cells. Our re-
cent studies show that in addition to causing toxicity, 4-
HNE induces defense m echanisms against oxidative stress
and protects the neighboring cells from apoptosis [14]. Its
concentration in cells is regulated by the alpha class gluta-
thione transferases, particularly GSTA4-4, that catalyze
its conjugation to glutathione (GSH) with high catalytic
efficiency [15-17]. In order to understand the exact role of
GSTA4-4 in protection of cells against acute 4-HNE to-
xicity, we isolated MEF from previously generated Gsta4
null mice [18]. In this study, we compared the effects of
electrophile/oxidants including 4-HNE(Ac)3, hydrogen
peroxide (H2O2), and N,N’-dimethyl-4,4’-bipyridinium
dichloride (paraquat) on Gsta4 null and WT MEF cells.
4-HNE(Ac)3 is a biologically inert pro-drug form of
*Conflicts of interest: The authors report no conflicts of interest and are
responsible for the content and writing of the paper.
#Corresponding author.
opyright © 2013 SciRes. OJApo
4-HNE that is advantageous for in vitro treatment of cells
[5,19]. 4-HNE is highly electrophilic and may interact
with cell surface proteins and components of culture me-
dium, whereas 4-HNE(Ac)3 is diffusible across the cell
membrane and upon enzymatic reaction with intracellular
hydrolases active 4-HNE is liberated, more closely mi-
micking intracellular 4-HNE formation. Sensitivity of Gsta4
null cells towards 4-HNE was further correlated with the
accumulation of 4-HNE-protein adducts, DNA fragmen-
tation, and activation of stress related kinases. In addition,
we tested if exogenous delivery of GSTA4-4 into Gsta4
null cells provides protection from 4-HNE toxicity by
reversing the course of stress kinase activation.
2. Materials and Methods
2.1. Materials
4-HNE(Ac)3 was prepared by the method of Neely et al.
[19]. DMEM cell culture medium, fetal bovine serum,
Penicillin/streptomycin, pho sph ate buffered saline (PBS),
proteinase K, 4% - 12% Bis-TrisNuPAGE gels, running
and transfer buffers, and SYBR green were purchased
from Invitrogen (Carlsbad, CA). BioTrek Protein De-
livery Regent was purchased from Stratagene. Antibodies
against caspase-3, JNK, ERK, and p38 MAPK were from
Cell Signaling Technology (Beverly, MA) and MAPK
inhibitors were purchased from EMD SERONO, INC.
(Rockland, MA). Caspase-Glo® kit for the detection of
caspase-3 , 8 , and 9 wer e purchased from Promega (Madi-
son, WI). Kinase inhibitors were obtained from Calbio-
chem, (EMD Biochemicals, Germany). CCK-8 kit was
purchased from Dojindo Molecular Technologies, Inc.
(Rockville, Maryland). Polyclonal antibodies against
mGSTA4-4 and hGSTA1-1 were raised in chicken and
rabbit respectively and antibody against DmGSTD1-1
was a kind gift from B. J. Cochrane, University of South
Florida, Tam pa,FL. All other reagents a nd chemicals were
purchased from Sigma-Aldrich (St. Louis, MO).
2.2. Mouse Embryonic Fibroblast Lines
MEF were harvested according to standard protocol [20,21].
Briefly, uteri were obtained at 13.5 days of pregnancy
from wild type (WT) and Gsta4 null mice in 129/Sv
background [18] and embryos isolated. Heads and vis-
cera were removed and bodies were minced then di-
gested with 0.25% trypsin /1 mM EDTA (GIBCO) for 5 -
10 min at 37˚C. A single cell suspension was obtained by
mixing digested bodies and complete DMEM medium.
Spontaneously immortalized cell lines were used for
most experiments at passage numbers less than 20. MEFs
were cultured until 80% - 85% co nfluent and passag ed at
a ratio of 1:2. Expression level of GSTA4-4 in WT and
Gsta4 null MEF was verified by western blot using anti-
mGSTA4-4 antibody.
2.3. Transient Transfection of Gsta4 Null MEF
with Control and mGSTA4-4 Expression
Gsta4 null MEF were transiently transfected with pRC/
CMV/Gsta4 and co ntrol plasmi d constructed earlier in our
lab [22] using Lipofectamine 2000 reagent (Life Techno-
logies, Grand Island, NY). After 24 h, media containing
transfection reagent and plasmid was replaced with com-
plete DMEM and cells allowed to recover for an addi-
tional 24 h before experiments. A portion of transiently
transfected cells were examined by Western blot for the
expression of mGSTA4-4 before cell viability assay (data
not shown).
2.4. Cell Viability Assay
Adherent MEF were trypsinized and pelleted by centri-
fugation at 500 × g for 5 minutes at 2˚C - 8˚C and washed
twice by suspending in 5 mL complete DMEM. Cell pellet
was resuspendedat 2 × 105 cells/ml in DMEM and 100
µL/well were seeded in 96 well plates and allowed to
recover for 16 - 18 hours before treatment. WT, Gsta4
null and transiently transfected Gsta4 null MEF cells were
treated with 4-HNE(Ac)3 (0 - 25 µM), and in a separate
experiment WT and Gsta4 null MEF cells were treated
with paraq uat (0 - 250 µM), and H2O2 (0 - 700 µM) for 24
hours and analyzed for viability by MTT assay using
CCK-8 kit.
2.5. Quantitation of 4-HNE-Protein Adducts by
4-HNE-protein adducts were quantitated by competitive
ELISA [23] using a polyclonal antibody against 4-HNE-
modified keyhole limpet hemocyanin, generously provid-
ed by Dr. Dennis R. Petersen, University of Colorado,
Denver. A debris free cell lysate from control and treated
MEF or known amounts of 4-HNE-casein were preadsor-
bed with anti 4-HNE antibody and added to a 96 well
immunoassay microplate pre-coated with 4-HNE modi-
fied casein (33 µg/ml per well). After incubation for 1 hr,
plate was washed with PBS (containing 0.05% Tween 20
and 0.25 mg/ml casein), HRP conjugated secondary anti-
body added to each well and incubated for an additional
30 min at RT. The plate was washed and color developed
by adding 100 µl/well of TMB One Solution (Promega,
Madison, WI). After blue color development (usually 4 -
5 min), the reaction was stopped by adding 1N HCl and
plate read at 450 nm. Samples were quantified using the
calibration curve constructed with known amounts of
competitor (4HNE-casein).
2.6. Determination of Caspase Activity
Activities of caspase-3, 8, and 9 in WT and Gsta4 null
Copyright © 2013 SciRes. OJApo
MEF treated with/without 4-HNE(Ac)3 were analyzed us-
ing Caspase-Glo® kit according to manufacturer’s in-
structions. Briefly, 2 × 104 null or WT cells were grown
for 24 h in white-walled 96 well plates compatible with
luminometer (Molecular Devices, Sunnyvale, CA). After
24 h cells were treated with 20 µM 4-HNE(Ac)3 for 2 h
with control cells receiving an equivalent amount of DMSO.
Cleavage of caspase-3 was also confirmed by Western
blot anal yses. For Wester n bl ot, 2 × 105 WT or Gsta4 null
cells were plated in 100 mm dishes and after 24 h of
incubation cells were treated with DMSO or 20 µM 4-
HNE(Ac)3 for different time points (0 - 5 hr). Following
treatment cells were lysed, clarified by centrifugation, and
25 µg protein/well separated on 4% - 12% NuPAGE gel.
Anti-caspase-3 antibody was used to detect pro and
cleaved caspas e-3 .
2.7. DNA Damage Determined by Comet Assay
DNA dama ge was assessed in 4-HNE(Ac)3 treated/control
WT and Gsta4 null MEF by “comet” assay (also called
single cell gel electrophoresis; SCGE) according to me-
thods described by Singh et al. [24] with slight modifica-
tions. In brief, control and treated cells were suspende d i n
liquid 0.5% low melting point agarose and spread on a
glass microscope slide coated with 1% (w/v) agarose.
Cells were ly sed ( 1% T ri t o n X -1 0 0 an d 1% sodium l a ury l
sarcosinate for 1 h at 4 degree C in dark) and DNA al-
lowed to unwind under alkaline conditions by covering
the slides with a 300 mM NaOH, 1 mM EDTA (pH > 13.0)
solution for 1 hr. Following unwinding, slides underwent
electro-phoresis (25 volts (~0.74 V/cm) for 20 min),
washed and stained with SYBR green for 5 min. Cells
were scored for DNA damage [25] by computerized im-
age analysis using CometScore™ Freeware (TriTek Cor-
poration, Sumerduck, VA;
2.8. Activation Analysis of Stress Kinases by
Western Blot
For the Western blot analyses, 2 × 105 cells were plated in
100 mm dishes and after 24 h of incubation treated with
inhibitors of JN K (SP600125, 50 µM), ERK (UO1 26, 20
µM), and p38 MAPK (SB202190, 2 µM) for 1h before
treatment wit h 20 µM 4-HNE(Ac)3. Afte r 2 h of treatment,
control and tre at ed cel ls w ere h arvest ed, ext ract s pre pare d
in RIPA buffer (Sigma-Aldrich, St. Louis, MO), and pro-
tein content determined by the Bradford method [26]. 25
µg of cell extracts were separated by SDS-PAGE in pre-
cast NuPage 4% - 12% Bis-Tris gels (Invitrogen, Carls-
bad, CA), and t he electroblot pro bed with po lycl onal anti-
bodies against phosphorylated JNK, ERK, and p38
MAPK. A peroxidase-coupled secondary antibody and
SuperSignal West Pico (Thermo scientific, Rockford, IL)
with chemiluminescent detection were used for visualiza-
tion of bands on a Bio-Rad imaging system (Bio-Rad
Laboratories, Hercul es, CA).
2.9. Effects of Kinase Inhibitors on MEF
To compare the effects of JNK, ERK and p38 MAPK
inhibitors on the viability of WT and Gsta4 null MEF, 2 ×
104 cells/well were plated in 96 well plates in complete
growth medium. After 24 h the cells were separately
treated with inhibitors of JNK (SP60 0125; 50 µM), ERK
(UO126; 20 µM), and p38 (SB202190; 2 µM) for 1 h and
then exposed to 20 µM 4-HNE(Ac)3. The viability of cells
after 24 h was determined by MTT assay as described
2.10. Delivery of Purified GSTs into Gsta4 Null
MEF Cells
The BioTrek Protein Delivery Regent (Stratagene) was
used to deliver purified recombinant mouse GSTA4-4,
Drosophila DmGSTD1-1 [27], and human GSTA1-1 ex-
pressed i n E. coli. Purified GSTs were dilut ed to 1 mg/mL
with PBS and 100 µl of the dilu ted protein solution tran-
sferred t o the tu be co ntaini ng ly ophil ized Bi oTre k reage nt,
mixed thor ou g hl y , an d i ncubate d at R T f or 5 min. Serum-
free medium was then added to a final volume of 500 µl.
Gsta4 null MEF cells approximately 50% - 60% confluent
were washed once with serum-free medium, 500 µl of
fresh serum-free medium added to each well, and 500 µl
of the BioTrek-protein mixture was added drop wise.
After 2 h complet e medium was ad ded and ce lls incubate d
at 37˚C and 5% CO2 in a humidified incubator for 16 h
before treatment with 20 µM 4-HNE(Ac)3. Control WT
and Gsta4 null MEF cells received reagent and/or treat-
ment to match protein delivered Gsta4 null MEF cells.
Cells were harvested 2 h af ter 4-HNE(Ac)3 treatment for
Western blot analy ses.
3. Results
3.1. Expression of GSTA4-4 in WT and Gsta4
Null MEF Cells
Expression of GSTA4-4 isozyme in WT and Gsta4 null
MEF cells was analyzed by Western blot using specific
antibody. The immunoblot presented in Figure 1(a) show-
ed robust expression of mGSTA4-4 in WT MEF, whereas
detectable expr essi on of t his isozyme was not obser ve d i n
Gsta4 null MEF.
3.2. Gsta4 Null MEF Are More Sensitive to
4-HNE and Oxidant Toxicity
In mammals, GSTA4-4 catalyzes conjugation additio n of
Copyright © 2013 SciRes. OJApo
reduced glutathione to 4-HNE, the majorendproduct of
peroxidative degradation of lipids and a commonly used
biomarker f or oxidative dam age in tissue [28]. Deletion of
Gsta4 was predicted to potentiate sensitivity towards 4-
HNE, and we also examined sensitivity of Gsta4 null
MEF towards other oxidants, H2O2 and paraquat (gener-
ates superoxide 2) known to be metabolized by catal-
ase and superoxide dismutases (SOD). Cytotoxicity of 4-
HNE(Ac)3 (0 - 25 µM), H2O2 (0 - 700 µM), and paraquat
(0 - 250 µM) were compared in WT and Gsta4 null MEF
cells by MTT assay, and IC50 values for each treatment
were calculated from the dose response curves (Table 1).
Cell viability curves (Figure 1(b)) indicated that Gsta4
null MEF cells were more sensitive than corresponding
WT cells to electrophilic stress elicited by 4-HNE(Ac)3, a
precursor which is converted in tracellularly to 4 -HNE [5,
19]. The functional role of mGSAT4-4 is further con-
firmed by the expe riment in which we compared cytotoxic
effects of 4-HNE(Ac)3 (0 - 25 µM) in control and m-
GSTA4-4 over-expressing Gsta4 null MEF. Results clear-
ly indicate that over expression of mGSTA4-4 rescues
Gsta4 null MEF and protect cells from 4-HNE toxicity
(Figure 1(b)). Reduced viability was also observed in
Gsta4 null MEF after exposure of cells to paraquat and
H2O2, both oxidants which initiate lipid peroxidation (Fig-
ures 1(c) and (d)).
3.3. Gsta4 Null MEF Cells Accumulate High
Levels of 4-HNE-Protein Adducts.
We have previously demonstrated that the tissues analy-
zed from Gsta4 null mice have high levels of 4-HNE
[18,29], known to interact with lysine, histidine, and cy-
steine residues of proteins and peptides [30]. To deter-
mine the status of 4-HNE-protein adducts in WT and
Gsta4 null MEF cells, we performed competitive ELISA
using antibodies against 4-HNE-protein adducts. Results
of these analyses (Figure 2) revealed the concentration of
4-HNE-protein adducts was significantly higher in Gsta4
null cells. These results are consistent with the high levels
of 4-HNE adducts we observed in liver, skeletal muscle
and white adipose tissue of Gsta4 null mice in 129/sv
background [29].
Table 1. IC50 values of 4-HNE(Ac)3, H2O2, and Paraquat
analyzed for MEF cells isolated from wild type and Gsta4
null mice. Data presented are Mean ± SD of three separate
experiments done in quadruplicates (n = 12).
IC50 (µM) ± SD
Treatment Wild type Gsta4 null
4-HNE(Ac)3 21.6 ± 1.65 11.7 ± 1.15
H2O2 527 ± 32 403 ±25
Paraquat 125 ± 15 80 ± 8
Figure 1. ( a): E xp re ss ion o f GS TA 4-4 in MEF c el ls p rep are d
from WT and Gsta4 null mice analyzed by Western blot;
(b)-(d): Viability of wild-type and Gsta4 null MEF cells ana-
lyzed with the CCK-8 kit after treatment with different con-
centrations of 4-HNE(Ac)3 (0 - 25 µM) , paraquat (0 - 250
µM) and H2O2 (0 - 700 µM).
Figure 2. Analysis of 4-HNE-protein adducts in control and
treated MEF cells by ELISA: Cells (2 × 106) were incubated
with 20 µM 4-HNE(Ac)3 for 2 h and subjected to ELISA
analysis using antibodies against 4-HNE protein adducts as
described in Methods section. Data presented are Mean ±
SD of two separate e xperiments done in triplicate (n = 6).
3.4. Gsta4 Null MEF Are More Sensitive to
4-HNE Induced Apoptosis
Previous studies have shown that 4-HNE initiates apopto-
tic cell death in a wide variety of cells via the Fas-
dependent extrinsic and mitochondria-mediated intrinsic
Copyright © 2013 SciRes. OJApo
pathways [14,31,32]. While in Fas-dependent extrinsic
apoptosis the initiator caspase-8 plays an important role,
caspase-9 has been shown to be involved in the mito-
chondria mediated apoptosis, h owever, both initiator cas-
pases can lead to the activation of the executioner or
effector caspases 3 and 7 [33]. Cytochrome C released
from mitochondria facilitates the cleavage of pro-caspase-
3 (37 kDa) to smaller fragments of 20, 17, and 12 kDa
[34]. Activation of caspase-3 analyzed by fluorescence
assay indicated that in Gsta4-null MEF cells, 4-HNE and
H2O2 mediated induction of caspase-3 was almost 2 fold
higher than WT MEF cells, indicating enhanced sensi-
tivity to 4-HNE-induced apoptosis (Figure 3(a)). This
observation was further confirmed by Western blot ana-
lysis (Figure 3(b)), which showed a time dependent in-
crease in the cleavage of caspase-3 in null MEF cells after
4-HNE(Ac)3 treatment not detected in MEF WT cells.
Likewise, the activation of caspase-8 and 9 was signi-
ficantly increased in MEF null cells treated with 4-HNE
(Figures 3(c) and (d)).
3.5. 4-HNE Causes DNA Damage
Caspases have been identified as key pro-apoptotic pro-
teins that can disrupt essential homeostatic processes and
initiate an orderly disassembly of cells, including degra-
dation of genomic DNA. Significant activation of cas-
pases in Gsta4 null cells by 4-HNE(Ac)3 was further cor-
related with strand breaks in DNA. The extent of DNA
damage in treated and control MEF cells was analyzed by
the alkaline comet assay [35]. As shown in Figure 4,
MEFs treated with vehicle alone did not show any signi-
ficant DNA damage. Substantial DNA fragmentation was
observed in both WT and Gsta4 null MEFs upon expos ure
to 20 µM 4-HNE(Ac)3 for 2 h, however, this damage was
significantly more pronounced in Gsta4 nu l l cells.
3.6. Activation and Phosphorylation of ERK,
JNK, and p38 MAPK Is Elevated in Gsta4
Null MEF
Treatment of hepatocytes or endothelial cells with 4-HNE
results in phosphorylation of ERK, JNK, and p38 MAPKs
[12,36], furthermore, pretreatment of bovine lung mic-
rovascular endothelial cells with inhibitors of MEK1/2,
JNK, or p38 MAPK only partially attenuated 4-HNE-
mediated barrier function and cytoskeletal remodeling
[12]. Western blot anal y si s (Figure 5) indicated that even
though 4-HNE treatment resulted in the activation and
phosphorylation of ERK, JNK, and p38 MAPK in both
WT and null MEF, the activation of these kinases was
only moderately more pronounced in null cells, which
could be attributed to elevated basal levels of 4-HNE.
Results showed that 4-HNE induced activation of these
protein kinases correlated with the increased sensitivity of
null MEF to oxidant ind uced apoptosis. To further corre-
Figure 3. 4-HNE or H2O2 induced activation of caspases in
MEF cells; (a) Activation of caspase-3 in MEF cells treated
with H2O2 (100 µM) and 4-HNE(Ac)3 (20 µM) measur ed by
ELISA; (b) Western blot analyses of 4-HNE induced activa-
tion of caspase3. Cells were treated with 4-HNE(Ac)3 (20 µM)
for 0 h - 5 hr, harvested, washed, and ly sed in RIPA buffer.
25 µg of protein from control and treated samples were
resolved by SDS-PAGE and immunobloted on nitrocellu-
lose membrane. Immunoblots were probed with caspase3
antibodies; (c) and (d). 4-HNE induced activation of cas-
pase-8 and 9 in MEF cells: WT and null MEF cells (5000
cells) wer e treated with 4-HNE(Ac)3 (20 µM) for 2 h. Control
and 4-HNE treated cells were analyzed for the activation of
caspase-8 and 9 by ELISA as describe d in the Methods sec-
tion. Data presented are Mean ± SD of two separate exper-
iments done in quadruplicates (n = 8).
late 4-HNE mediated induction of MAP kinases and in-
creased cytotoxicity in Gsta4 null MEF, we pre-treated
cells with selective and specific inhibitors of ERK (UO126)
[37], JNK (SP600125) [38], and p38 MAPK (SB 202190)
[39]. Pretreatment of MEF with kinase inhibitors blocked
the phosphorylation of stress related kinases in cells
treated with 4-HNE(Ac)3 (Figure 5) and partially abro-
gate the cytotoxic effect in Gsta4 null MEF (Figure 6).
Results clearly suggests that MAP kinases play an im-
portant role in 4-HNE mediated tox icity and cell death in
MEF and absence of GSTA4-4 potentiates the cytotoxic
effects of 4-HNE.
3.7. Delivery of GSTA4-4 into MEF Null Cells
Provides Protection from 4-HNE Induced
In order to ascertain the protective role of GSTA4-4
against oxidant toxicity, we delivered mGSTA4-4, Droso-
phila DmGSTD1-1, and hGSTA1-1 (catalytic efficiency
towards 4-HNE diminishes respectively) into Gsta4 null
Copyright © 2013 SciRes. OJApo
Figure 4. DNA damage determined by alkaline comet assay.
MEFs from wild-type and mGsta4 null mice were treated for
4 hr with vehicle or with 4-HNE(Ac)3. 60 cells from each tre-
atment were scored for DNA damage [25] using Comet-
Score ( Box: 25 - 75 percentile; whis-
kers: 10 - 90 percentile; horizontal line: median, circle:
mean. Untreated WT and mGsta4 null cells do not differ (p >
0.4), but cells treated with 20 µM 4-HNE(Ac)3 are signi-
ficantly different (p = 0.005, Wilcoxon rank-sum test).
Figure 5. Activation of MAP kinases in MEF cells after
treatment with kinase inhibitors and 4-HNE(Ac)3. Cells (2 ×
106) were treated separately with the SP600125 (JNK in-
hibitor), UO126 (ERK inhibitor) and SB202190 (p38 inhi-
bitor) and exposed to 4-HNE(Ac)3 (20 µM) for 2 hr. Cells
treated only with 4-HNE(Ac)3 and vehicle alone were used as
controls. E xtra cts of con trol an d tre ated c ells we re su bje cted
to Western blot analyses and probed separately with anti-
bodies against pERK [anti-pp44/42(Thr202/Tyr204)], p38
[anti-phospho-Thr180/Tyr204] and pJNK [anti-pJNK(Thr183/
Tyr185)]. Antibodies against β actin were used to ascertain
equal loading of proteins. Appropriate lanes in the Western
blot have been marked.
MEF using Stratagene Bio Trek delivery system [40].
After verifying successful delivery of GSTs by Western
blot (Figure 7(a)), we compared the effect of 4-HNE on
the viability of GST isozyme delivered null MEF cells.
Results of these studies indicated that while-cells deliv-
ered with mGSTA4-4 isozyme showed significant resis-
tance to 4-HNE(Ac)3, DmGSTD1-1 and hGSTA1-1 deliv-
ered cells did not show significant alteration in viability
upon treatment with 4-HNE(Ac)3 (data not shown). Ef-
fects of 4-HNE on th e activation of JNK and p38 MAPK
were also compared in GST isozyme delivered null MEF
cells. Western blot analyses (Figure 7(b)) revealed that
delivery of mammalian and invertebrate GSTs into Gsta4
null MEF cells indeed resulted in the attenuation of 4-
HNE mediated activation of JNK, ERK1/2, and p38
MAPK in a manner dependent on the catalytic efficiency
of each towards 4-HNE. Murine GSTA4-4, which has the
highest catalytic efficiency towards 4-HNE (1500 S-1.
mM-1 [41]), shows the highest reversal of stress kinase
activati on; foll owed by Dm GSTD1-1 (399 S-1. mM-1 [27])
and hGSTA1-1 (58.8 S-1. mM-1 [42]). Reversal by 4-
HNE-metabolizing GSTs indicates that the increased act-
ivation of stress-related kinases in Gsta4 null MEFs is
most likely due to 4-HNE and the activation of these pro-
tein kinases contributes to 4-HNE-induced apoptotic sig-
4. Discussion.
It is widely recognized that GSTs play a major role in the
Figure 6. Effect of inhibitors of p38 (a ); ERK ( b) and J NK (c)
on viability of MEF cells:WT and null cells ( 2 × 104) were
seeded in a 96 well plate and treated with fixed concen-
tration of inhibitors for 1 h then treated with 10 µM of 4-
HNE(Ac)3 for 24 h. Control cells received equal volume of
DMSO. After completion of treatment cell viability was as-
sayed by MTT as described in the Methods section. Data
shown are Mean ± SD of two experiments with eight re-
plicate wells.
Copyright © 2013 SciRes. OJApo
Figure 7. (a) Verification of GST isozyme uptake in Gsta4
null MEF cells after delivery with BioTrek reagent: mGsta4-
4, hGSTA1-1, and DmGSTD1-1 were delivered into Gsta4
null MEF cells using BioTrek (BT) reagent as described in
Methods section. Delivery of each isozyme in Gsta4 null
MEF was confirmed by Western blot analyses of GST tran-
sfected and control cell extracts (BT reagent treated and
untreated). (b) Effect on stress kinase activation by exo-
genous introduction of GST protein into Gsta4 null MEF
cells: Lane 1: untreated wild-type cells. Lane 2: wild-type
cells treated with protein delivery reagent only. Lane 3:
wild-type cells treated with protein delivery reagent and 20
µM 4-HNE(Ac)3. Lanes 4-10: mGsta4 null cells. Lane 4:
untreated. Lane 5: treated with 20 µM 4-HNE(Ac)3. Lane 6:
treated with delivery reagent only. Lane 7: treated with
delivery reagent and 20 µM 4-HNE(Ac )3. Lanes 8-10: treat-
ed with 20 µM 4-HNE(Ac)3 with delivery reagent plus 100
µg/dish of mGSTA4-4 (lane 8), hGSTA1-1 (lane 9), or
DmGSTD1-1 (lane 10), respectively. The core comparison:
delivery of no protein (lane 7) or the three GSTs into KO
cells (lane 8-10) exposed to 4-HNE(Ac)3.
regulation of intracellular 4-HNE levels and in defense
mechanisms against oxidative stress. The alpha class GST
isozymes GSTA1-1, GSTA2-2 , and GSTA3-3 efficiently
catalyze the GSH dependent reduction of fatty acid hy-
droperoxides [28,43] and limit the formation of 4-HNE,
while the isozyme GSTA4-4 exhibits high catalytic effici-
ency for conjugating 4-HNE to GSH for its metabolism
and disposition [41]. Thus, the alpha class GSTs limit the
cellular accumulation of 4-HNE and related unsaturated
lipid aldehydes asso ciated with oxidative damage to cells
via GSH peroxidase and GSH con jugating activities. The
Gsta4 null mouse model generated by us [18] has pro-
vided significant in sight into the physiolog ical and patho-
logical roles of 4-HNE. The ob served hypersensitivity of
Gsta4 null MEF cells to oxidants and the correlation of
this sensitivity with increased accumulation of 4-HNE-
protein adducts is consistent with previous findings [44,
45], suggesting that oxidative damage to cells leads to
significant increases in 4-HNE adducts and overexpres-
sion or induction of GSTA4-4 prevents such accretion
[13]. Rapid release of reactive oxygen species such as
superoxide radical and hydrogen peroxide during oxid-
ative stress leads to formation of the lipid peroxidation
product 4-HNE, which could be responsible for the im-
pairment of downfield protective mechanisms in the cel-
lular system [46]. Increased sensitivity of Gsta4 null MEF
towards unrelated substrates (H2O2 and paraquat) of
GSTA4-4 during in vitro treatment is possibly due to
excessive production of 4-HNE by treated cells. The rad-
ical-initiated reaction with polyunsaturated fatty acids is
unique since it results in a chain reaction [47], thus, 4-
HNE formation will be accelerated despite the protective
effects of catalase and supero xide dism uta se in Gsta4 null
cells. For this reason, cytotoxic effects of other oxidants
are not surprising. These results clearly indicate that GS-
TA4-4 is an important enzyme and it plays a pivotal
protective role du ri n g c hr o nic and acute oxidative stress.
Apoptosis is characterized by cell shrinkage, chrom atin
condensation, and blebbing of cellular components pro-
ducing apoptotic bodies [48-50]. The process can be sum-
marized as initiation by an apoptosis inducing agent,
cleavage of pro-caspases constituting a family of aspar-
tate-specific cysteine proteases resulting in a caspase cas-
cade, and culminating with the cleavage of proteins by
executioner ca spases and cel l death [51,52]. Supraphysio-
logical concentrations of 4-HNE are known to induce
apoptosis in most cell types studied to date and is induced
via the death receptor Fas-mediated extrinsic and the
mitochondria mediated intrinsic apoptotic p athway [52,53].
Increased apoptosis accompanied by increased 4-HNE-
protein a dducts an d DNA dama ge in Gsta4 null MEF cells
supports the pro-apoptotic role of 4-HNE. Furthermore,
an accelerated activat ion of caspase -3, caspase- 8, and cas-
pase-9 is consistent with our previous findings [14,31]
suggesting that 4-HNE induces apoptosis via both the ex-
trinsic and intrinsic pathways in a cell type independent
manner. 4-HNE(Ac)3 activated caspase-3 in Gsta4 null
cells possibly targets key enzymes responsible for DNA
repair and fragmentation to execute apoptosis [51]. The
observed increase in DNA fragmentation assessed by
comet assay in treated Gsta4 null cells positively corre-
lates with the execution er role of caspase-3.
ERK, JNK, and p38 MAPK are vital and central ele-
ments of the MAPK family, mediating multiple signaling
cascades initiated by stress, cytokines, and growth factors.
ERK is the final enzyme of the MAPK pathway which
transmits signals into the nucleus and chronic activation
of ERK can i n duce apoptosis [54]. Consi st e nt wi t h earl i e r
studies [12,31,45], treatment with 4-HNE resulted in the
Copyright © 2013 SciRes. OJApo
activation and phosphorylatio n of JNK, ERK1/2, and p38
MAPK in MEF cells. Higher basal levels of activated/
phosphorylated JNK, ERK1/2, and p38 MAPK observed
in Gsta4 null MEFs accompanied by increased 4-HNE
levels further correlates the role of 4-HNE in the activa-
tion of these ki nases. Even thou gh stres s ki nase act ivation
is known to play an important role in the mechanisms of
apoptosis, cell survival/proliferation, and inflammation,
their ultimate effect on cellular fate is still controversial.
For example, activation of different isoforms of JNK
under various stimuli has been shown to affect both apo-
ptotic and pro-survival signaling [32,55]. Our results
showing that pretreatment of MEF with inhibitors of
MAPK and JNK only partially abrogated the sensitivity of
MEF null cells to 4-HNE(Ac)3 (Figure 6) sugge st 4- HN E
also exerts its toxicity through undefined mechanisms in
addition to MAPK and JNK mediated apop tosis.
Functional differences between WT and cells lacking
GSTA4-4 could be triggered by di fferi ng l e vel s of 4-HNE,
by a change in the concentration of mGSTA 4-4 subst rates
other than 4-HNE, or by non-catalytic functions of mGS-
TA4-4, such as direct binding to proteins. GSTs of the Pi
and Mu classes are known to act as stress sensors by se-
questering signaling kinases under normal condition s and
releasing them in response to stress [56-62]. GSTs may
also serve an anti-apoptotic role, one example is a novel
plant GST shown to suppress Bax, and thus affect apop-
tosis [63-65]. To distinguish between the various modes
of GST action, we directly introduce mGSTA4-4 (which
has 4-HNE-conjugating ability), the phylogenetically di-
stant Drosophila DmGSTD1-1 (which is also capable of
conjugating 4-HNE, albeit with a lesser catalytic effi-
ciency than mGSTA4-4) [27], and hGSTA1-1 (which is
closely related to mGSTA4-4 but lacks significant activity
toward 4-HNE) into Gsta4 null cells. mGSTA4-4 and, to a
lesser extent, consistent with its lower activity, DmG-
STD1-1 were able to abrogate the activation of p38
MAPK and JNK (Figure 7) by 4-HNE(Ac)3, whereas
hGSTA1-1 had no effect. Drosophila DmGSTD1-1 be-
longs to a different family of GSTs than mGSTA4-4 [27],
and is unlikely to enter into specific protein-protein in-
teractions in a mammalian system. Thus, the only known
property of the three enzymes that correlates with the abil-
ity to prevent stress kinase activation is conjugation of 4-
HNE, indicating that 4-HNE mediates kinase activation.
Together, results of present studies clearly demonstrate
that 4-HNE significantly contributes to the cytotoxicity
and that GSTA4-4 plays a crucial ro le in nullifying acute
4-HNE toxicity by converting it to non-electrophilic glu-
tathione conjugate g lutathionyl-HNE (GS-HNE).
5. Concluding Remarks.
GSTA4-4 is a key enzyme that regulates 4-HNE concen-
tration in mammalian cellular systems. Our findings clear-
ly suggest that the impairment of 4-HNE conjugation in
Gsta4 null MEF increases the sensitivity toward s 4-HNE
by activating caspases and stress-activated kinases. Fur-
thermore, the positive relationships between DNA da-
mage, increased 4-HNE-protei n adduct accumulation, and
apoptosis in Gsta4 null MEF upon treatment with 4-
HNE(Ac)3 were demonstrated. These results suggest that
4-HNE ind uced apoptosis of Gsta4 null MEF is associated
with the enhanced accumulation of 4-HNE-protein ad-
ducts, DNA damage, and the activation of caspases-3, 8
and 9. We also demonstrated that exogenous delivery of
GST isozymes with medium to high catalytic efficiency
towards 4-HNE prevents stress-related kinase activation
upon treatment with HNE(Ac)3. Thus, we conclude that
GSTA4-4 is a major 4-HNE metabolizing enzyme in the
mouse, which protects cells from 4-HNE toxicity during
acute oxidative stress.
6. Acknowledgements.
This work was supported in part by National Institutes of
Health grants R01 AG028088 and AG032643 (to Piotr
Zimniak and Sharda Singh), Pilot and Exploratory Stud-
ies Program grant from Claude Pepper Older Americans
Independence Center (to Sharda P. Singh), and a grant
from Patricia Rogers Joslin Foundation for Pancreatic
Cancer Rsearch (to Yogesh C. Awasthi).
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