Acrolein consumption exacerbates myocardial ischemic injury and blocks
nitric oxide-induced PKCε signaling and cardioprotection
Guang-Wu Wanga,b, Yiru Guob, Thomas M. Vondriskaa, Jun Zhanga, Su Zhangb, Linda L. Tsaia,
Nobel C. Zonga, Roberto Bollib, Aruni Bhatnagarb, Sumanth D. Prabhub,c,⁎
aDepartment of Physiology and the Cardiovascular Research Laboratories, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
bInstitute of Molecular Cardiology, Department of Medicine/Cardiology, University of Louisville, Louisville, KY, USA
cMedical Service, Louisville VA Medical Center, Louisville, KY, USA
Received 7 December 2007; received in revised form 21 March 2008; accepted 22 March 2008
Available online 8 April 2008
Aldehydes are common reactive constituents of food, water and air. Several food aldehydes are potentially carcinogenic and toxic; however,
the direct effects of dietary aldehydes on cardiac ischemia-reperfusion (IR) injury are unknown. We tested the hypothesis that dietary consumption
of aldehydes modulates myocardial IR injury and preconditioning. Mice were gavage-fed the α, β-unsaturated aldehyde acrolein (5mg/kg) or
water (vehicle) 24h prior to a 30-min coronary artery occlusion and 24-hour reperfusion. Myocardial infarct size was significantly increased in
acrolein-treated mice, demonstrating that acute acrolein exposure worsens cardiac IR injury. Furthermore, late cardioprotection afforded by the
nitric oxide (NO) donor diethylenetriamine/NO (DETA/NO; dose: 0.1mg/kg × 4, i.v.) was abrogated by the administration of acrolein 2h prior to
DETA/NO treatment, indicating that oral acrolein impairs NO donor-induced late preconditioning. To examine potential intracellular targets of
aldehydes, we investigated the impact of acrolein on mitochondrial PKCε signaling in the heart. Acrolein-protein adducts were formed in a dose-
dependent manner in isolated cardiac mitochondria in vitro and specific acrolein-PKCε adducts were present in cardiac mitochondrial fractions
following acrolein exposure in vivo, demonstrating that mitochondria are major targets of aldehyde toxicity. Furthermore, DETA/NO
preconditioning induced both PKCε translocation and increased mitochondrial PKCε localization. Both of these responses were blocked by
acrolein pretreatment, providing evidence that aldehydes disrupt cardioprotective signaling events involving PKCε. Consumption of an aldehyde-
rich diet could exacerbate cardiac IR injury and block NO donor-induced cardioprotection via mechanisms that disrupt PKCε signaling.
Published by Elsevier Inc.
Keywords: Acrolein; Diethylenetriamine/NO; Myocardial infarction; PKC epsilon
The extent of ischemic injury suffered by the heart depends
upon several factors including the duration of ischemia and the
time of reperfusion. In addition, biochemical responses intrinsic
to the myocardium modulate the outcome of ischemic insults.
Extensive experimental and clinical work has shown that ische-
mic preconditioning or pharmacological preconditioning with
agents such as adenosine or nitroglycerin decreases myocardial
ischemia-reperfusion (IR) injury [1,2]. Triggers of precondi-
tioning activate complex signaling pathways that ultimately
strengthen the resistance of the heart to ischemia. The inherent
vulnerability of the heart to IR injury and its ability to mount a
preconditioning response are, however, susceptible to environ-
mental influences. Myocardial resistance to ischemic injury, for
instance, is enhanced by physical exercise [2,3], caloric
restriction , and alcohol consumption  and preconditioning
mechanisms are disrupted by aging , caffeine  or treatment
with drugs such as cyclooxygenase-2 inhibitors . However,
whether reactive constituents of diet affect myocardial sensi-
tivity to ischemia or preconditioning is unknown.
Aldehydes are highly reactive components of food and wa-
ter. More than 300 different aldehydes have been identified in
Available online at www.sciencedirect.com
Journal of Molecular and Cellular Cardiology 44 (2008) 1016–1022
⁎Corresponding author. Medicine/Cardiology, University of Louisville, ACB,
3rd Floor, 550 South Jackson Street, Louisville, KY 40202, USA. Tel.: +1 502
852 7959; fax: +1 502 852 7147.
E-mail address: email@example.com (S.D. Prabhu).
0022-2828/$ - see front matter. Published by Elsevier Inc.
various foods  (see Online Supplement, Table 1). Additional
aldehydes are generated during frying and cooking. Indeed,
because they are natural products of lipid peroxidation and
glucose oxidation, aldehydes are generated in high concentra-
tions by heating and cooking fats, oils, and sugars . At least
36 different aldehydes are also present in water , and with
the exception of metals, aldehydes are considered to be the
major pollutants in drinking water  (Online Supplement,
Table 1). By themselves, aldehydes are highly reactive poten-
tial carcinogens [9,11]. They form covalent adducts with DNA
Aldehydes have also been shown to depress myofilament
sensitivity and cardiac contraction , inhibit mitochondrial
respiration , and alter ion-channel conductance pathways
. Nevertheless, the cardiovascular toxicity of ingested
aldehydes has not been directly studied and their effects on
myocardial responses to ischemia are unknown.
dietary consumption of aldehydes affects myocardial ischemia-
reperfusion injury and preconditioning. Because toxicological
studies of complex aldehyde mixtures in food are difficult to
interpret, we used acrolein as the prototypical dietary aldehyde.
High levels of acrolein have been detected in several foods
including cheese, donuts, fish, bread, potatoes, and alcoholic
beverages [9,19] (Online Supplement, Table 1). The concentra-
tion of acrolein is particularly high in cigarette smoke and heated
oils . Our study shows that at concentrations comparable to
human consumption, acrolein worsens infarct size following IR
injury and blocks nitric oxide-induced cardioprotection in mice
via a mechanism that appears to involve disruption of protein
foods rich in aldehydes could increase myocardial susceptibility
to IR injury and abolish cardioprotective signaling.
The experimental protocols described herein were per-
formed in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals (Publication
2.1. Myocardial ischemia/reperfusion surgery and infarct size
ICR mice were subjected to myocardial ischemia and
reperfusion as previously described . Individual protocols
are illustrated in Fig. 1. In Group I, mice were fed either acrolein
(5mg/kg) or water by gavage, and 24h later were anesthetized
with pentobarbital sodium (50mg/kg body wt i.p.), intubated,
and ventilated with 100% oxygen. The chest was opened by a
left thoracotomy between ribs three and four and a 8-0 silk
suture was placed under the left anterior descending coronary
artery 1 to 3mm from the tip of the left atrial appendage.
Ischemia was induced by ligation of the suture (a 1 to 2mm
section of PE-10 tubing was placed between the suture and the
artery to prevent damage to the vessel). Following a 30-min
occlusion, the suture was removed, the chest wall closed, and
the heart was allowed to reperfuse for 24h. The heart was then
Fig. 1. Experimentalprotocolforstudiesofmyocardialischemia-reperfusioninjuryandnitricoxide-mediatedcardioprotection.Threegroupsofmiceunderwent30min
of coronary occlusion followed by 24 h of reperfusion. In Group 1, 24 h before occlusion (Day 0) the mice were fed acrolein (5 mg/kg, p.o.). Twenty four hours after
acrolein, the mice were subjected to coronary occlusion and reperfusion as indicated. In Group II, the mice received 4 intravenous boluses of DETA/NO (0.1 mg/kg)
before DETA/NO or PBS treatment. In both Groups II and III, 24 h after DETA/NO or acrolein treatment, the mice were either subjected to 30-min coronary occlusion
followed by 24-hour reperfusion, or their hearts were excised for PKC studies as indicated.
1017G.-W. Wang et al. / Journal of Molecular and Cellular Cardiology 44 (2008) 1016–1022
excised and perfused postmortem as described . The
infarcted region was delineated by perfusion with a 1% solution
of 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate
buffer (pH 7.4, 37°C). Non-infarcted tissue would take up
TTC (red), whereas infarcted regions would be TTC-free
(white). To delineate the occluded-reperfused coronary vascular
bed, the coronary artery was tied at the site of the previous
occlusion and the aortic root perfused with a 5% solution of
phthalo blue dye. Hence, the nonischemic region (region not at
risk) would be stained blue whereas the risk region would not
stain blue. Infarct size (white area) was measured by video-
planimetry with NIH Image software and expressed as a per-
centage of the region at risk (red plus white area). The region at
risk was expressed as a percentage of the total left ventricle
2.2. Acrolein administration
The dose-range of human aldehyde exposure estimated from
maximal daily consumption was 7mg/kg aldehyde/day (Online
Supplement, Table 2). The estimate of acrolein consumption
from just 8 different types of foods was 0.1mg/kg/day (Online
Supplement, Table 3). The approximate daily consumption of
unsaturated aldehydes (such as acrolein) was estimated to be
5mg/kg, whereas that of saturated aldehydes (such as form-
aldehyde and acetaldehyde) was 2mg/kg. Based on these
estimates a 5mg/kg dose of acrolein, representing the expected
unsaturated aldehyde intake, was chosen for this study. Eight
week old male mice (ICR) were gavage-fed acrolein (in 200μL
water) or the same volume of water (vehicle). Free acrolein was
prepared daily by acid hydrolysis (pH 3.0) of diethyl acetal
acrolein (Sigma) in 0.1N HCl for 1h at room temperature (RT).
Mice were housed in a pathogen-free room at 24°C, 55–65%
relative humidity, and with a 12:12-hour light:dark cycle. Ani-
mals had free access to food and water.
2.3. DETA/NO-induced cardioprotection
This protocol has been previously shown to induce a late
phase of pharmacological preconditioning in rabbits 
and mice . Mice were given four consecutive intravenous
bolus doses of either the NO donor diethylenetriamine/NO
(DETA/NO, 0.1mg/kg every 25min for a total dose 0.4mg/kg)
or PBS (vehicle) on Day 0 (Group II; Fig. 1). In an additional
group (Group III), the mice were fed acrolein 2h before DETA/
NO or PBS treatment.
2.4. Subcellular fractionation, immunoblotting, and
Western immunoblotting analysis of PKCε was performed as
described . Briefly, 24h after acrolein administration and
DETA/NO preconditioning, mouse hearts were homogenized to
obtain total myocardial lysates. Cytosolic and particulate or
mitochondrial fractions were obtained by differential centrifu-
gation as previously described [22,23]. Cytosolic contamination
of the mitochondrial fraction was less than 0.5% as measured by
lactate dehydrogenase activity. Proteins were separated on a
10% SDS-PAGE gel and immunoblotted using anti-PKCε mo-
noclonal antibodies (1:1000; BD Transduction Labs).
2.5. Assay for formation of acrolein-protein adducts in cardiac
Mitochondria were isolated from adult mouse hearts by en-
Fig. 2. Acute acrolein administration increases infarct size and blocks cardioprotection. A, Acrolein exposureincreases infarct size in the naive myocardium. Male ICR
mice were gavage-fed 5 mg/kg acrolein 24 h prior to a 30-min left coronary artery occlusion and 24-hour reperfusion and the infarct size was determined (⁎pb0.05 vs.
vehicle). B, Cardioprotection by DETA/NO is blocked by acrolein. Preconditioning with the NO donor DETA/NO (0.1 mg/kg×4 i.v.) significantly reduces infarct size
(⁎pb0.05 vs. vehicle). However, this protection is blocked by administration of acrolein 2 h prior to DETA/NO treatment (#pb0.05 vs. DETA/NO).
1018 G.-W. Wang et al. / Journal of Molecular and Cellular Cardiology 44 (2008) 1016–1022
as previously described [22,23]. Isolated mitochondria (50μg
protein) were incubated with different concentrations of acrolein
(0,1,3,10,30, 100,300,and 600μmol/L) for30minat37°C, and
acrolein-protein adducts were detected by immunoblotting
(1:5000; anti-acrolein-lysine monoclonal antibody was the ge-
nerous gift of Dr. Koji Uchida).
To detect the formation of acrolein-PKCε adducts, im-
munoprecipitation was carried out as described . Brief-
at 4°C. After washing three times with lysis buffer, the samples
acrolein-lysine monoclonal antibody).
Data are reported as mean ± standard error measurement
(SEM). Infarct sizes and protein expression were analyzed with
a one-way ANOVA followed by Student's t-tests for unpaired
3.1. Acrolein exacerbates myocardial IR injury
Mice gavage-fed 5mg/kg acrolein displayed no overt sign of
toxicity. There were no changes in liver enzymes, urine
composition, blood electrolytes, or echocardiographically
measured cardiac function (data not shown). Indeed mice
could be maintained on this dose of acrolein for up to one week
with no mortality. To test the hypothesis that aldehyde
consumption is detrimental to the heart, myocardial infarct
sizes were determined in mice subjected to a 30-min coronary
artery occlusion and 24-hour reperfusion following acrolein
administration (Fig. 2A). The data show that acute acrolein
exposure significantly increased infarct size as compared to
vehicle-treated mice (51.6 ± 1.4% vs. 37.7 ± 6.2%, p b 0.05).
The risk region as a percentage of total LV was equivalent
between the groups (vehicle 49.6 ± 12.8%; acrolein 47.1 ±
9.5%, p = NS).
3.2. Acrolein blocks NO donor-induced cardioprotection
We next examined whether acrolein impairs pharmacolo-
gical preconditioning using a model of NO donor-induced
cardioprotection (Fig. 2B). Mice administered the NO donor
DETA/NO 24h prior to a 30-min coronary artery occlusion
and 24-hour reperfusion exhibited significantly reduced
infarct size compared to control mice (22.2 ± 3.1% vs.
41.4 ± 8.2%; p b 0.05), demonstrating a late preconditioning
effect. In contrast, administration of acrolein 2h prior to
DETA/NO effectively abolished the cardioprotection afforded
by the NO donor (infarct size 44.8 ± 6.2%; p b 0.05 vs.
DETA/NO alone). The risk region as a percentage of total
LV was comparable between groups (vehicle 40.3 ± 3.1%;
DETA/NO 43.2 ± 2.5%; acrolein + DETA/NO 36.7 ± 4.5%,
p = NS).
3.3. Acrolein abrogates PKCε translocation and blocks NO
donor-induced increase in mitochondrial PKCε expression
To determine the mechanism by which aldehydes abolish
NO-dependent cardioprotection, translocation of PKCε was
examined in all groups of mice (Fig. 3A). Analogous to ob-
servations in rabbits [21,25], DETA/NO induced a significant
translocation of PKCε 24h after its administration (particu-
late fraction increased from 23.4 ± 5.7% to 36.7 ± 3.7%;
measurements given as percentage of total PKCε expression,
p b 0.001 vs. vehicle). Translocation, however, was blocked by
treatment with acrolein 2h prior to DETA/NO administration
(particulate fraction 15.3 ± 2.0%, p b 0.001 vs. DETA/NO).
Next, the expression of PKCε was examined in mitochondrial
fractions. As seen in Fig. 4A, DETA/NO increased mitochon-
driallocalizationof PKCε (241.6±3.1% of vehicle; pb0.05vs.
vehicle). Analogous to PKCε translocation, the increase in
mitochondrial expression of PKCε was also blocked if acrolein
was administered prior to DETA/NO (28.8±7.4% of vehicle;
pb0.05 vs. DETA/NO and vs. vehicle). Fig. 4B demonstrates
the purity of isolated mitochondrial fractions, as assessed
by sarcolemmal (Na+/K+ATPase), mitochondrial (cytochrome
C oxidase IV), and cytosolic (aldose reductase) markers.
Cytosolic contamination of the mitochondrial fraction was
negligible. Sarcolemmal contamination was less than 5%.
Fig. 3. A, Translocation of PKCε is blocked by acrolein. Cytosolic and
particulate distribution of PKCε was determined by Western immunoblotting
(IB) in all groups. When given prior to vehicle, acrolein had no effect on PKCε
distribution at 24 h, but when administered prior to DETA/NO, acrolein sig-
nificantly attenuated DETA/NO-induced PKCε translocation (⁎pb0.001 vs.
vehicle;#pb0.001 vs. DETA/NO; n=3–6 per group).
1019 G.-W. Wang et al. / Journal of Molecular and Cellular Cardiology 44 (2008) 1016–1022
3.4. Acrolein-protein adducts form in cardiac mitochondria
Lastly, the ability of acrolein to directly modify cardiac pro-
teins was examined in cardiac mitochondria. In vitro, aldehyde-
protein adducts formed in isolated cardiac mitochondria in a
dose-dependent fashion (Fig. 4C) while specific acrolein-PKCε
adducts were detected in cardiac mitochondrial fractions after
acrolein exposure in vivo (Fig. 4D), suggesting that the mito-
chondria are highly susceptible to aldehyde toxicity.
The major findings of this study are that oral acrolein
consumption increases myocardial IR injury and abolishes the
cardioprotective effects of nitric oxide. These novel results have
several important implications. First, the consumption of
acrolein and related aldehydes in aldehyde-rich foods could
significantly affect the myocardial sensitivity to ischemia. The
signaling mechanisms by which these changes occur appear to
involve attenuated PKCε translocation and decreased mito-
chondrial PKCε expression, as well as direct aldehyde-protein
adduct formation in cardiac mitochondria. Second, the dele-
terious effects of aldehydes also include the abrogation of NO-
dependent pharmacological cardioprotection, suggesting that
aldehyde consumption can interfere with the protective effects
of nitrate drug therapy for coronary artery disease. Third, these
findings support the notion that acrolein may act at PKCε-
dependent foci within cardioprotective signaling networks to
block the development of the infarct-sparing phenotype.
We interpreted our results to signify that acrolein both exa-
cerbates infarct size following ischemia and reperfusion and
blocks NO donor-induced preconditioning and cardioprotec-
tion (Fig. 2). An alternate explanation could be that of an offset
phenomenon, i.e. that DETA/NO attenuated acrolein-induced
injury. We feel that this was much less likely given for two
infarct size between the group treated with acrolein alone vs.
the group treated with acrolein prior to DETA/NO. Second,
acrolein administration also suppressed the translocation and
mitochondrial localization of PKCε induced by DETA/NO. As
there is strong evidence that subcellular redistribution of PKCε
is a critical event essential for the production of NO donor-
induced pharmacological preconditioning [21,26–28], this
observation indicates that acrolein directly disrupted NO-
dependent preconditioning pathways (and argues against an
offset phenomenon). Indeed, as PKCε has a well-established
role in cardioprotection [21,24,29], translocation of this iso-
form in response to the same dose of DETA/NO that induces
Fig. 4. Acrolein toxicity targets the cardiac mitochondria. Cardiac mitochondria were isolated and Western immunoblotted (IB) for PKCε expression. A, Acrolein
treatment results in decreased mitochondrial PKCε expression. DETA/NO-induced augmentation of mitochondrial PKCε expression at 24 h was blocked by the
administration of acroleinprior toDETA/NO treatment.B, Purityof isolated mitochondrialfractions.Total homogenateandcytosolic andmitochondrial fractionswere
subjected to Western blotting for sarcolemmal (Na+/K+ATPase) mitochondrial (cytochrome C oxidase IV), and cytosolic (aldose reductase) markers. Cytosolic
contamination of the mitochondrial fraction was negligible. Sarcolemmal contamination of the mitochondrial fraction was less than 5%. C, Formation of acrolein-
protein adducts in isolated cardiac mitochondria. Isolated mitochondria were incubated with different concentrations of acrolein (0, 1, 3, 10, 30, 300, and 600 μmol/L).
A dose-dependent formation of acrolein-protein adducts was observed. D, Formation of acrolein-PKCε adducts in cardiac mitochondria. Cardiac mitochondria were
isolated at the end of the in vivo experiments and immunoprecipitation was performed. Acrolein-PKCε adducts were observed in the mitochondria following acrolein
administration. Furthermore, the adducts formed evenin the presence of DETA/NO after acroleintreatment, indicating that DETA/NO had no effect on their formation.
1020 G.-W. Wang et al. / Journal of Molecular and Cellular Cardiology 44 (2008) 1016–1022
cardioprotection and PKCε translocation in the rabbit  was
not unexpected. In addition, the finding that translocation of
PKCε was blocked by the same dose of acrolein that prevented
cardioprotection further corroborates the critical mechanistic
link between NO, PKCε, and cardioprotection. The data herein
support the idea that PKCε plays a necessary role in protecting
the murine heart against infarction, since reduced translocation
in response to NO following acrolein treatment was accom-
panied by loss of the cardioprotective phenotype.
Our data indicate that NO-induced cardioprotection
increases localization of PKCε to the mitochondria. Given the
multitude of cardioprotective processes known to depend on the
mitochondria, this finding has significance regarding signaling
transduction by PKCε at this organelle. Previous studies have
shown that PKCε physically interacts with components of the
mitochondrial permeability pore to inhibit mitochondrial
permeability transition . The observation that cardioprotec-
tion provided by DETA/NO was associated with mitochondrial
PKCε translocation further supports the view that mitochondrial
association of PKCε may be an essential mechanism of
preconditioning. Intriguingly, treatment with acrolein before
DETA/NO administration was sufficient to block the increase in
PKCε in the mitochondria. Finally, the finding that acrolein
forms direct adducts with proteins including PKCε in cardiac
mitochondria, taken together with previous data demonstrating
that aldehydes interfere with mitochondrial function [15,17,30],
supports the notion that this organelle is a target for aldehyde
toxicity in the heart.
The observation that aldehyde consumption exacerbates
myocardial ischemia/reperfusion injury and blocks cardiopro-
tection highlights the vulnerability of the ischemic heart to
environmental influences. Exposure toenvironmentalpollutants
is an emerging risk factor for cardiovascular disease  as
several studies suggest that acute exposure to polluted air
increases myocardial sensitivity to ischemia and arrhythmias
. Indeed, in a recent analysis, ischemic heart disease was the
largest specific cause of death associated with pollutant ex-
posure, which accounted for a one-quarter of such death .
Statistically significant associations were also observed for
arrhythmias, heart failure and cardiac arrest, but surprisingly no
positive correlations were observed with respiratory diseases,
indicating that the diseased heart is highly sensitive to en-
vironmental pollutants. Hence, thepresentdata, demonstrating a
direct link between a specific ubiquitous dietary component and
myocardial sensitivity to ischemic injury, further underscore the
unique vulnerability of the heart to xenobiotic toxicity and
suggest that reactive xenobiotics in the diet may be heretofore
unrecognized modulators of myocardial ischemia-reperfusion
Even though our data support a strong link between al-
dehyde consumption and sensitivity to ischemia in mice, the
human susceptibility to food aldehydes remains unknown. The
dose-range of acrolein used in this study was similar to the
expected human consumption; however, aldehyde toxicity is
also a function of aldehyde metabolism. Aldehydes such as
acrolein are extensively metabolized by cardiovascular tissues,
kidney and liver [15,19,34,35], and their toxicity depends upon
the total aldehyde detoxification capacity which itself may
be under the control of environmental factors, such as disease
or age. Even within experimental animals sensitivity to acro-
lein varies. The LD50of acrolein in mice is 40 mg/kg, whereas
in rabbits it is 7 mg/kg . Whether humans are more or less
sensitive to acrolein is not known, but additional work is
clearly warranted to ascertain human sensitivity and to deter-
mine whether the aldehyde content of food (analogous to
the cholesterol or fat content) is a contributing factor to the
severity of heart disease. In this regard it is interesting to point
out that humans display wide variations in the levels of
glutathione-S-transferase (GST) , an enzyme involved in
aldehyde detoxification [15,19]. GST-deficient and null-
genotypes in human populations are associated with increased
risk for cancer  and dietary regulation of DNA damage
. It is possible that similar associations may exist for
ischemic heart disease.
Acrolein and related aldehydes are not only common food
constituents but also ubiquitous pollutants present in high
amounts in coal, wood, cotton, cigarette smoke, automobile
exhaust, and industrial waste [9,19]. Hence, even though it
remains unclear whether the effects of inhaled and ingested
aldehydes are similar, our findings raise the possibility that
consistent with the high cardiovascular toxicity associated
with the aldehyde-containing components of polluted air or
cigarette smoke . Further experimental, clinical and epide-
miological studies are needed to address these issues.
In summary, we provide the first line of evidence that
reactive aldehydes in diet can influence myocardial sensitivity
to ischemia and block NO-mediated cardioprotection related, at
least in part, to the disruption of the activation and mito-
chondrial localization of PKCε. The vulnerability of cardiopro-
tective signaling to dietary aldehydes points towards a complex
interplay between environment and genetic susceptibility, much
of which remains currently unknown.
This study was supported in part by AHA Grant 0465137Y
(RB, AB, and SDP), ES-12062 (AB), ES-11860 (AB and SDP),
VA Merit Grant (SDP), and the Laubisch Endowment at UCLA.
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