Hum Exp Toxicol Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells

http://het.sagepub.com/Human & Experimental Toxicology

http://het.sagepub.com/content/early/2012/01/09/0960327111432500The online version of this article can be found at:

DOI: 10.1177/0960327111432500

published online 12 January 2012Hum Exp ToxicolPant and S. Jahan

M A. Siddiqui, V. Kumar, M. P. Kashyap, M. Agarwal, A. K. Singh, V. K. Khanna, A. A. Al-Khedhairy, J. Musarrat, A. B.Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells

Published by:

http://www.sagepublications.com

can be found at:Human & Experimental ToxicologyAdditional services and information for

http://het.sagepub.com/cgi/alertsEmail Alerts:

http://het.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- Jan 12, 2012Proof >>

at UNIV OF LOUISVILLE on January 14, 2012het.sagepub.comDownloaded from

http://het.sagepub.com/

http://het.sagepub.com/content/early/2012/01/09/0960327111432500

http://www.sagepublications.com

http://het.sagepub.com/cgi/alerts

http://het.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://het.sagepub.com/content/early/2012/01/09/0960327111432500.full.pdf

http://online.sagepub.com/site/sphelp/vorhelp.xhtml


Original Paper

Short-term exposure of4-hydroxynonenal inducesmitochondria-mediated apoptosisin PC12 cells

MA Siddiqui1, V Kumar2, MP Kashyap2, M Agarwal2,AK Singh2, VK Khanna2, AA Al-Khedhairy1, J Musarrat1,AB Pant2 and S Jahan2

Abstract4-Hydroxynonenal (4-HNE) is one of the most reactive aldehydic by-products of lipid peroxidation. Therole of 4-HNE in the etiology of various neurodegenerative disorders including cerebral ischemia/reper-fusion, Alzheimer’s disease, Parkinson’s disease, etc. has been documented. We and others have reportedthat long-term toxic insults of 4-HNE triggers apoptotic signals and oxidative stress in various cells.However, the status of apoptosis following short-term exposure and underlying mechanisms has not beenexplored so far. We studied the apoptotic changes in PC12 cells receiving short-term exposure of4-HNE. A significant dose-dependent induction in reactive oxygen species (ROS) and early responsemarkers (c-Fos, c-Jun, and GAP-43) were observed in cells exposed to 4-HNE (10, 25, and 50 mM) for1h. Following the exposure of PC12 cells to 4-HNE, the levels of protein and messenger RNA expres-sions of P53, Bax, and caspase 3 were significantly upregulated, whereas the levels of Bcl2 was downre-gulated. We could record the apoptotic signals and ROS generation in PC12 cells receiving 4-HNEexposure for such a short period of time. Induction in the expression and activity of caspase 3 has alsoindicated the mitochondrial mediation in the apoptosis induction.

KeywordsPC12 cells, 4-hydroxynonenal, reactive oxygen species (ROS), apoptosis

Introduction

Aldehydic products of lipid peroxidation of biological

membranes have been reported as important etiologi-

cal factors in numerous neurodegenerative disor-

ders.1–3 Of these, 4-hydroxynonenal (4-HNE), a

long-chain a, b-unsaturated aldehyde, is known to

be most toxic.4,5 Higher concentrations of 4-HNE are

cytotoxic and causes oxidative stress-mediated cell

death in a variety of cell types including PC12

cells.6–9 The low-level exposure of 4-HNE also

modulates intracellular signaling by activating the

mitogen activated protein kinases (MAPK), stress-

activated protein kinase and c-Jun N-terminal protein

kinase cascades, and inhibiting the nuclear factor k-B

activity.7,10,11 We and others have shown the associa-

tion of 4-HNE-induced oxidative stress-mediated

cytotoxicity/genotoxicity with dopamine (DA-D2),

cholinergic (muscarinic), benzodiazepine, and seroto-

nin (5-HT)-2A receptors in cultured cells of human

and animal origins.8,12 The expression of GSTP1-1

and increased levels of intracellular Caþþ are also

associated with 4-HNE exposure in cells.9

1 Department of Zoology, College of Science, King SaudUniversity, Riyadh, Saudi Arabia2 In Vitro Toxicology Laboratory, Indian Institute of ToxicologyResearch, Lucknow, India

Corresponding author:AB Pant, In Vitro Toxicology Laboratory, Indian Institute ofToxicology Research, PO Box 80, MG Marg, Lucknow 226001,Uttar Pradesh, IndiaEmail: [email protected]; [email protected]

Human and Experimental Toxicology1–10

ª The Author(s) 2011Reprints and permission:

sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0960327111432500

het.sagepub.com



In our earlier studies, a concentration-dependent

metabolism of 4-HNE in PC12 cells was observed in

the form of glutathionyl conjugates and 4-Hydroxy-

trans-2-nonenoic acid (HNA) and 1,4-Dihydroxy-2-

nonene (DHN) metabolites within 1 h of exposure.13

Although, 4-HNE has been reported to induce apopto-

sis14–16 and necrosis,17–19 the underlying mechanisms

following short-term exposure of 4-HNE is not known.

Thus, the present study was carried out to test whether

the expression of selected apoptosis markers in PC12

cells, receiving short-term exposure of 4-HNE is

altered. Our results show significant dose-dependent

alterations in reactive oxygen species (ROS), and early

responses to specific apoptotic markers in cells exposed

to 4-HNE. Furthermore, enhanced expression and activ-

ity of caspase 3 following 4-HNE exposure to cells indi-

cates the mitochondrial mediation in the apoptosis

induction. Together, the results from these studies may

provide a mechanism through which 4-HNE induces

apoptosis in neuronal cells.

Materials and methods

Cell culture

PC12 cells used in the study were originally procured

from National Centre for Cell Sciences, Pune, India,

and grown in Nutrient Mixture/F-12 (Hams) supple-

mented with 2.5% fetal bovine serum (FBS), 15% horse

serum, 0.2% sodium bicarbonate, and antibiotic/anti-

mycotic solution (100�, 1 ml/100 ml of medium;

Invitrogen, Life technologies, Staley Road, Grand

Island, NY 14072, USA). The cells were maintained

in 5% CO2–95% atmosphere under high humidity at

37�C. Cells were assessed for cell viability by trypan

blue dye exclusion assay as described earlier by Pant

et al.20 and batches showing viability of more than

95% were used in the experiments.

Reagents and consumables

All the specified chemicals, reagents, and diagnostic

kits were procured from Sigma Chemical Company

Pvt. Ltd. (St. Louis, Missouri, USA), unless otherwise

stated. Nutrient mixture F-12 Hams, antibiotics/anti-

mycotics solution (100�), FBS (Staley Road, Grand

Island, NY, USA), and horse serum were purchased

from Gibco BRL (Kamstrupvej 90, Denmark-4000,

Roskilde, USA). Culture wares and other plastic

consumables used in the study were procured from

Nunc (Denmark). Milli-Q water was used in all the

experiments. 4-HNE, an unsaturated aldehyde, was

generously gifted by Dr Sanjay Srivastava, Depart-

ment of Cardiology, University of Louisville (Ken-

tucky, USA).

Experimental design

PC12 cells were exposed to various concentrations

(1–50 mM) of 4-HNE for 1 h. Cells receiving 4-

HNE insult were then analyzed for ROS generation

and to determine the changes in the expression of

early response proteins (c-Fos, c-Jun, and GAP-43)

as well as selected marker genes associated with

mitochondria-mediated apoptosis using real time-

polymerase chain reaction (RT-PCR). The possible

associations of these proteins in 4-HNE-induced

apoptosis were also assessed using immunoblot–

Western blot analysis.

ROS generation

ROS generation was assessed using 2,7-

dichlorodihydrofluorescein diacetate (DCFH-DA;

Sigma Aldrich (St. Louis, Missouri, USA), USA) dye

as a fluorescence agent following the protocol earlier

described by us.21 In brief, cells (5 � 104 per well)

were allowed to adhere to poly-L-lysine-coated

eight-well chamber slide flasks. Following the expo-

sure of 4-HNE for 1 h, cells were washed with

phosphate-buffered saline (PBS) and incubated for

the next 30 min in DCFH-DA (20 mM) containing

incomplete culture medium in the dark. Then, the

slides were analyzed for intracellular fluorescence

using an upright fluorescence microscope (Nikon

Eclipse 80i (Yokohama, Japan) equipped with Nikon

DS-Ri1 12.7 megapixel camera). Image analysis soft-

ware Leica Qwin 500 (Leica Microsystem, Wetzlar,

Germany) was used to quantify the change in the

fluorescence and data were expressed as percentage

change in the fluorescence area.

Transcriptional changes

Alterations in the messenger RNA (mRNA) expres-

sion of apoptosis marker genes (P53, Bax, Bcl2, and

caspase 3) were studied following the protocol as

described earlier by us.21,22 In brief, cells (1 � 106)

were allowed to grow in six-well culture plates unless

reached to 80–85% confluence. Following the expo-

sure of cells to 4-HNE for 1 h, total RNA was isolated

from both experimental and control sets using GeneE-

lute mammalian total RNA Miniprep Kit (catalog no.

RTN-70, Sigma). The purity and yield of RNA was

2 Human and Experimental Toxicology



assessed by Nanodrop ND-1000 Spectrophotometer

V3.3 (Nanodrop Technologies Inc., Wilmington, Dela-

ware, USA). RNA quality in terms of integrity was also

checked by running RNA onto 2% denaturing agarose

gel. Total RNA (2 mg) was reverse-transcribed into

cDNA by SuperScript III first-strand cDNA synthesis

Kit (catalog no. 18080-051, Invitrogen Life Science,

USA). qRT-PCR was performed by SYBR Green dye

(ABI, Applied Biosystem, 850 Lincoln Centre Drive

Foster City California, USA) using ABI PRISM1

7900HT Sequence Detection System (Applied Biosys-

tems, USA). Real-time reactions were carried out in tri-

plicate. Specificity of primer sets and genomic DNA

contamination were assessed for all the samples by ana-

lyzing the melting curve and no template control

(NTCs), respectively. GAPDH was used as internal con-

trol to normalize the data. 4-HNE-induced alterations in

mRNA expression were expressed in terms of relative

quantity.

Protein expression (Western blotting)

Alterations in the expression of selected marker pro-

teins (P53, Bax, Bcl2, and activated caspase 3) associ-

ated with apoptosis were carried out by Western

immunoblotting as described earlier.16 In brief, after

respective exposure, cells were washed twice with

ice-cold PBS (pH 7.4) and centrifuged at 1000 rpm for

10 min. Cell pellets were lysed using cell lysis reagent

(catalog no. C2978, Sigma) in the presence of 1� pro-

tein inhibitor cocktail (catalog no. P8340; Sigma). An

equal amount (50 mg/well) of proteins was loaded in

10% Tricine–sodium dodecyl sulfate gel and electro-

phoresis was carried out. Following electrophoresis,

protein bands from the gel were transferred on polyvi-

nylidene fluoride membrane (Millipore, Bedford,

Massachusetts, USA) in an electrophoresis transfer

apparatus (BioRad, Hercules, California, USA). The

membrane was blocked overnight in Tris-Buffered

Saline Tween-20 (TBST) (30 mM Tris, pH 7.5,

150 mM NaCl, and 0.1% Tween 20) containing 5%nonfat milk. Membranes were then probed with pri-

mary antibodies specific for Bax (1:500, Santa Cruz,

USA), Bcl2, activated caspase 3 (1:1000, Cell Signal-

ing Technology (CST), USA), and b-actin (1:2000,

Santa Cruz). After several washings with TBST, mem-

brane was incubated with horseradish peroxidase-

conjugated secondary antibody (goat anti-rabbit for

Bax and cleaved-caspase 3, goat anti-mouse for all

others) for 2 h at room temperature. Then, the blots

were developed using 3,30,5,50-tetramethylbenzidine

(TMB)–hydrogen peroxide (H2O2; Sigma Weltevreden

Park, Johannesburg, 1715, S.A.). Pictures of specific

band were taken by gel documentation system

(Alpha Innotech, USA) and densitometric analysis

was done by AlphaEase� FC StandAlone V. 4.0.0

software.

Protein expression (Immunocytochemistry)

Immunocytochemical localization for early response

gene proteins (c-Fos, c-Jun, and GAP-43) was carried

out using specific antibodies following the protocol.22

In brief, 4-HNE exposed cells were fixed in 4%paraformaldehyde for 10 min. Cells were washed

with PBS thrice and incubated with 0.5% H2O2

(w/v) in methanol to quench endogenous peroxidase.

Nonspecific binding sites were blocked by incubating

cells with 0.5% bovine serum albumin and

0.1% Triton X-100 in phosphate buffer saline (PBS)

for 2 h. Cells were then incubated in primary mono-

clonal antibodies specific for c-Fos, c-Jun, and Gap-

43 proteins (1:200, Sigma Chemicals Company Pvt.

Ltd.) for 1 h. Following the washing with PBS, cells

were again reincubated with goat anti-rabbit horse-

radish peroxidase conjugate secondary antibody

(1:500) for 2 h. Cells were washed with PBS to

remove any unbound secondary antibody and incu-

bated with diaminobenzidine hydrochloride for 5–

15 min to develop the color. Cells were visualized and

images were captured using an upright microscope

(Nikon Eclipse 80i equipped with Nikon DS-Ri1

12.7 megapixel camera). Image analysis software

Leica Qwin 500 was used to quantify the percentage

change in the area of expression of protein early

response proteins. Similar experiments in cells with-

out any exposure were run under parallel and served

as controls.

Caspase 3 activity

4-HNE-induced alterations in the activity of caspase 3

were monitored using kits (Biovision, catalog no.

K106, USA). Following 4-HNE exposure for 1 h,

cells were pelleted, resuspended in prechilled extrac-

tion buffer (50 ml), and incubated for 10 min on ice.

Then, the samples were centrifuged for 5 min at 500g

and the clear supernatant (50 ml per well) transferred

to 96-well culture plates. Assay buffer (50 ml) and

substrate conjugate (5 ml) were added and mixed

well. Immediately after the completion of 2 h incu-

bation at 37�C in dark, the contents were mixed

thoroughly and read for absorbance at 400 nm. The

Siddiqui MA et al. 3



values of exposed groups were compared with

unexposed control sets and the data expressed as

a percentage of control.

Statistical analysis

Results are expressed as mean + standard error

(SE) of the values obtained from three independent

experiments, and in each experiment, a minimum

of four replicates were used. Statistical analysis

was performed using one-way analysis of variance,

and post hoc Dunnett’s test was applied to compare

values between control and treated groups. The

values of p < 0.05 were considered as statistically

significant.

Results

ROS generation

Statistically significant (p < 0.001) ROS generation

was observed in PC12 cells receiving 4-HNE (10, 25,

and 50 mM) exposure for 1 h (Figure 1(a) and (b)). The

increase in ROS generation was dose dependent, i.e.

127.0 + 8.6%, 146.0 + 9.8%, and 178.0 + 12.1%of unexposed control sets following the exposure of

10, 25, and 50 mM of 4-HNE, respectively.

0

20

40

60

80

100

120

140

160

180

200

Control 1 µM 10 µM 25 µM 50 µM

RO

S ge

nera

tion

(% c

ontr

ol)

Various concentrations of 4-HNE

*

**

**

I II III

VIV

(b)

(a)

Figure 1. (a) 4-HNE-induced ROS generation in PC12 cells. Cells were exposed to various concentrations of 4-HNE for1 h. ROS generation was studied using dichlorofluorescin diacetate dye. Images were taken using Nikon fluorescencemicroscope (model 80i) attached with 12.7 Megapixel Nikon DS-Ri1 digital CCD cool camera. (I): unexposed cells (con-trol); (II): cells exposed to 4-HNE (1 mM); (III): cells exposed to 4-HNE (10 mM); (IV) cells exposed to 4-HNE (25 mM); and(V) cells exposed to 4-HNE (50 mM). (b) Relative quantification of ROS generation in PC12 cells following various con-centrations of 4-HNE for 1 h. Quantification of fluorescence images of intracellular ROS was done using Leica Q Win500image analysis software. *p < 0.01, **p < 0.001 versus control. 4-HNE: 4-hydroxynonenal; ROS: reactive oxygen species.




Transcriptional changes in apoptosis markers

The alterations in the levels of mRNA expression of

selected apoptosis marker genes in PC12 cells

exposed to 4-HNE for 1 h are presented in Figure

2(a) to (d). Significant (p < 0.001) upregulation in the

expression of proapoptotic genes, i.e. caspase 3

(3.55 + 0.15-fold), Bax (3.12 + 0.25-fold), and

p53 (2.96 + 0.21-fold), was observed at 50 mM con-

centration, whereas antiapoptotic gene Bcl2

(0.25 + 0.01-fold) was significantly downregulated

at this dose. In general, 4-HNE-induced alterations

were dose dependent at 10, 25, and 50 mM concentra-

tions, while the lowest concentration used, i.e. 1 mM

was found to be ineffective.

Translational changes in apoptosis markers

To further determine whether these changes in the

apoptotic marker genes are happening at the level of

protein expression, we carried out immunoblot analy-

ses for these proteins. The results of our immunoblot

analyses are shown in Figure 3(a) to (d). Cells

exposed to 4-HNE (10, 25, and 50 mM) for 1 h showed

a significant upregulation in the expressions of Bax

(1.5 + 0.15, 2.6 + 0.19, and 2.4 + 0.2-fold), caspase

3 (1.4 + 0.13, 2.1 + 0.2, and 3 + 0.26-fold), and p53

(1.8 + 0.13, 1.7 + 0.14, and 2.1 + 0.16-fold),

whereas the expression of Bcl2 protein was downregu-

lated by 0.8 + 0.06, 0.4 + 0.03, and 0.2 + 0.019-fold,

respectively. Similar to transcriptional changes,

0

0.5

1

1.5

2

2.5

3

3.5


Fol

d ch

ange

in th

e le

vel o

f p53

mR

NA

Concentrations of 4-HNE

**

**

*

0

0.5

1

1.5

2

2.5

3

3.5

4


Fol

d ch

ange

in th

e le

vels

of bax

mR

NA


**

**

**

0

0.2

0.4

0.6

0.8

1

1.2


Fol

d ch

ange

in t

he le

vel o

f bc

l 2 m

RN

A


**

**

*

0

0.5

1

1.5

2

2.5

3

3.5

4


Fol

d ch

ange

in th

e le

vel o

f cas

pase

-3m

RN

A


**

**

**

(a)

(c)

(b)

(d)

Figure 2. RT-PCR analysis to study the alterations in the expression of mRNA of various genes, namely, P53 (a), Bax (b),Bcl-2 (c), and caspase 3 (d) in PC12 cells exposed to different concentrations of 4-HNE for 1 h. The data provided aremean + SE from three separate experiments. *p < 0.05, **p < 0.001. RT-PCR: real-time polymerase chain reaction;4-HNE: 4-hydroxynonenal.




4-HNE-induced alterations at the protein level were

dose dependent and exposure of 1 mM for 1 h was

found to be ineffective.

Translational changes in early response markers

Cells exposed to 4-HNE (10, 25, and 50 mM) for 1 h

showed significant upregulation in the expressions of

c-Fos (8.87- + 0.62-, 19.67- + 1.2-, and 27.94-

+ 1.9-fold), c-Jun (8.67- + 0.64-, 19.52- + 1.2-, and

27.27- + 1.7-fold), and GAP-43 (16.57- + 1.2-,

26.27- + 1.9-, and 30.08- + 2.1-fold), respectively.

However, there were no alterations in the expression

of these gene proteins in cells expose to 4-HNE at

1 mM concentration for 1 h (Figure 4(a) and (b)).

Activity of caspase 3

Highlights of 4-HNE-induced alterations in the

activity of caspase 3 are shown in Figure 5. The

activity was found to be increased with increasing

doses of 4-HNE. The affects were statistically sig-

nificant for 4-HNE exposures at 25 and 50 mM

(1.77- + 0.15- and 2.39- + 0.23-fold of unexposed

controls, respectively).

p53

β-actin

0

0.5

1

1.5

2

2.5

Control 10 µM 25 µM 50 µM

Fol

d ch

ange

**

**

4-HNE (µM) 0 10 25 50

Bax

β-actin

4-HNE (µM) 0 10 25 50

0

0.5

1

1.5

2

2.5

3


Fol

d ch

ange

****

*

0

0.2

0.4

0.6

0.8

1

1.2


Fol

d ch

ange

Bcl-2

β-actin

**

**

*

4-HNE (µM) 0 10 25 50

00.5

11.5

22.5

33.5


Fol

d ch

ange

Activated Caspase-3

β-actin

**

**

*

4-HNE (µM) 0 10 25 50

(a)

(c)

(d)

(b)

Figure 3. 4-HNE-induced alterations in the protein expression of apoptosis markers, namely, P53 (a), Bax (b), Bcl-2 (c),and activated caspase 3 (d) in PC12 cells. Cells were exposed to various concentrations of 4-HNE for 1 h. Cells wereharvested and subjected to Western blotting using respective antibodies. Relative optical density was determined by den-sitometric analysis. Quantification was done with a gel documentation system (Alpha Innotech) with the help of AlphaEaseFC StandAlone V. 4.0 software. Data are expressed as mean + SE. *p < 0.05, **p < 0.01. 4-HNE: 4-hydroxynonenal.




Discussion

4-HNE-induced apoptosis and oxidative stress are

documented in neuronal cells.7,23 We have also

reported earlier that 4-HNE at higher concentrations

induce cytotoxicity in PC12 cells, which was found

to be associated with the altered levels of neurotrans-

mitter receptors such as dopamine, cholinergic,

serotonin, and benzodiazepine receptors.8 In addition,

we have reported the oxidative stress-mediated cyto-

toxic responses in PC12 cells exposed to various

doses of 4-HNE.9 In our earlier studies, a

concentration-dependent metabolism of 4-HNE in

PC12 cells has also been demonstrated within 1 h of

exposure in the form of glutathionyl conjugates and

HNA and DHN metabolites.13 In this study, we are

reporting the expressional changes in apoptosis marker

genes in PC12 cells exposed to various concentrations

of 4-HNE for such a short period of 1 h only.

ROS generation is considered to be one of the key

signals for oxidative stress-induced apoptosis .24 In

the present investigation, significant dose-dependent

generation of ROS was observed in PC12 cells

exposed to 4-HNE for 1 h. Our results confirm the

findings of Feng et al.25 and Uchida et al.26 who have

also reported ROS generation-mediated oxidative

stress in cells exposed to higher concentrations (25

and 50 mM) of 4-HNE. However, in their experi-

ments, the exposure periods were more than 1 h.

Similar to our findings, they have also shown that

lower doses of 4-HNE, i.e. <1 mM are ineffective.

Induced levels of ROS are well-known etiological

factors associated with oxidative stress and are

known to cause cell death via apoptosis in a variety

of cell types.21,27,28 Thus, ROS generation in the

present study may be a mediator for 4-HNE-

induced apoptosis in neuronal cells.

The involvement of mitochondrial chain com-

plexes in ROS-induced apoptotic changes in cyto-

plasm has been reported.29 Such apoptotic changes

are known to follow different pathways.24,30 The

Control

1 μμM

10 μμM

25 μμM

50 μμM

c-Fos GAP-43c-Jun

35


****

15

20

25

30 ****

***

*

0

5

10

15

% C

hang

e in

the

expr

essi

on o

fea

rly

resp

onse

gen

e pr

otei

ns

**

c-fos

Early responce gene protein

GAP-43c-jun

(b)

(a)

Figure 4. (a) Representative microphotographs of immu-nocytochemical localization of early response gene proteinsin PC12 cells following the exposure of 4-HNE (1–50 mM)for 1 h. (b) Expression of early response genes in PC12 cellsfollowing 4-HNE (1–50 mM) exposure for 1 h. All valuesrepresent the mean + SE obtained from the images of atleast 20 microscopic fields and analyzed by Leica Q-Win500 image analysis software. *p < 0.05, **p < 0.001. 4-HNE: 4-hydroxynonenal.

0

0.5

1

1.5

2

2.5

3

Control 1 µM 10 µM 25 µM 50 µM Postive control

*

**

*

Fol

d ch

ange

Figure 5. Induction in the activity of caspase 3 in PC12cells exposed to 4-HNE (1–50 mM) for 1 h. Cells exposedto camptothecin (1 mg/mL) for 24 h were used as positivecontrol. *p < 0.05; **p < 0.001. 4-HNE: 4-hydroxynonenal.




mitochondrial-dependent apoptotic pathway is also

known to be involved in 4-HNE-induced cytotoxicity

in various cells31,32 including PC12 cells.16,23 We

observed that 4-HNE significantly upregulates the

expression (mRNA and protein) of P53, Bax, caspase

3, and downregulated Bcl2 in a dose-dependent man-

ner. In general, transcriptional changes were well

coordinated with translational changes and with phy-

siological activity of caspase 3. The upregulation of

nuclear P53 protein is known to play an important role

in minimizing DNA damage by inducing transcrip-

tional reprogramming, which eventually leads to

controlled cell death.33–35 However, higher level of

cytoplasmic P53 protein interacts with mitochondria,

thereby promoting mitochondrial membrane permea-

bilization36 and plays an important role in the regula-

tion of apoptosis.37 This cytoplasmic P53 protein has

been suggested to induce proapoptotic members of

the Bcl2 family such as Bax and Bak, and their displa-

cement with antiapoptotic Bcl2 proteins.38 Thus, the

alterations in the expression profile of marker genes

in this study indicates that P53 triggers the mitochon-

drial apoptotic cascade in PC12 cells exposed to

4-HNE. Similar to our findings, 4-HNE-induced

apoptosis in other cell types has also been suggested

through the P53-dependent intrinsic pathway.11,39 The

induction of P53 by 4-HNE followed by the activation

of caspase 3 and the onset of apoptosis has also been

reported by Li et al.40 Association of 4-HNE-induced

apoptosis with P53 accumulation suggests that the

process of apoptosis elicited by 4-HNE may be due

to the changes in the availability or sensitivity of

specific regulatory pathways that are activated by the

4-HNE.

Initially, P53 is activated in response to DNA dam-

age by 4-HNE, and these upregulated levels of P53

promote induction in the expression of proapoptotic

Bax, while downregulating the expression of antia-

poptotic Bcl2 protein. This imbalance in the ratio of

Bax/Bcl-2 could lead to the dissipation in mitochon-

drial membrane potential. Finally, dysfunctional

mitochondria could release cytochrome-c in cytosol,

which could activate caspase 3 via the activation of

procaspase 9. In the case of 4-HNE-induced apopto-

sis, an imbalance in Bax/Bcl-2 protein may be an

upstream event followed by mitochondrial-mediated

activation of caspase 3, a final executer caspase,

which leads to cell apoptosis. Activation of P53 is

associated with a rapid increase in its levels and with

an increased ability to bind DNA and mediate tran-

scriptional activation of apoptotic signals.21,41

Induction in the levels of early response gene pro-

teins such as c-Fos, c-Jun, and GAP-43, which are

integral components of transcription factor AP-1, has

been discussed as a regulator of cell death, survival,

and regeneration.42 In our present study, exposure of

4-HNE-induced expression of c-Fos, c-Jun, and

GAP-43 shows the effects of 4-HNE as a toxicant and

could be well correlated with previously reported

induced expression of these genes in the appearance

of neurotoxicity.22,43 Oxidation products of 4-HNE

thereby may have a role in regulating cell death/

survival as in the case of neurodegeneration.

Together, our data provide insights to the induction

of apoptotic damages in neuronal cells following

short-term exposure (1 h) of 4-HNE, which may help

understand the possible cellular and molecular

mechanisms involved in 4-HNE-induced neuronal

damage.

Funding

Financial support was provided by Council of

Scientific and Industrial Research, New Delhi, India

(SIP-08).

Acknowledgements

The authors thank the Director, Indian Institute of

Toxicology Research, Lucknow, India, for his keen interest

in the present work and technical laboratory assistance of

Mr Rajesh Misra.

Declaration of Conflict of Interest

The authors declared no conflicts of interest.

References

1. Lu Z, Li CM, Qiao Y, Yan Y and Yang X. Effect of

inhaled formaldehyde on learning and memory of

mice. Indoor Air 2008; 18: 77–83.

2. Cai J, Chen J, He H, Yin Z, Zhu Z and Yin D. Carbonyl

stress: malondialdehyde induces damage on rat hippo-

campal neurons by disturbance of Caþ homeostasis.

Cell Biol Toxicol 2009; 25: 435–445.

3. Butterfield DA, Bader Lange ML and Sultana R.

Involvements of the lipid peroxidation product,

HNE, in the pathogenesis and progression of

Alzheimer’s disease. Biochim Biophys Acta 2010;

1801: 924–929.

4. Sakai T, Shimizu Y and Kawahara S. Effect of

NaCl on the lipid peroxidationderived aldehyde,

4-hydroxy-2-nonenal, formation in boiled pork. Biosci

Biotechnol Biochem 2006; 70: 815–820.




5. Lopachin RM, Geohagen BC and Gavin T. Synaptoso-

mal toxicity and nucleophilic targets of 4-hydroxy-2-

nonenal. Toxicol Sci 2009; 107: 171–181.

6. Arakawa M, Ishimura A, Arai Y, Kawabe K, Suzuki S,

Ishige K, et al. NAcetylcysteine and ebselen but not

nifedipine protected cerebellar granule neurons against

4-hydroxynonenal-induced neuronal death. Neurosci

Res 2007; 57: 220–229.

7. Abdul HM and Butterfield DA. Involvement of PI3K/

PKG/ERK1/2 signaling pathways in cortical neurons

to trigger protection by co-treatment of acetyl-L-carni-

tine and alpha-lipoic acid against HNE-mediated oxi-

dative stress and neurotoxicity implications for

Alzheimer’s disease. Free Radic Biol Med 2007; 42:

371–384.

8. Siddiqui MA, Singh G, Kashyap MP, Khanna VK,

Yadav S, Chandra D, et al. Influence of cytotoxic doses

of 4-hydroxynonenal on selected neurotransmitter

receptors in PC-12 cells. Toxicol In Vitro 2008; 22:

1681–1688.

9. Siddiqui MA, Kashyap MP, Khanna VK, Yadav S and

Pant AB. NGF induced differentiated PC12 cells as in

vitro tool to study 4-hydroxynonenal induced cellular

damage. Toxicol In Vitro 2010; 24: 1681–1688.

10. Srivastava S, Ramana KV, Tammali R, Srivastava SK

and Bhatnagar A. Contribution of aldose reductase to

diabetic hyperproliferation of vascular smooth muscle

cells. Diabetes. 2006; 55: 901–910.

11. Sharma R, Sharma A, Dwivedi S, Zimniak P,

Awasthi S and Awasthi YC. 4-Hydroxynonenal

self-limits fas-mediated DISC-independent apoptosis

by promoting export of Daxx from the nucleus to

the cytosol and its binding to Fas. Biochemistry

2008; 47: 143–156.

12. Yadav UC, Ramana KV, Awasthi YC and Srivastava

SK. Glutathione level regulates HNE-induced geno-

toxicity in human erythroleukemia cells. Toxicol Appl

Pharmacol 2008; 227: 257–264.

13. Siddiqui MA, Kashyap MP, Khanna VK, Gupta VK,

Tripathi VK, Srivastava S, et al. Metabolism of

4-hydroxy trans 2-nonenal (HNE) in cultured PC12

cells. Ann Neurosci 2008b; 15: 60–68.

14. Zhang H, Court N and Forman HJ. Submicromolar

concentrations of 4-hydroxynonenal induce glutamate

cysteine ligase expression in HBE1 cells. Redox Rep

2007; 12: 101–106.

15. Jang YJ, Kim JE, Kang NJ, Lee KW and Lee HJ.

Piceatannol attenuates 4-hydroxynonenal-induced

apoptosis of PC12 cells by blocking activation of

c-Jun N-terminal kinase. Ann N Y Acad Sci 2009;

1171: 176–182.

16. Siddiqui MA, Kashyap MP, Kumar V, Al-Khedhairy

AA, Musarrat J and Pant AB. Protective potential of

trans-resveratrol against 4-hydroxynonenal induced

damage in PC12 cells. Toxicol In Vitro 2010b; 24:

1592–1598.

17. Gallagher EP, Huisden CM and Gardner JL.

Transfection of HepG2 cells with hGSTA4 provides

protection against 4-hydroxynonenal-mediated oxida-

tive injury. Toxicol In Vitro 2007; 21: 1365–1372.

18. Borovic S, Cipak A, Meinitzer A, Kejla Z, Perovic D,

Waeg G and Zarkovic N. Differential sensitivity to

4-hydroxynonenal for normal and malignant mesench-

ymal cells. Redox Rep 2007; 12: 50–54.

19. Miranda CL, Reed RL, Kuiper HC, Alber S and Ste-

vens JF. Ascorbic acid promotes detoxification and

elimination of 4-hydroxy-2(E)-nonenal in human

monocytic THP-1 cells. Chem Res Toxicol 2009; 22:

863–874.

20. Pant AB, Agarwal AK, Sharma VP and Seth PK. In

vitro cytotoxicity evaluation of plastic biomedical

devices. Hum Exp Toxicol 2001; 20: 412–417.

21. Kashyap MP, Singh AK, Siddiqui MA, Kumar V, Tri-

pathi VK, Khanna VK, et al. Caspase cascade regu-

lated mitochondria mediated apoptosis in

monocrotophos exposed PC12 cells. Chem Res Toxicol

2010; 23: 1663–1672.

22. Kashyap MP, Singh AK, Kumar V, Tripathi VK,

Srivastava VK, Agarwal M, et al. Monocrotophos

induced apoptosis in PC12 cells: role of xenobiotic

metabolizing cytochrome P450s. PLoS ONE 2011;

6: e17757.

23. Raza H and John A. 4-hydroxynonenal induces mito-

chondrial oxidative stress, apoptosis and expression

of glutathione S-transferase A4-4 and cytochrome

P450 2E1 in PC12 cells. Toxicol Appl Pharmacol

2006; 216: 309–318.

24. Kroemer G, Galluzzi L and Brenner C. Mitochondrial

membrane permeabilization in cell death. Physiol Rev

2007; 87: 99–163.

25. Feng Q, Kumagai T, Torii Y, Nakamura Y, Osawa T

and Uchida K. Anticarcinogenic antioxidants as inhibi-

tors against intracellular oxidative stress. Free Radic

Res 2001; 35: 779–788.

26. Uchida Y, Ohba K, Yoshioka T, Irie K, Muraki T

and Maru Y. Cellular carbonyl stress enhances the

expression of plasminogen activator inhibitor-1 in rat

white adipocytes via reactive oxygen

species-dependent pathway. J Biol Chem 2004; 279:

4075–4083.

27. Fortunato JJ, Agostinho FR, Reus GZ, Petronilho FC,

Dal-Pizzol F and Quevedo J. Lipid peroxidative




damage on malathion exposure in rats. Neurotox Res

2006; 9: 23–28.

28. Sompol P, Ittarat W, Tangpong J, Chen Y, Doubins-

kaia I, Batinic-Haberle I, et al. A neuronal model of

Alzheimer’s disease: an insight into the mechanisms

of oxidative stress-mediated mitochondrial injury.

Neuroscience 2008; 153: 120–130.

29. Galluzzi L, Morselli E, Kepp O, Tajeddine N and

Kroemer G. Targeting p53 to mitochondria for cancer

therapy. Cell Cycle 2008; 7: 1949–1955.

30. Segui B and Legembre P. Redistribution of CD95 into

the lipid rafts to treat cancer cells? Recent Pat Antican-

cer Drug Discov 2010; 5: 22–28.

31. Jin Xu, Ji L-D and Xu L-H. Lead-induced apoptosis in

PC 12 cells: involvement of p53, Bcl-2 family and cas-

pase-3. Toxicol Lett 2006; 166: 160–167.

32. Vaillancourt F, Fahmi H, Shi Q, Lavigne P, Ranger P,

Fernandes JC and Benderdour M. 4-Hydroxynonenal

induces apoptosis in human osteoarthritic chondrocytes:

the protective role of glutathione-S-transferase. Arthritis

Res Ther 2008; 10: R107.

33. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S,

Brown JP, et al. Requirement for p53 and p21 to sus-

tain G2 arrest after DNA damage. Science 1998; 282:

1497–1501.

34. Bargonetti J and Manfredi JJ. Multiple roles of the tumor

suppressor p53. Curr Opin Oncol 2002; 14: 86–91.

35. Cui Q, Yu JH, Wu JN, Tashiro S, Onodera S, Minami

M and Ikejima T. P53-mediated cell cycle arrest and

apoptosis through a caspase-3- independent, but

caspase-9-dependent pathway in oridonin-treated

MCF-7 human breast cancer cells. Acta Pharmacol Sin

2007; 8: 1057–1066.

36. Moll UM, Wolff S, Speidel D and Deppert W. Tran-

scription independent pro-apoptotic functions of p53.

Curr Opin Cell Biol 2005; 17: 631–636.

37. Yu J and Zhang L. The transcriptional targets of p53 in

apoptosis control. Biochem Biophys Res Commun

2005; 331: 851–858.

38. Galluzzi L, Blomgren K and Kroemer G. Mitochon-

drial membrane permeabilization in neuronal injury.

Nat Rev Neurosci 2009; 10: 481–494.

39. Awasthi YC, Sharma R, Sharma A, Yadav S, Singhal

SS, Chaudary P, et al. Self-regulatory role of

4-hydroxynonenal in signaling for stress-induced pro-

grammed cell death. Free Radic Biol Med 2008; 45:

111–118.

40. Li D, Hinshelwood A, Gardner R, McGarvie G and

Ellis EM. Mouse aldo-keto reductase AKR7A5

protects V79 cells against 4-hydroxynonenal-induced

apoptosis. Toxicology 2006; 226: 172–180.

41. Lakin ND and Jackson SP. Regulation of p53 in

response to DNA damage. Oncogene 1999; 18,

7644–7655.

42. Vollgraf U, Wegner M and Richter-Landsberg C.

Activation of AP-1 and nuclear factor-kappaB tran-

scription factors is involved in hydrogen

peroxide-induced apoptotic cell death of oligodendro-

cytes. J Neurochem 1999; 73: 2501–2509.

43. Vaudano E, Rosenbald C and Bjorklund A. Injury

induced c-Jun expression and phosphorylation in the

dopaminergic nigral neurons of the rat: correlation

with neuronal death and modulation by

glial-cell-line derived neurotropic factor. Eur J Neu-

rosci 2001; 13: 1–14.




Hum Exp Toxicol Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells

Documents