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

http://het.sagepub.com/content/30/8/860The online version of this article can be found at:

 DOI: 10.1177/0960327110382130

2011 30: 860 originally published online 27 August 2010Hum Exp ToxicolMA Siddiqui, MP Kashyap, AA Al-Khedhairy, J. Musarrat, VK Khanna, S. Yadav and AB Pant

in PC12 cells-estradiol against co-exposure of 4-hydroxynonenal and 6-hydroxydopamineβProtective potential of 17

  

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:  

http://het.sagepub.com/content/30/8/860.refs.htmlCitations:  

What is This? 

- Aug 27, 2010 OnlineFirst Version of Record 

- Jul 19, 2011Version of Record >>

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

Protective potential of 17β-estradiolagainst co-exposure of 4-hydroxynonenaland 6-hydroxydopamine in PC12 cells

MA Siddiqui1, MP Kashyap2, AA Al-Khedhairy1,J Musarrat1, VK Khanna2, S Yadav2 and AB Pant2

Abstract4-hydroxynonenal (4-HNE) and 6-hydroxydopamine (6-OHDA)-mediated damage in dopaminergic neurons iswell documented. Protective potential of steroidal hormone (17b-estradiol) has also been suggested.However, therapeutic potential of such promising hormone is hampered due to complex brain anatomy andphysiology. Thus, the present investigations were studied to suggest the applicability of dopamine expressingPC12 cells as in vitro tool to screen the pharmacological potential of 17b-estradiol against 4-HNE and6-OHDA. MTT assay was conducted for cytotoxicity assessment of both 4-HNE (1 mM to 50 mM) and6-OHDA (10�4 to 10�7 M). Non-cytotoxic concentrations, that is, 4-HNE (1 mM) and 6-OHDA (10�6 M) wereselected to study the synergetic/additive responses. PC12 cells were found to be more vulnerable towardsco-exposure of individual exposure of 4-HNE and 6-OHDA, even at non-cytotoxic concentrations. Then, cellswere subjected to pre-treatment (24 hours) of 17b-estradiol (1 mM), followed by a permutation of combina-tions of both 4-HNE and 6-OHDA. Pretreatment of 17b-estradiol was found to be significantly effective againstthe cytotoxic responses of 4-HNE and 6-OHDA, when the damage was at lower level. However, 17b-estradiolwas found to be ineffective against higher concentrations. Physiological-specific responses of PC12 cells against4-HNE/6-OHDA and 17b-estradiol suggest its applicability as first tier of screening tool.

KeywordsPC12 cells, 4-HNE, 6-OHDA, 17b-estradiol, cytotoxicity

Introduction

Lipid peroxidation is well documented as one among

the key factors involved in the etiology of various

neurodegenerative disorders including stroke,1

Alzheimer’s disease,2 and Parkinson’s disease.3 The

higher concentrations of 4-hydroxynonenal, an unsa-

turated aldehydic product of o-6 polyunsaturated

fatty acids, produced during toxic insults is known

to cause neurodegeneration and neurotoxicity in

variety of cells.4-7 The role of 6-hydroxydopamine

(6-OHDA), a hydroxylated form of dopamine, in the

pathogenesis of Parkinson’s disease is well estab-

lished.8,9 It is a widely used chemical to investigate

the neurotoxic effects on dopaminergic neurons and

usually thought to cross cell membrane through dopa-

mine uptake transporters to inhibit mitochondrial

respiration and to generate intracellular reactive

oxygen species.10-12

The neuroprotective potential of estrogens, primarily

known as female sex hormones, is well documented.13

Early estrogen therapy has been suggested as one

among the strategy to reduce the risk of neurodegenera-

tive disorders such as Alzheimer’s disease,14,15

ischemic stroke,16 and Parkinson’s disease.17 Neuropro-

tective role of estrogens has been reported in a number

of experimental models of acute cerebral ischemia.18

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

Corresponding author:A B Pant, In Vitro Toxicology Laboratory, Indian Institute ofToxicology Research, PO Box: 80, MG Marg, Lucknow-226001,UP, IndiaEmail: abpant@rediffmail.com

Human and Experimental Toxicology30(8) 860–869

ª The Author(s) 2010Reprints and permission:

sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0960327110382130

het.sagepub.com

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

Estrogens-mediated neuroprotection has also been

demonstrated as an age-independent phenomenon in

female rats.19 Variety of neuronal cell types have been

employed to study the neuroprotective potential of

estrogens against number of toxic insults mainly includ-

ing H2O2,20 serum deprivation,21 oxygen-glucose depri-

vation,22 iron,23 amyloid peptide,24 excitotoxicity,25

and mitochondrial toxins such as 3-nitropropionic

acid,26 sodium azide,27 etc. 17b-Estradiol, a weak natu-

ral estrogen, has been recently evaluated for its protec-

tive potential through mitochondrial function against

ROS and H2O2 insult in cultured neuronal cell line.20,28

Thus, the present investigations were aimed to

study the protective potential of 17b-estradiol in

PC12 cells receiving experimental co-exposure of two

known neurotoxicants viz., 6-OHDA and 4-HNE,

which may be useful to understand the vulnerability

of brain cells in Parkinson’s patients.

Materials and methods

PC12 cells (a rat pheochromocytoma cell line) were

originally procured from National Centre for Cell

Sciences, Pune, India, and have been maintained at

In Vitro Toxicology Laboratory, Indian Institute of

Toxicology Research, Lucknow, India, as per the

standard protocol provided by the supplier. Briefly,

cells were cultured in Nutrient Mixture F-12 (Ham),

supplemented with 2.5% fetal bovine serum (FBS),

15% horse serum (HS), 0.2% sodium bicarbonate

and antibiotic, and antimycotic solution (100�,

1 mL/100 mL of medium, Invitrogen, Life Technol-

ogies, Carlsbad, CA, USA). Cells were grown in 5%CO2 – 95% atmosphere in high humidity at 37�C.

Each batch of cells was assessed for physiological

characteristics using established markers7,29 and cell

viability by trypan blue dye exclusion test30 prior to

experiments, and batches showing more than 95%cell viability were used in the present study. Cells

of passage number between 18 and 25 were used

in the present study.

Nutrient mixture F-12 Hams culture medium, anti-

biotics, fetal bovine, and horse serum were purchased

from Gibco BRL, Grand Island, NY, USA. Culture

wares and other plastic consumables were procured

from Nunc, Denmark. 4-hydroxy-trans-2-nonenal (4-

HNE) was generously gifted by Dr Sanjay Srivastava,

Associate Professor, Department of Cardiology,

University of Louisville, KY. All other specified che-

micals and reagents were purchased from Sigma

Chemical Company Pvt Ltd St Louis, Missouri, USA.

PC12 cells were exposed to various concentrations

of either of 4-HNE (1 mM to 50 mM) or 6-OHDA

(10�4 to 10�7 M) for a period up to 24 hours. To study

the prophylactic protection, cells were pretreated for

24 hours with 17b-estradiol at physiological concen-

tration (1 mM), then subjected to receive a permuta-

tion combination of both 4-HNE (1 mM to 50 mM)

and 6-OHDA (10�4 to 10�7 M) for 24 hours. Percent-

age loss in cell viability was assessed by MTT assay.

Percentage cell viability was assessed using the

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazo-

lium bromide (MTT) assay as described.7 Briefly,

cells (1 � 104) were allowed to adhere for 24 hours

under high humid environment in 5% CO2 at 37�Cin poly-L-lysine�coated 96-well culture plates. The

medium was then replaced with the medium contain-

ing either of 4-HNE (1 mM to 50 mM) or 6-OHDA

(10�4 to 10�7) or combination of both 4-HNE and

6-OHDA, for a period up to 24 hours. After the

respective exposure, MTT (5 mg/mL of stock in PBS)

was added (10 mL/well in 100 mL of cell suspension),

and plates were incubated for 4 hours. At the end of

incubation period, the reaction mixture was carefully

taken out and 200 mL of dimethyl sulfoxide (DMSO)

was added to each well by pippeting up and down sev-

eral times until the content gets homogenized. The

plates were kept on rocker shaker for 10 min at room

temperature and then read at 550 nm using Multiwell

Microplate Reader (Synergy HT, USA and Bio-Tek,

Bio-Tek Instruments, Winooski, VT, USA). Untreated

sets were also run under identical conditions and

served as basal control.

Statistical analysis

The results are expressed as mean and standard

error of means (SEM) for six wells of at least three

experiments, as indicated in the figures. One-way

ANOVA and Student’s t test were employed to

detect differences between the groups of treated

and control. p < 0.05 was taken to indicate signif-

icant differences.

Results

Data on cytotoxic responses of 4-HNE and/or 6-OHDA

and prophylactic protection of 17b-estradiol in cul-

tured PC12 cells are summarized in Figures 1�8.

A concentration-dependent decrease in percentage

cell viability was recorded following experimental

exposure of 4-HNE in PC12 cells. Maximum reduc-

tion in mitochondrial activity, that is, 50% + 2.7%

Siddiqui MA et al. 861

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

Figure 1. Effect on percentage cell viability in PC12 cells following various concentrations of 4-hydroxynonenal (4-HNE)for 24 hours. Values are mean + SE of four independent experiments.

Figure 2. Effect on percentage cell viability in PC12 cells following various concentrations of 6-hydroxydopamine(6-OHDA) for 24 hours. Values are mean + SE of four independent experiments.

862 Human and Experimental Toxicology 30(8)

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

of control was recorded with 50 mM of 4-HNE expo-

sure, followed by 25 mM (36% + 2.4%) and 10 mM

(16% + 0.7%), respectively. In general, the effect of

1 mM HNE was found to be insignificant on mito-

chondrial activity. Exposure (24 hours) of 4-HNE at

25 and 50 mM was found to be cytotoxic, whereas,

0

20

40

60

80

100

120

Control 1μM HNE 17β-Estradiol +1μM HNE

1μM HNE + 10-6 M 6-OHDA

17β-Estradiol +1μM HNE + 10-

6 M 6-OHDA

Concentrations

% C

ell v

iabi

lity

p < 0.001

p < 0.01

Figure 3. Effect of pretreatment of 17b-estradiol (1 mM) on percentage cell viability of PC12 cells exposed to 1 mM of4-hydroxynonenal (4-HNE) and 10�6 M of 6-hydroxydopamine (6-OHDA). PC12 cells were exposed to 17b-estradiol(1 mM) for 24 hours prior to the addition of HNE and/or 6-OHDA to the culture medium. Values are mean + SE of fourindependent experiments.

Figure 4. Effect of pretreatment of 17b-estradiol (1 mM) on percentage cell viability of PC12 cells exposed to 10 mM of4-hydroxynonenal (4-HNE) and 10�6 M of 6-hydroxydopamine (6-OHDA). PC12 cells were exposed to 17b-estradiol(1 mM) for 24 hours prior to the addition of HNE and/or 6-OHDA to the culture medium. Values are mean + SE of fourindependent experiments.

Siddiqui MA et al. 863

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

10 mM could be considered as cytostatic and 1 mM

non-cytotoxic (Figure 1). 6-OHDA was able to pose

statistically significant reduction in mitochondrial

activity at 10�4 and 10�5 M concentrations. Whereas,

lower concentrations of 6-OHDA (10�6 and 10�7 M)

used in the study could not cause significant detri-

mental effects in PC12 cells, thus considered as

non-cytotoxic concentrations (Figure 2).

Data on the protective potential of 17b-estradiol fol-

lowing experimental co-exposure of 4-HNE (1 mM)

and 6-OHDA (10�6 M) are summarized in Figure 3.

Statistically highly significant (p < 0.001), that is,

30% + 1.9% reduction in the percentage cell viability

was recorded. Interestingly, pretreatment of 17b-

estradiol (1 mM) was found to be significantly effec-

tive to protect the cells against the co-exposure of

4-HNE and 6-OHDA (Figure 3).

Although, the protective response of 17b-estradiol

was statistically significant in the cells co-exposed

to 4-HNE (10 mM) and 6-OHDA (10�6 M), the mag-

nitude was less pronounced when compared with the

cells exposed to non-cytotoxic concentrations of both

4-HNE and 6-OHDA (Figures 3 and 4). Severity of

damage got more significant and prominent with the

increasing concentrations of 4-HNE, that is, 25 and

50 mM in co-exposure groups with 6-OHDA (10�6

M). Pretreatment of 17b-estradiol was found to

increase the percentage cell viability significantly

(p < 0.01) when compared with co-exposed groups;

however, the values were significantly (p < 0.001)

lower than the untreated control (Figures 5 and 6).

In other sets of experiments, cells were co-exposed

with 6-OHDA at variable concentrations, that is, 10�4

and 10�5 M and non-cytotoxic concentration of 4-HNE,

that is, 1mM. Adverse effect on the mitochondrial activ-

ity was found largely due to cytotoxic concentrations

of 6-OHDA (10�4 and 10�5 M). No synergism in

response due to co-exposure of 6-OHDA with 4-HNE

(1 mM) was recorded. Pretreatment of 17b-estradiol

against the co-exposure of 6-OHDA and 4-HNE at

these concentrations was found to be insignificantly

effective (Figures 7 and 8).

Discussion

Higher concentrations of 4-HNE are known to be

associated with inhibition of several cellular functions

such as membrane transport, microtubule formation,

Figure 5. Effect of pretreatment of 17b-estradiol (1 mM) on percentage cell viability of PC12 cells exposed to 25 mM of4-hydroxynonenal (4-HNE) and 10�6 M of 6-hydroxydopamine (6-OHDA). PC12 cells were exposed to 17b-estradiol(1 mM) for 24 hours prior to the addition of HNE and/or 6-OHDA to the culture medium. Values are mean + SE of fourindependent experiments.

864 Human and Experimental Toxicology 30(8)

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

0

20

40

60

80

100

120

Control 50 μM HNE 17 β-Estradiol +50μM HNE

50μM HNE + 10-6 M 6-OHDA

17β-Estradiol + 50μM HNE + 10-

6 M 6-OHDA

% C

ell v

iabi

lity

Concentrations

P < 0.01

P < 0.01

P < 0.05

P < 0.05

Figure 6. Effect of pretreatment of 17b-estradiol (1 mM) on percentage cell viability of PC12 cells exposed to 50 mM of4-hydroxynonenal (4-HNE) and 10�6 M of 6-hydroxydopamine (6-OHDA). PC12 cells were exposed to 17b-estradiol(1 mM) for 24 hours prior to the addition of HNE and/or 6-OHDA to the culture medium. Values are mean + SE of fourindependent experiments.

Figure 7. Effect of pretreatment of 17b-estradiol (1 mM) on percentage cell viability of PC12 cells exposed to 1 mM of4-hydroxynonenal (4-HNE) and 10�4 M of 6-hydroxydopamine (6-OHDA). PC12 cells were exposed to 17b-estradiol(1 mM) for 24 hours prior to the addition of HNE and/or 6-OHDA to the culture medium. Values are mean + SE of fourindependent experiments. NS: non-significant.

Siddiqui MA et al. 865

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

and mitochondrial respiration.6,7 Cytotoxic responses

of 4-HNE have been reported in variety of cell sys-

tems including HepG2 cells,30 V79 cells,31 cerebellar

granule neurons,5 and primary cultures of normal

human osteoblasts.32 In the present investigations,

results are consistent with earlier studies and have

shown significant cytotoxic responses of 4-HNE

at 25 and 50 mM concentrations in PC12 cells. The

physiological stress was recorded even at lower

concentration (10 mM) of 4-HNE. Lower concen-

trations have also been reported to exert cytotoxi-

city by inducing signaling pathway for the

production of cytokines linked with cellular injury

and apoptosis.33 Possible involvement of oxidative

stress either by generating reactive oxygen and

nitrogen species or by the stress activated transcrip-

tion factors such as NF-kappa B, AP-1, and P53

has also been reported.34,35 Mitochondrial catabo-

lism involves in MTT assay, as 4-HNE or its meta-

bolites are reactive in nature and may interact

either directly to the proteins36 or oxidative stress

signaling pathways.37 HNE at 1 mM was found to

be ineffective in reducing the percentage cell viabi-

lity in PC12 cells. It might be correlated with the

earlier studies demonstrating the normal physiolo-

gical concentrations of 4-HNE in the plasma and

erythrocytes of human and experimental animals

ranged between 1 and 10 mM.38

Higher concentrations (10�4 and 10�5 M) of

6-OHDA were found be cytotoxic to PC12 cells

under our experimental conditions; whereas, 10�6

M and lower concentration did not cause any sig-

nificant decrease in percentage cell viability. Our

findings are consistent with earlier reports showing

6-OHDA induced mitochondrial abnormalities in

the pathogenesis of experimental model of Parkin-

son’s disease.39 6-OHDA is known to enter through

both dopaminergic and noradrenergic neurons and

induce damages to the catecholaminergic pathways

of both peripheral and central nervous systems.40

Therefore, it is one among the widely accepted

neurotoxicants to develop in vitro and in vivo

experimental models of Parkinson’s disease.10,41

6-OHDA-induced apoptotic cell death, mitochon-

drial dysfunction-mediated release of cytochrome

c, and the activation of caspase 3 have also been

reported in PC12 cells.11,42

Data indicates that 4-HNE (1 mM) has statistically

significant synergistic response to 6-OHDA (10�6 M)

cytotoxicity, even at non-toxic concentrations, where

independent exposure of either of 4-HNE or 6-OHDA

does not cause any significant cytotoxicity in PC12 cells

Figure 8. Effect of pretreatment of 17b-estradiol (1 mM) on percentage cell viability of PC12 cells exposed to 1 mM of4-hydroxynonenal (4-HNE) and 10�5 M of 6-hydroxydopamine (6-OHDA). PC12 cells were exposed to 17b-estradiol(1 mM) for 24 hours prior to the addition of HNE and/or 6-OHDA to the culture medium. Values are mean + SE of fourindependent experiments. NS: non-significant.

866 Human and Experimental Toxicology 30(8)

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

(Figure 3). The magnitude of this synergism was found

to be increased further with increased concentrations of

4-HNE. Thus, suggestive that co-exposure of 4-HNE

and 6-OHDA makes cellular system more vulnerable

under experimental conditions. This might be due to

comparatively faster 4-HNE-induced cellular changes,

which could lead an early physiological impairment in

the cells and this compromised state of cells made them

more vulnerable towards the 6-OHDA exposure. How-

ever, no synergism could be apparent in cells co-

exposed to non-cytotoxic concentration of 4-HNE

(1 mM) and toxic doses of 6-OHDA (10�4 and 10�5

M). As lower concentrations of 4-HNE are reported to

metabolize very fast and converted into non-toxic or

low-toxic metabolites in to short span of time,43,44 thus,

the loss of percentage cell viability was largely due to

6-OHDA when observed at 24 hours of exposure.

A pretreatment of 24 hours of 17b-estradiol at

physiological concentration (1 mM) was found to

be protective against damages induced at lower

concentrations by 4-HNE and 6-OHDA in PC12

cells. Our data are consistent with the earlier stud-

ies showing that 17b-estradiol exposure stabilizes

mitochondrial potential against oxidative stress26

and mutant presenilin-1.45 Mounting evidence sug-

gests that estrogens, acting as mitochondrial ener-

gizers by targeting mitochondrial sites to inhibit

opening of permeability transition pores (20), inhi-

bit the mitochondrial calcium uniport,46 increase

mitochondrial-specific proteins expression,47 cause

recovery of ATP production,20 and up-regulate the

antiapoptotic protein Bcl-2.48-50 Our findings could

also be supported by earlier findings demonstrating

the protective potential of 17b-estradiol in SK-N-SH,

a human neuroblastoma, by reducing lipid peroxida-

tion and 4-HNE production.20

Our data suggest that PC12 cells are vulnerable

to independent exposure of 4-HNE and 6-OHDA at

higher concentrations and co-exposure of both makes

cellular system more vulnerable even at non-cytotoxic

concentrations. Pretreatment of 17b-estradiol at phy-

siological concentration was found to be protective

against lower concentrations of both independent

and co-exposure of 4-HNE and 6-OHDA in PC12

cells.

Acknowledgment

The authors are grateful to Director, Indian Institute of

Toxicology Research, Lucknow, for his keen interest in the

present work. The technical laboratory assistance by Mr.

Rajesh Misra is also acknowledged.

Funding

Financial support for this study was obtained from Council of

Scientific & Industrial Research, New Delhi, India (SIP-08).

References

1. Adibhatla RM, Hatcher JF. Lipid oxidation and perox-

idation in CNS Health and disease: from molecular

mechanisms to therapeutic opportunities. Antiox Redox

Signal 2010; 12: 125–169.

2. Dmitriev LF. The involvement of lipid radical cycles

and the adenine nucleotide translocator in neurodegen-

erative diseases. J Alzheimer’s Dis 2007; 11: 183–190.

3. Liang LP, Huang J, Fulton R, Day BJ, and Patel M. An

orally active catalytic metalloporphyrin protects against

1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine neuro-

toxicity in vivo. J Neurosci 2007; 27: 4326–4333.

4. Piga R, Saito Y, Yoshida Y, and Niki E. Cytotoxic

effects of various stressors on PC12 cells: involvement

of oxidative stress and effect of antioxidants. Neuro-

toxicology 2007; 28: 67–75.

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

Ishige K, et al. N-Acetylcysteine and ebselen but not

nifedipine protected cerebellar granule neurons against

4-hydroxynonenal-induced neuronal death. Neurosci

Res 2007; 57: 220–229.

6. Abdul HM, Butterfield DA. Involvement of PI3K/PKG/

ERK1/2 signaling pathways in cortical neurons to trig-

ger protection by co-treatment of acetyl-L-carnitine and

alpha-lipoic acid against HNE-mediated oxidative

stress and neurotoxicity: implications for Alzheimer’s

disease. Free Radic Biol Med 2007; 42: 371–384.

7. 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.

8. Mazzio EA, Reams RR, and Soliman KF. The role of

oxidative stress, impaired glycolysis and mitochon-

drial respiratory redox failure in the cytotoxic effects

of 6-hydroxydopamine in vitro. Brain Res 2004;

1004: 29–44.

9. Inden M, Kitamura Y, Kondo J, Hayashi K, Yana-

gida T, Takata K, et al. Serofendic acid prevents 6-

hydroxydopamine-induced nigral neurodegeneration

and drug-induced rotational asymmetry in hemi-

parkinsonian rats. J Neurochem 2005; 95: 950–961.

10. Blum D, Torch S, Lambeng N, Nissou M, Benabid AL,

Sadoul R, et al. Molecular pathways involved in the

neurotoxicity of 6-OHDA, dopamine and MPTP: con-

tribution to the apoptotic theory in Parkinson’s disease.

Prog Neurobiol 2001; 65: 135–172.

Siddiqui MA et al. 867

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

11. Hanrott K, Gudmunsen L, O’Neill MJ, and Wonnacott S.

6-hydroxydopamine-induced apoptosis is mediated via

extracellular auto-oxidation and caspase 3-dependent

activation of protein kinase C delta. J Biol Chem 2006;

281: 5373–5382.

12. Wang T, Zhang QJ, Liu J, Wu ZH, and Wang S. Firing

activity of locus coeruleus noradrenergic neurons

increases in a rodent model of Parkinsonism. Neurosci

Bull 2009; 25: 15–20.

13. Kulkarni J, de Castella A, Fitzgerald PB, Gurvich CT,

Bailey M, Bartholomeusz C, et al. Estrogen in severe

mental illness: a potential new treatment approach.

Arch Gen Psychiatry 2008; 65: 955–960.

14. Bagger YZ, Tanko LB, Alexandersen P, Qin G, and

Christiansen C. Early postmenopausal hormone ther-

apy may prevent cognitive impairment later in life.

Menopause 2005; 12: 12–17.

15. McClean J, Nunez JL. 17alpha-Estradiol is neuropro-

tective in male and female rats in a model of early

brain injury. Exp Neurol 2008; 210: 41–50.

16. Huppmann S, Romer S, Altmann R, Obladen M, and

Berns M. 17beta-estradiol attenuates hyperoxia-

induced apoptosis in mouse C8-D1A cell line. J Neu-

rosci Res 2008; 86: 3420–3426.

17. Morissette M, Di Paolo T. Effect of estradiol on striatal

dopamine activity of female hemiparkinsonian mon-

keys. J Neurosci Res 2008; 87: 1634–1644.

18. Cheng MY, Sun G, Jin M, Zhao H, Steinberg GK, and

Sapolsky RM. Blocking glucocorticoid and enhan-

cing estrogenic genomic signaling protects against

cerebral ischemia. J Cereb Blood Flow Metab 2009;

29: 130–136.

19. Wise PM, Dubal DB, Wilson ME, Rau SW, Bottner M,

and Rosewell KL. Estradiol is a protective factor in the

adult and aging brain: understanding of mechanisms

derived from in vivo and in vitro studies. Brain Res

Brain Res Rev 2001; 37: 313–319.

20. Wang X, Dykens JA, Perez E, Liu R, Yang S, Covey

DF, et al. Neuroprotective effects of 17beta-estradiol

and nonfeminizing estrogens against H2O2 toxicity in

human neuroblastoma SK-N-SH cells. Mol Pharmacol

2006; 70: 395–404.

21. Saengsirisuwan V, Pongseeda S, Prasannarong M,

Vichaiwong K, and Toskulkao C. Modulation of insu-

lin resistance in ovariectomized rats by endurance

exercise training and estrogen replacement. Metabo-

lism 2009; 58: 38–47.

22. Zhang H, Xie M, Schools GP, Feustel PF, Wang W,

Lei T, et al. Tamoxifen mediated estrogen receptor

activation protects against early impairment of hippo-

campal neuron excitability in an oxygen/glucose

deprivation brain slice ischemia model. Brain Res

2009; 1247:196–211.

23. Zhao Y, Hu I, and Jin W. Transformation of oxidation

products and reduction of estrogenic activity of

17beta-estradiol by a heterogeneous photo-Fenton

reaction. Environ Sci Technol 2008; 42: 5277–5284.

24. Hruska Z, Dohanich GP. The effects of chronic estra-

diol treatment on working memory deficits induced

by combined infusion of beta-amyloid (1–42) and ibo-

tenic acid. Horm Behav 2007; 52: 297–306.

25. Perrella J, Bhavnani BR. Protection of cortical cells by

equine estrogens against glutamate-induced excito-

toxicity is mediated through a calcium independent

mechanism. BMC Neurosci 2005; 6: 34.

26. Wang J, Green PS, and Simpkins JW. Estradiol protects

against ATP depletion, mitochondrial membrane poten-

tial decline and the generation of reactive oxygen species

induced by 3-nitroproprionic acid in SK-N-SH human

neuroblastoma cells. J Neurochem 2001; 77: 804–811.

27. Regan RF, Guo Y. Estrogens attenuate neuronal injury

due to hemoglobin, chemical hypoxia, and excitatory

amino acids in murine cortical cultures. Brain Res

1997; 764: 133–140.

28. Zhang P, Li HF, Tian ZF, Qiu XQ, Wu JX, and Jia ZJ.

Effects of phytoestrogens and 17beta-estradiol on

vasoconstriction elicited by reactive oxygen species.

Pharmazie 2007; 62: 378–381.

29. Galbiati F, Volonte D, Gil O, Zanazzi G, Salzer JL.,

Sargiacomo M, et al. Expression of caveolin-1 and -2

in differentiating PC12 cells and dorsal root ganglion

neurons: caveolin-2 is up-regulated in response to cell

injury. Proc Natl Acad Sci U S A 1998; 95: 10257–

10262.

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

vitro cytotoxicity evaluation of plastic biomedical

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

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

Ellis EM. Mouse aldo-keto reductase AKR7A5 pro-

tects V79 cells against 4-hydroxynonenal-induced

apoptosis. Toxicology 2006; 226: 172–180.

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

Waeg G, et al. Differential sensitivity to

4-hydroxynonenal for normal and malignant mesench-

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

33. Shi Q, Vaillancourt F, Cote V, Fahmi H, Lavigne P,

Afif H, et al. Alterations of metabolic activity in

human osteoarthritic osteoblasts by lipid peroxidation

end product 4-hydroxynonenal. Arthritis Res Ther

2006; 8: 159.

34. Chung FL, Pan J, Choudhury S, Roy R, Hu W, and

Tang MS. Formation of trans-4-hydroxy-2-nonenal-

868 Human and Experimental Toxicology 30(8)

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from

and other enal-derived cyclic DNA adducts from

omega-3 and omega-6 polyunsaturated fatty acids and

their roles in DNA repair and human p53 gene muta-

tion. Mutat Res 2005; 531: 25–36.

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

Yadav S, Chandra D, et al. Oxidative stress mediated

cellular responses in 4-hydoxynonenal exposed PC12

cells. J Ecophysiol Occup Health 2007; 7: 97–109.

36. VChoudhary S, Xiao T, Srivastava S, Zhang W, Chan

LL, Vergara LA, et al. Toxicity and detoxification of

lipid-derived aldehydes in cultured retinal pigmented

epithelial cells. Toxicol Appl Pharmacol 2005; 204:

122–134.

37. Dwivedi S, Sharma A, Patrick B, Sharma R, and

Awasthi YC. Role of 4-hydroxynonenal and its meta-

bolites in signaling. Redox Rep 2007; 12: 4–10.

38. Srivastava S, Dixit BL, Cai J, Sharma S, Hurst HE,

Bhatnagar A, et al. Metabolism of lipid peroxidation

product, 4-hydroxynonenal (HNE) in rat erythrocytes:

role of aldose reductase. Free Radic Biol Med 2000;

29: 642–651.

39. Seth K, Agrawal AK, Aziz MH, Ahmad A, Shukla Y,

Mathur N, et al. Induced expression of early response

genes/oxidative injury in rat pheochromocytoma

(PC12) cell line by 6-hydroxydopamine: implication for

Parkinson’s disease. Neurosci Lett 2002; 330: 89–93.

40. Bove J, Prou D, Perier C, and Przedborski S. Toxin-

induced models of Parkinson’s disease. NeuroRx

2005; 2: 484–494.

41. Chaturvedi RK, Shukla S, Seth K, Chauhan S, Sinha C,

Shukla Y, et al. Neuroprotective and neurorescue

effect of black tea extract in 6-hydroxydopamine-

lesioned rat model of Parkinson’s disease. Neurobiol

Dis 2006; 22: 421–434.

42. Eminel S, Klettne A, Roemer L, Herdegen T, and

Waetzig V. JNK2 translocates to the mitochondria and

mediates cytochrome c release in PC12 cells in

response to 6-hydroxydopamine. J Biol Chem 2004;

279: 55385–55392.

43. Finkelstein EI, Ruben J, Koot CW, Hristova M, and

van der Vliet A. Regulation of constitutive neutrophil

apoptosis by the alpha,beta-unsaturated aldehydes

acrolein and 4-hydroxynonenal. Am J Physiol Lung

Cell Mol Physiol 2005; 289: 1019–1028.

44. Duarte AI, Santos MS, Oliveira CR, and Rego AC.

Insulin neuroprotection against oxidative stress in cor-

tical neurons–involvement of uric acid and glutathione

antioxidant defenses. Free Radic Biol Med 2005; 39:

876–889.

45. Mattson MP, Robinson N, and Guo Q. Estrogens stabi-

lize mitochondrial function and protect neural cells

against the pro-apoptotic action of mutant presenilin-

1. Neuroreport 1997; 8: 3817–3821.

46. Carmen D, Lobaton LV, Hernandez-SanMiguel E,

SantoDomingo J, Moreno A, Montero M, et al. Modu-

lation of mitochondrial Ca2þ uptake by estrogen recep-

tor agonists and antagonists. Br J Pharmacol 2005;

145: 862–871.

47. Razmara A, Duckles SP, Krause DN, and Procaccio V.

Estrogen suppresses brain mitochondrial oxidative stress

in female and male rats. Brain Res 2007; 1176: 71–81.

48. Nilsen J, Diaz BR. Mechanism of estrogen-mediated

neuroprotection: regulation of mitochondrial calcium

and Bcl-2 expression. Proc Natl Acad Sci U S A

2003; 100: 2482–2487.

49. Burris TP, Krishnan V. Estrogen: a mitochondrial

energizer that keeps on going. Mol Pharmacol 2005;

68: 956–958.

50. Stirone C, Duckles SP, Krause DN, and Procaccio V.

Estrogen increases mitochondrial efficiency and

reduces oxidative stress in cerebral blood vessels. Mol

Pharmacol 2005; 68: 959–965.

Siddiqui MA et al. 869

at UNIV OF LOUISVILLE on January 31, 2012het.sagepub.comDownloaded from