ORIGINAL PAPER

Effects of Selenium on Calcium Signaling and Apoptosisin Rat Dorsal Root Ganglion Neurons Induced by Oxidative Stress

Abdulhadi Cihangir Uguz • Mustafa Nazıroglu

Received: 20 February 2012 / Revised: 8 March 2012 / Accepted: 16 March 2012 / Published online: 3 April 2012

� Springer Science+Business Media, LLC 2012

Abstract Ca2? is well known for its role as crucial sec-

ond messenger in modulating many cellular physiological

functions, Ca2? overload is detrimental to cellular function

and may present as an important cause of cellular oxidative

stress generation and apoptosis. The aim of this study is to

investigate the effects of selenium on lipid peroxidation,

reduced glutathione (GSH), glutathione peroxidase (GSH-

Px), cytosolic Ca2? release, cell viability (MTT) and

apoptosis values in dorsal root ganglion (DRG) sensory

neurons of rats. DRG cells were divided into four groups

namely control, H2O2 (as a model substance used as a

paradigm for oxidative stress), selenium, sele-

nium ? H2O2. Moderate doses and times of H2O2 and

selenium were determined by MTT test. Cells were pre-

terated 200 nM selenium for 30 h before incubatation with

1 lM H2O2 for 2 h. Lipid peroxidation levels were lower

in the control, selenium, selenium ? H2O2 groups than in

the H2O2 group. GSH-Px activities were higher in the

selenium groups than in the H2O2 group. GSH levels were

higher in the control, selenium, selenium ? H2O2 groups

than in the H2O2 group. Cytosolic Ca2? release was higher

in the H2O2 group than in the control, selenium, sele-

nium ? H2O2 groups. Cytosolic Ca2? release was lower in

the selenium ? H2O2 group than in the H2O2. In conclu-

sion, the present study demonstrates that selenium induced

protective effects on oxidative stress, [Ca2?]c release and

apoptosis in DRG cells. Since selenium deficiency is a

common feature of oxidative stress-induced neurological

diseases of sensory neurons, our findings are relevant to the

etiology of pathology in oxidative stress-induced neuro-

logical diseases of the DRG neurons.

Keywords Calcium ion release � Selenium �Oxidative stress � Dorsal root ganglion neurons � Apoptosis

Introduction

Oxidative stress represents an imbalance status between

excessive production of reactive oxygen species (ROS),

oxygen-derived radical species and biological system’s

scavenging ability to detoxify the reactive agents. Most of

the ROS are formed as a result of the mitochondrial

respiratory chain pathways but can also be formed exoge-

nously [1]. Antioxidants such as glutathione (GSH), glu-

tathione peroxidase (GSH-Px) and related enzymes are

believed to play critical roles in protecting cells from

hazardous oxygen species [2]. GSH-Px, one of the major

intracellular antioxidant enzymes, detoxifies hydrogen

peroxide (H2O2) to water and also scavenge other perox-

ides [3].

Selenium is an essential dietary trace element which

plays an important role in a series of biological function. It

has been previously demonstrated that selenium plays an

important role in the continuation of the physiological

functions of the nervous system such as signal transduction

and development [4]. It also acts as a cofactor for the GSH-

Px enzyme and is also incorporated into the selenoproteins

that are involved in antioxidant defenses [5]. Selenium is

incorporated in mammalian proteins as selenocysteine or

selenomethionine, both of which are dietary forms of

selenium, although selenomethionine is the major form [6].

A. C. Uguz � M. Nazıroglu (&)

Department of Biophysics, Faculty of Medicine, Suleyman

Demirel University, Dekanlık Binasi, 32260 Isparta, Turkey

e-mail: mnaziroglu@med.sdu.edu.tr

A. C. Uguz � M. Nazıroglu

Neuroscience Research Center, Suleyman Demirel University,

Isparta, Turkey

123

Neurochem Res (2012) 37:1631–1638

DOI 10.1007/s11064-012-0758-5

Even though at high concentrations it can be toxic to the

biological systems, at low concentration of selenium is

implicated as neuronprotective agents in several neuronal

diseases including epilepsy [4, 7] and pain [6]. The neu-

roprotecive effects of selenium are attributed to its ability

to inhibit apoptosis [8, 9] and to modulate Ca2? influx

through ion channels [5, 9].

Dorsal root ganglions (DRG) are nodules on a dorsal

root that contains cell bodies of neurons in afferent spinal

nerves. They transducers noxious stimuli into electric

impulses, conduct impulses and modulate the synaptic

transmission in central nervous system [10]. Sensory neu-

ronopathies and neuropathic pain are common chronic

clinical conditions that affect million of people all around

the world. Sensory neuronopathies are caused by nerve

injury or diseases such as diabetes and cancer which

damage peripheral nerves. As a result of this damage DRG

neurons become more excitable. Sensory neuronopathies

are proposed to being characterised by the primary

degeneration of DRG neurons [11, 12].

We investigated the effects of selenium supplementation

on cytosolic Ca2? levels, apoptosis and oxidative stress

parameters in cultured DRG neurons.

Materials and Methods

Chemicals

All chemicals (cumene hydroperoxide, KOH, NaOH, thio-

barbituric acid, 1,1,3,3-tetraethoxypropane, 5,5-dithiobis-2

nitrobenzoic acid, tris-hydroxymethyl-aminomethane, glu-

tathione, butylhydroxytoluol,Triton X-100 and ethylene

glycol-bis[2-aminoethyl-ether]-N,N,N,N-tetraacetic acid

[EGTA]) were obtained from Sigma-Aldrich (St. Louis,

MO, USA) and all organic solvents (n-hexane, ethyl

alcohol) were purchased from Merck (Darmstadt, Ger-

many). Fura-2 acetoxymethyl ester was purchased from

Invitrogen (Carlsbad, CA, USA). All reagents were ana-

lytical grade. All reagents except the phosphate buffers

were prepared daily and stored at ?4 �C. Reagents were

equilibrated at room temperature for half an hour before

an analysis was initiated or reagent containers were refilled.

Phosphate buffers were stable at ?4 �C for 1 month.

APOPercentageTM assay kit was purchased from Biocolor

(Belfast, Northern Ireland). Collegenase IV was bought

from Worthington Inc. (USA).

Animals

Twenty-four female Wistar-albino rats with 21–30 days of

age were used for the experimental procedures. Rats were

allowed 1 week to acclimatize to the surroundings before

beginning any experimentation. Animals were housed in

individual plastic cages with bedding. Standard rat food

and tap water were available ad libitum for the duration of

the experiments. The temperature was maintained at

22 ± 2 �C. A 12/12 h light/dark cycle was maintained,

with lights on at 06.00, unless otherwise noted. Experi-

mental protocol of the study was approved by the ethical

committee of the Medical Faculty of Suleyman Demirel

University (Protocol Number; 2009: 16-02). Animals were

maintained and used in accordance with the Animal Wel-

fare Act and the Guide for the Care and Use of Laboratory.

Preparation of DRG Samples

The animals were anesthetized by ether asphyxiation in

accordance with SDU Experimental Animal legislation.

DRG neurons (T13-L5) were carefully dissected from

peripheral nerve root. DRG neurons were collected and

incubated in Dulbecco’s modified Eagle’s medium

(DMEM, Gibco, Istanbul, Turkey) with 1 % penicillin–

streptomycin (Sigma, Istanbul, Turkey) in 500 ml of

DMEM. The connective tissue was removed and ganglia

were treated with collegenase IV (0.28 ml in DMEM), and

tyripsin (25,000 units/ml in DMEM, for 45 min at 37 �C

and in an atmosphere containing 5 % CO2). After disso-

ciation with a sterile syringe, the cell suspension was

centrifuged at 3,500 rpm and seeded into 25 cm2 sterile

flasks.

Study Groups

DRG neurons of each animal were divided into four groups

as follows:

Group I (n = 6) was control group and the DRG neurons

were incubated (37 �C and 5 % CO2) for 24 h with

normal medium.

Group II (n = 6) was H2O2 group and the DRG neurons

were incubated with 1 lM H2O2 (37 �C, %5 CO2) for

2 h.

Group III (n = 6) was selenium (Se) group and the DRG

neurons were incubated with 200 nM Se for 30 h.

Group IV (n = 6) was Se ? H2O2 group and initially

the DRG neurons were pre-incubated with 200 nM Se

for 30 h and then they were incubated with 1 lM H2O2

for 2 h.

At the end of the experiments, half of the DRG samples

were immediately used for Ca2? signaling and apoptosis

analyses. The remaining neurons were washed with phos-

phate buffer (pH 7.4) and then frozen at -33 �C. GSH,

GSH-Px and lipid peroxidation analyses were performed

within 1 week.

1632 Neurochem Res (2012) 37:1631–1638

123

Determination of Moderate Incubation Doses

of Selenium by Cell Viability (MTT) Assay

Cell viability was evaluated by the MTT assay based on the

ability of viable cells to convert a water-soluble, yellow

tetrazolium salt into a water-insoluble, purple formazan

product. The enzymatic reduction of the tetrazolium salt

happens only in living, metabolically active cells but not in

dead cells. DRG cells were seeded in tubes at a density of

2 9 106/tube and subsequently exposed to several con-

centrations of sodium selenite (50 nM–1 lM) and H2O2

(1 lM–1 mM) at different incubation times (1–48 h for

sodium selenite and 0.5–24 h for H2O2) at 37 �C. After the

treatments, the medium was removed and MTT was added

to each tube and then incubated for 90 min at 37 �C in a

shaking water bath. The supernatant was discarded and

DMSO was added to dissolve the formazan crystals.

Treatments were carried out in duplicate. Optical density

was measured in an spectrophotometer at 490 and 650 nm

and presented as the fold increase over the pretreatment

level (experimental/control).

Measurement of Cytosolic Ca2? Concentration

([Ca2?]c)

The DRG cells were loaded with 4 lM Fura-2/AM in

loading buffer with 5 9 106 cells/ml for 45 min at 37 �C

in the dark, washed twice, incubated for an additional

30 min at 37 �C to complete probe de-esterification and

resuspended in loading buffer at a density of 3 9 106 cells/

ml according to a procedure published elsewhere [9]. The

four groups were exposed to H2O2 for stimulating intra-

cellular Ca2? release. Fluorescence was recorded from

2-ml aliquots of a magnetically stirred cellular suspension

at 37 �C using a spectrofluorometer (Carry Eclipse; Varian,

Sydney, Australia) with excitation wavelengths of 340 and

380 nm and emission at 505 nm. Changes in [Ca2?]c were

monitored using the Fura-2/AM 340/380 nm fluorescence

ratio and calibrated according to the method of Gry-

nkiewicz et al. [13]. In the experiments where calcium-free

medium is indicated, Ca2? was omitted and 2 mM EGTA

was added. Ca2? release was estimated using the integral

of the rise in [Ca2?]c for 40 s after addition of H2O2. Ca2?

release is expressed in nanomoles, taking a sample every

second as previously described [9].

Apoptosis Assay

The APOPercentageTM assay (Biocolor Ltd., Belfast,

Northern Ireland) was performed according to the

instructions provided by Biocolor Ltd. and elsewhere [14].

The APOPercentageTM assay is a dye-uptake assay,

which stains only the apoptotic cells with a red dye. When

the membrane of apoptotic cell loses its asymmetry, the

APOPercentage dye is actively transported into cells,

staining apoptotic cells red, thus allowing detection of

apoptosis by spectrophotometer [15].

Lipid Peroxidation (LP) Determinations

Lipid peroxidation levels in the DRG neurons cultures were

measured with the thiobarbituric-acid reaction by the

method of Placer et al. [16]. Thiobarbituric acid-reactive

substances were quantified by comparing the absorption to

the standard curve of malondialdehyde (MDA) equivalents

generated by acid-catalyzed hydrolysis of 1,1,3,3-tetra-

methoxypropane. LP values in DRG neurons were expressed

as micromoles per gram of protein.

Reduced Glutathione (GSH), Glutathione Peroxidase

(GSH-Px) and Protein Assay

The GSH content of the DRG neurons samples was mea-

sured at 412 nm using the method of Sedlak and Lindsay

[17]. The samples were precipitated with 50 % trichlor-

acetic acid and then centrifuged at 1,000g for 5 min. The

reaction mixture contained 0.5 ml of supernatant, 2.0 ml of

Tris–EDTA buffer (0.2 M; pH 8.9) and 0.1 ml of 0.01 M

5,50-dithio-bis-2-nitrobenzoic acid. The solution was kept

at room temperature for 5 min, and then read at 412 nm

using a spectrophotometer (Shimadzu UV-1800, Kyoto,

Japan). GSH-Px activities of DRG neurons samples were

measured spectrophotometrically at 37 �C and 412 nm

according to the method of Lawrence and Burk [18]. The

protein content in DRG neurons samples was measured by

the method of Lowry et al. [19] with bovine serum albumin

as the standard.

Statistical Analysis

Data are expressed as means ± SEM of the numbers of

determinations. Statistical significance was analyzed using

the SPSS program (9.05; SPSS, Inc., Chicago, IL, USA).

To compare the different treatments, statistical significance

was calculated by the Mann–Whitney U test. P \ 0.05 was

considered to indicate a statistically significant difference.

Results

Determination of Moderate Doses of H2O2

and Selenium in DRG Neurons by Cell Viability (MTT)

Test

The effects of H2O2 and selenium on the MTT in DRG

neurons were shown in Figs. 1 and 2, respectively. Cells

Neurochem Res (2012) 37:1631–1638 1633

123

were incubated with increasing concentrations of H2O2

(1 lM–1 mM) for six different time periods. The toxic

effect of H2O2 started at the 1 mM concentration at 2 h

after H2O2 incubation (P \ 0.001). For 24 h of incubation,

the toxic effect started at a higher concentration of H2O2

(mM) exposure. Hence we found the toxic concentration of

H2O2 as 1 mM. Then we investigated the toxic concen-

tration and duration of selenium exposure in DRG neuron

culture. Cells were incubated 5 different concentrations of

selenium (50 nM–1 lM) for six different times (1–48 h)

and the MTT test was performed from the samples. Mod-

erate dose and time of selenium were found 200 nM at

30 h by the MTT test.

Effects of Moderate and High Concentrations

of Selenium and Treatment H2O2 on the MTT Test

The effect of moderate and high concentrations of selenium

and H2O2 on the MTT levels in DRG neurons is shown in

Figs. 1 and 2. The neurons were pre-incubated with

200 nM selenium, for 30 h and then stimulated with 1 lM

H2O2 for 2 h. MTT levels decreased significantly

(P \ 0.05 and 70 %) in 1 mM H2O2 (group A), and the

cells did not tolerate high doses of H2O2 (1 mM).

However, cell viability decreased at the 100 lM con-

centration of H2O2 (P \ 0.05). Cell viability was also

recovered in the 100 lM group by selenium supplemen-

tation. However, selenium was not able to affect on cell

viability in the 1 mM group. Cells were pre-incubated with

1 mM and 100 lM H2O2 for 10 h. Cell viability decreased

by 70 % (P \ 0.001) and 25 % (P \ 0.05) at 1 mM and

100 lM H2O2, respectively.

Effects of Moderate Concentrations of Selenium

and H2O2 on Ca2? Release

Effects of moderate concentrations of selenium and H2O2

on Ca2? release in DRG neurons are shown in Figs. 3 and

4. The neurons were pre-incubated with 200 nM selenium

for 30 h and then stimulated by 100 lM H2O2. Ca2?

release level was increased by 100 lM H2O2. Ca2? release

into cells was significantly (P \ 0.001) lower in the

200 nM selenium and selenium ? H2O2 groups than in the

H2O2 group.

Effects of Selenium Treatment on Apoptosis

Effects of selenium treatment on apoptosis of DRG neurons

are shown in Fig. 5. Apoptosis levels were significantly

(P \ 0.001) higher in 50, 100 and 500 nM selenium groups

than in H2O2 group although its level was lower in 200 nM

selenium group than in control, 50, 100 and 500 nM

selenium groups. Hence, we observed sufficient and mod-

erate dose of selenium as 200 nM on oxidative stress-

Fig. 1 Effect of H2O2 on DRG neurons viability (MTT). Cells were

incubated with increasing concentrations of H2O2 (1 lM–1 mM) for

various times (0.5–24 h). The toxic effect of H2O2 started at 10 lM

concentration and 10 h after incubation (P \ 0.05). We found the

tolerable dose of H2O2 to be 1 lM for 2 h in the cell culture system

Fig. 2 Effects of selenium concentrations on DRG neurons viability

(MTT). There was statistical significance at five concentrations of

selenium between 50 nm and 1 lM. The moderate dose of selenium

was 1 lM for 10 h incubation time

Fig. 3 Calcium mobilization in DRG neurons. In the selenium

groups, cells were incubated with 200 nM of selenium (sodium-

selenite) during 30 h. Fura-2-loaded DRG neurons were stimulated

with 100 lM H2O2 in the presence of extracellular calcium

([Ca2?]o = 1.2 mM ? 2 mM EGTA was added). The traces shown

are representative of six separate experiments

1634 Neurochem Res (2012) 37:1631–1638

123

induced apoptosis in DRG neurons. This finding was also

supported by synergic MTT results of current study.

Effects of Selenium Treatment on Lipid Peroxidation

(LP) Levels

The effects of selenium on LP levels in DRG neurons are

shown in Fig. 6. LP levels were significantly (P \ 0.01)

higher in the H2O2 group than in the control, selenium and

selenium ? H2O2 groups. However, LP levels were sig-

nificantly (P \ 0.01) lower in the selenium (P \ 0.05).

Hence, we found that selenium induced protective effects

against oxidative stress-induced LP levels.

Effects of Selenium Treatment on GSH

and GSH-Px Values

The effects of selenium on GSH and GSH-Px values in

DRG neurons are shown in Figs. 6 and 7 respectively.

GSH-Px activity and GSH levels in DRG neurons were

Fig. 4 Effects of selenium (sodium-selenite) treatments on intracel-

lular Ca2? release induced by 100 lM H2O2. DRG neurons were

incubated with 200 nM Se during 30 h. For H2O2 group DRG neurons

were incubated with 1 lM H2O2. The graphics shown are the areas

under Ca2? release curves and representatives six separate experi-

ments. aP \ 0.001 versus to control, bP \ 0.001 versus to H2O2

group, cP \ 0.001 versus to Se group

Fig. 5 Levels of apoptosis in DRG neurons. DRG neurons were

incubated with 4 different dosages of selenium (sodium-selenite) then

stimulated with 1 lM H2O2 to confirm findings with MTT test.

Values are presented as ±SEM of six separate experiments, which

were carried out by duplicate. aP \ 0.001 versus to control,bP \ 0.001 versus to 50 nM group, cP \ 0.001 versus to 100 nM

group, dP \ 0.001 versus to 200 nM group

Fig. 6 Lipid peroxidation (MDA) levels in DRG neurons. DRG

neurons were incubated with 200 nM selenium (sodium-selenite) for

30 h and 1 lM H2O2 for 2 h. Values are represented as mean-

s ± SEM of six separate experiments. aP \ 0.001 versus to control,bP \ 0.001 versus to H2O2 group, cP \ 0.001 versus to Se group

Fig. 7 Levels of GSH levels in DRG neurons. DRG neurons were

incubated with 200 nM selenium (sodium-selenite) for 30 h and

1 lM H2O2 for 2 h. Values are represented as means ± SEM of six

separate experiments. *P \ 0.05 compared to Se values. aP \ 0.001

versus to control, bP \ 0.001 versus to H2O2 group, cP \ 0.001

versus to Se group, dP \ 0.05 versus to H2O2 group

Neurochem Res (2012) 37:1631–1638 1635

123

significantly (P \ 0.05) lower in the H2O2 group than in

the control, selenium and selenium ? H2O2 groups. GSH-

Px and GSH values were significantly higher in the sele-

nium and selenium ? H2O2 groups than in the H2O2

(P \ 0.01) and control (P \ 0.05) groups (Fig. 8).

Discussion

The H2O2 is the endogen source of the free radicals and

causes oxidative stress in cellular mechanisms [1]. Sele-

nium is a co-factor in GSH-Px enzyme. GSH is used as a

substrate to synthesis the GSH-Px [6]. If the free radical

production increases proportional to the consumption of

GSH-Px enzyme activities, GSH levels are also declining.

Recently, we observed decrease in Ca2? release in GSH

depleted DRG neurons [20] although N-acetylcysteine

incubation induced protective effects on Ca2? release and

oxidative stress in GSH depleted DRG neurons [21].

Hence, selenium may also protective effects on the oxi-

dative values, Ca2? release and apoptosis in the DRG

neurons. In the current study, we aimed to investigate the

effects of selenium on cytosolic Ca2? release, GSH, GSH-

Px, LP on DRG neurons. According to our knowledge this

is the first study which clarifies the effects of selenium on

DRG neurons against H2O2 induced oxidative stress model.

Selenium administrations both in various experimental

models, in animals and humans, caused an increased level

of GSH and GSH-Px activity [5, 9, 21]. Oxidative stress

represents an imbalance status between excessive produc-

tion of ROS, other radical species and biological system’s

scavenging ability to detoxify the reactive intermediates.

Most of the ROS are formed as a result of the mitochon-

drial respiratory chain pathways but can also be formed

exogenously [1]. Hence GSH and GSH-Px and related

enzymes are believed to play critical roles in protecting

cells from hazardous oxygen species [2].

We found that LP values were increased in H2O2 group

than in control, selenium, selenium ? H2O2 groups

although GSH levels were lower in H2O2 group than in

control, selenium, selenium ? H2O2 groups. With neu-

ropathy progression, antioxidants potentially decrease and

LP levels increase. Excessive production of free radicals

due to oxidative stress may primarily stimulate the voltage-

gated Ca2? channels. Stimulation of many other ion

channels by oxidative stress causes flow of calcium ions

into cytosol and will cause depolarization in mitochondria.

Depolarization of mitochondria triggers free radical gen-

eration [23, 24].

In the current study, we confirm that DRG neurons

exhibit Ca2? ion selective channel-dependent ROS gener-

ation and Ca2? influx following cytosolic GSH depletion,

as previously reported [20, 21]. In addition, we present

evidence showing that cytosolic GSH depletion generates

lipid peroxidation and decreases GSH-Px activity. It is well

known that increase in cytoplasmic Ca2? may jointly

enhance mitochondrial ROS generation through depolar-

ization of mitochondria [22]. In response to increase in

cytosolic Ca2? through activation of Ca2? cation channels,

may incur Ca2?-induced respiratory impairment, potenti-

ating free radical generation, inflicting structural damage to

mitochondria and ultimately apoptotic cell death if the

Ca2? influx is not inhibited by antioxidants. We observed

that, [Ca2?]c release was higher in H2O2 group than in

selenium and selenium ? H2O2 groups. This situation, in

the same correlation line with other studies which per-

formed with H2O2, in H2O2 triggered apoptotic pathways

with antioxidant property, selenium induced protective

effect on apoptotic pathways [25–27].

Similarly, Koistinaho et al. [25] reported that, incuba-

tion of DRG cells with selenium will cause reduction in the

LP levels which will delay age dependent malformation.

Molecular and cellular pathways of pain are not well elu-

cidated yet [28]. There is growing consensus, driven by

both clinical and laboratory studies, demonstrate that

excessive free radical production and oxidative stress may

play a critical role in pathophysiology of DRG neurons

[28, 29]. In vivo studies demonstrated that free radicals

directly join exitotoxicity mechanisms and antioxidants

will be useful in exitotoxicity induced neuronal diseases

[20, 21, 30]. Mitochondrial dysfunction and oxidative

stress are both reason and consequence of the neuropathy

in DRG neurons [31]. DRG neurons are important for

pathophysiology of pain mechanism. The pathogenesis of

Fig. 8 GSH-Px activity in DRG neurons. DRG neurons were

incubated with 200 nM selenium (sodium-selenite) for 30 h and

1 lM H2O2 for 2 h. Values are represented as means ± SEM of six

separate experiments. *P \ 0.05 compared to Se values. aP \ 0.001

versus to control, bP \ 0.001 versus to H2O2 group, cP \ 0.001

versus to Se group

1636 Neurochem Res (2012) 37:1631–1638

123

most common two disorders of the peripheral nervous

system, namely neuropathic pain and diabetic polyneu-

ropathy, has been associated with aberrant Ca2? channel

expression and function [32].

The activity of selenium is strictly dependent on its

serum and tissue concentrations; while the lower concen-

trations induce cell growth, the higher concentrations

inhibit growth and induce cell death [6]. Uguz et al. [9]

have also investigated the effects of different selenium

concentrations in HL-60 cells, where they have demon-

strated that at low concentrations (200 nM) selenium

induces a mild endoplasmic reticulum (ER) stress whereas

this stress is much more severe at higher concentrations

(1 lM). Uezono et al. [33] reported that treatment of cul-

tured bovine adrenal chromaffin cells with selenium for

24 h caused decreased in neurotransmitter contents and

Ca2? release, association with cell damage, at concentra-

tions over 30 and 300 lM. They observed also that sele-

nium even at higher concentrations (1 mM) did not affect

any stimulus-induced Ca2? influx. These studies have

demonstrated the dose dependent effects of selenium in

mediating oxidative stress via modulating the Ca2? release

from the ER [8].

In conclusion, we demonstrated that DRG cells, in

oxidative stress model created by H2O2, selenium induced

protective effect against oxidative stress and suppressed

apoptosis of the neurons through regulation of cytosolic

Ca2? release. Furthermore, we observed that oxidative

stress and irregularities in intracellular Ca2? release caused

by H2O2 should be improved by selenium. Our study

supports the neuroprotective effect of selenium. As anti-

oxidant element, selenium may useful in the treatment of

DRG neurons dependent pain and disorders.

Acknowledgments MN and ACU formulated the present hypothe-

sis. MN made critical revision to the manuscript. ACU was respon-

sible to make the analysis and writing report. The study was partially

supported by the Scientific Research Project Unit of Suleyman

Demirel University (1881-D-09). The abstract of the study was sub-

mitted in 14th International Symposium on Trace Elements in Man

and Animals, Enshi, Hubei, China.

References

1. Davies MJ (2003) Singlet oxygen-mediated damage to proteins

and its consequences. Biochem Biophys Res Commun 305:

761–770

2. Kockar MC, Nazıroglu M, Celik O et al (2010) N-acetylcysteine

modulates doxorubicin-induced oxidative stress and antioxidant

vitamin concentrations in liver of rats. Cell Biochem Funct

28:673–677

3. Nazıroglu M, Kilinc F, Uguz AC et al (2010) Oral vitamin C and

E combination modulates blood lipid peroxidation and antioxi-

dant vitamin levels in maximal exercising basketball players. Cell

Biochem Funct 28:300–305

4. Wirth EK, Conrad M, Winterer J et al (2010) Neuronal seleno-

protein expression is required for interneuron development and

prevents seizures and neurodegeneration. FASEB J 24:844–852

5. McKenzie RC, Arthur JR, Beckett GJ (2002) Selenium and the

regulation of cell signaling, growth, and survival: molecular and

mechanistic aspects. Antioxid Redox Signal 4:339–351

6. Schweizer U, Brauer AU, Kohrle J, Nitsch R, Savaskan NE

(2004) Selenium and brain function: a poorly recognized liaison.

Brain Res Rev 45:164–178

7. Nazıroglu M (2009) Role of selenium on calcium signaling and

oxidative stress-induced molecular pathways in epilepsy. Neu-

rochem Res 34:2181–2191

8. Savas S, Briollais L, Ibrahim-zada I, Jarjanazi H, Choi YH,

Musquera M, Fleshner N, Venkateswaran V, Ozcelik H (2010)

A whole-genome SNP association study of NCI60 cell line panel

indicates a role of Ca2? signaling in selenium resistance. PLoS

ONE 5:e12601

9. Uguz AC, Naziroglu M, Espino J et al (2009) Selenium modu-

lates oxidative stress-induced cell apoptosis in human myeloid

HL-60 cells through regulation of calcium release and caspase-3

and -9 activities. J Membr Biol 232:15–23

10. Wang W, Gu J, Li YQ et al (2011) Are voltage-gated sodium

channels on the dorsal root ganglion involved in the development

of neuropathic pain? Mol Pain 7:16

11. Sghirlanzoni A, Pareyson D, Lauria G (2005) Sensory neuron

diseases. Lancet Neurol 4:349–361

12. Devor M (2009) Ectopic discharge in abeta afferents as a source

of neuropathic pain. Exp Brain Res 196:115–128

13. Grynkiewicz C, Poenie M, Tsien RY (1985) A new generation of

Ca2? indicators with greatly improved fluorescence properties.

J Biol Chem 260:3440–3450

14. Beales IL, Ogunwobi OO (2010) Microsomal prostaglandin E

synthase-1 inhibition blocks proliferation and enhances apoptosis

in oesophageal adenocarcinoma cells without affecting endothe-

lial prostacyclin production. Int J Cancer 126:2247–2255

15. Li GY, Fan B, Zheng YC (2010) Calcium overload is a critical

step in programmed necrosis of ARPE-19 cells induced by high-

concentration HO. Biomed Environ Sci 23:371–377

16. Placer ZA, Cushman L, Johnson BC (1966) Estimation of prod-

ucts of lipid peroxidation (malonyl dialdehyde) in biological

fluids. Anal Biochem 16:359–364

17. Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound,

and nonprotein sulfhydryl groups in tissue with ellman’s reagent.

Anal Biochem 25:192–205

18. Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in

selenium-deficient rat liver. Biochem Biophys Res Commun

71:952–958

19. Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein mea-

surement with the folin phenol reagent. J Biol Chem 193:

265–275

20. Nazıroglu M, Ozgul C, Cig B, Dogan S, Uguz AC (2011) Glu-

tathione modulates Ca(2?) influx and oxidative toxicity through

TRPM2 channel in rat dorsal root ganglion neurons. J Membr

Biol 242:109–118

21. Ozgul C, Nazıroglu M (2012) TRPM2 channel protective prop-

erties of N-acetylcysteine on cytosolic glutathione depletion

dependent oxidative stress and Ca(2?) influx in rat dorsal root

ganglion. Physiol Behav 106(2):122–128

22. Chakraborti T, Das S, Mondal M et al (1999) Oxidant, mito-

chondria and calcium: an overview. Cell Signal 11:77–85

23. Roy SS, Hajnoczky G (2009) Fluorometric methods for detection

of mitochondrial membrane permeabilization in apoptosis.

Methods Mol Biol 559:173–190

24. Liu X, Hajnoczky G (2009) Ca2?-dependent regulation of mito-

chondrial dynamics by the miro-milton complex. Int J Biochem

Cell Biol 41:1972–1976

Neurochem Res (2012) 37:1631–1638 1637

123

25. Koistinaho J, Alho H, Hervonen A (1990) Effect of vitamin E and

selenium supplement on the aging peripheral neurons of the male

sprague-dawley rat. Mech Ageing Dev 51:63–72

26. Yuan H, Lan T, Lin J (2005) Modulation of nano-selenium on

tetrodotoxin-sensitive voltage-gated sodium currents in rat dorsal

root ganglion neurons. Conf Proc IEEE Eng Med Biol Soc

5:4846–4849

27. Yuan H, Lin J, Lan T (2006) Effects of nano red elemental

selenium on sodium currents in rat dorsal root ganglion neurons.

Conf Proc IEEE Eng Med Biol Soc 1:896–899

28. Vincent AM, Edwards JL, McLean LL et al (2010) Mitochondrial

biogenesis and fission in axons in cell culture and animal models

of diabetic neuropathy. Acta Neuropathol 120:477–489

29. Fischer LR, Glass JD (2010) Oxidative stress induced by loss of

Cu, Zn-superoxide dismutase (SOD1) or superoxide-generating

herbicides causes axonal degeneration in mouse DRG cultures.

Acta Neuropathol 119:249–259

30. Nazıroglu M (2011) TRPM2 cation channels, oxidative stress and

neurological diseases: where are we now? Neurochem Res

36:355–366

31. Vincent AM, Hinder LM, Pop-Busui R et al (2009) Hyperlipid-

emia: a new therapeutic target for diabetic neuropathy. J Peripher

Nerv Syst 14:257–267

32. Fernyhough P, Calcutt NA (2010) Abnormal calcium homeo-

stasis in peripheral neuropathies. Cell Calcium 47:130–139

33. Uezono Y, Toyohira Y, Yanagihara N, Wada A, Taniyama K

(2006) Inhibition by selenium compounds of catecholamine

secretion due to inhibition of Ca2? influx in cultured bovine

adrenal chromaffin cells. J Pharmacol Sci 101:223–229

1638 Neurochem Res (2012) 37:1631–1638

123