/freeradbiomed

Free Radical Biology & M

Review Article

Cadmium induces reactive oxygen species generation and lipid peroxidation

in cortical neurons in culture

E. Lopez, C. Arce, M.J. Oset-Gasque, S. Canadas, M.P. Gonzalez *

Instituto de Bioquımica (Centro Mixto CSIC-UCM), Facultad de Farmacia, 28040-Madrid, Spain

Received 18 November 2004; revised 20 October 2005; accepted 28 October 2005

Available online 21 November 2005

Abstract

Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders

related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons

in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with

reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a)

depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid

peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action.

This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular

glutathione levels and induced catalase and SOD activation in these cells.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Cortical neurons; Cadmium toxicity; Membrane potential; ATP depletion; ATP release; ROS formation; Lipid peroxidation; Free radical

Contents

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chemical materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cell isolation and culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Measurement of the glial contamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Assessment of cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Determination of neuronal apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Lactate dehydrogenase (LDH) release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Determination of ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Mitochondrial membrane potential determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ROS measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Phosphatidylcholine and phosphatidylserine liposome preparation . . . . . . . . . . . . . . . . . . . . . . . . . . .

Measurement of TBARS in liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Measurement of TBARS in neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0891-5849/$ - s

doi:10.1016/j.fre

Abbreviation

EMEM, minimu

dichlorodihydro

mitochondria m

* Correspondi

E-mail addr

edicine 40 (2006) 940 – 951

www.elsevier.com/locate

ee front matter D 2005 Elsevier Inc. All rights reserved.

eradbiomed.2005.10.062

s: ROS, reactive oxygen species; TMRM, tetramethylrhodamine methyl ester; 2,7-DCF-DA, 2,7-dichlorodihydrofluorescein diacetate; FITC,

m essential Eagle’s medium; FCS, fetal calf serum; HS, horse serum; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; H2DCF, 2,7-

fluorescein; PC, phosphatidylcholine; PS, phosphatidylserine; TBA, 2-thiobarbituric acid; TBARS, 2-thiobarbituric acid-reactive substance; MMP,

embrane potential; SIN-1, 3-morpholinosydnonimine; SOD, superoxide dismutase.

ng author. Fax: +91 394 17 82.

ess: pilarg@farm.ucm.es (M.P. Gonzalez).

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E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951 941

Glutathione determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Measurement of catalase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Superoxide dismutase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cadmium treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Data presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Action of cadmium on cellular viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Quantification of ROS induced by cadmium in cortical neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of cadmium on mitochondrial membrane potential (MMP) . . . . . . . . . . . . . . . . . . . . . . . . . . .

Action of cadmium on ATP intracellular levels and ATP release. . . . . . . . . . . . . . . . . . . . . . . . . . . .

Measurement of ATP intracellular content and ATP release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Measurement of lipid oxidation induced by cadmium in liposome PC/PS and cortical neurons . . . . . . . . . . . .

Cadmium action on intracellular reduced glutathione levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of cadmium on catalase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of cadmium on superoxide dismutase (SOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950

Introduction

Oxidative stress may be defined as an alteration in the

steady-state balance between oxidant and antioxidant agents in

the cells. When these intracellular sources of ROS are

increased, several physiological processes may be disturbed,

including increased permeability of the blood–brain barrier,

tubulin alterations, and perturbation in synaptic transmission

[1]. In brain, oxidative stress is responsible for several

degenerative diseases such as amyotrophic lateral sclerosis

[2], Alzheimer disease, and Down syndrome [3].

Exogenous agents such as photochemical smog, ozone,

pesticides, xenobiotics, and ionizing radiation are all known

to generate radicals/oxidants [4]. Cadmium, an environmental

contaminant, is a toxic transition metal and, although

generally it is associated with Zn2+, both ions have different

behavior in living cells. Zn2+ is an essential element in almost

all biological systems; however, Cd2+ can induce several toxic

effects, depending on the concentration and the exposure

time.

There are several sources of human exposure to cadmium,

including employment in primary metal industries, production

of certain batteries, and consumption of tobacco products [5].

The molecular mechanism for the toxic effects of cadmium

involves interferences with antioxidant enzymes [6], alteration

in thiol proteins [7,8], inhibition of energy metabolism [9],

alteration in DNA structure [10], and affectation of some

enzyme activities [11–13].

Cadmium accumulates and proves to be very toxic in many

organs, such as kidney [14], liver [15], lung [16,17], testis, and

bone, and the blood system [18]. However, there are not many

studies related to the action of cadmium on the central nervous

system. Although, Vorobjeva [19] described several neurolog-

ical disorders in workers exposed to cadmium and Hart et al.

[20] observed that when these workers presented high levels of

this ion in urine, they displayed lower attention levels and less

memory than workers with low cadmium levels in urine.

Beside that, there are reports which consider the cadmium

intoxication as a possible etiological factor of neuro-degener-

ative diseases [21].

The neurological pathologic effect of cadmium in experi-

mental animals includes cerebral bleeding and cerebral edema

in the neonatal mouse [22,23], hyperactivity [24], and

increased aggressiveness in juvenile rats [25,26]. Ali et al.

[27] indicated that exposure to cadmium produces a dose–

response effect on activity in rats of 3–12 days born from rats

exposed to cadmium in the drinking water. Popieluch et al. [28]

observed a diminished ability for training and learning in rats

exposed to cadmium during their neonatal life. Antonio et al.

[29] found that cadmium administered to pregnant Wistard rats

induced a decrease in the levels of several enzymes in the

brain, including acetylcholinesterase, ATP-ase, catalase, alka-

line phosphatase, and acid phosphates. Bar-Sela et al. [30]

suggested a cause effect relationship between cadmium

exposure and amyotrophic lateral sclerosis in workers exposed

to cadmium in a nickel–cadmium battery factory and Mendez-

Armenta et al. [31] indicated lipid peroxidation in some brain

regions of developing rats exposed to cadmium. All these

findings suggest that cadmium is toxic, which may induce

cerebral damage. On the other hand, previous results from our

group showed that cadmium induces apoptosis and necrosis in

cortical neurons, depending on both the cadmium concentra-

tion and the exposure time. These authors also observed that

the apoptosis induced by cadmium in cortical neurons is

mediated by the caspase pathway [32].

As cadmium produces cellular death in cortical neurons in

culture the existence of oxidative stress and lipid peroxidation

mediated by cadmium has been checked in this paper.

Material and methods

Chemical materials

Tetramethylrhodamine methyl ester (TMRM) and 2,7-

dichlorodihydrofluorescein diacetate (2,7-DCF-DA) were pur-

chased from Sigma Chemical Company (Barcelona, Spain).

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951942

ATP reagents were from Bio Orbit Company. FITC-conjugated

anti-rabbit IgG and rabbit anti-GFAP were obtained from

Pharmigen (Beckton Dickinson). Minimum essential Eagle’s

medium (EMEM) was supplied by Bio-Whittaker Company

(MD). Fetal calf serum (FCS) and horse serum (HS) were from

Sera-Lab (Sussex, England). Other chemicals were reagent

grade products from Merck (Darmstadt, Germany).

Cell isolation and culture

Fetal rat brains of 19 days of gestation were used. Brain

neurons were obtained following the procedure described by

Segal [33] with minor modifications. Neonatal brain tissues

were removed from rats and placed on 35 mm3 glass petri dishes

containing 2 ml of ice-cold EMEM. Fetal cortex was easily

dissociated by triturating in EMEM, using a sterile, long-neck

Pasteur pipette. Isolated neurons were suspended in EMEM

containing 0.3 g/L of glutamine, 0.6% glucose, 5% FCS, 5%HS,

100 U/ml of penicillin, and 100 Ag/ml of streptomycin, at a cell

density of 1�106 cell/ml. The cells were placed on plastic petri

dishes coated with 10 Ag/ml of poly-d-lysine. Incubations were

made in a humidified incubator with 5% CO2/95% air at 37-C.After 72 h, the incubation medium was replaced by fresh

medium to which 10 AM of cytosine arabinoside was added to

prevent overgrowth of contaminating glial cells. The cells were

used after 7 days of culture. Cell viability was checked by the

trypan blue exclusion and it was routinely higher than 95%. Cell

purity was checked by first staining cells with cresyl violet to

identify neurons and secondly with the specific anti-GFAP

antibody to identify the percentage of cells that were glial cells.

Measurement of the glial contamination

Glial contamination was measured as in Lopez et al. [32].

Assessment of cell viability

Cell viability was determined by the crystal violet tests. Fol-

lowing treatment with the cadmium, cortical neurons were washed

twice with PBS and then exposed for 20 min to 0.2% crystal violet

in 2% ethanol. Thereafter, cells were washed with distilled water

until the excess dye was eliminated. Cells were dried and then

lysed with 1% SDS and absorbance was measured at 560 nm.

Determination of neuronal apoptosis

Cortical neurons treated with cadmium or untreated were

washed twice with PBS and harvested with trypsin-EDTA

(0.25% trypsin, 1 mM EDTA). The detached and adherent cells

were pooled and pelleted by centrifugation at 2000g for 5 min.

The cells were washed with PBS, suspended in 200 Al of ice-cold PBS, and fixed with 500 Al of 70% (v/v) ethanol at �20-Cfor 2 min. The cells were centrifuged and suspended in 400

Al PBS and 5 Al RNAse A (20 mg/ml) for 30 min at 37-C.Finally, 25 Al of propidium iodide (0.1% in PBS) was added

and the cells were scored for apoptosis on a Becton Dickinson

FACS scan flow cytometer (Becton Dickinson).

Lactate dehydrogenase (LDH) release

LDH activity is measured as the rate of decrease of the

absorbance at 340 nm, resulting from the oxidation of NADH to

NAD.

For the determination of LDH, the culture medium was

collected after 24 h of treatment and the neurons were washed

with PBS and then lysed with 0.1 M Tris-HCl (pH 7.4)

containing 0.1% Triton X-100. LDH activity was measured

both in culture medium and in cell lysate, following the

addition of 1 mM pyruvate and 0.2 mM h-NADH. LDH

release is given as percentage of LDH in the culture medium

with respect to the total LDH (the sum between LDH in the

culture medium and LDH inside the cells.

Determination of ATP

ATP analysis was performed using the luciferase reaction.

The cellular extract was cooled to 0-C, and aliquots were

assayed immediately with firefly luciferase/D-luciferin and 2

mM of EDTA in 0.1 M Tris-acetate buffer, pH 7.5. The

increase in chemiluminescence was recorded in a Bio Orbit

1251 luminometer. When the release of ATP was measured,

ATP content was checked in the culture medium and inside the

cells. The ATP release was calculated as percentage of ATP in

the culture medium with respect to the total ATP (the sum

between ATP in the culture medium and ATP inside the cells.

Mitochondrial membrane potential determination

Mitochondrial membrane potential was measured according

to Lalitha Tenneti et al. [34] with a few modifications.

Cadmium-treated neurons were washed with PBS and incu-

bated for 30 min with 500 nM TMRM dissolved in Locke

medium (140 mM NaCl, 4.4 mM KCl, 2.5 mM CaCl2, 1.2 mM

MgSO4, 1.2 mM KH2PO4, 4 mM NaHCO3, 5.5 mM glucose,

0.58 mM ascorbic acid, and 10 mM Hepes, adjusted to pH 7.5).

Then the cells were washed with PBS and the fluorescence was

measured with an FL600-BioTek spectrofluorimeter using

filters of 530/25 nm excitation and 590/35 nm emission.

ROS measurement

To assay the ROS formation 2,7-dichlorodihydrofluorescein

diacetate (H2DCF-DA) was used. H2DCF-DA enters into the

cells, where it is transformed in 2,7-dichlorodihydrofluorescein

(H2DCF) by the action of intracellular stearases. H2DCF is

oxidized to fluorescent DCF by hydrogen peroxide. Before

cortical neurons were treated with several cadmium concentra-

tions, the incubation medium was removed and the cells were

washed twice with Locke medium. Then cells were incubated

with H2DCF-DA (5 AM) in incubation medium for 50 min.

H2DCF-DA was removed and cells were treated with the

indicated cadmium concentrations. After that, the medium was

removed and the cells were washed with PBS. Fluorescence

was measured in an FL600-BioTek spectrofluorometer with

filters of 485/20 nm excitation and 530/25 nm emission Results

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951 943

were expressed as arbitrary fluorescent units (AFU)/mg

protein.

Phosphatidylcholine and phosphatidylserine liposome

preparation

Phosphatidylcholine (PC) and phosphatidylserine (PS) (60:40)

were suspended in chloroform and the mixture was dried for 15

min in a Buchi vaporizer. Phospholipids were suspended (2.5 mg

phospholipid/ml) in Tris-HCl buffer 20 mM (pH 7.4). The

suspension was shaken for 1 min and incubated for 45 min at

45-C.The liposomes were diluted at 0.5 mg/ml in Tris-HCl.

Measurement of TBARS in liposomes

2-Thiobarbituric acid (TBA) reacts with the products

formed during lipid peroxidation to generate a complex which

can be quantified spectrophotometrically [35].

Liposomes were incubated with the indicated toxins for 90

min. Then, 0.1 ml of butylated hydroxytoluene (4% in ethanol

p/v) was added. The liposomes were dispersed in 0.25 ml of

SDS (3% w/v) and 0.5 ml of 2-thiobarbituric acid (1%, dis-

solved in 0.05 M NaOH) plus 0.5 ml of HCl (25% v/v) were

added. This mixture was incubated at 100-C for 15 min. After

cooling, the formed TBARS were extracted with 3 ml of n-

butanol and centrifuged at 2000g for 10 min. The butanolic

phase fluorescence was measured at 515 nm excitation and 555

nm emission.

The TBARS concentration was expressed as nanomole of

malondialdehyde. The malondialdheyde standard curve was

prepared from 1,1,3,3,-tetraetoxypropane (10 mM) dissolved in

H2SO4 (1%, v/v) and kept in the dark for 2 h. After hydrolysis,

the standard was diluted at 1:50 with H2SO4 (1%) (v/v) and the

Fig. 1. Cadmium effect on cell viability in cortical neurons. Figure inset refers to t

experiments from cells of different cultures, each one performed in triplicate. *Stat

between cells cultured in the absence and presence of vitamin C. ns, not significan

malondialdehyde concentration was determined knowing that

the (245 nm = 13,700 M�1 cm�1.

Measurement of TBARS in neurons

The membrane lipid peroxidation in neurons was assayed

according to Fraga et al. [36]. After neurons were incubated

with cadmium, the cultured medium was removed and cells

were washed twice with PBS, and 0.1 ml of ethanol solution

of butylated hydroxytoluene (4%), 0.5 ml of SDS (3%), and

0.3 ml of phosphotungstic acid (10%) was added to the

neurons. The suspension was shaken and incubated at 100-Cfor 45 min, and 1 ml of 2-thiobarbituric acid (0.7%) and 2

ml of HCl (0.1 N) were added. Once the suspension was

cooled, 3 ml of n-butanol was added, shaken, and

centrifuged at 2000g for 10 min. Fluorescence was measured

in the butanol phase at 515 nm excitation and 555 nm

emission.

Glutathione determination

The concentration of glutathione was determined accord-

ing to Hissin and Hilf [37]. Once the cells were treated with

cadmium they were washed with PBS. The cells were rinsed

with sodium phosphate (0.1 M) plus EDTA (5 mM) (pH

8.0) and sonicated. H3PO4 (25%) was added and this sus-

pension was centrifuged for 30 m at 13.000 rpm. Super-

natants were taken and reduced glutathione was measured as

follows: To 100 Al of each sample was added 200 Al of 40mM N-ethylmaleimide and 100 Al of ortho-phthalaldialde-

hyde. Fluorescence was measured at 350 nm excitation and

420 nm emission, after 15 min of incubation at room

temperature.

he action of vitamin C on cell viability. Data are means T SE of two separate

istical signification between control and cell treatment; &statistical signification

t, *, & P < 0.05, **, && P < 0.01, and *** P < 0.001.

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951944

Measurement of catalase activity

After cadmium treatment, 50 mM of potassium phosphate

(pH 7.2) was added to the cells. They were rinsed from the

plates and sonicated. The cellular suspension was centri-

fuged in an Eppendorf tube at 13.000 rpm for 4 min and at

4-C, and catalase activity was measured in the supernatant

according to Aebi [38]. Results were expressed as increment

of optical density per minute per milligram protein.

Superoxide dismutase activity

After the treatment, cells were rinsed from the plates

using 50 mM Tris-KCl (pH 8.2) and sonicated. The cellular

suspension was centrifuged at 13,000 rpm for 4 min at 4-C.Superoxide dismutase was measured in supernatants accord-

ing to Marklund and Marklund [39].

Cadmium treatment

The cells were incubated during variable periods of time

with cadmium chloride at concentrations between 100 nM and

100 AM, in a serum-free medium or in a medium with 10%

serum (5% FCS + 5% HS).

Data presentation

Data are expressed as means T SE of three or four

independent experiments with different batches of cells, each

one performed in duplicate or triplicate. Statistical comparisons

were made using one-way analysis of variance (ANOVA)

Fig. 2. Cadmium effect on cellular death type. Effect of vitamin C on [A] apoptos

Results are means T SE of three separate experiments each one performed in triplica

signification between cells cultured in the absence and presence of vitamin C. NS

(Scheffe’s F test) followed in some instance by a two-way

ANOVA test. A P value of <0.05 was considered significant.

Results

Action of cadmium on cellular viability

Cadmium at concentrations from 10 to 100 AM induces

cellular death detected by the crystal violet test (Fig. 1A).

Lower cadmium concentrations did not produce any significant

effect. This death was produced in about 40% of the total cells

and was performed in the absence and presence of serum in the

treatment medium. The death induced by 50 AM cadmium was

reversed, in part, by vitamin C, an antioxidant agent (Fig. 1,

inset).

The neuronal death induced by cadmium was performed by

an apoptotic process according to the results obtained by flow

cytometry (Fig. 2A) and by a necrotic mechanism judging by

the LDH release (Fig. 2B). The neuronal apoptosis was

completely abolished by vitamin C while the necrotic one

was not.

Since vitamin C protected against cellular death the

possibility of oxidative stress in this process could be possible.

In order to check this, the action of cadmium on ROS

formation was checked.

Quantification of ROS induced by cadmium in cortical neurons

Cortical neurons were incubated with cadmium concen-

trations from 100 to 100 AM for 24 h, in the presence and

absence of serum. Cadmium induced a significant increase

is, measured by flow cytometry; and [B] necrosis, measured by LDH release.

te. *Statistical signification between control and cell treatment; &, NS, statistical

or ns, not significant; ***, &&& P < 0.001.

Fig. 3. Action of cadmium on ROS formation. Cortical neurons were cultured for 24 h with the indicated cadmium concentrations in the absence and in the presence

of serum. Figure inset shows the effect of vitamin E on ROS formation when neurons were cultured with 50 AM cadmium in the absence of serum. Results are

means T SE of three separate experiments, each one performed in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001.

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951 945

in ROS formation under both conditions (Fig. 3), although

in a serum-free medium, this effect was higher with respect

to the control. This could mean that serum protects against

ROS formation. ROS production was evidenced at cadmium

concentrations lower than those which produced cellular

Fig. 4. Cadmium effect on membrane mitochondrial potential. Cortical neurons wer

using TMRM (A) in a serum-free medium and (B) in the presence of serum. Data

performed in triplicate. Results are presented as ratios of AFU with respect to the

death, measured by the crystal violet test (see Fig. 1). ROS

formation mediated by 50 AM cadmium was blocked by

vitamin E, an antioxidant agent (Fig. 1, inset).

The breakdown in mitochondria membrane potential and

the dysfunction in ATP formation may produce ROS

e exposed to several cadmium concentrations for 24 h and MMP was measured

are means T SE of two experiments from cells of different cultures each one

control. *P < 0.05, ***P < 0.001.

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951946

generation and, as consequence, oxidative stress, cellular

dysfunction, and cellular death [40]. As cadmium’s ability to

induce oxidative stress has been evidenced in several cells

[41] and we found ROS formation induced by cadmium in

cortical neurons, the implication of this ion in the induction

of mitochondrial membrane breakdown and decrease in the

ATP content was checked.

Effect of cadmium on mitochondrial membrane potential

(MMP)

MMP breakdown was determined after cortical neurons

were exposed to several cadmium concentrations (100 nM to

100 AM), for 24 h and in the absence (Fig. 4A) and

presence (Fig. 4B) of serum. Cadmium concentrations of 1

AM decreased the TMRM fluorescence, which indicates

mitochondrial membrane depolarization. This effect was

dose dependent. The fall in MMP was higher when the

cadmium treatment was performed in the absence of serum.

This could signify that against, the serum presents protector

action. The mitochondrial membrane depolarizations started

at higher cadmium concentrations (1 AM) than those which

produce ROS formation (100 nM) (see Fig. 3).

Action of cadmium on ATP intracellular levels and ATP release

As a breakdown of the ATP content may lead to ROS

formation and lipid peroxidation the ATP intracellular levels

and the lipid peroxidation were measured in cortical neurons

exposed to cadmium.

Measurement of ATP intracellular content and ATP release

ATP intracellular content was measured on neurons

exposed to several cadmium concentrations (100 nM to 100

Fig. 5. Cadmium effect on [A] ATP intracellular content and [B] ATP release. Data a

performed in triplicate. Ns, not significant, *P < 0.05 and ***P < 0.001.

AM) for 24 h both in the absence and in presence of serum in

the treatment medium (Fig. 5A). Cadmium concentrations of

100 nM to 1 AM did not affect the intracellular ATP content

in these cells. However, Cd2+ doses from 10 AM in absence

of serum and 50 AM in presence of serum reduced the

intracellular ATP concentration, indicating mitochondria

dysfunction.

This breakdown in the intracellular ATP levels was

accompanied by ATP release (Fig. 5B) which presented an

inverse profile, that is, the ATP release coincides with the fall

in ATP content. This indicates that the decrease in intracellular

ATP may be due not only to a lesser synthesis of ATP by the

mitochondria but also to a destruction of both the mitochon-

drial and the cytosolic membranes. This could be responsible

for the death by necrosis found by us.

Death by necrosis is induced when the cellular membrane is

breaking and the cellular content is released outside the cells.

Cellular membranes are damaged by lipid peroxidation; thus,

the cadmium action was measured using this parameter.

Measurement of lipid oxidation induced by cadmium in

liposome PC/PS and cortical neurons

To evaluate whether the effect of cadmium on lipid

peroxidation was due to cadmium ‘‘per se’’ or to an indirect

mechanism mediated through the ROS formation, two experi-

ments were performed:

1. Cortical neurons in culture were treated with 100 AM Cd2+

for 24 h in a serum-free medium and TBARS formation was

checked.

2. Liposomes of phosphatidylcoline and phosphatidylserine

were incubated for 90 min at 37-C with: (a) 25 AM Fe2+, an

ion which induces lipid peroxidation; this experiment was

performed in the absence and presence of reduced glutathi-

re means of three separate experiments from cells of different cultures, each one

Fig. 6. Action of cadmium on lipid peroxidation. (A) Lipid peroxidation induced by 100 AM cadmium in cortical neurons (24 h in the absence of serum). (B) Lipid

peroxidation induced by the indicated agents in liposomes. Ns, not significant, ***P < 0.001.

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951 947

one; (b) 0.5 mM SIN-1 (3-morpholinosydnonimine), a

peroxynitrite donor inductor of lipid peroxidation; and (c)

100 AM cadmium.

When cortical neurons were treated with 100 AM cadmium,

in a serum-free medium and lipid peroxidation formation was

measured, a great increase in TBARS concentration was found

(Fig. 6A).

When TBARS concentrations were measured in liposomes,

100 AM cadmium did not induce increases of TBARS

Fig. 7. Effect of different cadmium concentrations on intracellular reduced

glutathione. Cortical neurons were treated for 24 h, in the absence and presence

of serum, with the indicated cadmium concentrations. Data are means T SE of

three experiments from cells of different cultures, each one performed in

triplicate. Ns, not significant, *P < 0.05, ***P < 0.001. &&P < 0.01 (statistical

signification with respect to both controls, with and without serum).

concentration with respect to control (Fig. 6B). However, 25

AM Fe2+, which is an initiator of lipid peroxidation and may be

considered as a positive control, increased the TBARS

concentration about 30 times over that of control. Reduced

glutathione did not modify the Fe2+ action. The amount of 0.5

mM 3-morpholinosydnonimine, which it is known as an in-

ductor of lipid peroxidation, significantly increased the TBARS

levels about 3 times compared to that of control; however, in this

case reduced glutathione reversed the SIN-1 effect.

Cadmium action on intracellular reduced glutathione levels

After we studied the action of cadmium on ROS and lipid

peroxidation we investigated whether this ion has some

influence on antioxidant systems. Cortical neurons were

treated for 24 h with Cd2+ concentrations between 100 nM

Fig. 8. Cadmium action on catalase activity. Cells were treated for 24 h in the

absence and presence of serum, with the indicated cadmium concentrations.

Data are means T SE of three different experiments from cells of different

cultures, each one performed in triplicate. Ns, not significant, ***P < 0.001.&&&P < 0.001 (statistical signification with respect to both controls, with and

without serum).

Fig. 9. Cadmium action on superoxide dismutase activity. Cells were treated for

24 h in the absence and presence of serum, with the indicated cadmium

concentrations. Data are means T SE of three different experiments from cells

of different cultures, each one performed in duplicate. Ns, not significant, *P <

0.05, **P < 0.01, ***P < 0.001. &&&P < 0.001 (statistical signification with

respect to both controls, with and without serum).

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951948

and 10 AM in the absence and presence of serum. The

cadmium treatment induced a decrease of the reduced

glutathione levels (Fig. 7). This effect started at 1 AMcadmium when the cadmium exposure was in a serum-free

medium and at 10 AM Cd2+ in a medium with serum. The

reduction of glutathione by cadmium, in the absence of serum,

was dose-dependent. When the effect induced by a similar

cadmium concentration in the absence and presence of serum

was compared, the glutathione depletion was higher in a

serum-free medium than in a medium with serum. In control

cells, the only serum deprivation significantly decreased the

glutathione levels.

Fig. 10. Representation of a summary related to the mechanism by which (a) cadm

resistant to cadmium toxicity.

Effect of cadmium on catalase activity

Cortical neurons were exposed to cadmium concentrations

between 100 nM and 100 AM and for 24 h, in the absence and

presence of serum. Lower cadmium concentrations did not

affect the catalase activity. Under these conditions (Fig. 8) it

was found that Cd2+ concentrations of 50–100 AM increased

the catalase activity when the cadmium treatment was

performed in the absence of serum. However, in a medium

with serum, the catalase activity was not affected. It was also

observed that catalase activity was higher in a serum-free

medium than in a medium with serum, indicating that only the

serum deprivation induces catalase activation.

Effect of cadmium on superoxide dismutase (SOD)

Cadmium doses between 50 and 100 AM increased SOD

activity (Fig. 9), but in this case the action was performed both

in the absence and in the presence of serum in the treatment

medium. In both cases there was a dose-dependent increase in

SOD that was caused by cadmium doses of 50–100 AM. The

serum deprivation induced increases in SOD activity (see

control values in Fig. 9).

Discussion

Our results show that in cortical neurons, cadmium exposure

induced cellular death, which was, in part, reversed by vitamin

C, an antioxidant agent. This indicated that oxidative stress

could be implicated in the mechanism by which cadmium

induces death in cortical neurons. The death induced by

cadmium, in these cells, is mediated by two mechanisms,

ium induces neuronal death in cortical neurons and (b) in neurons which are

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951 949

apoptotic and necrotic. The apoptosis produced by cadmium

was reversed by vitamin C while the necrosis was not affected

by this antioxidant molecule. It also appears that in the

apoptotic mechanism mediated by cadmium, but not in the

necrotic mechanisms, oxidative stress could be implicated. The

ability of cadmium to induce oxidative stress in cortical

neurons is aided by the induction of ROS by this cation.

Cortical neurons treated with cadmium ions at concentrations

between 1 and 100 AM, in either the absence or in the presence

of serum in the treatment medium, generated ROS. The

induction of ROS in these cells type could be mediated by

mitochondria alterations because cadmium produces a break-

down of the mitochondrial membrane potential. The decreases

in ATP levels and in the mitochondria membrane potential

began at 10 and 50 AM cadmium ion, respectively, while the

ROS formation was detected at lower doses (100 nM or 1

AM). These results likely indicate that ROS formation occurs

or it is detectable before the toxic events on mitochondrial

function that lead to the breakdown in mitochondrial

potentials. The ability of cadmium ion to induce ROS

formation has been described by several authors in different

cells types [42–47].

The decreases in the ATP intracellular levels, at the highest

concentrations of cadmium used, were very remarkable and

they were accompanied by ATP release, indicating mitochon-

drial and cytosolic membrane breaking, both indicative of a

necrotic mechanism of death which has been demonstrated

with our results. On the other hand, the fall in the ATP levels

could be due to a breakdown in the mitochondrial electron

transport as Wang et al. [48] suggest.

Caldmium also induces lipid peroxidation in cortical

neurons but this does not seem to be mediated by cadmium

per se, but as consequence of the ROS increment induced by

this ion, since in our liposome preparation, a membrane model

[49], the only cadmium treatment did not induce lipid

peroxidation. However, Fe2+, a lipid peroxidation inductor

[50], and SIN-1, a peroxynitrite donor which induces lipid

peroxidation, produce lipid peroxidation in liposomes. These

results agree with those found by Casalino et al. [51] who

found that vitamin E, an antioxidant agent, reversed the lipid

peroxidation induced by cadmium in rat kidney mitochondrial

fractions.

On the other hand Kowaltowski et al. [52] propose that lipid

oxidation is induced by ROS and in the scientific literature

exist data which associate the ROS formation induced by Cd2+

with the process of lipid peroxidation [41,53]. Data which

deserve remark are that all parameters studied (ROS formation,

ATP content, and ATP release, as well as mitochondrial

membrane potential dysfunction) were lower in the presence

than in the absence of serum, indicating a serum protection

against oxidative stress. This protection could be mediated by

sequestration of cadmium by thiolic groups of serum proteins

and in this case the free cadmium should be smaller and as

consequence the effect should be also lower. This hypothesis

may be helped by our prior work in which we demonstrated

that in a medium with serum, the cadmium transport inside the

neurons was lower than in presence of serum [32].

Beside cellular death mediated by cadmium ion, there are

some cortical neurons which are resistant to the action of this

cation (survival neurons). The cellular survival in the presence

of the toxic effect of ROS formation is based on the

equilibrium between the toxic action and the cells ability to

protect themselves against ROS action. Taking this into

account, we studied the possible defense mechanisms induced

in living cortical neurons. The central nervous system (CNS)

possesses antioxidant enzymes as SOD, which are expressed in

higher proportion than catalase [54,55].

The accumulation of hydrogen peroxide in brain is a big

problem because this tissue contains a great amount of Fe2+

and Cu2+ cations [56], which may catalyze the formation of

hydroxyl radicals and induce lipid peroxidation [57]. On the

other hand, reduced glutathione is an antioxidant agent, which

is found in brain at high concentrations [55,58]. Our results

indicate that in cortical neurons, exposure to cadmium induces

an increase in SOD and catalase activity at the highest Cd2+

concentrations (50–100 AM). This seems to suggest that there

are a group of neurons which are resistant to cadmium because

they are able to induce enzyme against oxidative stress which

produces this cation. The increases in these enzymatic activities

could be due to an increase in the enzyme expression, since it is

known that oxidative damages induce a cellular response,

which tries to compensate the overload of the ROS formation

[59]. Another possibility is that cadmium acts directly on the

enzyme activity.

Regarding the cadmium effect on antioxidant enzymes,

there are contradictory results [60–62]. This diversity of results

may be because the effect of this cation varies in function to:

(a) the cellular type, (b) the Cd2+ concentration used, and (c)

the exposure time. We have also observed that in rat survival

cortical neurons, Cd2+ decreases the intracellular reduced

glutathione levels. In the literature, controversies exist as to

whether cadmium induces increases or decreases in the

intracellular glutathione levels [63–65], but the general

conclusion that we consider is that cells try to protect

themselves against the cadmium toxic effect and one of the

most important systems in the brain is the reduced glutathione.

Several papers describe that, as a consequence of the cadmium

treatment, a toxic effect appears which correlates with

glutathione depletion [48,66,67]. Our results are in accordance

with the results found by Figuereido-Pereira et al. [66] in HT4

and mesencephalic cells. These authors found that, in these

cells, cadmium treatment promotes a dose-dependent decrease

of reduced glutathione.

All these results are summarized as indicated in Fig. 10.

Cadmium ion enters inside the cortical neurons, but this entry

is lower in the presence of serum because serum proteins

kidnap cadmium, and the effect should be lower. Once inside

the cells, cadmium induces cellular death in a group of

cortical neurons (about 40%), but has no effect in some cells.

The death mechanism is produced by mitochondrial cadmium

toxicity with fall in ATP, breakdown of mitochondrial

membrane potential, and ROS formation. This ROS produc-

tion induces apoptosis and lipid peroxidation with disruption

of cellular membranes and necrosis. The survival process is

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951950

mediated by the start of antioxidant mechanisms (activation of

SOD and catalase and depletion of reduced glutathione).

Acknowledgments

The CAYCIT 98-0121 and CAM 08.8/0012/1998 supported

this work. E. Lopez is a recipient of Fellowships from

Ministerio de Educacion y Ciencia and UCM, respectively.

We thank M. Garcıa Maurino for helping us in the culture

preparations.

References

[1] Evans, P. H. Free radicals in brain metabolism and pathology. Br. Med.

Bull. 49:577–587; 1995.

[2] Hirano, A. Cytopathology of amyotrophic lateral sclerosis. Adv. Neurol.

56:91–101; 1991.

[3] Sinet, P. M.; Ceballos-Picot, I. Role of free radicals in Alzheimer’s and

Down’s syndrome. In: Free Radicals in the brain: aging, neurological

and mental disorders. Berlin: Springer Verlag; 1992: 91–98.

[4] Halliwell, B. Reactive oxygen species in living systems: source,

biochemistry and role in human disease. Am. J. Med. 91:14–21; 1991.

[5] National Toxicology Program. Tenth report on carcionogenesis. Re-

search Triangle Park, NC: Department of Health and Human Services;

2000: III-42– III-44.

[6] Hussain, T.; Shukla, G. S.; Chandra, S. V. Effects of cadmium on su-

peroxide dismutase and lipid peroxidation in liver and kidney of growing

rats: in vivo and in vitro studies. Pharmacol. Toxicol. 60:355–359; 1987.

[7] Belyaaeva, E. A.; Korotkov, S. M. Mechanism of primary Cd2+-induced

rat liver mitochondria dysfunction: discrete modes of Cd2+ action on

calcium and thiol-dependent domains. Toxicol. Appl. Pharmacol.

192:56–68; 2003.

[8] Li, W.; Zhao, Y.; Chou, I. N. Alterations in cytoskeletal protein

sulfhydryls and cellular glutathione in cultured cells exposed to cadmium

and nickel ions. Toxicology 77:65–79; 1993.

[9] Muller, L. Consequences of cadmium toxicity in rat hepatocytes:

mitochondrial dysfunction and lipid peroxidation. Toxicology 40:

285–295; 1986.

[10] Coogan, T. P.; Bare, R. M.; Waalkes, M. P. Cadmium-induced DNA

strand damage in cultured liver cells: reduction in cadmium genotoxicity

following zinc pretreatment. Toxicol. Appl. Pharmacol. 113:227–233;

1992.

[11] Jay, D.; Zamorano, R.; Munoz, E.; Gleason, R.; Boldu, J. L. Study of the

interation of cadmium with membrane-bound succinate dehydrogenase.

J. Bioenerg. Biomembr. 23:381–389; 1991.

[12] Watjen, W.; Benters, J.; Haase, H.; Schwede, F.; Jastorff, B.; Beyers-

mann, D. Zn2+ and Cd2+ increase the cyclic GMP level in PC12 cells by

inhibition of the cyclic nucleotide phosphodiesterase. Toxicology 157:

167–175; 2001.

[13] Casalino, E.; Calzaretti, G.; Sblano, C.; Landriscina, C. Molecular

inhibitory mechanism of antioxidant enzimes in rat liver and kidney by

cadmium. Toxicology 179:37–50; 2002.

[14] Goyer, R. A. Mechanisms of lead and cadmium nephrotoxicity. Toxicol.

Lett. 46:153–162; 1989.

[15] Goering, P. L.; Fisher, B. R.; Kish, C. L. Stress protein synthesis induced

in rat liver by cadmium precedes hepatotoxicity. Toxicol. Appl.

Pharmacol. 122:139–148; 1993.

[16] Manca, D.; Richard, A. C.; Tra, H. V.; Chevalier, G. Relation between

lipid peroxidation and inflammation in the pulmonary toxicity of

cadmium. Arch. Toxicol. 68:364–369; 1994.

[17] Shukla, G. S.; Chiu, J. F.; Hart, B. A. Cadmium-induced elevations in

the gene expression of the regulatory subunit of gamma-glutamylcys-

teine synthetase in rat lung and alveolar epithelial cells. Toxicology

151:45–54; 2000.

[18] Sarkar, S.; Yadav, P.; Bhatnagar, D. Lipid peroxidative damage on

cadmium exposure and alteration in antioxidant system in rat erythro-

cytes: a study with relation to time. J. Trace Elem. Med. Biol. 11:8–13;

1997.

[19] Vorobjeva, R. S. Investigation of the nervous system function in workers

exposed to cadmium oxide. Zh. Nevrol. Psikhiatri. S. S. Im. Korsakova

57:385–389; 1957.

[20] Hart, R. P.; Rose, C. S.; Hamer, R. M. Neuropsychological effects of

occupational exposure to cadmium. J. Clin. Exp. Neuropsychol.

11:933–943; 1989.

[21] Viaene, M. K.; Roels, H. A.; Leenders, J.; De Groof, M.; Swerts, L. S.;

Lison, D.; Hasschelein, R. Cadmium: a possible etiological factors in

peripheral polyneuropathy. Neurotoxicology 20:7–16; 1999.

[22] Gabbiani, G.; Baic, C.; Dezeil, C. Toxicity of cadmium for the central

nervous system. Exp. Neurol. 18:154–160; 1967.

[23] Webster, W. S.; Valois, A. A. The toxicity effects of cadmium on the

neonatal mouse central nervous system. J. Neuropathol. Exp. Neurol.

40:247–257; 1981.

[24] Smith, M. J.; Pihl, R. O.; Garber, B. Postnatal cadmium exposure and

long-term behaviour changes in the rat. Neurobehav. Toxicol. Teratol.

4:283–287; 1982.

[25] Holloway, W. R.; Thor, D. H. Cadmium exposure in infancy affects

activity and social behaviour of juvenile rats. Neurotoxicol. Teratol.

10:135–142; 1988.

[26] Baranski, B. Behavioural alterations in offspring of female rats

repeatedly exposed to cadmium oxide by inhalation. Toxicol. Lett.

22:53–61; 1984.

[27] Ali, M. M.; Murthy, R. C.; Chandra, S. V. Developmental and long-term

neurobehavioral toxicity of low levels in uterus cadmium exposure in

rats. Neurobehav. Toxicol. Teratol. 8:463–468; 1986.

[28] Popieluch, I.; Felinska, W.; Szkilnik, R.; Brus, R.; Shani, J.

Protective effect of ethanol, administered to pregnant rats, on

learning and memorizing, after cadmium intoxication. Pharmacol.

Commun. 5:91–100; 1995.

[29] Antonio, M. T.; Corredor, L.; Leret, M. L. Study of the activity of several

brain enzimes like markers of the neurotoxicity induced by perinatal

exposure to lead/or cadmium. Toxicol. Lett. 143:331–340; 2003.

[30] Bar-Sela, S.; Reingold, S.; Richter, E. D. Amyotrophic lateral sclerosis in

a battery-factory worker exposed to cadmium. Int. J. Occup. Environ.

Health 7:109–112; 2001.

[31] Mendez-Armenta, M.; Villeda-Hernandez, J.; Barroso-Moguel, R.;

Nava-Ruiz, C.; Jimenz-Capdeville, M. E.; Rıos, C. Brain regional

lipid peroxidation and metallothionein levels of developing rats

exposed to cadmium and dexamethasone. Toxicol. Lett. 144:151–157;

2003.

[32] Lopez, E.; Figueroa, S.; Oset-Gasque, M. J.; Gonzalez, M. P. Apoptosis

and necrosis: two distinct events induced by cadmium in cortical neurons

in culture. Br. J. Pharmacol. 138:901–911; 2003.

[33] Segal, M. Rat hippocampal neurons in culture: responses to electrical

and chemical stimuli. J. Neurophysiol. 50:1249–1264; 1983.

[34] Tenneti, L.; Mc D’Emilia, D.; Troy, C. M.; Liption, S. A. Liption Role of

caspase in N-methyl-d-aspartate-Induced apoptosis in cerebrocortical

neurons. J. Neurochem. 71:946–959; 1998.

[35] Quinlan, G. S.; Halliwell, B.; Moorhouse, C. P.; Gutteridge, J. M. C.

Action of lead (II) and aluminium (III) ions on iron-stimulated lipid

peroxidation in liposomes, erythrocytes and rat liver microsomal

fractions. Biochem. Biophys Acta 962:196–200; 1988.

[36] Fraga, C. G.; Oteiza, P. I.; Golup, M. S.; Gershwin, M. E.; Keen, C. L.

Effects of aluminium on brain lipid peroxidation. Toxicol. Lett.

51:213–219; 1990.

[37] Hissin, P. J.; Hilf, R. A. Fluorometric method for determination of

oxidized and reduced glutathione in tissues. Anal. Biochem. 74:214–226;

1976.

[38] Aebi, H. Catalase. Hydrogen peroxide: hydrogen-peroxide oxide

reductase (EC 1.1.1.1.6). In: Methods Enzymatic Analysis, third ed.,

Volume III. 1987: 273–282.

[39] Marklund, S.; Marklund, G. Involvement of superoxide anion radical in

the auto oxidation of pyrogallol and a convenient assay for superoxide

dismutase Eur. J. Biochem. 47:469–474; 1974.

E. Lopez et al. / Free Radical Biology & Medicine 40 (2006) 940–951 951

[40] Zamzami, N.; Marchetti, P.; Castedo, M.; Decaudin, D.; Macho, A.;

Hirch, T.; Susin, S. A.; Petit, P. X.; Mignotte, B.; Kroemer, G.

Sequential reduction of mitochondria transmembrane potential and

generation of reactive species in early programmed cell death. J. Exp.

Med. 182:367–377; 1995.

[41] Yang, C. F.; Shen, H. M.; Shen, Y.; Zhuang, Z. X.; Ong, C. N. Cadmium-

induced oxidative cellular damage in human foetal lung fibroblasts

(MRC-5 cells). Environ. Health Perspect. 105:712–716; 1997.

[42] Koizumi, T.; Shirakura, H.; Kumagai, H.; Tatsumoto, H.; Suzuki, K. T.

Mechanism of cadmium-induced cytotoxicity in rat hepatocytes:

cadmium-induced active oxygen-related permeability changes of the

plasma membrane. Toxicology 114:125–134; 1996.

[43] Hassoun, E. A.; Stohs, S. J. Cadmium-induced production of superoxide

anion and nitric oxide, DNA single strand breaks and lactate dehydro-

genase leakage in J774A cell cultures. Toxicology 122:219–226; 1996.

[44] Koizumi, T.; Li, Z. G. Role of oxidative stress in single-dose, cadmium

induced testicular cancer. J. Toxicol. Environ. Heath 32:25–36; 1992.

[45] Shen, Y.; Sangiah, S. Na+, K+-ATP-ase, glutathione and hydroxyl free

radicals in cadmium chloride-induced testicular toxicity in mice. Arch.

Environ. Contam. Toxicol. 29:174–179; 1995.

[46] Thevenod, F.; Friedmann, J. M.; Katsen, A. D.; Hauser, I. A. Up-

regulation of multidrug resistance P-glycoprotein via nuclear factor-

kappaB activation protects kidney proximal tubule cells from cadmium-

and reactive oxygen species-induced apoptosis. J. Biol. Chem.

275:1887–1896; 2000.

[47] Almazan, G.; Liu, H. N.; Khorchid, A.; Sundarajan, S.; Martinez-

Bermudez, A. K.; Chemtob, S. Exposure of developing oligodendrocytes

to cadmium causes HSP72 induction, free radical generation, reduction

in glutathione levels and cell death. Free Radic. Biol. Med. 29:858–869;

2000.

[48] Wang, J.; Fang, J.; Leonard, S. S.; Rao, K. M. K. Cadmium inhibits the

electron transfer chain and induces reactive oxygen species. Free Rad.

Biol. Med. 36:1434–1443; 2004.

[49] Bangham, A. D.; Standish, M. M.; Watkinns, J. C. Diffusion of

univalent ions across the lamellae of swollen phospholipids. J. Mol.

Biol. 13:238–252; 1965.

[50] Kusimoto, M.; Inoue, K.; Nojima, S. Effect of ferrous ion and ascorbate-

induced lipid peroxidation on liposomal membranes. Biochem. Biophys.

Acta 646:169–178; 1981.

[51] Casalino, E.; Sblano, C.; Landriscina, C. Enzyme activity alteration by

cadmium administration to rats: the possibility of iron involvement in

lipid peroxidation. Arch. Biochem. Biophys. 346:171–179; 1997.

[52] Kowaltowski, A. J.; Castilho, R. F.; Vercesi, A. E. Mitochondrial

permeability transition and oxidative stress. FEBS Lett. 495:12–15;

2001.

[53] Pourahmad, J.; O’Brien, P. J. A comparison of hepatocyte cytotoxic

mechanisms for Cu2+ and Cd2+. Toxicology 143:263–273; 2000.

[54] Gaal, T.; Mezes, M.; Noble, R. C.; Dixon, J.; Speake, B. K.

Development of antioxidant capacity in tissues of the chick embryo.

Comp. Biochem. Physiol. 112B:711–716; 1995.

[55] Hussain, S.; Slikker, W., Jr.; Ali, S. F. Age-related changes in antioxidant

enzymes, superoxide dismutase, catalase, glutathione peroxidase and

glutathione in different regions of mouse brain. Int. J. Dev. Neurosci.

13:811–817; 1995.

[56] Moos, T. Developmental profile of non-heme iron distribution in the

rat brain during ontogenesis. Brain Res. Dev. Brain Res. 87:203–213;

1995.

[57] Oubidar, M.; Boquillon, M.; Marie, C.; Bouvier, C.; Beley, A.; Bralet, J.

Effect of intracellular iron loading on lipid peroxidation of brain slices.

Free Radic. Biol. Med. 21:763–769; 1996.

[58] Nanda, D.; Tolputt, J.; Collard, K. J. Changes in brain glutathione levels

during postnatal development in the rat. Brain Res. Dev. Brain Res.

94:238–241; 1996.

[59] Dalton, T. P.; Shetrtze, R. H. G.; Puga, A. Regulation of gene

expression by reactive oxygen. Annu. Rev. Pharmacol. Toxicol.

39:67–101; 1999.

[60] Kostic, M. M.; Ognjanovic, B.; Dimitrijevic, S.; Zikic, R. V.; Stajn, A.;

Rosic, G. L.; Zivlovic, R. V. Cadmium-induced changes of antioxidant

and metabolic status in red blood cells of rats: in vivo effects. Eur. J.

Haematol. 51:86–92; 1993.

[61] Salovsky, P.; Shopova, V.; Dancheva, V.; Marev, R. Changes in

antioxidant lung protection after single intra-tracheal cadmium acetate

instillation in rats. Hum. Exp. Toxicol. 11:217–222; 1992.

[62] Zikic, R. V.; Stajn, A. S.; Pavlovic, S. Z.; Ognjanovic, B. I.; Saicic, Z. S.

Activities of superoxide dismutase and catalase in erythrocytes and

plasma transaminases of goldfish (Carassius auratus gibelio Bloch)

exposed to cadmium. Physiol. Res. 50:105–111; 2001.

[63] Quig, D. Cysteine metabolism and metal toxicity. Altern. Med. Rev.

3:262–270; 1998.

[64] Gaubin, Y.; Vaissade, F.; Croute, F.; Beau, B.; Soleilhavoup, J.; Murat, J.

Implication of free radicals and glutathione in the mechanism of

cadmium-induced expression of stress proteins in the A549 human lung

cell-line. Biochem. Biophys. Acta 1495:4–13; 2000.

[65] Kamiyama, T.; Miyakawa, H.; Li, J. P.; Akiba, T.; Liu, J. H.; Liu, J.;

Marumo, F.; Sato, C. Effects of one year cadmium exposure on livers

and kidneys and their relation to glutathione levels. Res. Commun. Mol.

Pathol. Pharmacol. 88:177–186; 1995.

[66] Figuereido-Pereira, M. E.; Yakushin, S.; Cohen, G. Disruption of the

intracellular sulfhydyl homeostasis by cadmium-induced oxidative stress

leads to protein thiolation and ubiquitination on neuronal cells. J. Biol.

Chem. 273:12703–12709; 1998.

[67] Figuereido-Pereira, M. E.; Yakushin, S.; Cohen, G. The ubiquitinin/pro-

teosome pathway: friend or foe in zinc- cadmium, and H2O2-induced

neuronal oxidative stress. Mol. Biol. Rep. 26:65–69; 1999.