Synergistic Neurotoxic Effects of Arsenic and Dopamine in ... · (10 lg/ml in distilled water) and...

TOXICOLOGICAL SCIENCES 102(2), 254–261 (2008)

doi:10.1093/toxsci/kfm302

Advance Access publication December 13, 2007

Synergistic Neurotoxic Effects of Arsenic and Dopamine in HumanDopaminergic Neuroblastoma SH-SY5Y Cells

Shaik Shavali1 and Donald A. Sens

Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202

Received October 9, 2007; accepted December 8, 2007

Parkinson’s disease is an environmentally influenced, neurode-

generative disease of unknown origin that is characterized by the

progressive loss of dopaminergic neurons in the substantia nigra

pars compacta of the brain. Arsenic is an environmental

contaminant found naturally in ground water, industrial waste,

and fertilizers. The initial goal of the present study was to

determine if a mixture of arsenite (As13) and dopamine (DA)

could cause enhanced degeneration of dopaminergic neuronal

cells. Additional goals were to determine the mechanism

(apoptosis or necrosis) of As- and DA-induced cell death and if

death could be attenuated by antioxidants. The cell culture model

employed was the SH-SY5Y neuroblastoma cell line that has been

shown to possess differentiated characteristics of dopaminergic

neurons. The results demonstrated that a mixture of As13 and DA

was synergistic in producing the death of the SH-SY5Y cells when

compared with exposure to either agent alone. A mixture of 10mM

As13 and 100mM DA produced almost a complete loss of cell

viability over a 24-h period of exposure, whereas, each agent alone

had minimal toxicity. It was shown that necrosis, and not

apoptosis, was the mechanism of cell death produced by exposure

of the SH-SY5Y cells to the mixture of As13 and DA. It was also

demonstrated that the antioxidants, N-acetylcysteine, and Sulfor-

aphane, attenuated the toxicity of the mixture of As13 and DA to

the SH-SY5Y cells. This study provides initial evidence that As13

and DA synergistically can cause enhanced toxicity in cultured

neuronal cells possessing dopaminergic differentiation.

Key Words: arsenic; cell death; dopamine; dopamine-quinone;

oxidative stress; Parkinson’s disease.

Parkinson’s disease (PD) is a progressive neurodegenerative

disorder that currently affects approximately 1.5 million people

in North America (Fahn and Przedborski, 2000). The in-

dividual with PD exhibits resting tremor, muscular rigidity, and

postural instability as major symptoms. The disease is associated

with the loss of dopaminergic neurons in the nigro-striatal region

of the brain and it is estimated that 60–80% of such neurons are

lost prior to the onset of visible disease symptoms (Przedborski,

2005a). These dopaminergic neurons are remarkable for the

presence of dopamine (DA), DA transporters (DAT), vesicular

monoamine transporters, and DA receptors. The cause for the

loss of dopaminergic neurons in the nigro-striatal region is

unknown, but post-mortem examination of brains from PD

patients shows that dopaminergic neurons in that region

experience increased oxidative stress (Alam et al., 1997;

Dexter et al., 1989; Floor and Wetzel, 1998; Przedborski and

Ischiropoulos, 2005b). Similar studies have shown other

biochemical abnormalities including loss of mitochondrial

complex 1 enzyme activity, a decrease in glutathione levels,

increase in iron content, and activation of microglia (Bharath

et al., 2002; Kim and Joh, 2006; Mann et al., 1994). Lewy-

bodies, alpha-synuclein positive protein aggregates, are also

commonly seen in degenerating nigral neurons (Forno, 1996).

The factors responsible for the generation of oxidative stress in

DA neurons and the mechanisms of DA neuron cell death have

not been elucidated in PD. Environmental, and not genetic,

factors are implicated as causative in late onset PD, which

begins typically after the age of 50 years. This was strongly

suggested by a genetic study in twins which observed that

monozygotic–dizygotic concordance rates were indistinguish-

able, implying a lack of genetic influence and a strong

probability of environmental influence (Tanner et al., 1999).

As reviewed by Brown et al. (2005), epidemiological studies

have shown an association of PD with farm occupation and

residence, exposure to pesticides and insecticides, residence in

rural locations, the use of well water for drinking, and exposure

to metals. However, in all cases there have been other studies

that have shown no association of these factors with PD.

A possible role of Arsenic (As), one of the environmental

toxicants has not been explored in the etiology of PD. The

Agency for Toxic Substances and Disease Registry (ATSDR)

lists As as one of the top seven most toxic substances present in

the environment (ATSDR, 2000). It is estimated that several

million people world wide are suffering from As toxicity

resulting from anthropogenic release into the environment

(Centeno et al., 2002). A possible role for As in PD is suggested

because ground water that is contaminated with As, agricultural

products and fertilizers are major sources of As in the

environment; factors placing rural populations at a higher risk

1 To whom correspondence should be addressed at School of Medicine and

Health Sciences, University of North Dakota, 501 North Columbia Road,

Grand Forks, ND 58202. Fax: (701) 777-3108. E-mail: sshavali@medicine.

nodak.edu.

� The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For Permissions, please email: [email protected]

for exposure to As. There is also evidence to suggest that As can

affect the peripheral, as well as, the central nervous system (CNS)

and it has been suggested that As could play a significant role in

causing neurological diseases (Rodrıguez et al., 2003). In addi-

tion, animal studies have shown that As can cross the blood brain

barrier, accumulate in different regions of the brain including the

striatum (Itoh et al., 1990), alter neurotransmitter synthesis and

release, and decrease locomotor activity (Itoh et al., 1990;

Rodrıguez et al., 2003). The development of the CNS in neonatal

rats is also affected by As and As has been shown to cause

neuronal death in adult rat brain (Chattopadhyay et al., 2002).

The first goal of the present study was to determine if

a mixture of Asþ3 and DA would cause enhanced degeneration

of dopaminergic neuronal cells when compared with either agent

alone. The cell culture model employed was SH-SY5Y

neuroblastoma cell line that has been shown to possess some

specific characteristics of dopaminergic neurons in the brain (Lee

et al., 2000). We also used other cell lines, urothelial (UROtsa),

human embryonic kidney (HEK) cells, and HEK cells that stably

overexpressed with human DAT as nondopaminergic cells. The

other goals of the study were to determine the mechanism

(apoptosis or necrosis) of As- and DA-induced cell death and if

death could be attenuated by treatment with antioxidants.

MATERIALS AND METHODS

Cell culture. The SH-SY5Y human neuroblastoma cell line was obtained

from the American Type Culture Collection (Manassas, VA). Human embryonic

kidney (HEK-293) cells and HEK cells stably overexpressed with human DAT

(HEK-DAT) were obtained from the laboratory of Dr Bertha K. Madras, Harvard

Medical School, Southborough, MA. SH-SY5Y, HEK, and HEK-DAT cells

were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented

with 10% fetal bovine serum, streptomycin, and penicillin. The UROtsa human

immortalized urothelial cell line was grown in DMEM containing 5% vol/vol

fetal calf serum as described previously by this laboratory (Rossi et al., 2001). All

cell lines were incubated at 37�C in a 5% CO2: 95% air atmosphere. The cells

were fed fresh growth medium every 3 days, and when confluent, SH-SY5Y,

HEK, and HEK-DAT cells were subcultured at a 1:5 ratio and the UROtsa cells

were subcultured at a 1:4 ratio using trypsin-ethylenediaminetetraacetic acid

(EDTA). All experiments were performed in triplicate.

Cell viability studies. Cell viability, as a measure of cytotoxicity, was

determined by measuring the capacity of the cells to reduce MTT (3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (Rossi

et al., 2002). Triplicate cultures treated with DA, As, DA þ As, DA transporter

blockers and DA receptor antagonists were analyzed after 24 h of incubation.

The MTT assay was used to determine the effects of Asþ3 (sodium arsenite),

DA, Asþ3 and DA, and various inhibitors on the viability of the SH-SH5Y,

HEK, and HEK-DAT and UROtsa cells. DA, DAT blockers, and DA-receptor

antagonists, MTT, Sulforaphane, and N-acetylcysteine (NAC) were all

purchased from Sigma (St Louis, MO). Briefly, cells were grown in six-well

plates and treated with graded series of Asþ3 (5–20lM) and DA (50–400lM)

concentrations for 24 h. In separate experiments, cells were also treated with

combinations of Asþ3 and DA. Control samples were treated with an equal

volume of phosphate-buffered saline (PBS). As Mazindol was dissolved in

DMSO (dimethyl sulfoxide), other control samples were treated with DMSO.

To test the neuroprotective effects of antioxidants against Asþ3 and DA

toxicity, the cells were coincubated with NAC or preincubated with

sulforaphane for 24 h before exposure to Asþ3 and DA. To test whether

inhibitors of DA transporters and DA receptors could block the toxicity of Asþ3

and DA, SH-SY5Y cells were pretreated for 1 h with DA transporter blockers

(Mazindol and GBR-12909) and DA receptor antagonists (SCH-23390 and

U-99194) followed by treatment with the mixture of Asþ3 and DA for 24 h.

Mechanism of As13-and DA-induced cell death. Experiments were

performed to determine if cell death of SH-SH5Y exposed to Asþ3 and DA

occurred by a necrotic or apoptotic mechanism. The effect of Asþ3 and DA on

the number of fragmented (apoptotic) nuclei of SH-SH5Y cells was visualized

microscopically using 4’,6-diamidino-2-phenylindole (DAPI)–stained nuclei as

described previously by this laboratory (Tarnowski et al., 1993). After 12 h of

incubation, wells containing the monolayers were rinsed with PBS, fixed for 15

min in 70% ethanol, rehydrated with 1 ml PBS, and stained with 10 ll DAPI

(10 lg/ml in distilled water) and visualized under the microscope (Axiovert 35,

Zeiss, Germany). The images were recorded with a digital camera (SPOT

Diagnostic Instruments, Inc., MI) attached to the microscope operated with

Paxit image analysis software (Paxcam, Villapark, IL).

In similar experiments, the presence or absence of DNA laddering was used to

confirm the presence or absence of apoptosis for SH-SH5Y cells (Somji et al.,

2006). Briefly, at four different time points (0, 6, 12, and 24 h), adherent and

detached cells were collected and combined from each well, centrifuged, and the

pellet resuspended in lysis buffer. The cell lysate was centrifuged and the

supernatant was incubated with 200 lg/ml proteinase K for 1 h at 50�C. The DNA

was extracted with phenol:chloroform:isoamyl alcohol (25:24:1 vol/vol/vol) and

precipitated overnight with absolute ethanol in the presence of 20 lg glycogen.

The DNA pellet was washed twice with 70% alcohol, air dried, and dissolved in

Tris–EDTA buffer. After treatment with ribonuclease A for 1 h at 50�C, the DNA

was loaded onto a 2% (wt/vol) agarose gel containing ethidium bromide.

It was also determined if Asþ3 and DA induced the activation of caspase-3 in

SH-SH5Y cells using assay procedures previously described by this laboratory

(Somji et al., 2006). Briefly, 10 lg of total cellular protein was separated on 12%

sodium dodecyl sulfate–containing polyacrylamide gel and electrophoretically

transferred to a hybond-P polyvinylidine difluoride membrane (Amersham

Biosciences). Membranes were blocked in Tris-buffered saline containing 0.1%

Tween-20 (TBS-T) and 5% (wt/vol) nonfat dry milk for 1 h at room temperature.

After blocking, the membranes were probed with primary antibody to Caspase-3

(Cell Signaling Technology, Danvers, MA) overnight at 4�C in antibody dilution

buffer (TBS-T containing 5% nonfat dry milk). Following three washes, the

membrane was incubated with the secondary antibody for 1 h at room

temperature. The blots were visualized using the Phototope-HRP Western blot

detection system (Cell Signaling Technology, Danvers, MA). Further, the

membrane was stripped and reprobed with antibodies to b-actin (Stressgen, Ann

Arbor, MI) to determine equal loading of the protein in each lane.

The release of lactate dehydrogenase (LDH) from cells was determined by

the Cyto Tox 96 assay kit (Promega) as described previously (Somji et al.,

2006). Briefly, 50 ll of the cell culture supernatant was transferred to a 96-well

enzymatic assay plate. Reconstituted substrate mix (50 ll) was added to each

sample and the enzymatic reaction was allowed to proceed for 30 min at room

temperature in the dark. The assay was stopped by adding 50 ll of the stop

solution (1M acetic acid) and the plate was read at 490 nm using an enzyme-

linked immunosorbent assay plate reader.

Statistics. Data obtained from the experiments were analyzed using Graph

Pad Prism software. Experiments were performed in triplicate and results

presented as the mean ± SEM. One-way and two-way analysis of variance

followed by post hoc test (Tukey) were used to assign significance. The

significance was considered when p value was less than 0.05.

RESULTS

Toxicity of DA, Arsenite, and Mixtures of DA and Arsenite onSH-SY5Y Cells

Initial experiments were performed to determine an exposure

level of both DA and Asþ3 that were the minimal level

ARSENIC & DOPAMINE TOXICITY IN SH-SY5Y CELLS 255

necessary to elicit a repeatable, but small, significant loss in

SH-SY5Y cell viability. The SH-SY5Y cells were exposed to

a graded series of DA concentrations and cell viability

determined using the MTT assay following 24 h of exposure.

It was shown that a concentration of DA of 100lM was the

minimal level that would elicit a significant loss of cell viability

(Fig. 1A). A similar determination was performed on

SH-SY5Y cells exposed to Asþ3 and it was shown that

a concentration of Asþ3 of 10lM was the minimal level that

would elicit a significant loss of cell viability (Fig. 1B). These

two exposure levels of Asþ3 and DA were then used to

determine the effect of combining the two chemicals on the

viability of the SH-SY5Y cells (Fig. 1C). It was shown that the

combination of DA and Asþ3 resulted in a significant increase

in toxicity to the SH-SY5Y cells when compared with

exposure to either agent alone. The viability of the SH-SY5Y

cells was decreased to 9.4 ± 1.13% compared with control cells

when exposed to the combination of Asþ3 and DA. In contrast,

each agent alone resulted in a decrease in cell viability to 79.1 ±0.5% for Asþ3 and 69.5 ± 7.5% for DA when compared with

control cells. The increased loss of cell viability due to the

combination of Asþ3 and DA was significant when compared

with either agent alone (p < 0.001). These results show that

exposure to a combination of Asþ3 and DA has increased toxicity

to dopaminergic SH-SY5Y cells when compared with either

agent alone.

Two experiments were designed to determine if the effects

of combined exposure to Asþ3 and DA was specific for cells

with dopaminergic differentiation. The first of these was

exposure of a human bladder epithelial cell line of urothelial

origin (UROtsa) to Asþ3, DA, and a combination of Asþ3 and

DA. It was demonstrated that the UROtsa cell line exhibited

a similar pattern of Asþ3 toxicity (Fig. 2). In contrast, the

UROtsa cells were shown to be very resistant to DA toxicity,

with levels of 600lM DA having no effect on cell viability

(data not shown). The lack of a DA effect on cell viability was

also observed even if the time course of exposure was extended

over 12 days with continued re-exposure to DA every 3 days.

With this limitation in mind, it was shown that the combination

of Asþ3 and DA had no effect on UROtsa cell viability over

that found for Asþ3 alone (Fig. 2). A second effort was made to

FIG. 1. (A) Effect of various concentrations of DA on SH-SY5Y cell

viability. Cells were treated with DA in the range of 50–400lM for 24 h and

cell viability was determined by measuring the capacity of the cells to reduce

MTT to formazan. Viability is expressed as percent of control. Values are

expressed as mean ± SEM. **p < 0.01, ***p < 0.001 are significantly different

from control groups. (B) Effect of various concentrations of As on SH-SY5Y

cell viability. Cells were treated with As in the range of 0–20lM for 24 h and

the cell viability was determined by MTT assay. Values are expressed as mean

± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 are significantly different from

control groups. (C) Effects of As, DA, and mixture of As and DA on SH-SY5Y

cell viability. Cells were treated with As (10lM), DA (100lM), mixture of As

and DA or saline treatment (control) separately for 24 h and the cell viability

was determined by MTT assay. Values are expressed as mean ± SEM. *p <

0.05, **p < 0.01, ***p < 0.001 are significantly different from control groups,

respectively. ###p < 0.001 is significantly different from As and DA groups,

respectively.

256 SHAVALI AND SENS

assess the effect of a DA and Asþ3 mixture on the viability of

a second nondopaminergic cell line. The wild type HEK cell

line and the HEK cell line stably transfected with the human

DAT were exposed to Asþ3, DA, and mixtures of the two

chemicals. Similar to that found with UROtsa cells, both the

wild type HEK cells and the HEK cells expressing the DA

transporter exhibited a similar pattern of Asþ3 toxicity, with

10lM Asþ3 being at the threshold of producing cell death

within 24 h of exposure (Fig. 2). Both the HEK cell lines were

also resistant to DA, with exposure to 600lM DA having no

effect on HEK cell viability (data not shown). Like the UROtsa

cells, a combination of Asþ3 (10lM) and DA (100lM) had no

effect on HEK cell viability over that found for Asþ3 alone

(Fig. 2). These results suggest that dopaminergic differentiation

is required for the cells to have enhanced susceptibility to

mixtures of Asþ3 and DA.

Toxicity of DA and Asþ3 Mixtures and DA TransporterBlockers and DA Receptor Antagonists

Studies have shown that the SH-SY5Y cells express both

DAT and DA receptors (Lee et al., 2000). The DATs and DA

receptors are molecules that are unique in dopaminergic

neurons of the brain. In the dopaminergic system, DA can be

transported into the presynaptic neurons by a reuptake system

utilizing the DAT. On the other hand, DA acts on DA receptors

to activate the signal transduction pathways and DA receptor

antagonists could block this action. We thought that either

DAT inhibitors or DA receptor antagonists could block the loss

of cell viability induced by the mixture of As and DA.

Therefore, the goal of this set of experiments was to determine

if either DAT inhibitors or DA receptor antagonists could

prevent the toxicity induced by mixtures of Asþ3 and DA on

SH-SY5Y cells. The DAT inhibitors tested were GBR-12909

and Mazindol, and the D1 and D2/D3 receptor antagonists that

were tested were SCH-23390 and U-99194. The results of this

analysis demonstrated that neither the DAT inhibitors nor the

DA receptor antagonists tested decreased the toxicity of

a mixture of Asþ3 and DA on SH-SY5Y cells (Fig. 3). There

was a trend that Mazindol and U-99194 may have had a small

potentiating effect on the toxicity of Asþ3 and DA. None of the

agents tested had any toxic effects on SH-SY5Y cells (Fig. 3).

Mechanism of Cell Death in SH-SY5Y Cells Exposed to Asþ3,DA, and a Mixture of Asþ3 and DA

The effects of Asþ3 and DA exposure on SH-SY5Y cells

were determined as a function of fragmented nuclei as

identified by DAPI staining, Caspase 3 activation, formation

of a DNA ladder, and the release of LDH into the growth

medium. The nuclei of SH-SY5Y cells was monitored using

DAPI staining at 12 h during the time course of exposure to the

mixture of Asþ3 and DA. The results demonstrated that there

was no increase in profiles of fragmented nuclei observed in

the Asþ3- and DA-treated cells over those noted to occur

spontaneously in control cells (Fig. 4). Likewise, no frag-

mented nuclei were observed in SH-SY5Y cells treated with

Asþ3 or DA alone (Fig. 4). That apoptosis was not the

mechanism of Asþ3 plus DA SH-SY5Y cell death as suggested

by absence of fragmented nuclei was further confirmed by

FIG. 3. Effects of DAT blockers, GBR-12909 (GBR) and Mazindol (Maz)

and DA D1 (SCH-23390) and D2/3 (U-99194) receptor antagonists on As and

DA-induced loss of SH-SY5Y cell viability. Cells were pretreated with DAT

blockers or DA-receptor antagonists for 1 h followed by treatment with the

mixture of As and DA for 24 h. After the incubation, the cell viability was

determined by MTT assay. Values are expressed as mean ± SEM. ***p < 0.001

is significantly different from control group. DAT blockers and DA-receptor

antagonists were not effective in blocking the toxic effects induced by As

and DA.

FIG. 2. Effects of As, DA, and mixture of As and DA on dopaminergic

(SH-SY5Y) and nondopaminergic urothelial (UROtsa), human embryonic

kidney (HEK) cells, and HEK cells that overexpressed with human DAT

(HEK-DAT). Cells were treated separately with As, DA, and a mixture of As

and DA or saline treatment (Control) for 24 h. The cell viability was determined

by MTT assay. Values are expressed as mean ± SEM. *p < 0.05 is significance

between control and As-treated SH-SY5Y, urothelial, HEK, and HEK-DAT

groups, respectively. **p < 0.01 is significant between control and DA group,

whereas ***p < 0.001 is significant between control and mixture of As and DA

treated SH-SY5Y group. No loss in cell viability was observed in urothelial,

HEK, and HEK-DAT cells in response to the mixture of As and DA.


determining both nuclear fragmentation and Caspase 3 activa-

tion. These determinations were performed on the SH-SY5Y

cells at the midpoint between the initiation of cell death

(rounding of the cells) and a total loss of cell viability (detach-

ment of cells from the surface). Using this window of viability

as a guide, it was shown that exposure of the SH-SY5Y cells

to the mixture of Asþ3 and DA failed to produce a DNA ladder

or the activation of Caspase 3 (Figs. 5 and 6).

In contrast to apoptotic mode of cell death, the SH-SY5Y

cells were shown to release LDH into the growth medium

following exposure to a mixture of Asþ3 and DA (Fig. 7). The

release of LDH by the SH-SY5Y cells was significantly

elevated within 1.5 h of exposure to the mixture of Asþ3 and

DA (p < 0.01) when compared with control cells or cells

treated with Asþ3 or DA alone (Fig. 7). The time course of

exposure demonstrated that LDH levels increased for the

SH-SY5Y cells treated with the mixture of Asþ3 and DA until

release was almost 100% of control, accounting for the total

loss of cell viability (Fig. 7).

Effect of Antioxidants on Asþ3 and DA Induced SH-SY5YCell Death

NAC and Sulforaphane were tested for their ability to inhibit

the death of SH-SY5Y cells treated with a mixture of Asþ3 and

DA. NAC acts as a free radical scavenger due to its thiol group

and also indirectly enhances the synthesis of glutathione,

a compound that reduces oxidative stress (Martınez et al.,1999). On the other hand, the nicotinamide adenine di-

nucleotide phosphate (reduced):quinone reductase is an

enzyme which induced by sulforaphane catalyzes the two-

electron reduction of quinone to the redox-stable hydroquinone

(Cavelier and Amzel, 2001; Joseph et al., 2000). It was

demonstrated that both agents reduced the loss of viability of

the SH-SY5Y cells that resulted from the treatment of the cells

with a mixture of Asþ3 and DA. The coincubation of the cells

exposed to a mixture of Asþ3 and DA with 100lM, 1mM, and

10mM concentrations of NAC resulted in significant decreases

FIG. 4. Nuclear morphology of SH-SY5Y cells treated with As 10lM (B), DA 100lM (C), mixture of As and DA (D) or saline treatment (A). Cells were

treated for 12 h and stained with nuclear dye DAPI. Nuclear morphology was visualized by fluorescent microscopy. No fragmented nuclei were observed in any of

the treated groups.

FIG. 5. Western blot analysis of cleaved caspase-3 in SH-SY5Y cells

treated with the mixtures of As and DA. Cells were treated with the mixture of

As (10lM) and DA (100lM) for different time periods (0, 3, 6, 12, and 24 h).

At each time point, cells were washed with PBS and lysed in CHOPS lysis

buffer. Proteins were separated by gel-electrophoresis, transferred onto

nitrocellulose membrane and probed with antibodies to caspase-3 that

recognizes pro-caspase-3 as well as cleaved caspase-3 fragments. Lane 1:

protein standard marker. Lane 2: 0 h (Control). Lane 3: 3 h. Lane 4: 6 h. Lane

5: 12 h; Lane 6: 24 h. No change was observed in pro-caspase-3 (35 kDa)

expression by the mixture of As and DA. Further, no cleaved caspase-3

fragments were observed (19 and 17 kDa) by As and DA.


of cell death in a concentration dependent manner (Fig. 8). It

was also demonstrated that cell death was significantly

inhibited when the SH-SY5Y cells were preincubated with

sulforaphane for 24 h prior to addition of the Asþ3 and DA

mixture (Fig. 8).

DISCUSSION

The initial goal of this study was to determine if a mixture of

Asþ3 and DA would cause enhanced degeneration of

dopaminergic neuronal cells when compared with either agent

alone. The results clearly demonstrated that a mixture of Asþ3

and DA was synergistic in its ability to elicit the death of

SH-SY5Y cells, a human neuroblastoma cell line that retains

dopaminergic differentiation. Specificity of enhanced toxicity

of the mixture to cells with dopaminergic differentiation was

suggested by the finding that the combination of Asþ3 and DA

demonstrated no increased toxicity to bladder epithelial cells

(UROTsa) or renal epithelial cells (HEK and HEK-DAT).

There are very few cell culture systems that model neural

dopaminergic differentiation and the SH-SY5Y cells have been

widely used as a model system (Lee et al., 2000). There are

limitations of this model, mainly the malignant origin from

a childhood cancer. With this limitation in mind, the results of

the present study could have major implications regarding the

pathogenesis of PD because the dopaminergic neurons that are

located in the substantia nigra pars compacta are progressively

lost in PD via oxidative stress dependent mechanisms.

Increasing evidence suggests that the selective vulnerability

of dopaminergic neurons in PD is due to oxidative metabolism

of DA and consequent oxidative stress (Ahlskog, 2005;

Weingarten and Zhou, 2001). The etiological factors that are

responsible for the pathogenesis of PD are currently unknown,

however, environmental factors such as heavy metals and

pesticides are high on the list of suspected toxicants (Brown

et al., 2005; Gorell et al., 1997, 1998). Furthermore, it has been

found that PD is associated with consumption of well water

and living in rural areas, both associated with an increased risk

of exposure to As (Brown et al., 2005; Hubble et al., 1993).

Thus, the finding that Asþ3 can potentiate the toxicity of DA in

dopaminergic cells of neuronal origin is in agreement with the

presence of As in many of the environments associated with the

development of PD. Although no direct association of As with

PD has been shown, As is neurotoxic in both humans and

FIG. 6. Agarose gel-electrophoresis of DNA extracted from SH-SY5Y

cells treated with mixtures of As (10lM) and DA (100lM) for different time

periods (0, 6, 12, and 24 h). The DNA was extracted with phenol:chlor-

oform:isoamyl alcohol (25:24:1 vol/vol/vol) and precipitated overnight with

absolute ethanol in the presence of 20 lg glycogen. The DNA was loaded and

separated in a 2% (wt/vol) agarose gel containing ethidium bromide. The DNA

was visualized under ultraviolet light and images were recorded in gel-

documentation system (NeucleoTech, CA). The DNA fragments of 180–200

base pairs which are hallmarks for apoptosis were absent from the DNA

extracted from As þ DA–treated SH-SY5Y cells.

FIG. 7. Effects of As, DA and a mixtures of As and DA on LDH release.

SH-SY5Y cells were treated with As (10lM), DA (100lM) or a mixture of As

and DA for various time periods (up to 24 h). At each time point, culture

medium was taken and released LDH levels were measured with an enzymatic

assay, which results in the conversion of a tetrazolium salt into a red formazan

product (Promega, Madison, WI). Values are expressed as mean ± SEM. *p <

0.05, **p < 0.01 and ***p < 0.001 are significantly different from control

group. #p < 0.05, ##p < 0.01 and ###p< 0.001 are significantly different from As

group. {{p < 0.01 and {{{p < 0.001 are significantly different from DA group.

Significantly elevated LDH levels were observed at all time points by the cell

group treated with the mixture of As and DA compared to control group.


animal models. Developmental exposure to As is associated

with neural tube defects and exencephaly (Wlodarczyk et al.,1996). A decrease in locomotor activity and behavioral

disorders has been shown to occur in rats exposed to As

(Rodrıguez et al., 2003). In the rat nervous system As exposure

has been shown to cause oxidative damage to both lipids and

proteins (Garcıa-Chavez et al., 2006; Samuel et al., 2005).

Exposure to low levels of As has also been shown to activate

nuclear factor-kappaB and AP1 in mesencephalic cells in

culture (Felix et al., 2005). These studies show that As can

elicit neurotoxicity and strengthen the potential for As to be

a possible cofactor in the development and progression of PD.

The second goal of this study was to determine the

mechanism (apoptosis or necrosis) of cell death that a mixture

of Asþ3 and DA elicited in the SH-SY5Y cells. It was shown

that the mixture of Asþ3 and DA elicited a necrotic mechanism

of cell death in the SH-SY5Y cells. This was based on the time

course of LDH release from the cells that reached 100% of

control values and a failure to demonstrate fragmented nuclei,

the formation of a DNA ladder and activation of Caspase 3.

The finding of a necrotic mechanism of cell death is in

agreement with studies that show neuroinflammation as

a commonly observed phenomenon in several neurological

diseases including PD. The glial cell response to brain injury

involves a neuroinflammatory process in which cytokines,

major effectors of the inflammatory cascade, play a key role in

cell damage (Allan and Rothwell, 2001). Furthermore,

activated microglia and increased levels of proinflammatory

cytokines have consistently been identified in PD brains

(McGeer et al., 1988; Mogi et al., 1994). A necrotic

mechanism of cell death as shown in the present study would

be associated with the generation of an inflammatory process.

Lastly, the present study shows that the cell death induced by

the mixture of Asþ3 and DA on SH-SY5Y cells could be

attenuated by antioxidants, suggesting increased oxidative

stress is responsible for the loss of cell viability. Evidence

for this was that the antioxidants NAC and sulforaphane both

prevented the loss of cell viability caused by mixtures of Asþ3

and DA. The mechanism(s) underlying the ability of Asþ3 and

DA to increase oxidative stress and dopaminergic neuron cell

death is currently unknown. We presume that Asþ together

with DA may produce highly toxic free radicals such as DA-

quinone (Sulzer and Zecca, 2000) or 6-hydroxydopamine in

SH-SY5Y cells. In support of this hypothesis, it has been

observed that 5-cysteinyl-catechols (5-cysteinyl-DA) are sig-

nificantly elevated in the substantia nigra of PD patients

compared to controls (Spencer et al., 1998). Further, it is

interesting to note that As can accumulate into the striatum of

mice along with other brain regions when administered through

drinking water (Itoh et al., 1990). As the striatum is the region

where the DA concentration is specifically higher, we presume

that this region may particularly be susceptible to the mixtures

of As and DA toxicity.

The results from our study also indicate that the toxicity

induced by a mixture of Asþ and DA is partly mediated via

intracellular events, because the DA-quinone reductase, an

intracellular enzyme which was induced by sulforaphane

completely prevented the loss of SH-SY5Y cell viability.

Further, NAC, which increases the levels of intracellular

glutathione also prevented the loss of cell viability. Therefore,

these results suggest that the neurotoxic effects by the mixture

of Asþ and DA are mediated partly through intracellular

events, although the extracellular mechanisms are not com-

pletely ruled out. Determination of free radical species that are

generated by the mixture of Asþ and DA, and delineating the

signaling pathways that are responsible for dopaminergic

neuronal cell death would be significant aspects in future

studies.

In conclusion, the present study provides initial evidence

that Asþ and DA can synergistically cause enhanced oxidative

stress and induce cell death in cultured neuronal cells

possessing dopaminergic differentiation.

ACKNOWLEDGMENTS

We thank Dr Bertha K. Madras, Harvard Medical School

(MA) for providing the Human embryonic kidney cells (HEK-

293) that stably overexpressed with human DAT.

FIG. 8. Effects of NAC and sulforaphane (Sul) against loss of SH-SY5Y

cell viability induced by the mixture of As and DA. Cells were coincubated

with various concentrations of NAC (100lM to 10mM) along with the mixture

of As and DA for 24 h. In other experiments, cells were preincubated with Sul

(0.1–5lM) for 24 h and then followed by incubation with the mixture of As and

DA for further 24 h. Cell viability was determined by MTT assay after the

incubation. Values are expressed as mean ± SEM. ***p < 0.001 is significantly

different from control group. ##p < 0.01 and ###p < 0.001 are significantly

different from As þ DA group. {{{p < 0.001 is significantly different from

As þ DA group. All concentrations of NAC and Sul tested were able to prevent

the loss of cell viability induced by the mixture of As and DA.


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