ToxSci Advance Access originally published online on December 13, 2007
Toxicological Sciences 2008 102(2):254-261; doi:10.1093/toxsci/kfm302
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Synergistic Neurotoxic Effects of Arsenic and Dopamine in Human Dopaminergic Neuroblastoma SH-SY5Y Cells
Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202
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{at}medicine.nodak.edu.
Received October 9, 2007; accepted December 8, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Parkinson's disease is an environmentally influenced, neurodegenerative 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 (As+3) 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 As+3 and DA was synergistic in producing the death of the SH-SY5Y cells when compared with exposure to either agent alone. A mixture of 10µM As+3 and 100µM 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 As+3 and DA. It was also demonstrated that the antioxidants, N-acetylcysteine, and Sulforaphane, attenuated the toxicity of the mixture of As+3 and DA to the SH-SY5Y cells. This study provides initial evidence that As+3 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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 individual 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 indistinguishable, 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 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 (Rodríguez et al., 2003). In addition, 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; Rodríguez 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 As+3 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 + 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 As+3 (sodium arsenite), DA, As+3 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 As+3 (5–20µM) and DA (50–400µM) concentrations for 24 h. In separate experiments, cells were also treated with combinations of As+3 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 As+3 and DA toxicity, the cells were coincubated with NAC or preincubated with sulforaphane for 24 h before exposure to As+3 and DA. To test whether inhibitors of DA transporters and DA receptors could block the toxicity of As+3 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 As+3 and DA for 24 h.
Mechanism of As+3-and DA-induced cell death.
Experiments were performed to determine if cell death of SH-SH5Y exposed to As+3 and DA occurred by a necrotic or apoptotic mechanism. The effect of As+3 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 µl DAPI (10 µg/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 µg/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 µg 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 As+3 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 µg 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 β-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 µl of the cell culture supernatant was transferred to a 96-well enzymatic assay plate. Reconstituted substrate mix (50 µl) 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 µl 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toxicity of DA, Arsenite, and Mixtures of DA and Arsenite on SH-SY5Y Cells
Initial experiments were performed to determine an exposure level of both DA and As+3 that were the minimal level 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 100µM 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 As+3 and it was shown that a concentration of As+3 of 10µM was the minimal level that would elicit a significant loss of cell viability (Fig. 1B). These two exposure levels of As+3 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 As+3 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 As+3 and DA. In contrast, each agent alone resulted in a decrease in cell viability to 79.1 ± 0.5% for As+3 and 69.5 ± 7.5% for DA when compared with control cells. The increased loss of cell viability due to the combination of As+3 and DA was significant when compared with either agent alone (p < 0.001). These results show that exposure to a combination of As+3 and DA has increased toxicity to dopaminergic SH-SY5Y cells when compared with either agent alone.
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Two experiments were designed to determine if the effects of combined exposure to As+3 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 As+3, DA, and a combination of As+3 and DA. It was demonstrated that the UROtsa cell line exhibited a similar pattern of As+3 toxicity (Fig. 2). In contrast, the UROtsa cells were shown to be very resistant to DA toxicity, with levels of 600µM 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 As+3 and DA had no effect on UROtsa cell viability over that found for As+3 alone (Fig. 2). A second effort was made to assess the effect of a DA and As+3 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 As+3, 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 As+3 toxicity, with 10µM As+3 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 600µM DA having no effect on HEK cell viability (data not shown). Like the UROtsa cells, a combination of As+3 (10µM) and DA (100µM) had no effect on HEK cell viability over that found for As+3 alone (Fig. 2). These results suggest that dopaminergic differentiation is required for the cells to have enhanced susceptibility to mixtures of As+3 and DA.
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Toxicity of DA and As+3 Mixtures and DA Transporter Blockers 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 As+3 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 As+3 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 As+3 and DA. None of the agents tested had any toxic effects on SH-SY5Y cells (Fig. 3).
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Mechanism of Cell Death in SH-SY5Y Cells Exposed to As+3, DA, and a Mixture of As+3 and DA
The effects of As+3 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 As+3 and DA. The results demonstrated that there was no increase in profiles of fragmented nuclei observed in the As+3- and DA-treated cells over those noted to occur spontaneously in control cells (Fig. 4). Likewise, no fragmented nuclei were observed in SH-SY5Y cells treated with As+3 or DA alone (Fig. 4). That apoptosis was not the mechanism of As+3 plus DA SH-SY5Y cell death as suggested by absence of fragmented nuclei was further confirmed by determining both nuclear fragmentation and Caspase 3 activation. 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 (detachment 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 As+3 and DA failed to produce a DNA ladder or the activation of Caspase 3 (Figs. 5 and 6).
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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 As+3 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 As+3 and DA (p < 0.01) when compared with control cells or cells treated with As+3 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 As+3 and DA until release was almost 100% of control, accounting for the total loss of cell viability (Fig. 7).
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Effect of Antioxidants on As+3 and DA Induced SH-SY5Y Cell Death
NAC and Sulforaphane were tested for their ability to inhibit the death of SH-SY5Y cells treated with a mixture of As+3 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 (Martínez et al., 1999
). On the other hand, the nicotinamide adenine dinucleotide 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 As+3 and DA. The coincubation of the cells exposed to a mixture of As+3 and DA with 100µM, 1mM, and 10mM concentrations of NAC resulted in significant decreases 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 As+3 and DA mixture (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The initial goal of this study was to determine if a mixture of As+3 and DA would cause enhanced degeneration of dopaminergic neuronal cells when compared with either agent alone. The results clearly demonstrated that a mixture of As+3 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 As+3 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 As+3 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 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 (Rodríguez et al., 2003). In the rat nervous system As exposure has been shown to cause oxidative damage to both lipids and proteins (García-Chávez 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 As+3 and DA elicited in the SH-SY5Y cells. It was shown that the mixture of As+3 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 As+3 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 As+3 and DA. The mechanism(s) underlying the ability of As+3 and DA to increase oxidative stress and dopaminergic neuron cell death is currently unknown. We presume that As+ 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 significantly 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 As+ 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 As+ and DA are mediated partly through intracellular events, although the extracellular mechanisms are not completely ruled out. Determination of free radical species that are generated by the mixture of As+ 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 As+ and DA can synergistically cause enhanced oxidative stress and induce cell death in cultured neuronal cells possessing dopaminergic differentiation.
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ACKNOWLEDGMENTS |
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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.
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