brain research 1608 (2015) 157–166

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N-acetylcysteineamide protects against manganeseinduced toxicity in SHSY5Y cell line Yasaswi Maddirala1, Shakila Tobwala1, Nuran Ercaln Department of Chemistry, Missouri University of Science and Technology, Rolla, MO 65409, USA

art i cle i nfo

ab st rac t

Article history:

Manganese (Mn) is an essential trace element required for normal cellular functioning.

Accepted 4 February 2015

However, overexposure of Mn can be neurotoxic resulting in the development of

Available online 12 February 2015

manganism, a syndrome that resembles Parkinson's disease. Although the pathogenetic

Keywords:

basis of this disorder is unclear, several studies indicate that it is mainly associated with

Oxidative stress

oxidative stress and mitochondrial energy failure. Therefore, this study is focused on (1)

N-acetylcysteineamide

investigating the oxidative effects of Mn on neuroblastoma cells (SHSY5Y) and (2)

GSH

elucidating whether a novel thiol antioxidant, N-acetylcysteineamide (NACA), provides

SHSY5Y cells

any protection against Mn-induced neurotoxicity. Reactive oxygen species (ROS) were

Neurotoxicity

highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione (GSH), malondialdehyde (MDA), and activities of glutathione reductase (GR) and glutathione peroxidase (GPx) were altered in the Mn-treated groups. Loss of mitochondrial membrane potential, as assessed by flow cytometry and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. However, pretreatment with NACA protected against Mn-induced toxicity by inhibiting lipid peroxidation, scavenging ROS, and preserving intracellular GSH and mitochondrial membrane potential. NACA can potentially be developed into a promising therapeutic option for Mn-induced neurotoxicity. This article is part of a Special Issue entitled SI: Metals in neurodegeneration. & 2015 Elsevier B.V. All rights reserved.

1.

Introduction

Manganese is widely used in industries such as mining, welding, automotive mechanics, and dry cell battery manufacturing (Gerber et al., 2002). In addition to the industries, other sources of manganese in the atmosphere include natural processes such as continental dust, volcanic gas and dust, and forest fires. Manganese is a constituent for the activity of several

mitochondrial enzymes such as glutamine synthetase, pyruvate carboxylase and mitochondrial superoxide dismutase, an important enzyme in antioxidant defense system. Hence, it is an essential element for normal functioning of mitochondria in humans (Anderson, 2004), therefore, it is crucial for regulation of many biological functions. However, it can be neurotoxic at high doses leading to manganese-induced Parkinsonism, also known as manganism. Over-exposure to manganese may lead to its

n Correspondence to: Department of Chemistry, Missouri University of Science and Technology, 400 West 11th Street, 142 Schrenk Hall, Rolla, MO 65409. Fax: þ573 341 6033. E-mail address: [email protected] (N. Ercal). 1 Shakila Tobwala and Yasaswi Maddirala are co-first authors and have contributed equally to the work being described.

http://dx.doi.org/10.1016/j.brainres.2015.02.006 0006-8993/& 2015 Elsevier B.V. All rights reserved.

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accumulation within mitochondria in the basal ganglia of the brain, resulting in decreased dopamine (DA) release by dopaminergic neurons in globus pallidus (GP) (Normandin et al., 2002). Although the cellular and molecular mechanisms underlying manganese neurotoxicity are not completely understood, oxidative stress (specifically mitochondrial oxidative stress) has been implicated as a contributing mechanism (Dobson et al., 2004; Erikson et al., 2004a; Gunter et al., 2006; Martinez-Finley et al., 2013; Roth et al., 2003; Simonian et al., 1996; Tamm et al., 2008). It has been proposed that accumulation of Mn in mitochondria (Chen et al., 2001; Galvani et al., 1995), interferes with oxidative phosphorylation and, thereby, leads to excessive production of reactive oxygen species (ROS). The resulting oxidative stress induces mitochondrial permeability transition (MPT), which is characterized by opening of permeability transition pores, leading to increased permeability and, consequently, to collapse of the inner mitochondrial membrane potential and mitochondrial energy failure (Castilho et al., 1995; Halestrap et al., 1997). A possible mechanism for Mninduced MPT is the mitochondrial overload of calcium resulting from the inhibition of Ca2þ efflux as Mn is transported into the mitochondria via Ca2þ transporters (Dubinsky et al., 1998; Gavin et al., 1990). A direct interference of Mn2þ with oxidative phosphorylation by binding to the F0F1 ATPase has also been reported (Malecki, 2001). Major antioxidant defense networks in the mitochondria include superoxide dismutase, glutathione peroxidase, and GSH, since they lack catalase. Glutathione (GSH) is the most abundant and crucial intracellular thiol antioxidant in the central nervous system (CNS). Since oxidative stress and mitochondrial dysfunction are implicated in the pathogenesis, a logical approach to counteract Mn-induced toxicity would be the use of an effective cell permeating GSH prodrug, which would neutralize and alleviate the cumulative oxidative damage. Indeed, NAC, a GSH prodrug, has previously been used in both in vitro (Marreilha dos Santos et al., 2008; Stredrick et al., 2004; Yoon et al., 2011; Zhang et al., 2008) and in vivo (Hazell et al., 2006; Martinez Banaclocha, 2000) for Mn-induced toxicity and has shown protection against Mn-induced toxicity. Unfortunately, NAC is negatively charged at physiological pH, limiting its ability to cross cell membranes and, therefore, requires higher doses and longer treatment times. However, N-acetylcysteineamide's (NACA) characteristics as a drug were improved over NAC by neutralizing the carboxylic group of NAC, which makes the NACA molecule more lipophilic and, therefore, enhances its ability to penetrate cellular membranes (Grinberg et al., 2005). In addition, our own studies demonstrated that NACA is better than NAC in alleviating oxidative stress (Tobwala et al., 2013, 2014). The enhanced ability to penetrate cells allows NACA to be administered at a lower dose than NAC, giving the drug a greater therapeutic index and lowering the risk of side effects that traditionally have been associated with higher doses of NAC (Cotgreave, 1997). NACA is an excellent source of sulfhydryl groups that can be converted by the cells into metabolites capable of stimulating GSH synthesis. The molecule has direct antioxidant action via its free thiol group, reacting and detoxifying ROS. NACA acts as a carrier of NAC and its antioxidant and free radical scavenging abilities are equal to or better than those of NAC (Ates et al., 2008; Penugonda et al., 2011; Tobwala et al., 2014). Previous studies have shown that

NACA is lipophilic and can cross membranes, chelate Cu2þ, scavenge free-radicals, and protect against oxidative stress (Banerjee et al., 2009; Carey et al., 2011; Grinberg et al., 2005; Penugonda et al., 2011; Tobwala et al., 2013; Zhang et al., 2009, 2012). Promising results with NACA in various oxidative stressrelated disorders encouraged us to investigate the ability of NACA to protect against Mn-induced toxicity. Therefore, the present study was undertaken to evaluate the protective role of NACA against Mn-induced toxicity in human neuroblastoma SHSY5Y cells. In addition, the SHSY5Y cell line is catecholaminergic and synthesizes dopamine, a primary neurotransmitter in areas that accumulate manganese, such as the substantia nigra and globus pallidus. Our data showed that NACA protects against Mn-induced toxicity by replenishing intracellular GSH, scavenging ROS, inhibiting lipid peroxidation, and preserving mitochondrial membrane potential. NACA can potentially be developed into a promising therapeutic option for Mn-induced neurotoxicity.

2.

Results

2.1.

Effect of NACA and MnCl2 on SHSY5Y cell viability

To assess cytotoxicity of NACA, the SHSY5Y cells were incubated with different concentrations of NACA for different time periods (Fig. 1A). NACA was nontoxic upto 750 mM for 4 h treatment and was chosen for further studies. A dose- and time-dependent decrease in cell viability was observed in SHSY5Y cells upon exposure to Mn (Fig. 1B), which was confirmed using a Calcein AM assay. Based on the dose- and time-response relationship, an 800 mM concentration of MnCl2, which decreased cell viability by about 50% in 24 h, was determined to be optimal for evaluating the protective effects of NACA.

2.2. Protective effect of NACA against Mn-induced cytotoxicity To study the protective effects of NACA against Mn-induced toxicity, SHSY5Y cells were pretreated for 4 h with 750 mM NACA, followed by incubation with 800 mM of MnCl2 for 24 h. The cell viability was then measured using the Calcein AM Assay. There was a significant increase in the cell viability in the NACA treatment group (Fig. 2).

2.3. Protective effect of NACA on MnCl2-induced ROS generation To assess the role of oxidative stress in Mn-induced toxicity, ROS levels were measured after treatment of SHSY5Y cells with 750 mM NACA followed by incubation with 800 mM of MnCl2 for 24 h. A four-fold increase in ROS was observed upon treatment with 800 mM of MnCl2. However, treatment with NACA reduced the ROS to near control levels (Fig. 3).

2.4.

Effect of NACA on intracellular glutathione levels

To further elucidate the mechanism by which Mn induces cell death and damage, we investigated its effects on GSH. Fig. 4 shows the effect of Mn on cellular GSH levels in SHSY5Y cells

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Fig. 3 – ROS levels in SHSY5Y cells after treatment with 800 μM MnCl2 and 750 μM NACA. ROS levels were measured after 1 h of treatment for all groups. An 800 μM concentration of MnCl2 significantly increased the ROS level. Pretreatment with 750 μM of NACA returned the ROS level to near that of the control level. *p r0.05 compared to the control group, and #pr 0.05 compared to the MnCl2 group. The graph is representative of five replicates and the values reported are mean7SD. Fig. 1 – Cell viability of SHSY5Y cells. (A) Cytotoxicity induced by NACA. (B) Cytotoxicity induced by MnCl2. Cell viability was quantified using Calcein AM assay. The plot is representative of five replicates and the values reported are mean.

Fig. 2 – Protective effect of NACA against Mn-induced cytotoxicity. Cell viability was quantified by a Calcein AM assay 24 h after exposure to MnCl2, following a 4-h pretreatment with NACA. Treatment with MnCl2 (800 μM) alone was seen to significantly decrease cell viability. NACA (750 μM) showed significant protection against Mn-induced cell toxicity. *p r0.05 compared to the control group and #pr0.05 compared to the MnCl2 group. The graph is representative of five replicates and the values reported are mean7SD.

in the presence and absence of NACA. A 24 h exposure with 800 μM of MnCl2 decreased the GSH level to 50% of that of the control. A treatment with 750 μM of NACA significantly increased the GSH levels close to the control.

Fig. 4 – Intracellular GSH levels in SHSY5Y cells after treatment with 800 μM MnCl2 and 750 μM NACA. Exposure to MnCl2 (800 μM) significantly decreased intracellular GSH levels. Pretreatment with NACA (750 μM), 4 h before the addition of MnCl2, prevented such a dramatic decrease. *pr 0.05 compared to the control group and #pr 0.05 compared to the MnCl2 group. The graph is representative of quadruplets and the values reported are mean7SD.

2.5.

Effect of NACA on lipid peroxidation

Malondialdehyde (MDA) was used as an index of lipid peroxidation. MnCl2-treated cells had significantly higher levels (about two-fold) of thiobarbituric acid (TBA)–MDA complex, as compared to those of the control (Fig. 5). Treatment with NACA completely reduced this increase, with TBA–MDA levels becoming nearly the same as those of the control, and with a p value of o0.05, as compared to that of the control.

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2.6. Effect of NACA on the activities of antioxidant enzymes GR and GPx In addition to GSH, antioxidant enzymes, such as GR and GPx, also play a major role in combating oxidative stress and, therefore, the protective effects of NACA were evaluated by measuring the activities of these enzymes. Activity of both GR and GPx was significantly lower in the MnCl2-only group than in the control group, while NACA treatment eliminated this decrease (Table 1). Activities of GPx and GR did not differ between the control group and the NACA-only group.

2.7. (ΔΨm)

Effect of NACA on mitochondrial membrane potential

Since mitochondrial oxidative stress is implicated in Mninduced toxicity, the protective effects of NACA were evaluated by measuring the changes in mitochondrial membrane potential (ΔΨm). A decrease in ΔΨm was demonstrated by using the membrane-permeative potentiometric dye JC-1, which exhibits potential dependent accumulation in mitochondria, indicated by the emission shift from green ( 529 nm) to red ( 590 nm). In healthy cells with high ΔΨm, JC-1 forms J-aggregates emitting intense red fluorescence, whereas in unhealthy cells with low

Fig. 5 – MDA levels in SHSY5Y cells after treatment with 800 μM MnCl2 and 750 μM NACA. MnCl2 (800 μM) induced a significant increase in the MDA level. Pretreatment with 750 μM of NACA decreased lipid peroxidation significantly. *pr0.05 compared to the control group and #pr0.05 compared to the MnCl2 group. The graph is representative of quadruplets and the values reported are mean7SD.

ΔΨm, JC-1 remains in the monomeric form emitting green fluorescence. Hence, ΔΨm is indicated by the red/green fluorescence intensity ratio. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was used as a positive control to separate the cells with healthy and unhealthy mitochondria by gating (Fig. 6A). Cells exposed to MnCl2 had 40% loss in ΔΨm compared to control, however NACA protected the cells by preserving the mitochondrial ΔΨm (Fig. 6B).

2.8.

Effect of NACA on ATP levels

The primary function of mitochondria is to synthesize ATP, which requires a balanced mitochondrial proton electrochemical potential. An extensive depletion of ΔΨm results in the collapse of mitochondrial ATP synthesis. Therefore, ATP levels were measured as an additional index for mitochondrial function. Incubation of cells with MnCl2 resulted in a significant decrease ( 25%) in the levels of ATP compared to control, whereas treatment with NACA significantly eliminated this depletion of ATP levels significantly (po0.05) compared to the MnCl2 only group (Fig. 7).

3.

Discussion

Widespread use of manganese in various industries may pose potential health risks. Although manganese is an essential trace element required for normal brain functioning, exposure to a high concentration of manganese may lead to manganism, a neurological disease with Parkinsonism as a cardinal clinical feature (Huang et al., 1989; Olanow, 2004). One possible mechanism of manganese-induced neurotoxicity is oxidative stress (Simonian et al., 1996; Taylor et al., 2006), particularly in dopaminergic neurons (Benedetto et al., 2009). Oxidative stress is a deleterious condition that results from insufficient scavenging of ROS, which are generated by a myriad of biochemical reactions. ROS lead to functional alterations in proteins, DNA, and lipids. Normally, these species are eliminated by intracellular antioxidant systems. However, under unusual stress, these defenses may not be sufficient to deter oxidative damage. As oxidative stress has been implicated in Mn-induced toxicity, researchers investigated the neuroprotective effect of antioxidants like NAC in both in vitro (Chen et al., 2002) and in vivo (Hazell et al., 2006) studies, Silymarin in in vitro and in vivo systems (Chtourou et al., 2010), and NAC, GSH and vitamin C in mitochondria isolated from Sprague Dawley rat

Table 1 – Glutathione reductase and glutathione peroxidase activity (mU/mg protein) in SHSY5Y cells after treatment with Mn and NACA. Groups

Glutathione reductase

Glutathione peroxidase

Control NACA-only MnCl2-only NACAþMnCl2

22.074.00 21.072.00 7.0073.00a 19.072.00b

26.974.24 28.171.24 16.575.39a 22.871.00b

All experiments were performed in triplicates, and the values reported are mean7SD. a Significantly different from the control group at po 0.05. b Significantly different from the MnCl2 group at po 0.05.

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Fig. 6 – Effects of NACA and MnCl2 on mitochondrial membrane potential (ΔΨm). (A) Fluorescence histograms of SHSY5Y cells using the potential-dependent aggregate forming lipophilic cation JC-1 after different treatments. (B) Quantitative analysis of flow cytometry data. Mitochondria in MnCl2-treated cells showed a loss of ΔΨm. However, pretreatment with NACA preserved ΔΨm similar to that of the control group. The graph is representative of quadruplets and the values reported are mean7SD.

brain (Zhang et al., 2004). NACA was previously reported to effectively protect dopaminergic neurons in experimental models of Parkinson's disease (Bahat-Stroomza et al., 2005) and, therefore, we evaluated the protective role of NACA in Mninduced toxicity. A significant increase in total ROS upon Mn treatment was observed in our study, which substantiates the role of oxidative

stress in the pathogenesis (Fig. 3). Our data are in agreement with other studies that have reported an increase in the production of ROS upon Mn treatment (Ali et al., 1995; Chtourou et al., 2010; Gunter et al., 2006; HaMai et al., 2001; Latronico et al., 2013; Yoon et al., 2011). One of the important effects of oxidative stress and free radical generation is decreased levels of cellular antioxidants, like GSH, which is

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Fig. 7 – ATP levels in SHSY5Y cells after treatment with 800 μM MnCl2 and 750 μM NACA. ATP levels were measured with the bioluminescence assay (Promega). Exposure to MnCl2 significantly decreased the ATP levels. Pretreatment with 750 μM of NACA increased it to near control levels. *pr0.05 compared to the control group and #pr0.05 compared to the MnCl2 group. The graph is representative of quadruplets and the values reported are mean7SD.

mainly responsible for maintaining the cellular redox status in cells. GSH scavenges free radicals and H2O2 and neutralizes toxic metabolites by condensing with them both enzymatically and nonenzymatically (Meister, 1995). In addition, GSH helps to maintain the cysteine residues in proteins in their reduced state, which is crucial for protein structure and activity. Alterations in brain GSH metabolism have been implicated in the pathogenesis of Mn-induced neurotoxicity (Liccione et al., 1988). Significant decreases in GSH levels (Fig. 4) and cell viability (Fig. 2) were observed in our study, following exposure to Mn, indicating that oxidative stress was responsible for reduced cell viability. A possible explanation for the decrease in GSH levels under oxidative stress is the reduced activity of the enzymes involved in GSH synthesis and/or GR activity. Some studies have indicated that loss of GSH will directly affect the activity of the GSH-dependent enzyme GR. This enzyme plays an important role in GSH homeostasis: it regenerates GSH from GSSG. It has been reported that, under oxidative stress, protein sulfhydryl groups are lost (Wu et al., 2004) which are believed to be essential for enzyme activity (Agarwal et al., 1999). Under such circumstances, GSH is not regenerated; thus, depletion of cellular GSH indicates that the cells are undergoing oxidative stress. Decreased activity of GR (Table 1) seen in our experiments, upon Mn treatment, supports our hypothesis of GSH depletion as a mechanism for cell death in Mn-treated SHSY5Y cells. Furthermore, a significant reduction in the activity of GPx (Table 1) was observed following Mn treatment. A decrease in GPx activity may have been partially due to diminished GSH levels that GPx needs as a substrate. Treatment with NACA increased GSH levels and cell viability as well as GR and GPx activity in the Mn-treated group, indicating that NACA had replenished the GSH levels in these cells and prevented oxidative stress-induced cell death. The protective effects of NACA could also be mediated by NACA's ability to scavenge free radicals, increase GSH biosynthesis by providing cysteine, reducing GSSG to GSH in a

thiol exchange reaction, and/or by supplying the sulfydryl groups that can stimulate GSH biosynthesis (Issels et al., 1988). Our results are in line with previous studies that reported a decrease in GSH levels upon Mn treatment in animal models and in cell cultures (Chtourou et al., 2010; Desole et al., 1995; Erikson et al., 2004b; Erikson et al., 2007; Marreilha dos Santos et al., 2008, 2011, 2012; Seth et al., 2002; Zhang et al., 2008). Erikson et al. (2004b) reported significant decreases in GSH levels in the striatum of Mn-exposed rats. In another study, Erikson et al. (2007) reported opposite responses in GSH metabolism in two brain regions exposed to similar Mn concentrations. Age-dependent responses in GSH metabolism upon exposure to high doses of Mn have also been reported in animals (Desole et al., 1995; Erikson et al., 2004b). A similar decrease in GPx activity has also been reported previously upon Mn exposure (Liccione et al., 1988). A decrease in GSH and an increase in ROS tend to set off a cascade of further oxidative damage. Lipid peroxidation is one of the key mechanisms by which ROS induce cell death. The double bonds in fatty acids undergo lipid peroxidation in the presence of free radicals and form stable by-products, such as malondialdehyde (MDA), which are used as markers of lipid peroxidation. SHSY5Y cells that were exposed to Mn had increased levels of MDA (Fig. 5), compared to those of the controls, indicating increased lipid peroxidation in the SHSY5Y cells. These results are in line with other studies which have reported increased lipid peroxidation upon Mn treatment (Chtourou et al., 2010; Cordova et al., 2012; Marreilha dos Santos et al., 2012; Milatovic et al., 2009; Zhang et al., 2008). Concomitant reduction of GSH levels (a substrate for glutathione peroxidase) might have hampered the decomposition of lipid peroxides in Mn-treated cells, thus increasing the MDA levels. NACA was able to break lipid peroxidation chain reaction by supplying an adequate amount of GSH, as a substrate for glutathione peroxidase, to effectively decompose lipid peroxides, thus reducing MDA levels. Mitochondrial dysfunction is hypothesized to be involved in Mn-induced neurotoxicity (Heron et al., 2001; Zhang et al., 2008; Zhang et al., 2004). Mitochondrial membrane potential (MMP) drives the synthesis of ATP and plays an important role in cell survival (Muramatsu et al., 2007). Loss of MMP is an early sign of mitochondrial dysfunction and often precedes many other signs of cell injury. A decrease in the ΔΨm (change in MMP) after treatment was detected and assessed in our studies using flow cytometry. In addition to this, we also observed a decrease in ATP levels after Mn treatment. Loss of ΔΨm interferes with the production of ATP, the cell's main source of energy, because mitochondria must have an electrochemical gradient to provide the driving force for ATP production. Decrease in ΔΨm and ATP levels seen in our study are consistent with disrupted mitochondrial function (Figs. 6 and 7). Similar decrease in mitochondrial membrane potential (Rao et al., 2004; Yoon et al., 2011; Zhang et al., 2008) followed by decrease in ATP levels (Milatovic et al., 2011) after Mn treatment have been reported by other researchers. However, NACA pretreatment restored the mitochondrial membrane potential, which could be due to the alleviation of oxidative stress induced by Mn and, thereby, preserving mitochondrial bioenergetic capacity. In summary, our data indicate that Mn exposure causes oxidative stress and mitochondrial dysfunction in SHSY5Y cells.

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NACA, a GSH prodrug, protected SHSY5Y cells against Mninduced oxidative damage, possibly by a variety of mechanism. These include scavenging of ROS; increasing the activity of the detoxification enzyme, glutathione peroxidase (GPx); by providing GSH, which is a limiting substrate for GPx activity; and halting the production of further ROS and lipid peroxidation. GSH might also provide protection by recycling other key antioxidants like vitamin E and vitamin C. Therefore, we conclude that Mn-induced cytotoxicity in SHSY5Y cells is associated with oxidative stress and GSH-prodrugs provide protection from Mn in this cell line. These encouraging in vitro results indicate that NACA can potentially be developed into a promising therapeutic option for Mn-induced neurotoxicity. Our future studies will focus on studying the protective effects of NACA in animal models. Further investigations will help to determine NACA's efficacy in Mn-induced toxicities and will provide additional information on NACA's therapeutic potential.

4.

Experimental procedures

4.1.

Materials

MnCl2 (Cat no: AC22361-0100,Z99.9% pure) was purchased from Fisher Scientific (Houston, TX). N-(1-pyrenyl)-maleimide (NPM, Cat no: P7908, Z98.5% pure) was purchased from SigmaAldrich (St. Louis, MO). N-acetylcysteineamide (NACA,Z99.9% pure) was provided by Dr. Glenn Goldstein (David Pharmaceuticals, New York, NY, USA). High performance liquid chromatography (HPLC) grade solvents were purchased from Fisher Scientific. All other chemicals were purchased from SigmaAldrich (St. Louis, MO).

4.2.

Cell culture and study design

Human neuroblastoma cell line, SHSY5Y cells (ATCC; # CRL2266), were cultured in Dulbecco's modified Eagle's medium (DMEM) in humidified 5% CO2/95% air at 37 1C. DMEM was supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin. Culture medium was changed once every 4 days and SHSY5Y cells at passages 22–27 were used in this study. All assays were performed in quadruplets. For cytotoxicity studies the SHSY5Y cells were seeded in a 96-well plate, at a density of approximately 1.5  104 cells/well and were allowed to attach for a day. To assess MnCl2 cytotoxicity, cells were incubated with different concentrations of MnCl2 (in serum free media) ranging from 250–2000 mM for different time periods (1 h, 3 h, 9 h, 12 h, 24 h, and 48 h). A 50% decrease in cell viability was observed when cells were treated with 800 mM MnCl2 for 24 h, and, therefore this concentration and dosing time of MnCl2 was chosen for further studies. When cells were pretreated with different concentrations of NACA (250 mM, 500 mM, 750 mM, 1000 mM) for different time periods (2 h, 4 h, 8 h, 12 h, 24 h, and 48 h) followed by treatment with 800 mM MnCl2, maximum protection was seen with 750 mM NACA at 4 h, and, therefore, we chose 750 mM NACA for all other studies. Oxidative stress parameters, including, GSH, MDA, activities of glutathione reductase (GR) and glutathione peroxidase (GPx), and mitochondrial permeability were measured after the cells were treated in 25 cm2 tissue culture flasks, as described here. After

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the flasks were 85–90% full, the flasks were divided into the following four groups: 1) control, 2) NACA-only, 3) MnCl2-only, (4) NACAþMnCl2. In the pretreatment group, serum free media containing 750 mM of NACA was added 4 h prior to the addition of MnCl2. After pretreatment, the media in the control and NACA-only groups were replaced with plain media, while both of the remaining two groups received media containing MnCl2 for 20 h. The cells were harvested and centrifuged at 1750 rpm for 8 min to obtain cell pellets, which were then further processed for appropriate assays. All assays were performed in quadruplets.

4.3.

Determination of cell viability

The SHSY5Y cells were seeded in a 96-well plate, at a density of approximately 1.5  104 cells/well and were allowed to attach for a day. The protective effects of NACA were studied by dividing the cells into four groups as described in Section 4.2. After the treatment, the medium was discarded and a Calcein AM assay kit (Biotium, Inc. CA) was used to measure cell viability relative to the control group. The cells were washed three times with PBS, and 100 mL of 2.0 mM Calcein AM in PBS were added to each well for 30 min at 37 1C. The fluorescence was measured with an excitation wavelength at 485 nm and an emission wavelength of 530 nm, using a microplate reader (FLUOstar, BMG Labtechnologies, Durham, NC, USA).

4.4.

Intracellular ROS measurement

Intracellular ROS generation was measured using a wellcharacterized probe, 2ˊ, 7ˊ-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Wang et al., 1999). H2DCF-DA is hydrolyzed by esterases to dichlorodihydrofluorescein (H2DCF), which is trapped within the cell. This non-fluorescent molecule is then oxidized to fluorescent dichlorofluorescein (DCF) by the action of cellular oxidants. SHSY5Y cells were seeded at a density of approximately 1.5  104 cells/well in a 96-well plate; once the plate reached 80% confluence, the cells were incubated with MnCl2 for 24 h. In the groups with NACA treatment, media containing 750 mM NACA was added and incubated for 4 h. H2DCF-DA stock solution (in DMSO) of 20 mM was diluted in PBS without serum to yield a 20 mM working solution. Cells were washed twice with PBS and then incubated with H2DCF-DA working solution for 30 min in a dark environment (37 1C incubator). The cells were washed and then fluorescence was determined at 485 nm excitation and 520 nm emission, using a microplate reader (Fluostar, BMG Labtechnologies, Durham, NC, USA).

4.5.

Intracellular GSH determination

Intracellular GSH levels were determined by reverse phase HPLC, according to the method developed in our laboratory (Winters et al., 1995). The SHSY5Y cell samples were homogenized in serum borate buffer (100 mM Tris–HCl, 10 mM borate, 5 mM serine, and 1 mM diethylenetriaminepentaacetic acid) pH 7.4 and centrifuged. Twenty microliters of this homogenate were added to 230 ml of HPLC grade water and 750 ml of NPM (1 mM in acetonitrile). The resulting solutions were incubated at room temperature for 5 min. The reaction was stopped by adding 10 ml

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of 2 N HCl. The samples were then filtered through a 0.45 mm filter (Advantec MFS, Inc. Dublin, CA, USA) and injected onto the HPLC system. 5 ml of the sample were injected for analysis using a Thermo Finnigan TM Spectra SYSTEM SCM1000 Vacuum Membrane Degasser, Finnigan TM SpectraSYSTEM P2000 Gradient Pump, Finnigan TM SpectraSYSTEM AS3000 Autosampler, and Finnigan™ SpectraSYSTEM FL3000 Fluorescence Detector (λex ¼330 nm and λem ¼376 nm). The HPLC column was a Reliasil ODS-1 C18 column (Column Engineering, Ontario, CA, USA). The mobile phase was 70% acetonitrile and 30% water and was adjusted to a pH of 2.5 through the addition of 1 ml/L of both acetic and o-phosphoric acids. The NPM derivatives were eluted from the column isocratically at a flow rate of 1 ml/min.

4.6.

Determination of lipid peroxidation

Malondialdehyde (MDA) is a thiobarbituric acid reactive substance (TBAR). The extent of cellular lipid peroxidation was determined by measuring concentrations of TBA–MDA complex. Briefly, the cell pellets were homogenized in SBB. To 0.350 ml of cell homogenate, 0.550 ml of 5% trichloroacetic acid (TCA) and 0.100 ml of 500 ppm butylated hydroxytoluene (BHT) in methanol were added. The samples were then heated in a boiling water bath for 30 min. After cooling on ice, the samples were centrifuged. The supernatant fractions were mixed 1:1 with saturated thiobarbituric acid (TBA). The samples were again heated in a boiling water bath for 30 min. After cooling on ice, 0.50 ml of each sample was extracted with 1 ml of n-butanol and centrifuged to facilitate the separation phases. The resulting organic layers were first filtered through a 0.45 mm filter and transferred to a 96-well plate for analysis. Fluorescence was determined at 510 nm excitation and 590 nm emission, using a microplate reader (FLUOstar, BMG Labtechnologies, Durham, NC, USA).

4.7.

Determination of glutathione reductase activity

Glutathione reductase (GR) is the enzyme responsible for recycling GSSG into GSH via a reduction mechanism, utilizing both GSSG and NADPH as a substrate. The activity of this enzyme was determined using a commercial kit from OxisResearch (Portland, OR, USA). The oxidation of NADPH to NADPþ was accompanied by a decrease in absorbance at 340 nm, providing a spectrophotometric means for monitoring the enzyme activity of GR. The activity of GR in cells was determined by adding homogenate to a solution containing both GSSG and NADPH and then recording the absorbance as a function of time at 340 nm. The rate of decrease in the A340 was directly proportional to the GR activity in the sample.

4.8.

Determination of glutathione peroxidase activity

Glutathione peroxidase (GPx) catalyzes the reduction of a variety of ROOH and H2O2, using GSH as a reducing agent. The GPx 340™ assay (a test kit from OxisResearch) was used as an indirect measure of the activity of GPx. Glutathione disulfide (GSSG) produced upon reduction of an organic peroxide by GPx, was recycled to its reduced state by the enzyme glutathione reductase. The oxidation of NADPH to NADPþ was accompanied by a decrease in absorbance at 340 nm

(A340), providing a spectrophotometric means for monitoring GPx enzyme activity. The molar extinction coefficient for NADPH is 6220 M  1 cm  1 at 340 nm. To measure the GPx activity, cell homogenate was added to a solution containing glutathione, glutathione reductase, and NADPH. The enzyme reaction was initiated by adding the substrate, tert-butyl hydrogen peroxide, and the absorbance was recorded at 340 nm. The rate of decrease in the A340 was directly proportional to the GPx activity in the sample.

4.9.

Measurement of ATP levels

Total ATP was quantified using a commercially available luciferin–luciferase assay kit (Abcam, Cambridge, MA, USA). 10 mM ATP stock standard was prepared by reconstituting the lyophilized ATP standard with deionized water. A series of dilutions ranging from 0 to 1  10  13 M ATP were made, and used as working standards to prepare a calibration curve. Aliquots of 100 ml of working standards were pipetted into a clean 96-well microplate. Additionally, 100 ml of cell lysate were placed into the 96-well microplate. 50 ml of the detergent were added to each well, and the plate was shaken for 5 min in an orbital shaker at 700 rpm. 50 ml of reconstituted substrate solution were added to each well and the microplate was shaken again for 5 min in an orbital shaker at 700 rpm. Plates were kept in the dark for 10 min; luminescence was measured using a luminometer (FLUOstar, BMG Labtechnologies, Durham, NC, USA).

4.10. (ΔΨm)

Determination of mitochondrial membrane potential

Mitochondrial membrane potential was determined using a JC-1 assay kit for flow cytometry (Molecular Probes, Eugene, OR, USA), as previously described (Mao et al., 1999) with minor modifications. Briefly, the cell pellets were homogenized in 1 ml of warm phosphate-buffered saline at approximately 1  106 cells/ml. JC-1 fluorescent dye was prepared as a stock solution (200 mM) by dissolving the contents of one vial in 230 ml of the DMSO provided. For positive control, 2 ml of 50 mM CCCP were added and incubated at 37 1C for 5 min. 10 ml of 200 mM JC-1 (2 mM final concentration) were added to the cells in all the groups and incubated at 37 1C in the dark for 30 min. The stained cells were washed, resuspended in 500 ml PBS, and analyzed by flow cytometry (BD Accuri C6 flow cytometer, San Jose, CA, USA) using an argon laser of 488 nm as excitation, with filters emitting at 530/30 nm in FL1 channel and 585/42 nm in FL2 channel. A minimum of 50,000 cells per sample were analyzed.

4.11.

Determination of protein

Protein levels of the cell samples were measured by the Bradford method (Bradford, 1976). Bovine serum albumin was used as the protein standard.

4.12.

Statistical analysis

All reported values were represented as the mean7S.D. of quadruplets. Statistical analysis was performed using the GraphPad Prism software (GraphPad, San Diego, CA). Statistical significance was ascertained by one-way analysis of

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variance, followed by Tukey's multiple comparison tests. Values of po0.05 were considered significant.

Acknowledgments The authors appreciate the efforts of Barbara Harris in editing the manuscript. Dr. Ercal is supported by Richard K. Vitek endowment.

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N-acetylcysteineamide protects against manganese-induced toxicity in SHSY5Y cell line.

Manganese (Mn) is an essential trace element required for normal cellular functioning. However, overexposure of Mn can be neurotoxic resulting in the ...
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