Materials and Methods 1. Cell Culture - Involvement of oxidative stress-mediated ERK1/2 and p3

Murine neuroblastoma cell line: Neuro-2a (CCL-131, American Type Culture Collection, Manassas, VA, USA) was culture in a humidified chamber with a 5%

CO2–95% air mixture at 37 °C and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%

penicillin-streptomycin (Gibco/Invitrogen, Carlsbad, CA, USA), and all assays were conducted within 10-15 cell passages after unfreezing the cells from the liquid nitrogen (to keep the best morphological and biological characteristics).

2.2. Cell viability

Neuro-2a cells were seeded at 2×10⁴ cells/well in 96-well plates and allowed to adhere and recover overnight. The cells were changed to fresh media and then incubated with MeHg (1-5 μM; Sigma-Aldrich, St. Louis, MO, USA) in the absence or presence of NAC (1 mM) or specific MAPK inhibitors (20 M, Sigma-Aldrich) for

24 h. After incubation, the medium was aspirated and fresh medium containing 30 μL of 2 mg/mL 3-(4, 5-dimethyl thiazol-2-yl-)-2, 5-diphenyl tetrazolium bromide (MTT;

Sigma-Aldrich) was added. After 4 h, the medium was removed and replaced with blue formazan crystal dissolved in dimethyl sulfoxide (100 μL; Sigma-Aldrich).

Absorbance at 570 nm was measured using a microplate reader (Bio-Rad, model 550, Hercules, CA, USA).

2.3. Determination of reactive oxygen species (ROS) production

ROS generation was monitored by flow cytometry using the peroxide-sensitive fluorescent probe: 2’, 7’-dichlorofluorescin diacetate (DCFH-DA, Molecular Probes, Inc, Eugene, OR, USA), as described Chen et al., 2010. In brief, cells were

coincubated with 20M DCFH-DA at 37 ℃. After incubation with the dye, cells were resuspended in ice-cold phosphate buffered saline (PBS) and placed on ice in a dark environment. The intracellular peroxide levels were measured by flow cytometer (FACScalibur, Becton Dickinson, Sunnyvale, CA), that emitted a fluorescent signal at 525 nm. Each group was acquired more than 10000 individual cells.

2.4. Analysis of intracellular GSH contents

Neuro-2a cells were seeded at 2×10⁵ cells/well in a 24-well plate and allowed to adhere and recover overnight. The cells were changed to fresh media and incubated with MeHg in the absence or presence of NAC (1 mM, Sigma-Aldrich) for 24 h. Then, cells were washed twice with PBS, and then a new medium which contained 60 M monochlorobimane (mBCL, a sensitive fluorescent probe, Sigma-Aldrich) was added and incubated for further 30 min at 37 ℃. After loading the culture cells with mBCL, the supernantants were discarded, cells were washed twice with PBS, and the measurement the intracellular GSH levels were performed as described previously (Yen et al., 2007).

2.5. Flow cytometric analysis of sub-G1 DNA content

Cells were seeded (in the same manner as for intracellular GSH analysis) and incubated with MeHg. After 24 h incubation, the cells were detached, collected, and washed with PBS, and the analysis of sub-G1 DNA content was performed as described previously (Lu et al., 2011). The cells were subjected to flow cytometry analysis of DNA content (FACScalibur, Becton Dickinson). Nuclei displaying hypodiploid, sub-G1 DNA contents were identified as apoptotic. The sample of each group was collected more than 10000 individual cells.

2.6. Determination of Mitochondrial Membrane Potential(MMP)

Cells were seeded (in the same manner as for intracellular GSH analysis) and exposure to MeHg in the absence or presence of NAC (1 mM) or specific MAPK inhibitors (20 M, Sigma-Aldrich). After 6 or 24 h incubation, cells were loaded with 100 nM 3,3`-di-hexyloxacarbocyanine iodide (DiOC₆, Molecular Probes, Inc) or 30 min at 37 ℃, and then trypsinized, collected, and washed twice with PBS. MMP was analyzed by FACScan flow cytometer (excitation at 475 nm and emission at 525 nm, Becton Dickinson)(Chen et al., 2006). CaspACE^TM fluorometric activity assay (Promega Corporation, Madison, WI, USA) as previously described (Lu et al., 2011). Protein levels of cell lysate samples were determined using the bicinchoninic acid protein assay kit with an absorption band of 570 nm (Pierce, Rockford, IL, USA) to normalize the cell numbers between control and different MeHg-treated groups.

2.8. Western blot analysis

Neuro-2a cells were seeded at 1×10⁶ cells/well in a 6-well plate and allowed to adhere and recover overnight. The cells were changed to fresh media and incubated with MeHg in the absence or presence of NAC (1 mM, Sigma-Aldrich) or specific MAPK inhibitors (20 M, Sigma-Aldrich) for different time intervals. After treatment, the expression of protein activation or phosphorylation was evaluated by the standard

protocols performance of Western blot (as previously described in Chen et al., 2010) using the specific antibodies, including: anti-cleaved caspase-3, -7, and -9, PARP, phosphor-p38 and phosphor-ERK1/2 (Cell Signaling Technology, Inc., Beverly, MA, UAS), anti-Bcl-2, Bax, p38, and ERK1/2 (Santa Cruz Biotechnology, Inc., CA, USA), and -tubulin (Epitomics, Inc., Burlingame, CA, USA).

2.9. Animal preparation

Randomly bred, normal male ICR mice (4weeks old, 20-25 g) were obtained from the Animal Center of College of Medical, National Taiwan University. All experiments were carried out according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC), and the care and use of laboratory animals were conducted in accordance with the guidelines of the Animal Research Committee of China Medical University (http://cmurdc.cmu.edu.tw/LA/index.php).

Mice were randomly assigned to pretreatment groups, weighed, and administrated MeHg or vehicle (oral application by gavaged). These groups were given 0 and 50

g/kg/day MeHg in the absence or present of NAC (150 mg/kg/day) for 7 consecutive

weeks. Each group contained more than 12 mice (n = 12~15). All experimental mice were deep anesthesia by an intraperitoneal injection of pentobarbital (80 mg/kg) and the whole blood samples were collected from an eyehole vessel. Whole blood sample were centrifuged at 3,000 × g for 10 min, and plasma was obtained, and LPO levels was assayed immediately. At the same time, mice were sacrificed by decapitation under pentobarbital anesthesia, and the cerebral cortex were quickly removed and stored at liquid nitrogen until use, and then was analysis of LPO levels, GSH content, and apoptosis-related genes expression.

2.10. Lipid peroxidation(LPO) analysis

Neuro-2a cells were seeded (in the same manner as for Western blot analysis) and exposure to MeHg alone or in combination with NAC (1 mM, Sigma-Aldrich).

After 24 h incubation, cells were harvested and homogenized in ice-cold 20 mM Tris-HCl buffer, pH 7.4, containing 0.5 mM butylated hydroxytoluene to prevent sample oxidation. The cerebral cortex was weighted and homogenized separately in ice-cold 20 mM Tris-HCl buffer, pH 7.4 (100 mg tissue/ml buffer), then homogenized sample was assayed immediately. LPO levels of equal volumes sample were

2.11. Measurement of glutathione (GSH) levels in the cerebral cortex

The cerebral cortex was homogenated with an isotonic buffer (25 mM Hepes. pH 7.4, containing 250 mM sucrose) and then centrifuged at 1,000 × g at 4 ℃. Each sample was discarded the pellet and added 10× lysis buffer to the supernant, and placed on ice for 10 min. After centrifuged, the supernant was performed the measurement of GSH levels using Glutathione assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. The protein concentration of each sample was determined using the bicinchoninic acid protein assay kit (Pierce). GSH level was expressed as nanomoles (nmol) GSH per microgram protein and estimated from the standard curve.

2.12. Real-time quantitative reverse-transcribed polymerase chain reaction (RT-PCR)

analysis

The expression of apoptosis-related genes was evaluated by real-time quantitative RT-PCR, as previously described (Lu et al., 2010). Briefly, intracellular total RNA was extracted from the cerebral cortex using RNeasy kits (Qiagen, Hilden, Germany) and reverse transcribed into cDNA using the AMV RTase (reverse transcriptase enzyme; Promega Corporation, Pty. Ltd.) according to the manufacture’s instructions. Each sample (2 1 cDNA) was tested with Real-time Sybr Green PCR reagent (Invitrogen, USA) with mouse specific primers (PCR primers for the examined genes were listed in Table 1.) in a 25-l reaction volume, and amplification was performed using an ABI StepOnePlus sequence detection system (PE, Applied replicates in each PCR run, and their average CT was used for relative quantification analyses (the relative quantification method utilizing real-time PCR efficiencies (Pfaffl et al., 2002)). TF expression data were normalized by subtracting the mean of reference gene C_T value from their C_T value (ΔC_T). The Fold Change value was calculated using the expression 2^-ΔΔCT, where ΔΔCT represents ΔCT-condition of interest – ΔCT-control. Prior to conducting statistical analyses, the fold change from the mean of the control group was calculated for each individual sample.

2.13. Statistical analysis

Data are presented as means ± standard deviations (S.D.) The significance of difference was evaluated by the Student’s t-test. When more than one group was

compared with one control, significance was evaluated according to one-way analysis of variance (ANOVA) was used for analysis, and the Duncans’s post hoc test was applied to identify group differences. The P value less than 0.05 was considered to be significant. The statistical package SPSS, version 11.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.

3. Results

3.1. MeHg exposure induces LPO, depletion of GSH levels, and changes in apoptosis-related gene expression in the cerebral cortex of mice

Our previous study indicated that exposure to MeHg (50 g/kg/day) caused neurophysiological dysfunctions and biological changes in mice brain, which was accompanied with significant mercury accumulation (Huang et al., 2008). However, the toxicological effects and possibly molecular mechanism of MeHg-induced neurotoxicity have not been understood. Therefore, we investigated the LPO and GSH levels in the plasma and/or cerebral cortex of MeHg-treated mice (50 g/kg/day, by oral gavage). The results revealed that LPO levels in the plasma and cerebral cortex significantly increased after MeHg exposure for 7 consecutive weeks (Supplement Figure 1), whereas the measurement of GSH levels in the cerebral cortex markedly decreased (Figure 1).

We further investigated whether MeHg-induced oxidative stress could cause apoptosis in the cerebral cortex of mice. To address this issue, we analyzed apoptosis-related gene expression by using real-time quantitative RT-PCR. As shown in Figure 2, there was significant down-regulation of Bcl-2 (anti-apoptotic gene) and up-regulation of Bax, Bak, and p53 (pro-apoptotic genes) expression in the cerebral cortex of mice after MeHg exposure for 7 consecutive weeks (Figure 2A).

Furthermore, these changes were accompanied by marked up-regulation of caspase-3, caspase-7, and caspase-9 expressions (Figure 2B). Antioxidant NAC (150 mg/kg/day) effectively prevented MeHg-induced toxic responses (Supplement Figure 1, Figures 1 and 2).

3.2. MeHg induces cell death, ROS production, and intracellular GSH depletion in

Neuro-2a cells

To investigate whether the mechanisms involved in MeHg-induced neurotoxicity, we explored the in vitro effects of MeHg in Neuro-2a cells. The cell viability of Neuro-2a cells was markedly decreased after 24 h MeHg (1-5 M) treatment, and the LD50 was determined to be approximately 3 M (Figure 3A).

To further evaluate the effects of MeHg on oxidative stress damage, we treated the cells with MeHg (3 and 5 M) and measured ROS generation, LPO production, and intracellular GSH levels. After exposure of Neuro-2a cells to MeHg for 0.5-2 h, the intracellular ROS levels were found to be significantly increased by using DCF fluorescence probe as an indicator of ROS formation (Figure 3B). Incubation of cells with MeHg for 24 h also produced remarkably high malondialdehyde (MDA) levels in the cell membrane (3 M MeHg, 3.08 ± 0.07; 5 M MeHg, 3.91 ± 0.08; control, 1.92 ± 0.08 nmole-MDA/mg protein)(Figure 3C). Moreover, the levels of intracellular GSH (a principal cellular protective thiol against oxidative stress-induced toxicity, and determined using an mBCl fluorescent probe) was significantly decreased after treatment with MeHg (3 and 5 M, not 1 M) for 24 h (3 M MeHg, 61.31 ± 3.13%;

5 M MeHg, 30.60 ± 4.07% of control)(Figure 3D). These MeHg-induced responses could be reversed by NAC (1 mM) (Figures 3B, 3C, 3D, and 3E).

3.3 MeHg causes cell death via mitochondria-dependent apoptosis pathways in Neuro-2a cells

To investigate the involvement of apoptosis in MeHg-induced Neuro-2a cell cytotoxicity, we analyzed the sub-G1 hypodiploid cell population (as an indicator of apoptosis) by flow cytometry. Cells treated with MeHg (3 and 5 M) for 24 h exhibited a significant increase in the sub-G1 hypodiploid cell population (Figure 4A).

Moreover, caspase-3 activity (an integral step in the majority of apoptotic events) was

also markedly induced after treatment of Neuro-2a cells with MeHg (Figure 4B).

These results indicated that exposure of Neuro-2a cells to MeHg could induce apoptosis.

Next, we explored whether MeHg-induced apoptosis was mediated through the mitochondrial dysfunction. To show that MeHg affected mitochondrial permeability transition, MMP was analyzed using flow cytometry with the cationic dye DiOC6. As shown in Figure 5A, exposure of Neuro-2a cells to MeHg (3 M) markedly induced MMP loss (for 6 and 24 h). We also investigated cytochrome c release from the mitochondria into the cytosol in MeHg-treated Neuro-2a cells. Cells exposure to MeHg (3 M) for 6 h effectively increased cytochrome c release in the cytosol fraction, and this increase in the level of cytochrome c was more significant after 24 h MeHg exposure (Figure 5B). Antioxidant NAC (1 mM) could effectively reverse the MeHg-induced responses (Figures 5A and B). Moreover, we further examined the changes in the expression of Bcl-2 family proteins. Treatment of Neuro-2a cells with MeHg (3 M) significantly decreased the expression of Bcl-2 and increased the level of Bax, which led to a marked shift in the pro-apoptotic (Bax)/anti-apoptotic (Bcl-2) expression ratio toward an apoptosis-associated state (Figure 5C).

To further investigate whether MeHg-induced the activation of cysteine proteases, which are important biomarkers of the apoptotic process representing both the initiation and execution of cell death, the expressions of caspase cascades were detected by Western blot analysis. As shown in Figure 5D, it increased the activation of caspase-3 and caspase-7 in Neuro-2a cells treated with MeHg (3 M) for 6-24h.

The MeHg-induced caspase-3 activity in Neuro-2a cells could be reversed by NAC (Figure 4B). MeHg also significantly increased the level of cleaved product (active form) of PARP as well as the expression of upstream caspase-9 (Figure 5D).

3.4. Effects of MeHg on activation of ERK- and p38-MAPK in Neuro-2a cells

To further evaluate the involvement of MAPKs signals in responses triggered by MeHg-induced apoptosis, the expressions of MAPKs activation were examined. As shown in Figure 6A, exposure of neuro-2a cells to MeHg (3 M) significantly increased the levels of phosphorylation of ERK1/2- and p38-MAPK (for 0.5-2 h), but not that of JNK. To determine the relationship between MeHg-induced apoptotic signaling transduction in Neuro-2a cells and MAPKs activation, the cells were pretreated with the specific ERK inhibitor PD98059, p38 inhibitor SB203580, and JNK inhibitor SP600125. It was found that MeHg-induced neuronal cell cytotoxicity was attenuated by the ERK1/2 and p38 inhibitors (20 M), but not by the JNK inhibitor (Figure 6B, a). NAC, ERK and p38 inhibitors could prevent MeHg-induced ERK1/2- and p38-MAPK activation (Figure 6B, b and c). Loss of MMP and the increase of caspase-3 activity induced by MeHg-treated Neuro-2a cells could also be effectively reversed by ERK and p38 inhibitors (Figures 6C and D). Furthermore, MeHg-induced increase in ERK1/2- and p38-MAPK protein phosphorylation was observed in the cerebral cortex of MeHg-treated mice (50 g/kg/day, for 7 consecutive weeks), which could be prevented by NAC (Supplement figure 2). These results indicate that ROS-mediated ERK1/2 and p38-MAPK activation play a crucial role in MeHg-induced neuronal cell apoptosis.

4. Discussion

Many studies have indicated that MeHg is a potent neurotoxicant affecting both the developing and mature central nervous system, and can cause severe neuropathophysiological disorders with exposure to high concentrations (0.5-40 ppm in drinking water or 0.2-2 mg/kg/day, for more than 7 consecutive days) in vivo system, including loss of neurons in the calcarine cortex, cerebellar Purkinje, and granule cells, leading to vision, motion, or postural abnormalities (Carvalho et al., 2007; Chuu et al., 2007; Dare et al., 2003; Goulet et al., 2003; Onishchenko et al., 2007). Exposure to MeHg (20-50 g/kg) in mice, which was the possible dosage from food ingested in MeHg-contaminated areas (8.03-174 g-MeHg/kg, even more than 200g-MeHg/kg), caused severe neurotoxic injuries has also reported, that

accompanied with significant mercury accumulation (113.0-241.8 ng-mercury/g wet.

wt.) and oxidative stress generation in the brain regains (Grandjean et al., 1992;

Huang et al., 2008 and 2011; Maramba et al., 2006; Qiu et al., 2008). Oxidative stress, which has demonstrated to strongly induce under MeHg-exposed conditions and play a key role for cascade activation during mercury-induced injury, is involved in the progression of brain and/or neuronal cell dysfunction and death in mammals (Dreiem et al., 2005; Shanker et al., 2004 Yin et al., 2007). Of late, an increasing number of studies have suggested that oxidative stress, which disturbs the physiological functions of neuronal cells and causes apoptosis, is linked to the progression of neurodegenerative diseases (Branham et al., 2004; Loh et al., 2006), and clinical studies also described that significant and higher concentrations of mercury are detected in the blood and/or brain regions of Alzheimer’s disease patients compared to healthy peoples; these suggest a decisive role of mercury in the origin or progression of neurodegenerative diseases (Gerhardsson et al., 2008; Hock et al., 1998; Mutter et

al., 2007). Despite several studies showing that MeHg can produce neurotoxic injuries by inducing oxidative stress in mammals, the role of ROS and the precise signaling mechanisms underlying MeHg-induced neuronal degeneration and cell death are still unclear. In this study, our results revealed that MeHg significantly induced LPO production (as an indicator for oxidative stress damage formation) in the plasma and cerebral cortex of mice exposed to MeHg (50 g/kg/day) for 7 consecutive weeks, which was accompanied with GSH depletion. Meanwhile, the results also revealed that MeHg significantly altered apoptosis-related genes expression, including anti-apoptotic (Bcl-2), pro-apoptotic (Bax, Bak, and p53), and caspase cascades (caspase-3, caspase-7, and caspase-9) in the cerebral cortex of MeHg-treated mice.

Moreover, MeHg (1-5 M) exposure significantly decreased cell viability, increased ROS production, depleted intracellular GSH, and caused apoptotic events (increase in sub-G1 hypodiploid cell population and caspase cascades activation) in Neuro-2a cells. The antioxidant NAC could effectively prevent, but not fully reverse, these MeHg-induced responses. These findings indicate that oxidative stress is caused by MeHg exposure, and it may be involved in MeHg-induced neuronal degeneration and cell apoptosis. Furthermore, Fujimura et al (2009) has reported that the expression of Rac1 (Rho-family protein) can be down-regulated and ultimately led to apoptosis in MeHg-exposed neuronal cells. Tofighi et al (2011) has also indicated that MeHg induces caspase-independent cell death via parallel activation of calpains and lysosomal proteases in hippocampal neurons. However, the critical roles of these signals in MeHg-induced neurotoxicity will still investigation in the future.

ROS can elicit oxidative stress, which triggers cell death; thus, it can be implicated in various neurodegenerative conditions resulting from metal-induced neurotoxicity. (Bush, 2000; Rana, 2008). ROS is also known to cause mitochondrial dysfunction (as being a key mechanism in apoptosis) by oxidative stress-induced

apoptosis (Chen et al., 2006, Lu et al., 2011). Mitochondria are highly sensitive to the effects of oxidative stress, and 2 major events have been noted in oxidative stress-induced apoptosis involving mitochondrial dysfunction. One of the events is alteration in the mitochondrial membrane permeability and the subsequent depolarization of MMP; while the other event is the release of mitochondria-associated proteins (including cytochrome c, Apaf-1, and apoptosis-inducing factor) from the intermembrane space of mitochondria into the cytosol (Chen et al., 2010; Kroemer et al., 1997; Lu et al., 2010). Moreover, the biological function of Bcl-2 family proteins has been demonstrated to regulate mitochondrial-dependent apoptosis while balancing anti- and pro-apoptotic members to arbitrate life/death decisions. Therefore, the ratio of Bcl-2 to Bax is a pivotal factor that determines whether apoptosis will occur in the cells exposed to injurious chemicals (Cheng et al., 2007; Pradelli et al., 2010). Here, we found that MeHg could capable of inducing apoptotic cell death in Neuro-2a cells, which was accompanied with trigger MMP depolarization and cytochrome c release. Moreover, treatment with 3 M MeHg significantly increased Bax and decreased Bcl-2 protein expression in Neuro-2a cells, and resulted in an increase of Bax/Bcl-2 ratio that might contribute to the promotion of MeHg-induced apoptosis. These MeHg-induced neuronal cells apoptotic responses could be prevented by the antioxidant NAC. Therefore, these results implicate that MeHg-induced oxidative stress-regulated Neuro-2a cell apoptosis involves in the mitochondria-dependent apoptotic pathway.

MAPKs, including ERK1/2, JNK, and p38, play critical roles as mediators of cellular responses to extracellular stimuli, such as the proliferation, differentiation, survival, and death of nervous cells (Chang and Karin, 2001; Chen et al., 2009;

Cowan and Storey, 2003). MAPKs activation is also involved in apoptosis and may play a pivotal role in the progress of neurodegenerative diseases (Kim and Choi, 2010;

Miloso et al., 2008). Some studies have indicated that oxidative stress is an important risk factor in the development of Alzheimer’s disease via MAPK signaling activations and downstream-regulated apoptosis in neuronal cells exposed to many injurious agents (Margues et la., 2003; Puig et al., 2004). To our knowledge, however, a limited number of studies have investigated the role of MAPKs in MeHg-induced neuronal cell apoptosis. In the present study, we found that MeHg induced the activation of ERK1/2- and p38-MAPK, but not that of JNK, in Neuro-2a cells. Pretreatment of cells with the antioxidant NAC, specific ERK inhibitor PD98059, and p38 inhibitor SB203580, but not JNK inhibitor SP600125, attenuated MeHg-induced cytotoxicity

在文檔中 Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury (頁 29-73)