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Environmental Toxicology and Pharmacology 26 (2008) 13–21

Neuroprotective effects of chlorogenic acid against apoptosis of PC12 cells induced by methylmercury Yongjin Li a,b , Wei Shi c , Yindong Li d , Yong Zhou b , Xuefeng Hu a , Chunmei Song b , Hongbo Ma b , Changwen Wang b , Yong Li a,∗ a

Department of Nutrition and Food Hygiene, Health Science Center, Peking University, Beijing 100083, PR China b School of Public Health, Jilin Medical College, Jilin 132013, PR China c School of Nursing, Jilin Medical College, Jilin 132013, PR China d Shunyi Center for Disease Control and Prevention; Beijing 101300, PR China Received 14 October 2007; received in revised form 20 December 2007; accepted 21 December 2007 Available online 19 January 2008

Abstract Chlorogenic acid (CGA) widely exists in edible and medicinal plants. We aimed to evaluate the effect of CGA on the protection from apoptosis by methylmercury (MeHg) in PC12 cells. Cell viability was evaluated by MTT assay. Apoptosis was assayed by flow cytometry detection. Caspase-3 activity was measured by confocal microscopy. Intracellular GSH levels were determined by bicinchoninic acid protein assay. Intracellular reactive oxygen species (ROS) was assessed by means of chloromethyl-dihydrodichlorofluorescein diacetate. Glutathione peroxidase (GPx) activity was determined by UV. In order to elucidate the action of CGA, the protective effects of CGA were compared to Vit.E. CGA was effective at protecting PC12 cells against MeHg-induced damage in dose-dependent manner. CGA not only suppressed the generation of ROS, the decrease of activity in GPx and the decrease of GSH, but also attenuated caspase-3 activation in PC12 cells by MeHg. CGA eventually protected PC12 cells against MeHg-induced apoptosis. The results highlighted that CGA may exert neuroprotective effects through its antioxidant actions. © 2008 Published by Elsevier B.V. Keywords: Chlorogenic acid; Neurotoxicity; Methylmercury; Free radicals; PC12 cells

1. Introduction MeHg is a significant environmental contaminant with an established risk to human health (Aschner et al., 2000; Shanker and Aschner, 2003; Virtanen et al., 2007). Due to its easily crossing of the blood–brain barrier and accumulation in the cerebellum, cerebral cortex, and retina (Erie et al., 2005; Herculano

Abbreviations: BSA, bovine serum albumin; CGA, chlorogenic acid; CM-H2 DCFDA, chloromethyl-dihydrodichlorofluorescein diacetate; FITC, fluorescein isothiocyanate; FCM, flow cytometry; GPx, glutathione peroxidase; GSH, glutathione; MeHg, methylmercury; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; PI, propidium iodide; ROS, reactive oxygen species; TAE, Tris/acetate/EDTA buffer; Vit.E, Vitamin E. ∗ Corresponding author at: Department of Nutrition and Food Hygiene, Health Science Center, Peking University, Beijing 100083, PR China. Tel.: +86 10 82801177; fax: +86 10 82801177. E-mail addresses: [email protected] (Y. Li), [email protected] (Y. Li). 1382-6689/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.etap.2007.12.008

et al., 2006), long-term low level MeHg intake causes a range of subclinical to mild clinical neurobehavioral abnormalities, including psychomotor coordination (Lebel et al., 1998; Carta et al., 2003; Auger et al., 2005) and sensory deficits (Lebel et al., 1998; Canto Pereira et al., 2005). Investigators have presented work on induction of apoptosis by MeHg in multiple cell types in vitro (Gatti et al., 2004; Sakaue et al., 2005), and studies also demonstrated that MeHg oxidant is associated to its neurotoxic damage (Aschner, 2007; Dave et al., 1994; Shanker and Aschner, 2001; Shanker et al., 2003, 2004; Yin et al., 2007). Antioxidant phytochemicals from edible and medicinal plants, have been proposed as the major dietary antioxidants providing health benefits (Kasai et al., 2000; Garc´ıa-Alonso et al., 2006; Rangkadilok et al., 2007). CGA is such polyphenolic antioxidant as protective functional factor and beneficial in oxidative stress-related diseases (Delcy et al., 2002, 2006; Jin et al., 2005; Suzuki et al., 2006; Wang et al., 2007). In order to investigate the antioxidant of CGA against MeHg in PC12 cells, we used MeHg as neuronal oxidative agent, and PC12

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2.4. Assessment of apoptosis

Fig. 1. Structure of chlorogenic acid (Gonthier et al., 2003).

cells were preincubated with or without CGA were exposed to different MeHg dose for different time. In our research we evaluated the activity of GPx, caspase-3, reactive oxygen species (ROS), and GSH level were also measured. We found that CGA protected PC12 cells from MeHg-induced apoptosis by a mechanism that was related to GSH, ROS, GPx and caspase-3. The data implied that the extraneous antioxidants may contribute to the sensitivity of neuronal cells to oxidative injury. CGA could be of considerable preventive to some free radical associated health problems of metal-induced neurotoxicity, as well as several neurodegenerative diseases and ageing. Furthermore, the finding gave fresh light helpful for understanding the healthenhancing potential of polyphenols from edible and medicinal plants. 2. Materials and methods 2.1. Reagents Chlorogenic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), anti-caspase-3 antibody, Vitamin E (Vit.E), propidium iodide (PI) PI, penicillin and streptomycin were obtained from Sigma (St. Louis, MO, USA). RPMI 1640 medium was purchased from Gibco Laboratories (USA). PC12 cell line was obtained from institute of Biochemistry and Cell Biology (IBCB, Shanghai Institutes for Biological Sciences, Academy of Sciences Shanghai, China). Methylmercury chloride was purchased from Merck (Darmstadt, Germany). Fetal calf serum and horse serum were obtained from Sijiqing Biotechnology Co. Ltd. (Hang Zhou, China). Chlorogenic acid, as described previously, a repert (Gonthier et al., 2003) gives the chemical structures of these crocins (Fig. 1).

Cells were incubated with MeHg in the dose 2.5 ␮M for 0–48 h to estimate time-course effect, and in 0–6 ␮M to estimate dose-dependent effect. Cells were preincubated with 0.05–1.35 ␮M CGA followed by exposure to 2.5 ␮M of MeHg for 24 h. Aliquots (100 ␮l) were removed from the cell cultures, centrifuged at 2000 × g for 10 min. Flow cytometry (FCM) detection of cell death, apoptotic and necrotic cells were quantified by Annexin V binding and PI uptake. The membrane events were analyzed by measuring the binding of FITC–Annexin V protein to the phospholipid phosphatidylserine present on the external surface of the apoptotic cell membrane. The assay was performed with a two colour analysis of FITC-labeled Annexin V binding and PI uptake using the Annexin V–FITC apoptosis detection kit. Positioning of quadrants on Annexin V/PI dot plots was performed and live cells (Annexin V−/PI−), early/primary apoptotic cells (Annexin V+/PI−), late/secondary apoptotic cells (Annexin V+/PI+) and necrotic cells (Annexin V−/PI+) were distinguished (Vermes et al., 1995). Therefore, the total apoptotic proportion included the percentage of cells with fluorescence Annexin V+/PI− and Annexin V+/PI+. Briefly, after the treatment period (24 h), the harvested cells were resuspended in 1 ml binding buffer. An aliquot of 100 ␮l was incubated with 5 ␮l Annexin V–FITC and 10 ␮l PI for 15 min in dark at room temperature and 400 ␮l PBS was added to each sample. The fluorescein-5-isothiocyanate (FITC) and PI fluorescence were measured through FL-1 filter (530 nm) and FL-2 filter (585 nm), respectively, on BD-LSR flow cytometer using Cell Quest software and 10,000 events were acquired.

2.5. Assay for caspase-3 activity by confocal microscopy Cells were incubated with MeHg in the dose 0–6 ␮M for 6 h, or were preincubated with 0.15 ␮M CGA or 75 ␮M Vit.E followed by exposure to 2.5 ␮M MeHg for 0–15 h. Cells plated on glass coverslips were washed with PBS, fixed in 2% p-formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. All preparations were treated with 3% bovine serum albumin (BSA) to saturate nonspecific binding. Monolayers were then incubated with anti-caspase-3 antibody (1:200 dilution) overnight at 48 ◦ C. After three washes for 10 min each in PBS with 0.2% BSA, the coverslips were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100 dilution) for 30 min and washed three times for 5 min each time in PBS with 0.2% BSA. Then PI was applied to the coverslips for 5 min at room temperature. After washes, samples were analyzed by confocal immunofluorescence microscopy using a Leica Microsystems equipment (Leica Microsystems Heidelberg GmbH).

2.2. Cell culture and treatment PC12 cell line was routinely grown in RPMI 1640 with antibiotics (penicillin 100 IU/ml, and streptomycin 10 ␮g/ml) containing 10% inactivated horse serum and 5% inactivated fetal bovine serum onto 25 cm2 flasks and maintained in humidified atmosphere containing 5% of CO2 at 37 ◦ C. In all the experiments the cells were preincubated with or without CGA or Vit.E for 24 h, prior to experiment, the cells were cultured in the presence or absence of MeHg for the different indicated times.

2.3. Analysis of cell viability The MTT assay method was used to assess cell viability. Cells were seeded onto 96-well plates at the density of 1 × 105 cells/well and, after 24 h, incubated with MeHg in the dose 2.5–7.5 ␮M, or preincubation with 0.05–1.35 ␮M CGA or 25–100 ␮M Vit.E followed by exposure to 5 ␮M of MeHg for 24 h.Briefly, cells in 96-well plates were rinsed with phosphate-buffered saline (PBS), and MTT 500 ␮g/ml were added to each well and incubated for 1 h and then processed according to Mosmann. Survival rate was calculated from the relative absorbance at 570 nm, and expressed as percentage of control (Peng et al., 1998).

Fig. 2. Chlorogenic acid prevented loss of PC12 cell viability. Cell viability was measured by the reduction of MTT activity. (A) MeHg exposure for 24 h caused loss of PC12 cell viability; *P < 0.05 compared with control without MeHg. (B) Preincubation with chlorogenic acid or Vit.E for 24 h prevented cell death induced by MeHg at 24 h; *P < 0.05 compared with MeHg group (MeHg only) without chlorogenic acid or Vit.E pretreatment. Data are means ± S.E.M. of three independent experiments.

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Fig. 3. Chlorogenic acid prevented MeHg-induced apoptosis in PC12 cells. The percentage of cells with apoptotic nuclei was quantified by FCM. (A) Time-course study was performed during 0–48 h after exposure to 2.5 ␮M of MeHg; (B) 24 h incubation with MeHg caused a dose-dependent apoptosis of PC12 cells; *P < 0.05 compared with control without MeHg. (C) Flow cytometric histograms of chlorogenic acid and Vit.E effects on apoptotic rate (low right quadrant in figs) of PC12 cells treated with MeHg. After incubation, cells were labeled with Annexin V–FITC and PI. (C1) 2.5 ␮M MeHg only; (C2) MeHg +0.05 ␮M chlorogenic acid; (C3) MeHg +0.15 ␮M chlorogenic acid; (C4) MeHg +0.45 ␮M chlorogenic acid; (C5) MeHg +1.35 ␮M chlorogenic acid; (C6) MeHg +75 ␮M Vit.E. (D) Treatment with chlorogenic acid and Vit.E followed by exposure to 2.5 ␮M MeHg significantly decreased the percentage of apoptotic cells; *P < 0.05, compared with group treated with MeHg only. Data are means ± S.E.M. of three independent experiments.

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2.9. Statistical analysis Data are presented as means ± S.E.M. Statistical analysis of the data for multiple comparisons was performed by analysis of variance (one-way ANOVA or MANOVA). For single comparison, the significance of differences between means was determined by t test. A value of P < 0.05 or P < 0.01 was considered statistically significant.

3. Results 3.1. CGA prevented MeHg-induced loss of PC12 viability

Fig. 3. (Continued ).

2.6. Quantification of intracellular GSH levels To determine cellular levels of GSH, PC12 cells were seeded at density of 4 × 105 cell/ml in 25 cm2 flasks. Cells were treated for 24 h with 1–5 ␮M MeHg, or pretreatment with CGA (0.05–1.35 ␮M) or Vit.E (75 ␮M) followed by exposure to 2.5 ␮M of MeHg for 24 h. Cells were collected by centrifugation and washed twice in ice cold PBS. Cells were resuspended in 400 ␮l of 5% (w/v) metaphosphoric acid and centrifuged at 4500 × g and 4 ◦ C, for 10 min. Samples were processed and the protein content was determined at the same experimental times for bicinchoninic acid protein assay. Results were expressed as percentage of controls (Gatti et al., 2004).

2.7. Evaluation of intracellular reactive oxygen species generation Intracellular generation of ROS was assessed by means of chloromethyldihydrodichlorofluorescein diacetate (CM-H2 DCFDA). This probe passively diffuses into cells where it is retained following the acetyl groups cleavage by intracellular esterases. Oxidation of the molecule yields a fluorescent signal, which is proportional to intracellular ROS production. Cells were seeded at the density of 2 × 105 cells/ml in 25 cm2 flasks and incubated with MeHg 0–7.5 ␮M for 1 h, or Pretreatment with CGA (0.05–1.35 ␮M) or Vit.E (75 ␮M) followed by exposure to 2.5 ␮M of MeHg. At the end of treatment cells were centrifuged and resuspended in complete fresh medium containing 10 ␮M of CM-H2 DCFDA. After 1 h fluorescence emitted at 530 nm was measured using a microplate reader. Results were expressed as percentage of controls.

2.8. Assay for GPx activity Cells were incubated with MeHg in the dose 0–7.5 ␮M for 24 h, or were preincubated with 0.05–1.35 ␮M CGA or 75 ␮M Vit.E followed by exposure to 2.5 ␮M MeHg for 24 h. GPx activity determination is based on reports of Paglia and Valentine (1967). In the presence of GSH reductase and NADPH, the oxidized GSH is immediately converted to the reduced form with the concomitant oxidation of NADPH to NADP+ . The decrease in absorbance was measured at 37 ◦ C. For the assay, 1000 ␮l of working reagent (GSH 4 mM, GSH reductase 1 U/l, and NADPH 0.34 mM in 50 mM phosphate buffer pH 7.2 containing 4.3 mM EDTA) and 40 ␮l of 0.18 mM cumene hydroperoxide was added to 20 ␮l of sample or water. The indicated concentrations are those yielded in the test for a final volume of 1060 ␮l in a semi-micro quartz glass cuvette. The activity of GPx was measured at 340 nm. The contribution of spontaneous NADPH oxidation was subtracted from the overall reaction rate. GPx activity was determined by nanomole of NADPH oxidized per minute per milligram of protein. Results were expressed as percentage of control.

The loss of cell viability in culture is generally measured by the reduction of MTT activity. In our MTT assay, exhibited cell viability was reduced by 54–12% and a concentrationdependent decrease of cell viability (with decrease of absorbance of formazan) (Fig. 2A). Preincubation of PC12 cells with CGA (0.05–1.35 ␮M) for 24 h reduced this cell damage in a concentration-dependent manner, as evidenced by its effect on the increase in cell viability by 45–66% (Fig. 2B). Vit.E (25–100 ␮M), known free radical scavenger, also prevented MeHg-induced cell death and increased cell viability by 40–59% (Fig. 2B). 3.2. CGA suppressed MeHg-induced apoptosis in PC 12 cells Quantitative analysis of apoptotic cells was assessed after exposure by FCM. Time-course study was performed. PC12 cells were exposed to 2.5 ␮M MeHg from 0 to 48 h. Only 16.2% apoptotic cells were noted at 6 h treatment, and this value continued to increase with the time, by 24 h reaching to 50.3% (Fig. 3A). The 24 h of exposure to MeHg was chosen for subsequent experiments. Exposure of PC12 cells by 24 h, dose-dependent apoptotic cells significantly increased with the increase in MeHg dose (Fig. 3B). At a concentration of 5 and 5.5 ␮M, there was 79% and 82% of cells exhibiting apoptotic after 24 h of exposure (Fig. 3B). Treatment with CGA (0.00–1.35 ␮M) and Vit.E (75 ␮M) before MeHg exposure significantly decreased the percentage of apoptotic cells in a dose-dependent manner. Amount as 0.15, 0.45, and 1.35 ␮M of CGA significantly decreased the percentage of apoptotic cells by 43.1 ± 1.3%, 40.1 ± 1.8%, and 36.5 ± 2.2% of that in PC12 cells without CGA treatment (50.3 ± 2.9%), respectively (Fig. 3C and D). 3.3. Effects of CGA on MeHg-induced caspase-3 activation (by confocal microscopy) In this study, we evaluated caspase activity by assessing relative levels of activated caspase-3, using the FITC-conjugated secondary antibodies. Caspase-3 was activated in PC12 cells by MeHg treatment. Exposure of MeHg for 6 h caused the increase of caspase-3 activity in a dose-dependent manner (Fig. 4A). As shown in Fig. 4B1, caspase-3 activity was up-regulated after 0.5 h of MeHg treatment, and it reached a maximum at 6 h of treatment. When PC12 cells were treated with CGA for 24 h

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prior to exposing to MeHg, the caspase-3 activity was reduced (Fig. 4B1 and B2). In order to determine whether CGA attenuated caspase-3 activity by its antioxidant property or whether suppression of free radical production was sufficient to prevent apoptosis, we pretreated PC12 cells with Vit.E before exposure to MeHg. Our results showed that Vit.E (75 ␮M) suppressed caspase-3 activity (Fig. 4B1 and B2). 3.4. CGA prevented oxidative stress induced by MeHg 3.4.1. Quantification of intracellular GSH levels Changes in intracellular-reduced GSH measured at 24 h after the MeHg exposure are plotted in Fig. 5A. A dose-dependent decrease with the dose increase of MeHg, GSH level (% of control) of PC12 cell treated by 2.5 and 5 ␮M MeHg decreased to

Fig. 4. Effects of chlorogenic acid on MeHg-induced caspase-3 activation in PC12 cells. Caspase-3 activity was estimated by confocal microscopy. (A) Effects of MeHg on caspase-3 activities in PC12 cells treated with MeHg for 6 h; *P < 0.05 compared with control without MeHg treatment. (B) Effects of chlorogenic acid and Vit.E on caspase-3 activities by MeHg. Where indicated, cells were treated with 0.15 ␮M chlorogenic acid and 75 ␮M Vit.E for 24 h prior to exposing to 2.5 ␮M MeHg for 0–15 h. After 0–15 h, cells were fixed and permeabilized. Immunofluorescence was carried out with the rabbit anti-caspase-3 antibody, followed by FITC-labeled anti-rabbit antibody. Finally, PI was added to dye nuclei. Double indirect immunofluorescence microscopy was performed on a Leica Scanning Confocal microscope. (B1) At 6 h bar, 20 ␮m. From left row to the right were FITC, PI and merged images of caspase-3, respectively. (B2) PC12 cells were either untreated or pretreated with 0.15 ␮M chlorogenic acid or 75 ␮M Vit.E followed by exposure to 2.5 ␮M MeHg for 0-15 h; P < 0.05, MANOVA test, as compared CGA, Vit.E proup with group treated with 2.5 ␮M MeHg only. Data are means ± S.E.M. of three independent experiments.

Fig. 5. Effects of chlorogenic acid on GSH level in PC12 cells at 24 h after MeHg exposure. (A) Intracellular GSH values dependent changes in MeHg levels in PC12 cells. (B) Pretreatment with chlorogenic acid (0.05–1.35 ␮M) or Vit.E (75 ␮M) followed by exposure to 2.5 ␮M of MeHg increased intracellular GSH values; *P < 0.05, **P < 0.01 compared with group treated with MeHg only. Data are means ± S.E.M. of three independent experiments.

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37 and 19% of that in control without MeHg. Pretreatment with chlorogenic acid (0.05–1.35 ␮M) or Vit.E (75 ␮M) followed by exposure to 2.5 ␮M of MeHg increased intracellular GSH values (Fig. 5B). 3.4.2. Quantification of intracellular accumulation of oxygen-free radicals in PC12 cells To evaluate the dose-dependent ROS generation, dichlorofluorescein oxidation was measured fluorometrically at 1 h of cell incubation in the presence of MeHg 1–7.5 ␮M. A significant increase in fluorescence from 3 to 23% of control without MeHg (Fig. 6A). Pretreatment with CGA or Vit.E for 24 h suppressed the generation of ROS followed by exposure of 2.5 ␮M MeHg for 1 h (Fig. 6B). 3.5. MeHg-induced decrease of GPx activity Fig. 7 shows the effect of CGA on the activity of GPx induced by MeHg. Exposure to1-7.5 ␮M of MeHg for 24 h decreased

Fig. 7. The effect of MeHg on GPx activity in PC12 cells. (A) Exposure to 2.5, 5, 7.5 ␮M of MeHg for 24 h decreased the activity of GPx, *P < 0.01 compared with control. (B) The effect of chlorogenic acid on GPx activity in PC12 cells exposed to MeHg. Pretreatment with chlorogenic acid (0.05–1.35 ␮M) or Vit.E (75 ␮M) increase GPx activity in PC12 cells followed by exposure to 2.5 ␮M of MeHg for 24 h. **P < 0.05, *P < 0.01 vs. group treated with MeHg only. Data are means ± S.E.M. of three independent experiments.

the activity of GPx by 95–84%. compared with control cells (Fig. 7A), 2.5, 5 and 7.5 ␮M MeHg significantly decreased the activity of GPx by 86%, 83% and 84%, respectively. However, preincubation of cells with CGA (0.05–1.35 ␮M) and Vit.E (75 ␮M) increased GPx activity by 87–96% and 94% (Fig. 7B). 4. Discussion

Fig. 6. Chlorogenic acid or Vit.E prevented oxidative stress induced by MeHg in PC12 cells. The accumulation of oxygen-free radicals was estimated by fluorescence assay. (A) Concentration-dependent changes in MeHg levels in PC12 cells at 1 h after MeHg exposure; *P < 0.05 compared with group treated with control without MeHg. (B) PC12 cells pretreated with chlorogenic acid (0.05, 1.35 ␮M) or Vit.E (75 ␮M) followed by exposure to 2.5 ␮M of MeHg, ROS levels suppressed; *P < 0.05, **P < 0.05 compared with group treated with MeHg only. Data are means ± S.E.M. of three independent experiments.

There is abundant evidence highlighting the fact that MeHg is a significant environmental contaminant with an established risk to human health (Aschner et al., 2000; Shanker and Aschner, 2003; Virtanen et al., 2007) and has a high affinity for thiols, which results in the depletion of intracellular GSH leading to accumulation of ROS (Campbell et al., 2001; Berntssen et al., 2003; Shanker and Aschner, 2003; Kaur et al., 2007; Yin et al., 2007). And ROS accumulation could be pathogenetically relevant as co-factor contributes to the etiology of several neurodegenerative diseases (Wang and James, 1999; Gatti et al., 2004). Studies demonstrated that MeHg-dependent inhibition

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of glutamate, cystine and cysteine uptake, adversely affect intracellular GSH content, redox status in astrocytes and support a role for astrocytic ROS in MeHg-associated neurotoxic damage (Aschner, 2007; Dave et al., 1994; Shanker and Aschner, 2001; Shanker et al., 2003, 2004; Yin et al., 2007). Investigators have presented work on induction of apoptosis by MeHg in multiple cell types in vitro (Gatti et al., 2004; Sakaue et al., 2005). Data from this study showed that treatment with MeHg resulted in a significant increase of ROS level and decrease of GSH level in dose-dependent manner. This was consistent with previous descriptions of MeHg-induced generation of free radicals (Gatti et al., 2004). It is well known that mercurials are able to interact with and deplete sulfhydryl groups from biomolecules and this is closely related to their neurotoxic effects (Aschner and Clarkson, 1988; Shanker et al., 2004; James et al., 2005; Franco et al., 2006; Bridges and Zalups, 2006). Oxidative stress induced by MeHg may affect not only thiol groups (Aschner et al., 2000), but also other functions (Gatti et al., 2004) and leaded to apoptosis. In our research, mercury may be a trigger for the depletion of antioxidant GSH by the production of reactive oxygen species. ROS induced lipid, protein, and DNA oxidation. Generation of ROS in the cytoplasm of cells may increase the mitochondrial hydrogen peroxide production and lipid peroxidation of mitochondrial membrane, resulting in loss of membrane integrity and cell necrosis or apoptosis. CGA is indeed being absorbed and metabolized by humans (Olthof et al., 2001). Epidemiological, biological and biochemical data also concur to support the beneficial role of CGA and other dietary polyphenolic compounds in human health. In particular, CGA has been reported to exert inhibitory effects on carcinogenesis in the large intestine, liver, tongue and a protective action on oxidative stress in vivo. Other research has examined the protective effects of phenolic CGA against oxidative damage (Kasai et al., 2000). A study strongly suggested that CGA can protect RGC-5 cells against oxidative stress-induced death (Nakajima et al., 2007). In the present study, we used CGA to inhibit MeHg-induced oxidative stress in PC12 cells. We found that CGA was effective at protecting PC12 cells against MeHg-induced cell damage (Fig. 2B). We further found that CGA, not only suppressed the generation of ROS (Fig. 6B), decrease of GSH (Fig. 5B) and Gpx (Fig. 7B), but also attenuated caspase-3 activation (Fig. 4B1 and B2), and eventually protected against MeHg-induced apoptosis (Fig. 3C and D). Other kinds of antioxidants against methylmercury toxicity also gave the neuroprotective effects (Ganther, 1980). Clinical trials with antioxidants have also shown the efficacy in slowing the progression of disease from neurotoxicity (Morris et al., 1998; Grundman, 2000). To determine whether suppression of free radical production was sufficient to prevent apoptosis, we employed Vit.E, an agent known as a free radical scavenger, to examine its effects on PC12 apoptotic cell death induced by MeHg. We demonstrated that Vit.E suppressed cell death (Fig. 2B), apoptosis (Fig. 3C and D), caspase-3 activation (Fig. 4B1 and B2) by MeHg exposure. It has also been proposed that Vit.E inhibited the increase of ROS (Fig. 6B), decrease of GSH level (Fig. 5B) and GPx by MeHg exposure in PC12 cells (Fig. 7B). Findings ascribed in this study sug-

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gested that CGA may act on reactive oxygen species to inhibit apoptosis from MeHg neurotoxicity by its antioxidant property. We especially observed a significant increase in GPx activity and GSH level in PC12 cells preincubated with CGA followed by MeHg exposure. Thus, as regards GPx activity, they might exert their protective effect by delaying the consumption of GSH and other cellular antioxidants, during the process of CGA or Vit.E detoxication. For the experiments, some oxidants such as tert-butylhydroperoxide, H2 O2 were chosen as oxidative stressor because it has been shown to efficiently increase the activity of endogenous enzymes in culture cells (Alia et al., 2005; Chen et al., 2006; Baron et al., 2006; Dai et al., 2007). Plant polyphenolic compounds, panduratin A and sylibin, as well as plant extracts showing high antioxidant activity have been shown to reverse the GSH-depleting effect of oxidant tert-butylhydroperoxide in hepatocyte systems (Yau et al., 2002; Sohn et al., 2005). And higher phenolic content of berry juices not only increases antioxidant activities, but also strongly inhibited activated-macrophage NO production, induced tumor necrosis factor-alpha production else well (Tri et al., 2006). Our research data also suggests that CGA might have acted as antioxidant. In conclusion, the findings we reported, in conjunction with data reported by other investigators, clearly suggested that CGA may protect PC12 cells against apoptosis by oxidative stress from MeHg exposure, and our data indicated that CGA may exert neuroprotective effects through its antioxidant actions. The results obtained highlighted the potential of CGA in offering protection against oxidative stress and supported the fact that CGA may provide health benefits. Acknowledgements This work was supported by Mega-projects of Science Research for the 11th Five-year Plan Foundation of Ministry of Science and Technology of the People’s Republic of China (No. 2006BAD27B08). We thank Drs. Zhi Ming Li and Dong Wei Zhang for their useful advice and technical support. And we are grateful to Dr. Tom McDonnell for his English revision. References Alia, M., Ramos, S., Mateos, R., Bravo, L., Goya, L., 2005. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J. Biochem. Mol. Toxicol. 19 (2), 119–128. Aschner, M., 2007. Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes. Brain Res. 1131, 1–10. Aschner, M., Clarkson, T.W., 1988. Distribution of mercury 203 in pregnant rats and their fetuses following systemic infusions with thiol-containing amino acids and glutathione during late gestation. Teratology 38 (2), 145– 155. Aschner, M., Yao, C.P., Allen, J.W., Tan, K.H., 2000. Methylmercury alters glutamate transport in astrocytes. Neurochem. Int. 37 (2–3), 199– 206. Auger, N., Kofman, O., Kosatsky, T., Armstrong, B., 2005. Low-level methylmercury exposure as a risk factor for neurologic abnormalities in adults. Neurotoxicology 26, 149–157.

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