Hyperoside prevents oxidative damage induced by hydrogen peroxide in lung fibroblast cells via an antioxidant effect

Biochimica et Biophysica Acta 1780 (2008) 1448–1457

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Hyperoside prevents oxidative damage induced by hydrogen peroxide in lung fibroblast cells via an antioxidant effect Mei Jing Piao a, Kyoung Ah Kang a, Rui Zhang a, Dong Ok Ko a, Zhi Hong Wang a, Ho Jin You b, Hee Sun Kim c, Ju Sun Kim d, Sam Sik Kang d, Jin Won Hyun a,⁎ a

Department of Biochemistry, School of Medicine, Institute of Medical Science, Cheju National University, Jeju-si 690-756, Korea Department of Pharmacology, College of Medicine, Chosun University, Gwangju 501-759, Korea Department of Neuroscience, College of Medicine, Ewha Womans University, Seoul 110-783, Korea d Natural Products Research Institute and College of Pharmacy, Seoul National University, Seoul 110-460, Korea b c

a r t i c l e

i n f o

Article history: Received 24 May 2008 Received in revised form 10 July 2008 Accepted 25 July 2008 Available online 15 August 2008 Keywords: Hyperoside Reactive oxygen species Apoptosis Lipid peroxidation Protein carbonyl DNA damage

a b s t r a c t We elucidated the cytoprotective effects of hyperoside (quercetin-3-O-galactoside) against hydrogen peroxide (H2O2)-induced cell damage. We found that hyperoside scavenged the intracellular reactive oxygen species (ROS) detected by fluorescence spectrometry, flow cytometry, and confocal microscopy. In addition, we found that hyperoside scavenged the hydroxyl radicals generated by the Fenton reaction (FeSO4 + H2O2) in a cell-free system, which was detected by electron spin resonance (ESR) spectrometry. Hyperoside was found to inhibit H2O2induced apoptosis in Chinese hamster lung fibroblast (V79-4) cells, as shown by decreased apoptotic nuclear fragmentation, decreased sub-G1 cell population, and decreased DNA fragmentation. In addition, hyperoside pretreatment inhibited the H2O2-induced activation of caspase-3 measured in terms of levels of cleaved caspase-3. Hyperoside prevented H2O2-induced lipid peroxidation as well as protein carbonyl. In addition, hyperoside prevented the H2O2-induced cellular DNA damage, which was established by comet tail, and phospho histone H2A.X expression. Furthermore, hyperoside increased the catalase and glutathione peroxidase activities. Conversely, the catalase inhibitor abolished the cytoprotective effect of hyperoside from H2O2-induced cell damage. In conclusion, hyperoside was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing antioxidant enzyme activity. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Hyperoside (quercetin-3-O-galactoside) is a flavonoid compound, which is mainly found from hypericum perforatum L. [1]. Hyperoside has been shown to possess various biological functions against ROSinduced damage, such as the antidepressant effect by inhibiting nitric oxide synthase in rat blood and cerebral homogenate [2], the inhibitory effect of linoleic acid peroxidation or deoxyribose degradation induced by ROS [1], the partial uncoupling effect of oxidative phosphorylation in cardiac mitochondria [3], and the protection of PC 12 rat pheochromocytoma against cytotoxicity induced by hydrogen peroxide and tert-butyl hydroperoxide [4]. The lung is a highly vascularized organ which facilitates the uptake of oxygen and the release of carbon dioxide. Due to its high ratio of surface area to air, the lung is vulnerable to pathogens, pollutants, oxidants, gases, and toxicants that are inhaled from air, thus making it susceptible to oxidative stress [5]. ROS are known to cause oxidative modification to DNA, proteins, lipids, and small intracellular mole-

⁎ Corresponding author. Tel.: +82 64 754 3838; fax: +82 64 726 4152. E-mail address: [email protected] (J.W. Hyun). 0304-4165/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2008.07.012

cules. Lipids, including pulmonary surfactants, react with ROS to produce lipid peroxides, which cause increased membrane permeability and the inactivation of surfactants [6]. In turn, proteins reacting with ROS lead to decreased protein synthesis due to the modification of proteins or ribosomal translocation, ultimately resulting in impaired cellular metabolism [7]. In addition, ROS was found to damage nucleic acids by modifying purine and pyrimidine bases and causing DNA strand breakage [8]. The oxidative stress induced by the overproduction of ROS in lung causes many clinical conditions including cancer, asthma, cystic fibrosis, ischemia-reperfusion injury, drug-induced lung toxicity, and aging [9–11]. The antioxidant effect of hyperoside in lung has not been reported until now. Furthermore, the precise mechanisms behind the cytoprotective effect of hyperoside against oxidative stress have also not been clearly determined. To the best of our knowledge, the present study was the first to report on the cytoprotective effect of hyperoside against cell damage induced by hydrogen peroxide in lung fibroblast cells and the likely protective mechanisms involved. Our results demonstrated that hyperoside protected cells from hydrogen peroxide-induced damage by scavenging ROS and the induction of antioxidant enzymes. As a result, this study suggests that hyperoside may have therapeutic properties against the various oxidative stress-related lung diseases.

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2. Materials and methods 2.1. Reagents The hyperoside (quercetin-3-O-galactoside, Fig. 1) compound was purchased from Fluka Co. (Buchs, Switzerland). 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA), Hoechst 33342, 5, 5dimethyl-1-pyrroline-N-oxide (DMPO), and 3-amino-1, 2, 4 triazol (ATZ) were purchased from the Sigma Chemical Company (St. Louis, MO, USA), and thiobarbituric acid from BDH Laboratories (Poole, Dorset, UK). The primary anti-catalase antibody was purchased from Biodesign International Company (Saco, Maine, USA), and the primary anti-phospho histone H2A.X antibody from Upstate Biotechnology (Lake Placid, NY, USA), and the primary anti-caspase 3 and poly ADPribosyl polymerase (PARP) antibodies from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA). 2.2. Cell culture The lung fibroblast cell is reported to be sensitive to oxidative stress response [12]. To study the effect of hyperoside on oxidative stress, the Chinese hamster lung fibroblast cells (V79-4) were used. The cells were maintained at 37 °C in an incubator, with a humidified atmosphere of 5% CO2, and cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 µg/ml) and penicillin (100U/ml). 2.3. Detection of hydroxyl radical Hydroxyl radicals were generated by the Fenton reaction (H2O2 + FeSO4), which were then quickly reacted with a nitrone spin trap, 5, 5dimethyl-1-pyrroline-N-oxide (DMPO). The resultant DMPO-OH adducts were detected using an ESR spectrometer. The ESR spectrum was recorded using JES-FA ESR spectrometer (JEOL, Tokyo, Japan), at 2.5 min after being mixed in a phosphate buffer solution (pH 7.4) with 0.2 ml of 0.3 M DMPO, 0.2 ml of 10 mM FeSO4, 0.2 ml of 10 mM H2O2, and 5 µM hyperoside. The parameters of the ESR spectrometer were set at the following conditions: magnetic field of 336.5 mT, power of 1.00 mW, frequency of 9.4380 GHz, modulation amplitude of 0.2 mT, gain of 200, scan time of 0.5 min, scan width of 10 mT, time constant of 0.03 s, and a temperature of 25 °C [13,14]. 2.4. Intracellular reactive oxygen species (ROS) measurement The DCF-DA method was used to detect the levels of intracellular ROS [15]. The V79-4 cells were seeded in a 96 well plate at 2 × 104 cells/ well. Sixteen hours after plating, the cells were treated with hyperoside at concentrations of 1, 2.5, and 5 µM. After 30 min, 1 mM of H2O2 was added to the plate. The cells were incubated for an additional 30 min at 37 °C. After the addition of 25 µM of DCF-DA solution for 10 min, the fluorescence of 2′, 7′-dichlorofluorescein was detected using a Perkin Elmer LS-5B spectrofluorometer and using flow

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cytometry (Becton Dickinson, Mountain View, CA, USA), respectively. The image analysis for the generation of intracellular ROS was achieved by seeding the cells on a cover-slip loaded six well plate at 2 × 105 cells/well. Sixteen hours after plating, the cells were treated with hyperoside at 5 µM. After 30 min, 1 mM of H2O2 was added to the plate. After changing the media, 100 µM of DCF-DA was added to each well and was incubated for an additional 30 min at 37 °C. After washing with PBS, the stained cells were mounted onto microscope slide in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on a confocal microscope. 2.5. Cell viability To determine the effect of hyperoside on the cell viability in H2O2 treatment, the cells were treated with hyperoside for 1 h at 5 µM. Next, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. Fifty µl of the [3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium] bromide (MTT) stock solution (2 mg/ml) was then added into each well to attain a total reaction volume of 200 µl. After incubating for 4 h, the plate was centrifuged at 800 ×g for 5 min and the supernatants were aspirated. The formazan crystals in each well were dissolved in 150 µl of dimethylsulfoxide and read at A540 on a scanning multi-well spectrophotometer [16]. To determine the effect of catalase inhibitor on the cell viability, cells were pretreated with final 20 mM ATZ for 1 h, followed by 1 h of incubation with hyperoside and exposure to 1 mM H2O2 for 24 h and the cell viability was measured using MTT test. 2.6. Nuclear staining with Hoechst 33342 The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. 1.5 µl of Hoechst 33342 (stock 10 mg/ml), a DNA specific fluorescent dye, was added to each well and incubated for 10 min at 37 °C. The stained cells were then observed under a fluorescent microscope, which was equipped with a CoolSNAP-Pro color digital camera, in order to examine the degree of nuclear condensation. 2.7. Flow cytometry analysis The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. Flow cytometry was performed to determine the content of apoptotic sub G1 hypo-diploid cells [17]. The cells were harvested, and fixed in 1 ml of 70% ethanol for 30 min at 4 °C. The cells were washed twice with phosphate buffered saline (PBS), and then incubated for 30 min under dark condition at 37 °C in 1 ml of PBS containing 100 µg propidium iodide and 100 µg RNase A. The flow cytometric analysis was performed and the proportion of sub G1 hypo-diploid cells was assessed by the histograms generated using the computer program, Cell Quest and Mod-Fit. 2.8. DNA fragmentation The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. Cellular DNA fragmentation was assessed by using a cytoplasmic histone-associated DNA fragmentation kit from Roche Diagnostics (Mannheim, Germany) according to the manufacturer's instructions. 2.9. Lipid peroxidation assay

Fig. 1. Chemical structure of hyperoside (quercetin-3-D-galactoside).

The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. Lipid peroxidation was assayed by the thiobarbituric acid reaction [18]. The cells were then washed with cold PBS, scraped, and homogenized in

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ice-cold 1.15% KCl. One hundred µl of the cell lysates was mixed with 0.2 ml of 8.1% sodium dodecylsulfate,1.5 ml of 20% acetic acid (adjusted to pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid (TBA). The mixture was made up to a final volume of 4 ml with distilled water and heated to 95 °C for 2 h. After cooling to room temperature, 5 ml of n-butanol and pyridine mixture (15:1, v/v) was added to each sample and shaken. After centrifugation at 1000 ×g for 10 min, the supernatant fraction was isolated, and the absorbance was measured spectrophotometrically at 532 nm. The amount of thiobarbituric acid reactive substance (TBARS) was determined using standard curve with 1,1, 3, 3-tetrahydroxypropane. 2.10. Protein carbonyl formation The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. The amount of carbonyl formation in protein was determined using an

Oxiselect™ protein carbonyl ELISA kit purchased from Cell Biolabs (San Diego, CA, USA) according to the manufacturer's instructions. Cellular protein was isolated using protein lysis buffer (50 mM Tris (pH 7.5), 10 mM EDTA (pH 8), 1 mM PMSF) and quantified using a spectrophotometer. 2.11. Comet assay A Comet assay was performed to assess oxidative DNA damage [19,20]. Cells were treated with hyperoside at 5 µM for 30 min later, 1 mM H2O2 was added and incubated for 15 min. The mixture was centrifuged at 13,000 ×g for 5 min and the cell pellet (0.5 × 105 cells) was mixed with 100 µl of 0.5% low melting agarose (LMA) at 39 °C and spread on a fully frosted microscopic slide that was pre-coated with 200 µl of 1% normal melting agarose (NMA). After solidification of the agarose, the slide was covered with another 75 µl of 0.5% LMA and then immersed in

Fig. 2. The effects of hyperoside on intracellular ROS and cell-free hydroxyl radicals. The cells were treated with hyperoside at concentrations of 1, 2.5, and 5 µM. After 30 min, 1 mM of H2O2 was added to the plate. After an additional 30 min, the intracellular ROS generated was detected by spectrofluorometry (A) and flow cytometry (B) after the DCF-DA treatment. FI indicates the fluorescence intensity of DCF-DA. (C) The representative confocal images illustrate the increase in red fluorescence intensity of DCF produced by ROS in H2O2-treated cells compared to the control and the lowered fluorescence intensity in H2O2-treated cells with hyperoside (original magnification × 400). The measurements were made in triplicate and the values were expressed as means ± SE. ⁎Significantly different from hyperoside untreated cells (p b 0.05). (D) Hydroxyl radicals generated by the Fenton reaction (H2O2 + FeSO4) were reacted with DMPO, and the resultant DMPO-OH adducts were detected by ESR spectrometry.

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determined. Aliquots of the lysates (40 µg of protein) were boiled for 5 min and electrophoresed in 10% sodium dodecylsulfate-polyacrylamide gel. The blots in the gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then incubated with the primary antibodies. The membranes were further incubated with the secondary immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA). Protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK), and then exposed onto X-ray film. 2.13. Immunocytochemistry The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. The cells plated on coverslips were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 2.5 min. The cells were treated with blocking medium (3% bovine serum albumin in PBS) for 1 h and incubated with anti-phospho histone H2A.X antibody diluted in blocking medium for 2 h. Immunoreactive primary phospho histone H2A.X antibody was detected with a 1:500 dilution of FITCconjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Glove, PA, USA) for 1 h. After washing with PBS, the stained cells were mounted onto microscope slides in mounting medium with DAPI (Vector, Burlingame, CA, USA). Images were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on a confocal microscope. 2.14. Catalase activity The cells were seeded at 1 × 105 cells/ml, and at 16 h after plating, the cells were treated with hyperoside for 3 h. The harvested cells were suspended in 10 mM phosphate buffer (pH 7.5) and then lysed on ice by sonication twice for 15 s. Triton X-100 (1%) was then added to the lysates which was further incubated for 10 min on ice. The lysates were centrifuged at 5000 ×g for 30 min at 4 °C to remove the cellular debris and the protein content was determined. For detection catalase activity, 50 µg of protein was added to 50 mM of phosphate buffer (pH 7) containing 100 mM of H2O2. The reaction mixture was incubated for 2 min at 37 °C and the absorbance was monitored at 240 nm for 5 min. The changes in absorbance with time were proportional to the breakdown of H2O2. The catalase activity was expressed as U/mg protein where 1 U of enzyme activity was defined as the amount of enzyme required to breakdown of 1 µM of H2O2 [21]. Fig. 2 (continued).

lysis solution (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Trion X-100, and 10% DMSO, pH 10) for 1 h at 4 °C. The slides were then placed in a gel-electrophoresis apparatus containing 300 mM NaOH and 10 mM NaEDTA (pH 13) for 40 min to allow DNA unwinding and the expression of the alkali labile damage. An electrical field was applied (300 mA, 25 V) for 20 min at 4 °C to draw negatively charged DNA toward an anode. After electrophoresis, the slides were washed three times for 5 min at 4 °C in a neutralizing buffer (0.4 M Tris, pH 7.5) and then stained with 75 µl of ethidium bromide (20 µg/ml). The slides were observed using a fluorescence microscope and image analysis (Komet, Andor Technology, Belfast, UK). The percentage of total fluorescence in the tail and the tail length of the 50 cells per slide were recorded. 2.12. Western blot The cells were harvested, washed twice with PBS, lysed on ice for 30 min in 100 µl of a lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP 40] and then centrifuged at 13,000 ×g for 15 min. The supernatants were collected from the lysates and the protein concentrations were

Fig. 3. The effect of hyperoside on H2O2-induced cell damage. The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate. After an incubation of 24 h, cell viability was determined by the MTT assay. ⁎Significantly different from control cells (p b 0.05). ⁎⁎Significantly different from H2O2 treated cells (p b 0.05).

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2.15. Glutathione peroxidase activity Fifty µg of the protein was added to 25 mM phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM NaN3, 1 mM glutathione (GSH), 0.25 U of glutathione reductase, and 0.1 mM NADPH. After incubation for 10 min at 37 °C, H2O2 was added to the reaction mixture at

a final concentration of 1 mM. The absorbance was measured at 340 nm for 5 min. The activity of glutathione peroxidase was measured as the rate of NADPH oxidation [22,23]. Glutathione peroxidase activity was expressed as U/mg protein, and 1 U of enzyme activity was defined as the amount of enzyme required to oxidize 1 mM NADPH.

Fig. 4. The effect of hyperoside on H2O2-induced apoptosis. The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate. After an incubation of 24 h, (A) the apoptotic body formation was observed under a fluorescent microscope after Hoechst 33342 staining. The apoptotic bodies are indicated with arrows. (B) The apoptotic subG1 DNA content was detected by flow cytometry after propidium iodide staining and (C) DNA fragmentation was quantified by ELISA kit. The measurements were made in triplicate and the values were expressed as means ± SE. ⁎Significantly different from control cells (p b 0.05). ⁎⁎Significantly different from H2O2-treated cells (p b 0.05). (D) After the cell lysates were electrophoresed, caspase 3 and PARP were detected by a specific antibody.

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Fig. 5. The effect of hyperoside on lipid peroxidation. The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. Lipid peroxidation was assayed by measuring the amount of TBARS formation. The measurements were made in triplicate and the values were expressed as means ± SE. ⁎Significantly different from control cells (p b 0.05). ⁎⁎Significantly different from H2O2-treated cells (p b 0.05).

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treated cells in comparison to H2O2 alone treated cells; 56% of the cells were viable in hyperoside pretreated and H2O2 treated cells as compared to 42% of the cells viable in H2O2 alone treated group. Moreover, to evaluate the cytoprotective effects of hyperoside on apoptosis induced by H2O2, the nuclei of the V79-4 cells were stained with Hoechst 33342 and assessed by microscopy. The microscopic pictures in Fig. 4A revealed that the control cells had intact nuclei, while the H2O2-treated cells showed significant nuclear fragmentation, which is indicative of apoptosis. However, when the cells were treated with hyperoside for 1 h prior to H2O2 treatment, a decrease in nuclear fragmentation was observed. As shown in Fig. 4B, an analysis of the apoptotic sub-G1 DNA content in the H2O2-treated cells revealed a 65% increase in the apoptotic sub-G1 DNA content. Moreover, treatment with 5 µM of hyperoside decreased the apoptotic subG1 DNA content to 37%. The treatment of cells with H2O2 was found to increase the levels of cytoplasmic histone-associated DNA fragmentation compared to the control group. However, treatment with 5 µM of hyperoside decreased the degree of DNA fragmentation (Fig. 4C). Next, we examined the activity of caspase 3 by western blot, since caspase 3 is known as an executioner of apoptosis [24]. Hyperoside inhibited the H2O2-induced active form of caspase 3 (17 and 19 kDa), which is further demonstrated by the cleavage of PARP (89 kDa) (Fig. 4D). These results suggest that hyperoside protects cell viability by inhibiting H2O2-induced apoptosis.

2.16. Statistical analysis All the measurements were made in triplicate and all values were represented as means ± standard error (SE). The results were subjected to an analysis of the variance (ANOVA) using the Tukey test to analyze the differences. p b 0.05 was considered to be significant. 3. Results 3.1. The effect of hyperoside on ROS scavenging The intracellular ROS scavenging activity of hyperoside in V79-4 cells after H2O2 treatment was detected by the DCF-DA assay. The fluorescence spectrometric data revealed that the intracellular ROS scavenging activity of hyperoside was in a dose dependent pattern; 30% at 1 μM, 70% at 2.5 µM, and 84% at 5 µM. (Fig. 2A). However, there was no clear difference of ROS scavenging effect at 5, 25, 50, and 100 µM of hyperoside (data not shown). Therefore, we determined to choose 5 µM as an optimal dose of hyperoside for further study. Moreover, the fluorescence intensity of DCF-DA staining was measured using a flow cytometer and a confocal microscope. The level of ROS detected using a flow cytometer revealed a fluorescence intensity value of 422 for the ROS stained by DCF-DA fluorescence dye in H2O2 with hyperoside (5 µM) treated cells, compared to a fluorescence intensity value of 527 in H2O2 treated cells (Fig. 2B). Moreover, the results of confocal microscopy revealed that hyperoside reduced the red fluorescence intensity with H2O2 treatment (Fig. 2C), thus indicating a reduction in ROS generation. In addition, the hydroxyl radicals generated by the Fenton reaction (FeSO4 + H2O2) in a cell-free system was detected by ESR spectrometery. The ESR data revealed that a signal was not observed for the control and hyperoside at 5 µM, however, the signal of the hydroxyl radical increased up to 4893 in the FeSO4 + H2O2 system. Hyperoside treatment decreased hydroxyl radical signal to 2345 (Fig. 2D).

3.3. The effect of hyperoside against the damage of cellular components induced by H2O2 treatment H2O2-induced damage to cellular components is the important lesions responsible for the loss of cell viability. The effect of hyperoside on the damage of membrane lipid, protein, and cellular DNA in H2O2treated cells was investigated. As shown in Fig. 5, V79-4 cells exposed to H2O2 revealed an increase in lipid peroxidation, which was substantiated by the generation of TBARS. However, hyperoside prevented the H2O2-induced peroxidation of lipids. The protein carbonyl formation serves as a biomarker for cellular oxidative damage [25]. Moreover, the protein carbonyl content in cells increased significantly after H2O2 treatment, and hyperoside prevented the H2O2-induced protein carbonyl formation (Fig. 6). Moreover, damage to cellular DNA induced by H2O2 exposure was detected by an alkaline comet assay, and by phospho histone-H2A.X expression. The exposure of cells to H2O2 was found to increase the tail length and percentage of

3.2. The effect of hyperoside on H2O2-induced cell death The protective effect of hyperoside on cell survival in H2O2-treated cells was measured using the MTT test. Cells were treated with hyperoside at 5 µM for 1 h prior to the addition of H2O2. The cell viability was determined 24 h later by the MTT assay. As shown in Fig. 3, cell survival increased by 14% in hyperoside pretreated and H2O2

Fig. 6. The effect of hyperoside on protein carbonyl formation. The cells were treated with hyperoside at 5 µM. After 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 24 h. Protein oxidation was assayed by measuring the amount of carbonyl formation. The measurements were made in triplicate and the values were expressed as means ± SE. ⁎Significantly different from control cells (p b 0.05). ⁎⁎Significantly different from H2O2-treated cells (p b 0.05).

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Fig. 7. The effect of hyperoside on DNA damage. Cells were treated with hyperoside at 5 µM for 30 min later, 1 mM H2O2 was added and incubated for 15 min. (A) percentage of cellular DNA damage were detected by an alkaline comet assay. ⁎Significantly different from control cells (p b 0.05). ⁎⁎Significantly different from H2O2-treated cells (p b 0.05). The cells were treated with hyperoside at 5 µM. After 1 h,1 mM of H2O2 was added to the plate. After an incubation of 24 h, (B) the cell lysates were electrophoresed and phospho histone H2A.X protein was detected by a specific antibody. (C) The confocal image shows that the FITC-conjugated secondary antibody staining indicates the location of phospho histone H2A.X (green) by the anti-phospho histone H2A.X antibody. The DAPI staining indicates the location of the nucleus (blue), and the merged image indicates the location of the phospho histone H2A.X protein in the nucleus.

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DNA in the tails of the cells. Moreover, when the cells were exposed to H2O2, the percentage of DNA in the tail increased to 59%, whereas treatment with hyperoside at 5 µM decreased the percentage of DNA in the tail to 38% (Fig. 7A). The phosphorylation of the nuclear histone H2A.X, a sensitive marker for breaks of double stranded DNA [26], increased in the H2O2-treated cells, as shown by western blot and immuno-fluorescence image (Figs. 7B and C). However, hyperoside treatment in H2O2-treated cells decreased the expression of phospho H2A.X. These results suggest that hyperoside protects cell viability by inhibiting the damage of cellular components induced by H2O2. 3.4. The effect of hyperoside on catalase and glutathione peroxidase The activity of catalase and glutathione peroxidase was measured to investigate whether the radical scavenging activity of hyperoside is mediated by catalase and glutathione peroxiase, which convert H2O2 into molecular oxygen and water [27]. We found that hyperoside increased catalase activity in a concentration dependent manner (Fig. 8A). To confirm the activation of catalase by hyperoside in terms of the protein expression, a western blot analysis was performed. As shown in Fig. 8B, the protein expression of catalase significantly increased in the presence of hyperoside at 5 µM. To determine the effect of catalase on hyperoside-induced cytoprotection from H2O2induced damage, the cells were pretreated with ATZ, which is a specific inhibitor of catalase [28]. As shown in Fig. 8C, ATZ treatment abolished the protective effect of hyperoside in H2O2-damaged cells. In addition the induction of catalase, hyperoside increased glutathione peroxidase activity (Fig. 8D). 4. Discussion We found hyperoside to decrease the intracellular ROS as well as increase antioxidant enzyme activity. Hyperoside was reported to permeate the cell membrane as well as inhibit free radical formation and the propagation of free radical reactions by chelating transition metal ions in the cells [1,4]. Hyperoside possess the intrinsic feature of low bioavailability because its sugar moiety lowers the permeability of the cell membrane. It has been reported that flavonoid glycosides are hydrolyzed to their corresponding aglycones before entering the biological system [29,30], which means that hyperoside itself could be detected in cells with small amount [4]. Therefore, the antioxidant effect of hyperoside may be in its aglycone or biotransformed form. Hyperoside has a polyphenolic structure, which may be related to the antioxidant property of hyperoside. In our system, the antioxidant effect of hyperoside might involve dual actions: (1) direct action on ROS radical scavenging, as shown by ESR data, and (2) indirect action via induction of catalase and glutathione peroxidase activities. It is known that flavonoids have antioxidant activity by a free radical scavenging mechanism with the formation of less reactive phenoxyl radicals by donation of electron or hydrogen [31,32]. Another antioxidant mechanism of flavonoids is based on the ability to chelate transition metal like iron, thereby suppressing the hydrogen peroxidedriven Fenton reaction. Iron chelating properties and radical scavenging activity of flavonoids are closely related; iron is chelated by the flavonoid and the reactive oxygen species which are formed in its vicinity are subsequently scavenged by the flavonoids [33]. In that case, the flavonoids would have a double, synergistic action, which would make it a powerful antioxidant [34]. For example, one of the well Fig. 8. The effects of hyperoside on catalase and glutathione peroxidase. The catalase activity (A) is expressed as the average enzyme U/mg protein ± SE. ⁎Significantly different from the control (p b 0.05). (B) The cell lysates were electrophoresed and the protein expression of catalase was detected by a specific antibody. (C) After treatment of ATZ, hyperoside or/and H2O2, the cell viability was determined by the MTT assay. ⁎Significantly different from control cells (p b 0.05). ⁎⁎Significantly different from H2O2treated cells (p b 0.05). (D) The glutathione peroxidase activity is expressed as the average enzyme U/mg of protein ± SE. ⁎Significantly different from the control (p b 0.05).

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known radical scavengers, quercetin showed highly effective chelating activity on transition metals [35]. Chelating properties of flavonoids depend on the structural features like catechol group on the B-ring, 4carbonyl, and 5-hydroxyl group, and Fenton-induced oxidation is strongly inhibited by flavonoids with 3′, 4′-catechol, 4-carbonyl, and 5hydroxyl group [36–38]. Thus on the basis of structural criteria accounting for metal chelation, hyperoside might have possible metal binding sites between the 5-hydroxyl and 4-carbonyl group, or between 3′ and 4′-hydroxyl group, thereby chelating metal ions. Although the antioxidant activity of flavonoids is believed to be caused by a combination of iron chelation and free radical scavenging activities, there are somewhat controversial results in the literature and the outcome largely depends on the experimental conditions and the type of assay used [34]. Therefore, further investigation on the contribution of iron chelation to the antioxidant activity of hyperoside is remained for further study. The influence of iron chelation on the antioxidant activity of hyperoside needs to be compared in biological relevant assay system between dependent and independent of iron individually. Catalase is located in the peroxisome and it converts H2O2 into molecular oxygen and water. Similarly, glutathione peroxidase is a selenoprotein and also converts H2O2 into molecular oxygen and water. Catalase and glutathione peroxidase play an important role in the cellular protection from oxidative stress-induced cell damage [39– 41]. The V79-4 cells exposed to H2O2 exhibited distinct features of apoptosis, including nuclear fragmentation, sub G1-hypo-diploid cells, and DNA fragmentation, and the activation of caspase 3. However, cells that were pretreated with hyperoside, had a significant reduction in the percentage of apoptotic cells. These findings suggest that hyperoside inhibits H2O2-induced apoptosis via its antioxidant effect. H2O2 is one of the major reactive oxygen species related to oxidative stress. It facilely penetrates into cells and proceeds to react with intracellular ions such as iron and copper, to generate the highly reactive hydroxyl radicals which successively attack cellular components including lipids, proteins, and DNA to cause various oxidative damages [42,43]. H2O2-induced cell membrane lipid peroxidation is one of the most important lesions responsible for the loss of cell viability. Moreover, oxidative damage of amino acid residues in proteins results in the formation of carbonyl derivatives [44]. In addition, the oxidative damage of DNA involves both base modification and DNA strand breakage, which ultimately may lead to genetic alteration [45]. In the present study, considerable amounts of lipid, protein, and DNA damage were produced in V79-4 cells after H2O2 exposure, and it was shown that the hyperoside pretreatment significantly reduced lipid, protein, and DNA damage, which consequently protected the cells from cell death. It is thus invoked that hyperoside-treated cells modulate the cellular antioxidant potential by either acting as a free radical scavenger or activating antioxidant enzyme activity. Further studies are underway to elucidate the precise mechanism of the hyperoside effect for the modulation of cellular antioxidant defenses or the various pathways leading to cell death. Acknowledgements This research was supported by the DNA repair regulation with disease program [M1063901] of the Korea Science and Engineering Foundation (KOSEF) and by the program of Basic Atomic Energy Research Institute (BAERI) which is a part of the Nuclear R and D programs grant from the Ministry of Science and Technology of Korea. References [1] Y. Zou, Y. Lu, D. Wei, Antioxidant activity of a flavonoid-rich extract of Hypericum perforatum L. in vitro, J Agric. Food Chem. 52 (2004) 5032–5039. [2] L. Luo, Q. Sun, Y.Y. Mao, Y.H. Lu, R.X. Tan, Inhibitory effects of flavonoids from Hypericum perforatum on nitric oxide synthase, J. Ethnopharmacol. 93 (2004) 221–225.

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