Chemico-Biological Interactions 207 (2014) 7–15

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Enhanced antioxidant effect of caffeic acid phenethyl ester and Trolox in combination against radiation induced-oxidative stress Hua Bai 1, Rui Liu 1, Hong-Li Chen, Wei Zhang, Xin Wang, Xiao-Di Zhang, Wen-Li Li, Chun-Xu Hai ⇑ Department of Toxicology, Shaanxi Provincial Key Lab of Free Radical Biology and Medicine, The Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi’an 710032, China

a r t i c l e

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Article history: Received 13 June 2013 Received in revised form 17 October 2013 Accepted 28 October 2013 Available online 6 November 2013 Keywords: Caffeic acid phenethyl ester Trolox Radiation Oxidative stress Nrf2

a b s t r a c t Combinations of antioxidants are believed to be more effective than single antioxidant because when antioxidants are combined they support each other synergistically to create a magnified effect. Discovering the enhancer effects or synergies between bioactive components is valuable for resisting oxidative stress and improving health benefits. The aim of this study was to investigate a possible cooperation of natural antioxidant caffeic acid phenethyl ester (CAPE) with synthetic antioxidant Trolox in the model systems of chemical generation of free radicals, lipid peroxidation of microsomes and radiation-induced oxidative injury in L929 cells. Based on the intermolecular interaction between CAPE and Trolox, the present study shows a synergistic effect of CAPE and Trolox in combination on elimination of three different free radicals and inhibition of lipid peroxidation initiated by three different systems. CAPE and Trolox added simultaneously to the L929 cells exerted an enhanced preventive effect on the oxidative injury induced by radiation through decreasing ROS generation, protecting plasma membrane and increasing the ratios of reduced glutathione/oxidized glutathione and the expression of key antioxidant enzymes mediated by nuclear factor erythroid 2 p45-related factor 2 (Nrf2). Our results showed for the first time that administration of CAPE and Trolox in combination may exert synergistic antioxidant effects, and further indicate that CAPE and Trolox combination functions mainly through scavenging ROS directly, inhibiting lipid peroxidation and promoting redox cycle of GSH mediated by Nrf2-regulated glutathione peroxidase and glutathione reductase expression. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction It is now widely accepted that oxidative stress induced by reactive oxygen species (ROS) is involved in the pathogenesis of various diseases ranging from cancer to degenerative diseases. Consequently the role of antioxidants in the prevention and treatment of diseases has received much attention of scientists, clinicians and general public [1–6]. Since the activity of endogenous antioxidants cannot be deliberately increased, it would be reasonable to increase cell antioxidant capacity using exogenous antioxidant compounds, derived from the diet, in order to increase cell defenses against oxidative stress. Besides being effective alone, accumulating studies have suggested that supplementation with a combination of antioxidants may be more effective than that with single antioxidants considering the multiform and different chemistry and biochemistry of ROS, the interaction between antioxidants and the different antioxidant mechanisms [2,7–10]. In addition, many single antioxidants would need to be taken in ⇑ Corresponding author. Tel.: +86 02983374879; fax: +86 02984774879. 1

E-mail address: [email protected] (C.-X. Hai). Co authors.

0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.10.022

dangerously high doses to create a significant antioxidant effect. Accordingly, the search for newer combination of natural antioxidants, especially of food origin, has ever since increased. Caffeic acid phenethyl ester (CAPE) is one of the major components of honeybee propolis and its structure is similar to flavonoids. CAPE has been demonstrated to have some biological and pharmacological properties, such as antioxidant, antiinflammatory, anticarcinogenic and immunomodulatory activities [11]. CAPE was shown to exert its antioxidant activity by suppressing lipid peroxidation (LPO), scavenging the reactive oxygen species, inhibiting xanthine oxidase and nitric oxide synthase activities, and preventing the consumption of superoxide dismutase activity [12–15]. Because flavonoids are consumed with other antioxidants in the diet, their combination may prove to be more beneficial [16]. Yet until now, few studies have ever been done to examine the combination antioxidant effect of CAPE and other antioxidants in vitro and in vivo. Therefore, the present investigation was undertaken to determine in vitro protective effect of CAPE in combination with Trolox, a water-soluble analog of vitamin E. Trolox is a powerful free radical scavenger and the carboxyl group present within the structure imparts water solubility which makes the use of Trolox more

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advantageous over other active antioxidants (e.g., vitamin E) which are only lipid-soluble [17]. In this work, we have investigated the direct scavenging effect of CAPE and Trolox on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radicals and superoxide anions, which are general indexes of antioxidant activity. And using rat liver microsomal membrane models, we also have evaluated the antioxidant activity of CAPE and Trolox in inhibiting LPO induced by three different sources of free radicals. In addition, we have evaluated the cytoprotective effect of CAPE and Trolox against radiation-induced oxidative injury in L929 cells.

2. Materials and methods 2.1. Materials and reagents Nuclear factor E2-related factor 2 (Nrf2), CuZn superoxide dismutase (CuZnSOD), Mn superoxide dismutase (MnSOD), LaminB, and b-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Glutathione peroxidase (Gpx), Glutathione reductase (GR) and catalase (CAT) antibody were purchased from Abcam (Cambridge, MA). CAPE, Trolox, cuminehydroperoxide (CHP), D-glucose 6-phosphate, glucose 6-phosphate dehydrogenase, 2,20 -diphenyl-1-picrylhydrazyl (DPPH) and dihydroethidium (DHE) were purchased from Sigma Chemical Co. (St. Louis, MO). Thiobarbituric acid (TBA) was obtained from Merck (Germany). Reduced form of nicotinamideadenine dinucleotide phosphate tetrasodium salt (NADPHNa4) was purchased from Roche. Other chemicals and reagents used in this study, unless indicated, were also from Sigma.

2.2. UV–vis absorption spectra UV–vis absorption spectra were measured by using a Shimadzu UV–vis spectrophotometer at room temperature. The mixtures were prepared by mixing CAPE & Trolox compound directly in water at room temperature.

2.3. DPPH radical-scavenging activity The stable free radical-scavenging activity was determined by the DPPH assay as we described previously [18]. 200 ll of 100 lM DPPH in methanol was mixed with 20 ll samples (CAPE, Trolox, or CAPE and Trolox) dissolved in ethanol and kept in dark for 20 min. The quantity of DPPH remaining in the mixed solution was measured at 517 nm. Ethanol without the sample was employed as a control. The DPPH radical-scavenging activity was calculated according to the following formula: (%) inhibition ratio = [(Abscontrol  Abssample)/Abscontrol]  100. 2.4. Hydroxyl radical-scavenging activity Co(II)/EDTA-induced luminolchemiluminescence measurements were carried out as previously described [19]. 1 ml boric acid buffer solution (0.05 M, adjusted to pH 9 with 1 M NaOH) containing 1 mg/ml EDTA and 0.2 mg/ml CoCl26H2O was vortexed for 15 s with 50 ll of luminol solution (5.6  104 M) in carbonic acid (0.5 M, adjusted to pH 10 with 1 M NaOH) with (I) or without (I0) 50 ll of sample in a glass cuvette. Then, 25 ll of H2O2 aqueous solution (5.4  103 M) was added and the mixture was vortexed again for 10 s and taken fleetly into the detecting instrument. The 30 s chemiluminescence intensity was recorded (each measurement repeated six times). The inhibitory effect was calculated as: (%) inhibition ratio = (I0  I)/I0  100.

2.5. Superoxide anion radical-scavenging activity Xanthine oxidase and xanthine-induced luminolchemiluminescence measurements were carried out as previously described [19]. 0.6 ml luminol solution (0.1 mM) in carbonic acid (0.5 M, adjusted to pH 10 with 1 M NaOH) was vortexed for 15 s with 0.6 ml 140 lM xanthine in carbonic acid (0.5 M, adjusted to pH 10 with 1 M NaOH) containing 120 lM FeSO47H2O with (I) or without (I0) 50 ll of sample in a glass cuvette. Then, 10 ll of xanthine oxidase solution (4 U/ml in 50 mM phosphoric acid, adjusted to pH 7.8, containing 10 mM EDTA) was added and the mixture was vortexed again for 10 s and taken fleetly into the detecting instrument. The 30 s chemiluminescence intensity was recorded (each measurement repeated six times). The inhibitory effect was calculated as: (%) inhibition ratio = (I0  I)/I0  100. 2.6. Preparations of liver microsomes Rat liver microsomes were prepared by standard differential centrifugation techniques as previously described [20]. Briefly, male Sprague–Dawley rats (180–220 g) were killed by decapitation. The livers were quickly removed, washed with ice-cold saline and weighed. One gram of liver was homogenized with 4 ml of icecold 0.1 M Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged at 14,000g for 30 min. The supernatant was collected and centrifuged at 165,000g for 60 min. The resultant microsomes were washed with 0.1 M Tris–HCl buffer (pH 7.4) and suspended in the same buffer. Aliquots of microsomal suspensions were stored at 80 °C. 2.7. Vitamin C/Fe2+-induced lipid peroxidation Lipid peroxidation of microsomes was performed as described previously [21]. The microsomes (2 mg microsomal protein/ml) were pre-incubated with (I) or without (I0) different samples in the presence of 0.1 M Tris–HCl buffer (pH 7.4) and FeSO4 (50 lM). LPO was initiated by adding ascorbate (5 mM), and the samples were then incubated at 37 °C for 15 min. The extent of lipid peroxidation was detected by the TBA method using trichloroacetic acid (10% w/v) and TBA (0.67% w/v) measuring the absorbance at 535 nm [22]. The thiobarbituric acid reactive substances (TBARS) were calculated as malondialdehyde (MDA) equivalents. Appropriate controls were performed to discard any possible interference with the assay. The inhibitory effect on vitamin C/Fe2+-induced LPO was calculated as: (%) inhibition ratio = (I0  I)/I0  100. 2.8. Cuminehydroperoxide (CHP)-induced lipid peroxidation The microsomes (2 mg microsomal protein/ml) were pre-incubated with (I) or without (I0) different samples in the presence of 0.1 M Tris–HCl buffer (pH 7.4). LPO was induced by CHP (1 mM), and the samples were then incubated at 37 °C for 15 min. TBARS were determined as described above. 2.9. CCl4/NADPH-induced lipid peroxidation The reaction mixture contained 2 mg microsomal proteins and a NADPH-generating system (0.2 mM NADP+, 4 mM glucose 6phosphate and 0.8 units glucose-6-phosphate dehydrogenase) in the 0.1 M Tris–HCl buffer (pH 7.4) with (I) or without (I0) testing samples. LPO was initiated by CCl4:DMSO (1:4, v/v) [23]. After 15 min incubation at 37 °C, TBARS were determined as described above.

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2.10. Cell culture and treatments The mouse fibroblast cell lines L929 (American Type Culture Collection, Rockville, MD, USA) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). All cells were incubated in a 5% CO2 atmosphere at 37 °C. When cells reached 60–70% confluence in 6-well, 24-well or 96-well plate, the medium was removed and cells were rinsed with PBS. Then the cells were exposed to 20 Gy radiation using a cobalt60 apparatus (gamma energy, with a distance of 50 cm from the source to the surface). The dose rate of radiation was 777.76 cGy/ min. Control cells were subjected to sham-radiation. After radiation, cells were placed into serum-free medium containing CAPE and/or Trolox. CAPE and Trolox was dissolved in ethanol at a concentration of 50 mM, and then freshly diluted with culture medium to the 50 lM working concentration. Culture medium with ethanol served as the control. Cells were incubated for another 4 h before collected for further experimental procedures. 2.11. Cell viability assays Cell viability was measured using a microculturetetrazolium (MTT) assay. Briefly, cells were treated with 0.5 mg/ml MTT at 37 °C. Four hours later, the formazan crystals were dissolved in DMSO, and the absorption values were determined at 492 nm on an automated Bio-Rad 550 microtiter plate reader.

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materials. To test GSSG, sample was incubated at room temperature with 0.04 M N-ethylmaleimide solution for 30 min to prevent interference of GSH with measurement of GSSG. Then 100 ll of sample was placed in a glass centrifuge tube. 1.9 ml of 0.1 M NaOH and 100 ll of 0.1% o-phthalaldehyde were added and mixed. To test GSH, 0.1 M phosphate buffer (pH 8.0, 5 mM EDTA) was used as the diluent instead of 0.1 M NaOH. After being kept at room temperature for 40 min, the mixture was measured with a fluorometric spectrophotometer at 420 nm with excitation at 350 nm. Commercially procured GSSG or GSH was used to establish a standard curve. The results were expressed in micromoles GSSG or GSH per gram protein. 2.15. Western blotting Equal amounts of total and nuclear proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis and electro-transferred onto a nitrocellulose membrane. Membranes were incubated with antibodies to Nrf2, MnSOD, CuZnSOD, CAT, Gpx, GR. The secondary antibodies used for detection were HRPconjugated anti-rabbit and anti-mouse IgG. Immunoreactive bands were detected by an enhanced chemiluminescence kit (Millipore, USA) and results were quantitated using an image analyzer Quantity One System (BIO-RAD, USA). Proteins were assayed by the method of Lowry. b-Actin and LaminB were used as loading control for total and nuclear fractions, respectively.

2.12. ROS determination 2.16. Statistical analysis Intracellular ROS were assessed using DHE as previously described [24]. L929 cells were cultured on plastic coverslips in 24well plates. DHE (5 lM) was added as a fluorescent indicator of ROS and incubated for 30 min in a humidified chamber at 37 °C. Then coverslips were washed with PBS, fixed with 4% paraformaldehyde, and mounted to glass slides. Images were obtained with an Olympus DP70 fluorescence microscope. Relative fluorescence intensity was quantified using Image Pro 6.0 software. A minimum of three independent samples was analyzed per treatment. ROS levels were expressed as percentage of control. 2.13. Determination of lipid peroxidation The lipid peroxidation products in the cell were determined by measuring TBARS. The method of Yagi [25] was used with some modifications. Briefly, 20 ll of sample was placed in a glass centrifuge tube. 4.0 ml of 1/12 N H2SO4 was added and mixed gently. Then, 0.5 ml of 10% phosphotungstic acid was added and mixed. After allowing it to stand at room temperature for 5 min, the mixture was centrifuged at 1600g for 10 min. The supernatant was discarded and the sediment mixed with 2.0 ml of 1/12 N H2SO4 followed by 0.3 ml of 10% phosphotungstic acid. The mixture was centrifuged at 1600g for 10 min. The sediment was then suspended in 1.0 ml of distilled water and 1.0 ml of 0.67% (w/v) TBA reagent was added. The reaction mixture was heated at 95 °C for 60 min. After cooling with tap water, 5.0 ml of n-butanol was added and the mixture was shaken vigorously. After centrifugation at 1600g for 15 min, the n-butanol layer was taken for fluorometric measurement at 553 nm with excitation at 515 nm. 1,1,3,3-Tetraetoxypropane was used as the primary standard. 2.14. Determination of oxidized glutathione (GSSG), reduced glutathione (GSH) GSH and GSSG were determined according to the protocol described previously [26]. When the pH is 8 or 12, ophthaldialdehyde may react with GSH or GSSG, respectively, generating fluorescent

All values were expressed as means ± SD. The results were analyzed by one-way ANOVA followed by LSD test for multiple comparisons. Factorial analysis (Two-way ANOVA) was used to assess synergy. Differences were considered statistically significant at P < 0.05. 3. Results 3.1. UV–vis absorption spectrum of CAPE and Trolox In order to unequivocally investigate that CAPE and Trolox may form complex and show interaction between them, UV–vis absorption spectrum of CAPE, Trolox and their mixture were measured with the concentration of 1.0 M at room temperature on Shimadzu UV–vis spectrophotometer. As shown in Fig. 1, CAPE (1.0 lM) had a maximum absorption (0.443) peak at 330 nm and one shoulder absorption (0.345) peak at 306 nm, while Trolox (1.0 lM) only had a single maximum absorption (0.106) peak at 290 nm. Since the mixture of CAPE (0.5 lM) and Trolox (0.5 lM) showed a shifted maximum absorption (0.260) peak at 294 nm and one shoulder absorption (0.234) peak at 330 nm, which clearly indicated the intermolecular interaction between CAPE and Trolox together with comparing the half absorption (0.213) of individual CAPE (0.323) + Trolox (0.102) at 294 nm. 3.2. Scavenging effect on free radicals by CAPE and Trolox in combination Fig. 2A shows the activities of CAPE, Trolox and their mixture in reducing DPPH free radical. CAPE or Trolox alone and their combination caused concentration-dependent (from 25 to 200 lM) inhibition of DPPH when compared with control. The estimated half maximal inhibitory concentrations (IC50) were 221.4 ± 12.3 lM (Trolox), 154.9 ± 10.1 lM (CAPE) and 93.0 ± 16.5 lM (CAPE + Trolox). Further analyses showed a synergistic effect of CAPE and

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The estimated IC50 were 1.37 ± 0.21 lM (Trolox), 0.506 ± 0.083 lM (CAPE) and 0.258 ± 0.041 lM (CAPE + Trolox). Combination of CAPE and Trolox had a synergistic effect at the concentration of 0.125 lM (Trolox, 5.3 ± 4.3%; CAPE, 22.0 ± 1.6%; Trolox + CAPE, 35.4 ± 2.5%) and 0.25 lM (Trolox, 5.3 ± 5.5%; CAPE, 30.4 ± 2.1%; Trolox + CAPE, 52.2 ± 3.5%).

3.3. Inhibition of lipid peroxidation in rat liver microsomes by CAPE and Trolox in combination

Fig. 1. Absorption spectrum of CAPE (black closed square), Trolox (blue open circle) and the mixture of CAPE & TROLOX (red closed circle) in water at the concentration of 1.0 lM, 294 and 330 nm were indicated under green straight line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Trolox in combination at the concentration of 100 lM (Trolox, 23.5 ± 0.7%; CAPE, 30.4 ± 1.6%; Trolox + CAPE, 56.8 ± 1.3%). As shown in Fig. 2B, CAPE or Trolox alone and their combination showed concentration-dependent (from 0.0125 to 0.2 lM) hydroxyl radical-scavenging activity when compared with control. The estimated IC50 were 0.1 ± 0.01 lM (Trolox), 0.06 ± 0.008 lM (CAPE) and 0.026 ± 0.005 lM (CAPE + Trolox). Combination of CAPE and Trolox had a synergistic effect at the concentration of 0.025 lM (Trolox, 13.3 ± 2.6%; CAPE, 24.6 ± 4.3%; Trolox + CAPE, 57.2 ± 3.5%) and 0.05 lM (Trolox, 26.4 ± 4.2%; CAPE, 49.4 ± 5.2%; Trolox + CAPE, 88.7 ± 8.4%). As shown in Fig. 2C, CAPE or Trolox alone and their combination showed concentration-dependent (from 0.125 to 2 lM) superoxide anion radical-scavenging activity when compared with control.

In the vitamin C/Fe2+-induced LPO system (Fig. 3A), CAPE given alone concentration-dependently reduced the extent of TBARS formation compared with the control, whereas Trolox given alone was without effect. Combination of CAPE and Trolox had a dosedependent synergistic effect at the concentration of 6.25 lM (Trolox, 0.5 ± 4.2%; CAPE, 1.9 ± 2.8%; Trolox + CAPE, 10.6 ± 1.8%), 12.5 lM (Trolox, 2.4 ± 2.4%; CAPE, 13.0 ± 3.4%; Trolox + CAPE, 15.9 ± 3.8%) and 25 lM (Trolox, 5.3 ± 3.4%; CAPE, 38.7 ± 1.9%; Trolox + CAPE, 51.7 ± 4.0%). However, this effect was not observed at higher doses (50 lM) of the combination. In the CHP-induced lipid peroxidation system (Fig. 3B), the inhibitory effect of CAPE on the TBARS formation was concentration-depended and the highest effects were seen for CAPE’s concentration 50 lM. Combination of CAPE and Trolox had a synergistic effect at the concentration of 12.5 lM (Trolox, 2.9 ± 1.0%; CAPE, 9.6 ± 2.0%; Trolox + CAPE, 15.4 ± 1.3%) and 25 lM (Trolox, 0.7 ± 0.8%; CAPE, 17.6 ± 1.2%; Trolox + CAPE, 33.1 ± 1.8%), whereas Trolox alone was without effect at these doses. In the CCl4/NADPH-induced LPO system (Fig. 3C), a dose-dependent anti-lipid peroxidation activity of CAPE were observed (from 6.25 to 50 lM), whereas a promoting LPO activity of Trolox (from 6.25 to 25 lM) were observed. A synergistic effect of TBARS inhibition were again observed when CAPE + Trolox was given at doses of 12.5 lM (Trolox, 10.7 ± 3.4%; CAPE, 32.0 ± 1.5%; Trolox + CAPE, 49.3 ± 2.5%), 25 lM (Trolox, 14.7 ± 2.8%; CAPE, 39.3 ± 1.6%; Trol-

Fig. 2. Effects of CAPE, Trolox and their combination on scavenging free radicals. DPPH radicals (A), hydroxyl radicals (B), and superoxide anion radicals (C) were induced by different models. The values are expressed as mean ± SD (n = 6). The symbol ⁄ signifies enhanced effect (P < 0.05).

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Fig. 3. Effects of CAPE, Trolox and their combination on lipid peroxidation in rat liver microsomes initiated by vitamin C/Fe2+ (A), CHP (B), or CCl4/NADPH (C). The symbol ⁄ signifies enhanced effect (P < 0.05).

ox + CAPE, 66.7 ± 1.4%), and 50 lM (Trolox, 4.0 ± 1.9%; CAPE, 51.3 ± 1.9%; Trolox + CAPE, 82.0 ± 1.0%). 3.4. Effect of CAPE and Trolox in combination against radiation-induced oxidative injury in L929 cells L929 cells were used to investigate the protective effect of CAPE and Trolox on irradiation-induced cytotoxicity and oxidative stress. As showed in Fig. 4A, cell viability decreased markedly in the cells treated by radiation (41.0 ± 5.3%). CAPE and Trolox played a protective role against the toxicity induced by radiation. The cell viability in the combination group (82.0 ± 10.1%) was significant higher compared to both CAPE (64.0 ± 7.9%) and Trolox (55.0 ± 11.4%) alone. Changes of ROS level were analyzed using the DHE probe. As shown in Fig. 4B and C a highest fluorescent intensity was observed in the radiation group (76.0 ± 21.4%), as expected. The treatment with CAPE or Trolox significantly decreased ROS level compared with that of the radiation group. The combination of these compounds had a stronger inhibition (29.5 ± 16.3%) than CAPE (47.4 ± 23.5%) or Trolox (62.3 ± 10.4%) individually (P < 0.05). TBARS is an index of lipid peroxidation and oxidative stress. As shown in Fig. 4D, radiation markedly increased the content of TBARS from 280.0 ± 23.4 to 1634.0 ± 98.4 lM/g protein, which was inhibited by CAPE (645.0 ± 56.8 lM/g protein) and Trolox (567.0 ± 45.5 lM/g protein) significantly. The content of TBARS was significantly decreased in the combination group (356.0 ± 47.6 lM/g protein) compared CAPE alone and Trolox alone. The decrease of GSH/GSSG ratio is considered to be a marker of oxidative damage and could be attributed to GSH depletion and GSSG expulsion. As shown in Fig. 4E and F, radiation markedly decreased the content of GSH and increased the content of GSSG, which was reversed by CAPE and Trolox. The GSH and GSSG content in the combination group were significant lower than that of single agent group (P < 0.05). We further calculate the ratios of GSH/GSSG. As shown in Fig. 4G, radiation decreased GSH/GSSG

ratio significantly (43.7 ± 6.4) compared with that of control group (191.0 ± 10.3).When the cells were treated with a combination of CAPE and Trolox, GSH/GSSG ratio (102.3 ± 15.3) had a stronger increase than CAPE (83.8 ± 9.2) or Trolox (75.9 ± 10.3) separately (P < 0.05). 3.5. Effect of CAPE and Trolox in combination against radiation-induced changes of antioxidant enzymes In order to study the mechanism of enhanced antioxidant activity of CAPE and Trolox in combination, the expression of Nrf2 and antioxidant enzymes was determined using western blotting. As shown in Fig. 5, Nrf2 expression in the radiated group decreased significantly compared with that of the control group. Corresponding changes of the expression of GR, Gpx, CuZnSOD and MnSOD were observed simultaneously. On contrary, the expression of CAT was up-regulated by radiation. Compared to the radiation group, the expression level of Nrf2, GR, Gpx, CuZnSOD and MnSOD in CAPE-treated and Trolox-treated cells was increased significantly. Specifically, when the cells were treated with a combination of CAPE and Trolox, Nrf2, GR and Gpx level had a stronger increase than CAPE or Trolox separately (P < 0.05). Although many treatments were statistically significant compared with the radiation control, no more enhancer effects were found in CAT, MnSOD and CuZnSOD levels (P > 0.05). 4. Discussion An interesting area in functional food science is discovering the enhancer effects or synergies between bioactive components to create foods that have improved health benefits. In the present study, we have a special interest in the combination administration of CAPE and Trolox. Although the antioxidant capacity of each agent separately has been well proved in the earlier studies, no available data has provided evidence of their interactions with each other in biological systems until now. By measuring UV–vis

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Fig. 4. Effects of CAPE, Trolox and their combination on cell viability (A), ROS generation (B and C), TBARS content (D), GSH content (E), GSSH content (F) and GSH/GSSG ration (G) in L929 cells induced by radiation. R, radiation; C, CAPE; T, Trolox. The values are expressed as mean ± SD (n = 6). a–eMean values with unlike letters were significantly different and the symbol ⁄ signifies enhanced effect (P < 0.05).

absorption spectrum, our work unequivocally indicated the intermolecular interaction between CAPE and Trolox (Fig. 1). However, we now cannot give a clear map of such weak interaction we concluded from UV spectra, maybe intermolecular H-bonding or pi–pi stacking. Based on this, we carried out a series of in vitro models to assess if this combination exerts enhancer antioxidant effects in biological systems. Just as that different free radicals have subtle preferences for target molecules, antioxidants also have preferential target radicals. For example, glutathione can react directly with hydrogen peroxide, superoxide, hydroxyl & alkoxyl radicals and hydroperoxides. Vitamin E has a particular role in preventing oxidative modification of low density lipoprotein and Coenzyme Q10 favors peroxyl radicals. In order to evaluate the preferential target radicals of CAPE and Trolox, three chemical free radical-generating models were used. Our results showed that Trolox and CAPE were

effective in scavenging DPPH, hydroxyl radicals and superoxide anions initiated by DPPH, Co(II)/EDTA and xanthine oxidase. The Trolox was less potent than CAPE (Fig. 2). The chemistry underlying this activity results mainly from hydrogen atom transfer or single electron transfer reactions, or both involving hydroxyl groups. The position of hydroxyl groups and other features in the chemical structure of Trolox and CAPE are important for their antioxidant and free radical scavenging activities. Our results further showed that the potency of Trolox as a free radical scavenger is markedly increased when combined with CAPE at the indicated concentration (Fig. 2).This synergistic effect of free radical scavenging activities of Trolox and CAPE could be explained, at least in part, by the weak interaction we observed from UV spectra. Biological membranes are important targets for ROS due to their high concentration of polyunsaturated fatty acids [27]. The CAPE and Trolox were tested for their antioxidant activities by

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Fig. 5. Effect of CAPE, Trolox and their combination on the protein expression of Nrf2 and related antioxidant enzymes in L929 cells induced by radiation. (A) Western blot representative of three independent experiments is shown. Protein loading was determined with antibodies anti b-actin for cytosol and LaminB for nuclei. (B) The values from densitometry of Nrf2 and CuZnSOD, MnSOD, CAT, GR, Gpx were normalized to the level of LaminB and b-actin protein, respectively. R, radiation; C, CAPE; T, Trolox. The data were expressed as the means ± SD of three independent experiments. a–eMean values with unlike letters were significantly different and the symbol ⁄ signifies enhanced effect (P < 0.05).

measuring their abilities to inhibit LPO induced by vitamin C/Fe2+, CHP or CCl4/NADPH in rat liver microsomes. A combination of CAPE and Trolox was found to result in a markedly significant decrease in MDA compared to each alone, suggesting a synergistic antioxidant effect of CAPE and Trolox (Fig. 3). It is should be noted that a prooxidative effect of Trolox is observed under some indicated concentrations in these LPO models. Similar prooxidative effect of vitamin C has also been observed in our previous study [19]. An antioxidant is a chemical that is oxidized itself and thereby reducing other chemical such as ROS. Once they get oxidized, some of them can move repeatedly from the reduced form to free radical and back again. However, many things such as the redox potential of the individual molecule and the inorganic chemistry of the cell can influence the redox cycle of an antioxidant and decide its antioxidant or pro-oxidant characteristic [28]. For example, the atocopherol (a-TocH) is the major and most active chain-breaking antioxidant in lipid. Our studies show, however, that a-TocH can be a strong prooxidant for the extracted microsomal lipid itself. We proposed that a steady flux of alkylperoxyl radicals (ROO) generated from inorganic (vitamin C/Fe2+), organic (CHP) or enzymatic (CCl4/NADPH) initiator induced lipid peroxidation in microsome which was faster in the presence of a-TocH than in its absence. The reaction of ROO with a-TocH forces a-Toc to propagate a radical chain via its reaction with PUFA lipid (LH) within the particle (a-Toc + LH + O2 ? a-TocH + LOO). Termination of this radical

chains occurs when the a-Toc radical is eliminated by other agents such as CAPE in this study. Antioxidants work synergistically as a team, with one recharging or rejuvenating the other through the process of electron donation. Accumulating studies have proved that taking only one single antioxidant is not only no benefit, but could potentially be dangerous. For example, findings from a large-scale study indicate taking a vitamin E supplement may increase the risk of prostate cancer by 17 percent. In contrary, supplementation with antioxidant-rich foods was proved to be more effective than that with single antioxidants in lowering urinary excretion of oxidatively damage DNA. In our present study, the prooxidative effect of Trolox was totally counteracted when CAPE was added simultaneously to the system, indicating that Trolox and CAPE could work more safely and effectively together than in isolation. In the following experiments, we were to employ gamma radiation as an oxidant, which was widely used to induce oxidative stress in vivo and in vitro [29–31]. Exposure to ionizing radiation produces oxygen-derived free radicals in the tissue environment including hydroxyl radicals, superoxide anion radicals and other oxidants such as hydrogen peroxide [32]. The overproduction of free radicals following radiation overwhelms the antioxidant defense system’s ability to eliminate them and then oxidative stress occurs. In the present study, radiation induced ROS production caused obvious cytotoxicity to cultured L929 cells (Fig. 4).

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because its catalytic cycle requires the interaction of two H2O2 molecules with a single active site, which is less likely at low H2O2 concentrations [36]. Therefore, CAT is not expected to play a significant role in eliminating low levels of H2O2. This might explain why CAT expression is upregulated by radiation but went down again when radiation generated ROS was reduced remarkably by CAPE, Trolox and their combination. Also, unlike CAT and Gpx, SOD could not be considered as a beneficial antioxidant enzyme to a certain extent when it detoxifies O 2 but produce another ROS. So the over expression of SOD was undesirable when H2O2 could not be further detoxified efficiently. Overall, CAPE and Trolox in combination ameliorates the oxidative injury in L929 cells induced by radiation through increasing the expression of GR and Gpx and then the redox cycle of GSH mediated by Nrf2. We believed this function was achieved by the mutual regulation between ROS and the AREs systems. Fig. 6. CAPE and Trolox work synergistically as a team to perform enhanced antioxidant actions.

5. Conclusion A significant increase of MDA and GSSG contents and decrease of GSH content and GSH/GSSG ratio were also observed (Fig. 4). When the L929 cells was treated with CAPE, Trolox or combinations, the above indicators of oxidative stress were improved and combination therapy showed a stronger protective activity compared to single treatment modalities (Fig. 4). The above results indicated that, in addition to elimination of ROS and inhibition of LPO, redox cycle of GSH may be involved in the synergistic effect of CAPE and Trolox against oxidative processes induced by radiation. Cells are equipped with enzymatic and nonenzymatic antioxidant systems to eliminate ROS and maintain redox homeostasis. The basic leucine-zipper transcription factor-Nrf2 has been shown to play a vital role in protecting cells from oxidative stress [33]. In response to oxidative stress, Nrf2 translocates to the nucleus and binds to antioxidant-response elements (AREs) in the promoters of target genes, leading to transcriptional induction of several cellular defense genes including SOD, CAT, Gpx and GR. SOD was demonstrated to have ROS-metabolizing activity and can efficiently and specifically catalyze dismutation of O 2 to O2 and H2O2. Further conversion of H2O2 to H2O occurs through the action of CAT, a heme-based enzyme that is normally localized in the peroxisome. H2O2 also can be converted to O2 through coupled reactions with the conversion of GSH to oxidized GSSG, catalyzed by GPX. Furthermore, GR was required in the recycling of GSSG to keep the balance of GSH and GSSG. Obviously, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GR and Gpx [34]. In the present study, CAPE, Trolox or combinations reversed the decrease of Nrf2, Gpx, GR induced by radiation and combination therapy again showed a stronger effect of up-regulation compared to single treatment, which was consistent with the changes of GSH/GSSH ratio (Fig. 5). We supposed that the enhanced increase of GSH/GSSH induced by CAPE and Trolox in combination was probably due to the Nrf2 regulated activation of GR and Gpx. However, the enhanced effect on expression of CAT and SOD were not observed by combinations as compared to single treatment (Fig. 5). In bacteria and unicellular eukaryotes, the induced expression of detoxifying enzymes exhibits biphasic response to ROS concentrations [35]. Although they regulate intracellular ROS levels, they are at the same time regulated by ROS themselves. For example, Gpx seem to easily scavenge H2O2 by coupling its reduction to H2O with oxidation of GSH. With increasing cellular concentration of H2O2, Gpx can, however, become overoxidized, reducing their scavenging capacity. This suggests that under such circumstances an antioxidant Gpx pool would be somewhat depleted just as observed under radiation. Oppositely, CAT is not efficient in eliminating low levels of H2O2

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