Article pubs.acs.org/crt
Induction of Oxidative DNA Damage by Flavonoids of Propolis: Its Mechanism and Implication about Antioxidant Capacity Yi-Chih Tsai,† Yi-Hsiang Wang,† Chih-Chiang Liou,† Yu-Cun Lin,‡ Haimei Huang,‡ and Yin-Chang Liu*,† †
Institute of Molecular Medicine and ‡Institute of Biotechnology, National Tsing-Hua University, Hsin-Chu, Taiwan 30013 S Supporting Information *
ABSTRACT: Propolis from beehives is commonly used as a home remedy for various purposes including as a topical antiseptic. Despite its antioxidant capacity, propolis induces oxidative DNA damage. In exploring the underlying mechanism, we found that the induction of oxidative DNA damage is attributed to the hydrogen peroxide (H2O2) produced by propolis. The formation of H2O2 can take place without the participation of cells but requires the presence of transition metal ions such as iron. Flavonoids such as galangin, chrysin, and pinocembrin that are commonly detected in propolis have the capacity to induce oxidative DNA damage, and that capacity correlates with the production of H2O2, suggesting the involvement of flavonoids in propolis in this process. On the basis of these results, we propose that the flavonoids of propolis serve as temporary carriers of electrons received from transition metal ions that are relayed to oxygen molecules to subsequently generate superoxide and H2O2. In addition, propolis induces oxidative DNA damage that is subject to repair, and propolis-treated cells show a lower level of DNA damage level when challenged with another oxidative agent such as amoxicillin. This is reminiscent of an adaptive response that might contribute to the beneficial effects of propolis.
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INTRODUCTION Propolis is a beehive product and is a mixture that contains mainly the resinous substances of plants collected by the insects to build their shelters.1 Despite the risk of allergic effects,2,3 propolis is commonly used as a topical antiseptic or inflammation suppressor on mucosal surface areas, such as the mouth and throat.4 The antimicrobial activity of propolis may be a direct effect or is mediated via the stimulation of the immune system.5 In addition, propolis has been shown to have antitumor and antioxidation activities.6−8 Most of those biological activities have been associated with flavonoids or phenolic constituents of propolis.6,7,9 By serendipity, we found that propolis can inhibit gap filling during the repair of UVC-induced DNA lesions.10 That type of DNA damage is repaired by nucleotide excision repair,11 which consists of a cascade of events including initial damage recognition, dual incision to excise the damage containing oligonucleotide, gap filling, and ligation.12 The step of gap filling can be blocked by DNA synthesis inhibitors such as hydroxyurea and ara-C.13,14 Such blockage results in the accumulation of repair intermediates with the gap, which can be detected by methods such as single cell gel electrophoresis, also called the comet assay.15,16 If the DNA synthesis inhibitors are removed, the gap filling will be quickly restored. The restoration of gap filling was found to be delayed by propolis, and that delay has been correlated with the induction of oxidative DNA damage induced by propolis.10 Oxidative DNA lesions can be detected by the comet assay with incubation of © 2011 American Chemical Society
formamido pyrimidine glycosylase (Fpg) and endonuclease III (Endo III), which are bacterial enzymes that recognize oxidized purines and pyrimidines, respectively.17,18 In this study, we characterize the underlying mechanism of such damage induction. Our results indicate that reactive oxygen species, such as hydrogen peroxide (H2O2), are formed through redox reactions involving transition metal ions. As H2O2 can be formed in a cell-free system, a model showing the formation of H2O2 outside of cells is proposed. In addition, we show that cells pre-exposed to propolis acquire resistance to a subsequent challenge of oxidative stress. This adaptive response is discussed in the context of the antioxidant activity of propolis.
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MATERIALS AND METHODS
Cell Cultures. The cells used in this study include AGS human gastric adenocarcinoma cells19 and CL 1-0 human lung adenocarcinoma cells20 that were used in the experiments regarding adaptation (as noted in the text). Both cell lines were originally obtained from the American Type Culture Collection (Manassas, VA, USA), and were grown in 1× RPMI medium (Gibco, Grand Island, NY, USA). All cell culture media were supplemented with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Biet Haemek, Israel) and 0.03% glutamine, and cells were grown at 37 °C in a water-saturated atmosphere containing 5% CO2. Received: September 30, 2011 Published: December 7, 2011 191
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Chemicals and Propolis. Glutathione, β-carotene, butylated hydroxytoluene, ferrous ammonium sulfate hexahydrate, xylenol orange, pinocembrin, galangin, chrysin, trans-cinnamic acid, catalase, and deferoxamine were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Propolis was obtained from New Zealand (Triple Treasure ltd., Christchurch, New Zealand). Propolis, pinocembrin, galangin, chrysin, and trans-cinnamic acid were dissolved in dimethyl sulfoxide (DMSO) before use. Detection of Oxidative DNA Damage with the Comet Assay. Conventional comet assays (single-cell gel electrophoresis) were performed as described previously21 with modifications.22 Preparation of the cell-containing gel (on microscopic slides) and the subsequent cell lysis were carried out as described previously.22 After cell lysis, the slides were washed three times with deionized water. The slides were incubated with Endo III and Fpg (Trevigen, Gaithersburg, MD, USA; 2 unit of each enzyme per slide in buffer with 10 mM Tris-HCl, pH 7.4) as described previously.17 A coverslip was applied, and the slides were incubated at 37 °C for 1 h in a sealed box containing a piece of wet tissue paper. After the incubation, the slides were then denatured in 0.3 N NaOH and 1 mM EDTA for 20 min. Electrophoresis was carried out in the same denaturation solution at 25 V and 300 mA for 25 min. Each slide was rinsed briefly in water, blotted, and then transferred to 0.4 M Tris-HCl, pH 7.5. DNA was stained by adding 20 μL of propidium iodide (50 μg/mL) onto the slide. A coverslip was then applied, and the slide was examined using a fluorescence microscope (Axioplan 2, Zeiss Co, Thornwood, NY, USA). Images of at least 50 cells per slide were recorded with a closedcircuit display camera (CoolSNAP). The migration of DNA from the nucleus of each cell was measured with a computer program (http:// tritekcorp.com) and is expressed as % DNA in the tail. Detection of Cellular Oxidative Stress. Cellular oxidative stress was detected based on the increase of fluorescence as result of the oxidation of a nonfluorescent probe 2′,7′-dichlorfluorescein-diacetate (DCFH-DA).23 In brief, cells were fed with DCFH-DA (25 μM) for 1 h and then were treated with propolis or compounds of interest for indicated periods (0−1 h) before being examined with a fluorescence microscope (IX71, Olympus Co., excitation 488 nm, emission 525 nm). Measurement of H2O2 by the Fox Assay. The measurement of H2O2 is based on the formation of the blue color Fe3+−phenol complex as the result of oxidation of Fe2+ by H2O2.24 In brief, 0.9 mL of Fox reagent (see below) was mixed with 0.1 mL of the sample tested. After 30 min or less, the absorbance at 562 nm of the mixture was recorded, and the corresponding level of H2O2 was obtained based on a standard curve with H2O2. The Fox reagent (per 100 mL) contains 90% methanol, 10% H2SO4 (250 mM), 88 mg of butylated hydroxytoluene, 9.8 mg of ferrous ammonium sulfate hexahydrate, and 7.6 mg of xylenol orange. Data Analysis. All experiments were performed independently at least three times. Data are expressed as means ± SE. Student’s t test was used for statistical analyses.
Figure 1. (A) Oxidative stress in propolis-treated cells. AGS cells were incubated with DCFH-DA for 1 h and were then treated with 0.3 μg/mL propolis in DMSO or with 20 μM H2O2 for 30 min before being examined with a fluorescence microscope. Control: DMSO alone. Fractions of fluorescence-positive cells are indicated at the bottom. Bar = 35 μm. (B) β-Carotene and GSH blocked the propolis-induced oxidative DNA damage. AGS cells in overnight culture at 50% confluency were treated with 0.3 μg/mL propolis (prop.) with or without 18 μM β-carotene (β-car.) or 1 mM GSH for 3 h before being analyzed for oxidative DNA damage with the comet assay. Control: DMSO alone. The asterisk (*) indicates statistical significance (p < 0.01; n = 3) between the indicated experiments. Error bars for SE are shown.
suspect that the mediator of the propolis-induced oxidative DNA damage is H2O2. To test if H2O2 is formed in propolis-treated cell cultures, we included catalase, a H2O2 hydrolyzing enzyme, in the medium and found that inclusion of that cell impermeable enzyme to the cell cultures abolished the induction of oxidative DNA damage by propolis (Figure 2A). The condition treated with H2O2 was used as a control. The formation of H2O2 was confirmed by Raman spectrometric analysis (Figure S1, Supporting Information), and the level of H2O2 produced was quantified by the Fox reaction (Figure 2B, see the curve with serum; the curve without serum will be discussed below). Since the induction of oxidative DNA damage was observed in cell cultures grown in serum-containing media, it is of interest to determine if serum is involved in the induction. Exclusion of serum from the experimental system impaired the induction of oxidative DNA damage (Figure 3A, second column from the left), indicating that the serum is crucial. Consistently, the level of H2O2 produced is low in serum-free conditions (Figure 2B, curve without serum). As serum
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RESULTS Propolis Causes Oxidative DNA Damage Only in the Presence of Fe2+ Ions. Despite its antioxidant capacity, propolis may induce oxidative DNA damage (ref 10 and data herein). Consistently, oxidative stress was elevated in propolistreated cells (Figure 1A). To determine the underlying mechanism of that, we tested whether the propolis-induced oxidative stress could be suppressed by antioxidants, such as β-carotene and glutathione (GSH). Both of those antioxidants blocked the induction of oxidative DNA damage by propolis (Figure 1B). As GSH is cell impermeable,25 our finding suggests that the mediator that led to oxidative DNA damage was formed outside of cells. As the immediate reactive species causing oxidative DNA damage is the hydroxyl radical (·OH), and since ·OH is primarily formed through the reaction of hydrogen peroxide (H2O2) and transition metal ions,26 we 192
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Figure 2. (A) Effect of catalase on propolis-induced oxidative DNA damage. AGS cells in overnight culture at 50% confluency were treated with 0.3 μg/mL propolis or with 20 μM H2O2 in medium with or without catalase (50 unit per mL) for 1 h before being analyzed for oxidative DNA damage with the comet assay. The asterisks (*) indicate statistical significance (p < 0.01; n = 3) between the indicated experiments. (B) Quantitative analysis of H2O2 induced by propolis. The level of H2O2 was measured by mixing 0.9 mL of Fox reagent and 0.1 mL of sample with or without 10% FBS (i.e., ± serum) and the indicated concentrations of propolis. Propolis was allowed to incubate with the serum for 15 min before the sample solution was mixed with the Fox reagent. Error bars for SE are shown.
Figure 3. (A) Effect of serum or iron(II) on propolis-induced oxidative DNA damage. AGS cells in overnight culture at 50% confluency were treated with 0.3 μg/mL propolis under the specified conditions for 3 h before being analyzed for oxidative DNA damage with the comet assay. Columns 1 and 2 (from the left): cells were treated with propolis in medium with or without 10% FBS. Symbols: serum for 10% FBS and SF for serum-free, respectively. Column 3: cells treated with propolis and deferoxamine (DFO-B, 100 μM) in serum-containing medium. Columns 4 and 5: cells treated with propolis in serum-free (SF) medium containing FeSO4 or FeCl3 at 50 μM. (B) Propolis-induced oxidative DNA damage shows the dose dependence of iron(II) in the range 0−50 μM. AGS cells were treated with 0.3 μg/mL propolis in serum-free medium containing FeSO4 at the indicated concentrations for 1 h before being analyzed with the comet assay. (C) Supplementation with the transition metal ions of Fe2+ or Cu2+ but not Zn2+ restores the propolis-induced oxidative DNA damage. AGS cells were treated with 0.3 μg/mL propolis in medium without serum but containing FeSO4 (50 μM) or CuSO4 or ZnSO4 (both at 100 μM) for 3 h before being analyzed with the comet assay. The asterisks (*) indicate statistical significance (p < 0.01; n = 3) between the indicated experiments. Error bars for SE are shown.
contains proteins and metal ions (particularly iron ions), it is desirable to know their involvement in the induction of oxidative DNA damage. Deferoxamine (DFO-B), an iron chelator, was found to effectively inhibit the induction of oxidative DNA damage (Figure 3A, third column from the left), indicating that iron is essential. Consistently, iron supplementation of the serum-free culture system restored the induction of oxidative DNA damage (Figure 3A, fourth column from the left) and occurred in a dose-dependent manner in the range of 0−50 μM FeSO4 (Figure 3B). However, only FeSO4 but not FeCl3 restored the induction of oxidative DNA damage (Figure 3A, fifth column from the left), suggesting that Fe2+ but not Fe3+ had that effect. Supplementation with copper was also effective but at a lower level (Figure 3C). The effect of zinc was barely detectable, suggesting that typical transition metal ions, such as iron or copper, in the serum are responsible for the induction effect. Phenolic Compounds of Propolis Induce Oxidative Stress. Previous studies have indicated that propolis is a mixture of compounds including flavonoids. The flavonoid content of the propolis sample used in our study was estimated to be close to 10% in reference to pinocembrin27 (data not shown). To determine whether flavanoids are involved in the
induction of oxidative DNA damage, representative flavonoids, such as pinocembrin, chrysin, and galangin, plus nonflavonoid compounds such as cinnamic acid10,28 were tested. As shown in Figure 4A, pinocembrin, chrysin, and galangin, but not cinnamic acid, had the capacity to induce oxidative DNA damage. Among the compounds tested, cinnamic acid is the only one lacking a phenolic group. The induction of oxidative 193
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Figure 4. (A) Effects of flavonoids of propolis on oxidative DNA damage. The procedures were similar to the procedures described for Figure 3 except that AGS cells were treated with the indicated flavonoids at 20 μM in DMSO for 1 h before being analyzed for oxidative DNA damage with the comet assay. Control: DMSO alone. (B) Flavonoids of propolis induce the formation of H2O2. The level of H2O2 produced was measured by mixing 0.9 mL of Fox reagent and 0.1 mL of sample containing 10% FBS and the indicated flavonoids at 100 μM. The asterisks (*) indicate statistical significance (p < 0.01; n = 3) between the indicated experiments. Error bars for SE are shown.
DNA damage by these compounds can be correlated with their capacity to elicit H2O2 measured by the Fox reaction (Figure 4B). Pre-Exposure to Propolis Renders Cells Resistant to the Subsequent Induction of Oxidative DNA Damage. As reported in our previous study,10 the induction of oxidative DNA damage by propolis is transient, i.e., the damage is subject to repair (also shown in Figure 5A and Figure S2, Supporting Information). We speculated that such an induction of oxidative DNA damage might be beneficial to cells to meet the challenge of subsequent oxidative stress as a result of the cellular response to DNA damage. We tested that idea with amoxicillin, a mild oxidative stress inducer.29 The results (Figure 5A) indicate that induction of oxidative DNA damage by propolis conferred cells the capacity to resist challenge by amoxicillin. Similar results were found if amoxicillin was replaced with propolis (Figure S3, Supporting Information). Such an acquired resistance or adaptation requires de novo protein synthesis, as the capacity was markedly attenuated by protein synthesis inhibitors such as cycloheximide (Figure 5B).
Figure 5. (A) Cellular adaptive response to propolis-induced oxidative DNA damage. AGS cells in overnight culture at 50% confluency were treated with or without 0.3 μg/mL propolis overnight (ca. 16 h; O/N) and then were treated with 2.5 mM amoxicillin (amox.) at the indicated time point. Cells were harvested for the comet assay at the indicated time points. Symbols: solid and dotted curves show with or without pretreatment of propolis, respectively. (B) De novo protein synthesis is required for the adaptive response. Similar to the condition described for panel A, AGS cells were treated with propolis overnight and then were treated with amoxicillin for 1 h before being analyzed with the comet assay, except for the use of cycloheximide (HX,10 μg/mL) to inhibit protein synthesis in the indicated experiments. (C) Glutathione reductase is essential to the adaptive response. The experimental scheme was similar to that described for panel A except that CL1-0 cells or their derivatives with GR RNAi knockdown were used. The asterisks (*) indicate statistical significance (p < 0.01; n = 3) between the indicated experiments. Error bars for SE are shown. 194
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indicates that the induction of oxidative DNA damage by phenolic compounds occurs within 1 h, suggesting that the effect is less likely due to the DNA−polyphenol−metal ion complex described in the model of Hadi et al.31 Our study indicates that Cu2+ ions are capable, although less effective than Fe2+ ions, of inducing oxidative DNA damage. To replace Fe2+ with Cu2+ in the model, the electron must flow first from the phenolic compounds to copper, when Cu2+ would be reduced to Cu1+. The electron would then transfer from Cu1+ to O2 to generate superoxide. According to our model, other phenolic compounds, such as amoxicillin and colcemid, may also serve the same function as electron carriers. This is consistent with our previous observations with amoxicillin and colcemid,29,33 which also showed their capacity to induce oxidative DNA damage. In this study, the Fox assay was used to measure H2O2.24 We have found that the levels of H2O2, produced by propolis or other phenolic compounds tested, appeared lower than the expected values based on oxidative DNA damage. As the level of H2O2 is proportional to the formation of the Fe3+−phenol complex,34 which may be disturbed by phenolic compounds, this could explain the discrepancy. In this study, we demonstrate that pretreatment with propolis renders cells tolerant to subsequent oxidative stress challenges. A similar adaptive survival response has been reported by Surh and Na in their study on the cytotoxicity of tetrahydropapaveroline (THP), an isoquinoline.35 The adaptation is linked with the increased expression of heme oxygenase1, which in turn is regulated by NF-E2-related factor 2 (Nrf2).36 In our study, GR was found to be crucial to the propolis-induced adaptation (Figure 5C). In this experiment, CL1-0 but AGS cells were used simply because of the availability of the GR knockdown clone. GR appears to be regulated by Nrf2 as an increase of GR can be correlated with the activation of Nrf2.37 A recent study on the effect of oxidative stress on insulin resistance indicates that acute but not chronic oxidative stress benefits health as a result of the restoration of insulin sensitivity and glucose uptake in muscle cells.38 This example provides clinical implications for the adaptation. In summary, in this study we use experimental approaches to characterize the mechanism underlying the induction of oxidative DNA damage by propolis. The model we propose for this induction consists of the formation of H2O2 outside of cells through a series of redox reactions, where the phenolic compounds of propolis serve as temporary electron porters. A brief induction of oxidative stress by propolis may be beneficial to health as it confers cells with resistance to subsequent challenge.
Knockdown of the expression of glutathione reductase (GR) (Figure S4, Supporting Information), the enzyme that regenerates cellular GSH from oxidized GSH,30 completely eliminated the adaptation response (Figure 5C). As knockdown of GR did not render cells more sensitive to amoxicillin (data not shown), we consider that GR is likely involved in this adaptation mechanism.
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DISCUSSION Propolis is an antioxidant; however, it may also act as a prooxidant and can induce oxidative DNA damage under certain circumstances. In this study, we show that transition metal ions are required for propolis-induced oxidative DNA damage and that flavonoid components of propolis, which have been associated with the antioxidant capacity of propolis, can cause the DNA damage. Our data indicate that reactive oxygen species such as H2O2 are produced in cell culture and in a cell free system with representative phenolic compounds of propolis and iron. On the basis of the results of our study and the current knowledge of DNA damage, we propose a model (Figure 6) to
Figure 6. Model for the induction of oxidative DNA damage by phenolic compounds of propolis and subsequent adaptation. Superoxide is converted to H2O2 by reacting with protons (H+) presumably in water. H2O2 may diffuse to nuclei and through the Fenton reaction may be converted to hydroxyl radicals (·OH), which are responsible for causing oxidative DNA damage.
describe how propolis induces oxidative DNA damage. In this model, the phenolic compounds of propolis serve as temporary carriers of electrons in a series of redox reactions, in which the electrons of ferrous ions are relayed to oxygen molecules and generate superoxide after which H2O2 is formed. The relatively stable H2O2 molecules are highly permeable, and some of them may reach nuclei and be converted to highly reactive ·OH radicals catalyzed by metal ions through the Fenton reaction. Regions of DNA which are adjacent to ·OH are subjected to damage. The pro-oxidant effect of phenolic compounds has been investigated, and the possible underlying mechanisms have been described.31,32 Hadi et al. studied DNA degradation with plant polyphenols and hypothesized that DNA breakage occurs through redox reactions involving a DNA−polyphenol− Cu2+ ternary complex.31 However, Halliwell et al. were among the first to demonstrate the formation of H2O2 outside of cells in their study on the cytotoxicity of dopamine.32 Although our model agrees with the results by Halliwell et al. regarding the formation of H2O2, our model now clearly indicates the important role of metal ions. A previous study on the metabolism of propolis indicates that flavonoids of propolis may be absorbed into the body;9 however, it remains unclear how readily the phenolic compounds enter cells. Our study
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ASSOCIATED CONTENT S Supporting Information * Surface-enhanced Raman spectrometric analysis; propolisinduced oxidative DNA damage is subject to repair; cellular adaptation to propolis-induced oxidative DNA damage; and glutathione reductase (GR) activity of cell extracts of CL1-0 cells and the GR RNAi knockdown strain. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected]. 195
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Funding This work was supported partly by the NTHU-CMU Joint Research Program (98N2446E1); NTHU Research Boost Program (98N2937E1); and National Science Council, Taiwan (NSC 98-2311-B-007-008-MY3) to Y.C.L.
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ACKNOWLEDGMENTS We thank Drs. M. C. Kao (IMM, NTHU) and Lily Wang (ICM, NTHU) for their critical review of the study. ABBREVIATIONS Fpg, formamido pyrimidine glycosylase; Endo III, endonuclease III; GSH, glutathione; DFO-B, deferoxamine; HX, cycloheximide; GR, glutathione reductase
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REFERENCES
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