|Year : 2016 | Volume
| Issue : 2 | Page : 990-994
Evaluation of the radioprotective effect of Turkish propolis on foreskin fibroblast cells
Can Ozgur Yalcin1, Yuksel Aliyazicioglu2, Selim Demir3, Ibrahim Turan4, Zumrut Bahat5, Sema Misir6, Orhan Deger2
1 Department of Pharmaceutical Toxicology, Institute of Health Sciences, Ankara University, 06100 Ankara, Turkey
2 Department of Medical Biochemistry, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey
3 Department of Medical Biochemistry, Institute of Health Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey
4 Department of Genetic and Bioengineering, Faculty of Engineering and Natural Sciences, Gumushane University; Traditional Medicine Practice and Research Center, Gumushane University, 29100 Gumushane, Turkey
5 Department of Radiation Oncology, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey
6 Department of Medical Biochemistry, Institute of Health Sciences, Karadeniz Technical University, 61080 Trabzon; Department of Biochemistry, Faculty of Pharmacy, Cumhuriyet University, 58140 Sivas, Turkey
|Date of Web Publication||25-Jul-2016|
Department of Medical Biochemistry, Faculty of Medicine, Karadeniz Technical University, 61080 Trabzon
Source of Support: This work was supported by the Foundation of Scientific
Research of Karadeniz Technical University (No: 2008.114.001.5),
Trabzon, Turkey, Conflict of Interest: None
Aim of Study: Propolis is a resinous bee product, rich of polyphenolic compounds and flavonoids. It is known that in different geographic zones its chemical composition varies due to the different plant sources. Many biological properties including antimicrobial, antioxidative, anti-inflammatory, antitumoral, antigenotoxic, antimutagenic, cytostatic activities have been ascribed to propolis. These biological effects are predominantly attributed to its content of polyphenols. In this study, we aimed to evaluate the radioprotective effect of ethanolic extract of Turkish propolis. (EETP) against γ-ray-induced DNA damage on fibroblast cells using comet assay for the first time.
Materials and Methods: Fibroblast cells were pretreated 15 and 30 min with concentrations of 100, 200 and 300 μg/mL EETP then they were exposed to 3 Gy γ-rays. Amifostine (synthetic aminothiol compound) was used as a positive control.
Results: The results showed a significant decrease in γ-ray-induced DNA damage on cells treated with EETP and amifostine when compared to only irradiated cells. (P < 001).
Conclusion: It was concluded that EETP prevent γ-ray-induced DNA damage in fibroblast cells and might have radioprotective activity.
Keywords: Comet assay, DNA damage, ionizing radiation, polyphenols, radioprotective agents, Turkish propolis
|How to cite this article:|
Yalcin CO, Aliyazicioglu Y, Demir S, Turan I, Bahat Z, Misir S, Deger O. Evaluation of the radioprotective effect of Turkish propolis on foreskin fibroblast cells. J Can Res Ther 2016;12:990-4
|How to cite this URL:|
Yalcin CO, Aliyazicioglu Y, Demir S, Turan I, Bahat Z, Misir S, Deger O. Evaluation of the radioprotective effect of Turkish propolis on foreskin fibroblast cells. J Can Res Ther [serial online] 2016 [cited 2020 Jan 20];12:990-4. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/990/154050
| > Introduction|| |
Propolis is one of the bee products that honeybees collect it from exudates and buds of various plants, and it is used to fill gaps and to seal parts of the hive. Propolis has been used for centuries in traditional medicine since ancient times. In recent years, many biological properties, including antimicrobial, antiviral, antigenotoxic, anticancer, anti-inflammatory, antioxidant, hepatoprotective, immunostimulating, and cytostatic activities have been described for propolis. Chemical composition of propolis mainly depends on the feature of climate and geography of harvest region. Major constituents of propolis are polyphenols, flavonoids, organic acids, vitamins, and minerals. Flavonoids are considered good antioxidant molecules that play crucial roles in the scavenging of reactive oxygen species (ROS), which may cause chronic diseases such as neurodegenerative disorders, atherosclerosis, cancer. Radiation is one of the main sources of ROS. People usually may expose to radiation from both natural and man-made sources. Several alterations have occurred in the cellular macromolecules (especially DNA) when cells are exposed to radiation., Genotoxicity, chromosomal abnormalities, and gene mutations occur in the cell as a result of DNA damage originating from higher ROS levels. Radiation-induced DNA damage happens mainly due to water radiolysis around of the DNA and production of oxygen-centered free radical, which includes superoxide anion, hydroxyl and peroxyl radicals, hydrogen peroxide, and nitric oxide.,, ROS, are highly mutagenic molecules and cause oxidative damage to cellular macromolecules resulting in the DNA strand breaks (DSBs, SSBs), base modifications, and genetical alterations. It was known that polyphenols numerously decrease the harmful effects of ionizing irradiation at the molecular, cellular and/or tissue level due to are able to scavenge ROS and to chelate metal ions.,,
In our previous studies, we observed that of ethanolic extract of Turkish propolis (EETP) inhibit the respiratory burst within K-562 cells and protect fibroblast cells from H2O2-induced DNA damage probably by their antioxidant potentials.,
Thus, in this study, we aimed to evaluate the radioprotective effect of EETP against γ-ray-induced DNA damage on fibroblast cells by using comet assay for the first time.
| > Materials and Methods|| |
Ethanol and NaCl were purchased from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) was purchased from Amresco (Solon, USA), penicillin-streptomycin and tyripsin/EDTA solution from Gibco (Paisley, Scotland), Dulbecco's modified eagle medium (DMEM), low melting point agarose (LMA) and normal melting agarose (NMA) were from Lonza (Verviers, Belgium), fetal bovine serum (FBS) and polylysine from Biochrom (Berlin, Germany), phosphate buffer saline (PBS) tablet from Medicago (Uppsala, Sweden), sodium hydroxide from Riedal De Haen (Seelze, Germany), amifostine was purchased from (MedImmune Pharma, Bedford, OH, USA) and all other chemicals are analytical grade from Sigma (St. Louis, MO, USA).
Preparation of ethanolic extract of Turkish propolis/amifostine solutions
Propolis samples which were obtained by Trabzon Agricultural Development Cooperative, were produced by honeybees (Apis mellifera L.) in the region of Yomra, Trabzon, Turkey rich in genus of Picea, Fagus, Castenea, and Rhododendron. Briefly, 0.5 g propolis was dissolved in 20 mL absolute ethanol. After vortexing, it was incubated at 60°C and 150 rpm for 24 h by continuous mixing. After incubation, extracts were filtered from filter paper, 0.22 µm filters. Prepared 25,000 µg/mL stock ethanolic extract of propolis (EEP) was aliquoted and used for experiments.
As a positive control group, a well-known radio and chemoprotective agent Amifostine was used. Amifostine solutions prepared at concentration of 4, 7, and 14 mM in PBS.
Foreskin fibroblast cells (ATCC, CRL-2522) were maintained in DMEM containing 10% FBS, 1% penicillin and streptomycin in T-75 flasks, with 5% CO2 supply at 37°C in an incubator. Cells were passaged when they reached 70-80% growth in flasks. All experiments were carried out using foreskin fibroblast cells between the 5th and 10th culture passage.
Cell viabilities were evaluated by the trypan blue method through all experiments as described previously. Briefly, after treatments, every cell suspension was mixed with an equal volume of 0.4% trypan blue solution. The cells were analyzed in a counting chamber under invert microscope. At least 100 cells were counted per experimental group, and viable and nonviable cells (appear blue) were recorded.
Following pretreatment, cells were exposed to γ-rays from 60 Co source (Alcyon-II P, General Electric) at room temperature. For this purpose, T-25 flasks containing fibroblast cells were placed in an acrylic phantom (dimensions: 30 × 30x15 cm 3), in depth of 5.5 cm, and it was placed transversally to the axis of irradiation. Radiation field was 25 × 25 cm 2, and the distance between the surface of phantom and source of radiation was 80 cm. Total time of exposure to radiation and the absorbed dose was 600s and 3 Gy, respectively. Irradiated cells were immediately stored on ice and within 15 min transferred in the laboratory where comet assay was carried out.
A total of 2 × 105 fibroblast cells were cultured in several individual culture T-25 flasks. After 24 h, a pilot experiment was performed to decide the optimum gamma radiation dose of 3 Gy then fibroblast cells were treated as follows.
- Negative control group: The cells of this group had no treatment with compounds of experiments
- Radiation alone group: The cells of this group were exposed to only 3 Gy of gamma radiation
- EETP + Radiation group: The cells of this group were pretreated with 100, 200 and 300 μg/mL concentrations of EETP for 15 and 30 min before exposure to 3 Gy gamma radiation
- Positive control group (Amifostine + Radiation): The cells of this group were pretreated with 4, 7 and 14 mM concentrations of amifostine for 15 and 30 min before exposure to 3 Gy gamma radiation.
After various treatments, levels of DNA damage of these cells were determined by comet assay. Each assay was performed in triplicate (n = 3) and all experiments data were pooled.
Alkaline version of the comet assay basically as described by Singh et al. was preferred and carried out in this study with slight modification. A total of 40 μL of cell suspension was mixed with 80 μL of LMA in a polypropylene tube, spread on a slide which is previously coated with polylysine and NMA, closed with a coverslip and incubated at +4°C for 10 min. After this time, coverslips were removed and slides were incubated in freshly prepared lysis buffer (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris-HCl [pH 10.0], and 1% Triton X-100) for 1 h at + 4°C to lyse the cells. Then, the slides were placed in a horizontal electrophoresis unit and treated with chilled alkaline electrophoresis solution (0.3 M NaOH, 10 mM EDTA, [pH 13.1]) for 30 min to unwind DNA. Next, electrophoresis was conducted for 20 min at 22 V (1 V/cm) and 300 mA. After electrophoresis, cells were neutralized in buffer (Tris, pH 7.4) for 15 min and incubated in ethidium bromide for 20 min. Finally, slides were covered with coverslips and examined at 40×magnification by using a fluorescence microscope (Nikon Eclipse E800, New York, USA). Three slides were prepared for each experiment. 100 cells from each of the slides were scored for DNA damage. Slide scoring was performed on a blind basis, with the scorer blind to the treatment conditions for each slide. Examined cells were classified according to tail length between 0 and 3, from nondamaged to most damaged. The few images of comets (containing no head or with a very wide tail) were excluded from the analysis since they probably represent dead cells. All slides were scored with the following formula  with a maximum damage possibility of 300:
Score = (1 × n1) + (2 × n2) + (3 × n3) (n = number of cells in each class analyzed).
Statistical analysis was performed using SPSS (version 13.0.1, Licence number: 9069727, Chicago, IL) for Windows. The results were evaluated by analysis of variance (ANOVA) and post hoc Tukey test. Values are given as the mean ± SD. P < 0.05 was regarded as significant.
| > Results|| |
Treatment of fibroblast cells with residual doses of gamma radiation caused increasing DNA damage in a linear dose-dependent manner as measured by comet scores [Figure 1]. Gamma radiation dose of 4 Gy and overdoses cells were scored + 300 by visual analysis. Therefore, measurable maximum gamma radiation treatment dose was chosen as 3 Gy.
|Figure 1: Gamma radiation dose-dependent comet scores in fibroblast cells (n=3). aSignificantly different from the negative control group (P<0.05)|
Click here to view
The comet results of the pretreatment of EETP (100, 200, 300 μg/mL) and amifostine (4, 7, 14 mM) groups are shown in [Table 1] and [Table 2], respectively. Both EETP and amifostine pretreatments showed significant decreasing comet scores compared to radiation alone (3 Gy irradiated cells) group. However, the radioprotective effects of EETP and amifostine were not dose-dependent; EETP and amifostine pretreated cell groups were not statistically significant from each other.
|Table 1: Concentration and pretreatment time of EETP-dependent comet scores in irradiated fibroblast cells (n=3)|
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|Table 2: Concentration and pretreatment time of amifostine-dependent comet scores in irradiated fibroblast cells (n=3)|
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| > Discussion|| |
Due to its generation of ROS, radiation induces ROS-mediated reactions, resulting in various alterations (especially DNA damage) in the cell. Several synthetic compounds have been used as radioprotectors against these deleterious effects of radiation. However, their practical applicability remained limited owing to their high toxicity at the optimum protective dose. The use of flavonoids as potential radioprotectors is of increasing interest because of their high antioxidant activity and wideness in the natural product. The wide spectrum of propolis activities mainly attributed to its contents of polyphenols. Especially, antioxidant properties of propolis and its polyphenolic components are related to their ability to scavenge ROS and to chelate metal ions. Moreover, many studies reported beneficial properties of propolis in vivo and in vitro including antimicrobial, anti-inflammatory, antigenotoxic, immunostimulatory and carcinostatic activities, among others.,,,,,,,,
In the present study, radioprotective effect of Turkish propolis was evaluated against γ-ray-induced DNA damage in human fibroblast cell lines. Studies thus far have not reported the radioprotective effect of Turkish propolis. In our study, pretreatment of fibroblast cells with a concentrations of 100, 200, and 300 μg/mL EETP diminished the harmful effects of radiation on DNA as visualized by comet assay.
The comet assay which is one of the methods for detection of DNA damage, has advantages for evaluating DNA damage in respect to other methods (sister chromatid exchange, alkaline elution, and micronucleus (MN) assays) because it is rapid, easy, and sensitive method, and detects several classes of DNA injury, such as SSBs, DSBs, alkali-labile sites, and crossings. For these reasons, alkaline version of the comet assay basically as described by Singh et al. was preferred and carried out in this study with slight modification. Under alkaline conditions, increased DNA migration is proportional with enhanced levels of DNA damage.
During the recent years, there have been many studies carried out on propolis and plant flavonoids. However; there is limited study about radioprotective capability of propolis in the literature. To the best of our knowledge from literature, the mechanism of the radioprotective effect of propolis from a different region could be explained by its ability to scavenge ROS and activate DNA repair enzymes.,,,,,
In our previous study, we investigated the best solvent for Turkish propolis using concentrations of total polyphenols and flavonoids, assays of ferric reducing power and total antioxidant status in extracts prepared with water, ethanol, DMSO, glycerol, and acetone. Each extract of propolis was also analyzed qualitatively by HPLC method. We observed that EETP has the highest antioxidant status than the others and Turkish propolis rich in flavonoid compound of quercetin. For this reason in this study, we have preferred EETP for evaluating its possible radioprotective effect.
Benkovic et al. investigated that the radioprotective effects of ethanolic extract of Croatian propolis and quercetin on 4 Gy γ-ray-induced DNA damage in human lymphocytes using comet assay, MN and chromosomal aberration (CA) tests. The results were then compared with the known chemical radioprotector aminoethyl isothiourea (AET). They showed that 30 min pretreatment with AET provided better radioprotection than 100 μg/mL EEP and 50 μg/mL quercetin. With a prolonged incubation time, AET significantly increased levels of DNA damage compared to EEP and quercetin. Similarly in our study, parallel concentrations of EETP were effective in the irradiated cell lines.
Similar to our findings, Montoro et al. demonstrated that the capability of EEP to decrease significantly the radiation-induced chromosome damage in human cells exposed to 2 Gy γ-rays. The protection of EEP was found concentration-dependent, with a maximum protection beyond 120 μg/mL of EEP pretreatment. Moreover, higher concentrations of propolis have shown no additional protection.
In a study from Croatia, Benkovic et al. reported a radioprotective effect of water-soluble derivative of propolis and its flavonoid constituents caffeic acid, chrysin and naringin in 4 Gy gamma-irradiated human lymphocytes. Radioprotective effects of tested compounds were comparable with AET. By the CA, MN tests and comet assay results WDSP and its flavonoid constituents did not exhibit toxic effects, however, 30 min pretreatment resulted in significantly decreasing levels of radiation-induced DNA damage.
Prasad et al. reported that 30 min pretreatment with concentrations of 1, 5, 10 μg/mL ferulic acid which is one of the cinnamic acid derivative of propolis, protect human lymphocytes against the 4 Gy gamma radiation-induced MN, CA formations and lipid peroxidation. Rithidech et al. reported that antioxidant effective flavonoid compound of apigenin pretreatment degreased MN formation in 2 Gy gamma radiation-induced chromosomal damage in human lymphocytes. Zhang et al. found that flavonoid compound of morin able to reduce the intracellular ROS generated by γ-irradiation and protected cellular components against DNA damage, membrane lipid peroxidation and recovered cell viability via inhibition of apoptosis. Jeon et al. showed that flavonoids compound naringin elevate CAT, SOD and GPx mRNA synthesis; these effects of flavonoids and polyphenols are probably responsible for the observed radioprotection of EETP against γ-ray-induced DNA damage.
Similar to flavonoids, dietary antioxidants such as vitamins have protective effects against radiation-induced DNA damage. In the study of Konopacka and Rzeszowska-Wolny reported that natural products with antioxidant effect like Vitamin C, E, and β-carotene reduce the 2 Gy γ-ray-induced MN formation in the cultured human peripheral blood lymphocytes.
In this investigation, amifostine was used as a positive control. The physiochemical mechanisms of amifostine mainly attributed to its cytoprotective efficacy include the ability to scavenging ROS through donation of hydrogen atoms and the induction of intracellular hypoxia. Pretreatment with amifostine 15 and 30 min both showed radioprotective effect at concentrations of 4, 7, and 14 mM. Similarly, in the study of Kopjar et al. pretreatment with amifostine at concentration of 7.7 mM have shown radioprotective effect on 2 Gy γ-ray inducted human lymphocytes. In other study, Mozdarani et al. have reported that amifostine pretreatment 15 min at concentrations of 2, 4, and 6 mM degreased MN formation in 6 Gy gamma radiation-induced human blood lymphocytes.
Although, in recent years, there are increasing relevance and studies about propolis, the exact mechanism of biological activities of propolis still remains unknown. Its biological activities are attributed to its polyphenolic contents and approximately 200 different compounds have been determined in propolis so far. Our data show that EETP may have radioprotective effect due to strong antioxidant properties. However, further investigations are needed to clarify the molecular mechanism(s) of the radioprotective effect of propolis and its constituents regarding the use of propolis as functional foods and ingredients in pharmaceuticals, nutraceuticals, medicine, and natural protective agent.
| > Acknowledgments|| |
The authors gratefully acknowledge to Prof. Dr. Murat Erturk, Department of Microbiology, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey for kindly providing foreskin fibroblast cell line.
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[Table 1], [Table 2]