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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 3  |  Page : 512-516

Radioprotective effect of grape seed extract against gamma irradiation in mouse bone marrow cells


1 Department of Radiology, Faculty of Paramedicine, AJA University of Medical Sciences; Department of Radiology, School of Allied Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Radiology, Faculty of Paramedicine, AJA University of Medical Sciences; Department of Medical Physics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
3 Department of Radiology, Faculty of Paramedicine, AJA University of Medical Sciences, Tehran, Iran

Date of Web Publication29-May-2019

Correspondence Address:
Dr. Valiallah Saba
Department of Radiology, Faculty of Paramedicine, AJA University of Medical Sciences, Etemadzadeh Avenue, Fatemi Street, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_243_17

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 > Abstract 


Introduction: Ionizing radiations produce free radicals which are often responsible for DNA damage or cell death. Grape seed extract (GSE) is a natural compound having an antioxidant that protects DNA, lipids, and proteins from free radical damages. In this study, radioprotective effect of the GSE has been investigated in mouse bone marrow cells using micronucleus test.
Materials and Methods: Four groups of mice were investigated in this study: Mice in Group 1 were subjected to injection of distilled water with no irradiation. Mice in Group 2 were exposed to 3 Gy gamma radiation after the injection of distillated water. Mice in Group 3 were injected with 200 mg/kg of the GSE without any irradiation. In another group, mice were exposed to three gray gamma irradiation after the injection of GSE. Animals were killed, and slides were prepared from the bone marrow cells 24 h after irradiation. The slides were stained with May Grunwald–Giemsa method and analyzed microscopically. The frequency of the micronucleated polychromatic erythrocytes (MnPCEs), micronucleated normochromatic erythrocyte (MnNCEs), and polychromatic erythrocyte/polychromatic erythrocyte + normochromatic erythrocyte (PCE/PCE + NCE) ratios was calculated.
Results: Injection of GSE significantly decreased the frequency of MnPCEs (P < 0.0001) and MnNCEs (P < 0.05) and increased the ratio of PCE/PCE + NCE (P < 0.0001) compared to the irradiated control group.
Discussion and Conclusions: GSE could reduce clastogenic and cytotoxic effects of gamma irradiation in mice bone marrow cells; therefore, it can be concluded that the GSE is a herbal compound with radioprotective effects against gamma irradiation. Free radical scavenging and the antioxidant effects of the GSE probably are responsible mechanisms for the GSE radioprotective effects.

Keywords: Bone marrow cells, grape seed extract, irradiation, micronucleus assays, radiation protection


How to cite this article:
Targhi RG, Banaei A, Saba V. Radioprotective effect of grape seed extract against gamma irradiation in mouse bone marrow cells. J Can Res Ther 2019;15:512-6

How to cite this URL:
Targhi RG, Banaei A, Saba V. Radioprotective effect of grape seed extract against gamma irradiation in mouse bone marrow cells. J Can Res Ther [serial online] 2019 [cited 2019 Oct 21];15:512-6. Available from: http://www.cancerjournal.net/text.asp?2019/15/3/512/229520




 > Introduction Top


Ionizing radiation could produce reactive free radicals such as hydroxyl radicals, hydrogen radicals, and toxic substances (i.e., hydrogen peroxide) in the living tissues. When free radicals interact with critical macromolecules, such as DNA, proteins, or membranes, irreversible damage occurs, leading to cell death.[1]

Over the recent years, the investigations have focused on the evaluation of plant products as radioprotectors, due to their efficacy and low toxicity.[2] The radioprotective effect of plant extract is due to their large number of active constituents, such as antioxidants, immune stimulants, and compounds with antimicrobial activity.[2]

The grape seed extract (GSE) has been widely used in medicine.[3],[4],[5] One of the GSE futures is its antioxidant activity which is greater than the Vitamins C, E, and betacarotene.[6] GSE has been reported to be rich in polyphenols containing monomeric flavanols: catechin, epicatechin and epigallocatechin, or dimers, trimers, and polymers with seven or more flavanol units: the proanthocyanidins.[7]

Natural grape seed proanthocyanidins have polyphenolic flavonoids which include oligomeric proanthocyanidins. These proanthocyanidins have demonstrated biological, pharmacological, therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stresses.[8]

In this study, we investigated the radioprotective effect of the GSE against gamma irradiation by micronucleus assay in Balb/c mice.


 > Materials and Methods Top


Animals

Adult male Balb/c mice (6–8 weeks old) with the weights of 25–30 g were purchased from the Pasteur Institute (Tehran, Iran). The animals were maintained in good conditions at the university animal house and were fed with the standard mouse pellet and water ad libitum. All of the animals were kept under the controlled lighting condition (light: dark, 12:12 h) and temperature (22°C ± 2°C). Experimental protocols were approved by the Institutional Animal Ethics Committee.

Treatment

GSE was dissolved in distilled water, and a 200 mg/kg of GSE which dissolved in water was injected intraperitoneally to the experimental animals 1 h before gamma irradiation. The control group received the same volume of distilled water.

Irradiation

The mice were irradiated by a cobalt-60 gamma radiation source (Teratron 780, Canada). Mice were placed in ventilated Plexiglas cages. The source-to-skin distance was 70 cm. The mice were exposed to 3 Gy whole-body gamma irradiation with a dose rate of 54 cGy/min at room temperature (23°C ± 2°C).

Experimental design

The animals (32 mice) were randomly divided into four groups (eight mice in each group):

  • Group I – The distilled water was intraperitoneally injected to these animals instead of the GSE without any irradiation
  • Group II – Animals of this group were exposed to 3 Gy gamma radiation after the injection of distilled water
  • Group III – GSE was injected intraperitoneally to these animal group members without any irradiation to assess the toxicity of the GSE
  • Group IV – Animals of this group were intraperitoneally injected by 200 mg/kg of GSE before gamma irradiation.


Twenty-four hours after the treatment or irradiation, the animals of these four groups were anesthetized with ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively, intraperitoneal [i.p].) and were sacrificed by cervical dislocation. Both femurs of these animals were removed for micronucleus assay.

Micronucleus assay

The mouse bone marrow micronucleus test was carried out according to the method described by Schmid.[9] The bone marrow from the femurs was flushed in the form of a fine suspension and placed in a centrifuge tube containing fetal bovine serum. The cells were collected by centrifugation at 1000 rpm for 10 min at 4°C. Bone marrow smears were prepared and the slides were placed in room temperature, and smears were stained with May–Grunwald/Giemsa after 24 h of air drying. According to this method, polychromatic erythrocytes (PCEs) stained reddish-blue and normochromatic erythrocytes (NCEs) stained orange, while nuclear materials were dark purple. Eight mice were used for each experimental group, and a total of 8000 PCEs were scored per each group to determine the percentage of micronucleated polychromatic erythrocytes (MnPCEs), micronucleated normochromatic erythrocytes (MnNCEs), and the ratio of PCE/PCE + NCE. The ratio of PCE/PCE + NCE was determined for each group to assess the radiation effects with or without GSE on bone marrow proliferation.[1]

Statistical analysis

Statistical analysis was performed using SPSS 16.0 software (IBM Corporation, Chicago, IL, USA). The one-way ANOVA analysis and the Turkey's honest significant difference test were used for multiple data comparisons.


 > Result Top


Effect of gamma irradiation on mice bone marrow cells

The frequency of MnPCE and MnNCE increased significantly in a group that only irradiated with 3 Gy gamma irradiation in comparison with the control group. The number of micronucleus in PCE cells highly increased (P < 0.00001); however, this parameter did not remarkably increase in the NCE cells (but it is significant compared to the control group) (P < 0.02); this was due to the more radiation sensitivity of PCE cells compared to the NCE cells. The ratio of PCE/PCE + NCE significantly decreased by 3 Gy gamma irradiation (P < 0.00001). The results are shown in [Table 1] and [Figure 1].
Table 1: Effects of the grape seed extract on the formation of radiation-induced micronuclei in polychromatic erythrocytes, normochromatic erythrocytes cells, and the ratio of polychromatic erythrocytes/polychromatic erythrocytes + normochromatic erythrocytes in mice bone marrow exposed to 3Gy gamma irradiation (values are means±standard error of the mean for each group)

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Figure 1: Frequency of micronucleated polychromatic erythrocyte in the control group, irradiated group, and the groups that received 200 mg/kg of grape seed extract before irradiation. Error bars indicate standard errors of mean values

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These data showed that 3 Gy gamma irradiation induced genotoxicity and cytotoxicity in mouse bone marrow cells.

Effects of the grape seed extract on mice bone marrow cells

GSE was injected to the animals and frequencies of MnPCE, MnNCE, and PCE/PCE + NCE ratios were evaluated in bone marrow cells after 24 h.

Injection of the 200 mg/kg of the GSE, without any irradiation, did not significantly change the frequency of MnPCE, MnNCE, and PCE/PCE + NCE ratios, compared to the control group (P > 0.05). These results indicated that the GSE did not have clastogenic and cytotoxic effects on mouse bone marrow cells [Table 1] and [Figure 2].
Figure 2: Frequency of micronucleated normochromatic erythrocyte in the control group, irradiated group, and the groups that received 200 mg/kg of grape seed extract before irradiation. Error bars indicate standard errors of mean values

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Effect of gamma irradiation + grape seed extract on mice bone marrow cells

Administration of GSE 1 h before irradiation caused a reduction in the frequency of MnPCE (P < 0.0001) and MnNCE (P < 0.03), but an increase in the ratio of PCE/PCE + NCE (P < 0.0001) compared to irradiation without GSE administration [Table 1] and [Figure 3]. These results indicate that GSE reduced clastogenic and cytotoxic effects of radiation.
Figure 3: The ratio of erythrocyte/polychromatic erythrocyte + normochromatic erythrocyte in the control group, irradiated group, and the groups that received 200 mg/kg of grape seed extract before irradiation. Error bars indicate standard errors of mean values

Click here to view



 > Discussion Top


Radiation can induce oxidative stress through generation of reactive oxygen species, resulting in imbalance of pro-oxidants and antioxidants in the cells, which is suggested to culminate in cell death.[10] Intracellular generation and accumulation of free radicals in the stressed cells overcome the natural antioxidant defense, causing damage to biological macromolecules, including nucleic acids, proteins, and lipids.[11] It is obvious that a large number of DNA-single-strand and double-strand breaks are due to the damages of free radicals. DNA-double-strand break leads to chromosome breakage and causes micronucleus to indicate the chromosomal breakage.[12]

It was showed that antioxidant agents (such as potent antioxidant plant) neutralized the free radical species so they can inhibit the side effects induced by irradiation.[3] Many plants were the subject for investigations to find herbal radioprotectors.[13],[14] In several investigations, radioprotective effects of some plants were evaluated by micronucleus assay in mice bone marrow cells.[2],[15],[16] Injection of plant extracts (Triphala, Hippophae rhamnoides, Mangifera indica, Panax ginseng, Mentha piperita, Tinospora cordifolia, Aeglemarmelos, Naringin, and Spirulina)[17] before any irradiation to the mice resulted in the reduction of a mortality and symptoms of radiation sickness compared to the irradiated controls.[18]

The radioprotective effects of hesperidin (HES), a flavonone glucoside, were investigated by using the micronucleus test for anticlastogenic and cell proliferation activity.[16] All doses of HES significantly reduced the frequencies of MnPCEs and increased PCE/PCE + NCE ratio in mice bone marrow compared with nondrug-treated irradiated control (P = 0.0001). There was a drug dose–response effect of HES in reducing MnPCE and increasing the PCE/PCE + NCE ratio in bone marrow cells. The maximum reduction in MnPCE was observed in mice treated with HES at a dose of 80 mg/kg

The radioprotective effect of hawthorn (Crataegus microphylla) fruit extract against genotoxicity induced by gamma irradiation has been investigated in mouse bone marrow cells,[15] and a single i.p administration of hawthorn extract at doses of 25, 50, 100, and 200 mg/kg 1 h before gamma irradiation (2 Gy) reduced the frequencies of MnPCEs and increased the PCE/PCE + NCE ratio in mice bone marrow compared with the nondrug-treated irradiated control (P < 0.02–0.00001). The maximum reduction in MnPCEs was observed in mice treated with extract at a dose of 200 mg/kg.

The antioxidant activity and free radical scavenging have been attributed to flavonoids, orientin, and vicenin.[19] Radioprotective and antioxidative effects of various other natural products have also been reported,[20] and oxidative damage induced by pathophysiological agents such as photosensitization and radiation in rat liver membrane was examined with two medicinal plant extracts, namely, Andrographis paniculata and Swertia chirata. Results showed that simultaneous addition of both the extracts (50 μg/ml) could significantly prevent the increased levels of products of lipid peroxidation.

The radioprotective effects of Mentha oil (Mentha piperita Linn.) against radiation-induced hematological alterations in peripheral blood and the survival of Swiss albino mice were studied. Mentha oil 40 μL/animal/day for 3 consecutive days when fed orally before whole-body gamma irradiation (8 Gy) showed protection of the animals in terms of the survival percentage and hematological parameters in mice. Only 17% of the mice died within 30 days in the experimental group (Mentha oil pretreated irradiated) compared to the control group in which 100% mortality was observed.[18]

In the present study, the radioprotective effect of the GSE was investigated. We observed that the GSE significantly reduced the number of MnPCE and MnNCE that were induced by gamma radiation in mouse bone marrow cells. It also increased the ratio of PCE/PCE + NCE, which was reduced by radiation. Our results are consistent with the results obtained by the other researchers.[17],[21],[22],[23],[24]

The GSE has powerful antioxidant compounds such as flavonoids, proanthocyanidins, and polyphenols such as gallic acid, catechin, and epicatechin.[21] Bagchi et al. showed that the grape seed proanthocyanidins are safe, novel, highly potent, and bioavailable free radicals and antioxidant scavengers and possess a broad spectrum of health benefits.[22] In another study, they discovered that the grape seed proanthocyanidins had significantly greater protection against free radicals, free radical-induced lipid peroxidation, and DNA damages compared to Vitamins C and E and β-carotene.[23] Such a remarkable spectrum of biochemical and cellular functions holds promise for the prevention and treatment of a variety of human disorders caused by oxidative stresses.

Some studies have reported an enhancing effect of GSE or its polyphenolic constituents on several antioxidant enzymes such as glutathione superoxide dismutase, catalase, and other detoxifying/antioxidant enzymes. They have also shown that the GSE prevents free radical-induced DNA damages.[24]

Heba Hosny showed that the GSE could increase the endogenous antioxidant defense mechanism in a rat and thereby protect the animals from radiation-induced hepatotoxicity.[25] Data obtained from this study indicate that GSE could increase the endogenous antioxidant defense mechanism in rat and thereby protect the animals from radiation-induced hepatotoxicity.[25]

Injection of the 200 mg/kg GSE (1 h before irradiation) reduced the frequency of MNPCE and MNNCE cells with a dose reduction factor of 2.19–2.81 and increased the ratio of PCE/PCE + NCE.

Higher doses of GSE are toxic and administration of GSE before radiation is more effective than after radiation.[22],[26],[27] Hence, the dose of 200 mg/kg of GSE was administered 1 h before irradiation in the current study to have higher protection against radiation. Other irradiation sources such as electron, proton, or other ionizing radiations with different energies and intensities can be used in the similar experiments. Another subject that can be studied is the mechanisms which are responsible for the GSE radioprotective effect.


 > Conclusions Top


The results (from this study) indicated that the GSE was an effective herbal compound for decreasing the clastogenic and cytotoxic effects of gamma irradiation. Free radical scavenging and the antioxidant effects of the GSE probably are the responsible mechanisms for the GSE radioprotective effects. Injection of GSE significantly reduced the frequency of MnPCEs (P < 0.0001) and MnNCEs (P < 0.05) and increased the ratio of PCE/PCE + NCE (P < 0.0001) compared to the irradiated control group. It should be mentioned that genotoxicity or toxic effects of the GSE were not observed in the mice bone marrow cells.

Financial support and sponsorship

This study was financially supported by a grant from AJA University of Medical Sciences (Tehran, Iran).

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Mansour HH. Protective role of grape seed extract against radiation induced oxidative stress in rats: Role of endogenous antioxidants. J Rad Res Appl Sci 2011;4:895-909.  Back to cited text no. 18
    
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