Investigation of the radioprotective efficacy of hesperidin against gamma-radiation induced cellular damage in cultured human peripheral blood lymphocytes

Mutation Research 676 (2009) 54–61

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Investigation of the radioprotective efficacy of hesperidin against gamma-radiation induced cellular damage in cultured human peripheral blood lymphocytes K.B. Kalpana, N. Devipriya, M. Srinivasan, Venugopal P. Menon ∗ Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608002, Tamil Nadu, India

a r t i c l e

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Article history: Received 23 October 2008 Received in revised form 20 February 2009 Accepted 20 March 2009 Available online 31 March 2009 Keywords: Hesperidin Comet assay Dicentric aberration DNA fragmentation assay Lymphocytes Micronuclei TBARS Gamma-radiation

a b s t r a c t The present study was aimed to evaluate the radioprotective efficacy of hesperidin (HN), a flavonone glycoside against ␥-radiation-induced cellular damage in cultured human peripheral blood lymphocytes. Different concentrations of HN (3.27, 6.55, 9.83, 13.10, 16.38 and 19.65 ␮M) were pre-incubated with lymphocytes for 30 min prior to ␥-irradiation [4Gy] and the micronuclei (MN) scoring, dicentric aberration and comet assay were performed to fix the effective dose of HN against ␥-irradiation induced cellular damage. The results indicated that among all the concentrations, 16.38 ␮M concentration of HN showed optimum protection by effectively decreasing the MN frequencies, dicentric aberrations and comet attributes. Based on the above results, 16.38 ␮M concentration of HN was fixed as the effective dose to further investigate its radioprotective efficacy which was then carried out by pre-incubating lymphocytes with 16.38 ␮M concentration of HN, exposing the lymphocytes to different doses (1, 2, 3 and 4Gy) of radiation and investigating radiation induced genetic damage (MN, dicentric aberration, comet assay, DNA fragmentation assay) and biochemical changes (changes in the level of enzymic and non-enzymic antioxidants, lipid peroxidation). The results indicated a dose dependent increase in both genetic damage and thiobarbituric acid reactive substances (TBARS), accompanied by a significant decrease in the antioxidant status compared to HN treated groups which modulated the toxic effects through its antioxidant potential. Thus the current study shows HN to be an effective radioprotector against ␥-radiation induced in-vitro cellular damage in lymphocytes. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The application of ionizing radiation in cancer treatment as an alternative to surgery was recognized soon after the discovery of X rays in 1895 by Roentgen and has been used for several decades in curative and palliative treatments of cancerous solid tumors [1]. In particular, ␥-radiation has been used in radiotherapy for several decades, and it is known that its exposure gives rise to genomic instability leading to mutagenesis, carcinogenesis, and cell death [2]. Exposure of gamma rays is also known to induce a variety of cellular and sub-cellular damage in many living organism at sub-lethal doses [3]. ␥-Radiation can be absorbed directly by DNA, leading to ionization of both the nucleobases and sugar in a mechanism described as the direct effect of generating single and tandem

∗ Corresponding author at: Department of Biochemistry and Biotechnology, Chairman of Science, Center for Micronutrient Research, Annamalai University, Annamalainagar 608002, Tamil Nadu, India. Tel.: +91 04144 238343; fax: +91 04144 239141. E-mail addresses: [email protected], [email protected] (V.P. Menon). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.03.005

DNA damage [4]. However, approximately 65% of the DNA damage is caused by the indirect effect of free radicals such as hydroxyl radicals (• OH) that are formed from the radiolysis of surrounding water molecules and that successively attack DNA [5]. Most of the effort to understand the molecular basis of DNA damage by ␥-radiation has been performed using alkaline single-cell gel electrophoresis (commonly referred to as the alkaline comet assay) [6], cytokinesis-block micronucleus method [7], chromosomal aberrations and DNA fragmentation assay. Apart from the genetic damage, lipid peroxidation (LPO) is also considered as a critical event during ionizing radiation induced damage [8]. Lipid peroxidation has been found to increase with increase in radiation dose in rat liver mitochondria, microsomes and splenic lymphocytes [9]. To maintain the redox balance and in-order to protect them from free radicals action, living cells have evolved an endogenous antioxidant defense mechanism which includes non-enzymatic entities like glutathione, ascorbic acid and also enzymes like superoxide dismutase, catalase, glutathione peroxidase, etc. [10]. Although endogenous cellular antioxidants act in concert to eliminate ROS accumulation in a physiological state, under pathological conditions, ROS overload might exceed the cellular antioxidant capacity,

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2.4. Isolation of lymphocytes

Fig. 1. Molecular structure of Hesperidin [MW: 610.57 daltons].

affecting critical biological macromolecules and triggering oxidative stress [11]. For these reasons, the search for new radioprotectors that are less toxic than the currently available compounds is crucially needed to develop better strategies for protecting normal cells from radiation induced damage. Much of the attention given to flavonoid compounds comes from the results of epidemiological studies that suggest high fruit and vegetable consumption is associated with a decreased risk of several types of cancer, including breast, colon, lung, larynx, pancreas, oral and prostate cancer [12]. These suggested protective effects of flavonoids, together with their potent antioxidative and free-radical scavenging activities observed in in-vitro studies [13] have increased the public’s interest in the use of flavonoids for their potential health benefits. Hesperidin (HN), a flavanone-type flavonoid, is found abundant in citrus fruit [14]. The peel and the membranous parts of these fruits have the highest HN concentrations. HN (Fig. 1) is comprised of the flavanone (a class of flavonoids) hesperitin and the disaccharide rutinose. HN has been reported to exert a wide range of pharmacological effects [15] which includes antioxidant, anti-inflammatory, anti-allergic, hypolipidemic, vasoprotective and anticarcinogenic actions [16]. HN molecular formula is C28 H34 O15 , and its molecular weight is 610.57 daltons. However, only few studies have been carried out on the in-vitro radioprotective effect of HN. Hence the aim of our present study was to investigate the radioprotective effect of HN on ␥-radiation induced cellular damage in cultured human peripheral blood lymphocytes. 2. Materials and methods 2.1. Chemicals Hesperidin [CAS registry number: 520-26-3], Cytochalasin-B (Cyt-B), heat inactivated fetal calf serum (FCS), colchicine, ethidium bromide, histopaque-1077, low melting agarose (LMA), normal melting agarose (NMA), Roswell Park Memorial Institute (RPMI-1640) media, thiobarbituric acid (TBA), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), 5 ,5 -dithio (bis)-2-nitrobenzoic acid (DTNB), nicotinamide adenine dinucleotide (NAD), triton X-100, ethylene diamine tetra-acetic acid (EDTA) and sodium sarcosinate were purchased from M/s. Sigma Chemical Co., St Louis, USA. Other chemicals like penicillin, streptomycin, l-glutamine and reduced glutathione (GSH) were purchased from Himedia, Mumbai. Phytohemagglutinin M (PHA-M) was purchased from GIBCO-BRL, USA. Giemsa stain was obtained from EMerck, Germany. DNA extraction solution was purchased from Genei, Bangalore. All other chemicals and solvents were of analytical grade and obtained from S.D. Fine chemicals, Mumbai. 2.2. Preparation of drug HN (1 mg/ml) was dissolved in 0.01% dimethyl sulfoxide (DMSO) and was used as the stock solution. The stock solution was then diluted with sterile distilled water (milli-Q water) to arrive at a final concentration of 3.27, 6.55, 9.83, 13.10, 16.38 and 19.65 ␮M concentration of HN. 0.2% DMSO was used as a sham control. 2.3. Irradiation 60 Co-gamma rays in a Phonex teletherapy unit (GVN Hospitals, Trichy, Tamil Nadu, India) were used for the irradiation purpose. Lymphocytes (with or without hesperidin pre-treatment) were exposed to different doses of radiation, depending upon the requirement of the present study.

Blood samples were aseptically collected in heparinized sterile glass tubes from median cubital vein of nonsmoking healthy individuals (22–25 years). Written consent was obtained from each one of them. Lymphocytes were isolated from blood using Ficoll–Histopaque (Sigma, USA) and cultured as described previously [17]. Blood was diluted 1:1 with phosphate buffered saline (PBS) and layered onto the Histopaque in the ratio of 4:3 (blood + PBS: Histopaque). The blood was centrifuged at 400 × g for 30 min at room temperature. The lymphocyte layer was removed and washed twice in PBS at 250 × g for 10 min each, and then washed with RPMI-1640 media. The concentration of viable cells was established by trypan blue dye exclusion study and hemocytometer was used to count the number of viable cells. The viable cells were then suspended in (RPMI-1640) supplemented with NaHCO3 (7.5% (w/v)), 20% FCS, 200 mM l-glutamine, penicillin 100 units/mL and streptomycin 100 ␮g/ml were in a 15 ml conical tube. PHA-M (0.2 ml) was added to the culture to initiate cell division. Cells were incubated at 37 ◦ C in a humidified 5% CO2 atmosphere. Typically, each culture consisted of an initial density of 1 × 106 cells in 2 ml culture medium. 2.5. Experimental design Our present study was classified into two stages. The first stage was carried out to fix the effective dose of HN by carrying out MN, dicentric aberrations and comet assay. The effective dose was then used to study the radioprotective effect of HN in cultured human lymphocytes in vitro. 2.6. Treatment of lymphocytes After 24 h following culture initiation, the lymphocytes were treated with different concentrations of HN for 30 min depending upon our study. In the control group, DMSO alone was used. During treatment, normal lymphocytes with/without HN showed no variation in their viability. Thirty minutes after HN treatment, all the lymphocyte culture tubes excluding the control lymphocytes were exposed to 60 Co ␥-rays (as per required to the study). The samples soon after irradiation were kept at 37 ◦ C for 1 h to allow for possible damage-repair, a process simulating the in vivo situation and then transported to the laboratory in ice condition. 2.7. Study design 1 2.7.1. Selection of effective dose of HN To determine the effective dose of HN, lymphocytes were incubated with graded drug doses (3.27, 6.55, 9.83, 13.10, 16.38 and 19.65 ␮M concentrations) of HN and exposed to 4Gy gamma irradiation because it has been known to induce a high frequency of micronuclei in human lymphocytes exposed in vitro [18]. Cultured human lymphocytes were divided into 8 groups; in each group 6 individual samples were processed (n = 6). Sham Control: The cultured lymphocyte received 0.2% DMSO Irradiation Control: The culture of this group was exposed to 4Gy gamma irradiation HN + irradiation: The cultures of these groups were treated with different doses (3.27, 6.55, 9.83, 13.10, 16.38 and 19.65 ␮M) of HN for 30 min before exposure to 4Gy gamma irradiation 2.7.1.1. Micronuclei assay and scoring. The presence of micronuclei (MN) in a binucleated cell was assayed by blocking the cell at cytokinesis stage as described by Fenech and Morely [19]. To 0.5 ml of the lymphocyte culture (treated/untreated), 5 ml culture medium (RPMI-1640), supplemented with NaHCO3 (7.5%, w/v), 20% fetal calf serum, 200 mM l-glutamine, penicillin 100 units/mL and streptomycin 100 ␮g/mL in a 15 mL conical tubes were added. About 0.2 mL of PHA-M was added to the culture to initiate cell division. Cyt-B was added to the culture at 44 h. The cells were further incubated at 37 ◦ C for another 28 h. The cells were harvested, cast on a pre-cooled slide and stained with Giemsa. Binucleated cells (BNC) surrounded by cytoplasm were scored for the presence of MN according to the criteria of Countryman and Heddle [20]. In each group a total of 6000 (1000 cells from six individual experiments) BNC were scored and the frequency of cells with one (MN1), two (MN2) and three (MN3) micronuclei were recorded. The total number of MN in each group was derived from (1 × MN1) + (2 × MN2) + (3 × MN3). The data are presented as the number of MN/1000 binucleate cells. 2.7.1.2. Dicentric (DC) aberration and scoring. Lymphocyte culture was set as described for MN up to the stage of addition of PHA-M. Colchicine (Sigma) at a final concentration of 0.1 ␮g/5 ml was added at 67 h to block the cells at metaphase stage. The cells were harvested at 72 h, and were given hypotonic treatment for 10 min and transferred to a pre-cooled slide. The slides were stained with 10% Giemsa, mounted with DPX and examined under 10 × 100 oil immersion to score DC aberration [21]. In each group, a total of 600 (100 cells from each experiments) were scored and the frequencies of cells with one (DC1), two (DC2) and three (DC3) dicentric were recorded. The total number of DC in each group was derived from (1 × DC1) + (2 × DC2) + (3 × DC3). The data are presented as the number of DC/100 cells.

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Fig. 2. Dose dependent effect of HN on MN and DC frequencies induced by 4Gy gamma radiation. Values are given as mean ± SD of six experiments in each group. a P < 0.01 compared to radiation alone; b P < 0.05 compared to group 3; c P < 0.01 compared to group 4; d P < 0.01 compared to group 5; e P < 0.01 compared to group 6; f P < 0.01 compared to group 7.

2.7.1.3. Alkaline single-cell microgel electrophoresis (Comet assay). Once viabilities were ensured more than 80%, the lymphocytes were subjected to the Comet assay by the method of Singh [22]. In brief, frosted microscopic slides were covered with 200 ␮L of 1% normal melting agarose (NMA) in PBS at 45 ◦ C and kept at 4 ◦ C for 10 min to allow the agarose to solidify. The second layer of 200 ␮L of 0.5% low melting agarose (LMA) containing approximately 106 cells of lymphocytes (treated/untreated) were added. After solidification for 15 min on ice, the slides were placed in the chilled lysing solution containing 2.5 M NaCl, 100 mM Na2+ EDTA, 100 mM Tris–HCl, at pH 10 and 1% DMSO, 1% triton X 100 and 1% sodium sarcosinate for 1 h at 4 ◦ C. The slides were removed from the lysing solution and placed on a horizontal electrophoresis tank filled with alkaline buffer (300 mM NaOH, 1 mM Na2+ EDTA and 0.2% DMSO, pH ≥ 13) for 20 min. The electrophoresis was carried out for 20 min. After electrophoresis, the slides were washed gently with 0.4 M Tris–HCl buffer (pH 7.4) and the slides were stained with 50 ␮L of propidium iodide and visualized using a Nikon fluorescent microscope. The images (45–55 cells/slide) were captured with high performance Nikon camera. The quantification of the DNA strand breaks of the stored images were done using the CASP software by which all comet attributes could be obtained directly. 2.8. Study design 2 2.8.1. Investigation of radioprotective efficacy of HN The radioprotective effect of HN in cultured human lymphocytes was carried out by performing biochemical estimations, micronuclei, comet assay, looking for dicentric (DC) aberrations and DNA fragmentation assay. To perform the assays, lymphocytes cultures were set up as described early and divided into the following groups. Sham control: the culture received 0.2% DMSO as vehicle HN control: HN (effective dose selected from study design 1) pretreated lymphocytes Radiation control: The cultured lymphocytes were exposed to different doses of (1, 2, 3 and 4Gy radiation) HN + irradiation: The culture of this group was treated with HN (effective dose) before exposure to different doses of radiation (1, 2, 3 and 4Gy). 2.8.1.1. Biochemical assays. Lymphocytes were suspended in 130 mM KCl plus 50 mM PBS containing 10 ␮M dithiothreitol and centrifuged at 20,000 × g for 15 min (4 ◦ C). The supernatant was taken for biochemical estimations. In each group six samples (n = 6) were processed. The level of lipid peroxidation was determined by analyzing TBA-reactive substances according to the protocol of Nichans and Samuelson [23]. The pink-colored chromogen formed by the reaction of TBA with breakdown products of lipid peroxidation was measured. Superoxide dismutase (SOD) activity was assayed by the method of Kakkar et al., [24], based on the inhibition of the formation of NADH–PMS–NBT complex. Catalase (CAT) activity was assayed by the procedure of Sinha [25], quantifying the hydrogen peroxide after reacting with dichromate in acetic acid. The activity of glutathione peroxidase (GPx) was assayed by the method of Rotruck et al. [26], a known amount of enzyme preparation was allowed to react with hydrogen peroxide (H2 O2 ) and GSH for a specified time period. Then the GSH content remaining after the reaction was measured. The total GSH content was measured by the method of Ellman [27]. This method was based on the development of a yellow color when 5 ,5 -dithiobis 2-nitrobenzoic acid was added to compounds containing sulfhydryl groups. 2.8.1.2. DNA fragmentation assay. DNA gel electrophoresis was performed in order to verify DNA fragmentation [28]. After treatments, cellular DNA from different

groups was isolated using DNA extraction kit according to the manufacturer’s instruction (Bangalore, Genei). Briefly, 1 ml of DNA extraction solution was added to 107 cells either in pellet or suspension and the cells was lysed with repeated pipetting. Pellet the homogenate for 15 min at 10,000 rpm at 37 ◦ C. Following centrifugation, transfer the resulting supernatant to a fresh tube and precipitate DNA by adding equal volumes of 100% ethanol. The DNA is quickly visible as cloudy precipitate which is then transferred into a fresh tube, dried, resuspended in TE buffer and quantitated spectrophotometrically at OD260 nm [29]. Two micrograms of DNA per lane was electrophoresed on a 2% agarose gel stained with ethidium bromide (2.5 ␮g/ml) at 50 V for approximately 2 h. The gel was then documented (Gel Doc, Bio Rad) and the intensity of the bands was determined (Quantity one software). 2.9. Statistical analysis Statistical analysis was performed using Student’s t test. The values were mean ± SD for six samples in each group. The values of P < 0.05 and P < 0.01 were considered to be significant.

3. Results 3.1. Selection of effective dose of HN The alteration in the levels of MN and DC frequencies in the lymphocytes is shown in Fig. 2. Gamma irradiated lymphocytes showed a significant elevation in the levels of MN and DC frequencies whereas pre-incubation of HN with lymphocytes prior to gamma radiation significantly decreased the levels of MN and DC frequencies in a concentration dependent manner (3.27, 6.55, 9.83, 13.10, 16.38 and 19.65 ␮M). However, optimum protection was observed at the concentration of 16.38 ␮M as evidenced by a significant decrease in the level of MN and DC counts (59.85 ± 4.56 and 25.02 ± 1.90) when compared to all other concentrations. Damage to cellular DNA was determined by the alkaline comet assay (Fig. 3). Exposure of lymphocytes to ␥-radiation resulted in an increase in comet attributes such as tail length, tail moment, olive tail moment and % DNA in the tail. When compared to the gamma irradiated lymphocytes, pretreatment with HN decreased the comet parameter at all concentrations (3.27, 6.55, 9.83, 13.10, 16.38 and 19.65 ␮M). Among all concentrations, 16.38 ␮M concentration of HN was found to be more effective in decreasing the tail length (23 ± 1.75), tail moment (7.00 ± 0.53), olive tail moment (2.17 ± 0.16) and % DNA in tail (8.12 ± 0.61). Based on these observed results of decreasing micronuclei frequencies, dicentric aberrations and comet attributes, 16.38 ␮M concentration of HN was selected as the effective dose in preventing damage against gamma irradiation and was used to further evaluate its radioprotective efficacy.

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Fig. 3. Dose dependent effect of HN on tail length, tail moment, olive tail moment and % DNA in tail induced by 4Gy radiation. Values are given as mean ± SD of six experiments in each group. a P < 0.05 compared to radiation alone; b P < 0.01 compared to radiation alone; c P < 0.05 compared to group 3; d P < 0.05 compared to group 4; e P < 0.05 compared to group 5; f P < 0.01 compared to group 6; g P < 0.01 compared to group 7.

3.2. Radioprotective efficacy of HN 3.2.1. Micronuclei and Dicentric aberration assay The frequencies of MN and DC in human lymphocytes induced by various doses of radiation and in combination with hesperidin are shown in Fig. 4. A dose dependent increase in the total MN and DC frequencies was observed in the gamma irradiated (1, 2, 3 and 4Gy) groups. When compared to their corresponding radiation groups, pre-treatment of HN (16.38 ␮M) prior to different doses of radiations (1, 2, 3 and 4Gy) showed a significant (P < 0.01) decrease in the MN and DC frequencies and the protection offered by HN was approximately at the range of 80-85%, which is the biological index for the detection of inhibiting potential of HN on cellular toxicity induced by gamma irradiation. HN alone treated lymphocytes did not show any index of MN frequencies.

3.2.2. Comet assay The alteration in the levels of comet attributes is shown in Fig. 5. Gamma irradiated groups showed a dose dependent increase in the levels of all comet parameters (tail length, tail moment, olive tail moment and % DNA in the tail), whereas pretreatment of hesperidin (16.38 ␮M) with lymphocytes prior to 1, 2, 3 and 4Gy irradiations showed a significant decrease in the levels of comet attributes. In HN alone treated lymphocytes, we observed no significant increase in the comet formation when compared to sham control. 3.2.3. Endogenous antioxidant status The levels of enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) and the activities of non-enzymatic antioxidant: reduced glutathione (GSH) are shown in Figs. 6 and 7. The results indicated that gamma irradiated lymphocytes showed a significant decrease in the levels of

Fig. 4. Inhibitory effect of HN on MN and DC frequencies induced by different doses of radiation. Values are given as mean ± SD of six experiments in each group. a P < 0.1 compared to group 1; b P < 0.01 compared to their corresponding radiation groups.

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Fig. 5. Protection of cellular DNA (i–iv) of human peripheral blood lymphocytes from different doses of gamma radiation by HN as measured by tail length, tail moment, olive tail moment and % DNA in tail. Values are given as mean ± SD of six experiments in each group. a P < 0.1 compared to group 1; b P < 0.01 compared to their corresponding radiation groups.

both enzymatic and non-enzymatic antioxidant status when compared to hesperidin pre-treated groups which showed an increased antioxidant status indicating that pretreatment with hesperidin restored the antioxidant status to near normal. In addition, HN alone treated lymphocytes did not show any alteration in the antioxidant status. 3.2.4. Lipid peroxidation Changes in the level of lipid peroxidative index are shown in the Fig. 8. Exposure to different doses of ␥-irradiation (1, 2, 3 and 4Gy) resulted in a significant increase in the levels of TBARS, which was

effectively modified by pretreatment with hesperidin at the dose 16.38 ␮M prior to ␥-irradiation which indicates the antioxidant potential of hesperidin. In hesperidin alone treated lymphocytes, we observed (P < 0.1) significant difference when compared to control lymphocytes. 3.2.5. DNA fragmentation assay The results of DNA fragmentation assay is shown in Fig. 9. The results indicated that gamma irradiated groups (lane 3) showed an increase in the length of DNA fragments indicating the damage caused by ␥-irradiation which is repaired by treatment of DNA with

Fig. 6. Changes in the activities of SOD, CAT and GPx (i–iii) in normal, ␥-irradiated and HN pretreated lymphocytes. Ua , 50% inhibition of nitroblue tetrazolium reduction in 1 min, Ub , ␮moles of hydrogen peroxide consumed per minute, Uc , ␮moles of GSH utilized/min. Values are given as mean ± SD of six experiments in each group. a P < 0.1 compared to group 1; b P < 0.01 compared to their corresponding radiation groups.

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Fig. 7. Changes in the levels of GSH in normal, ␥-irradiated and HN pretreated lymphocytes. a P < 0.1 compared to group 1; b P < 0.01 compared to their corresponding radiation groups.

Fig. 8. Antiperoxidative effect of HN. The Values are given as mean ± SD of six experiments in each group. a P < 0.1 compared to group 1; b P < 0.01 compared to their corresponding radiation groups.

16.38 ␮M HN (lane 4) prior to ␥-irradiation. Thus the result clearly indicated that pretreatment with hesperidin restored DNA damage to near normal indicating the efficacy of our drug. In addition, HN alone treated DNA (lane 2) did not show any damage compared to normal (lane1) indicating the protective nature of our drug. 4. Discussion

Fig. 9. Protective effect of HN in normal, ␥-irradiated and HN pretreated lymphocytes as measured by DNA fragmentation assay. Lane 1 – Control; lane 2 – 4Gy irradiation + Hesperidin; lane 3 – 4Gy irradiation; lane 4 – 4Gy + Hesperidin [10 ␮g/ml].

Radiation has been considered an enigma to the general public and the use of radiation for therapeutic and other purposes has always been associated with some skepticism. Since radiation therapy is the treatment of choice for a majority of cancer patients, search for efficient radioprotectors are needed to protect normal tissues when ␥-irradiation is used to induce irreversible damage in nearby targeted cancer cells. In the present study, we observed a dose-dependent increase in the levels of genetic damages (MN, DC, DNA fragmentation) in all ␥-irradiated groups. Mainly three biologic endpoints have been used to determine radiosensitivity in vitro. After in vitro irradiation of cells, chromosomal aberrations [30] and repair of radiation-induced DNA damage (e.g., singleand double-strand breaks) [31] have been measured and the data obtained have been compared with different types of clinical side effects. Thus, the characterization of DNA repair in lymphocytes might be a suitable approach to predict clinical radiation reactions. In particular, the alkaline single-cell microgel electrophoresis assay (or comet assay) has been shown to be useful for the assessment of DNA damage and repair within epidemiologic studies, because it is a fast and reliable assay that needs only a small number of cells [32]. Since the DNA damage produced is directly proportional to the radiation dose, any change in radiation dose should be reflected in a proportional change in the comet measurement. Thus the size and shape of the Comet and the distribution of DNA

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within the Comet have been correlated with the extent of DNA damage [33]. In cells exposed to ionizing radiation, DNA lesions can be produced in isolation (termed the singly damaged sites) by the action of a single hydroxyl radical or in clusters (termed the multiply damaged sites) by bursts of hydroxyl radicals [34]. While the singly damaged sites are similar to those produced by endogenous reactive oxygen species (ROS) during oxidative metabolism, the formation of multiply damaged sites appears to be a unique feature of ionizing radiation. Furthermore, it has been widely accepted that DC aberrations appear as a result of double-strand breaks and mis-repaired DNA [35]. A cytological consequence of the induction of these chromosomal aberrations is the formation of micronuclei (MN) that are observed in interphase cells. The cytokinesis-block micronucleus (CBMN) assay has been widely used to assess the in vitro radiation-induced chromosomal damage, and satisfactory dose relationships have been reported [36]. MN frequency was also shown to be a reliable biomarker in many biomonitoring studies among human populations therapeutically [37] exposed to ionizing radiation. It is also documented that one of the effects of oxidative stress-induced cytotoxicity in cells is DNA fragmentation caused by apoptosis [38]. Our study on the selection of effective dose of hesperidin against ␥-radiation shows that among all concentrations (3.27, 6.55, 9.83, 13.10, 16.38 and 19.65 ␮M), 16.38 ␮M concentration of hesperidin was found to offer optimum protection to lymphocytes against 4Gy irradiation. The exact mechanism for the optimum protection of hesperidin is unclear, still it could be suggested that at lower concentrations hesperidin might not be enough to quench all the radicals generated by ionizing radiation and at higher concentration hesperidin might have reacted with some other ligands in the system and thus might not be completely available for quenching the free radicals. Other studies have shown that cimetidine (1 mM) [39] and vanillin (0.5 mM) [40] significantly reduced the micronuclei frequencies and comet parameters respectively. However, the results of the present study demonstrate that the concentration of hesperidin, which provides optimum protection from gamma irradiation is very low (16.38 ␮M) when compared to the above-mentioned radioprotectors. Thus, 16.38 ␮M concentration of hesperidin was selected as the effective dose that protected lymphocytes against gamma radiation induced cellular damage. Our results show that hesperidin-pretreated lymphocytes decreased the genetic damages (MN, DC frequencies, comet parameters and DNA fragmentation) when compared to ␥-irradiated groups. The exact mechanism by which hesperidin develops anticlastogenic potential is still unknown. But this may be due to the effective antioxidant potential of hesperidin. As seen in Fig. 1, the overall antioxidant potential of hesperidin depends on the number and arrangement of the hydroxyl groups and the extent of structure conjugation [41]. They can donate hydrogen atom from their hydroxyl groups and stabilize the phenoxy radical formed by delocalization of the unpaired electron within the aromatic structure. It was well-known that aromatic compounds containing hydroxyl groups, especially those having an O-dihydroxy group on ring B, appears to be important scavengers as reported for flavonoids [42]. Thus these poly-phenols are excellent scavengers of free radicals due to the high reactivity of their hydroxyl substituents [43]. In addition to their antioxidant property, hesperidin acts as metal chelating agents and inhibits the superoxide-derived Fenton reaction, which was an important source of the most reactive hydroxyl radicals. Thus hesperidin could have decreased the DNA damage during exposure to ␥-irradiation by effectively scavenging the free radicals that produce damage to DNA. The spatial arrangement of substituents in hesperidin as shown in Fig. 1 was perhaps a greater determinant of antioxidant activity than the backbone structure alone. Both the configuration and the total number of hydroxyl groups substantially influence several mechanisms of antioxidant

activity of hesperidin. Another potential explanation for the protective effect of hesperidin on DNA was the direct interaction of hesperidin with DNA [44]. Previous studies have also reported that hesperidin protects mice against ␥-irradiation induced DNA damage in bone marrow cells [45]. Endogenous antioxidants are a group of substances when present at low concentrations compared to oxidized substrates significantly inhibit or delay oxidative processes, while being oxidized themselves. Antioxidant enzymes such as SOD, CAT and GPx are important in providing protection from radiation exposure [46], the proper balance of the enzymes in specific cells and in the whole organism required for maximum radioprotection. Therefore, a reduction in the activity of these enzymes can result in a number of deleterious effects due to the accumulation of superoxide radicals and H2 O2 . Reduced glutathione (GSH) participates non-enzymatically in protection against radiation damage [47]. Membrane lipids are the major targets of ROS and the free radical chain reaction thus initiated causes extensive membrane lipid peroxidation. Lipid radicals (L• ) are believed to be formed by the reaction of • OH radicals generated by ionizing radiation with polyunsaturated fatty acids (LH) which subsequently reacts with oxygen to form lipid peroxyl radical (LOO• ) after undergoing molecular rearrangement of conjugation in double bonds and eventually a chain reaction is initiated on irradiation in oxygenated condition [48]. The increase in the levels of lipid peroxidation products such as malondialdehyde, hydroperoxides, TBARS and conjugated dienes are the indices of membrane lipid damage. The mechanisms involved in many human diseases such as hepatotoxicities, hepatocarcinogenesis, diabetes, malaria, acute myocardial infarction and skin cancer include lipid peroxidation as a main source of membrane damage. Hence the mechanistic study of membrane damage induced by reactive oxygen species in relation to human diseases and their possible prevention by antioxidants constitutes an active area of research in recent years. Moreover enhanced levels of lipid peroxidation induced by radiation are accompanied by a decrease in the activities of SOD, CAT and GPx [49]. In our study, we observed a significant decrease in the level of enzymatic antioxidant (SOD, CAT, GPx), non-enzymatic antioxidant (GSH) and an increase in the level of lipid peroxidative index TBARS in gamma-irradiated cells. Earlier, we have reported that the increase in the dose of radiation depletes the antioxidant status and increases lipid peroxidation in the lymphocytes [50,51]. But pretreatment of lymphocytes with hesperidin prior to radiation exposure increased the antioxidant status at both enzymic and non-enzymic levels and decreased the level of TBARS. This effect was due to its antioxidant property and shows that hesperidin acts as a good scavenger against free radical generation and thereby inhibits lipid peroxidation [52]. It was also reported that hesperidin offers protection by terminating the lipid peroxidative side chain rather than scavenging extra cellular non-lipid radicals that initiate lipid peroxidation [53]. Thus, the increase in the antioxidant status during hesperidin pretreatment might have further decreased the attack of free radicals on biomolecules including DNA and membrane lipids and thereby decreased the deleterious effects of radiation on lymphocytes. Moreover hesperidin alone pretreated lymphocytes (Drug Control) do not show any biochemical alterations and chromosome aberrations indicating the protective nature of the drug. Thus from the results obtained, we conclude that hesperidin is an efficient radioprotector which protects human peripheral lymphocytes from the pleiotrophic damaging effects of gamma radiation. Previous reports have also shown the beneficial effect of hesperidin on lipopolysacccharide-induced hepatotoxicity [54] apoptotic effect of hesperidin through caspase 3 activation in human colon cancer cells [55] and antioxidant status of hesperidin during nicotine-induced lung toxicity in experimental animal model [56].

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