Biochemical and Molecular Mechanisms of Radioprotective Effects of

Article pubs.acs.org/JAFC

Biochemical and Molecular Mechanisms of Radioprotective Effects of Naringenin, a Phytochemical from Citrus Fruits Sumit Kumar and Ashu Bhan Tiku* Radiation and Cancer Therapeutics Laboratory, School of Life Science, Jawaharlal Nehru University, New Delhi, India 110067 ABSTRACT: The present study was aimed to evaluate the radioprotective effects of naringenin in vivo using Swiss albino mice as a model system. Oral administration of 50 mg/kg body weight of naringenin for 7 days prior to radiation exposure protected mice against radiation-induced DNA, chromosomal and membrane damage. Naringenin pretreatment also increased the antioxidant status of irradiated mice. Multiple factors operating at cellular and molecular levels led to increased endogenous spleen colonies and survival of mice. Although naringenin induces apoptosis in cancer cells we found that it can protect against radiation-induced apoptosis in normal cells by modulating the expression of p53, Bax, and Bcl-2. The results from the present study indicate that naringenin inhibits the NF-kB pathway and down regulates radiation-induced apoptotic proteins resulting in radioprotection at the cellular, tissue and organism levels. KEYWORDS: naringenin, antioxidants, flavonoids, free radical scavenging, apoptosis, radiation-protection

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INTRODUCTION Ionizing radiation induces biological effects via a series of molecular events, set off by reactive oxygen species (ROS). These free radicals, •OH, HO2−, eaq, O2•−, and H3O+, can cause oxidative damage to biological molecules including DNA and membranes resulting in the dysfunction/malfunctioning of biological processes and sometime even cell death.1 However, radiation has varied applications ranging from killing of cancer cells as in the case of radiotherapy, to diagnostic uses, food preservation, agriculture, industry, and power generation.1 In fact clinical usefulness of radiation as a means of cancer treatment is well appreciated as nearly 50% of cancer patients receive it during a course of treatment.1 Because of anatomical location and an unclear boundary, normal tissues may also get damaged and affect the quality of life for survivors.2 Also, risks are associated with radiation exposure from occupational or accidental events such as the one that happened in “Fukushima” recently. Therefore, a need for an effective radio-modifier that provides protection from radiation exposure in normal cells and tissues is increasing day by day. The development of pharmacological approaches for protection of normal tissues is the need of the hour. Although synthetic radioprotectors like sulfhydral containing free radical scavenging compounds were discovered in the beginning of the nuclear era, an ideal radioprotector yet remains elusive because of various reasons.1,2 In the last two decades the evaluation of bioactive natural compounds from plants has attracted considerable attention. Besides acting as free radical scavengers to neutralize radiation-induced free radicals these compounds have low toxicity, easy availability, cost benefit, differential effects on cancer and normal cells, and time tested medical effectiveness.2 Studies using catechins (EGCG, ECG, and EGC) and flavonoids such as resveratrol have been shown to protect against radiation-induced damage.3,4 Unfortunately, the catechins bioavailability is shown to be very low (0.1−1.1%) in humans and also they rapidly oxidize (80% is in just 1 h).5 © XXXX American Chemical Society

Similarly, the bioavailability of resveratrol is also very poor as t1/2 and Cmax values have been reported to 3.3 h and 23.5 ng/ mL after administering 200 mg of resveratrol in single oral dose.6 Furthermore, because of many adverse report on catechins and resveratrol, their potential clinical usefulness remains doubtful.7,8 Citrus fruit production reached 82 million tons in 2009−10, and thus stimulated the research on its main constituents, naringenin (grapefruit) and hesperidin (sweet orange).9 Moreover naringenin and hesperidin have received wide attention because of their high occurrence in other foods also.9 The hesperetin and naringenin Cmax values in plasma are reported to be 2.2 μmol/L (t1/2, 2.2 h) and 5.99 μmol/L (t1/2, 2.2 h), respectively, after administering the dose of hesperetin and naringenin equivalent to 126 mg and 199 mg in humans; similarly the much relevant “area under the plasma concentration−time curve in 24 h” (AUC0−24) was found to be much better for naringenin (27.7 μmol.h/L) than for hesperetin (10.3 μmol.h/L).10 Moreover due to its antihyperglycemic and antihyperlipidemic property, naringenin has received more attention over hespertin in recent time. Naringenin is present as naringenin and naringenin glycosides in nature and the former is absorbed directly from the intestine; while the latter needs to be deglycosylated by intestinal bacteria before being absorbed.11 Orally fed naringenin is mainly present in plasma in the form of free naringenin and monoglucuronide derivatives (mainly at the fourth and seventh positions).12,13 While at the organ level, naringenin is mainly dominant in the liver and brain while monoglucuronide forms in adipose tissues.12,14 Naringenin, a nontoxic molecule with a median lethal dose of >5000 mg/kg body weight in mice is reported to have protective effects against various types of aliments.11−15 In vitro Received: October 29, 2015 Revised: January 28, 2016 Accepted: January 29, 2016

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DOI: 10.1021/acs.jafc.5b05067 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry studies, also show that naringenin protects the HaCaT cells against the UV radiation-induced damage by promoting the DNA repair and induces melanin production in murine melanoma (B16−F10) cell lines.16,17 The protection of DNA probably lies in the capacity of naringenin to scavenge free radicals as well as to modulate the expression of enzymes involved in repair of damaged DNA.18 Naringenin has been reported to protect against oxidative stress and inflammation also.10−15 Therefore, the present study was designed to evaluate the radioprotective effects of naringenin in whole body irradiated mice (WBI), and we tried to elucidate the mode of action at the biochemical and molecular level.

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MATERIALS AND METHODS

Chemicals. Naringenin, reduced glutathione (GSH), bovine serum albumin (BSA), thiobarbituric acid (TBA), 5,5-dithio-bis(2-nitrobenzoic acid (DTNB), 1 chloro-2-4-dinitrobenzene (CDNB), primary mice antibody (p53, Bax, Bcl-2), paraformaldehyde, antifade medium, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), normal agarose, low melting agarose, ethidium bromide (EtBr), acridine orange, Triton X-100, and 3(4,5-dimethyl thiozol-2-yl (MTT) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Primary mice βactin and secondary antimice Ig-G-HRP were purchased from Santa Cruz Biotechnology, Texas, USA. RPMI medium-1640, fetal bovine serum (FBS) and antibiotics were purchased from Himedia Laboratories, Mumbai, India. Plasmid pBR322 was purchased from Banglore Genei, Bangalore, India. All other chemicals used were of analytical grade. Plasmid Relaxation Assay for in Vitro Studies. Plasmid relaxation assay was used to determine the extent of DNA damage in acellular system in terms of proportion of super coiled and open circular from of DNA in gel. pBR322 (50 ng) in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8) was co-incubated with naringenin (dissolved in DMSO, final concn 0.002%), for 30 min at 4 °C and then exposed to γ-radiation. After irradiation, samples were further incubated for 30 min at 4 °C and then analyzed by electrophoresis in 1% agarose gel. The DNA bands were quantified using imager inbuilt spot denso band analysis software (Alpha Innotech Corp, San Leandro, CA, USA) and result was expressed as % change of supercoiled form into open circular form. Metal Chelating Activity. Metal ion chelating activity was performed as described by Moore and co-workers.19 Animals. Six to seven week old random-bred male Swiss albino male mice (25−30 g) were used for the present study. They were housed in standard cages at a university animal house (T, 22 ± 2.5 °C; 12 h light/dark cycle; RH, 65%) and given food and drinking water ad libitum. All studies were performed adhering to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, constituted by the Animal Welfare Division, Government of India and Institutional Animal Ethics Committee on the use of animals in scientific research. A total 24 animals were divided into four groups as detailed in Figure 1. The required amount of naringenin was suspended in 100 μL of carboxy-methyl cellulose (0.5% in distilled water) and given by oral gavage to mice. Naringenin was given at a dose of 50 mg/kg body weight daily for 7 days. Irradiation of Mice. Mice were placed inside the ventilated Perspex containers (2 h after the last feeding of naringenin) and subjected to whole body irradiation (WBI) using 60Co in a gamma chamber (240 TBq 60Co Model 4000A) obtained from the isotope division of the Bhabha Atomic Research Centre (BARC), Mumbai, India, at a dose rate of 2.14 Gy/min. Following irradiation animals were housed in animal house under normal condition and euthanized by cervical dislocation before sacrificing at the desired time point for the examination of different parameters. Animals were segregated into different groups. Experimental protocol is given in Figure 1.

Figure 1. Diagrammatic representation of experimental design and methodology used in the present study. Animal Survival Study. To study the effectiveness of naringenin in radioprotection, an animal survival study was carried out as described earlier.20 Micronucleus Analysis. Blood from mice (six each) of different treatment groups was collected from retro-orbital plexus using a glass capillary at the seventh day postirradiation and processed as described earlier.21 In brief, a thin film of blood was made on a glass slide immediately after collection, and slides were kept in fixing solution (fixative: Sorensen buffer A: 0.05 M KH2PO4; Sorensen buffer B: 0.05 M Na2HPO4·2H2O, pH 6.8, 30 μg/mL SDS; and 1% glutaraldehyde) for 5 min, washed with PBS and stained for five min with a mixture of 1:3 solution A (0.1 mL of Triton X-100, 8 mL of 1.0 N HCl, 0.877 g of NaCl, and 91 mL of distilled water ) and Solution B [37 mL of 0.1 M anhydrous citric acid, 63 mL of 0.2 M Na2HPO4 (pH 6.0), 0.877 g of NaCl, 34 mg of EDTA disodium salt, and 0.6 mL of acridine orange (1 mg/mL)] The slides were washed with PBS and scanned using a fluorescent microscope (Olympus Microscopes, Tokyo, Japan). The frequency of the micronuclei was calculated as follows: micronuclei frequency = no. of cells with micronuclei/total no. of cells × 100 Histopathological Analysis. The liver and other organs were dissected out from mice, at 6 and 24 h postirradiation, perfused with 0.9% NaCl, fixed in 10% formalin solution, dehydrated in ascending grades of alcohol, embedded in paraffin, and cut into 5 μm thick sections using microtome. The sections were stained with hematoxylin-eosin and mounted in a neutral DPX medium. The sections were examined under a light microscope (Nikon, Tokyo, Japan), and images were captured. For immuno-histopathological studies five micrometer thick sections were made using a cryostat (Leica Biosystems) and dipped in blocking buffer (10% in rabbit serum, 0.3% Triton X-100) for 45 min at room temperature. The sections were dipped in primary monoclonal antibody solution, washed in buffer [washing buffer (0.1% BSA in PBS)] and incubated with Alexa Fluor-488 conjugated secondary antibody (Invitrogen, Waltham, Massachusetts, USA). The sections were washed twice in washing buffer, stained with DAPI solution, and semidried before mounting on a slide using antifade medium. Protein expression was visualized using a fluorescence microscope (Olympus BX51, Olympus Microscopes, Tokyo, Japan). Protein Expression Analysis by Western blot. Whole cell protein lysates of liver tissues were prepared in RIPA (radioimmunoprecipitation assay) buffer and protein concentration was determined using the Quick Start Bradford Protein kit (Biorad, Hercules, California, USA). For nuclear extract, perfused hepatic tissue B


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Journal of Agricultural and Food Chemistry

Figure 2. (a) Effect of radiation on plasmid pBR322 DNA damage; (b) graph representing band intensity of panel a; (c) effect of different concentrations of naringenin on plasmid DNA; (d) protective effects of naringenin on radiation-induced DNA damage at 5 Gy; (e) protective effects of naringenin on radiation induced DNA damage at 30 Gy; (f) bar diagram representing band intensity of panels c−e. Notation: OC, open circular; SC, supercoiled; C, control; R, radiation control; R, 5 Gy; R′, 30 Gy; D1, 1nM NG (naringenin); D2, 10 nM NG; D3, 100 nM NG; D4, 1 μM NG; D5, 10 μM NG. Band image is representative from three independent experiments; Data is expressed as mean ± SD of three independent experiments. (∗) p < 0.05 vs C; (#) p < 0.05 vs R. was minced and homogenized in homogenization buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1× protease inhibitors). Triton-x 100 (0.4%) was added to the homogenate and kept in ice for 30 min with intermittent shaking. Homogenate was centrifuged at 15000g for 5 min at 4 °C and the supernatant was saved as cytoplasmic extract. The pellet was washed once with homogenization buffer followed by addition of the nuclear extraction buffer (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 0.1 mM EDTA, 0.1 mM EGTA, 1× protease inhibitors) in the pellet. The sample was kept in ice for another 30 min with intermittent shaking and centrifuged at 15000g for 15 min at 4 °C. The supernatant was collected and stored at −70 °C for further analysis of nuclear proteins. Extracted protein was separated in 12% SDS−PAGE gel and transferred to a nitrocellulose membrane. The blots were probed with primary antibody (p53, Bax, Bcl-2, NF-κB and β-actin) followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The bands were detected using ECL reagents, and the images were permanently captured in X-ray films using developer and fixer. The band intensity was quantified using MyImageAnalysis (Thermo scientific) software and results were normalized with respect to loading control. Transmission Electron Microscopy (TEM). Fixed splenocytes [2% glutaraldehyde (Merck KGaA, Darmstadt, Germany)] solution in PBS)] were placed over a copper grid with a laser carbon film and allowed to dry in air. Cells were scanned (10000× magnification) using JEOL-JEM-2100F TEM (JEOL Inc., Peabody, Massachusetts, USA) operating at 200 kV. Endogenous Spleen Colony Assay. Endogenous spleen colony assay were carried out as described earlier.20 Biochemical Analysis. For biochemical studies liver tissues of six different animals from each group were perfused in 0.9% ice cold NaCl and homogenized in 0.1 M phosphate buffer (pH 7.0) using Potter

Elvehjem homogenizer. The homogenates were divided into two portions to measure the GSH and cytosolic enzymes. GSH was measured from one portion as described by Moron.22 The other portion of the homogenate was further processed by spinning at 32000g for 20 min at 4 °C, and the supernatant was further centrifuged at 100000g for 60 min at 4 °C. The resulting supernatant was used to measure the superoxide dismutase,23 catalase,24 and glutathione peroxidase activity,25 whereas the pellet (microsomes) was resuspended in 0.15 M KCl and 10 mM Tris-HCl buffer (pH 7.4) to measure the lipid peroxidation.26 Protein was quantified by the folin Lowry method.27 LDH Assay. The LDH (lactate dehydrogenase) level in hepatic extracellular space was measured to check the status of damaged cells. Hepatic tissue (200 mg) was homogenized in a dounce homogenizer in 5 mL PBS (1×). Cell suspension was then centrifuged at 500g for 10 min at 4 °C. The supernatant was collected and used for LDH measurement as described earlier.24 Isolation of Splenocytes. Splenocytes were prepared and cultured as described in our previous paper.20 Comet Assay. Splenocytes were harvested 6 h after irradiation and around 10000 cells were immobilized in a layer of low melting agarose over a microscopic glass slide having an area of 2 × 1 in. The cells were lysed (lysing solution: 2.5 M NaCl, 100 mM EDTA, 100 mM Tris base, 1% Triton X-100, and 10% DMSO pH 10) for 2 h at 4 °C and subjected to electrophoresis for 30 min at 0.74 V/cm in alkaline electrophoresis buffer (1.2% NaOH, 0.037% EDTA). The DNA was stained with EtBr (5 μg/mL), visualized under fluorescence microscope and the comet parameters were estimated using comet score 15 software (TriTek Corp., VA, USA). Apoptosis Assay in Splenocytes. Apoptosis in splenocytes was detected by staining cells in a mixture of EtBr/AO solution (200 μg/ mL of AO in PBS; 200 μg/mL EtBr in PBS) for 2 min. The cells were visualized under fluorescence microscope (Olympus BX51, Olympus C


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Figure 3. Effect of naringenin on radiation-induced DNA damage: (a) microphotographs showing comet formation in splenocytes; (b) table representing changes observed in comet parameters; (c) microphotographs depicting the representative micronuclei formed in the peripheral blood of mice after 7 days of irradiation (5 Gy) (arrow represents micronuclei); (d) bar graph showing the micronuclei frequency in different treatment groups; (e) Fe2+ ions binding with naringenin measured by 2,2′-bipyridyl in triplicate. Each data point represents mean ± SD of 250 (b) and 1500 (d) cells from six independent experiment. (∗) p < 0.05 vs control; ($) p < 0.05 represents the linear (pairwise) change. Microscopes, Tokyo, Japan) and live, apoptotic, and necrotic cells were differentiated on the basis of emitted florescence, that is, green, yellow, and red, respectively. Data Analysis. Mean and standard deviation was calculated using Graph Pad Prism 5.03. A statistical test was done using one-way ANOVA followed by Mann−Whitney rank sum test. A p value less than 0.05 was considered significant. For survival studies log-rank (Mantel-Cox) test was used, and p values less than 0.05 were considered as significant.

retarded migration in agarose gel electrophoresis. When double-stranded plasmid pBR322 was exposed to different doses of radiation, single-strand breaks were induced in DNA as shown by an increase in the relative intensity of the bands corresponding to the open circular form. The results presented in Figure 2 panels a and b show that there is a radiation dosedependent increase in the open circular form (OC). Naringenin alone (1 nmol−10 μmol) did not show any effect on plasmid DNA (Figure 2c). The protective effect of naringenin on radiation-induced strand breaks in plasmid pBR322 DNA was tested at different concentrations (1 nmol−10 μmol). In the presence of, naringenin the extent of DNA damage decreased significantly in a concentration-dependent manner. In naringenin (10 μmol) pretreated plasmid samples the percentage of SC decreased from 22 to 16% at 5 Gy and from 35 to 19%, at 30 Gy of radiation exposure (Figure 2d−f). The vehicle

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RESULTS AND DISCUSSION Effect of Naringenin on Radiation-Induced Strand Breaks in Plasmid DNA. The protective role of naringenin was evaluated using pBR322 plasmid DNA damage assay in terms of its ability to limit DNA strand breaks generated on exposure to ionizing radiations. This assay is based on conversion of supercoiled (SC) double-stranded DNA molecules into open circular (OC) form, which displays D


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Table 1. Effect of Naringenin (50 mg/kg body wt) Pretreatment for 7 Days on Hepatic Parameters of Whole Body Irradiated (5 Gy) Mice after 6 ha treatment groups biochemical parameter

sham control (0.5% CMC)

radiation control (5 Gy)

naringenin control (50 mg/kg body wt)

naringenin (50 mg/kg body wt) + radiation (5 Gy)

reduced glutathioneb lactate dehydrogenasec superoxide dismutased catalasee glutathione peroxidasef lipid peroxidationg

4.97 ± 0.35 11.09 ± 0.93 5.95 ± 0.57 36.77 ± 1.93 0.0725 ± 0.0078 0.0147 ± 0.0021

3.22 ± 0.31 14.70 ± 1.05a 4.31 ± 0.32a 27.90 ± 2.92a 0.0536 ± 0.0096a 0.0340 ± 0.0014a

6.04 ± 0.23 11.56 ± 1.07 6.01 ± 0.21 37.26 ± 2.74 0.0815 ± 0.0117 0.0153 ± 0.0016

5.19 ± 0.30 12.08 ± 0.91a 5.06 ± 0.62 34.18 ± 1.23a 0.072 ± 0.006a 0.0209 ± 0.0017a

a Each data point represents mean ± SD of six separate observations. * p < 0.05 vs control, # p < 0.05 vs radiation control. bμmol of GSH formed/g of liver tissue; cSpecific activity in μmol NADH oxidized/min/mg protein; dSpecific activity (U)/min/mg protein; eμmol H2O2 consumed/min/mg protein; fSpecific activity (U)/min/mg protein; gnmol of MDA formed/mg protein.

radiation-induced DNA damage in vivo in peripheral leukocytes.34,35 The radioprotective effect of naringenin on DNA damage was probably due to its antioxidant property, however repair of radiation-induced DNA damage could be an additional mechanism. Naringenin has also been reported to activate the DNA repair enzymes such as 8-oxoguanine-DNA glycosylase 1 (hOGG1), apurinic/apyrimidinic endonuclease and DNA polymerase beta (DNA poly beta).18,36 The decrease in DNA damage observed in vivo can also be due to chelating of the metal ions such as Fe2+ resulting in reduced production of free radicals via the Fenton reaction (hydroxyl radical via irondependent reaction) (Figure 3e). Protective Effect of Naringenin on Radiation-Induced Lipid Peroxidation and Redox Status. Apart from DNA, radiation also targets the membrane, resulting in oxidation of the polyunsaturated fatty acids in a chain reaction “lipid peroxidation (LP)” and eventually resulting in loss of membrane integrity.37 In the present study naringenin pretreatment to mice mitigated the radiation-induced lipid peroxidation by 41%. Reduction in the peroxidative damage could be attributed to better redox status as naringenin has been reported to reduce the lipid peroxidation via a glutathione-dependent pathway in α-tocopherol deficient liver microsomes.32 In the present study we also observed an increased level of glutathione in naringenin-treated unirradiated mouse groups, but no significant change in the antioxidant enzyme profile was noticed. However, in naringenin pretreated irradiated groups, an increase in level of SOD and catalase by 17.4% and 27.9% (p < 0.05), respectively, was observed in comparison to irradiated controls (Table 1). The reduction in the radiation-induced membrane damage could be attributed to the polar nature of naringenin and the presence of the 4′-hydroxyl group which have enabled it to stick to the membrane, resulting in reduction of the peroxidation.38,39 The reduction of radiation-induced damage was further substantiated by an observed reduction in the level of LDH release in the extracellular spaces, a marker of necrotic events (Table 1). Previously catchins and flavonoids have been shown to reduce the radiation induced oxidative damage by modulating the cellular antioxidant enzyme.40,41,44 One major system that responds to oxidative stress to restore the redox balance involves genes coordinated by antioxidant response elements (ARE), primarily activated by Nrf2. The role of naringenin in modulation of cellular antioxidant enzyme levels via activation of the nrf2-ARE pathway cannot be ruled out.42

(DMSO 0.002%) did show any effect on plasmid DNA (data not shown). The ROS and free radicals produced by radiation (OH•, O2•−, eaq.−, H2O2, etc.) play a central role in the radiationinduced damages,1 and since plasmid DNA is a repair deficient system the reduction in radiation-induced DNA damage can be due to direct scavenging of the free radicals from the medium. Several in vitro studies have demonstrated that the chemical structures of individual flavonoids, more specifically the number and position of hydroxyl groups, affect their capacity to act as antioxidants.28−31 The presence of hydroxyl groups in the ortho position or a hydroxyl group and an oxo group at proximal carbon positions has been shown as the main structural requirement for the greater capacity of flavonoids to protect DNA against oxidative damage.28−30 Therefore, the presence of a 4-OH group in the B ring of naringenin, possessing electron donating properties, could be quenching radiation-induced free radicals and eliminating the free radicals before their interaction with DNA.31,32 Moreover interaction of flavanoids with DNA bases has been shown to reduce the radiation-induced direct damage.33 Therefore, naringenin can protect DNA via denying access of free radicals to it as well as free radical scavenging. Protection against Radiation-Induced Genotoxicity in Mice. Radiation-induced DNA damage in mice pretreated with naringenin for 7 days and exposed to 5 Gy of radiation was evaluated by comet as well as micronucleus assay. The comet assay evaluates DNA strand breaks, utilizing the DNA migration as a measure of the DNA damage. The single cell gel electrophoresis or comet assay showed that total comet length increased by 3.27 fold and comet area by 6.18 on exposure to 5 Gy of radiation in comparison to control (Figure 3a,b). However, in naringenin pretreated mice radiationinduced comet length and area reduced by 60% and 42%, respectively. The DNA in the tails of comets also reduced by 49.5% in naringenin pretreated mice in comparison to the radiation control group. Some basal level of the DNA damage in the form of a few comets was also observed in the unirradiated control group of mice. Further micronucleus assay performed in blood samples of the mice also showed a protective effect. Naringenin was found to reduce the number of micronuclei in pretreated mice to almost half (Figure 3d) in comparison to radiation alone control groups. No significant difference was observed in the frequency of micronucleated cells in control and drug alone treated groups of mice. Chrysin, and other flavonoids like caffeic acid and naringin have also been reported to reduce the E


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Journal of Agricultural and Food Chemistry Radioprotective Effect of Naringenin on RadiationInduced Apoptosis. Radiation-induced apoptosis was assayed in splenocytes 24 h, postirradiation. A significant increase in the apoptotic splenocytes was observed in irradiated mice (Figure 4a,b). This was further confirmed by observing the ultrastructure of cells using TEM. Radiation treatment also increased the number of the autophagosomes along with chromatin fragmentations (Figure 4c). Naringenin treatment prior to radiation exposure was found to reduce the percentage of apoptotic cells by 61% in comparison to radiation control (Figure 4b). Naringenin has been reported to reduce radiationinduced apoptosis by inhibition of mitochondrial depolarization and release of AIF Endo-G from mitochondria in primary rat hepatocytes.43 From a mechanistic point of view, biological systems respond to radiation-induced damage by an increase in the levels of transcription factors like p53, which regulate the expression of a number of downstream effects or genes involved in apoptotic pathway such as Bax, p21, and Bcl-2 etc.16,47 Radiation-treated groups showed an increase in the level of the p53 in liver 6 h postirradiation (Figure 5a,b). The level of Bcl-2, an antiapoptotic protein was decreased after radiation exposure, and apoptotic protein Bax showed an increased level. However, in the naringenin pretreated mice the level of Bcl-2 was restored to the normal level, but the expression of Bax and p53 was decreased. The decrease in the level of the expression of p53 in naringenin pretreated mice was further confirmed by performing the IHC (immunohistochemistry) study of liver (Figure 5c). Besides p53, activation of NFκB (a transcription factor) as a part of DNA damage response is known to regulate a large no. of genes. It is also known to be induced by radiation; however, currently there is no consensus about its functional contribution in activation of the radiation response.45 It is one of the key proteins that modulate apoptotic response. Abundant reports dealing with cancer cells have shown that inhibition of NF-κB leads to radiosensitization.46 On the contrary Daroczi et al., (2009), using six different inhibitors have shown that targeting the canonical pathway (IKKβ/IκB/NF-κBp65) can result in radioprotective effects.45 In the present study, we observed an increase in the levels of NF-κB in radiation treated mice with a concommitent increase in apoptosis in liver and spleen (Figure 5d). However, naringenin pretreated mice exposed to radiation showed a reduction in NF-κB levels along with lower levels of apoptosis. In an earlier study epicatechin has been shown to ameliorate the radiation induced hepatotoxicity by inhibition of NF-kB and improving the hepatic redox status.40 NF-κB is induced either via a Toll-like receptor (TLR) mediated pathways or ROS. Therefore, we challenged the mice with LPS, which is known to induce it via a TLR pathway, so if naringenin reduces the expression of NF-κB via free radical scavenging, it may not interfere with the LPS induced NF-κB. However, interestingly we observed a reduction in the level of LPS induced NF-κB (Figure 5d). The reduction in the level of LPS induced NF-kB by naringenin indicates that probably naringenin suppresses the radiation-induced NF-kB via a receptor mediated pathway resulting in the reduction in the level of apoptotic proteins.47,48 Protective Effect of Naringenin on the RadiationInduced Organ and Organism Level Mortality. Damage at cellular level often leads to organ failure which is a significant component of the radiation syndrome. Histopathological studies of liver, kidney, and spleen showed that radiation exposure resulted in various changes in the histoarchitecture of

Figure 4. Effect of naringenin and radiation on splenocyte apoptosis after 24 h of whole body irradiation (5Gy): (a) photomicrographs depicting live, apoptotic and dead cells; (b) bar diagram showing percentage apoptotic and dead cells; (c) transmission electron micrograph of spleen cells shows typical radiation-induced cell death. Each data point represents mean ± SD of 1000 cells from five independent experiments. (∗) p < 0.05. C, chromatin; N, nucleus; n, nucleolus; Mvp, microvilli projection; L, lysosomes; Atg, autophagosomes/autolysosomes; Satg, secretary autolysosomes; Mtg, mitophagy. (C, control; R, radiation control (5 Gy); NG, drug control (50 mg/kg body wt); NG+R, naringenin + radiation.)

organs. Radiation exposure resulted in sinusoidal dilatation (black circle) and loss of a large number of cells as compared with control liver (Figure 5e). The normal spleen is made up of white pulp and red pulp, and after 24 h of irradiation the white F


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Figure 5. Effect of 7 days naringenin (50 mg/kg BW) pretreatment on expression of the p53, Bax, Bcl-2, and NF-kB (p65) and organs damage in whole body irradiated (5 Gy) mice: (a,b) blot shows level of p53, Bax, and Bcl-2 after irradiation, and bar diagram shows the mean band intensity; (c) immunohistochemistry photomicrographs shows level of p53 in liver; (d) expression level of NF-κB and its downstream genes, Bcl-2 and Bax in liver of mice, LPS (8 μg/g BW) was injected intraperitoneally at 7th day of naringenin treatment and mice scarified after 6 h; (e) histopathology of liver tissue showing morphological changes, black circle represents sinusoid; (f) histopathology of spleen showing morphological changes, black square represents megakaryocytes; (g) histopathology of kidney showing morphological changes, round dotted circle and black arrow showing atrophy and inflammatory cell infiltration, respectively. Protein expression and histopathological analysis (H&E staining) were performed after 6 and 24 h post irradiation, respectively. Each data point represents mean ± SD (n = 3). (*) p < 0.05 vs control. (#) p < 0.05 radiation control. C, control; R, radiation (5 Gy), NG, naringenin control; NG + R, naringenin+radiation. Western blot image or tissue micrograph is a representative from three independent experiments.

animal from mortality by hematopoietic reconstitution. The number of colonies formed indirectly gives an assessment of the surviving cells in the hematopoietic system. An average of 18.3 ± 3.05 colonies was observed per mouse exposed to 6 Gy (Figure 6a). The number of colonies increased to 32.1 ± 4.01/ mouse in the naringenin pretreated group. Naringenin alone or untreated mice did not show any colonies in their spleens. The 30 day survival studies showed that exposure of mice to radiation doses of 6 and 9 Gy resulted in death of 40% (6 Gy) and 80% (9 Gy) mice within 30 days after irradiation, whereas at 12 Gy all mice died by day 12 (Figure 6b). In naringenin pretreated mice, mortality was 30, 50, and 70% at 6, 9, and 12 Gy of radiation, respectively. On the basis of the above data the calculated LD50/30 for radiation control and naringenin + radiation increased from 6.6 to 9 Gy, respectively (Figure 6c), and DRF (dose reduction factor) was found to be 1.3.

pulp was decreased and a slight increased in the number of infiltrated megakaryocyte (square) was observed (Figure 5f). As compared to the control, examination of kidneys of WBI mice showed damage to glomeruli, tubules, and interstitial tissue. The tubules presented vacuoles, hydropic changes; atrophy (dashed circle) and inflammatory cell infiltrate (black arrow) (Figure 5g). However, naringenin pretreatment significantly reduced the radiation-induced damage in all the three investigated organs. The reduction in radiation induced damage in multiple organs seems to be due to NF-kB inhibition, as inhibiting the inflammatory markers is shown to protect the mice against radiation-induced injury in kidney, spleen, and liver, but free radical scavenging also cannot be ruled out.49 Radiation exposure also results in depletion of cell in the hematopoietic system, except for a few surviving stem cells that repopulate to form colonies in spleen and help in rescuing the G


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Figure 6. Effect of 7 days naringenin (50 mg/kg BW) pretreatment before whole body irradiation on mortality and proposed model of action: (a) micropictograph depicting spleen colonies (dorsal and ventral) in respective groups; bar graph shows mean ± SD of number of colonies per spleen on 11th day postirradiation; (b) radiation dose response; (c) dose reduction factor (LD50/30) of radiation; (d) putative molecular mechanism of radioprotection by naringenin in mice. Each data point represents mean ± SD from 10 mice.(∗) p < 0.05. C, control; R, radiation (5 Gy); NG, naringenin control; NG+R, naringenin+radiation.

Notes

Enhancers of hematopoietic cellularity, such as propolis extract/derivatives and acemannan have been reported to help in reducing the radiation-induced lethality.20,34,50 Cells from a single spleen colony have the ability to rescue a myelosuppressed mouse by repopulating myeloid and lymphoid cells. The increase in the number of spleen colonies in comparison to irradiated control helped to rescue the animals from radiation-induced mortality. Therefore, multiple effects operating at the cellular and molecular level following naringenin treatment translated into increased survival of mice (Figure 6d). The mechanism of radioprotection is comparable to catechins.40 This study shows that oral naringenin pretreatment for 7 days prior to radiation exposure results in increased survival of mice. The mechanism involved could be free radical scavenging, and increase in redox potential resulting in reduction of radiation-induced DNA and membrane damage. At molecular level naringenin reduced the radiation-induced apoptosis by modulating the expression of p53, Bax, and Bcl-2. Besides free radical scavenging, the protective effect could also be due to down regulation of radiation-induced NF-κB, which can influence various pathways from apoptosis to inflammation leading to enhancement of survival. Therefore, naringenin needs to be further evaluated for its clinical efficacy against radiation-induced damages.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.B.T. and S.K. would like to thank UGC and JNU, New Delhi, for providing the funds and infrastructure needed for present study. Further S.K. gratefully acknowledges the Council of Scientific and Industrial Research, New Delhi, for providing fellowship and contingency during the research work.

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ABBREVIATIONS USED SC , supercoiled; OC, open circular; ROS, reactive oxygen species

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