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Mutation Research 775–776 (2014) 1–6

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

jo ur nal home page: www.elsev ier .com/ locate /gentoxComm uni t y ad dress : www.elsev ier .com/ locate /mutres

ffects of barley �-glucan on radiation damage in the humanepatoma cell line HepG2

aleh Ghavamia, Bahram Goliaeia,∗, Bita Taghizadeha, Alireza Nikoofarb

Institute of Biochemistry & Biophysics, University of Tehran, Tehran, IranDepartment of Radiobiology, Tehran University of Medical Sciences, Tehran, Iran

r t i c l e i n f o

rticle history:eceived 12 January 2014eceived in revised form 30 July 2014ccepted 9 September 2014vailable online 23 September 2014

a b s t r a c t

Damage to normal tissue is an obstacle to radiotherapy of cancer. We have tested whether barley �-glucancan enhance radioprotection in the human hepatoma cell line HepG2. The cytotoxicity of �-glucan wasdetermined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. A clono-genic assay was used to study the sensitivity of cells to �-glucan, ionizing radiation (2–8 Gy), and thecombination of both treatments. Acridine Orange/ethidium bromide staining was used to examine induc-tion of apoptosis by �-glucan, radiation (6 Gy), and the combination. DNA strand breaks were assessed

eywords:-GlucanepG2adio-protectionNA damagesomet assay

by the comet assay. The MTT assay showed that treatment with �-glucan was not cytotoxic. Indeed, aslight increase in cell viability was observed. Pre-treatment with �-glucan, 1 �g/ml, for 72 h protectedHepG2 cells against radiation, as indicated by increased surviving fraction, reduced apoptosis, and fewerDNA strand breaks. These results show that barley �-glucan is a radioprotective agent.

© 2014 Elsevier B.V. All rights reserved.

poptosis

. Introduction

Ionizing radiation is a powerful treatment modality againstumor cells, but it is not tumor specific and may affect neighbor-ng normal cell as well [1–4]. Various polysaccharides, includinglucan, mannan, and glucomannan have anti-genotoxic, anti-xidative, immunomodulatory, anti-infective, and anti-tumoralroperties [5]. Barley �-glucan is a linear polysaccharide of �(1→3)nd �(1→4) linked d-glucose molecules. The chain conformationf �-glucan is altered at different concentrations [6–10], whichignificantly affects its bioactivity [11–13]. �-Glucan is known toeduce blood lipid levels, including cholesterol and triglycerides

14–16]. Polysaccharides have been used for decades to stimulatehe immune system and immune modulation by �-glucan is ben-ficial for cancer prevention and hematopoietic recovery [17–22].

Abbreviations: MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumromide; LMP, low-melting-point agarose; FBS, fetal bovine serum; DMSO,imethylsulfoxide; PBS, phosphate-buffered saline; SCGE, single cell gel elec-rophoresis; SEM, standard error of mean; AO/EB, acridine orange/ethidiumromide; LPS, lipopolysaccharide; TLR, Toll-Like Receptor; NFkB, Nuclear Factorappa B; MMS, methyl methane sulfonate.∗ Corresponding author at: Laboratory of Biophysics and Molecular Biology, Insti-

ute of Biochemistry and Biophysics, University of Tehran, PO Box 13145-1384,ehran, Iran. Tel.: +98 21 66498672; fax: +98 21 66956985.

E-mail addresses: goliaei@ibb.ut.ac.ir, foroughmand@ibb.ut.ac.ir (B. Goliaei).

ttp://dx.doi.org/10.1016/j.mrgentox.2014.09.005383-5718/© 2014 Elsevier B.V. All rights reserved.

The biological activity of �-glucan is mediated by pattern recogni-tion receptors located on immune and hematopoietic cells. Thus,the radio-protective properties of glucans are closely related tohematopoietic recovery [23–26]. However, �-glucan’s cellular andmolecular effects on other tissues are still unknown.

Liver injury during right chest radiotherapy has been reportedwidely. The liver is located under the right ribs, just beneath theright lung, and its protection from ionizing radiation is essential. Inthis study, we used the human hepatoma cell line (HepG2), widelyused as a model for in vitro cytotoxicity experiments [27–32], todetermine whether �-glucan could enhance DNA repair and exertbeneficial cyto- and radio-protective effects after treatment withX-rays.

2. Materials and methods

2.1. Drugs and reagents

The following chemicals were used in this study. �-Glucan (G6513) waspurchased from Sigma Aldrich (USA). Low-melting-point agarose (LMP), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and trypan blue wereobtained from Sigma Chemical (Germany). RPMI 1640 and fetal bovine serum (FBS)

were purchased from GIBCO (UK). Dimethylsulfoxide (DMSO), agarose, trypsin,Triplex 3, buffer salts, methyl hydrobenzoate, methyl green, and acetic acid wereobtained from Merck (Germany). Acridine orange and ethidium bromide dyeswere purchased from Hopkins & Williams Ltd. (Chadwell Health, UK) and Merck(Germany), respectively.

2 L. Ghavami et al. / Mutation Research 775–776 (2014) 1–6

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ig. 1. 2000 cells/cm2 were seeded into 24 well plates and treated with different coars) and 72 h (solid bars). Bars indicate means ± standard deviation of three indep

.2. Cell culture

The human hepatoma cell line HepG2 was obtained from the National Cell Bankf Iran (Pasteur Institute, Iran) and routinely sub-cultured in RPMI1640 mediumupplemented with 10% heat-inactivated fetal bovine serum (FBS), 200 �g/ml strep-omycin (Jaberebn-Hayan, Iran) and 500 �g/ml penicillin (Sigma, USA) at 37 ◦C in aumidified atmosphere and 5% CO7. Cells were maintained as monolayer cultures,nd subculturing was routinely performed when cells reached 80–90% confluency.

.3. Growth curve

Cells were plated at 2 × 104 cells/cm2 in T25 culture flasks (Nunc, Denmark)nd incubated overnight. �-Glucan was added (0, 1, or 100 �g/ml) and cells werencubated for 72 h. Cells were then thoroughly washed and plated at 2 × 104 cell/cm2

n a 24-well plate (Nunc, Denmark) and allowed to adhere overnight. Three wellsere counted every 24 h for nine days. Media were changed every two days. The

ssay was performed in triplicate.

.4. MTT assay

Briefly, 2 × 104 cells/cm2 were seeded into 96-well plates (Nunc, Denmark) andncubated overnight to ensure attachment of cells before �-glucan was added. Theollowing day, cells were treated with 1–400 �g/ml �-glucan and incubated for 24,8, or 72 h. After indicated times, cells were washed with PBS and MTT solution0.5 mg/ml, in PBS) was added to each well and incubated for another 4 h at 37 ◦C33]. Formazan crystals were dissolved in 100 �l DMSO, and the absorbance wasetermined at 570 nm using an Asys High-tech Eliza reader spectrophotometer. Theumber of viable cells was then calculated as follows:

umber of viable cells (%) = Abs of sample × 100Abs of control

The assay was performed in triplicate.

.5. Fluorescence microscope analysis of apoptosis and necrosis

Apoptosis and necrosis were analyzed by the differential uptake of fluores-ent DNA binding stains ethidium bromide and acridine orange (AO/EtBr). Acridinerange is a vital dye that stains both live and dead cells, whereas ethidium bromidenly stains those cells that have lost their membrane integrity. Briefly, both adherentnd floating cells were collected and stained with a mixture of AO (0.5 mg/ml) andtBr (0.5 mg/ml) solution (1:1, v/v). The stained cells were visualized by Axoscope 2lus fluorescence microscope (Zeiss, Germany) using 20× magnification. 200 cellsere analyzed to calculate the fraction of apoptotic and necrotic cells [34]. The assayas performed in triplicate.

.6. Irradiation

X-Irradiation was carried out at room temperature using Siemens PRIMUS linearccelerator (Germany) at Pars Hospital (Tehran, Iran). 72 h after �-glucan (1 �g/ml)reatment, cell culture flasks were replenished with 25 ml fresh medium. Cells were

rations of �-glucan (1, 5, 10, 100, 200, 400 �g/ml) for 24 h (open bar), 48 h (dottedt repetitions.

irradiated with doses of 2, 4, 6, or 8 Gy at a dose rate of 2 Gy/min. Control flaskswere sham-irradiated at the same time. �-Glucan was removed from cell culturebefore radiation exposure by replenishing cell culture completely. The assay wasperformed in triplicate.

2.7. Clonogenic survival assay

Control and pre-treated cells with 1 �g/ml �-glucan were trypsinized 30 minafter irradiation and appropriate numbers of cells were plated into 60 mm Petridishes (Nunc, Denmark) for survival analysis using clonogenic assay [35,36]. Cellswere incubated for 14–16 days to form colonies, fixed with formaldehyde (Merck),and stained with 2% crystal violet solution [37]. The plating efficiency and survivingfraction at each dose of irradiation was determined. All data points were the meansof three experiments.

Plating efficiency (PE) = Number of colonies countedNumber of cells seeded

× 100

Surviving fraction (SF) = Number of colonies countedNumber of cells seeded × (PE/100)

2.8. Comet assay

The alkaline phosphate comet assay was performed immediately after expo-sure to ionizing radiation [38–41]. For each experiment, a set of four slides wasprocessed simultaneously, including: negative or solvent control (C), positive con-trol, which was pretreated with 1 �g/ml �-glucan for 72 h (B), negative controlexposed to 6 Gy ionizing radiation (R6), and positive control exposed to 6 Gy ion-izing radiation and �-glucan (1 �g/ml) (B6). The assay was performed 0, 30, 60,120 min and 24 h after radiation exposure. After irradiation, cells were trypsinizedand suspended in 70 ml pre-warmed LMP agarose (0.5% in PBS) and 65 ml of thesuspension was deposited on a fully frosted slide which was pre-coated with 80 ml1% normal agarose in PBS. The agarose was allowed to set at 4 ◦C for 10 min. Theslides were then put into a tank filled with lysis buffer solution (2.5 M NaCl, 0.1 MEDTA, 10 mM Tris/HCl adjusted to pH 10, 1% Triton X-100 freshly added) for 1 hat 4 ◦C. To allow DNA unwinding, the slides were incubated in fresh electrophoresisbuffer (0.3 M NaOH and 1 mM EDTA, pH 13.6) for 30 min at 4 ◦C. The slides were thenplaced into a horizontal electrophoresis tank and electrophoresis was performed at16 V (1 V/cm, 300 mA) for 30 min at room temperature. After electrophoresis, theslides were placed in fresh neutralization buffer (0.4 M Tris/HCl adjusted to pH 7.5)

before staining with 50 �l ethidium bromide solution (20 �g/ml) and were observedat 20× magnification using a Zeiss Axoscope 2 fluorescence microscope (Germany)[42]. 150 randomly selected cells per slide were visually scored and analyzed usingimage analysis software (Tri Tek Comet Score 1.5). Data represent three independentexperiments.

L. Ghavami et al. / Mutation Research 775–776 (2014) 1–6 3

Table 1Doubling time of HepG2 cells treated with various concentrations of �-glucan.

�-Glucan concentration (�g/ml)

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Table 2� and � values obtained from survival curves in two different type of treatments.

value value

Radiation 0.182 0.001Radiation + �-glucan 0.043 0.063

Fig. 2. Effects of �-glucan on radio-sensitivity. Pre-treatment of HepG2 cells with

Fd(i

0 1 100

Doubling time (h) 37.573 ± 1.9 37.1 27.56 ± 1.3

.9. Statistical analysis

The data values are presented as means ± SEM and the t-test was performed tonalyze the statistical significance between samples. P < 0.05 is considered signifi-ant; * and ** represent P < 0.05 and 0.001, respectively.

. Results

.1. Cytotoxicity of ˇ-glucan

Fig. 1 shows the effect �-glucan (1–400 �g/ml) at three differentreatment times (24, 48 or 72 h) on viability of HepG2 cells. Noytotoxicity was observed under these conditions. The �-glucan-nduced increase in cell viability was concentration-independent.

.2. Effect of ˇ-glucan on cell proliferation

Growth curves of HepG2 cells treated with various concentra-ions of �-glucan were generated and used to calculate doublingimes. Results are shown in Table 1. Treatment of cells with 1 �g/ml-glucan did not have any significant effect on the doubling timef the cells, whereas 100 �g/ml �-glucan decreased the doublingime by 10 h (P < 0.05)

.3. Clonogenic assay

The effect of �-glucan on radiosensitivity of HepG2 cells ishown in Fig. 2. Control cells or cells treated with �-glucan (1 �g/ml,2 h) were irradiated with 0, 2, 4, 6, or 8 Gy. The linear-quadraticodel was used to analyze �-glucan’s effects on reproduction. This

ig. 3. Effects of �-glucan on HepG2 cells undergoing apoptosis following irradiation wecreased apoptosis after X-ray irradiation. Apoptotic cells were identified by the cond1 �g/ml), (c) 6 Gy radiation, (d) �-glucan + 6 Gy radiation. Green arrows indicate apoptotnterpretation of the references to color in this figure legend, the reader is referred to the

1 �g/ml �-glucan for 72 h protected the cells against radiation. The colony formingefficiency was determined and the survival curves were generated. Each date pointmeans results from three independent experiments.

analysis of radiation survival curves demonstrated that radiopro-tection was mainly due to decreased alpha (initial slope of survivalcurve). Table 2 shows the and values estimated from the survivalcurves.

3.4. Morphological observations by acridine orange and ethidiumbromide (AO/EB) staining

Fluorescent staining with acridine orange and ethidium bro-

mide (AO/EB staining) dyes was performed to evaluate the nuclearmorphology of cells and apoptosis induction by �-glucan. Cellspre-treated with �-glucan (1 �g/ml), radiation (6 Gy) and the com-bination were evaluated for apoptosis and necrosis (Fig. 3). As

ere assessed by fluorescence microscopy. �-Glucan treatment (1 �g/ml) for 72 hensation and fragmentation of their nuclei. (a) HepG2 control cells, (b) �-glucanic cells (early and late apoptotic cells) in contrast to red arrow (necrotic cells). (For

web version of the article.)

4 L. Ghavami et al. / Mutation Rese

Fig. 4. Effects of �-glucan on radiation induced apoptosis in HepG2 cell. �-Glucantreatment (1 �g/ml) for 72 h did not show significant differences compared tothe un-treated control group, whereas �-glucan treatment decreased number ofam

san

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poptotic cells after 6 Gy radiation. Each column represents the mean of triplicateeasurements out of three different experiments.

hown in Fig. 4, �-glucan pre-treatment protected HepG2 cellsgainst radiation induced apoptosis, whereas �-glucan itself didot induce apoptosis in un-irradiated control cells.

.5. Comet assay

The single cell gel electrophoresis (SCGE) results (Fig. 5) clearlyemonstrate that in both control and �-glucan-treated cells, DNAamage is repaired during the post-irradiation incubation. Inadiation-exposed cells in the absence of �-glucan, DNA dam-ge was detectable after 24 h, whereas in �-glucan-treated cells,amage decreased after 1 h post-irradiation incubation to levelsbserved in untreated control cells (Fig. 5). On the other hand,-glucan treated cells at zero time, immediately after radiation,

howed less damage than un-treated cells following radiationxposure. Results are shown in Fig. 6.

ig. 5. Results obtained by the comet assay: effects of �-glucan on radiation-nduced DNA damage at different incubation times after irradiation. HepG2 cells

ere pre-incubated for 72 h with 1 �g/ml �-glucan then exposed to 6 Gy X-rays.fter irradiation, cells were collected and analyzed for formation of DNA breaks byomet assay (30, 60, 90, 120 min and 24 h after radiation exposure). �-Glucan pre-reatment decreased the tail moment (indicating the degree of DNA strand breakage)o the level of control un-treated cells, whereas DNA breaks in 6 Gy irradiated cellsn the absence of �-glucan remained unrepaired even at 24 h post-irradiation incu-ation. Each column represents the mean triplicate measurements out of threeifferent experiments.

arch 775–776 (2014) 1–6

4. Discussion

Radiation is one of the most effective modalities for treating can-cer. Unfortunately, damage to normal cells is dose-limiting. Liverinjuries increase during right chest radiotherapy, because of theanatomical position of the liver. The search for agents that canprotect cells from ionizing radiation is of interest, and it is impor-tant to identify compounds that can distinguish between cancerousand normal cells [43]. �-Glucan can be used as an anticancer drug[44] but exerts no cytotoxic effect on normal cells [45]. In thisstudy, we selected HepG2 cells, well known for their capacity formetabolic activation of drugs and as a tool for chemical risk assess-ment [30,46], to investigate the biological properties of �-glucanand to examine whether �-glucan can protect these cells fromradiation. The MTT assay is a colorimetric assay widely used fordetermining cell viability. Since reduction of MTT can only occurin metabolically active cells, the level of activity is an indicationof viability. In the MTT assay results, no cytotoxicity was observedwith increasing �-glucan concentration (1–400 �g/ml) at 24–72 h.Cell viability increased independently from the �-glucan dose andtime of incubation.

�-Glucan demonstrates dose-dependent scavenging activityand proliferation induction at concentrations above 150 �g/ml[46–50]. �-Glucan showed two distinct pathways for proliferationenhancement. Since the effect was observed at 100 �g/ml �-glucanwhile no increase occurred at 1 �g/ml, the latter concentrationwas chosen for further assessments. �-Glucan pre-treatment alsoincreased the colony-forming ability of cells. Clonogenic ability ofcells is an indication of how many times a cell can divide after treat-ment with ionizing radiation. Comet assay analysis showed that�-glucan also significantly reduced DNA damage following irra-diation. Almost all DNA strand breaks were repaired 60 min afterX-irradiation treatment in �-glucan pre-treated cells, and someremaining damages (DNA double strand breaks or complex lesions)disappeared after 24 h of post-treatment incubation. Thus, it maybe concluded that the increase in cell viability with �-glucan as seenin the MTT assay and the increase in cell growth could be the resultof enhanced DNA repair. �-Glucan may activate specific repair sig-naling pathways via unknown receptors on the HepG2 cell surface.

LPS (lipopolysaccharide) affects cells via TLR4 (Toll Like Recep-tor 4) [51]. Recent studies provide evidence of TLR signaling in thehepatic non-immune cell population [52,53]. �-Glucan may acti-vate repair signaling via the TLR receptor. Consequently, it couldactivate the Nuclear Factor Kappa B (NFkB) signaling pathway inHepG2 cells. On the other hand, studies showed expression oftwo �-glucan receptor (�GR) transcripts in the mRNA of varioushuman tissues. These receptors are human homologs of murineDectin-1 receptor that are structurally and functionally similar tothe mouse receptor [54]. �-Glucan may activate a repair signalingpathway via this receptor or by a combination of both receptors.The MTT assay showed that 1 �g/ml �-glucan gave the highest via-bility and does not have an effect on cell proliferation; there may bea very limited number of receptors that bind to �-glucan. �-Glucandoes not induce receptor expression at concentrations > 100 �g/mlthrough 72 h treatment. Consequently, it has been shown that�-glucan affects HepG2 cells by two distinct pathways. At con-centrations > 100 �g/ml, the rate of cell proliferation increased,possibly via the TLR signaling pathway which activates the NFkBpathway, accompanied by antioxidant behavior at this concentra-tion. At 1 �g/ml, the “dectin-like receptor”, which is present at alimited number in liver cells, or other receptors that are not yetidentified, may be acting.

On the basis of mechanism of action, there are two typesof anti-mutagens: desmutagens and bio-antimutagens. Desmu-tagens interact directly with mutagens and block their effects;bio-antimutagens act after the damage has already been done, by

L. Ghavami et al. / Mutation Research 775–776 (2014) 1–6 5

Fig. 6. Effects of �-glucan on radiation induced DNA damages assessed by comet assay. HepG2 cells were pre-incubated for 72 h with 1 �g/ml �-glucan then exposed to 6 GyX-ray photon. Following irradiation, cells were collected and analyzed for the formation of DNA breaks by the comet assay in 6 different time after irradiation (0, 30, 60, 90,1 withw

mcsmacrasta

20 min and 24 h). Comet tail of cells after exposure to 6 Gy of X-irradiation treatedith �-glucan (B) or without �-glucan (C).

odulation of DNA replication [48]. �-Glucan extracted from Sac-haromyces cervisiae acts as a desmutagen toward methyl methaneulfonate (MMS) [50]. Based on the findings presented here, theechanism of action for 1 �g/ml barley �-glucan involves bio-

ntimutagenesis. This could be due to the action of unknownell-surface receptors or variability in �-glucan conformation. Theadical-scavenging activity of �-glucan may also be involved. The

bility of �-glucan to cause apoptosis was also investigated. Ourtudies revealed that pre-treatment with �-glucan decreases apop-otic cell death in accord with DNA repair observed by the cometssay.

�-glucan (B6) or without �-glucan (R6) compared to those of control cells treated

This study provides evidence that �-glucan possesses protec-tive properties and suggests that �-glucan could be consideredas a potential radio-protective agent in non-immune and non-hematopoietic cells. Further studies are needed to elucidate theexact mechanisms of �-glucan action and to identify the pertinentHepG2 receptors.

Conflict of interest statement

None declared.

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