In vivo and In vitro Genotoxic Effects of Zerumbone

CARYOLOGIA Vol. 63, no. 1: 11-17, 2010In vivo and In vitro Genotoxic Effects of ZerumboneAl-Zubairi 1,3 Adel S., Ahmad Bustamam Abdul 1,2, *, Mohammed Yousif 1 ,Siddig Ibrahim Abdelwahab 1 , Manal Mohamed Elhassan 1 and Syam Mohan 11Laboratory of Cancer Research MAKNA-UPM, Institute of Biosciences (IBS), University Putra Malaysia, Serdang,43400, Selangor DE, Malaysia.2Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, University Putra Malaysia, Serdang,43400, Selangor D.E., Malaysia.3Department of Biochemistry and Molecular Biology, Faculty of Medicine and Health Sciences, University ofSana’a, Sana’a, Yemen.Abstract — Zerumbone (ZER) is derived from Zingiber zerumbet smith from the Zingiberaceae family. It hasbeen shown to have anti-cancer and apoptosis-inducing properties against various human tumour cells. The aimof our study was to assess the genotoxic effects of ZER in cultured human peripheral blood lymphocytes, ChineseHamster Ovary (CHO) cells and rat bone marrow polychromatic erythrocytes (PCEs) using micronucleustest (MN). All in vitro treatments were carried out in the absence of any exogenous metabolic activation system.Mitomycin C (MMC) was used as a positive control for in vitro treatments, while cisplatin was used as a positivemicronucleus inducer in rat bone marrow PCEs. ZER at high concentrations induced an apparent significantincrease in the frequency of micronuclei in vivo (1000 mg/kg b.w) and in vitro (40 and 80 µM) compared toconcurrent control values. Our in vivo and in vitro cytogenotoxicity studies suggest that high doses of ZER maybe genotoxic and cytotoxic.Keywords: CHO, genotoxicity, human peripheral blood lymphocytes, micronucleus, MNPCEs, zerumbone.INTRODUCTION*Corresponding authors: phone: +603-89462124; fax:+603-89462101; e-mail: abustamam@putra.upm.edu.my;adelalzubairi@hotmail.com.Zingiber zerumbet smith is used in local traditionalmedicine as a cure for a number of illnesses,locally known as ‘lempoyang’ wild gingerbelongs to Zingiberaceae family. It is native toSouth East Asia but has been widely cultivatedplant in village gardens throughout the tropicaland subtropical area around the world andhas naturalized in some areas for its medicinalproperties. In some Southeast Asian countries,the rhizomes of the plant are employed as traditionalmedicines for anti-inflammation, whilethe young shoots and inflorescence are used ascondiments. Scientific research towards Zingiberzerumbet proved that it contained a suppressiveeffect which was conducted by a bioactivecompound, zerumbone. It has been shown thatzerumbone is one of the most promising chemopreventiveagents against colon and skin cancer(MURAKAMI et al. 2004). It was reported to suppresscolonic tumour marker formation in ratsand potentiated TRAIL-induced apoptosis inhuman HCT116 colon cancer cells (YODKEEREEet al. 2009). The compound was shown to inhibitthe proliferation of human colonic adenocarcinomacell lines in a dose-dependent manner,while the growth of normal human dermal andcolon fibroblast was less affected (MURAKAMI etal. 2004). Zerumbone was further demonstratedto inhibit both azoxymethane-induced rataberrant crypt foci and phorbol ester-inducedpapilloma formation in mouse skin a furtherindication of its efficacy to prevent colon andskin cancers (MURAKAMI et al. 2004; TANAKA etal. 2001). Recently, Sung et al., (2009) reportedzerumbone as modulator for osteoclastogenesis

12AL-ZUBAIRI, ABDUL, YOUSIF, ABDELWAHAB, ELHASSAN and MOHANinduced by RANKL and breast cancer (SUNG etal. 2009).Genotoxicity studies of both naturally occurringand synthetic substances are of great interestbecause of the widespread and often chronicuse of herbal remedies, of modern medicinalproducts, of food ingredients, as well as of otherhousehold and environmental chemicals. Manyplant products contain compounds known tocause various diseases or even death in animalsand humans (DEARFIELD et al. 2002; AMES andGOLD 1997; RASKIN et al 2002; RATES 2001). Syntheticsubstances present as environmental pollutants,and toxicants may cause similar effects(AMES and GOLD 1997; RATES 2001). Many naturaland synthetic compounds have been reportedto act as mutagens and/or carcinogens (VARGASet al. 1990).A variety of in vitro genotoxicity test systemshave been developed including the culturedmammalian cell systems such as human peripheralblood lymphocytes cells (PBL) or Chinesehamster ovary (CHO) cells (DEAN and DAN-FORD 1984; SCOTT et al. 1990), for the screeningof potentially mutagenic, carcinogenic and/orteratogenic agents. The CBMN assay in humanlymphocytes uses cytochalasin-B, an inhibitorof actins polymerisation, which prevents cytokinesiswhile permitting nuclear division (FENECHand MORLEY 1985). As a result, binucleated(BN) cells are produced, which are scored forthe presence of MN (FENECH 1993). The rodentbone-marrow MN test is the most widely usedshort-term in vivo assay for the identification ofgenotoxic effects such as chromosome damageand aneuploidy associated with mutagenesis andcarcinogenesis (VANDERKERKEN et al 1989).Taking into account the lack of informationabout in vivo and in vitro cytogenetic effects ofzerumbone, we decided to provide some data onthe cytogenetic activity of this compound. Herewe report the results obtained on the cytogeneticeffects of zerumbone in vivo using rat bonemarrow erythrocyte micronucleus (MNPCE),in vitro chromosomal aberrations assay and micronucleustest using human lymphocytes (PBL)and micronucleus test in cultured CHO cell.MATERIALS AND METHODSZerumbone extraction - Zerumbone was extractedin the laboratory of cancer research MAK-NA-UPM, University Putra Malaysia, from therhizomes of Zingiber zerumbet plant. The rhizomesobtained from the wet market in KualaLumpur, Malaysia. Zerumbone was extracted,isolated and purified using methanol extractionand column chromatography (CC) method. Theisolated and purified zerumbone crystals weresubjected to High Performance Liquid Chromatography(HPLC) and Liquid ChromatographyMass Spectrometry (LCMS) to confirm itspurity and molecular weight. Further, 13 C NMRand 1 H NMR analysis were conducted towardsthe zerumbone crystals to confirm its molecularstructure (Figure 1). A stock solution of zerumboneis prepared immediately before use in absoluteethanol (HmbG Chemicals).Chemicals - Mitomycin C (MMC), CytochalasinB (CB [CAS 4930-96-2] and Cisplatin [CAS15663-27-1] were obtained from Sigma, Geimsastain [CAS 67-56-1] and phytohaemagglutinin(PHA) were obtained from (Gibco, Germany)and Colcemid from (PAA Laboratories).Animals and their treatment for measurement ofMNPCE - Male Sprague-Dawley rats (6-8 weeksold) weighing 170-200g were obtained from theInstitute of Medical Research, Kuala Lumpur.The rats were maintained in group per cage atroom temperature (25±1 C) and 12-h light: 12-hdark cycle and were given food and water ad libitum.The animals were acclimatised for at least5 days prior to dosing and were divided into fivegroups containing 3-6 rats each. Three dose levelsof zerumbone (250, 500, 1000 mg/kg) weregiven intraperitoneal for 24h. Dose selection wasbased on preliminary experiments in which themaximum tolerable dose (MTD) was identifiedto be 1000 mg/kg body weight. An untreatedcontrol and a positive control (cisplatin 10 mg/kg) were also used to test the validity of the assay.The experiment complied with the guidefor Animal Care and Use Committee (ACUC),Faculty of Medicine and Health Sciences, UniversityPutra Malaysia.After 24h the animals were anesthetized withchloroform and sacrificed. For bone-marrowpreparations, both hind femora were isolatedand the adherent muscle removed. The epiphyseswere cut off and bone marrow cells wereflushed out with foetal bovine serum (PAA Laboratories).The suspension of bone marrow cellsand foetal bovine serum was centrifuged for 10min at 1000 rpm. The resulting sediment wasre-suspended in foetal bovine serum. Bone marrowsmears were prepared from the resultingcell suspension. After air-drying and fixing for10 min in absolute methanol, slides were stainedwith Giemsa-staining method. The slides were

IN VIVO AND IN VITRO GENOTOXIC EFFECTS OF ZERUMBONE 13Fig. 1 — Molecular structure of zerumboneanalyzed in a blinded fashion using a Nikonlight microscope. Both normochromatic erythrocytes(NCE) and polychromatic erythrocytes(PCE) were scored for bone marrow activityand PCEs were scored for micronuclei (MN).A total of 2000 polychromatic erythrocytes werescored per animal for MNPCE and 200 erythrocyteswere counted for the PCE: NCE ratioaccording to the OECD guideline for testing ofchemicals (mammalian erythrocyte micronucleustest), guideline No. 474 (OECD 1997). Forevery group of animals, the following parameterswere reported, the number of MN-containingcells/2000 PCE/animal, the number of PCE; thenumber of NCE and the NCE: PCE ratio.Micronucleus test in cultured human lymphocytes- Buffy coat (0.2 ml) was added to 5ml RPMI-1640 medium supplemented with 20% heat inactivatedfoetal bovine serum, antibiotics (penicillinand streptomycin) and L-glutamine (PAAlaboratories). Lymphocytes were stimulated byadding 2% phytohaemagglutinin (PHA) (Gibco).The cultured lymphocytes were incubatedfor 48h before treatment with zerumbone. Thecultures were incubated at 37°C for 72h andtreated with zerumbone at 5, 10, 20, 40 and80µM during the last 24h. A control untreatedculture, MMC treated and ethanol treated cultureswere established as well. Cytochalasin-B(6µg/ml) was added to arrest cytokinesis at 44hafter the start of the culture. Then, the cells wereharvested by centrifugation (1000rpm, 10min),and the pellet was re-suspended in a pre-warmedhypotonic solution of 0.075M KCI for 5 min.Cells were re-centrifuged and fixed three timesin cold methanol: acetic acid (3:1). Slides wereprepared by dropping and air-drying. Finallyslides were stained with 5% Giemsa (pH 6.8) inphosphate buffer for 10 min, washed in distilledwater, dried at room temperature and mounted.Scoring - The induction of MN was determinedin 1000 binucleated cells with the cytoplasmwell preserved. In a blind test, using a Nikonmicroscope, cells containing 1 micronuclei werescored. The criterion for the identification ofMN was according to Fenech (FENECH 1993). Ineach treatment, the numbers of mononucleated,binucleated, and polynucleated cells per 500cells were counted for cell cycle kinetic analysisand cytochalasin B proliferation index (CBPI) asdetermined in a blind test. Cells with well preservedcytoplasm, containing 1-4 nuclei, were

14AL-ZUBAIRI, ABDUL, YOUSIF, ABDELWAHAB, ELHASSAN and MOHANscored. The CBPI was calculated to determinepossible cytotoxic effects, according to OECDguideline number 487 (OECD 2004) using thefollowing formula:CBPI= MI + 2MII +3MIII+ 4MIV/Nwhere MI-MIV correspond to the numbers ofcells with one, two, three and four nuclei andN is the total number of cells (SURALLES et al.1995).Micronucleus test (MN) in CHO - Chinese hamsterovary (CHO) cells were purchased fromECACC (UK). The cells grow as an adherentmonolayer in appropriate tissue culture vesselsand were maintained in RPMI 1640 medium(PAA Laboratories, Germany) supplementedwith 10% foetal bovine serum (PAA Laboratories,Germany). The cells were incubated ina humidified tissue culture incubator at 37°Cand 5% CO 2. The overnight cell cultures wereexamined under an inverted microscope. Duplicatecultures were prepared for each test substanceconcentration and controls. Control cultureswere handled in a manner identical to thetreated ones. Mitomycin-C (Sigma) was used asa positive control. The treatment medium was 5ml of the cell culture medium with 10% foetalbovine serum, with the treatment concentrationor a control solution and the final concentrationswere 5, 10, 20, 40 and 80µM ZER. Thecells were cultured in the treatment medium for48h. After treatment, cells were washed twicewith 10 mL PBS, trypsinized with 0.05% trypsinand centrifuged for 5 min at 800 rpm. CHO cellswere then harvested and scored using the samemethod of human lymphocytes micronucleusmentioned above.Statistical analysis - Statistical analysis was performedusing SPSS 15. Rat bone marrow erythrocytesMN results were expressed as mean ±SE and were analyzed with one way analysis ofvariance, ANOVA, while the results from in vitrowork were analyzed using Chi square analysis.All statistical tests were performed at the p

IN VIVO AND IN VITRO GENOTOXIC EFFECTS OF ZERUMBONE 15respectively. These results reveal that, ZER has agenotoxic activity only after treatment with highdrug concentrations (40 and 80 µM), inducingan increase in the frequency of binucleated cellswith MN (BNMN) when compared to the concurrentuntreated control cells (P

16AL-ZUBAIRI, ABDUL, YOUSIF, ABDELWAHAB, ELHASSAN and MOHANably elevated the frequencies of MN formationscompared to the concurrent controls, both inhuman lymphocyte when treated for 24h aswell as in CHO cells when treated for 48h. Thepercentage of structurally damaged cells in theMMC (positive control) treatment groups wasstatistically increased compared to the solventcontrol data indicating the responsiveness of thecells in this test system.Analysis of the frequency of occurrence ofmicronuclei in treated cells provides a comparativelyrapid and sensitive indication of bothchromosomal aberrations and chromosome lossthat lead to numerical chromosomal anomalies.Micronuclei are cytoplasmic chromatin masseswith the appearance of small nuclei that arisefrom chromosome lagging at anaphase or fromacentric chromosomal fragments. They providea quantifiable measure of recent DNA injury thatresult from when acentric fragments or wholechromosomes are left behind the main nucleusat telophase. An increase in the prevalence ofMN in a population of cells indicates that chromosomedamage has occurred as a result of anexposure that caused either clastogenic or ananeuploidogenic effect (GONSEBATT et al. 1997;MAHATA et al. 2003). MN assay is a widely usedcytogenetic method to assess in vivo and in vitrochromosomal damage. However, the MN studyis the most reproducible one to show positiveeffects (ROBBIANO et al. 1998). Results of MNinduction in human lymphocytes revealed theability of this compound at high dose to act asaneugenic/clastogenic substance by inducingMN formation in human lymphocytes in vitro.The lethal dose (LD) of 2000 mg/kg b.w. wasdetermined in rat after intraperitoneal administrationof ZER. In the present experimental conditions,a significant increase in rat bone marrowmicronuclei was recorded at the highest dose(1000 mg/kg b.w.). The induction of micronucleatederythrocytes following exposure to highdose of ZER indicates a potential for clastogenicity.In the present study, the observed inhibitionof cell proliferation in the rat bone marrow illustratesthe cytotoxicity of ZER. These data indicatethe cytotoxic potential of ZER at higherexposure doses.Our results show that the increase in MN,and the decrease in the PCE:NCE ratio wasdose-dependent. We found that there is a linearrelationship between the ZER dose used and thefrequencies of micronuclei. It is concluded thatZER has a cytotoxic effect on bone marrow inrats, because the decrease in the ratio PCE:NCEwas observed at all doses compared with the control.Such a decreased ratio is often used as anindicator of bone marrow cytotoxicity or alterationsin erythropoiesis. In normal bone marrow,the PCE:NCE ratio is generally around 1:1.In conclusion our in vivo and in vitro cytogenotoxicitystudies suggest that high dosesof zerumbone used in the present investigationmay be genotoxic and cytotoxic. It is importantto carry out more investigations using variouscytogenetic tests under different experimentalconditions to assess more the genotoxic effectsof zerumbone.Acknowledgment — The authors would like toextend their utmost gratitude and appreciation toMOSTI (Ministry of Science, Technology and Innovation)and The National Cancer Council Malaysia(MAKNA) for providing the research grants, IRPANo: 06-02-04-0720-EA001. The authors also wouldlike to convey their thanks to UPM for their providingadditional support of this work.TABLE 3 — Frequencies of micronucleus (MN) formation, cell cycle kinetics and CBPI on Human PBL culturestreated for 24h with different concentrations of zerumbone, mitomycin C and untreated control.Cell cycle kinetics aTreatment BN MNi %MNi M1 M2 M3 % BN CBPIZII (µM) 0 1000 4 0.40 170 251 79 50.2 1.81810 1000 4.0 0.40 185 201 114 40.2 1.85820 1000 4.0 0.40 180 207 113 41.4 1.86640 1000 16.0* 1.60 203 222 75 44.4 1.74480 1000 75.0** 7.50 400 65 35 13.0 1.270EtOH 1000 3.0 3.00 175 248 77 49.6 1.804MMC (µg/ml) 1.2 1000 100** 10.00 384 82 34 16.4 1.300The numbers of mononucleated (M1), binucleated (M2), and polynucleated (M3) cell per 500 cells were quantitated for cellcycle kinetic analysis.*P < 0.05, significantly different from control.

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