Evaluation of multi endpoint assay t Source Mutat Res SO 2013 Feb 18 751 1 59 65[PMIDN23201538]

Mutation Research 751 (2013) 59–65Contents lists available at SciVerse ScienceDirectMutation Research/Genetic Toxicology andEnvironmentalMutagenesisj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /g e n t o x C o m m u n i t y a d d r e s s :w w w.e l s e v i e r.c o m /l o c a t e /m u t r esEvaluation of multi-endpoint assay to detect genotoxicity and oxidative stress in mice exposed to sodium fluorideManivannan J a ,Sonali Sinha b ,Manosij Ghosh b ,Anita Mukherjee a ,∗a 35Ballygunge Circular Road,Cell Biology and Genetic Toxicology,epartment of Genetics,University of Calcutta,Kolkata 700019,Indiab35Ballygunge Circular Road,Cell Biology and Genetic Toxicology,Centre of Advance Study,Department of Botany,University of Calcutta,Kolkata 700019,Indiaa r t i c l ei n f oArticle history:Received 25July 2012Received in revised form 28October 2012Accepted 11November 2012Available online 29 November 2012Key words:Comet assayBone marrow cellsChromosome aberrations DNA strand breaks MicronucleusOxidative damagea b s t r a c tFluoride compounds are naturally present in soil,water and food.The objective of this study was to investigate the genotoxic and oxidative damage induced by chronic fluoride exposure on mammalian cells in vivo .For this purpose,the genotoxic potential was investigated in bone marrow cells by the micronucleus test,chromosome aberration assay and comet assay (DNA strand breaks).In addition,DNA damage was evaluated in soft tissues and organs like spleen,liver and kidney cells.The oxidative damage was assessed by selective biochemical parameters by the measurement of lipid peroxidation,reduced glutathione (GSH),glutathione S-transferase (GST)and catalase (CAT)activity in liver.Adult Swiss albino male mice were exposed to sodium fluoride in drinking water at the concentrations of 4,12and 20mg/L for 30consecutive days.Control groups (vehicle and positive)were also included.Animals were sacri-ficed;bone marrow and soft tissue samples were collected and subjected to series of assays respectively.We observed that NaF exposure,at the various concentrations tested caused a significant increase in the frequency of micronucleus (MN)in polychromatic erythrocytes (PCEs),structural chromosome aberra-tions in bone marrow cells.With the exception of the spleen cells,DNA damage was observed in bone marrow cells as well as in kidney and liver cells.We found an increase in lipid peroxidation,and catalase activity as well as decrease in glutathione activity (GSH and GST)in liver of mice respectively which were exposed to sodium fluoride.In conclusion,the data obtained clearly documents that NaF exhibits genotoxic activity and enhanced oxidative damage in mouse model.© 2012 Elsevier B.V. All rights reserved.1.IntroductionSodium fluoride (NaF)has been used ubiquitously for decades,due to its specific and effective caries prophylactic property;as well as used for water fluoridation.Excess fluoride ingestion is the cause of fluorosis in human being.The incidence of fluorosis affecting young and old,men and women is not only confined to India,but occurs in 23other nations around the globe [1].In addition to well-known effects on the skeleton and teeth,fluorosis can adverselyAbbreviations:CAT,Catalase;SOD,superoxide dismutase;GST,glutathione S-transferase;ROS,reactive oxygen species;MMC,Mitomycin C;OSI,organo-somatic index;MN,Micronucleus test;PCE,polychromatic erythrocyte;NCEs,normochromatic erythrocytes;MN-PCE,micronucleated-polychromatic erythro-cytes;CA,Chromosome Aberration;TBARS,Thiobarbituric acid reactive species;MDA,malonadialdehyde;CDNB,Chloro-dinitrobenzene;PCE/NCE,polychromatic erythrocyte/normochromatic erythrocyte.∗Corresponding author at:35Ballygunge Circular Road,Cell Biology and Genetic Toxicology,Department of Genetics,University of Calcutta,Kolkata 700019,India.Tel.:+919831061998;fax:+9103324614849.E-mail address:anitamukherjee28@ (A.Mukherjee).affect many tissues and organs as exhibited by a broad array of symptoms and pathological changes [2–5].Based on the epidemiological,in vitro and in vivo studies in human,human cell lines and rodents respectively,the National Research Council-US report [6]on fluoride in drinking water noted that the genotoxic effects of fluoride at environmental concentra-tions are contradictory.A number of the genotoxicity studies done in vitro [7–11]using cell lines or in vivo [12,13]are contradictory to the results that showed lack of genotoxic potential [14–23].There are reports of increased chromosome aberrations in mice/rat bone marrow and testes in vivo ,but other studies,using similar protocols and dose ranges,have reported no induced chromosome damage [24].Zeiger et al.[24]in their review considered chromosome dam-age induced by fluoride in vivo as an unresolved issue.In humans [1,25,26]and in animal models [13],a close asso-ciation between chronic fluoride toxicity and increased oxidative stress has been reported.Numerous studies revealed that fluo-ride caused extensive oxidative stress in liver,kidney,brain and heart by increasing lipid peroxidation and reduced antioxidant enzyme activities like catalase (CAT),superoxide dismutase (SOD),and glutathione S-transferase (GST)[27–31].Other investigators1383-5718/$–see front matter © 2012 Elsevier B.V. All rights reserved./10.1016/j.mrgentox.2012.11.00660M.J et al./Mutation Research751 (2013) 59–65[22,27,32–34]however,have reported thatfluoride does not impair antioxidant systems.Research history offluoride so far demands for further evi-dences to conclude on the role offluoride as genotoxic through ROS(reactive oxygen species)production[35].Presented here are the results of an evaluation of multiple genotoxic endpoints(chro-mosome aberrations,micronucleus formations and DNA strand breaks),combined with biochemical assays in liver(measuring lipid peroxidation,reduced glutathione level-GSH,glutathione S-transferase-GST activity and catalase activity-CAT)performed in vivo in mice using the same dosing regimen.2.Material and methods2.1.ChemicalsSodiumfluoride(NaF,CAS No.7681-49-4),Bovine serum albumin(BSA),normal melting point agarose(NMA),low melting point agarose(LMPA),thiobarbituric acid (TBA,CAS no.504-17-6),ethidium bromide(EtBr,CAS no.1239-45-8),triton X-100 were purchased from Sigma–Aldrich Co.(USA).Roswell Park Memorial Institute medium(RPMI1640),Fetal bovine serum(FBS),Phosphate-buffered saline(PBS, Ca2+and Mg2+free),glutathione reduced(CAS No.70-18-8),1-chloro-2,4-dinitro benzene(CDNB,CAS No.97-00-7),Ellman’s reagent[5,5 -dithiobis-(2-nitrobenzoic acid)or DTNB,CAS No.69-78-3],2-nitrobenzioc acid(CAS No.69-78-3),dithiothre-itol(CAS No.3483-12-3),di-sodium salt of ethylenediaminetetra acetic acid(EDTA), were purchased from Hi Media,Mumbai,India.Giemsa(CAS No.51811-82-6),May-Gruenwald(CAS No.62851-42-7),tris buffer(CAS No.77-86-1),dimethyl sulfoxide (DMSO),trichloroacetic acid,methanol,hydrogen peroxide,acetic acid,potassium chloride,sodium citrate monohydrate,sodium hydroxide(NaOH),sodium chloride (NaCl)were purchased from Merck,India.2.2.Animal handling and careHealthy male Swiss-albino mice(8-12weeks old and weighing25–30g)that were randomly bred at the institutional animal house were used for the study.The animals were kept in cages with autoclaved paddy husk for bedding and maintained under standard laboratory conditions(14h:10h dark/light cycle,a temperature of (22±2◦C),and50–70%humidity).The animals were fed on with standard rodent pellets(consisting of crude protein,andfiber)and drinking water(containing NaF) ad libitum throughout the study.The ethical clearance for the use of animals in the study was obtained from the institutional animal ethics committee.The experiments were performed in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals(CPCSEA),India.2.3.Dose selectionAccording to World Health Organization(WHO)guidelines for drinking water, afluoride level of1.5mg/L is the desirable upper limit and for the US Environmental Protection Agency[36]it is4.0mg/L.India reduced the upper limit offluoride in drinking water from1.5to1.0mg/L with a rider that less is better[1].In India,water sample analysis revealsfluoride contaminant level range from0.2to20mg/L[37]. Based on the above information following concentrations viz.4,12and20mg/L of NaF were selected for the current study.2.4.Dose preparationDaily fresh solution of sodiumfluoride was prepared in double distilled water. The animals had free access to drinking water,containing different concentrations of NaF(4,12and20mg/L)prepared in distilled water.Control set of animals were provided with distilled water.To avoid additionalfluoride contamination from the water used,quantification offluoride ion was performed usingfluoride ion selec-tive electrodes(WTW,Germany).The effective concentration offluoride ion thus obtained was<0.087,1.6,6.1and8.6mg/L for control,4,12and20mg/L NaF con-centrations respectively.Mitomycin C(MMC),dissolved in physiological saline was used as positive control.2.5.Treatment scheduleThe animals were divided into5experimental groups,each of six male mice as follows:Group1–negative controls received distilled water in drinking water.Groups2,3,4–Animals were exposed to NaF in drinking water at the concen-trations4,12and20mg/L,respectively.Group5–Positive controls received a single ip.injection of MMC(2mg/kg body weight)24h before sacrifice.All animals except those of Group5were sacrificed on the thirtieth day of the experiment.Animals were sacrificed by cervical dislocation and the femur and other organs were removed.Micronucleus test and comet assay in multiple organs were conducted in same set of animals(n=6),whereas chromosome aberration test and the enzyme assays were performed in another set of animals(n=5)having identical groups.ano-somatic index of liverThe body weight of each animal was recorded before the treatment and also on the thirtieth day of the treatment.The weight of whole liver of respective group of animals was recorded.From these values the organo-somatic index(OSI)of liver was calculated by the following formula[38]:Organo-somatic index=(Weight of the organ/Total body weight on day 30th)×1002.7.Micronucleus test(MN)The micronucleus test was carried out in mouse femoral bone marrow cells and frequencies of micronucleated-polychromatic erythrocytes(MN-PCE)were eval-uated according to the method of Schmid[39],with minor modifications[40]. Animals were sacrificed by cervical dislocation on thirtieth day of the experiment. The femoral bone marrow cells were aspirated using syringe and needle(21G) with3.0ml of fetal bovine serum and centrifuged at800×g for10min.The super-natant was discarded;pellet was mixed,smeared on clean glass slides andfixed in methanol for5min.Thefixed smear was stained with undiluted May–Gruenwald stain for5min followed by diluted May–Gruenwald stain(1:1,v/v in distilled water) for3min.The slides were washed with distilled water,stained with Giemsa(10%, v/v in Sorenson buffer)for10min and observed under light microscope(Carl,Zeiss, Berlin,Germany).All slides were coded and scored blind.The incidence of micronu-cleated(MN)cells per500polychromatic erythrocyte(PCE)was determined for each animal and the percentage of PCEs with MN was calculated.Thousand erythrocytes were scored from each animal to calculate the ratio of polychromatic erythrocyte (PCEs)to normochromatic erythrocytes(NCEs)and the toxic effect of NaF to bone marrow cells was evaluated.2.8.Single cell gel electrophoresis(Comet assay)The alkaline comet assay was performed according to the method of Singh et al.[41]with minor modifications.Animals were sacrificed,liver,kidney,spleen and femurs were removed and then single cell suspension was prepared as previously described by Sasaki et al.[42].Briefly,the spleen,kidney and liver tissues were minced in0.075M NaCl solution containing0.024M Na2EDTA(pH7.5).Followed by the centrifugation,the cells were resuspended in phosphate buffered saline. The bone marrow cells were washed in0.075M NaCl solution containing0.024M Na2EDTA(pH7.5)and resuspended in phosphate buffered saline.Slides were pre-pared by mixing the cell suspension with1%low melting point agarose;layered on the slide base coated with1%normal melting point agarose and placed in a chilled lysing solution(2.5M NaCl,100mM Na2EDTA,10mM Trizma,10%DMSO,and1%Tri-ton X-100,pH10.0)at4◦C for1h.Then the slides were subjected to DNA unwinding in chilled alkaline solution(300mM NaOH and1mM Na2EDTA,pH>13)for20min and subsequently electrophoresis was performed at0.7V/cm and300mA at4◦C for 25min in freshly prepared electrophoresis buffer(1mM EDTA disodium salt and 300mM NaOH).After electrophoresis the slides were neutralized with Tris buffer (400mM,pH7.4).Slides were stained with20␮g/ml ethidium bromide(EtBr)and stored at4◦C in a humidified slide box until scoring.Slides were scored at afinal magnification of400×using an image analysis system(Komet5.5,Kinetic Imaging, Andor technology,Nottingham,UK)attached to afluorescence microscope(Leica, Germany)equipped with an attachment of a CCD camera.The comet parameters used to measure DNA damage in the cells were tail DNA(%).Images from150random cells(per animal)were analyzed as per the guidelines[43].2.9.Chromosome Aberration test(CA test)The chromosome aberration test was performed on mouse bone marrow cells with slight modifications[44].Animals were sacrificed by cervical dislocation on the thirtieth day of the experiment.One and half hours prior to sacrifice,animals were injected intraperitoneally with colchicine(4.0mg/kg bw).The femoral bone marrow cells were aspirated in RPMI1640media and centrifuged at800×g for 10min,the pellet was incubated in8.0ml of KCl(0.075M)at37◦C for30min,fol-lowed by centrifugation at800×g for5min.The cells werefixed in Carnoy’sfixatives (glacial acetic acid/methanol,1:3,v/v),washed thrice with Carnoy’sfixative at inter-vals of10min.The pellet was resuspended infixative and dropped on chilled slides from the height of2–2.5ft,and air dried.The slides were stained with freshly pre-pared Giemsa stain(8%,v/v in Sorenson buffer)for10min followed by washing with distilled water.A total number of50metaphases per animal were evaluated for chromosomal aberrations.The types of aberrations were scored and recorded strictly in accordance with the method of Tice and Ivett[45].The metaphase cells were scored at1000×magnification,with selection being based on uniform staining quality,lack of overlapping chromosomes and chromosome number(40±2chro-mosomes).Each chromosome aberration recorded was of the following types:G ,G , as chromatid and isochromatid gaps;B ,B ,as chromatid and chromosome breaks, RR as chromatid rearrangement.Responses were evaluated as the percentage ofM.J et al./Mutation Research751 (2013) 59–6561aberrant damaged metaphase cell(%DC)and as the number of aberrations per cell (CA/cell).Chromatid and chromosome gaps were recorded but were not included in calculations.For a count of the number of CA/cell,chromatid and chromosome breaks,and chromatid rearrangements(dicentric,ring,exchanges)were taken as one,regardless of the number of breakage events involved[45].2.10.Assay of lipid peroxidation2.10.1.Thiobarbituric acid reactive species(TBARS)levelThe lipid peroxidation level in the liver homogenates was measured using the levels of malonadialdehyde(MDA),which is the end product of lipid peroxida-tion and reacts with TBA as a TBARS to produce a pink colored complex that has peak absorbance at535nm according to Buege and Aust[46].Briefly,the tissue was homogenized with9volume of chilled physiological saline and centrifuged at8000×g for5min.1ml of supernatant was mixed with2ml TCA-TBA-HCl and heated for15min in a boiling water bath.The mixture was cooled on ice and centrifuged at10,000×g for15min.The absorbance of the resultant supernatant was read at535nm spectrophotometrically.The amount of TBARS was calculated using a molar extinction coefficient of1.56×105M−1cm−1expressed as nanomoles MDA/mg protein.2.11.Estimation of non-enzymatic antioxidants2.11.1.Reduced glutathione(GSH)levelReduced glutathione(GSH)level in the liver homogenate of NaF treated mice was estimated by the method of Sedlak and Lindsay[47].The assay is based on the reaction of GSH with DTNB(Ellman’s reagent)that gives2-nitro-5-thiobenzoic acida yellow colored compound.In brief,250␮l of tissue homogenate was added to1.76ml of distilled water and3.0ml of50%TCA solution to precipitate the protein, mixed and centrifuged at3000×g for10min.The resulting supernatant so obtained after centrifugation was then taken for GSH estimation.To the supernatant,2.0ml of0.4M tris buffer solution(pH8.9)and0.5ml of DTNB was added and then the intensity of yellow color was read at412nm.The GSH content in the samples were calculated using a molar extinction coefficient of14,150M−1cm−1and expressed as nanomoles/mg protein.2.12.Determination of glutathione S-transferase(GST)activityGlutathione S-transferase catalyzes the conjugation reaction with glutathione in thefirst step of mercapturic acid synthesis.The activity of GST was measured according to the method of Habig et al.[48].Chloro-dinitrobenzene(CDNB)was used as a substrate.The absorbance was measured spectrophotometrically at340nm.The specific activity of GST was estimated using extinction co-efficient9.6mM−1cm−1 expressed as nanomoles of GSH-CDNB conjugate formed/min/mg protein.2.13.Catalase(CAT)assayCatalase activity was determined according to the method of Aebi[49].The liver tissues were homogenized in phosphate buffer(pH7.0)and centrifuged at10,000×g for15min.The pellet was vortexed vigorously with phosphate buffer containing 1%Triton X-100and then centrifuged at10,000×g for15min.The reaction was initiated by addition of10mM H2O2solution to the supernatant and read at240nm. Catalase specific activity was calculated by using molecular extinction co-efficient of39.4M−1cm−1and represented as IU/mg protein.2.14.Protein estimationProtein quantification was carried out spectrophotometrically by the method of Lowry et al.[50]with bovine serum albumin as standard.2.15.Statistical analysisThe data were analyzed using the Statistical Programme–SigmaStat3.0(SPSS Inc.,Chicago,IL,USA).One-way analysis of variance(ANOVA)test,followed by Dunnett’s multiple comparison tests at P≤0.05,was done for single cell gel electrophoresis,biochemical estimations and organo-somatic index.For the chro-mosome aberration and micronucleus assay,student’s t-test was performed.The level of significance was established at P≤0.05.3.Results3.1.Oragano-somatic index of liverAdministration of NaF to the mice through drinking water for 30days alters the organo-somatic index(Fig.1)of liver of mice. 3.2.Micronucleus testThe effects of NaF on frequency of MN-PCEs and the PCE/NCE ratio in mouse bone-marrow cells are presented in Table1.A sig-nificant increase in the number of MN-PCEs was observed in mice exposed to NaF in all the treatment groups.In positive control group this was significantly higher than that of control and treated group demonstrating the sensitivity of the test system.The cytotoxic effect of NaF on bone marrow cell was tested by assessing polychro-matic erythrocyte/normochromatic erythrocyte(PCE/NCE)ratio.A statistically significant decrease in the PCE/NCE ratio was observed at concentrations12and20mg/L.3.3.Single cell gel electrophoresis(Comet assay)The alkaline comet assay was performed in multiple organs like liver,kidney,spleen and bone marrow cells.The exposure to vari-ous concentrations of NaF caused an increase in DNA strandbreaks Fig.1.62M.J et al./Mutation Research751 (2013) 59–65 Table1Micronucleated polychromatic erythrocyte(MN-PCEs)in the bone marrow cells ofmale mice exposed to different concentrations of sodiumfluoride.Na F concentrations(mg/L)%MN-PCE(mean±SEM)PCE/NCE (mean±SEM)0a0.09±0.003 2.28±0.02 40.14±0.014* 2.01±0.05 120.29±0.018* 1.59±0.02* 200.24±0.014* 1.58±0.02* MMC b 2.44±0.135*0.82±0.12*MN-PCEs,micronucleated polychromatic erythrocytes(1000cells/animal,N=6); PCEs,polychromatic erythrocytes;NCEs,normochromatic erythrocytes.*P≤0.05compared to control(Student’s t test).a Vehicle control.b Positive control–Mitomycin C(2mg/kg bw).leading to greater DNA migration out of the nucleus into the tail of the comet(Table2).The values did not yield a statistically signifi-cant increase in DNA damage in spleen cells.Significant increase in percent Tail DNA was observed in bone marrow(4,12and20mg/L), kidney(12and20mg/L)and liver(20mg/L)cells.3.4.Effect on Chromosome aberration frequencyThe chromosome aberrations observed in bone marrow metaphase cells of mice are presented in Table3.NaF treatment caused increase in the frequency of chromosome aberrations at all concentrations as compared to control.However,the values were significant at concentrations of4and12mg/L of NaF exposure. The major types of structural aberrations observed in this study were chromatid gaps and breaks,with a few chromosome breaks. The frequency of aberrations observed in positive control group was significantly higher than the vehicle control as well as of NaF exposed groups.3.5.Biochemical assaysThe results of lipid peroxidation,GSH,GST and catalase activity in the liver of NaF exposed mice are presented in Table4.Fluoride treatment caused an increase in the levels of lipid peroxidation, catalase activity(statistically significant at20mg/L)and decrease in the concentrations of GSH and GST activity significantly at vari-ous concentrations(4,12and20mg/L)of NaF when compared with the corresponding group of control animals.The observed increase in MDA level with increasing concentrations offluoride indicates the elevated lipid peroxidation in liver membrane because of the reactive oxygen species leading to membrane damage.Further the decrease in GSH level and GST activity(12and20mg/L)with increase influoride concentrations shows increased consumption of glutathione for the scavenging of reactive oxygen species(ROS).4.Discussion and conclusionThe systemic genotoxic effect related tofluoride toxicity was investigated in multiple organs of Swiss albino male mice.The investigations reported here used in vivo genotoxic test procedures for determination of-(1)the prevalence of chromosome damage as observed by the presence of micronuclei and type of chromosomal aberrations in mouse bone-marrow cells;and(2)the DNA damageTable2DNA damage expressed as Percent Tail DNA(mean±SEM)in spleen,liver,kidney and bone marrow cells of male mice exposed to different concentrations of sodiumfluoride. Test substance Concentration(mg/L)DNA damage(Tail DNA%)Spleen Liver Kidney Bone marrow Vehicle control– 6.96±0.4611.30±0.9711.10±0.867.29±0.94 NaF47.51±0.3112.19±1.0914.22±0.8011.22±0.99*127.86±0.5716.33±1.1418.01±0.77*12.12±0.67*208.34±0.4118.24±1.59*19.55±1.11*13.81±0.79* MMC2mg/kg bw13.56±1.28*33.89±1.94*26.06±1.07*25.59±2.34*N=6;MMC–Mitomycin C(2mg/kg bw).150cells/animal(N=6)/conc.*P≤0.05(one-way ANOVA).Table3Chromosome aberrations in bone marrow cells of male mice exposed to different concentrations of sodiumfluoride.Test substance Concentration(mg/L)Total chromosome aberrations a Aberrant cells(%)±SEM CA/cell±SEMG G B B RVehicle control–6–10–– 3.60±2.230.040±0.03 NaF43–30––10.00±1.55*0.100±0.02* 127–222–11.60±1.33*0.116±0.01*203–191–7.60±0.400.084±0.01 Mitomycin C2mg/kg bw102313–13.60±1.26*0.140±0.02*a50metaphase plates/animal(5animals/conc.);G Chromatid gap;G Chromosome gap;B Chromatid break;B Chromosome break;R Rearrangement.*P≤0.05compared to control(Student’s t test).Table4Contents of LPO,GSH,GST and Catalase activity in liver cells of male mice exposed to different concentrations of sodiumfluoride.NaF mg/L)LPO(nmol MDA/mg protein)GSH(nmol/mg protein)GST(CDNB-GSH conjugate/min/mg protein)Catalase activity(IU/mg protein)00.056±0.0126.66±0.9899.44±5.4843.42±3.3240.076±0.0120.48±0.83*72.75±9.04*38.91±1.98120.072±0.0117.49±0.77*61.82±6.15*47.27±1.70200.102±0.01*13.18±0.54*88.69±7.01*54.76±3.30*Values are expressed as mean±SEM,n=5animals/conc.*P≤0.05(one-way ANOVA).M.J et al./Mutation Research751 (2013) 59–6563in cells of bone marrow,liver,kidney and spleen by the single cell gel electrophoresis(comet assay).Since oxidative stress is impli-cated influoride toxicity,additional biochemical studies to measure the changes in lipid peroxidation,content of reduced glutathione, GST,and catalase were done.The data obtained from these assays clearly showed a signif-icant increase in the frequency of MN,structural chromosomal aberrations,in bone marrow cells and DNA fragmentation in bone marrow cells as well as in kidney and liver cells of mice that were exposed to various doses of NaF.Literature survey revealed that earlier investigations onfluoride genotoxicity reported signif-icant increased chromosome aberrations in mice and cultured cells [51–53].In recent years Chaurasia et al.[54]observed dose depend-ent increase in chromosomal aberrations in bone marrow cells of Swiss albino mice.Similar trend was noted in in vitro experiments in HL-60cells where reduced cell viability,decreased DNA and protein biosynthesis,and apoptosis was observed at high concen-trations(100-250mg/L);and no such effect at lower concentrations (0-50mg/L)was noted[55].In vitro experiment in human blood cultures exposed to NaF demonstrated significant increase in DNA breakage and the induction of chromosomal aberrations and MN [56].Tiwari et al.[57]also reported that lowfluoride concentra-tions caused increased chromosomal aberration and DNA damage in human peripheral blood cultures.Poddar et al.[13]found the action of NaF was more genotoxic at lower concentrations than at higher concentrations.They reported mitotic inhibition,chro-mosomal aberrations were more pronounced in mice that received relatively low dose of NaF(15mg/L)in drinking water over a period of3months[13].In the present study,we observed significant increase in the percentage of aberrant metaphases and chromo-somal aberrations in all the treated groups,the effect being more pronounced in mice that received12mg/L of NaF.Interestingly, a decrease in the severity of these parameters was noted at the highest NaF concentration(20mg/L)that remains unexplained at the moment.However one plausible explanation could be that MN assay detects both clastogenicity and aneugenicity whereas only clastogenic damages are consider in CA[58].Contradictoryfind-ings on the genotoxic potential of NaF in mammalian cells using in vitro and in vivo systems have also been reported[21,22,59–68]. Zeiger et al.,[59]reported no increase in aberrations in bone marrow cells and peripheral blood erythrocytes mice following administration of NaF in drinking water(100-400mg/L).Several in vivo studies,using sister chromatid exchange[16,65],micro-nucleus formation[17,18,20,63]and other endpoints[14,15,19] have also reported negativefindings.It has been found that there is no effect offluoride compounds on sister chromatic exchange in Chinese hamster ovary(CHO)cells and bone marrow(CHBM)cells [69]even at cytotoxic concentrations.No chromosomal aberrations were observed in human diploidfibroblast cells treated with rela-tively low concentration of NaF(5-10ppm)[70]and no mutagenic effects were observed in human EUE cells at10-150ppm of NaF [71].The disparity in various reports could be due to the differ-ences between in vitro and in vivo test systems for the assessment of the genotoxicity.It could also be due to additional variability like age,sex of the animal,tissue examined,dosage and mode of exposure.As far as DNA damage is concerned research onfluorosis depicted that NaF leads to increased olive tail moment,percentage DNA in comet tail and S-phase arrest in cell cycle studied in primary rat hippocampal neurons[72].Thesefindings are in agreement with our data demonstrating DNA damage(the percentage DNA in comet tail)in liver,kidney,and bone marrow cells.It was interesting to note that the DNA strand breaks were more frequent in bone mar-row cells and the spleen cells were least affected.The reason for such discrepancy is unknown at the moment and warrants further analysis.In addition,the present in vivo study addressed the relation-ship betweenfluoride-induced oxidative stress and DNA damage. An increase in the levels of lipid peroxidation,and decrease in the concentrations of GSH and GST activity was observed in liver of mice exposed to NaF.The GSH is the main intracellular non-protein thiol,which is a direct radical scavenger,and also acts as an essential co-factor for enzyme reducing oxidative stress[73]. The decrease in the level of GSH and GST in our study might be due to inability of the cells to generate enough GSH,due to severe cellular damage or due to greater utility in combating the oxida-tive stress.The oxidative stress enhanced ROS because of exposure of NaF might have raised the lipid peroxidation level and thereby enhanced the membrane damage.It is evident from the literature that NaF generates ROS by decreasing the activities of antioxidant enzymes and increasing lipid peroxidation thereby causing oxida-tive stress[1,5,26,28,29,31,34,68,73–76].Thus the observations of the present study suggest that NaF-induced oxidative stress could be one of the possible mechanisms underlying for DNA damage. This corroborates earlier reports on NaF-induced DNA damage and apoptosis in rats’brain,oral mucosal cells and hepatocytes caused by the induction of oxidative stress[12,77].In conclusion,NaF one of the most widely usedfluoride com-pounds,revealed significant increase in chromosome aberrations, MN,DNA fragmentation in bone marrow cells at concentrations of 4-20mg/L.As far as DNA damage is concerned the soft tissues were differentially affected,spleen was the least affected organ while kidney was found to be most susceptible followed by liver.The rise in MDA and subsequent decrease in reduced glutathione level and the GST activity in the liver suggest that oxidative damage is the major mode of action offluoride that could be one of the possi-ble mechanisms for genotoxic activity.Therefore,oxidative stress influoride genotoxicity necessitates more elaborate studies involv-ing other indicators of oxidative damage along with the cytogenetic endpoints.Conflict of interestsThe authors have declared that there is no conflict of interest. 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