Mutation Research 751 (2013) 5965

Contents lists available at SciVerse ScienceDirect

Mutation Research/Genetic Toxicology and

Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres

Evaluation of multi-endpoint assay to detect genotoxicity and oxidative stress in

mice exposed to sodium uoride
Manivannan J a , Sonali Sinha b , Manosij Ghosh b , Anita Mukherjee a,
a
b

35 Ballygunge Circular Road, Cell Biology and Genetic Toxicology, epartment of Genetics, University of Calcutta, Kolkata 700019, India
35 Ballygunge Circular Road, Cell Biology and Genetic Toxicology, Centre of Advance Study, Department of Botany, University of Calcutta, Kolkata 700019, India

a r t i c l e

i n f o

Article history:
Received 25 July 2012
Received in revised form 28 October 2012
Accepted 11 November 2012
Available online 29 November 2012
Key words:
Comet assay
Bone marrow cells
Chromosome aberrations
DNA strand breaks
Micronucleus
Oxidative damage

a b s t r a c t
Fluoride 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 uoride 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 uoride in drinking water at the concentrations of 4, 12 and 20 mg/L
for 30 consecutive days. Control groups (vehicle and positive) were also included. Animals were sacriced; 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 signicant increase in the
frequency of micronucleus (MN) in polychromatic erythrocytes (PCEs), structural chromosome aberrations 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 uoride. 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. Introduction
Sodium uoride (NaF) has been used ubiquitously for decades,
due to its specic and effective caries prophylactic property; as well
as used for water uoridation. Excess uoride ingestion is the cause
of uorosis in human being. The incidence of uorosis affecting
young and old, men and women is not only conned to India, but
occurs in 23 other nations around the globe [1]. In addition to wellknown effects on the skeleton and teeth, uorosis can adversely

Abbreviations: CAT, Catalase; SOD, superoxide dismutase; GST, glutathione

S-transferase; ROS, reactive oxygen species; MMC, Mitomycin C; OSI, organosomatic index; MN, Micronucleus test; PCE, polychromatic erythrocyte; NCEs,
normochromatic erythrocytes; MN-PCE, micronucleated-polychromatic erythrocytes; CA, Chromosome Aberration; TBARS, Thiobarbituric acid reactive species;
MDA, malonadialdehyde; CDNB, Chloro-dinitrobenzene; PCE/NCE, polychromatic
erythrocyte/normochromatic erythrocyte.
Corresponding author at: 35 Ballygunge Circular Road, Cell Biology and Genetic
Toxicology, Department of Genetics, University of Calcutta, Kolkata 700019, India.
Tel.: +91 9831061998; fax: +91 033 24614849.
E-mail address: anitamukherjee28@gmail.com (A. Mukherjee).
1383-5718/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.mrgentox.2012.11.006

affect many tissues and organs as exhibited by a broad array of

symptoms and pathological changes [25].
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 uoride in drinking water noted
that the genotoxic effects of uoride at environmental concentrations are contradictory. A number of the genotoxicity studies done
in vitro [711] using cell lines or in vivo [12,13] are contradictory to
the results that showed lack of genotoxic potential [1423]. 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 damage induced by uoride in vivo as an unresolved issue.
In humans [1,25,26] and in animal models [13], a close association between chronic uoride toxicity and increased oxidative
stress has been reported. Numerous studies revealed that uoride 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) [2731]. Other investigators

60

M. J et al. / Mutation Research 751 (2013) 5965

[22,27,3234] however, have reported that uoride does not impair

antioxidant systems.
Research history of uoride so far demands for further evidences to conclude on the role of uoride as genotoxic through
ROS (reactive oxygen species) production [35]. Presented here are
the results of an evaluation of multiple genotoxic endpoints (chromosome aberrations, micronucleus formations and DNA strand
breaks), combined with biochemical assays in liver (measuring
lipid peroxidation, reduced glutathione level-GSH, glutathione Stransferase-GST activity and catalase activity- CAT) performed
in vivo in mice using the same dosing regimen.

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.
2.6. Organo-somatic index of liver
The 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) 100
2.7. Micronucleus test (MN)

2. Material and methods

Sodium uoride (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 SigmaAldrich Co. (USA). Roswell Park Memorial Institute
medium (RPMI 1640), 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), Ellmans reagent [5,5 -dithiobis-(2-nitrobenzoic
acid) or DTNB, CAS No. 69-78-3], 2-nitrobenzioc acid (CAS No. 69-78-3), dithiothreitol (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), MayGruenwald (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.

The micronucleus test was carried out in mouse femoral bone marrow cells and
frequencies of micronucleated-polychromatic erythrocytes (MN-PCE) were evaluated according to the method of Schmid [39], with minor modications [40].
Animals were sacriced by cervical dislocation on thirtieth day of the experiment.
The femoral bone marrow cells were aspirated using syringe and needle (21 G)
with 3.0 ml of fetal bovine serum and centrifuged at 800 g for 10 min. The supernatant was discarded; pellet was mixed, smeared on clean glass slides and xed in
methanol for 5 min. The xed smear was stained with undiluted MayGruenwald
stain for 5 min followed by diluted MayGruenwald stain (1:1, v/v in distilled water)
for 3 min. The slides were washed with distilled water, stained with Giemsa (10%,
v/v in Sorenson buffer) for 10 min and observed under light microscope (Carl, Zeiss,
Berlin, Germany). All slides were coded and scored blind. The incidence of micronucleated (MN) cells per 500 polychromatic 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.

2.2. Animal handling and care

2.8. Single cell gel electrophoresis (Comet assay)

Healthy male Swiss-albino mice (8-12 weeks old and weighing 2530 g) 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 (14 h: 10 h dark/light cycle, a temperature of
(22 2 C), and 5070% humidity). The animals were fed on with standard rodent
pellets (consisting of crude protein, and ber) 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.

The alkaline comet assay was performed according to the method of Singh et al.
[41] with minor modications. Animals were sacriced, liver, kidney, spleen and
femurs were removed and then single cell suspension was prepared as previously
described by Sasaki et al. [42]. Briey, the spleen, kidney and liver tissues were
minced in 0.075 M NaCl solution containing 0.024 M Na2 EDTA (pH 7.5). Followed
by the centrifugation, the cells were resuspended in phosphate buffered saline.
The bone marrow cells were washed in 0.075 M NaCl solution containing 0.024 M
Na2 EDTA (pH 7.5) and resuspended in phosphate buffered saline. Slides were prepared by mixing the cell suspension with 1% low melting point agarose; layered on
the slide base coated with 1% normal melting point agarose and placed in a chilled
lysing solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Trizma, 10% DMSO, and 1% Triton X-100, pH 10.0) at 4 C for 1 h. Then the slides were subjected to DNA unwinding
in chilled alkaline solution (300 mM NaOH and 1 mM Na2 EDTA, pH >13) for 20 min
and subsequently electrophoresis was performed at 0.7 V/cm and 300 mA at 4 C for
25 min in freshly prepared electrophoresis buffer (1 mM EDTA disodium salt and
300 mM NaOH). After electrophoresis the slides were neutralized with Tris buffer
(400 mM, pH 7.4). Slides were stained with 20 g/ml ethidium bromide (EtBr) and
stored at 4 C in a humidied slide box until scoring. Slides were scored at a nal
magnication of 400 using an image analysis system (Komet 5.5, Kinetic Imaging,
Andor technology, Nottingham, UK) attached to a uorescence 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 from 150 random
cells (per animal) were analyzed as per the guidelines [43].

2.1. Chemicals

2.3. Dose selection

According to World Health Organization (WHO) guidelines for drinking water,
a uoride level of 1.5 mg/L is the desirable upper limit and for the US Environmental
Protection Agency [36] it is 4.0 mg/L. India reduced the upper limit of uoride in
drinking water from 1.5 to 1.0 mg/L with a rider that less is better [1]. In India, water
sample analysis reveals uoride contaminant level range from 0.2 to 20 mg/L [37].
Based on the above information following concentrations viz. 4, 12 and 20 mg/L of
NaF were selected for the current study.
2.4. Dose preparation
Daily fresh solution of sodium uoride was prepared in double distilled water.
The animals had free access to drinking water, containing different concentrations
of NaF (4, 12 and 20 mg/L) prepared in distilled water. Control set of animals were
provided with distilled water. To avoid additional uoride contamination from the
water used, quantication of uoride ion was performed using uoride ion selective electrodes (WTW, Germany). The effective concentration of uoride ion thus
obtained was <0.087, 1.6, 6.1 and 8.6 mg/L for control, 4, 12 and 20 mg/L NaF concentrations respectively. Mitomycin C (MMC), dissolved in physiological saline was
used as positive control.
2.5. Treatment schedule
The animals were divided into 5 experimental groups, each of six male mice as
follows:
Group 1 negative controls received distilled water in drinking water.
Groups 2, 3, 4 Animals were exposed to NaF in drinking water at the concentrations 4, 12 and 20 mg/L, respectively.
Group 5 Positive controls received a single ip. injection of MMC (2 mg/kg body
weight) 24 h before sacrice.
All animals except those of Group 5 were sacriced on the thirtieth day of the
experiment. Animals were sacriced by cervical dislocation and the femur and other
organs were removed. Micronucleus test and comet assay in multiple organs were

2.9. Chromosome Aberration test (CA test)

The chromosome aberration test was performed on mouse bone marrow cells
with slight modications [44]. Animals were sacriced by cervical dislocation on
the thirtieth day of the experiment. One and half hours prior to sacrice, animals
were injected intraperitoneally with colchicine (4.0 mg/kg bw). The femoral bone
marrow cells were aspirated in RPMI 1640 media and centrifuged at 800 g for
10 min, the pellet was incubated in 8.0 ml of KCl (0.075 M) at 37 C for 30 min, followed by centrifugation at 800 g for 5 min. The cells were xed in Carnoys xatives
(glacial acetic acid/methanol, 1:3, v/v), washed thrice with Carnoys xative at intervals of 10 min. The pellet was resuspended in xative and dropped on chilled slides
from the height of 22.5 ft, and air dried. The slides were stained with freshly prepared Giemsa stain (8%, v/v in Sorenson buffer) for 10 min followed by washing
with distilled water. A total number of 50 metaphases 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 at 1000 magnication, with selection being based on uniform staining
quality, lack of overlapping chromosomes and chromosome number (40 2 chromosomes). 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 of

M. J et al. / Mutation Research 751 (2013) 5965

aberrant 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].

61

1% Triton X-100 and then centrifuged at 10,000 g for 15 min. The reaction was
initiated by addition of 10 mM H2 O2 solution to the supernatant and read at 240 nm.
Catalase specic activity was calculated by using molecular extinction co-efcient
of 39.4 M1 cm1 and represented as IU/mg protein.
2.14. Protein estimation

2.10. Assay of lipid peroxidation

2.10.1. Thiobarbituric acid reactive species (TBARS) level
The lipid peroxidation level in the liver homogenates was measured using the
levels of malonadialdehyde (MDA), which is the end product of lipid peroxidation and reacts with TBA as a TBARS to produce a pink colored complex that has
peak absorbance at 535 nm according to Buege and Aust [46]. Briey, the tissue
was homogenized with 9 volume of chilled physiological saline and centrifuged
at 8000 g for 5 min. 1 ml of supernatant was mixed with 2 ml TCA-TBA-HCl and
heated for 15 min in a boiling water bath. The mixture was cooled on ice and
centrifuged at 10,000 g for 15 min. The absorbance of the resultant supernatant
was read at 535 nm spectrophotometrically. The amount of TBARS was calculated
using a molar extinction coefcient of 1.56 105 M1 cm1 expressed as nanomoles
MDA/mg protein.

Protein quantication was carried out spectrophotometrically by the method of

Lowry et al. [50] with bovine serum albumin as standard.
2.15. Statistical analysis
The data were analyzed using the Statistical ProgrammeSigmaStat 3.0 (SPSS
Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) test, followed by
Dunnetts multiple comparison tests at P 0.05, was done for single cell gel
electrophoresis, biochemical estimations and organo-somatic index. For the chromosome aberration and micronucleus assay, students t-test was performed. The
level of signicance was established at P 0.05.

3. Results

2.11. Estimation of non-enzymatic antioxidants

3.1. Oragano-somatic index of liver

2.11.1. Reduced glutathione (GSH) level
Reduced 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 (Ellmans reagent) that gives 2-nitro-5-thiobenzoic acid
a yellow colored compound. In brief, 250 l of tissue homogenate was added to
1.76 ml of distilled water and 3.0 ml of 50% TCA solution to precipitate the protein,
mixed and centrifuged at 3000 g for 10 min. The resulting supernatant so obtained
after centrifugation was then taken for GSH estimation. To the supernatant, 2.0 ml
of 0.4 M tris buffer solution (pH 8.9) and 0.5 ml of DTNB was added and then the
intensity of yellow color was read at 412 nm. The GSH content in the samples were
calculated using a molar extinction coefcient of 14,150 M1 cm1 and expressed as
nanomoles/mg protein.
2.12. Determination of glutathione S-transferase (GST) activity
Glutathione S-transferase catalyzes the conjugation reaction with glutathione
in the rst 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 at 340 nm. The
specic activity of GST was estimated using extinction co-efcient 9.6 mM1 cm1
expressed as nanomoles of GSH-CDNB conjugate formed/min/mg protein.

Administration of NaF to the mice through drinking water for

30 days alters the organo-somatic index (Fig. 1) of liver of mice.
3.2. Micronucleus test
The effects of NaF on frequency of MN-PCEs and the PCE/NCE
ratio in mouse bone-marrow cells are presented in Table 1. A signicant 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 signicantly 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 polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio. A
statistically signicant decrease in the PCE/NCE ratio was observed
at concentrations 12 and 20 mg/L.
3.3. Single cell gel electrophoresis (Comet assay)

2.13. Catalase (CAT) assay

Catalase activity was determined according to the method of Aebi [49]. The liver
tissues were homogenized in phosphate buffer (pH 7.0) and centrifuged at 10,000 g
for 15 min. The pellet was vortexed vigorously with phosphate buffer containing

Fig. 1.

The alkaline comet assay was performed in multiple organs like

liver, kidney, spleen and bone marrow cells. The exposure to various concentrations of NaF caused an increase in DNA strand breaks

62

M. J et al. / Mutation Research 751 (2013) 5965

Table 1
Micronucleated polychromatic erythrocyte (MN-PCEs) in the bone marrow cells of
male mice exposed to different concentrations of sodium uoride.
Na F concentrations (mg/L)

% MN-PCE
(mean SEM)

0a
4
12
20
MMCb

0.09
0.14
0.29
0.24
2.44

were chromatid gaps and breaks, with a few chromosome breaks.

The frequency of aberrations observed in positive control group
was signicantly higher than the vehicle control as well as of NaF
exposed groups.

PCE/NCE
(mean SEM)

0.003
0.014*
0.018*
0.014*
0.135*

2.28
2.01
1.59
1.58
0.82

0.02
0.05
0.02*
0.02*
0.12*

3.5. Biochemical assays

leading to greater DNA migration out of the nucleus into the tail of
the comet (Table 2). The values did not yield a statistically signicant increase in DNA damage in spleen cells. Signicant increase in
percent Tail DNA was observed in bone marrow (4, 12 and 20 mg/L),
kidney (12 and 20 mg/L) and liver (20 mg/L) cells.

The results of lipid peroxidation, GSH, GST and catalase activity

in the liver of NaF exposed mice are presented in Table 4. Fluoride
treatment caused an increase in the levels of lipid peroxidation,
catalase activity (statistically signicant at 20 mg/L) and decrease
in the concentrations of GSH and GST activity signicantly at various concentrations (4, 12 and 20 mg/L) of NaF when compared with
the corresponding group of control animals. The observed increase
in MDA level with increasing concentrations of uoride 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 (12 and 20 mg/L) with
increase in uoride concentrations shows increased consumption
of glutathione for the scavenging of reactive oxygen species (ROS).

3.4. Effect on Chromosome aberration frequency

4. Discussion and conclusion

The chromosome aberrations observed in bone marrow

metaphase cells of mice are presented in Table 3. NaF treatment
caused increase in the frequency of chromosome aberrations at all
concentrations as compared to control. However, the values were
signicant at concentrations of 4 and 12 mg/L of NaF exposure.
The major types of structural aberrations observed in this study

The systemic genotoxic effect related to uoride 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 damage

MN-PCEs, micronucleated polychromatic erythrocytes (1000 cells/animal, N = 6);

PCEs, polychromatic erythrocytes; NCEs, normochromatic erythrocytes.
*
P 0.05 compared to control (Students t test).
a
Vehicle control.
b
Positive control Mitomycin C (2 mg/kg bw).

Table 2
DNA damage expressed as Percent Tail DNA (mean SEM) in spleen, liver, kidney and bone marrow cells of male mice exposed to different concentrations of sodium uoride.
Test substance

Concentration (mg/L)

DNA damage (Tail DNA %)

Liver

Kidney

Vehicle control

6.96 0.46

11.30 0.97

11.10 0.86

7.29 0.94

NaF

4
12
20

7.51 0.31
7.86 0.57
8.34 0.41

12.19 1.09
16.33 1.14
18.24 1.59*

14.22 0.80
18.01 0.77*
19.55 1.11*

11.22 0.99*
12.12 0.67*
13.81 0.79*

MMC

2 mg/kg bw

13.56 1.28*

33.89 1.94*

26.06 1.07*

25.59 2.34*

Aberrant cells (%) SEM

CA/cell SEM

Spleen

Bone marrow

N = 6; MMC Mitomycin C (2 mg/kg bw). 150 cells/animal (N = 6)/conc.

*
P 0.05 (one-way ANOVA).
Table 3
Chromosome aberrations in bone marrow cells of male mice exposed to different concentrations of sodium uoride.
Test substance

Total chromosome aberrationsa

Concentration (mg/L)

G

G

B

B

Vehicle control

10

3.60 2.23

0.040 0.03

NaF

4
12
20

3
7
3

30
22
19

2
1

10.00 1.55*
11.60 1.33*
7.60 0.40

0.100 0.02*
0.116 0.01*
0.084 0.01

Mitomycin C

2 mg/kg bw

31

13.60 1.26*

0.140 0.02*

a
*

10






50 metaphase plates/animal (5 animals/conc.); G Chromatid gap; G Chromosome gap; B Chromatid break; B Chromosome break; R Rearrangement.
P 0.05 compared to control (Students t test).

Table 4
Contents of LPO, GSH, GST and Catalase activity in liver cells of male mice exposed to different concentrations of sodium uoride.
NaF mg/L)

LPO (nmol MDA/mg protein)

0
4
12
20

0.056
0.076
0.072
0.102

0.01
0.01
0.01
0.01*

GSH (nmol/mg protein)

26.66
20.48
17.49
13.18

Values are expressed as mean SEM, n = 5 animals/conc.

*
P 0.05 (one-way ANOVA).

0.98
0.83*
0.77*
0.54*

GST (CDNB-GSH conjugate/min/mg protein)

99.44
72.75
61.82
88.69

5.48
9.04*
6.15*
7.01*

Catalase activity (IU/mg protein)

43.42
38.91
47.27
54.76

3.32
1.98
1.70
3.30*

M. J et al. / Mutation Research 751 (2013) 5965

in cells of bone marrow, liver, kidney and spleen by the single cell
gel electrophoresis (comet assay). Since oxidative stress is implicated in uoride 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 significant 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 on uoride genotoxicity reported significant increased chromosome aberrations in mice and cultured cells
[5153]. In recent years Chaurasia et al. [54] observed dose dependent increase in chromosomal aberrations in bone marrow cells of
Swiss albino mice. Similar trend was noted in in vitro experiments
in HL-60 cells where reduced cell viability, decreased DNA and
protein biosynthesis, and apoptosis was observed at high concentrations (100-250 mg/L); and no such effect at lower concentrations
(0-50 mg/L) was noted [55]. In vitro experiment in human blood
cultures exposed to NaF demonstrated signicant increase in DNA
breakage and the induction of chromosomal aberrations and MN
[56]. Tiwari et al. [57] also reported that low uoride concentrations 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, chromosomal aberrations were more pronounced in mice that received
relatively low dose of NaF (15 mg/L) in drinking water over a period
of 3 months [13]. In the present study, we observed signicant
increase in the percentage of aberrant metaphases and chromosomal aberrations in all the treated groups, the effect being more
pronounced in mice that received 12 mg/L of NaF. Interestingly,
a decrease in the severity of these parameters was noted at the
highest NaF concentration (20 mg/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]. Contradictory ndings on the genotoxic potential of NaF in mammalian cells using
in vitro and in vivo systems have also been reported [21,22,5968].
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 (100-400 mg/L). Several
in vivo studies, using sister chromatid exchange [16,65], micronucleus formation [17,18,20,63] and other endpoints [14,15,19]
have also reported negative ndings. It has been found that there
is no effect of uoride 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 diploid broblast cells treated with relatively low concentration of NaF (5-10 ppm) [70] and no mutagenic
effects were observed in human EUE cells at 10-150 ppm of NaF
[71]. The disparity in various reports could be due to the differences 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 on uorosis
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]. These ndings 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 marrow cells and the spleen cells were least affected. The reason for
such discrepancy is unknown at the moment and warrants further
analysis.

63

In addition, the present in vivo study addressed the relationship between uoride-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 nonprotein 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 oxidative 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 oxidative stress [1,5,26,28,29,31,34,68,7376]. 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 used uoride compounds, revealed signicant increase in chromosome aberrations,
MN, DNA fragmentation in bone marrow cells at concentrations of
4-20 mg/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 of uoride that could be one of the possible mechanisms for genotoxic activity. Therefore, oxidative stress
in uoride genotoxicity necessitates more elaborate studies involving other indicators of oxidative damage along with the cytogenetic
endpoints.
Conict of interests
The authors have declared that there is no conict of interest.
Acknowledgements
The authors would like to thank the Board of Research in
Nuclear ScienceDepartment of Atomic Energy, Trombay (Sanction
No. 2009/37/7/BRNS) for funding this research work. The authors
would also like to acknowledge the reviewers and the editor for
their critique, which has helped us improve the quality of the
manuscript.
References
[1] A.K. Susheela, M. Bhatnagar, Reversal of uoride induced cell injury through
elimination of uoride and consumption of diet rich in essential nutrients and
antioxidants, Mol. Cell. Biochem. 234/235 (2002) 335340.
[2] A.K. Susheela, K.T. Das, Chronic uoride toxicity: a scanning electron microscopic study of duodenal mucosa, Clin. Toxicol. 26 (1988) 467476.
[3] N.J. Chinoy, R. Joseph, E. Sequeira, M.V. Narayana, Effects of sodium uoride on
the muscle and liver of albino rats, Ind. J. Environ. Biol. 1 (1991) 129134.
[4] A. Machalinska, B. Wiszniewska, J. Tarasiuk, B. Machalinski, Morphological
effects of sodium uoride on hematopoietic organs in mice, Fluoride 35 (2002)
231238.
[5] X. Guo, G. Sun, Y. Sun, Oxidative stress from uoride-induced hepatotoxicity in
rats, Fluoride 36 (2003) 2529.
[6] National Research Council-US, Fluoride in Drinking Water: A Scientic Review
of EPAs Standards, National Academies Press, Washington, DC, 2006.
[7] P.K. Gadhia, S. Joseph, Sodium uoride induced chromosome aberrations and
sister chromatid exchange in cultured human lymphocytes, Fluoride 30 (1997)
153156.
[8] A. Machalinska, A.M. Machoy-Mokrzynska, W. Marlicz, I. Stecewicz, B.
Machalinski, NaF-induced apoptosis in human bone marrow and cord blood
CD34 positive cells, Fluoride 34 (2001) 258263.

64

M. J et al. / Mutation Research 751 (2013) 5965

[9] J. Ha, Q. Chu, A. Wang, T. Xia, K. Yang, Effects on DNA damage and apoptosis
and p53 protein expression induced by uoride in human embryo hepatocytes,
Wei Sheng Yan Jiu 33 (2004) 400402.
[10] K. Liu, L. Ma, H. Yao, Y. Zhang, L. Li, G. Wang, Fluoride-mediated apoptosis and
disordering of cell cycle distributions during in vitro organ culture of mouse
fetal long bones, Fluoride 40 (2007) 1923.
[11] C.D. Anuradha, S. Kanno, S. Hirano, Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in uoride-induced apoptosis in HL-60
cells, Free Radic. Biol. Med. 31 (2001) 367373.
[12] L.F. He, J.G. Chen, DNA damage, apoptosis and cell cycle changes induced by
uoride in rat oral mucosal cells and hepatocytes, World J. Gastroenterol. 12
(2006) 11441148.
[13] S. Podder, A. Chattopadhyay, S. Bhattacharya, M.R. Ray, Differential in vivo genotoxic effects of lower and higher concentrations of uoride in mouse bone
marrow cells, Fluoride 41 (2008) 301307.
[14] S.I. Voroshilin, E.G. Plotko, E.Z. Gatiyatullina, E.A. Gileva, Cytogenetic effect of
inorganic uorine compounds on human and animal cells in vivo and in vitro,
Sov. Genet. (Engl. Transl.) 9 (1975) 492496.
[15] G.R. Martin, K.S. Brown, D.W. Matheson, H. Lebowitz, L. Singer, R. Ophaug,
Lack of cytogenetic effects in mice or mutations in Salmonella receiving sodium
uoride, Mutat. Res. 66 (1979) 159167.
[16] D. Kram, E.L. Schneider, L. Singer, G.R. Martin, The effects of high and low uoride diets on the frequencies of sister chromatid exchanges, Mutat. Res. 57
(1978) 5155.
[17] E. Gocke, M.T. King, K. Eckhardt, D. Wild, Mutagenicity of cosmetics ingredients
licensed by the European communities, Mutat. Res. 90 (1981) 91109.
[18] R. Albanese, Sodium uoride and chromosome damage (in vitro human lymphocyte and in vivo micronucleus assays), Mutagenesis 2 (1987) 497499.
[19] J.A. Skare, K.R. Schrotel, G.A. Nixon, Lack of DNA-strand breaks in rat testicular
cells after in vivo treatment with sodium uoride, Mutat. Res. 170 (1986) 8592.
[20] M. Hayashi, M. Kishi, T. Sofuni, M. Ishidate Jr., Micronucleus tests in mice on
39 food additives and eight miscellaneous chemicals, Food Chem. Toxicol. 26
(1988) 487500.
[21] Y. Li, A.J. Dunipace, G.K. Stookey, Lack of genotoxic effects of uoride in the
mouse bone-marrow micronucleus test, J. Dent. Res. 66 (1987) 16871690.
[22] D.A. Ribeiro, P.L.A. De Lima, M.E.A. Marques, D.M.F. Salvadori, Lack of DNA damage induced by uoride on mouse lymphoma and human broblast cells by
single cell gel (Comet) assay, Braz. Dent. J. 17 (2006) 9194.
[23] D.A. Ribeiro, M.E.A. Marques, D.M.F. Salvadori, Lack of effect of prior treatment
with uoride on genotoxicity of two chemical agents in vitro, Caries. Res 41
(2007) 239243.
[24] E. Zeiger, M.D. Shelby, K.L. Witt, Genetic toxicity of uoride, Environ. Mol.
Mutagen. 21 (1993) 309318.
[25] Inkielewicz, M. Rogowska, J. Krechniak, Lipid peroxidation and antioxidant
enzyme activity in rats exposed to uoride and ethanol, Fluoride 39 (2006)
5359.
[26] V. Ailani, R.C. Gupta, S.K. Gupta, K. Gupta, Oxidative stress in cases of chronic
uoride intoxication, Ind. J. Clin. Biochem. 24 (2009) 426429.
[27] D. Chlubek, Fluoride and oxidative stress, Fluoride 36 (2003) 217228.
[28] Krechniak, I. Inkielewicz, Correlations between uoride concentrations and
free radical parameters in soft tissues of rats, Fluoride 38 (2005) 293296.
[29] S. Chouhan, S.J.S. Flora, Effects of uoride on the tissue oxidative stress and
apoptosis in rats: Biochemical assays supported by IR spectroscopy data, Toxicology 254 (2008) 6170.
[30] K.P. Reddy, G. Sailaja, C. Krishnaiah, Protective effects of selenium on the uoride induced alterations in certain enzymes in brain of mice, J. Environ. Biol.
30 (2009) 859864.
[31] H.A. Hassan, A.F. Abdel-Aziz, Evaluation of free radical-scavenging and antioxidant properties of black berry against uoride toxicity in rats, Food Chem.
Toxicol. 48 (2010) 19992004.
[32] T. Kumar, E.M. Mohan, N. Ramesh, K.S. Pillai, B.P.K. Murthy, Toxicity of combination of uoride and monocrotophos 36% SL to Wistar rats, J. Environ. Biol. 19
(1998) 305311.
[33] B.G. Reddy, A.L. Khandare, P.Y. Reddy, G.S. Rao, N. Balakrishna, I. Srivalli, Antioxidant defence system and lipid peroxidation in patients with skeletal uorosis
and uoride intoxicated rabbits, Toxicol. Sci. 72 (2003) 363368.
[34] A. Chattopadhyay, S. Podder, S. Agarwal, S. Bhattacharya, Fluoride-induced
histopathology and synthesis of stress protein in liver and kidney of mice, Arch.
Toxicol. 85 (2011) 327335.
[35] H.H. Pant, M.V. Rao, Evaluation of in vitro anti-genotoxic potential of melatonin
against arsenic and uoride in human blood cultures, Ecotoxicol. Environ. Saf.
73 (2010) 13331337.
[36] EPA, Basic Information About Fluoride in Drinking Water, U.S. Enviromental Protection Agency, May 2012, website: http://water.epa.gov/drink/
contaminants/basic information/uoride.cfm
[37] S.K. Sharma, High uoride in groundwater cripples life in parts of India, in:
Diffuse Pollution Conference, Dublin, 2003, pp. 5152.
[38] Chirumari, P.K. Reddy, Dose-dependent effects of uoride on neurochemical
milieu in the hippocampus and neocortex of rat brain, Fluoride 40 (2007)
101110.
[39] W. Schmid, The micronucleus tests for cytogenetic analysis, in: A. Hollaender
(Ed.), Chemical Mutagens: Principles and Methods for their Detection, vol. 4,
Plenum Press, New York, 1976, pp. 3153.
[40] A. Mukherjee, A.K. Giri, A. Sharma, G. Talukder, Relative efcacy of short-term
tests in detecting genotoxic effects of cadmium chloride in mice in vivo, Mutat.
Res. 206 (1988) 285295.

[41] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantication of low levels of DNA damage in individual cells, Exp. Cell Res. 175
(1988) 184191.
[42] Y.F. Sasaki, S. Kawaguchi, A. Kamaya, M. Ohshita, K. Kabasawa, K. Iwamaa, K.
Taniguchi, S. Tsuda, The comet assay with 8 mouse organs: results with 39
currently used food additives, Mutat. Res. 519 (2002) 103119.
[43] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y.
Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelines
for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35
(2000) 206221.
[44] R.J. Preston, B.J. Dean, S. Galloway, H. Holden, A.F. McFee, M. Shelby, Mammalian
in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow
cells, Mutat. Res. 189 (1987) 157165.
[45] R.R. Tice, J.L. Ivett, Cytogenetic analysis of bone marrow damage, in: R.D. Irons
(Ed.), Toxicology of the Blood and Bone Marrow, Raven Press, New York, 1985,
pp. 119140.
[46] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52
(1978) 302311.
[47] J. Sedlak, R.N. Lindsay, Estimation of total protein bound and non-protein
sulphydryl groups in tissue with Ellman reagent, Anal. Biochem. 25 (1968)
192205.
[48] W.H. Habig, M.J. Pabst, W.D. Jakoby, Glutathione S-transferases, the rst enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 71307139.
[49] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121126.
[50] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with
the folin phenol reagent, J. Biol. Chem. 193 (1951) 265276.
[51] A.H. Mohamed, M.E. Chandler, Cytologial effects of sodium uoride on mice,
Fluoride 15 (1982) 110118.
[52] T. Tsutsui, K. Ide, H. Maizumi, Induction of unscheduled DNA synthesis in cultured human oral keratinocytes by sodium uoride, Mutat. Res. 140 (1984)
4348.
[53] T. Tsutsui, N. Suzuki, M. Ohmori, Sodium uoride induced morphological
and neoplastic transformation, chromosome aberrations, sister chromatid
exchanges, and unscheduled DNA synthesis in cultured Syrian hamster embryo
cells, Cancer. Res. 44 (1984) 938941.
[54] O.P. Chaurasia, C. Kumari, Anikita, Sangita, Genotoxic effects of ground water
salts rich in uoride, Cytologia (Tokyo) 72 (2007) 141144.
[55] J.S. Song, H.Y. Lee, E. Lee, H.J. Hwang, J.H. Kim, Cytotoxicity and apoptosis
induction of sodium uoride in human promyelocytic leukemia (HL-60) cells,
Environ. Toxicol. Pharmacol. 11 (2002) 8591.
[56] N.H. Kleinsasser, H. Weissacher, B.C. Wallner, E.R. Kastenbauer, U.A. Harreus,
Cytotoxicity and genotoxicity of uorides in human mucosa and lymphocytes,
Laryngohinootologie 80 (2001) 187190.
[57] H. Tiwari, M.V. Rao, Curcumin supplementation protects from genotoxic effects
of arsenic and uoride, Food Chem. Toxicol. 48 (2010) 12341238.
[58] G. Krishna, M. Hayashi, In vivo rodent micronucleus assay: protocol, conduct
and data interpretation, Mutat. Res. 455 (2000) 155166.
[59] E. Zeiger, D.K. Gulati, P. Kaur, A.H. Mohamed, J. Revazova, T.G. Deaton,
Cytogenetic studies of sodium uoride in mice, Mutagenesis 9 (1994)
467471.
[60] D.D. Jhala, N.J. Chinoy, M.V. Rao, Mitigating effects of some antidotes on uoride
and arsenic induced free radical toxicity in mice ovary, Food Chem. Toxicol. 46
(2008) 11381142.
[61] A.J. Dunipace, W. Zhang, T.W. Noblitt, Y. Li, G.K. Stookey, Genotoxic evaluation
of chronic uoride exposure: micronucleus and sperm morphology studies, J.
Dent. Res. 68 (1989) 15251528.
[62] Y. Li, A.J. Dunipace, G.K. Stookey, Absence of mutagenic and antimutagenic
activities of uoride in Ames Salmonella assays, Mutat. Res. 190 (1987)
229236.
[63] Y. Li, N.A. Heerema, A.J. Dunipace, G.K. Stookey, Genotoxic effects of uoride
evaluated by sister chromatid exchange, Mutat. Res. 192 (1987) 191201.
[64] Y. Li, A.J. Dunipace, G.K. Stookey, Genotoxic effects of uoride: a controversial
issue, Mutat. Res. 195 (1988) 127136.
[65] Y. Li, W. Zhang, T.W. Noblitt, A.J. Dunipace, G.K. Stookey, Genotoxic evaluation
of chronic uoride exposition: sister chromatid exchange study, Mutat. Res.
227 (1989) 159165.
[66] Y. Li, C.K. Liang, B.P. Katz, E.J. Brizendine, G.K. Stookey, Long-term exposure to
uoride in drinking water and sister chromatid exchange frequency in human
blood lymphocytes, J. Dent. Res. 74 (1995) 14681474.
[67] M.A.R. Buzalaf, D.M.F. Salvadori, M.E.A. Marques, E.E. Caroselli, A.L. Leite, E.A.
Camargo, D.A. Ribeiro, Absence of DNA damage in multiple organs after oral
exposure to uoride in Wistar rats, Bull. Environ. Contam. Toxicol. 77 (2006)
700706.
[68] D.A. Ribeiro, D.M.F. Salvadori, G.F. de Assis, M.E.A. Marques, Does uoride cause
DNA damage? An in vitro evaluation using rats oral mucosa cells, Braz. J. Oral.
Sci. 2 (2003) 268271.
[69] Y. Li, W. Zhang, T.W. Noblitt, A.J. Dunipace, G.K. Stookey, Genotoxic evaluation
of chronic uoride exposure: sister chromatid exchange study, Mutat. Res. 227
(1989) 159165.
[70] T. Tsutsui, Y. Tanaka, Y. Matsudo, A. Uehama, T. Smeya, F. Hamaguchi, H.
Yamamoto, M. Takahashi, No increases in chromosome aberrations in human
diploid broblasts following exposure to low concentration of sodium uoride
for long times, Mutat. Res. 335 (1995) 1520.

A. Gabelova, K. Ruppova., Cytotoxicity and genotoxicity testing

[71] D. Slamenova,
of sodium uoride on Chinese hamster V79 cells and human EUE cells, Mutat.
Res. 279 (1992) 109115.

M. J et al. / Mutation Research 751 (2013) 5965

[72] Zhang, A. Wang, T. Xia, P. He, Effects of uoride on DNA damage, S-phase cellcycle arrest and the expression of NF-B in primary cultured rat hippocampal
neurons, Toxicol. Lett. 179 (2008) 15.
[73] A. Bast, G.R., Haenen, C.J., Doelman, Oxidants, Antioxidants: State of Art, Am. J.
Med. 91 (C.J.) 125135.
[74] Z.Z. Guan, K.Q. Xiao, X.Y. Zeng, Y.G. Long, Y.H. Cheng, S.F. Jiang, Y.N. Wang,
Changed cellular membrane lipid composition and lipid peroxidation of kidney
in rats with chronic uorosis, Arch. Toxicol. 74 (2000) 602608.

65

[75] A.G. Wang, T. Xia, Q.L. Chu, M. Zhang, F. Liu, X.M. Chen, K.D. Yang, Effects of
uoride on lipid peroxidation, DNA damage and apoptosis in human embryo
hepatocytes, Biomed. Environ. Sci. 17 (2004) 217222.
[76] O. Barbier, L. Arreola-Mendoza, L.M.D. Razo, Molecular mechanism of uoride
toxicity, Chem. Biol. Interact. 188 (2010) 319333.
[77] J. Chen, X. Chen, K. Yang, T. Xia, H. Xie, Studies on DNA damage and apoptosis in rat brain induced by uoride, Chinese J. Prev. Med. 36 (2002)
222224.