Toxicology 254 (2008) 61–67

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Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Effects of fluoride on the tissue oxidative stress and apoptosis in rats:

Biochemical assays supported by IR spectroscopy data
Swapnila Chouhan, S.J.S. Flora ∗
Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India

a r t i c l e i n f o a b s t r a c t

Article history: The mechanism underlying the toxicity of fluoride still remains unknown. To investigate the effects of
Received 11 July 2008 different doses of fluoride on blood and tissue oxidative stress and apoptosis, we exposed male rats to three
Received in revised form 4 September 2008 doses of fluoride (10, 50 and 100 ppm in drinking water) for a period of 10 weeks. The results suggested
Accepted 5 September 2008
that exposure to 10 ppm fluoride significantly increased the level of reactive oxygen species (ROS) in blood
Available online 18 September 2008
accompanied by a decrease in glutathione (GSH) level. No evidences of oxidative stress in soft tissues
were seen. Fluoride (10 ppm) also decreased GSH/GSSG ratio significantly. Contrary to expectation, 50
Keywords:
and 100 ppm fluoride exposure did not produce a more pronounced toxicity in the soft tissues. However,
Fluoride
Dose-dependent study
we observed a significantly elevated concentration of ROS and depleted GSH level in blood. Exposure to
Oxidative stress fluoride did not produce any sign of apoptosis. To support our above mentioned biochemical observations
Biochemical variables and to suggest possible mechanism of action of fluoride, IR spectra of brain tissues were recorded. The
Infra red spectroscopic assay results of these spectra indicated significant shift in the characteristic peak of –OH group in animals
Fluoride concentration exposed to 10 ppm fluoride however at higher doses, the shift was minimal. It can thus be concluded that
Rats fluoride-induced toxicity is mediated through oxidative stress particularly at a comparatively lower level
of exposure however at the higher doses the mode of action still unclear and needs further investigation.
© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Safe drinking water is the primary need of every human being.
Although, groundwater is considered safe but it is also contam-
inated with soluble organic and inorganic materials. Common
inorganic contaminants are fluoride (F), nitrates and nitrites of
metals and various heavy metals like arsenic, lead, cadmium, mer-
cury, etc. Fluorides of several metals and nonmetals are important
industrial chemicals and are mainly used for aluminum production,
drinking water fluoridation and the manufacture of fluoridated
dental preparations (Lu et al., 2000). Fluoride exists in drinking
water in an ionic form and hence, rapidly passes through the
intestinal mucosa and interferes with metabolic pathways of living
system. Fluoride is toxic when consumed in excess but of bene-
fit when consumed within permissible limit (Guan et al., 2000).
The fluoride concentration in drinking water up to 1 ppm is safe for
human body but above this limit is considered deleterious to health.
Excess fluoride intake causes fluorosis, which is an endemic pub-
lic health problem in 22 nations around the globe including India.
Epidemiological investigations reveal that the IQ of children living
in high fluoride areas of Tianjin, Guizhou, and other provinces of
∗ Corresponding author. Tel.: +91 751 2344301; fax: +91 751 2341148.
E-mail addresses: sjsflora@hotmail.com, drde@sancharnet.net.in (S.J.S. Flora).
China is 8–12% lower than in children living in low fluoride areas
(Lu et al., 2000; Zhao et al., 1996). Excess fluoride exposure leads to
a condition known as fluorosis which is of three types, viz., skele-
tal fluorosis (affecting bones), dental fluorosis (affecting teeth), and
non-skeletal fluorosis affecting soft tissues such as muscles, liver,
kidney, lungs, blood, cells, reproductive cells, and gastrointestinal
mucosa, nervous system, etc.
Fluoride is a cumulative poison. On an average, only 50% of the
fluoride ingested by our body each day is excreted through the
kidneys while remaining accumulates in our bones, pineal gland,
and other tissues. Fluoride is biologically active even at its lower
concentrations. It interferes with hydrogen bonding (Emsley et al.,
1981) and inhibits numerous enzymes (Waldbott et al., 1978). Fluo-
ride is an anion with a rather small molecular weight, and it shows
its effect on the organism by combining with calcium ions (Ca2+ )
which may leads to hypocalcaemia. Fluoride under certain condi-
tion can virtually affect every phase of human metabolism. It can
readily penetrate cell membranes by simple diffusion and cause
adverse effects on cell metabolism and function.
Fluoride is a chemically active ionized element. It can affect
oxygen metabolism and induce the production of O2− free radi-
cals (Inkielewicz and Krechniak, 2004). Previous studies revealed
that fluoride induces excessive production of oxygen free radicals
and caused a decrease in biological activities of some antiox-
idant enzymes like super oxide dismutase (SOD), catalase and

0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2008.09.008
62 S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67
glutathione peroxidase (GPX ) (Shanthakumari et al., 2004; Wang et
al., 2003). However, the manner in which the whole body effects are
produced is still unclear. The adverse toxic effects of fluoride are due
to (a) enzyme inhibition, (b) collagen breakdown (c) gastric damage
and (d) disruption of immune system (Ahmed et al., 2000). More-
over various injurious effects are associated with chronic exposure
of 1.5 mg/l or more in drinking water, including neurological and
reproductive disorders (Ortiz et al., 2003). Many studies on labora-
tory animals over a range of fluoride concentration (0.1–250 mg/l
in drinking water) indicate that adverse reproductive and devel-
opmental effects occur at high fluoride concentration (Doull et al.,
2006). Fluoride is effective activator of the signal transduction path-
way regulated G proteins and can probably induce a conformational
change of G protein that regulate second messenger CAMP and Ca2+
ultimately leading to cell apoptosis (Susa, 1991). Fluoride is also
known to be an inhibitor/activator of numerous enzymes. Allergy,
hypersensitivity, gastrointestinal and skin irritation are known side
effects of fluoride ingestion.
Although, a relationship in humans and animal fluorosis par-
ticularly between free radical generation, lipid per oxidation and
antioxidant defence system has been investigated extensively how-
ever, the results are mostly contradictory. Soni et al. (1984) reported
that sodium fluoride at a low concentration of 5 and 20 mg/kg
body mass of rats caused an increase in lipid per oxidation in tis-
sues while, the higher dose caused an inhibition in the liver, lungs
and testes. Xiong et al. (2007) also studied dose effect relationship
between fluoride concentration in drinking water and damage to
soft tissue. They reported that 2.0 mg/l fluoride can cause dam-
age to liver but not the kidney. In a recent report by Chlubek et
al., incubation of sodium fluoride increased concentration (2, 5,
50 and 500 ␮M) with mitochondrial fraction from human placenta
induced MDA formation, but higher concentration of sodium fluo-
ride was less potent in raising levels of MDA (Chlubek et al., 1999).
Even though fluoride toxicity is increasingly being considered to be
important, very little information is available on the mechanism of
action of fluoride.
The purpose of this study was to assess the effects of different
concentration of fluoride on (i) oxidative stress and apoptosis (ii)
blood and soft tissue fluoride concentration and (iii) correlation of
fluoride exposure with the condition of oxidative stress. In addition
to it we also aimed to investigate possible mechanism of fluoride-
induced toxicity.
2. Materials and methods
2.1. Chemicals and reagents
Sodium Fluoride (NaF Molecular weight 41.99) was procured from Sigma Chem-
ical (USA). All other analytical laboratory chemicals and reagents were purchased
from Merck (Germany), Sigma (USA) or BDH chemicals (Mumbai, India). All the
samples were stored and refrigerated in desiccators to avoid oxidation and thermal
decomposition
2.2. Animals and treatments
Twenty-four, adult male albino rats of approximate weight 100–120 g were
obtained from animal house facility of Defence Research and Development Estab-
lishment (DRDE), Gwalior. Prior to dosing, rats were acclimatized for 7 days to
light from 06:00 to 18:00 h alternating with 12 h darkness. The animals were
housed in stainless steel cages in an air-conditioned room with temperature main-
tained at 25 ± 2 ◦ C. Rats were allowed standard chow diet (Amrut feeds, Pranav
Agro, New Delhi, India; metal contents of diet, in mg/kg dry weight, Cu 10.0, Zn
45.0, Mn 55.0, Co 5.0, Fe 75.0) throughout the experiment and water was given
ad libitum.
Animals were randomized into four groups of six animals per group and were
treated as below for 10 weeks:
Group I: Unexposed animals (received normal water)
Group II: Fluoride as sodium fluoride 10 ppm in drinking water
Group III: Fluoride as sodium fluoride 50 ppm in drinking water
Group IV: Fluoride as sodium fluoride 100 ppm in drinking water
After 10 weeks, exposure was stopped and animals were sacrificed under light
ether anesthesia, 48 h, after last dosing. Blood was collected in heparinized vials.
Liver, kidneys and brains were removed, washed with normal saline and all the
extraneous materials were removed before studying various parameters.
2.3. Biochemical assays
2.3.1. Blood ı-aminolevulinic acid dehydratase (ALAD)
The activity of blood ␦-aminolevulinic acid dehydratase (ALAD) was assayed
according to the procedure of Berlin and Schaller (1974). Total volume of 0.2 ml of
heparinized blood was mixed with 1.3 ml of distilled water and incubated for 10 min
at 37 ◦ C for complete hemolysis. After adding 1 ml of standard ␦-aminolevulinic
acid (ALA), the tubes were incubated for 60 min at 37 ◦ C. Enzyme activity was
stopped after 1 h by adding 1 ml of 10% trichloroacetic acid (TCA). After centrifu-
gation (1500 × g) of reaction mixture, equal volume of Ehrlich reagent was added to
the supernatant and the absorbance was recorded at 555 nm after 5 min.
2.3.2. Blood glutathione (GSH)
Blood GSH concentration was determined following the procedure of Ellman
(1959) and modified by Jollow et al. (1974). 0.2 ml of whole blood was added to
1.8 ml of distilled water and incubated for 10 min at 37 ◦ C for complete hemolysis.
After adding 3 ml of 4% sulphosalycylic acid, tubes were centrifuged at 2500 × g
for 15 min. 0.2 ml of supernatant was mixed with 0.4 ml of 10 mM solution of 5,5 -
dithiobis-(2-nitro benzoic acid) (DTNB) in the presence of 1 ml phosphate buffer
(0.1 M pH 7.4). The absorbance was recorded at 412 nm.
2.3.3. Clinical hematological variables
White blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), hematocrit
(Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin
concentration (MCHC) and platelet (PLT) counts were measured on Sysmex Hema-
tology Analyzer (model K4500).
2.3.4. Tissue reduced (GSH) and oxidized glutathione (GSSG) level
Liver, kidney and brain were assayed for GSH and GSSG contents according to
the method described by Hissin and Hilf (1973). Briefly, 0.25 g of tissue sample was
homogenized on ice with 3.75 ml of 0.1 M phosphate +0.005 M EDTA buffer (pH 8.0)
and 1 ml of 25% HPO3 which was used as a protein precipitant. The homogenate
(4.7 ml) was centrifuged at 1,00,000 × g for 30 min at 4 ◦ C. For the GSH assay, 0.5 ml
supernatant and 4.5 ml phosphate buffer (pH 8.0) were mixed. The final assay mix-
ture (2.0 ml) contained 100 ␮l supernatant, 1.8 ml phosphate - EDTA buffer and
100 ␮l O-phthaldehyde (OPT; 1000 ␮l/ml in absolute methanol, prepared fresh in
dark). After mixing, fluorescence was determined at 350 nm excitation wavelength
and 420 nm emission wavelength using a spectrofluorometer (Model RF 5000 Shi-
madzu, Japan).
For the GSSG assay, 0.5 ml supernatant was incubated at room temperature with
200 ␮l of 0.04 mol/l N-ethylmaleimide solution for 30 min. To this mixture, 4.3 ml
of 0.1 mol/l NaOH was added. A 100-␮l sample of this mixture was taken for the
measurement of GSSG using the procedure described above for GSH assay, except
that 0.1 mol/l NaOH was used as the diluent instead of phosphate buffer.
2.3.5. Reactive oxygen species (ROS) level in blood and tissues
Amount of ROS in blood, liver, kidney and brain was measured using 2 ,7 -
dichlorofluorescin diacetate (DCFDA) that gets converted into highly fluorescent
DCF by cellular peroxides (including hydrogen peroxide). The assay was performed
as described by Socci et al. (1999). Briefly, 5% RBC hemolysate was prepared and
diluted to 1.5% with ice-cold 40 mM Tris–HCl buffer (pH 7.4) and further diluted to
0.25% with the same buffer and placed on ice. The samples were divided into two
equal fractions. In one fraction 40 ␮l of 1.25 mM DCFDA in methanol was added for
ROS estimation. Another fraction in which 40 ␮l of methanol was added, served as
a control for tissue/hemolysate auto fluorescence. All samples were incubated for
15 min in a water bath at 37 ◦ C. Fluorescence was determined at 488 nm excitation
and 525 nm emission wavelength using a fluorescence plate reader (Tecan Spectra
Fluor Plus).
2.3.6. Determination of caspase activity
Caspase activity was determined in brain using a caspase inhibitor VAD-FMK
conjugated with FITC, which irreversibly binds with activated caspase in apop-
totic cells. The assay was performed using caspase detection kit of Calbiochem
cat. No. Q1A90. The fluorescence was determined at 485 nm excitation and 535 nm
emissions using a fluorescence reader. The results were expressed as arbitrary flu-
orescence intensity units (FIU at 540 nm).
2.3.7. Determination of ATPase activity
ATPase activity was determined in brain by the procedure of Seth and Tangari
(1966). In brief 10% tissue homogenate in 0.5 M Tris buffer (pH 8.0) was prepared
followed by addition of 0.01 M ATP. The reaction mixture was incubated for 15 min
 S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67 63

at 37 ◦ C and reaction was stopped by adding 10% TCA. Supernatant was estimated significantly in 50 and 100 ppm fluoride-exposed animals in dose-
for inorganic phosphate using the procedure of Fiske and Subbarow (1925). The dependent manner.
absorbance was recorded at 600 nm using spectrophotometer.
In contrary to these observations we did not obtained dose-
2.3.8. DNA damage studies in brain
dependent decline in blood ALAD activity (Table 1). ALAD activity
Agarose gel electrophoresis was carried out for the analysis of DNA fragmen- decreased significantly at the lowest dose of fluoride (10 ppm) of
tation (Yokozawa and Dong, 2001). DNA was isolated as described by Sambrook fluoride while it came to nearly normal value at the highest dose
et al. (2001). Then DNA was dissolved in TE buffer (Tris EDTA buffer, pH 8.0). The (100 ppm). Similar results we obtained in WBC counts, it decreased
DNA samples were electrophoresed on 0.7% agarose gel using TBE buffer (Tris-boric
significantly at the 10 ppm exposure level and came back to normal
acid-EDTA buffer, pH 8.3) at 40 V for 4 h. DNA was visualized by ethidium bromide
staining. Then the bands were visualized and analyzed in gel documentation system at 100 ppm exposure level. Platelet count also showed same type
(Bio-Rad Lab, USA). of observation marginal decrease was observed at 10 ppm fluoride
exposure whereas it increased with increase in the concentration of
2.3.9. Spectral analysis fluoride exposure. While other clinical variables remained almost
IR spectra of brain tissues were recorded using PerkinElmer FTR Spectrometer,
model BXII. The spectra were recorded under the range of 4000–400 cm−1 with
unaltered.
resolution of 4 cm−1 .
3.2. Effects on tissue oxidative stress
2.3.10. Elemental analysis
Fluoride concentration in blood, liver, kidney and brain was measured after wet
Since blood parameters indicate condition of oxidative stress
acid digestion using a Microwave Digestion System (CEM, USA, model MDS-2100).
Fluoride was measured in the digested blood and tissue samples using Orion ion we evaluated parameters related to oxidative stress in liver, kidney
analyzer (Orion, EA, USA). By constructing the cell using the fluoride ion-selective and brain of normal and exposed animals. We did not observe a
electrode and calomel reference electrode in a solution of fluoride at pH 5.35 significant elevated concentration of ROS in all three tissues except
adjusted with total ionic strength adjusting buffer (TISAB), the cell potential was a marginal increase in liver at 50 ppm exposure level. However it
determined. By measuring the cell potential for a series of fluoride standards like 0.1,
was an interesting finding of the experiment that in kidney there
1.0, 10.0, 100 and 1000 mg/l and the standard calibration graph was constructed by
plotting the cell potential versus log(F), it is possible to find out the unknown fluoride was exceptionally high concentration of ROS at the dose of 100 ppm
concentration from measured cell potential. The detection limit of the instrument fluoride (Fig. 1). To find out the condition of oxidative stress we
was 0.025–500 mg/l. evaluated the ratio of GSH: GSSG, which is a marker of oxidative
stress. Decline in this ratio indicate condition of oxidative stress.
2.4. Statistical analysis
Maximum alteration in GSH/GSSG ratio was observed at 10 ppm
The results are expressed as the mean ± SEM of number of observations. Com- exposure. In liver it was found to be 28% at 10 ppm, 34% at 50 ppm
parison of means were carried out using one way analysis of variance (ANOVA) and 41% at 100 ppm fluoride exposure. While in kidney it was 58% at
followed by Turkey’s post hoc test to compare means between the different treat- 10 ppm, 44% at 50 ppm and nearly 94% at 100 ppm level. It suggests
ment groups. Differences were considered significant at p < 0.05 unless otherwise
that at 100 ppm exposure level there was minimum decline in this
stated in the text.
ratio, suggestive least oxidative stress condition. In brain this ratio
did not altered significantly (Fig. 2).
3. Results
3.3. Effects on cell death
3.1. Effects of fluoride exposure on hematological variables
In order to find out fluoride-induced cell death we find out cas-
We evaluated effect of different doses of fluoride some
pase 3 activity in cells and observed no significant increase at any
selected blood biochemical variables indicative of alterations
of the dose of fluoride rather we observed decreased caspase activ-
in heme biosynthesis pathway and oxidative stress in rats
ity at 50 and 100 ppm fluoride exposure (Fig. 3A). To confirm that
(Table 1). It was observed that the blood GSH level decreased
apoptosis does not occur at these doses of fluoride we performed
significantly in all fluoride-exposed animals in dose-dependent
DNA damage studies and observed lack of DNA damage at all three
manner as compared to the normal animals. However 10 and
doses of fluoride (Fig. 4). We did not obtained significant alterations
50 ppm fluoride exposure did not show dose-dependent decline
in ATPase activity followed by fluoride exposure (Fig. 3B).
in GSH concentration. Decline in GSH level is related to decreased
antioxidant status and overproduction of ROS. Thus in our exper-
3.4. Effects on fluoride concentration
iment we evaluated concentration of ROS, which was increased
In toxicity studies it is expected that given amount of dose will
Table 1
accumulate inside the tissue however, in case of fluoride toxicity
Effect of different doses of fluoride on hematological variables in rats.
the mechanism might be different. In blood we observed a signifi-
Normal F (10) F (50) F (100) cant dose-dependent increase in the blood fluoride concentration
ALAD 9.84 ± 0.77 *
3.82 ± 0.78†
8.25 ± 0.39*
9.33 ± 0.39* except a marginal decline at the 10 ppm exposure level. However in
ROS 0.52 ± 0.04* 0.52 ± 0.03* 0.90 ± 0.05† 1.18 ± 0.63‡ other three tissues we observed a dose-dependent decline in tissue
GSH 5.65 ± 0.22* 4.77 ± 0.31† 4.62 ± 0.18† 3.79 ± 0.10‡ fluoride concentration as compared to normal. In liver it reduced
WBC 37.2 ± 1.9* 29.4 ± 3.11† 32.7 ± 0.39* 36.1 ± 2.78*
about 70% at 10 ppm, 66% at 50 ppm exposure and 61% at 100 ppm
RBC 7.28 ± 0.48* 8.35 ± 0.37* 8.01 ± 0.23* 6.45 ± 0.53*
HGB 12.6 ± 0.74* 15.04 ± 1.66* 13.33 ± 0.51* 11.35 ± 0.96* exposure level. In kidney the decrease was 47% at 10 and 50 ppm
HCT 37.6 ± 2.36* 45.3 ± 2.55* 42.1 ± 1.25* 34.7 ± 3.53* while 36% at 100 ppm exposure level. In brain the decrease was 72%
PLT 1030 ± 138.1* 801.6 ± 102.3* 946.8 ± 61.8* 865 ± 181.2* at 10 ppm, 68% at 50 ppm and 54% at 100 ppm exposure level (Fig. 5
Abbreviation used and units—ALAD - ␦-aminolevulinic acid dehydratase as ).
nmol/min/ml erythrocytes; GSH - glutathione as mg/ml; ROS - reactive oxygen
species as ␩moles/min/ml of RBC; WBC - white blood cells as ×103 /␮l; RBC - red 3.5. Evaluation of IR spectra
blood cells as ×106 /␮l; HGB - hemoglobin as g/dl; Hct - hematocrit as%; PLT - platelet
as ×103 /␮l.
Values are mean ± SE; n = 6. The results of present experiment indicate some interesting
* ,†,‡
Differences between values with matching symbol notations within each row findings, which are quite surprising. In order to find out the possi-
are not statistically significant at 5% level of probability. ble mechanism for such type of observation, i.e., absence of DNA
64 S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67

Fig. 1. Effects of different doses of fluoride exposure on ROS concentration in liver, kidney and brain of rats. Abbreviation used and units—ROS - reactive oxygen species as
␩moles/min/mg of protein. Values are mean ± SE; n = 6. *,† Differences between values with matching symbol notations within each column is not statistically significant at
5% level of probability.

damage and less pronounced toxicity at higher dose of fluoride
we evaluated IR spectra of brain tissue as brain is supposed to
be the target organ for fluoride toxicity and fluoride easily crosses
blood–brain barrier.
Table 2 indicates various characteristics peaks of IR spectra. The
IR region 4000–400 cm−1 is of great importance in studying an
organic compound since IR spectra contains large number of bands.
We obtained significant change in the peak at 3410 cm−1 in normal
tissue (Fig. 6A), which showed a significant shift towards lower fre-
quency side 3304 cm−1 at 10 ppm exposure level (Fig. 6B) and again
come towards normal side 3312 at 50 ppm (Fig. 6C) and 3385 at
100 ppm exposure level (Fig. 6D). The peak obtained at 1546 cm−1
is characteristic peak for lipids, which shifted towards lower fre-
quency side at 10 ppm fluoride exposure. Other peaks did not show
much shifting towards higher or lower frequency side.
the ability to interfere with the functions of the brain and other tis-
sues by direct and indirect means. In spite of many years research
little is known about the toxicity of fluoride mechanism and the
results produced conflicting findings (Chlubek, 2003). Thus the
present study was planned to investigate possible mechanism of
fluoride-induced oxidative stress at different doses and to further
impelled more research in this field.
Since reactive oxygen species (ROS) are implicated as important
pathologic mediators in many disorders, various studies have inves-
tigated whether oxidative stress is involved in the pathogenesis of
chronic fluorosis. The results of those studies are conflicting and
contradictory to one another. The impact of fluoride on free radical

4. Discussion

On the basis of information largely derived from histological,

chemical, and molecular studies, it is apparent that fluorides have

Fig. 2. Effects of different doses of fluoride exposure on GSH/GSSG ratio in liver,
kidney and brain of rats. Abbreviation used—GSH - reduced glutathione as mg/g
tissue; GSSG - oxidized glutathione as mg/g tissue; Values are ratio of GSH and
GSSG; n = 6. *,†,‡ Differences between values with matching symbol notations within
each column is not statistically significant at 5% level of probability.
Fig. 3. Alteration in caspase 3 activity (A) and ATPase activity (B) in brain after differ-
ent doses of fluoride exposure in mice. Values of caspase are expressed as arbitrary
FIU (540 nm) emission wavelength and ATPase activity is expressed as arbitrary
absorbance at 600 nm. Values are mean ± SE; n = 6. *,†,‡ Differences between values
with matching symbol notations within each column is not statistically significant
at 5% level of probability.
 S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67 65

and • O2 − . There is much evidence that free radicals are impor-

tant mediator of fluoride-induced toxicity (Sharma and Chinoy,
1998; Rzeuski et al., 1998), which is in agreement with the present
study. However in the soft tissues (liver, kidney and brain) we did
not obtained significantly elevated concentration of ROS which in
accordance with the previous findings (Reddy et al., 2003; Chlubek
et al., 2003). However exceptionally high concentration of kidney
ROS at 100 ppm exposure was obtained which might be attributed
to the more rapid elimination of fluoride at 100 ppm exposure level.
At this concentration fluoride can easily form H-bond with water
molecules. There was a good correlation between fluoride exposure
and ROS concentration (Fig. 7).
Elevated concentration of ROS results in impaired cellular
antioxidant defence system. GSH participate in the reaction that
destroys hydrogen peroxide, or organic peroxide, free radicals and
certain foreign compounds. Our group has previously reported that
fluoride exposure restricts antioxidant activity of GSH and inhibits
various enzymes, which require GSH as a cofactor (Mittal and Flora,
2006, 2007). We too observed significant decline in blood GSH
concentration on fluoride exposure. Chinoy and Patel (2000) also
reported decreased GSH level on fluoride exposure.
Fig. 4. Effects of different doses of fluoride exposure on DNA damage in brain of rats. Ratio between GSH/GSSG is a marker of oxidative stress. Chlubek
Lane 1: normal; Lane 2: fluoride 10 ppm; Lane 3: fluoride 50 ppm; Lane 4: fluoride et al. (1999), reported that at fluoride at relatively low concen-
100 ppm.
tration creates a condition of oxidative stress while at higher
concentration it act as an inhibitor of free radical production.
Our results are in accordance with this finding. We too observed
that at lowest dose of fluoride the decline in GSH/GSSG ratio was
maximum while increase in fluoride concentration leads to less
pronounced depletion of this ratio. The possible mechanism for
such type of observations still remains unresolved. Reddy et al.
(2003) evaluated the antioxidant defense system (both enzymatic
and non-enzymatic) and lipid peroxidation both in human beings
from an endemic fluorosis area (5 ppm fluoride in drinking water)
and in rabbits receiving water containing 150 ppm of fluoride for 6
months, and found that there is no significant difference in lipid per
oxidation, glutathione and vitamin C in blood of fluorosis patients
and fluoride-intoxicated rabbits as compared to the controls. They
Fig. 5. Effects of different doses of fluoride exposure on fluoride concentration in
also found that there are not any changes in the activities of cata-
blood, liver, kidney and brain of rats. Units: fluoride blood: mg/ml; fluoride tissue:
mg/g of wet tissue. Values are mean ± SE; n = 6. *,†,‡ Differences between values with lase, superoxide dismutase, glutathione peroxidase, or glutathione
matching symbol notations within each column is not statistically significant at 5% S-transferase in the blood due to fluoride intoxication (of rabbits)
level of probability. or fluorosis in human beings.
Overproduction of ROS leads to cell death or DNA damage how-
parameters in soft tissues has been investigated by many authors ever in the present experiment we did not obtained any sign of cell
(Kaushik et al., 2001; Guan et al., 2000). In our experiment we death or DNA damage. Ribeiro et al. (2006) reported that sodium
observed significant increase in blood ROS level at 50 and 100 ppm fluoride is not a genotoxic agent and absence of DNA damage on flu-
fluoride exposure, suggesting that free radicals were involved in oride exposure. It is assumed that these results may be due to the
oxidative stress. Wang et al. (1997) reported that production of OH• fact that fluoride is not capable of forming adducts on the bases of
and • O2 − radicals is dependent on fluoride concentration; super- DNA or those that intercalate into DNA secondary structure. These
oxide radicals prevail at high fluoride concentrations, whereas at results are consistent with those of previous studies (IARC, 1982;
low ones there is dominance of hydroxyl radicals generated in the Slamenova et al., 1992; Tong et al., 1988).
Haber–Weiss reaction with the participation of Fe2+ as well as H2 O2 Impaired heme biosynthesis pathway is attributed to the
decreased activity of ALAD which was observed in animals exposed
to fluoride. The results suggest maximum depletion at lowest dose
Table 2 while increase in the fluoride concentration leads to less pro-
Characteristics IR peaks (cm−1 ) of normal and exposed brain tissue.
nounced depletion in ALAD activity. Such observation is needed
Normal F (10) F (50) F (100) to be further studied to find out a possible mechanism.
539 538 541 541 It is evident from the above discussion that toxic effect of flu-
1083 1076 1072 1076 oride exposure does not follow a similar trend in all reported
1236 1238 1237 1237 literature. The mechanism is still unresolved. In this context this
1402 1399 1401 1401 was the first experiment in which we tried to find out some possible
1463 1459 1462 1460
1546 1543 1545 1544
mode of action of fluoride at cellular level. Our results of IR spec-
1655 1656 1655 1655 tra give preliminary information regarding the site of action and
2924 2926 2924 2925 possible interference with biomolecules. Fluoride is also known to
3410 3304 3312 3385 cross the cell membranes and to enter soft tissues. Impairment of
3841 3753 3752 3753
soft-tissue function has been demonstrated in fluoride-intoxicated
66 S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67
Fig. 6. IR spectra of brain tissue of animals normal (A), fluoride10 ppm (B), fluoride 50 ppm (C) and fluoride 100 ppm (D). The spectra was taken using IR spectrophotometer
under the range of 4000–400 cm−1 .
animals (Mullenix et al., 1995; Vani and Reddy, 2000; Rzeuski et al.,
1998). Thus we analyzed IR spectra of lyophilized brain tissue and
obtained various peaks of organic biomolecules. Strong peak in the
range of 3200–3600 cm−1 is characteristic for alcoholic/phenolic
–OH group (Haris and Chapman, 1992) which is highly prominent
in the present IR spectra of normal and exposed brain tissues. H-
bonding as well as resonance weakens the –OH bond and as a result
absorption takes place at lower frequency (Fujimoto and Jinno,
1992). In present experiment characteristic peak of –OH group was
obtained at 3410 cm−1 in normal brain tissue. Maximum shift in this
peak was obtained at 10 ppm exposure (3304 cm−1 ) shows high-
est H-bonding (–OH–F) which might be due to high concentration
of fluoride at in vivo sites. On the other hand at 50 ppm exposure
Fig. 7. Correlation between fluoride exposure level and ROS concentration in blood
of fluoride-exposed animals. The correlation was found to be r2 = 0.8367.
level the peak was obtained at 3312 cm−1 and at 100 ppm expo-
sure it shifted towards higher frequency side (3385 cm−1 ) and came
near to normal (3410 cm−1 ). H-bonding tends to broaden the peaks
and shift it towards lower wavelength side. Our results are in great
agreement with this rule of IR spectroscopy. These observations led
us to hypothesize that it may be due to ionic mobility of fluoride
ion. At low concentration fluoride ion easily crosses cell membrane
and reaches to the target site while at higher concentration reduced
ionic mobility is responsible for the less availability of fluoride ion.
These findings are also supported by concentration of fluoride in
blood and soft tissues. At higher doses fluoride did not interact with
biomolecules and hence we did not obtained a dose-dependent
increase in fluoride concentration in soft tissues. He and Chen
(2006) also reported increase in fluoride concentration in blood and
urine of fluoride-exposed animals. However, in blood significant
increase in fluoride concentration was observed in dose-dependent
manner.
5. Conclusion
Present study led us to conclude that fluoride exerts its toxic
effects possibly ascribed to the enhanced oxidative stress however
short term fluoride exposure did not generate signs of apoptosis.
One of interesting finding of the study is that at low concentration
it exerts more deleterious effects while at higher concentration the
toxic effects are less pronounced. The study also provides important
data about the possible mode of action of fluoride. However the
detailed mechanism, of course, needs further investigation.
 S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67
Conflict of interest
The authors declare that there is no conflicts of interest.
Acknowledgement
Authors thank Dr. R. Vijayaraghavan, Director of the establish-
ment for his support and encouragement.
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