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Toxicology 254 (2008) 61–67

Contents lists available at ScienceDirect

Toxicology

  j 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 / t o x i c o l

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

 Article history:

Received 11 July 2008

Received in revised form 4 September 2008

Accepted 5 September 2008

Available online 18 September 2008

Keywords:

Fluoride

Dose-dependent study

Oxidative stress

Biochemical variables

Infra red spectroscopic assay

Fluoride concentration

Rats

a b s t r a c t

The mechanism underlying the toxicity of fluoride still remains unknown. To investigate the effects of differentdosesof fluoride on bloodand tissue oxidative stressand apoptosis, we exposedmale ratsto three

doses of fluoride (10, 50 and 100 ppm in drinking water) for a period of 10 weeks. The results suggested

thatexposureto 10ppm fluoride significantlyincreased the level of reactiveoxygenspecies(ROS) in blood

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

and 100 ppm fluoride exposure did not produce a more pronounced toxicity in the soft tissues. However,

we observed a significantly elevated concentration of ROS and depleted GSH level in blood. Exposure to

fluoride did not produce any signof apoptosis. To support our above mentioned biochemical observations

and to suggest possible mechanism of action of fluoride, IR spectra of brain tissues were recorded. The

results of these spectra indicated significant shift in the characteristic peak of –OH group in animals

exposed to 10 ppm fluoride however at higherdoses, theshift was minimal. It canthus be concluded that

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 chemicalsand are mainly used for aluminumproduction,

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 to1 ppm is safe for

humanbody butabove thislimit is considereddeleterious 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;Zhaoet 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) andinhibitsnumerousenzymes (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

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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

for inorganic phosphate using the procedure of  Fiske and Subbarow (1925). The

absorbance was recorded at 600 nm using spectrophotometer.

  2.3.8. DNA damage studies in brain

Agarose gel electrophoresis was carried out for the analysis of DNA fragmen-

tation (Yokozawa and Dong, 2001). DNA was isolated as described by Sambrook

et al. (2001). Then DNA was dissolved in TE buffer (Tris EDTA buffer, pH 8.0). The

DNAsamples were electrophoresed on 0.7%agarose gel using TBE buffer (Tris-boric

acid-EDTA buffer, pH 8.3) at 40 V for 4 h. DNA was visualized by ethidium bromidestaining. Then thebands were visualized andanalyzed in geldocumentationsystem

(Bio-Rad Lab, USA).

  2.3.9. Spectral analysis

IR spectra of brain tissues were recorded using PerkinElmer FTR Spectrometer,

model BXII. The spectra were recorded under the range of 4000–400cm−1 with

resolution of 4 cm−1.

  2.3.10. Elemental analysis

Fluoride concentrationin blood, liver,kidney and brain wasmeasured after wet

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

analyzer (Orion, EA, USA). By constructing the cell using the fluoride ion-selective

electrode and calomel reference electrode in a solution of fluoride at pH 5.35

adjusted with total ionic strength adjusting buffer (TISAB), the cell potential was

determined. By measuring thecell potential fora series of fluoridestandards like0.1,

1.0, 10.0, 100 and 1000 mg/l and the standard calibration graph was constructed byplottingthecell potentialversuslog(F), it ispossible tofind outthe unknownfluoride

concentration from measured cell potential. The detection limit of the instrument

was 0.025–500 mg/l.

  2.4. Statistical analysis

The results are expressed as the mean ±SEM of number of observations. Com-

parison of means were carried out using one way analysis of variance (ANOVA)

followed by Turkey’s post hoc test to compare means between the different treat-

ment groups. Differences were considered significant at p < 0.05 unless otherwise

stated in the text.

3. Results

 3.1. Effects of fluoride exposure on hematological variables

We evaluated effect of different doses of fluoride some

selected blood biochemical variables indicative of alterations

in heme biosynthesis pathway and oxidative stress in rats

(Table 1). It was observed that the blood GSH level decreased

significantly in all fluoride-exposed animals in dose-dependent

manner as compared to the normal animals. However 10 and

50 ppm fluoride exposure did not show dose-dependent decline

in GSH concentration. Decline in GSH level is related to decreased

antioxidant status and overproduction of ROS. Thus in our exper-

iment we evaluated concentration of ROS, which was increased

 Table 1

Effect of different doses of fluoride on hematological variables in rats.

Normal F (10) F (50) F (100)

ALAD 9.84 ± 0.77* 3.82 ± 0.78† 8.25 ± 0.39* 9.33 ± 0.39*

ROS 0.52 ± 0.04* 0.52 ± 0.03* 0.90 ± 0.05† 1.18 ± 0.63‡

GSH 5.65 ± 0.22* 4.77 ± 0.31† 4.62 ± 0.18† 3.79 ± 0.10‡

WBC 37.2 ± 1.9* 29.4 ± 3.11† 32.7 ± 0.39* 36.1 ± 2.78*

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*

HCT 37.6 ± 2.36* 45.3 ± 2.55* 42.1 ± 1.25* 34.7 ± 3.53*

PLT 1030 ± 138.1* 801.6 ± 102.3* 946.8 ± 61.8* 865 ± 181.2*

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

blood cellsas×106/l; HGB- hemoglobin asg/dl; Hct- hematocrit as%; PLT- platelet

as ×103/l.

Values are mean±SE; n = 6.* ,†,‡Differences betweenvalueswith matchingsymbol notations within eachrow

are not statistically significant at 5% level of probability.

significantly in 50 and 100 ppm fluoride-exposed animals in dose-

dependent manner.

In contrary to these observations we did not obtained dose-

dependent decline in blood ALAD activity (Table 1). ALAD activity

decreased significantly at the lowest dose of fluoride (10 ppm) of 

fluoride while it came to nearly normal value at the highest dose

(100 ppm). Similar results we obtained in WBC counts, it decreased

significantly at the 10 ppmexposure level and came back to normal

at 100 ppm exposure level. Platelet count also showed same type

of observation marginal decrease was observed at 10 ppm fluoride

exposurewhereas it increased withincrease in the concentration of 

fluoride exposure. While other clinical variables remained almost

unaltered.

 3.2. Effects on tissue oxidative stress

Since blood parameters indicate condition of oxidative stress

we evaluated parameters related to oxidative stress in liver, kidney

and brain of normal and exposed animals. We did not observe a

significant elevated concentration of ROS in all three tissues except

a marginal increase in liver at 50 ppm exposure level. However it

was an interesting finding of the experiment that in kidney there

was exceptionally high concentration of ROS at thedose of 100 ppm

fluoride (Fig. 1). To find out the condition of oxidative stress we

evaluated the ratio of GSH: GSSG, which is a marker of oxidative

stress. Decline in this ratio indicate condition of oxidative stress.

Maximum alteration in GSH/GSSG ratio was observed at 10 ppm

exposure. In liver it was found to be 28% at 10ppm, 34% at 50 ppm

and41% at100ppm fluoride exposure. While in kidneyit was 58%at

10 ppm, 44% at 50ppmand nearly94% at 100ppm level. It suggests

that at 100 ppm exposure level there was minimum decline in this

ratio, suggestive least oxidative stress condition. In brain this ratio

did not altered significantly (Fig. 2).

 3.3. Effects on cell death

In order to find out fluoride-induced cell death we find out cas-pase 3 activity in cells and observed no significant increase at any

of the dose of fluoride rather we observed decreased caspase activ-

ity at 50 and 100 ppm fluoride exposure (Fig. 3A). To confirm that

apoptosis does not occur at these doses of fluoride we performed

DNA damage studies and observed lack of DNA damage at all three

doses of fluoride (Fig.4). We did not obtained significant alterations

in ATPase activity followed by fluoride exposure (Fig. 3B).

  3.4. Effects on fluoride concentration

In toxicity studies it is expected that given amount of dose will

accumulate inside the tissue however, in case of fluoride toxicity

the mechanism might be different. In blood we observed a signifi-

cant dose-dependent increase in the blood fluoride concentrationexcept a marginal decline at the10 ppmexposure level.Howeverin

other three tissues we observed a dose-dependent decline in tissue

fluoride concentration as compared to normal. In liver it reduced

about 70% at 10 ppm, 66% at 50 ppm exposure and 61% at 100 ppm

exposure level. In kidney the decrease was 47% at 10 and 50 ppm

while 36%at 100 ppmexposure level. In brainthe decrease was 72%

at 10 ppm, 68% at 50ppmand 54% at100ppmexposure level (Fig. 5

).

  3.5. Evaluation of IR spectra

The results of present experiment indicate some interesting

findings, which are quite surprising. In order to find out the possi-

ble mechanism for such type of observation, i.e., absence of DNA

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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 numberof bands.

We obtained significant change in the peak at 3410 cm−1 in normal

tissue (Fig. 6A), which showeda significant shift towards lowerfre-

quency side 3304cm−1 at 10 ppmexposure 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 ppmfluoride exposure.Other peaks did not show

much shifting towards higher or lower frequency side.

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.

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 mediatorsin many disorders,variousstudieshave 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

Fig.3. Alterationin caspase3 activity(A) andATPase 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.

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Fig.4. Effects of differentdosesof fluoride exposure on DNA damagein brain of rats.

Lane 1: normal; Lane 2: fluoride 10 ppm; Lane 3: fluoride 50ppm; Lane 4: fluoride100ppm.

Fig. 5. Effects of different doses of fluoride exposure on fluoride concentration in

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

matching symbol notations within each column is not statistically significant at 5%

level of probability.

parameters in soft tissues has been investigated by many authors

(Kaushik et al., 2001; Guan et al., 2000). In our experiment we

observed significant increase in blood ROS level at 50 and 100 ppm

fluoride exposure, suggesting that free radicals were involved in

oxidative stress. Wang et al.(1997) reported that production of OH•

and •O2− radicals is dependent on fluoride concentration; super-

oxide radicals prevail at high fluoride concentrations, whereas at

low ones there is dominance of hydroxyl radicals generated in the

Haber–Weiss reaction with the participationof Fe2+ aswellasH2O2

 Table 2

Characteristics IR peaks (cm−1) of normal and exposed brain tissue.

Normal F (10) F (50) F (100)

539 538 541 541

1083 1076 1072 1076

1236 1238 1237 1237

1402 1399 1401 1401

1463 1459 1462 1460

1546 1543 1545 1544

1655 1656 1655 1655

2924 2926 2924 2925

3410 3304 3312 3385

3841 3753 3752 3753

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

tothe more rapideliminationof fluoride at 100 ppmexposure level.

At this concentration fluoride can easily form H-bond with water

molecules.There wasa goodcorrelation 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 GSHas 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.

RatiobetweenGSH/GSSGis a marker of oxidative stress. Chlubek

et al. (1999), reported that at fluoride at relatively low concen-

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, andfoundthat 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. Theyalso found that there are not any changes in the activities of cata-

lase, superoxide dismutase, glutathione peroxidase, or glutathione

S-transferase in the blood due to fluoride intoxication (of rabbits)

or fluorosis in human beings.

Overproduction of ROS leads to cell death or DNA damage how-

ever in the present experiment we did not obtained any sign of cell

death or DNA damage. Ribeiro et al. (2006) reported that sodium

fluoride is nota genotoxic agent andabsenceof DNA damageon flu-

oride exposure. It is assumed that these results may be due to the

fact that fluoride is not capable of forming adducts on the bases of 

DNA or those that intercalate into DNA secondary structure. These

results are consistent with those of previous studies (IARC, 1982;

Slamenova et al., 1992; Tong et al., 1988).

Impaired heme biosynthesis pathway is attributed to thedecreasedactivity of ALAD which was observed in animals exposed

to fluoride. The results suggest maximum depletion at lowest dose

while increase in the fluoride concentration leads to less pro-

nounced depletion in ALAD activity. Such observation is needed

to be further studied to find out a possible mechanism.

It is evident from the above discussion that toxic effect of flu-

oride exposure does not follow a similar trend in all reported

literature. The mechanism is still unresolved. In this context this

was thefirstexperiment in which wetriedto findout some possible

mode of action of fluoride at cellular level. Our results of IR spec-

tra give preliminary information regarding the site of action and

possible interference with biomolecules. Fluoride is also known to

cross the cell membranes and to enter soft tissues. Impairment of 

soft-tissue function has been demonstrated in fluoride-intoxicated

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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 andReddy, 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 3200–3600cm−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 resonanceweakens the–OH bond andas a result

absorption takes place at lower frequency (Fujimoto and Jinno,

1992). In present experiment characteristic peak of –OH group was

obtainedat 3410cm−1 innormal braintissue. Maximum shiftin this

peak was obtained at 10ppm 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 r 2

= 0.8367.

level the peak was obtained at 3312cm−1 and at 100 ppm expo-

sureit shifted towards higherfrequency 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

agreementwith this rule of IR spectroscopy. These observationsled

us to hypothesize that it may be due to ionic mobility of fluoride

ion. At lowconcentration fluoride ion easily crosses cell membrane

andreachesto thetargetsite while at higherconcentration reduced

ionic mobility is responsible for the less availability of fluoride ion.

These findings are also supportedby concentration of fluoride in

blood andsoft tissues. Athigherdosesfluoride didnot interactwith

biomolecules and hence we did not obtained a dose-dependent

increase in fluoride concentration in soft tissues. He and Chen

(2006) also reportedincreasein fluoride concentration in bloodandurine 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 areless pronounced. The study alsoprovides important

data about the possible mode of action of fluoride. However the

detailed mechanism, of course, needs further investigation.

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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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