Upload
rogelio-gonzalez
View
217
Download
0
Embed Size (px)
Citation preview
8/8/2019 toxcology 2008
http://slidepdf.com/reader/full/toxcology-2008 1/7
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: [email protected] , [email protected] (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
8/8/2019 toxcology 2008
http://slidepdf.com/reader/full/toxcology-2008 2/7
8/8/2019 toxcology 2008
http://slidepdf.com/reader/full/toxcology-2008 3/7
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
8/8/2019 toxcology 2008
http://slidepdf.com/reader/full/toxcology-2008 4/7
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.
8/8/2019 toxcology 2008
http://slidepdf.com/reader/full/toxcology-2008 5/7
S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67 65
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
8/8/2019 toxcology 2008
http://slidepdf.com/reader/full/toxcology-2008 6/7
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 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.
8/8/2019 toxcology 2008
http://slidepdf.com/reader/full/toxcology-2008 7/7
S. Chouhan, S.J.S. Flora / Toxicology 254 (2008) 61–67 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.
References
Ahmed, S.,Hiyasat, A.,Elbetiecha,A.M.,Darmani, H.,2000. Reproductive toxic effectsof ingestion of sodium fluoride in female rats. Fluoride 33, 279–284.
Berlin, A., Schaller, K.H., 1974. European standardized method for the determinationof delta aminolevulinic dehydratase in blood. Z. Klin. Chem. Klin. Biochem. 12,389–390.
Chinoy, N.J., Patel,T.N.,2000. Theinfluence of fluorideand/oraluminium on freerad-ical toxicityin thebrain of female mice and beneficial effects of some antidotes.Fluoride 33, S8.
Chlubek, D., 20 03. Fluoride and oxidative stress. Fluoride 36, 217–228.Chlubek, D., Grucka-Mamczar, E., Birkner, E., Polaniak, R., Stawiarska-Pieta, B.,
Duliban, H., 2003. Activity of pancreatic antioxidative enzymes and mal-ondialdehyde concentrations in rats with hyperglycemia caused by fluorideintoxication. J. Trace Elem. Med. Biol. 17, 57–60.
Chlubek, D., Zawierta, J., Kazmierczyk, A., Kramek, J., Olszewska, M., Stachowska, E.,
1999.Effect of differentfluorideion concentrationson malondialdehyde (MDA)formation in themitochondrialfraction of human placental cells. Bromat.Chem.Toksykol. 32, 119–122.
Doull, J., Boekelheide, K., Farishian, B.G., Isaacson, R.L., Klotz, J.B., Kumar, J.V., Lime-back, H., Poole, C., Puzas, J.E., Reed, N.M.R., Thiessen, K.M., Webster, T.F., 2006.Fluoridein DrinkingWater: A ScientificReviewof EPA’sStandards. TheNationalAcademies Press, Washington, DC.
Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. 82, 70–77.Emsley,J., Jones, D.J., Miller,J.M.,Overill, R.E., Waddilove,R.A.,1981. Anunexpectedly
stronghydrogenbond: abinitiocalculationsand spectroscopicstudies of amide-fluoride systems. J. Am. Chem. Soc. 103, 24–28.
Fiske, C.H., Subbarow, Y., 1925. The colorimeteric determination of phosphates. J.Biol. Chem. 66, 375–379.
Fujimoto, C., Jinno, K., 1992. Chromatography/FT-IR spectrometry approaches toanalysis. Anal. Chem. 64, 476A.
Guan, Z.Z., Xiao, K.Q., Zeng, X.Y., Cheng, Y.H., Jiang, S.F., Wang, Y.N., 2000. Changedcellular membrane lipid composition and lipid peroxidation of kidney in ratswith chronic fluorosis. Arch. Toxicol. 74, 602–608.
Haris, P.I., Chapman, D., 1992.Does Fourier transform infrared spectroscopyprovide
useful information on protein structures? Trends Biochem. Sci. 17, 328.He, L.F., Chen, J.G., 2006. DNA damage, apoptosis and cell cycle changes induced by
fluoride in rat oral mucosal cells and hepatocytes. World J. Gastroenterol. 21,1144–1148.
Hissin, P.J., Hilf, R., 1973. A fluorometric method for the determination of oxidizedand reduced glutathione in tissue. Anal. Biochem. 74, 214–226.
IARC (International Agency for Research on Cancer) Monographs, 1982. Inorganicfluorides used in drinking-water and dental preparations. IARC Monogr. Eval.Carcinog. Risk Chem. Hum. 27, 237–303.
Inkielewicz, I., Krechniak, J., 2004. Fluoride effects on glutathione peroxidase andlipid peroxidation in rats. Fluoride 37, 7–12.
Jollow, D.J., Mitchell, J.R., Zamppaglione, Z., Gillette, J.R., 1974. Bromobenzeneinduced liver necrosis. Protective role of glutathione and evidence for3,4-bromobenzene oxide as the hepatotoxic metabolites. Pharmacology 11,151.
Kaushik,T.,Shyam,R., Vats, P.,Suri, S.,Kumria, M.M.L.,Sharma, P.C.,2001. Glutathionemetabolism in rats exposed to high-fluoride water and effect of spirulina treat-ment. Fluoride 34, 132–138.
Lu,Y., Sun, Z.R.,Wu, L.N.,Wang, X.,Lu,W.,Liu,S.S.,2000.Effect ofhighfluoridewateron intelligence in Children. Fluoride 33, 74–78.
Mittal, M., Flora, S.J.S., 2006. Effects of individual and combined exposure to sodiumarsenite and sodium fluoride on tissue oxidative stress, arsenic and fluoridelevels in male mice. Chem. Biol. Inter. 162, 128–139.
Mittal, M., Flora, S.J.S., 2007. Vitamin E protects oxidative stress and essential metalimbalance during concomitant exposure to arsenic and fluoride in male mice.
Drug Chem. Toxicol. 30, 263–281.Mullenix, P.J., Denbesten, P.K., Schunior, A., Kernan, W.J., 1995. Neurotoxicity of
sodium fluoride in rats. Neurotoxicol. Teratol. 17, 169–177.Ortiz, A.,Justo,P.,Sanz,A., Lorz,C.,Egido,J., 2003.Targetingapoptosisin acute tubular
injury. Biochem. Pharmacol. 66, 1589–1594.Reddy, G.B., Khandare, A.L., Reddy, P.Y., Rao, G.S., Balakrishna, N., Srivalli, I., 2003.
Antioxidant defense system and lipid peroxidation in patients with skeletalfluorosis and in fluoride-intoxicated rabbits. Biol. Trace Elem. Res. 92, 115–122.
Ribeiro,D.A., Lima,P.L.A.,Marques, M.E.A., Salvadori, D.M.F.,2006. Lackof DNA dam-age induced by fluoride on mouse lymphoma and human fibroblast cells bysingle cell gel (Comet) assay. Braz. Dent. J. 17, 91–94.
Rzeuski, R., Chlubek, D., Machoy, Z., 1998. Interactionsbetween fluoride and biolog-ical free radical reactions. Fluoride 31, 43–45.
Sambrook,J., Fritsch,E.F., Maniatis, T., 2001. Molecular Cloning:A LaboratoryManualthird edition, vol. 1. Cold Spring Harbor Laboratory Press, pp. 6.4–6.11.
Seth, P.K., Tangari, K.K., 1966. Biochemical effects of some newer salicylic acid con-geners. J. Pharm. Pharmacol. 18, 831–833.
Shanthakumari, D.,Srinivasalu,S., Subramanian, S.,2004. Effectsof fluorideintoxica-
tion on lipidperoxidation and antioxidant statusin experimental rats.Toxicology204, 219–228.Sharma, A., Chinoy, N.J., 1998. Role of free radicals in fluoride-induced toxicity in
liver and kidney of mice and its reversal. Fluoride 31, S26.Slamenova, D.,Gabelova, A., Ruppova, K., 1992. Cytotoxicityand genotoxicitytesting
ofsodium fluoride onChinesehamsterV79cellsandhumanEUEcells. Mut.Res.6, 109–115.
Socci, D.J., Bjugstad, K.B., Jones, H.C., Pattisapu, J.V., Arendash, G.W., 1999. Evidencethat oxidative stress is associated with the pathophysiology of inherited hydro-cephalus in the H-Tx rat model. Exp. Neurol. 155, 109–117.
Soni,M.G.,Kachole, M.S., Pawar, S.S.,1984. Alterationsin drugmetabolisingenzymesand lipid peroxidation in different rat tissues by fluoride. Toxicol. Lett. 21,167–172.
Susa, M., 1991. Heteromeric G proteins as fluoride targets in bone. Int. J. Mol. Med.3, 115–126.
Tong, C.C., McQueen,C.A., VedBrat,S., Williams, G.M., 1988.The lack of genotoxicityof sodium fluoride in a battery of cellular tests. Cell. Biol. Toxicol. 4, 173–186.
Vani, M.L., Reddy, K.P., 2000. Effects of fluoride accumulation on some enzymes of brain and astrocnemius muscle of mice. Fluoride 33, 17–26.
Waldbott, G.L., Burgstahler, A.W., McKinney, H.L., 1978. Fluoridation: The GreatDilemma. Coronado Press, Lawrence, Kansas, pp. 148–174.
Wang, A., Xia, T., Ran, P., Bai, Y., Yang, K., Chen, X., 2003. Effects of selenium andfluoride on apoptosis and lipid per-oxidation in human hepatocytes. Fluoride36, 45–46.
Wang, Y.Y., Zhao, B.L.,Li, X.J.,1997. Spintrapping technique studies on active oxygenradicalsfrom humanpolymorphonuclearleukocytes during fluoride-stimulatedrespiratory burst. Fluoride 30, 5–15.
Xiong, X.Z., Liu, J.L., He, W.H., Xia, T., He, P., Chen, X.M., Yang, K.D., Wang, A.G., 2007.Dose-effect relationship between drinking water fluoride levels and damage toliver and kidney functions in children. Environ. Res. 103, 112–116.
Yokozawa,T.,Dong,E., 2001. Role ofginsenoside-Rd in cisplatininducedrenal injury:special reference to DNA fragmentation. Nephron 89, 433–438.
Zhao, L.B., Liang, G.H., Zhang, D.N., Wu, X.R., 1996. Effect of a high fluoride watersupply on children’s intelligence. Fluoride 29, 190–192.