Arsenic and fluoride: Two major ground water pollutants

Indian Journal of Experimental Biology
Vol. 48, July 2010, pp. 666-678

Review Article

Arsenic and fluoride: Two major ground water pollutants

Swapnila Chouhan & S J S Flora*
Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior 474 002, India

Increasing human activities have modified the global cycle of heavy metals, non metals and metalloids. Both arsenic and
fluoride are ubiquitous in the environment. Thousands of people are suffering from the toxic effects of arsenicals and
fluorides in many countries all over the world. These two elements are recognized worldwide as the most serious inorganic
contaminants in drinking water. Many studies have reported as regards to simple fluorosis and arsenicosis, but the
knowledge of the joint action of these two elements is lacking and the results derived from previous studies were
inconclusive. Contradictory results were reported in experimental studies in which different joint actions such as
independent, synergistic and antagonistic effects were observed. This indicates that interaction mechanism of these two
elements is considerable complicated and requires extensive studies. When two different types of toxicants are
simultaneously going inside a human body they may function independently or can act as synergistic or antagonistic to one
another. Thus there is an urge to resolve the question that how arsenic and fluoride act in condition of concomitant exposure.
Although there have been reports in literature of individual toxicity of arsenic and fluoride however, there is very little
known about the effects following the combined exposure to these toxicants. This review focused on recent developments in
the research on the condition of individual exposure to arsenic and fluoride along with the recent updates of their combined
exposure to better understand the joint action of these two toxicants.

Keywords: Antioxidants, Arsenic, Chelators, Fluoride toxicity

Global pollution is increasing due to variations in for use as a suicidal and homicidal agent. Fluorine-
natural and anthropogenic activities leading to containing compounds are at the leading edge of
contamination of various terrestrial and aquatic many new developments in the life science industry.
ecosystems with metals, non metals, organic and Exposure to both arsenic and fluoride occurs
inorganic compounds. Uncontrolled discharge of environmentally from natural and anthropogenic
wastes, use of agricultural herbicides, pesticides, sources and occupationally in several industries,
insecticides and sewage disposal is some of the major including mining, pesticide, pharmaceutical,
contributors of contaminants. A large part of the beverages, food, glass and microelectronics. The
population in the industrialized world is exposed daily cause of combined occurrence of arsenic and fluoride
to a variety of chemicals. Industrial disposal contains in groundwater can be geogenic as well as
many toxic metals which are health hazardous. The anthropogenic. Ingestion of drinking water containing
pollution of groundwater by arsenic and fluoride high concentrations of F and As, primarily from
has been identified in many developing and natural contamination is the main source of human
some developed countries. The concurrent chronic environmental exposure worldwide1,2 . There are
poisonings with fluoride and arsenic is an emergent various fluoride compounds of arsenic which have
endemic disease in India and many other countries been recognized as a super acid system for organic
including China, Mexico, Argentina, and Bangladesh. synthesis e.g. HF/AsF5, and also as an electrolyte in
There are several places throughout the world where rechargeable gel batteries3,4. In addition to this
arsenic and fluoride both are present in ground water improper discharge of waste water, also increases the
at high concentration. Arsenic contamination of possibility of combined occurrence of arsenic and
groundwater in the West Bengal basin in India is fluoride in ground water. Although there have been
unfolding as one of the worst natural geo- reports in literature of individual toxicity of arsenic
environmental disaster to date. Arsenic is well known and fluoride, however, there is very little known about
the effects following the combined exposure to these
—————— toxicants. This review attempts to provide a
* Correspondent author–Telephone: 0751 2344301 comprehensive account of recent developments in the
Fax: 0751 2341148 research on the condition of individual exposure to
E-mail: [email protected]; [email protected]

CHOUHAN & FLORA: COMBINED EXPOSURE TO ARSENIC & FLUORIDE 667

arsenic and fluoride along with the recent updates of to cancer in kidney, liver, lungs, urinary bladder and,
their combined exposure to better understand the joint skin10. Trivalent arsenicals, including sodium arsenite
action of these two toxicants. and the more soluble arsenic trioxide, inhibit many
enzymes by reacting with biological ligands which
Arsenic posses available sulfur groups. Symptoms of acute
Arsenic is 33rd element in the periodic table. arsenic intoxication usually occur within 30 min of
exposure. Severe nausea and vomiting, colicky
It exists in the metallic state in nature in abdominal pain and profuse diarrhoea (bloody in
three allotropic forms and in several ionic forms. some cases), due to vasodilation with transudation of
Environmental arsenic exists mainly as sulphide fluid into the bowel lumen and sloughing leading to
complexes e.g. realgar (As2S2), orpiment (As2S3) and increased peristalsis11,12. The clinical features of acute
iron pyrites. Arsenic is the 20th most abundant arsenic poisoning include gastrointestinal discomfort
element in the earth crust known as a poison and (nausea, diarrhea, and abdominal pain), haemolysis,
human carcinogen. Arsenic is listed as the highest central and peripheral nervous system disorders
priority contaminant on the ATSDR/EPA priority list (headaches, weakness, delirium), and cardiovascular
of hazardous substances at Superfund sites5. In India, disorders (hypertension, shock). Major effects of
many areas from West Bengal have shown to be acute arsenic exposure in humans include haemolytic
affected, whereas Bihar is an emerging area with anaemia, hemoglobinuria, and jaundice that lead to
high arsenic contamination6. The possible methods renal failure. Chronic exposure to inorganic arsenic is
of exposure to arsenic are contact, ingestion and associated with irritation of the skin and mucous
inhalation. Ingestion of contaminated drinking membranes (dermatitis, conjunctivitis, pharyngitis,
water is the predominant source of significant and rhinitis). Bowen’s disease is a long-term
environmental exposure globally. complication of chronic arsenic. Chronic exposure
also results in fatigue and loss of energy,
Absorbed arsenic passes to bloodstream and inflammation of the stomach and intestines, kidney
distributed to organs/tissues after first passing through degeneration, cirrhosis of the liver, bone-marrow
the liver. Once absorbed, arsenic rapidly combines degeneration, and severe dermatitis. Death from acute
with the globin portion of haemoglobin and therefore arsenic poisoning is usually caused by irreversible
localises in the blood within 24 h. Arsenic circulatory insufficiency. Chronic toxicity is much
redistributes itself to the liver, kidney, spleen, lung more insidious and the diagnosis is often difficult to
and gastrointestinal tract, with lesser accumulation in establish.
muscle and nervous tissue7-9. After accumulation of
small dose of arsenic, it undergoes methylation Fig. 1—Major routes of exposure and various steps in the
mainly in the liver to monomethylarsonic acid and metabolism of arsenic
dimethylarsinic acid which are excreted along with
residual inorganic arsenic in the urine. Biologically,
the trivalent arsenite is significantly more active than
the pentavalent arsenate, including the ability to
induce gene amplification in mammalian cells.
Arsenate and arsenite have different fates in the body.
Arsenate enters the cell via the phosphate carrier
system. It can bind to polyphosphates like adenosine
di-phoshate, after that it is rapidly hydrolyzed.
Arsenite can bind to thiols such as glutathione (GSH)
and thiol containing proteins. Arsenic is cleared from
the body relatively rapidly and primarily through
kidney. Urine is the primary route of elimination for
both pentavalent and trivalent inorganic arsenicals.
Major routes of exposure and various steps in the
metabolism of arsenic is described in Fig. 1.

Toxicity— Exposure to inorganic arsenic causes
many adverse human health effects, including
cardiovascular, hepatic and renal diseases in addition

668 INDIAN J EXP BIOL, JULY 2010

Mechanism of toxicity—Trivalent inorganic ROS, reactive nitrogen species (RNS) also are
arsenite (As+3) acts as - thought to be directly involved in oxidative
damage to lipids, proteins and DNA in cells exposed
• React with molecules containing sulfhydryl to arsenic. Many recent studies have provided
groups such as glutathione (GSH), δ- experimental evidence that arsenic-induced
aminolevulinic acid dehydratase (ALAD) etc. generation of free radicals can cause cell damage
and form strong complex with vicinal thiols and death through activation of oxidative sensitive
groups thereby inhibiting the activity of signalling pathways17. These ROS and RNS
molecules. are capable of damaging a wide variety of
cellular macromolecules, including DNA, lipids,
• Inhibits PDH activity, perhaps by binding to and proteins. Finally, cellular signal transduction
the lipoic acid moiety. can be altered (e.g., activation of transcription
factors, changes of gene expression); cell growth,
• Methylated trivalent arsenicals such as MMAIII proliferation, and differentiation can be promoted;
are potent inhibitors of GSH reductase and and apoptosis leading to cell death or cancer
thioredoxin reductase. development can be induced18.

• Blocks the Krebs cycle and interrupt oxidative ROS may result in strand breakage, nucleic acid-
phosphorylation, resulting in a marked protein crosslinking, and nucleic base modification.
depletion of cellular ATP and eventually death Base modification, crosslinking of DNA–DNA and
of the metabolising cell. DNA–proteins, sister chromatid exchange, and single
or double-strand breakage may lead to the disruption
Pentavalent inorganic arsenate acts as - of transcription, translation, and DNA replication19.
• Undergoes reduction to form arsenite (AsIII). Genotoxic effects caused by arsenic are implicated
in carcinogenic outcomes20,21. Recent studies have
• Mimic phosphate in in-vivo system there by proposed two mode of action for arsenic induced
can replace phosphate in the sodium pump DNA damage, (i) inhibition of various enzyme
and the anion exchange transport system. involved in DNA repair e.g. poly ADP-ribose
polymerase-I (PARP-I), an important DNA repair
• Can form esters with glucose and enzyme22 and (ii) induction of ROS capable inflicting
gluconate, forming glucose-6-arsenate and DNA damage23.
6-arsenogluconate respectively. These
compounds resemble glucose-6-phosphate Fig. 2— Structure of some common chelating agents used against
and 6-phosphogluconate thus inhibit activity arsenic poisoning
of hexokinase.

• Uncouples in vitro oxidative phosphorylation
termed as arsenolysis.

Many studies have focused on arsenic induced
toxicity via generation of reactive oxygen
and nitrogen species in biological systems13.
Oxidative stress is a relatively new theory of
arsenic toxicity14,10. Arsenic and fluoride mediated
generation of reactive oxygen species involves
generation of superoxide (O2•–), singlet oxygen
(1O2), peroxyl radical (ROO•), nitric oxide (NO•)15.
Among these ROS, hydroxyl radical is generally
assumed to be the critical species that directly
attacks DNA (Fig. 2).

Dimethyl arsenic reacts with oxygen to
form dimethylarsenical radicals and superoxide
anoin. This dimethylarsenical radical combine
with molecular oxygen and generates
dimethylarsenic peroxyl radical. Hydroxyl radical
generates during this reaction then further
involves in oxidative stress16. In addition to

CHOUHAN & FLORA: COMBINED EXPOSURE TO ARSENIC & FLUORIDE 669

Arsenic is a well known human carcinogen of these chelating agents however, suffer from
that causes cancers in many human organs10. serious side effects. Meso 2,3-dimercaptosuccinic
It is generally suggested that arsenic shares acid (DMSA) is found to be one of the least toxic
many properties with tumor promoters. It acts by drugs that could be given orally. However hydrophilic
inducing intracellular signal transduction, activating and lipophobic properties of DMSA do not allow it to
transcription factors, and changing the expression pass through cell membrane. Recently some mono
of genes that are involved in promoting cell growth, and diasters of DMSA especially the higher
proliferation, and malignant transformation. Research analogoues have been developed and tried against
shows that arsenic significantly affects specific cases of experimental arsenic poisoning in in vitro
signal transduction molecules that are involved and in vivo both28,29. MiADMSA is a monoester
in mediating cellular proliferation or apoptosis, of DMSA with a straight and C-5 branched chain
including MAPKs, p53, AP-1, and NFκB. These amyl group thereby increasing the lipophilicity and
changes in cellular signaling pathways have been number of carbon atoms of the compound. Due to its
associated with both arsenic carcinogenicity10. The lipophilic nature it can easily cross the cell membrane
tumor suppressor gene p53, for example, has been and chelate arsenic from intracellularly and
linked to the DNA damage, cell cycle perturbations, extracellularly both. This is the best therapeutic part
and apoptosis that is seen with arsenic24,25. of this chelating agent. It also assumed that
MiADMSA could be able to decrease the oxidative
Treatment— Chelating agents are organic stress in tissue either by removing arsenic from the
compounds capable of linking together metal ions to target organs or by directly scavenging ROS through
form complex ring like structure called chelates. its sulfhydryl group. Flora and Mehta30 have also
Chelating agents have been used clinically as reported that MiADMSA does not show any major
antidotes for acute and chronic arsenic poisoning. alternation in heme synthesis pathway except for a
Chelators not only enhance excretion but also slight rise in the zinc protoporphyrin levels that
decrease the clinical signs of toxicity by preventing indicates mild anemia. MiADMSA has been seen to
metals from binding to cellular target molecules26. be slightly more toxic in terms of copper and zinc loss
Chelator form a complex with the respective toxic ion and some biochemical variables in the hepatic tissue
and these complexes reveal a lower toxicity and more in females as compared to male rats31.
easily eliminated from the body27. It includes 2,3-
dimercaprol (British Anti Lewisite, BAL), sodium Apart from synthetic chemical chelators, studies
2,3-dimercaptopropane-1-sulfonate (DMPS), meso have been carried out to explore natural antioxidants
2,3-dimerceptosuccinic acid (DMSA) (Fig. 3). Most against the toxic elements. Antioxidants (AOX) are
substances, which inhibit or delay oxidation of a
Fig. 3—Chemical structure of quercetin and silybin, active substrate. Antioxidant molecules are thought to play a
component of silymarin group crucial role in counteracting free radical induced
damage to macromolecules. Nutritional antioxidants act
through different mechanisms- (1) directly neutralize
free radicals; (2) reduce the peroxide concentrations
and repair oxidized membranes; and (3) quench iron to
decrease ROS production via lipid metabolism, short-
chain free fatty acids and cholesterol esters neutralize
ROS32. There is a wide range of antioxidants which
can counteract the condition of oxidative stress.
It includes vitamins, phenolic compounds (flavonoids),
carotenoids, hormones (melatonin, estradiol and
insulin). In addition, minerals such as selenium, zinc,
manganese, magnesium and copper are also involved
in hundreds of antioxidant roles in the body. Apart
from the free radical scavenging property, antioxidants
are known to regulate the expression of number of
genes and signal regulatory pathways and thereby may
prevent the incidence of cell death33.

670 INDIAN J EXP BIOL, JULY 2010

Vitamin C (ascorbic acid) acts as a scavenger maintenance of hepatic protein synthesis via RNA
of free radicals and plays an important role in activation and preservation of mitochondrial transport
regeneration of vitamin E (α-tocopherol). It scavenges function57-62. Chemical structure of quercetin and
the aqueous reactive oxygen species (ROS) by rapid silymarin are shown in Fig. 4
electron transfer that inhibits lipid peroxidation.
Flora et al.63 have studied herbal products/ extracts
Recent studies in rats using sodium arsenite, however, against several heavy metal toxicity. Besides,
indicate that vitamin C ameliorated arsenic-induced providing beneficial effects in eliminating body
toxicity34-36. Effect of vitamin C and E on arsenic burden of arsenic and reversing the altered
induced oxidative damage and antioxidant status has biochemical variables these herbal products intake
been studied37. Co-administration of vitamin C and E could also be useful in enhancing endogenous
antioxidant levels. Flora et al63 have also reported
to arsenic-exposed rats resulted in a reduction in the moderate chelating and antioxidant properties of
levels of lipid peroxidation, protein carbonyls and Moringa oleifera, Centella asiatica and Aloe vera
against arsenic during concomitant administration64-66.
hydrogen peroxide and an elevation in the levels These naturally occurring herbal products known to
of reduced glutathione38-40. Vitamins have been possess an effective arsenic removal property, either
known to alter the extent of DNA damage by reducing individually or in combination for the treatment of
TNF-α level and inhibiting the activation of caspase chronic arsenic toxicity67.
cascade in arsenic intoxicated animals41. Our group
There is also increased interest to find out a new
has also reported beneficial effects of vitamin E treatment strategies to minimize side effects and
supplementation during arsenic and fluoride to achieve maximum beneficial effects. Among
intoxication42. those strategies which are proposed, some include
combination therapy (co-administration of structurally
Flavonoids such as quercetin, hesperetin, different chelating agents, supplementation of an
naringenin, and epicatechin have been proposed to antioxidant with chelating agent, or co-administration

exert beneficial effects during cancer, cardiovascular Fig. 4— Combined toxicity of arsenic and fluoride
disease and neurodegenerative disorders43-46. The

precise mechanisms by which flavonoids exert their
beneficial remain unclear. However, recent studies
have speculated that their classical hydrogen-donating

antioxidant activity is unlikely to be the sole
explanation for cellular effects47-49.

Quercetin is one of the most frequently studied

dietary flavonoids and is ubiquitously present in
various vegetables, fruits, seeds, nuts, tea and red
wine. It is an excellent free radical scavenging
antioxidant47. It has been shown to have very potent
antioxidant and cytoprotective effects in preventing

endothelial apoptosis caused by oxidants. In addition,
quercetin is a more potent antioxidant than other
antioxidant nutrients, such as vitamin C, E and
β-carotene on a molar basis50-53.

Silymarin is also a polyphenolic antioxidant
flavonoid widely found in vegetable sources54,55. The

potential role of oxidative stress in pathogenesis
induced by arsenic suggests that antioxidants can be

considered as an alternative approach in mitigating
arsenic induced toxicity37. Silymarin has been proved
to be effective in restoring the diminished level

of antioxidants against arsenic induced toxicity in
in vitro56. The possible mechanism underlying the

protective properties of silymarin include prevention
of GSH depletion, destruction of free radicals,

CHOUHAN & FLORA: COMBINED EXPOSURE TO ARSENIC & FLUORIDE 671

of antioxidants with moderate chelating abilities diffusion. When ionic fluoride enters the acidic
beside antioxidant potential)67,68. Reports have environment of stomach lumen, it is largely converted
indicated that treatment with chelating agents into hydrogen fluoride74. It is rapidly distributed by
alone may not provide better clinical recoveries69, the systemic circulation to the intracellular and
but combinational therapies with antioxidants extracellular sites of tissues. However, ion normally
like n-nacetylcysteine27, α-lipoic acid70, captopril71, accumulates only in calcified tissues such as bone and
quercetin and also some herbal extracts72 have shown teeth. In blood ion is asymmetrically distributed
considerable promise in improving clinical recoveries. between plasma and blood cells, so that the plasma
Our group have also reported that co-administration concentration is approximately twice as high as that
of naturally occurring vitamins like vitamin E or associated with the cells75. Fluoride is distributed
vitamin C along with the administration of a from plasma to all tissues and organs. In humans and
thiol chelator like DMSA or MiADMSA may be laboratory animals, approximately 99% of the total
more beneficial in the restoring altered biochemical body burden of fluoride is retained in bones and teeth,
variables. Although, it has only limited role with remaining distributed in highly vascularized soft
in depleting arsenic burden14. Mishra et al have also tissues and the blood. Fluoride is concentrated to high
reported that combined administration of MiADMSA levels within the kidney tubules, so this organ has a
with M. oleifera provided better treatment than higher concentration than plasma76-78. Ingested
monotherapy with the thiol chelator in chronic arsenic fluoride that is not absorbed into the GI is excreted in
toxicity. Studies strongly support the theory that the faeces. Some fluoride is also lost from the body
combination therapy has a major role to play in future through sweat.
approach towards finding a safe, suitable and an
effective treatment for heavy metal poisoning72. Toxicity— Fluoride predominantly effects the skeletal
systems, teeth and also the structure and function
Fluoride of skeletal muscle, brain, and spinal cord79. General
Fluorine is the 13th most abundant element on symptoms of acute fluoride poisoning includes nausea,
salivation, vomiting, diarrhea and abdominal pain.
earth. It cannot exist outside a controlled Fluoride is also found to be involved in the alteration
environment without combining with other of metabolism of some essential nutrients which leads
substances to become fluorides. Three main to hyperkalemia, hypocalcemia, hypomagnesemia,
anthropogenic sources were identified as fertilizers, hypophosphatemia. Persistent fluoride serum level
combusted coal and industrial waste with phosphate leads to mineral homeostasis which ultimately causes
fertilizer being the most significance source of cellular damage. Symptoms of acute fluoride toxicity
fluoride73. There are ionizable and non-ionizable, have been summarized in Table 1.
organic and inorganic fluorides. Fluorine is probably
an essential element for animals and humans. Chronic fluoride toxicity occurs after the long-term
Low concentrations provide protection against ingestion of small amount of fluoride. It inhibits
dental caries, especially in children. Minimum the synthesis of DNA, protein and inhibits cell
concentration of fluoride in drinking water required proliferation and cytotoxic at high doses76,80.
to produce protective effects is approximately Symptoms of long term fluoride toxicity include
0.5 mg/L. emaciation, stiffness of joints and abnormal teeth and
bones. Other effects include lowered milk production
Soluble inorganic fluorides ingested through water and detrimental effects on reproduction. Fluoride is
and foods are almost completely absorbed from the known to cross the blood brain barrier and accumulate
gastrointestinal (GI) tract by a process of simple in the brain of animals exposed to high fluoride

Table 1— Symptoms of acute fluoride poisoning

Gastric Symptoms Electrolyte abnormalities Neurological effects Cardiovascular effects

Hypersalivation Hypocalcemia Headache Widening of QRS
Nausea Hypomagnesemia Tremors Various arrhythmias
Vomiting Tetanic contractions
Hyperkalemia Hyperactive reflexes Seizures Shock
Diarrohea Hypoglycemia Muscle weakness Muscular spasm Cardiac arrest
Abdominal pain

Dysphagia
Mucosal injury

672 INDIAN J EXP BIOL, JULY 2010

levels81. Recent studies have shown accumulation of damage or by directly diminishing the occurrence of
fluoride in the hippocampus of the brain causing oxidative damage by means of enzymatic (SOD,
degeneration of neurons, decreased aerobic Catalase, GPx, GR) and non-enzymatic antioxidants
metabolism and altered free-radical metabolism in (GSH, vitamins and several essential micronutrients).
liver, kidney, and heart82. Long term exposure to Some studies have shown lipid peroxidation (LPO) as
fluoride through various fluoride containing water and one of the molecular mechanisms involved in chronic
other products leads to development of fluorosis. fluoride-induced toxicity86. It may impair a variety of
Fluorosis is also known as a crippling and painful intra and extra mitochondrial membrane transport
disease. Fluorosis includes skeletal, dental and systems that may contribute to apoptosis. It leads to the
non-skeletal fluorosis. Dental fluorosis occurs during formation of secondary products such as conjugated
the period of enamel formation. It is linked to excessive dienes, hydrocarbon gases (ethane), and carbonyl
incorporation of fluoride into dental enamel and compounds (malondialdehyde) and decreased levels of
dentine, which prevents normal maturation of enamel. polyunsaturated fatty acid87. In addition to this ROS
Skeletal fluorosis is a pathological condition which (reactive oxygen species) is also found to play major
includes inhibition of bone hardening (mineralization), role during pathogenesis of fluoride. It may directly
causing the bones to become brittle and their oxidize amino acids leading to a loss of function of
tensile strength may be reduced83. Symptoms include proteins and a deactivation of enzymes88.
limited movement of joints, skeletal deformities, and
intense calcification of ligaments, muscle wasting and A possible mechanism of DNA damage induced by
neurological deficits76,84. fluoride is as follows— (i) fluoride has a dense
negative charge and is biochemically very active thus
Mechanism of toxicity— Fluoride leads to toxicity directly effect on DNA due to strong affinity for
as follows-- uracil and amide bonds by –NH···F–interactions89;
(ii) fluoride can combine stably with DNA by
• Binds calcium ions and may lead to covalent bonding, affecting the normal structure of
hypocalcemia which could further lead to DNA; (iii) fluoride can induce the production of free
osteoid formation radicals, which can damage DNA strands directly or
by lipid peroxidation initiated by free radicals90 ; and
• Disrupts oxidative phosphorylation, glycolysis, (iv) fluoride may depress enzyme activity, such as
coagulation, and neurotransmission (by binding DNA polymerase which might further affect the
calcium). process of DNA replication or repair and thereby
damage DNA91. However few studies have reported,
• Inhibits Na+/K+-ATPase, which may lead fluoride does not induce DNA damage, while others
to hyperkalemia by extracellular release of have observed the genotoxic potential of fluoride in
potassium. rats and human cells92-94. Effect of fluoride on DNA
damage in lymphocytes and its possible relation with
• Inhibits acetyl cholinesterase, which may be oxidative stress needs extensive research.
partly responsible for hyper salivation,
vomiting, and diarrhea (cholinergic signs). Fluoride was found to be an equivocal carcinogen
by the National Cancer Institute Toxicological
Exact mechanism of fluoride toxicity is not known. Program95. IARC evaluated that there is limited data,
It has been suggested that oxidative stress can be a which provide inadequate evidence about fluoride-
possible mechanism through which fluoride induces induced carcinogenicity. In a recent study, rats and
damage to the various tissues. Due to high mice given sodium fluoride in drinking-water at 11,
electronegativity, fluoride (F-) has a proclivity to form 45, or 79 mg/L have shown only the incidence of
strong hydrogen bonds, especially with –OH and –NH osteosarcomas in bones of male rats96.
moieties in biomolecules (Fig. 4). Fluoride is also
able to exert powerful influences on various enzymes Treatment—There is no safe and effective
and endocrine gland functions that affect or control treatment for the cases of chronic fluoride toxicity.
the status of oxidant/antioxidant systems in living However, the treatment for acute poisoning
organisms. Hydroxyl radicals were previously mainly relies on the use of antioxidants, vitamins
proposed as initiation of lipid peroxidation (LPO) and essential elements. Administration of some
through iron catalyzed Fenton reaction in
membranes85. The cell has several ways to alleviate
the effects of oxidative stress, either by repairing the

CHOUHAN & FLORA: COMBINED EXPOSURE TO ARSENIC & FLUORIDE 673

vitamins like vitamin C and E is known to revert approach to manage the menace. It is known that a
toxic effects induced by fluoride exposure42. The nutritionally rich diet may play an important role in
alleviating fluoride intoxication. It has been observed
mechanism of action of vitamin C might be due to its that supplementation of adequate dietary factors
powerful reducing action97,98. During fluoride toxicity, such as protein, calcium, magnesium, melatonin and
selenium may significantly reduce toxic effects of
generated free radicals attack the double bonds of fluoride in bone and other soft tissues106,107. These
substances decrease the absorption of fluoride and
polyunsaturated fatty acids initiating a chain reaction increase its removal from the body. In addition use of
green leafy vegetables, cumin seeds, milk, paneer,
and affect membrane integrity and cellular function. orange, vegetable oil, garlic, ginger, and pumpkin
must be promoted. Aluminium promotes the urinary
This chain reaction is inhibited by vitamin E excretion of fluoride, calcium and phosphate interfere
the gastrointestinal absorption of fluoride and borate
(α-tocopherol) by reacting with free radicals and enhances its eliminating process108.

converting itself into an α-tocopheroxyl radical which Co-exposure to arsenic and fluoride
is not harmful99. This α-tocopheroxyl radical, thus Few reports are available in literature which

formed is converted back to α-tocopherol by cytosolic suggest the effect of combined exposure to arsenic
vitamin C100. Thus, vitamins C and E show synergistic and fluoride on major organs. Li et al109 have studied
effects of arsenic-fluoride co-exposure on rat teeth
action in the recovery of altered variables suggestive and concluded that no detectable amount of arsenic
gets deposited in dentine and no combined effects of
of oxidative stress and organ damage by fluoride arsenic and fluoride have been seen on dental tissues.
exposure98. In addition to this certain non-vitamin However, some studies have also shown that
combined exposure to arsenic and fluoride is related
antioxidants such as Co-enzyme Q (Co-Q) and to distinct damage on the nerve system of the
offspring with decreased learning and memory
liponate have also been studied against fluoride ability110. Chinoy and Shah111 have reported altered
histology of cerebral hemisphere following combined
toxicity. CoQ is present in cells in two forms: arsenic-fluoride exposure, wherein the effects
produced by arsenic are more prominent as compared
oxygenated (ubiquinone) and a reduced form to fluoride. Genotoxic effects of combined exposure
to arsenic and fluoride have been reported to be
(ubiquinol). Only the reduced form demonstrates anti- more pronounced as compared to their individual
exposure100,112. Guizhou113 has suggested that the
oxidative properties. It reduces the concentration of toxicological effects of fluoride can be enhanced by
arsenic. More contradictory results have been reported
MDA in rabbit blood plasma, wherein it prevents the in different experimental studies in which different
joint actions such as independent, synergistic and
initiation and propagation of peroxidation of antagonistic effects have been observed108,114-116. This
indicates that the interaction mechanism of these two
polyunsaturated fatty acids, lipids, and phospholipids elements in relation to the development of endemic
of mitochondrial membranes101. Grucka-Mamczar disease is considerable complicated and can be
et al.102, have studied the effect of some vitamin affected by some uncertain factors. However, the
evidence of interactions between fluoride and arsenic
(vitamins A, C, and E) and non-vitamin antioxidants is inconclusive. Results from an experimental study
on rabbits have suggested that there is an antagonistic
(Coenzyme Q and liponate) on fluoride induced lipid effect between fluoride and arsenic108. Therefore, it is
quite probable that there are some interactions
peroxidation and found that these antioxidants are between fluoride and arsenic on some biological

most effective in counteracting the free radical

processes generated by sodium fluoride.

Flavonoid like quercetin has also been studied
against fluoride poisoning which support its beneficial
role on lipid peroxidation, serum cholesterol
level, triglycerides and total proteins in fluoride
intoxication103. Mixture of quercetin sulfonates has

been reported to stimulate and normalize tissue
respiratory activity and thus, can be helpful for the
prevention of fluoride toxicity in persons who are
exposed to excessive fluoride104. The antioxidative
action of quercetin is due to the presence of hydroxyl
groups in B ring of the molecule (Fig. 4), which

contain two catechol or three pyrogallol hydroxyls.
Combination therapy with chelator and an antioxidant
have never been used against fluoride toxicity.
However, combination of vitamin C and E has been
reported to protect endometrial tissue via their
anti-oxidant function on fluoride-induced damage105.

As such there is no effective treatment for fluorosis.
Therefore, prevention and control is the only

674 INDIAN J EXP BIOL, JULY 2010

indexes. Several studies have shown that combined Fluoride may be able to ameliorate the toxic effects
exposure of arsenic and fluoride causes oxidative of arsenic either through some strong bonding with
damage in the rat brain and also decreases activity arsenic or may be able to decrease its affinity for
of antioxidant enzymes and increased lipid active cell components. Decreased toxicity in arsenic-
peroxidation99-111. Effects of fluoride alone or in fluoride co-exposure can be explained on the basis of
combination with arsenic on antioxidant activities are ionization. Sodium fluoride is an ionic compound and
controversial117. Increased activity of SOD, catalase gets completely ionized in aqueous solution. Arsenic
and GPx and increased thiol status has also been has an empty d orbital of fairly low energy. Arsenic
reported following combined exposure to these reacts directly and readily with halogens and some
toxicants. There are few reports which suggest other non-metals. Arsenic predominately binds with
antagonism between arsenic and fluoride108,118. They halogen due to their electro negativity. In trivalent
have reported antagonistic effects following arsenic- oxidation state it shows SP3 hybridization and can
fluoride co-exposure on antioxidant system in liver form AsF3, while in pentavalent oxidation state it
and kidney of rats. It is therefore highly important to shows SP3d hybridization and forms AsF5. AsF3 exists
investigate the pattern and mechanism of combined in pyramidal structure, while AsF5 exists as trigonal
arsenic and fluoride exposure on different organs. bipyramidal structure. AsF5 is a potent ion acceptor
forming [AsF6]- ions or more complex species.
There are few reports suggesting effects of Therefore fluoride can suppress the ionization of
individual exposure to arsenic or fluoride on DNA sodium arsenite thereby reducing its toxicity. But still
damage and cellular deformities, and relatively little this hypothesis needs further investigations.
is known about their combined exposure on structure
and metabolism of various tissues. A loss of DNA Diagnosis of arsenic and fluoride toxicity— Three
integrity in the form of single strand breaks has been most commonly employed biomarkers used to
recorded during individual exposure of arsenic and identify and quantify arsenic exposure are total
fluoride. In arsenic and fluoride co-exposed animals arsenic in hair or nails, blood arsenic and total or
DNA damage has been found to be less pronounced speciated metabolites of arsenic in urine. Because
compared to their individual exposure indicated by arsenic accumulates in keratin-rich tissues such
decreased comet tail119. Arsenic- and fluoride both are as skin, hair and nails due to its high affinity for
known to cross blood brain barrier but during sulfhydryl groups, arsenic level in hair and nails
combined exposure ROS and TBARS level in brain may be used as an indicator of past arsenic exposure.
remain unaltered suggesting some interaction between Blood arsenic levels are highly variable. Blood
these toxicants thereby, inhibiting their free access in arsenic, normally less than 1 µg/dl may be elevated
brain tissue. Co-exposure to arsenic and fluoride also on acute intoxication, but it is rapidly cleared from
led to a significant recovery in depleted GSH level of the blood. The most important diagnostic test
tissues as compared to arsenic alone exposed animals, for detecting arsenic exposure is urine arsenic
which in turn again support the hypothesis that this determination. Since arsenic is rapidly metabolized
combination might have some antagonistic value. and excreted into the urine, total arsenic, inorganic
Concomitant exposure to arsenic and fluoride arsenic and the sum of arsenic metabolites (inorganic
(5mg/kg) has been found to show antagonistic effects arsenic + MMA + DMA) in urine have been used
on dopamine level and monoamine oxidase activity as biomarkers of recent arsenic exposure. The levels
(important neurotransmitters of our body), which of fluoride in plasma, serum and urine have
might be due to possible interaction between them. been considered useful biomarkers for fluoride
However synergistic effects have been observed exposure75,120,121. There are several methods for
during co-exposure to arsenic and higher dose of determining fluoride toxicity: Serum, urine, tooth
fluoride (10 mg/kg). Such observations could be enamel, bone and hair analyses. It has been
attributed to the possible interaction between arsenic suggested that the fluoride concentration in nails and
and fluoride, which is exclusively concentration hair can also be used as a marker of fluoride
dependent. It can also be suggested that at low exposure122,123. There is, however, no specific
concentration fluoride ions are sufficient enough to biomarker to examine the condition of combined
react with arsenic however at high concentration the arsenic-fluoride exposure.
effects are predominantly of free fluoride ions
compared to arsenic119.

CHOUHAN & FLORA: COMBINED EXPOSURE TO ARSENIC & FLUORIDE 675

Conclusion 11 ATSDR, Toxicological Profile for arsenic, Agency for Toxic
There is paucity of experimental data on the Substances and Disease Registry, ATSDR/TP-88/02, (U.S.
Public Health Service, Atlanta, GA) 1989
interactive effects of sodium arsenite and sodium
fluoride when administered concomitantly, on the 12 Huang Y Z, Qian X C, Wang G Q, Xiao B Y, Ren D D, Feng
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experimental evidence if the combined exposure will arsenism in Xinjiang, Chin Med J, 98 (1985) 219.
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Also the current management of acute and chronic 13 Valko M, Izakovic M, Mazur M, Rhodes C J & Telser J,
arsenic poisoning relies on supportive care and Role of oxygen radicals in DNA damage and cancer
chelation therapy however till date there is no incidence, Mol Cell Biochem, 266 (2004) 37.
effective treatment for chronic fluoride poisoning.
Thus an attempt should be made to explore the 14 Flora S J S, Bhadauria S, Kannan G M & Singh N, Arsenic
researches which provide information regarding the induced oxidative stress and role of antioxidant
mode of action to these toxicants and possible supplementation during chelation: A Review, J Environ Biol,
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15 Rin K, Kawaguchi K, Yamanaka K, Tezuka M, Oku N &
Acknowledgement Okada S, DNA- strand breaks induced by dimethylarsenic
The authors thank the Director for his support and acid, a metabolite of inorganic arsenics, are strongly enhanced
by superoxide anion radicals, Biol Pharm Bull, 18 (1995) 45.
suggessions. The author (SC) acknowledge DRDO,
India for the award of Senior Research Fellowship. 16 Yamanaka K, Hoshino M, Okanato M, Sawamura R,
Hasegawa A & Okada S, Induction of DNA damage by
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