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26
Copyright © 2006 C.M.B. Edition
Cellular and Molecular Biology
TM
53
, N°1, 26-47 ISSN 1165-158X
DO
I 10.1170/T773
2007 Cell. Mol. Biol.
TM
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S.J.S. FLORA, G. FLORA
1
, G. SAXENA AND M. MISHRA
Division of Pharmacology and Toxicology, Defence Research and Development Establishment,
Jhansi Road, Gwalior 474 002, India
Fax: +91 751 341 148; E-mail: sjsflora@hotmail.com
1
Department of Neurological Surgery, The Miami Project to Cure Paralysis,
University of Miami Miller School of Medicine, 1095 NW 14
th
Terrace (R-48), Miami, FL 33136, USA
Received July 19
th
, 2006; Accepted November
3
rd
, 2006; Published April 15
th
, 2007
Abstract – Health hazards caused by heavy metals have become a great concern to the population. Lead and arsenic
are one of the most important current global environmental toxicants. Their toxic manifestations are being considered
caused primarily due to the imbalance between pro-oxidant and antioxidant homeostasis and also due to a high
affinity of these metals for thiol groups on functional proteins. They also interfere with a number of other body
functions and are known to affect central nervous system (CNS), hematopoietic system, liver and kidneys and
produce serious disorders. They produce both acute and chronic poisoning, of which chronic poisoning is more
dangerous as its very difficult to revert back to normal condition after chronic exposure to these insidious metals
present in our life. Despite many years of research, we are still far from an effective treatment of chronic plumbism
and arsenicosis. Current approved treatment lies in the administration of chelating agents that forms an insoluble
complex with the metal and removes it. They have been used clinically as antidotes for treating acute and chronic
poisoning. The most widely used chelating agents are calcium disodium ethylenediamine tetra acetic acid
(CaNa
2
EDTA), D-penicillamine and British anti-lewisite (BAL). Meso 2,3 dimercaptosuccinic acid (DMSA), an
analogue of BAL, has been tried successfully in animals as well as in humans. But it is unable to remove the metal
from intracellular sites. Effective chelation therapy for intoxication by heavy metals depends on whether the
chelating agents are able to reach the intracellular site where the heavy metal is firmly bound. One of the important
approaches has been the use of combination therapy. This includes use of structurally different chelators or a
combination of an adjuvant/ antioxidant/ herbal extracts and a chelator to provide better clinical/ biochemical
recovery. A number of other strategies have been suggested to minimize the numerous problems. This article
presents the recent development made in this area with possible directions for future research.
Key words: Arsenic and lead poisoning, free radicals, oxidative stress, chelation therapy, chelating agents,
antioxidants, adjuvants, herbal extracts
INTRODUCTION
Heavy metal toxicity represents an
uncommon, yet clinically significant, medical
condition. The heightened concern for reduction
of environmental pollution that has been
occurring over the past 20 – 25 years has
stimulated active continuing research and
literature on the toxicology of heavy metals. If
unrecognized or inappropriately treated, heavy
metal toxicity can result in significant morbidity
and mortality. The periodic table contains 105
elements, of which 80 are considered metals.
Toxic effects in humans have been described for
less than 30 of these. Many metals are essential
to biochemical processes, and others have found
therapeutic uses in medicine. While the toxic
effects of these substances are a widespread
concern in the modern industrial context.
Abbreviations: CaNa
2
EDTA: calcium disodium
ethylenediamine tetra acetic acid; BAL: D-penicillamine
and British anti-lewisite; DMSA: Meso 2,3
dimercaptosuccinic acid.
However, occupational exposure to heavy metals
has accounted for the vast majority of poisonings
throughout human history. Hippocrates described
abdominal colic in a man who extracted metals,
and the pernicious effects of arsenic and mercury
among smelters were known even to
Theophrastus of Erebus (370-287 BC). Virtually
all metals can produce toxicity when ingested in
sufficient quantities, but there are several which
are especially important because either they are
so pervasive, or produce toxicity at such low
concentrations among which lead and arsenic are
known to be most common. Intentional or
unintentional ingestion of arsenic has been
notorious as a means of suicide and homicide.
Oxidative Stress/ Free radicals mediated toxicity
It has been observed that oxygen is both life-
sustaining and life-threatening inhalant. During
the past two decades, the evidence supporting the
deleterious effects of oxygen free radicals in
Arsenic and lead induced free radical generation
27
Copyright © 2006 C.M.B. Edition
many pathological processes has grown
considerably (1). Free radicals may play an
important role in several pathological conditions
of the CNS where they directly injure tissue and
their formation may also be a consequence of
tissue injury (1). Recently, attention has also
been focused on the contribution of oxygen free
radicals to brain dysfunction and brain cell death
after brain injury such as cerebral ischemia and
head trauma. Free radicals produce tissue damage
through multiple mechanisms, including excito-
toxicity, metabolic dysfunction, and disturbance
of intracellular calcium homeostasis (2). Free
radicals can be defined as molecules or molecular
fragments containing one or more unpaired
electrons. The presence of unpaired electrons
usually confers a considerable degree of
reactivity upon a free radical. Those radicals
derived from oxygen represent the most
important class of such species generated in
living systems (3). Oxidative stress, a condition
describing the production of oxygen radicals
beyond a threshold for proper antioxidant
neutralization has been implicated as an
important mechanism for arsenic and lead
induced toxicity.
Many studies have focused on metal-induced
toxicity and carcinogenicity, emphasising their
role in the generation of reactive oxygen and
nitrogen species in biological systems, and the
significance of this therein (3-9). Metal-mediated
formation of free radicals may cause various
modifications to DNA bases, enhanced lipid per-
oxidation, and changes in calcium and sulfhydryl
homeostasis.
ROS can be produced from both
endogenous and exogenous substances. Potential
endogenous sources include mitochondria,
cytochrome P-450 metabolism, peroxisomes, and
inflammatory cell activation (10). Mitochondria
have long been known to generate significant
quantities of hydrogen peroxide. The hydrogen
peroxide molecule does not contain an unpaired
electron and thus is not a radical species. Under
physiological conditions, the production of
hydrogen peroxide is estimated to account for
about ~2% of the total oxygen uptake by the
organism. However, it is difficult to detect the
occurrence of the superoxide radical in intact
mitochondria, most probably in consequence of
the presence of high SOD activity therein.
Generation of the superoxide radical by
mitochondria was first reported more than three
decades ago by Loschen and Flohe (11). After
the determination of the ratios of the
mitochondrial generation of superoxide to that of
hydrogen peroxide, the former was considered as
the stoichiometric precursor for the latter.
Mitochondria have been described as the
“power house” of the cell because they link the
energy-releasing activities of electron transport
and proton pumping with the energy conserving
process of oxidative phosphorylation to harness
the value of foods in the form of ATP.
Mitochondria generate approximately 2–3 nmol
of superoxide/min per mg of protein, the
ubiquitous presence of which indicates it to be
the most important physiological source of this
radical in living organisms (10). Since
mitochondria are the major site of free radical
generation, they are highly enriched with
antioxidants including GSH and enzymes, such
as superoxide dismutase (SOD) and glutathione
peroxidase (GPx), which are present on both
sides of their membranes in order to minimise
oxidative stress in the organelle (12). Superoxide
radicals formed on both sides of mitochondrial
inner membranes are efficiently detoxified
initially to hydrogen peroxide and then to water
by Cu, Zn-SOD (SOD1, localised in the
intermembrane space) and Mn- SOD (SOD2,
localised in the matrix).
The generation of various free radicals is
closely linked with the participation of redox-
active metals (7). The redox state of the cell is
largely linked to an iron (and sometimes copper)
redox couple and is maintained within strict
physiological limits. It has been suggested that
iron regulation ensures that there is no free
intracellular iron; however, in vivo, under stress
conditions, an excess of superoxide releases “free
iron” from iron-containing molecules. The
release of iron by superoxide has been
demonstrated for [4Fe–4S] cluster-containing
enzymes of the dehydratase-lyase family (13).
The released Fe(II) can participate in the Fenton
reaction, generating highly reactive hydroxyl
radical (Fe(II) +H
2
O
2
→Fe(III) + •OH+OH
−
).
Thus under stress conditions O
2
•−
acts as an
oxidant of [4Fe–4S] cluster-containing enzymes
and facilitates •OH production from H
2
O
2
by
making Fe(II) available for the Fenton reaction
(4-7). The superoxide radical participates in the
Haber-Weiss reaction (O
2
•−
+H
2
O
2
→ O
2
+ OH
•
+
OH
−
) which combines a Fenton reaction and the
reduction of Fe(III) by superoxide, yielding
Fe(II) and oxygen
(Fe(III) +O
2
•−→Fe(II) +O
2
) (Liochev and
Fridovich , 2002)
FLORA S.J.S. et al.
28
Copyright © 2006 C.M.B. Edition
The following article is a compilation of the toxic
effects of two important toxic heavy metals
namely arsenic and lead and the pharmacologic
agents and rationales used to treat them.
ARSENIC POISONING
Arsenic (usually as arsenic trioxide, As
2
O
3
) is
well known as a poison and has been discovered
to be a carcinogen in humans. Arsenic occurs
naturally in the environment as an element of the
earth’s crust. Arsenic is combined with other
elements such as oxygen, chlorine, and sulfur to
form inorganic arsenic compounds. Exposure to
higher-than-average levels of arsenic occurs
mainly in workplaces, near or in hazardous waste
sites, and areas with high levels naturally
occurring in soil, rocks, and water. Exposure to
arsenic at low levels for extended periods of time
can cause a discoloration of the skin and the
appearance of small corns or warts. Exposure to
high levels of arsenic can cause death. The
natural occurrence of arsenic in groundwater
constitutes a setback in the provision of safe
drinking water to millions of citizens in Asia.
There are millions of people at risk in the world
because they drink water containing carcinogenic
amounts of arsenic (14,15). Chronic exposure to
inorganic arsenic can lead to cancer of the skin,
lungs, bladder and liver if the exposure is via
ingestion (16). Lung cancer can occur if exposure
is by inhalation (17). The first case of arsenicosis
which was revealed in West Bengal in early
1980s was the outcome of ground water arsenic
poisoning in Bangladesh, and since its detection
in 1993, cases of arsenic poisoning have been
increasing in an alarming way. Arsenic is
reported to occur at high concentrations in the
water supply of communities in diverse countries
such as India, Nepal, Vietnam, China, Argentina,
Mexico, Chile, Taiwan, Mongolia and United
States of America. Recent reports proved that
number of countries and many states in India
(Uttar Pradesh, Bihar, Jharkhand, West Bengal,
Assam, Manipur and Bangladesh) in the Ganga-
Meghna-Brahmaputra (GMB) plain an area of
569,749 Km
2
, with a population of over 500
million are at risk from ground water arsenic
concentration and its health effects. The British
Geological Survey (BGS) in 2001 estimated that
46% of all (10 million) shallow tube wells in
Bangladesh are contaminated with arsenic at
concentrations exceeding the World Health
Organization’s (WHO) guideline concentration
of 0.01 mg/L.
The toxicity of this environmental toxicant is
complex and depends, in part, on its chemical
form, dose, route and duration of exposure,
degree of accumulation, rate of clearance and
animal species. Arsenic can exist in three
possible oxidation states: element (0), trivalent
(+3 e.g. Arsenite or -3 eg. Arsine) and
pentavalent (+5, eg. arsenate). Because it has
multiple and inter-convertible oxidation states,
arsenic can participate in a number of chemical
and biological reactions, including oxidation–
reduction reactions, acid–base reactions, covalent
interactions with most non-metals and metals and
methylation–demethylation reactions. In general,
inorganic forms of arsenic (eg. arsenite and
arsenate) are more toxic than organic forms (e.g.
methyl arsonate, dimethyl arsenite or
arsenobetaine). The toxicity of different arsenic
species varies in the order: arsenite > arsenate >
mono-methyl arsonate (MMA) > dimethyl
arsenite (DMA).
Both inorganic and organic arsenic are
absorbed from the gastrointestinal tract; however,
arsenic toxicity results from absorption of
trivalent and pentavalent inorganic arsenic. After
absorption, arsenic is cleared rapidly from the
blood and during its “first pass” phase it reaches
the liver where it is detoxified by conversion into
MMA and DMA. Arsenic metabolism is
characterized by two sequential reactions (18,19)
(Figure 1):
(a) The reaction of pentavalent arsenic to
trivalent arsenic in the presence of
glutathione(20);
(b) Oxidative methylation reaction, in which
the trivalent forms of arsenic are sequentially
methylated to form mono, di and trimethylated
products using S-adenosyl methionine (SAM) as
methyl donor and GSH as an essential co-factor.
Arsenic methylation occurs primarily in liver.
Many studies confirmed the generation of free
radicals during arsenic metabolism in cells (21).
Interestingly, some recent reports have provided
experimental evidence that arsenic-induced
generation of free radicals can cause cell damage
and death through activation of oxidative
sensitive signaling pathways (22). Arsenic-
mediated generation of reactive oxygen species is
a complex process which involves the generation
of a variety of ROS including superoxide (O
2
•−),
singlet oxygen (
1
O
2
), the peroxyl radical (ROO•),
nitric oxide (NO•), hydrogen peroxide (H
2
O
2
),
dimethylarsinic peroxyl radicals
Arsenic and lead induced free radical generation
29
Copyright © 2006 C.M.B. Edition
([(CH
3
)
2
AsOO•]) and also the dimethylarsinic
radical [(CH3)
2
As•].
Figure 1.
Figure showing the sequential reactions
of arsenic metabolism
The exact mechanism responsible for the
generation of all these reactive species is not yet
clear, but some workers have proposed the
formation of intermediary arsine species (21).
Another route to the production of H
2
O
2
was
suggested, involving the oxidation of As (III) to
As (V) which, under physiological conditions,
results in the formation of H
2
O
2
:
H
3
AsO
3
+H
2
O + O
2
→ H
3
AsO
4
+H
2
O
2
In recent studies concerning the mechanism of
arsenite toxicity in the brain it was reported that
some of its effects have been traced to the
generation of the hydroxyl radicals (23). The
time-evolution of the formation of the hydroxyl
radical in the striatum of both female and male
rats who underwent a direct infusion of different
concentrations of arsenite was investigated. The
treatment with arsenite induced significant
increases of hydroxyl radical formation. These
results support the participation of hydroxyl
radicals in arsenic-induced disturbances in the
central nervous system. Arsenic is a well-
established human carcinogen (24). Arsenic
compounds bind to SH groups and can inhibit
various enzymes, including glutathione
reductase. Studies support the hypothesis that
arsenic may act as a co-carcinogen-not causing
cancer directly, but allowing other substances,
such as cigarette smoke and UV radiation, to
cause DNA mutations more effectively (25)
(Figure 2). Arsenic is one of the few species
besides vinyl chloride that causes angiosarcoma,
which provides a good indication of the potency
of arsenic as a cancer-causing agent.
Figure 2. Figure showing the modes of
carcinogenicity of arsenic
LEAD POISONING
Occupational lead poisoning has been a
recognized health hazard for more than 2,000
years. Characteristic features of lead toxicity,
includes anemia, colic, neuropathy, nephropathy,
sterility and coma. Lead serves no useful biologic
function in the human body. Over the past
several years, concern has increased over the
health effects of low-level lead exposure and the
"normal" body burden of lead. In the
occupational setting, the present "no-effect" level
for lead exposure is currently being re-evaluated
as more sensitive measures of the physiologic
effects of lead are made available through
clinical investigations. The biochemical basis for
lead toxicity is its ability to bind the biologically-
important molecules, thereby interfering with
their function by a number of mechanisms
(Figure 3). Lead has been reported to impair
normal metabolic pathways in children at very
low blood levels (26,27). At least three enzymes
of the heme biosynthetic pathway are affected by
lead and at high blood lead levels the decreased
heme synthesis which leads to decreased
synthesis of hemoglobin. Blood lead levels as
low as 10 µg/dL have been shown to interfere
with one of the enzymes of the heme pathway, δ-
aminolevulinic acid dehydratase.
Accumulating evidences have shown that lead
causes oxidative stress by inducing the
generation of reactive oxygen species (ROS) and
weakening the antioxidant defence system of
cells (28-30). Depletion of cells’ major
sulfhydryl reserves seems to be an important
indirect mechanism for oxidative stress that is
induced by redox-inactive metals (5,31). When
GSH is reduced by lead, GSH synthesizing
systems start making more GSH from cysteine
via the γ-glutamyl cycle. GSH is usually not
FLORA S.J.S. et al.
30
Copyright © 2006 C.M.B. Edition
effectively supplied, however, if GSH depletion
continues because of chronic exposure (32).
Several enzymes in antioxidant defense system
may protect this imbalance but they also get
inactive due to direct binding of lead to the
enzymes’ active sites, if the sites contain
sulfhydryl group e.g. ALAD. Further, zinc which
usually serves as a cofactor of many enzymes
could be replaced by lead, thereby making the
enzyme inactive.
The increased lipid peroxidation and inhibition
of enzymes responsible to prevent such oxidative
damage have demonstrated lead induced
oxidative injury (33). Lead induced disruption of
the prooxidant/antioxidant balance could induce
injury via oxidative damage to critical
biomolecules. The possible mechanisms resulting
in the formation of free radicals include
generation of superoxide ion (34). A significant
decrease in the activity of tissue superoxide
dismutase (SOD), a free radical scavenger and
metalloenzyme (zinc/copper) on lead exposure
have been reported (35,36). This could be due to
an increase in lead concentration in these tissues
and their possible reaction with this enzyme (37)
thereby, reducing the disposal of superoxide
radicals. Catalase activity too has been shown to
increase in kidney.
Catalase is an efficient decomposer of H
2
O
2
and known to be susceptible to lead toxicity (38).
Lead induced decrease in brain GPx activity may
arise as a consequence of impaired functional
groups such as GSH and NADPH or selenium
mediated detoxification of toxic metals (39).
While, antioxidant enzyme glutathione S-
transferase (GST) is known to provide protection
against oxidative stress and the inhibition of this
enzyme on lead exposure might be due to the
depletion in the status of tissue thiol moiety.
These enzymes are important for maintaining
critical balance in the glutathione redox state.
Production of GSH is considered to be the first
line of defense against oxidative injury and free
radical generation where GSH functions as a
scavenger and a co-factor in metabolic
detoxification (40). GSH has carboxylic groups,
an amino group, a sulfhydryl group and two
peptide linkages as sites for the reaction of lead.
Its functional group, -SH plays an important role
in lead binding. Several reports have
demonstrated that GSH is decreased in the brain,
liver and eye lens of rats exposed to lead (31).
SYMPTOMS OF LEAD TOXICITY
Lead is known to cause acute, sub-chronic and
chronic toxicity. The most commonly used
biological marker is the concentration of lead in
blood. The concentration of lead in plasma is
very low and thus is not recommended.
Acute Toxicity
Acute lead toxicity occurs at blood levels of
100-120 µg/dL in adults and 80-100 µg/dL in
children. It results from inhalation of large
quantities of lead due to occupational exposure
among industrial workers and in children through
ingestion of large oral dose from lead based paint
on toys. The clinical symptoms of acute
poisoning are characterised by metallic taste,
abdominal pain, vomiting, diarrhoea, anaemia,
oliguria, collapse and coma.
Chronic toxicity
Symptoms of chronic toxicity may appear in
adults at blood lead levels of 40-60 µg/dL. This
is more common and can be described in three
stages of progression: The early stage is
characterised by loss of appetite, weight loss,
constipation, irritability, occasional vomiting,
fatigue, weakness, gingival lining on gums and
anaemia; The second stage is marked by
intermittent vomiting, irritability, nervousness,
tremors and sensory disturbances in the
extremities, most often accompanied by stippling
of red blood cells; and the third severe stage of
toxicity is characterised by persistent vomiting,
encephalopathy, lethargy, delirium, convulsions
and coma.
Figure 3 Figure depicting the mechanisms of lead
toxicity
It can thus be concluded that inhibitory effect
of lead on antioxidant enzymes and glutathione
appear to impair the cells’ antioxidant defenses
and render them more susceptible to oxidative
attacks.
Arsenic and lead induced free radical generation
31
Copyright © 2006 C.M.B. Edition
TREATMENT OF METAL
POISONING
Chelation therapy
Chelating agents are organic compounds
capable of linking together metal ions to form
complex ring-like structure called chelates.
‘Chelate’ is a Greek word meaning the claws of a
lobster. Chelators act according to a general
principle: the chelator forms a complex with the
toxic ion, and these complexes reveal a lower
toxicity and are more easily eliminated from the
body through the excretory system.
It is of great importance that the chemical
affinity of the complexing agent for the toxic
metal ion should be higher than the affinity of the
metal for the sensitive biological molecules.
Thus, chemical measurement of the stability
constants of the metal-complexes formed may
give a first indication of the effectiveness of a
particular chelating agent. An ideal chelating
agent should possess the characteristic like,
greater affinity for the toxic metal that has to be
chelated, low toxicity, rapid elimination of metal,
high water solubility, ability to penetrate cell
membrane, administered orally, ability to chelate
with natural chelating groups found in biological
system, minimal metabolism etc.
The metal chelate complexes have a
reduced tendency to undergo exchange
reactions once they are formed. However, it
is frequently advantageous to use a
preferred donor atom in a chelating agent of
lower density. It is also necessary to keep in
mind that the introduction of the chelating
agent into any intracellular space requires its
passage through the cell membrane. This
passage can be accomplished either (a) by
passing through the lipid part of the
membrane as an uncharged molecule or (b)
via utilizing one of the anion/cation
transport systems present in the membrane.
There is a hypothesis that large ion complex
with a positive charge will pass out of a cell
very slowly because of their inability to pass
through either the lipid portion of the
cellular membrane or the cation transport
system designed to move ions with +1 or a
+2 charge across the membrane. Another
important property of metal complexes is
the stereochemistry of the toxic metal ion.
Chelating agents tie up all the coordination
position of a metal ion (41-46). It should be
noted that metal chelating agents usually
contain more than one functional group, in
order to provide a chemical ‘claw’ to chelate
the toxic metal.
Conventional chelating agents
The most commonly used chelating agents
that have been the forerunners in chelation
therapy belong to the polyaminocarboxylic
groups. As the name indicates, these chelators
utilize the amino and the carboxylic groups to
scavenge the toxic metal from the system. In this
category, calcium disodium ethylene diamine
tetra acetic acid (CaNa
2
EDTA) is a derivative of
ethylene diamine tetra acetic acid (EDTA), a
synthetic polyamino-polycarboxylic acid was
used for the treatment of metal poisoning and had
been the mainstay of chelation therapy for many
years. Another member belonging to this family
is diethylene triamine pentaacetic acid DTPA is a
synthetic polyaminocarboxylic acid with
properties similar to EDTA (47). It can be
affirmed that EDTA does not penetrate cell
membranes and has a biological half-life of 50-
60 minutes; 90% is excreted within 6-8 hours
after administration. Renal clearance is mainly
through active tubular secretion without any
significant re-absorption. Variations in the pH
and diuresis do not affect the excretion rate (48).
CaNa
2
EDTA has the LD
50
value of 16.4 mmol/kg
in mouse (49). Intravenous administration of this
drug results in good absorption but very painful
at the injection site. Hence intravenous injection
could be given either by diluting in 5% dextrose
or saline (49). Hypocalcaemia is reported with
the administration of Na
2
EDTA. CaEDTA has
the major toxic effects on the renal system
causing the necrosis of tubular cells. Severe,
hydropic degeneration of proximal tubule cells
has also been reported. These lesions along-with
some alterations in the urine like hematuria,
proteinuria and elevated BUN are generally
reversible when the treatment ceases. Another
side effect of EDTA is its ability to chelate
various essential metals endogenous to the body,
zinc in particular (50,51). Zinc administration
during EDTA administration is generally
recommended to reduce toxicity (50).
It has been well established that
administration of EDTA during pregnancy can
result in teratogenic effects especially when
administered between days 11 to 14 at doses
comparable to humans (52). Tuchmann-
Duplessis and Mercier-Parort (53) were the first
to report teratogenic effect of EDTA. Absorption
FLORA S.J.S. et al.
32
Copyright © 2006 C.M.B. Edition
into the circulation, potential interaction with
essential trace elements, and the stress associated
with the administration of the compound were
suggested to be the possible factors involved in
the differences in EDTA-induced maternal and
developmental toxicity (54). Brownie et al. (52)
also reported teratogenic effects. Another
reported disadvantage of CaNa
2
EDTA is that it
redistributes lead to the brain. Cory Slechta et al.
(55) and Flora et al. (56) in separate studies
provided evidence that rat given lead as lead
acetate in their drinking water and then treated
with CaNa
2
EDTA mobilized lead from their
tissues and redistributed to brain and liver on the
first day of treatment. The large number of side
effects due to the administration of these
chelating agents prompted in the
commercialization of chelators containing thiol
or sulfhydryl groups.
D-Penicillamine (DPA) is 3,3
dimethylcysteine, a sulfhydryl containing amino
acid, first introduced in clinical practice by
Walshe (57) but was tried by Ohlsson (58) as an
antidote for low or mild lead poisoning. It can
penetrate cell membranes and then get
metabolized. It can be absorbed through the
gastro intestinal tract and thus can be
administered orally. Its absorption from the
gastrointestinal tract is between 40 to 70% (59).
It is fairly stable as its SH group is very resistant
to oxidation in vivo, attack from enzymes such as
cysteine desulfhydrase and L-amino acid
oxidase, compared to other monothiols.
Excretion of DPA through urine is very fast.
Small amount is also reported to cross hepatocyte
membrane and excreted through bile. However,
the major toxic effect of DPA is antagonizing
pyridoxine and inhibiting pyridoxine dependent
enzyme such as transaminases. Other toxic
effects include hypersensitive allergic reactions
like fever, skin rashes, leucopoenia and
thrombocytopenia (60). In few reports
nephrotoxic effects too have been observed along
with penicillin allergic reaction in sensitive
individual due to cross reactivity. Prolonged
treatment may also lead to anorexia, nausea,
vomiting in human. Apart from this, DPA is also
a well recognized teratogen and lathyrogen that
causes skeletal, palatal, cutaneous and pulmonary
abnormalities (61-63). As compared to other
chelators, the developmental toxicity of DPA is
abundant in both human and experimental
animals. First report on human embryopathy
associated with DPA was published by
Mjolnerod et al. (64). Author described the effect
of DPA on the infant with generalized connective
tissue defects including lax-skin, hyperflexibility
of joints, vein fragility, varicosities and impaired
wound healing, the child died at a age of 7
weeks. Since DPA chelated copper, it was
hypothesis that the drug might be teratogenic
(65). Various investigations were performed in
the early eighties to test the hypothesis (65-68)
and it was observed that when pregnant rats were
given DPA along with their diet, there was a high
incidence of malformations. The frequency of
reabsorption and the frequency and severity of
malformations increased in the rats in a dose
dependent manner (67). However, literature also
suggests that the administration of DPA during
pregnancy protects the mother from the relapse
of Wilson’s disease, while it would carry few
risks to the fetus (69). DPA have been tried
safely throughout pregnancy in women with
Wilson’s disease, suggesting that the excessive
copper stores improve tolerance (70). The
American Academy of Pediatrics (71)
recommends pencillamine use only when
unacceptable adverse reactions to both DMSA
and EDTA have occurred. However, Kreppel et
al. (72) reported that pencillamine was
ineffective in reducing arsenic burden in rats.
It is clear from above that most of the
conventional chelators are compromised with
many side effects and drawbacks and there is no
safe and effective treatment available for arsenic
and lead poisoning. In the recent past some
newer strategies were adopted to find a solution
to this problem. In the following paragraphs
some of these strategies have been discussed in
brief.
SYNTHESIS OF NEW CHELATORS
Thiol chelators:
In the early eighties it was shown that some
newer complexing agents like DMPS and DMSA
were effective against mercury, arsenic and lead
poisoning. When compared to BAL these newer
chelating agents were of significant lower
toxicity and moreover they could be administered
orally or intravenously (73). In addition to their
heavy metal chelating properties, these agents
have a dithiol group that may act as an oxygen
radical scavenger and thus inhibit lipid
peroxidation (74-76). Chemical structures of
some of the newer thiol chelators are
summarized in Figure 4.
Arsenic and lead induced free radical generation
33
Copyright © 2006 C.M.B. Edition
Sodium 2,3 dimercaptopropane 1-sulphonate
(DMPS)
DMPS was first introduced in Soviet Union
in the 1950s as `Unithiol'. DMPS is mainly
distributed in the extra cellular space; it may
enter cells by specific transport mechanism.
After i.p. injection of lethal doses the animals
were highly irritable for some minutes before
they became apathetic and breathing ceased
(49,77). DMPS is rapidly eliminated from the
body through the kidneys. The serum half-life is
about 20 to 60 minutes. Following oral
administration, about 60 to 30% of the
administered dose is absorbed in dogs (78) and
30 % in rats (49,79), and plasma peak levels are
reached after 30 to 45 minute (78). Rapid
oxidation of DMPS after intravenous
administration to disulfide forms is well reported
in blood (80,81). Fifteen minutes after iv
administration of DMPS (3 mg/kg) to humans
only 12% of the total DMPS was oxidized to
disulfides (82). DMPS is not involved in
important metabolic pathway and parts of
administered substance are excreted in an
unchanged form. By the parenteral route the LD
50
for various species is about 1 g/kg to 2 g/kg. No
major adverse effects following DMPS
administration in humans or animals have been
reported (83). However, a dose dependent
decrease in the copper contents was found in the
serum, liver, kidneys and spleen. Information
regarding the developmental toxicity of DMPS is
rather scarce. No abnormalities in the offspring
with chronic oral DMPS treatment are reported.
Oral administration of DMPS did not adversely
affect late gestation, parturition, or lactation in
mature mice and fetal and neonatal development
does not appear to be adversely affected(84).
DMPS although known for its antidotal
efficacy against mercury, it has been reported to
be an effective drug for treating arsenic
poisoning. This drug too can be administered
both orally and intravenously. An oral dose of
100 mg/kg thrice a day for 10-12 days is
effective against mild arsenic poisoning while no
recommendation for treating chronic arsenic
poisoning is available (85). In experimental
animals, i.p. administration of DMPS increased
the lethal dose of sodium arsenite in mice by four
folds. A quantitative evaluation of three drugs
reveals that DMPS is 28 times more effective
than BAL in arsenic therapy in mice (86), while
DMSA and DMPS are equally effective. DMPS
also appeared to be effective at least in reducing
the body lead and gold burden (79,87). In
children, 5 mg/kg per single dose should be
given (88). Oral DMPS treatment in adults may
be given with an initial dose of 100-300 mg and
continued with 100 mg every 6 or 8 hours. In
children, the oral dosage is 5 mg/kg/day.
Succimer or meso 2,3-dimercaptosuccinic acid
(DMSA)
The one chemical derivative of dimercaprol,
which has gained more and more attention these
days, is DMSA also known as Succimer.
Succimer is an orally active chelating agent,
much less toxic than BAL and its therapeutic
index is about 30 times higher (89). US FDA has
approved this compound in 1991 for the
treatment of children whose blood lead
concentration was above 45
µ
g/dL (90). The
empirical formula of DMSA is C
4
H
6
O
4
S
2
and its
molecular weight is 182.21. It’s a weak acid
soluble in water (49).
DMSA distribution is predominantly extra
cellular since it is unable to cross hepatic cell
membrane and excreted by the kidney with a
half-life of about two days (73). Over 95% of
blood DMSA is bound mainly to albumin
(73,91). DMSA appears to be transported by
plasma albumin. It has been reported that 2-4
hours after DMSA administration only 12% of
meso DMSA excreted in urine was unaltered
whereas about 88% oxidized to form disulfates
(DMSA attached to one or 2 cysteine molecules).
No mixed disulfates are found in the blood (91-
94). The absorption of DMSA after oral
administration is about 60%. Studies addressing
the possibility that DMSA may chelate metal
stored in the gut, because a significant percentage
of an oral dose is not absorbed, have yet to be
elucidated (92).
The LD
50
values of sodium salts of DMSA in
mice are: iv 2.4, im 3.8, ip 4.4 and po 8.5 g/kg,
respectively. Using a percutaneous route, the
acute LD
50
for rats and mice is about 2 g/kg.
Graziano et al. (95) reported that i.p.
administration of 200 mg/kg DMSA could
produce only a marginal change in growth but
did not elicit any appreciable change in
histopathological alterations in tissue or cause
hematological or biochemical change in blood.
No significant loss of essential metals like zinc,
iron, calcium or magnesium was observed. A
slight increase in transaminase activities in serum
of human and animals has been reported after
DMSA treatment (95,96). Adverse reaction to
DMSA includes gastrointestinal discomfort, skin
reaction, mild neutropenia and elevated liver
FLORA S.J.S. et al.
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Copyright © 2006 C.M.B. Edition
enzymes. No redistribution of lead also occurred
on DMSA administration in rats (55).
Orally administered DMSA caused no
marked adverse reactions but some sulphurous
odor in the mouth, weakness, abdominal
distension and anorexia. These reactions were
mild and disappeared quickly after withdrawal of
DMSA. No pathological findings were observed
in the blood, urine, ECG or ultrasonography of
liver and spleen.
It has also been found that DMSA resulted in
low maternal liver copper and calcium
concentration whereas high iron levels, the fetal
copper, calcium and zinc levels decreased (97).
Although the results suggest that DMSA induced
developmental toxicity was due to an induced
zinc deficiency, additional investigations showed
that the embryo/fetal toxicity of DMSA might be
mediated, at least in part, through altered fetal
copper metabolism (98). In contrast, the oral
route did not cause any adverse affects on the
offspring survival and development (99).
DMSA has been tried successfully in animal
as well as in few cases of human arsenic
poisoning (100). DMSA has been shown to
protect mice due to lethal effects of arsenic. A
subcutaneous injection of DMSA provided 80-
100% survival of mice injected with sc sodium
arsenite (101). Flora and Tripathi (100) also
reported a significant depletion of arsenic and a
significant recovery in the altered biochemical
variables of chronically arsenic exposed rats.
This drug can be effective if given by either oral
or i.p. route. Patients treated with 30 mg/kg
DMSA per day for 5 days showed significant
increase in arsenic excretion and a marked
clinical improvement. In a double blind,
randomized controlled trial study conducted on
few selected patients from arsenic affected West
Bengal (India) regions with oral administration
of DMSA suggested that DMSA was not
effective in producing any clinical or
biochemical benefits or any histopathological
improvements of skin lesions (102). In an
experimental study recently conducted, provided
an in vivo evidence of arsenic induced oxidative
stress in number of major organs of arsenic
exposed rats and that these effects can be
mitigated by pharmacological intervention that
encompasses combined treatment with N-
acetylcysteine and DMSA (103).
US FDA has recently licensed the drug
DMSA for reduction of blood lead levels. It was
reported that EDTA increases the lead content in
the brain due to redistribution (55). DMSA when
administered either alone or in combination with
EDTA decreases the lead concentration in the
brain (55,56). Besunder et al. (104) too recently
confirmed these findings in rats and
recommended the administration of DMSA and
EDTA to children hospitalized for combined
chelation therapy.
Esters of Succimer (DMSA)
A large number of esters of DMSA have
been synthesized for achieving optimal chelation
as compared to DMSA. These esters are mainly
the mono and dimethyl esters of DMSA that have
been studied experimentally with the aim of
enhancing tissue uptake of chelating agents (93).
In order to make the compounds more lipophillic
the carbon chain length of the parent DMSA was
increased by controlled esterification with the
corresponding alcohol (methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, pentyl, isopentyl and
hexyl). A large number of esters have been
synthesized and are being tried for the treatment
of metal poisoning. It has also been reported that
these mono and diesters have a better potential in
mobilizing cadmium and lead from the tissues in
mice (105,106). Rivera et al. (107) reported that
that the dimethyl ester of DMSA (meso-
DiMeDMSA) increased the excretion of
cadmium. They also reported that when rabbit
liver metallothionein was incubated with the
diester, 32% of the cadmium and 87% of zinc
bound metallothionein was removed from the
system (108). Although, the diester entered the
cell but it caused severe zinc depletion (108).
Singh et al. (109) examined the efficacies of
three diesters of DMSA and found that these
diesters were effective in reducing the soft organ
lead concentrations when compared to BAL.
Kreppel et al. (110) reported the therapeutic
efficacy of six analogues of DMSA in mice.
They administered mice with a single LD
80
dose
of arsenic trioxide followed by a single dose of
these six analogues of DMSA. They found that
meso 2,3-di(acetylthio) succinic acid (DATSA)
and 2,3-di(benzoylthio) succinic acid (DBTSA)
increased the survival rates by 29% and 43%
respectively when administered via gastric tube
(i.g) and 89% when administered
intraperitoneally (i.p). Administration of
dimethyl DMSA (DMDMSA) through i.g and i.p
and diethyl DMSA (DEDMSA), di-n-propyl
DMSA (DnPDMSA) and diisopropyl DMSA
(DiPDMSA) through i.g route did not reduce the
lethality. While the i.p. administration of
DnPDMSA increased the survival rate by 72%
Arsenic and lead induced free radical generation
35
Copyright © 2006 C.M.B. Edition
whereas DEDMSA and DiPDMSA increased it
by 86% (110). Kreppel et al. (111) also reported
the effects of 4 monoesters of DMSA in
increasing the survival and arsenic elimination in
various organs in mice. It was observed that all
the monoesters, MiADMSA (mono- isoamyl),
MnDMSA (mono n-amyl), MnBDMSA (mono
n-butyl) and MiBDMSA (mono i-butyl)
markedly decreased the arsenic content in most
of the organs as soon as 1.5 hrs after
administration. They found that MiADMSA and
MnADMSA were the most effective in
increasing the survival of mice (111). Similar
studies were also performed by Flora et al. (112)
where they investigated the effect of DMDMSA,
DEDMSA DiPDMSA and diidoamyl DMSA
(DiADMSA) on sub chronically arsenic treated
rats. The results suggested that the diesters
reduced the arsenic burden in blood and soft
tissue but were only moderately effective in
reversing the biochemical recoveries when
compared to DMSA (112).
Walker et al. (106) studied the effects of
seven different monoalkyl esters of DMSA on
the mobilization of lead in mice and observed
that after a single parenteral dose of the chelator
DMSA there was a 52% reduction in the lead
concentrations while with the monoesters the
reduction varied from 54% to 75%. Jones et al.
(105) reported the efficacy of ten different
monoesters through oral and i.p. route on
cadmium mobilization in mouse. Out of the ten
monoesters studied they found MiADMSA to be
the most effective in reducing the cadmium
concentrations from the liver and kidneys.
In all of the reported literature, it was
observed that the analogues of DMSA were
capable of crossing the membranes and were
more effective in reducing the metal burden in
acute and sub-chronic metal intoxication. Most
of the studies have also suggested that the
monoesters are more effective in treatment of
experimentally induced metal intoxication.
Monoisoamyl DMSA (MiADMSA)
Among these new chelators, monoisoamyl
ester of DMSA (MiADMSA; a C
5
branched
chain alkyl monoester of DMSA) has been found
to be the more effective than DMSA in reducing
cadmium and mercury burden (113,114). It is
reported that the toxicity of DMSA with LD
50
of
16 mmol/kg is much lower than the toxicity of
MiADMSA with LD
50
of 3 mmol/kg but lesser
than BAL (1.1 mmole/kg). The interaction of
MiADMSA and DMSA with essential metals is
same. Mehta and Flora (115) reported for the
first time the comparison of different chelating
agents (3 amino and 4 thiol chelators) on their
role on metal redistribution, hepatotoxicity and
oxidative stress in chelating agents induced
metallothionein in rats. We suggested that out of
all the 7 chelators, MiADMSA and DMSA
produced the least oxidative stress and toxicity as
compared to all other 5 chelators (115).
However, no reports are available about the
toxicity of this metal complexing agent except
for its developmental toxicity. No observed
adverse effect levels (NOAELs) for maternal and
developmental toxicity of MiADMSA were 47.5
mg/kg and 95 mg/kg/day respectively indicating
that MiADMSA would not produce
developmental toxicity in mice in the absence of
maternal toxicity (116). Bosque et al., (117)
reported that administration of MiADMSA
through the parenteral route to pregnant mice
during organogenesis produced maternal toxicity
at a dose of 95 and 195 mg/kg with a significant
decrease in the body weight and an increase in
the liver weights. They also reported that
MiADMSA caused embryo/fetotoxicity at a dose
of 190 mg/kg by significantly increasing the
embryo lethality and non-significant increase in
the skeletal defects. Taubeneck et al. (98) showed
that the developmental toxicity of DMSA is
mediated mainly through disturbed copper
metabolism and this may also be true for
MiADMSA. Recently, our group was the first to
report the toxicological data of MiADMSA when
administered in male and female rats (118-120)
through the oral as well as the intraperitoneal
route (25, 50 and 100 mg/kg /3 weeks). We
observed that there was no major alteration in the
heme biosynthesis pathway except for a slight
rise in the zinc protoporphyrin levels suggesting
mild anemia at the highest dose. The oral route of
administration was also seen to be better when
compared to the ip route based on the
histopathological studies of the liver and kidney
tissues. MiADMSA was seen to be slightly more
toxic in terms of copper loss and some
biochemical variable in the hepatic tissue in
females as compared to male rats. The studies
concluded that the administration of MiADMSA
in female rats is confounded with side effects and
may require caution during its use (118-120).
Since administration of a chelating agent during
pregnancy is always with caution, we studied the
effects of MiADMSA administration from day
FLORA S.J.S. et al.
36
Copyright © 2006 C.M.B. Edition
14 of gestation to day 21 of lactation at different
doses through oral and ip routes to examine the
maternal and developmental toxicity in the pups
(121). Results suggested that MiADMSA had no
effect on length of gestation, litter-size, sex ratio,
viability and lactation. No skeletal defects too
were observed following the administration of
the chelator. However, MiADMSA
administration produced some marginal maternal
oxidative stress at the higher doses (100mg/kg
and 200 mg/kg) based on thiobarbituric acid
reactive substances (TBARS) in RBCs and
decrease in the δ-aminolevulinic acid
dehydratase (ALAD) activity. MiADMSA
administration too caused some changes in the
essential metal concentration in the soft tissues
especially the copper loss in lactating mothers
and pups, which would be of some concern.
Apart from copper, changes too were observed in
the zinc concentrations in mothers and pups
following administration of MiADMSA. The
study further suggested that the chelator could be
administered during pregnancy as it does not
cause any major alteration in the mothers and the
developing pups (118). Since chelating agents
are administrable to individuals of all ages, we
investigated the effect of MiADMSA
administration in different age groups of male
rats (young, adult and old rats) based on the fact
that whether MiADMSA, a dithiol agent was a
pro-oxidant or an antioxidant (118). Results
suggested that MiADMSA administration
increased in activity of ALAD in all the age
groups and increased blood GSH levels in young
rats. MiADMSA also potentiated the synthesis of
MT in liver and kidneys and GSH levels in liver
and brain. Apart from this it also significantly
reduced the GSSG levels in tissues. MiADMSA
was found to be safe in adult rats followed by
young and old rats (118,119).
A large number of reports are available on
the therapeutic efficacy of the MiADMSA (122,
123). Pande et al. (122) found that MiADMSA
was effective in prevention and treatment of
acute lead intoxication. Walker et al. (106)
reported that MiADMSA administration reduced
the brain lead concentrations by 75% when
compared to 35% with DMSA whereas the ip
administration reduced kidney lead levels by
93% while oral administration reduced the
kidney lead by 94% (106). MiADMSA
completely prevented the testicular damage after
intraperitoneal administration of cadmium
chloride at a dose of 0.03 mmol/kg (113). Jones
et al. (105) reported that MiADMSA enhanced
the cadmium elimination through urine by 3.6%
compared to 0.02% of the controls and 24% in
faeces compared to 0.11% in controls.
Therapeutic effects of MiADMSA against
mercury burden have shown that MiADMSA is
capable of decreasing mercury concentration by
59% and 80% after two doses when compared to
DMSA (25% and 54% respectively). The total
corporal mercury burden of 29.25µg was reduced
to 21.06µg with DMSA after a single injection of
0.5 mmol/kg. The same dose of MiADMSA
effected a reduction to 12.09 µg (114). Belles et
al. (124) assessed the protective activity of
MiADMSA against methyl mercury-induced
maternal and embryo/fetal toxicity in mice. Oral
methyl mercury administration increased the
number of resorptions, decreased fetal weights
and increased skeletal abnormalities. MiADMSA
administration could not reverse the embryo
lethality but fetotoxicty was significantly reduced
by the administration of these agents at different
doses.
Recently, Flora et al. (125) reported the
effect of MiADMSA on the reversal of gallium
arsenide (GaAs) induced changes in the hepatic
tissue. Rats were exposed for 24 weeks with 10
mg/kg GaAs, orally, once daily and treated with
0.3 mmol/kg of MiADMSA or DMSA for two
courses. They observed that MiADMSA was
better than DMSA in mobilizing arsenic and in
the turnover of the GaAs sensitive biochemical
variables. Histopathological lesions, also
responded more favorably to chelation therapy
with MiADMSA. In another study, dose
dependent therapeutic potential of MiADMSA
was compared with monomethyl ester and
DMSA in sub-chronically GaAs treated rats and
it was found that MiADMSA was highly
effective in the reversal of altered biochemical
variables and in the mobilization of arsenic
(126).
Dose and route dependent efficacy of
MiADMSA against chronic arsenic poisoning
has also suggested that the chelator is highly
effective through oral route in reversing the
arsenic induced changes in the variables
indicative of oxidative stress in major organs as
well as in mobilization of arsenic (127). Kreppel
et al. (111) reported that MiADMSA was
effective in increasing the survival of arsenic
exposed mice when compared to its parent
DMSA.
Arsenic and lead induced free radical generation
37
Copyright © 2006 C.M.B. Edition
Figure 4. Figure showing the structures of
common chelating agents
Despite a few drawbacks/side effects
associated with MiADMSA, the above results
suggest that MiADMSA may be a future drug of
choice owing to its lipophilic character and the
absence of any metal redistribution. However,
significant copper loss requires further studies.
Moderate toxicity after repeated administration
of MiADMSA may be reversible after the
withdrawal of the chelating agent.
Role of Micronutrients or Adjuvants
One of the best measures to minimize heavy
metal exposure is by maintaining nutritional
health. Absorption of lead for an instance is
increased in subjects with deficiencies in iron,
zinc, vitamins (like thiamine); thus maintaining
good nutrition minimizes dietary absorption of
lead. A new trend in chelation therapy has also
emerged recently, which is to use combination
therapy instead of monotherapy with chelating
agents. Vitamins, essential metals or amino acid
supplementation during chelation therapy has
been found to be beneficial in increasing metal
mobilization and providing recoveries in number
of altered biochemical variables. Since the
defense of biological system against damage
caused by activated oxygen involves a battery of
interrelated protective agencies, the
micronutrients which have come to be regarded
as antioxidant nutrients lie functionally at the
heart of this protective mechanism and includes
vitamins such as α-tocopherol, ascorbic acid etc.
These antioxidants when given either alone or in
combination with a chelating agent proved to be
effective in mobilizing metal from soft as well as
hard tissue. It is now well known that most of the
heavy metals with special reference of lead and
arsenic cause their toxicity by the involvement of
reactive oxygen species (ROS). These metals
bind to biological molecules and produce
different free radicals that in turn attack the
building blocks of the biological systems. Recent
studies have shown that lead causes oxidative
stress by inducing the generation of reactive
oxygen species, reducing the antioxidant defense
system of cells via depleting glutathione,
inhibiting sulfhydryl dependent enzymes,
interfering with some essential metals needed for
antioxidant enzyme activities, or increasing
susceptibility of cells to oxidative attack by
altering the membrane integrity and fatty acid
composition. Consequently it is plausible that
impaired oxidant/ antioxidant balance can be
partially responsible for the toxic effects of lead.
The important role of heavy metals in oxidative
damage suggested a new mechanism for an old
problem, whether lead is involved in the
oxidative deterioration of biological
macromolecules. Although several mechanisms
have been proposed to explain the lead-induced
toxicity (128), none of the mechanisms have
been yet defined explicitly. Recent studies
suggest oxidative stress as one of the important
mechanisms of toxic effects of lead (129,130).
The oxidative stress has also been implicated to
contribute to lead associated tissue injury in the
liver, kidneys and brain (131,132). Indirect in
vivo evidence of oxidative involvement in lead
induced pathotoxicity was demonstrated by
alleviation of oxidative stress in the erythrocytes
after treatment with thiol containing proven
antioxidants, N-acetyl cysteine and a succimer in
lead exposed rats (130). Deficiency of several
essential nutrients namely vitamins and essential
elements, has been shown to exacerbate the toxic
effects of metals, and supplementation of such
nutrients ameliorates the toxicity. In addition to
the role of micronutrients in modifying metal
toxicity, these nutritional components can also
act as complimentary chelating agents
FLORA S.J.S. et al.
38
Copyright © 2006 C.M.B. Edition
(adjuvants) increasing the efficacy of a known
chelator, or by acting independently.
Calcium
Interaction between lead and calcium occurs
at several sites in the body, including cellular
mechanisms that regulate ion transport across
membrane (133). Calcium deficiency decreases
lead clearance and increases lead absorption
whereas, calcium excess only decreases lead
clearance slightly and has little effect on lead
absorption. Six and Goyer (134) reported that by
lowering dietary calcium deficiency from 0.7 to
0.1% significantly enhanced the body lead
burden of adult rat exposed to 200 ppm lead in
drinking water for 10 weeks. A significant
increase in tissue lead, urinary delta-
aminolevulinic acid (ALA) and renal intranuclear
lead inclusion bodies was also observed in lead
exposed rats consuming low calcium. Kostial et
al. (135) recommended adequate calcium (940
mg/day) especially for pregnant and lactating
women (to prevent bone resorption) and for
children (to enhance bone mass formation).
Further work in this area will be useful
particularly in view of few recent reports where,
it has been reported that coprophagy may be a
serious complication in the rat model system as
both calcium and lead may be recycled.
The mechanism by which calcium interferes
with lead absorption is not clear however; few
interesting studies using ligated isolated loop
technique suggest that calcium intake rather than
calcium status of the animals modulate lead
absorption. These studies also demonstrated that
at-least in part calcium appears to inhibit lead
absorption via competition for common binding
sites on intestinal binding proteins.
Iron
Iron functions mainly in the regulation of
oxidative processes. It is a component of heme
compounds that transport oxygen, cytochrome
that function in the electron transport chain and
metalloprotein (136,137). Subjects consuming
low iron diet had tissue lead concentration
significantly higher than subjects consuming
adequate iron. Further, excess iron uptake
decreased blood; femur and kidney lead
concentration while the low iron increased the
tissue lead concentration (138-141). Very limited
information of whether or not neurobehavioral
changes and cognitive impairment are more
extreme in iron deficient, lead toxic children than
in either condition, are available. The role of iron
and lead in haem synthesis is well-understood.
The cellular basis for greater susceptibility of
non-iron deficient animals to lead is that limited
iron in the mitochondria apparently enhances the
impairment by lead of iron utilisation for heme
synthesis. Additional studies have demonstrated
the capacity of MT to attenuate the lead-induced
inhibition of blood aminolevulinic acid
dehydratase (142). Existence of MT-like protein
in erythrocyte that binds lead and possibly
protects against lead toxicity by rendering lead
unavailability for retention in the target organs.
Zinc
When dietary zinc is increased over
requirement level, it reduces trace metal
absorption. Lead and zinc are competitive at
tissue sites, which would account for at-least part
of the protective effect of zinc on lead toxicity.
Victery et al. (143) examined the excretion of
lead, zinc and calcium in rats exposed to different
levels of lead. Adult male rats were fed Teklad
AIN-76 diet containing 5.2 g Ca and 0.0314 g Zn
/Kg and received 0, 200, 500 or 1000 ppm lead
as acetate. Brain zinc concentration decreased
significantly in animals while, plasma,
erythrocyte and kidney zinc levels remained
unchanged by lead exposure. We reported the
influence of orally supplemented zinc in
preventing lead intoxication in experimental
animals (144). Thus, the protective effect of zinc
against lead toxicity could be attributed to a
decrease in metal absorption in the
gastrointestinal tract. Zinc could also be
competing for and effectively reducing the
availability of binding sites for trace metal
uptake. Enhanced zinc also increases the renal
and hepatic contents of metallothionein and
causes detoxification through metal binding in
this form.
Arsenic is capable of inducing an increase in MT
levels suggesting the possible role of this
cysteine rich low molecular weight protein.
Kreppel et al. (145) however, reported that zinc
induced increase in MT do not seems to be
responsible for the protective role of pre-
administered zinc against arsenic induced
lethality. Zinc pre-treatment however afforded an
increase in arsenic elimination. Studies on the
effect of zinc on mercury exposure have focussed
mainly on inorganic mercury rather than organic
mercury. It is believed that zinc may reduce lipid
peroxidation by increasing the activities of
enzymes like glutathione peroxidase (GPx) to
Arsenic and lead induced free radical generation
39
Copyright © 2006 C.M.B. Edition
ameliorate the sign of mercury induced
neurotoxicity.
Selenium
Selenium is a required dietary element for
health but it is also a toxic material. It is an
integral component of ubiquitous enzyme
glutathione peroxidase, an antioxidase enzyme.
This enzyme together with superoxide dismutase,
catalase and vitamin E neutralises reactive
oxygen species (ROS). Selenium is known to
affect the distribution of many toxic metals. Role
of selenium in lead intoxication has rather been
controversial. Cerklewski and Forbes (146)
investigated the effect of low and high dietary
selenium on toxicity of dietary lead male rats and
suggested that low dietary levels mildly protects
against toxic effects of lead while, at high levels
it exaggerates the lead toxicity. Rastogi et al.
(147) observed that selenium and lead protect
against toxicity of each other. Enzymatic activity
of ALAD and Cytochrome P-450 in liver was
normal in rats exposed concomitantly to
selenium and lead. We also suggested that oral
administration of selenium could partly prevent
lead toxicity during the course of simultaneous
administration (148). Othman and El-Missiry
(149) reported that intramuscular injection of
selenium prior to lead exposure provided
prophylactic action against lead effects and
observed that selenium enhances the autooxidant
capacity of the cells by increasing the activity of
the superoxide dismutase, glutathione reductase
and glutathione content.
Interaction of arsenic and selenium promotes
the biliary excretion of exogenous selenium and
selenite also augments the excretion of arsenic
into bile. Few authors suggested that arsenic
augmented the hepatobiliary transport of
selenium and facilitated accumulation of
selenium in red blood cells. Selenium in turn
facilitated the biliary excretion of arsenic (150-
152). Glattre et al. (153) studied the distribution
and interaction of arsenic and selenium in rat
thyroid. Combined arsenic plus selenium
administered group exhibited same selenium
concentration as the sum of the mean selenium
concentration in the groups pre-treated with
arsenic or selenium alone suggesting both arsenic
and selenium accumulate in thyroid tissue. Post-
mortem examination of thyroid following arsenic
exposure indicated toxic changes whereas, only
minor changes were observed in selenium or
arsenic plus selenium treated group (153).
Copper
Copper is a component of the mitochondrial
electron transport chain, functions in iron
absorption and mobilization and maintenance of
neurotransmitter levels in brain (154,155).
Adequate intake of copper has been reported to
prevent lead-induced anemia, which was
developed when dietary copper was low (156).
However, in an conflicting report animals fed
low, adequate and high copper to the lead
exposed rats, exhibited a pronounced decrease in
lead contents in animals fed low calcium diet
while, high copper diet produced an increase
tissue lead accumulation. There are few reports
concerning cadmium-copper interaction. Dietary
copper supplementation reduces mortality rate
and severity of anemia, in experimental animals.
Cadmium exposure has been reported to produce
disturbances in the copper metabolism
particularly depletion of plasma copper and
copper sensitive ceruloplasmin. Beneficial effect
of copper has been attributed to the competition
between cadmium and copper for binding to the
metallothionein (MT). Copper may displace
cadmium for MT because of its higher affinity
for the protein (157,158).
Role of Antioxidants
Induction of reactive oxygen species by
metal and subsequent depletion of antioxidant
cell defenses can result in disruption of the pro-
oxidant / antioxidant balance in mammalian
tissues. In the event that oxidative stress can be
partially implicated in metal toxicity, a
therapeutic strategy to increase the antioxidant
capacity of cells may fortify the long term
effective treatment of metal poisoning. This may
be accomplished by either reducing the
possibility of metal interacting with critical
biomolecules and inducing oxidative damage, or
by bolstering the cells antioxidant defenses
through endogenous supplementation of
antioxidant molecules. Although many
investigators have confirmed lead induced
oxidative stress, the usefulness of antioxidants
along or in conjunction with chelation therapy
has not been extensively investigated yet.
Recently we (122) explored the therapeutic
efficacy of antioxidant along with a chelating
agent during the removal of lead in rats. Some
groups (50,159-163) investigated the ability of
some molecules with antioxidant activity to
prevent or treat experimental lead toxicity in
animals.
FLORA S.J.S. et al.
40
Copyright © 2006 C.M.B. Edition
The following part apprises with some
antioxidants that have been tried in treatment of
metal poisoning with special reference to lead
and arsenic (Figure 5).
N-Acetyl cysteine (NAC)
NAC is a thiol-containing antioxidant that
has been used to mitigate various conditions of
oxidative stress. Its antioxidant action is
believed to originate from its ability to stimulate
GSH synthesis, therefore maintaining
intracellular GSH levels and scavenging reactive
oxygen species (ROS) (164,165). Besides the
antioxidant potential, NAC also has some
chelating properties against lead (166). One of
the first reports by Pande et al., (122) suggested
that NAC could be used both as preventive as
well as a therapeutic agent along with
MiADMSA/DMSA in the prevention and
treatment of lead intoxication in rats. Pande et al
(122) reported that simultaneous administration
of NAC with succimer reversed the altered
ALAD and TBARS levels, increased the reduced
glutathione levels and decreased the lead levels,
apart from this the study too highlighted the
favorable response of NAC in post-exposure
treatment along with succimer (122). Combined
administration of NAC and succimer post arsenic
exposure led to a significant turnover in variables
indicative of oxidative stress and removal of
arsenic from soft organs (103). A recent report
suggested that co-administration of NAC along
with succimer in sub-chronically lead exposed
rats, reduced oxidative stress significantly by
lowering the TBARS levels, oxidized glutathione
levels along with the decrease in the lead burden
on the soft tissues especially the brain (167).
Melatonin
Melatonin, N-acetyl-5-methoxy triptamine, is
a hormonal product of the pineal gland that plays
many roles within the body including control of
reproductive functions, modulation of immune
system activity, limitation of tumorigenesis and
effective inhibition of oxidative stress (168).
One major function of melatonin is to scavenge
radicals formed in oxygen metabolism (168,169),
thereby potentially protecting against free radical
induced damage to DNA, proteins and
membranes (168,170). It has been shown that
melatonin stimulates the antioxidative enzyme
GPx in the brain, thus providing indirect
protection against free radical attack (171). In
animal experiments, metatonin prevented the
induction of free radical damage by a variety of
conditions including ingestion of toxins, ionizing
radiation, ischaemia, reperfusion and excessive
exercise (172-176). Melatonin has a molecular
weight of 232 and is both lipid (177,178) and
water soluble (179), although its solubility in
lipid is clearly greater.
α
-Lipoic acid (LA)
α-lipoic acid is a naturally occurring
antioxidant and is able to abate some of the toxic
effects of lead (180). It functions as a cofactor in
several multienzyme complexes (181). Its
reduced form, dihydrolipoic acid (DHLA), has
two free sulfhydryl groups and the two forms
LA/DHLA possess a great antioxidant potential
(182). Both LA and DHLA (i) have the ability to
scavenge some reactive species (ii) can
regenerate other antioxidants (i.e. vitamins E and
C and GSH) from their radical or inactive forms,
and (iii) have metal chelating activity. Lipoic
acid also have an advantage over NAC in
opposing GSH loss, since LA is effective in a
micromolar range while millimolar NAC is
needed for a similar effect (183). The capability
of LA to cross the blood brain barrier (184) is an
extra advantage because the brain is an important
target in lead poisoning.
Vitamin E (
α
-tocopherol)
Various vitamins have been found to reduce
the toxic manifestation of lead (185,186). Dietary
oral supplementation with these vitamins often
lessens the severity of lead poisoning by
inhibiting the lead absorption or interaction at the
macromolecular site of physiological action
(186-188).
The antioxidant function of vitamin E has
also been proposed in cadmium induced brain
damage (189). It also appears that the protective
effect of vitamin E in lead toxicity is attributed
mainly to its antioxidant property. Anemia,
splenomegaly and increased fragility of red blood
cells in lead toxicity of vitamin E deficient rats
have been reported (190-193). Vitamin E which
is a low molecular mass antioxidant interact
directly with the oxidizing radicals (194,195) and
protect the cells from reactive oxygen species
(196). The lipid soluble, non-enzymatic
antioxidant, α-tocopherol checks the lipid
peroxidation through limiting the propagation of
chain reaction of lipid peroxidation (197). Lead
poisoning has been shown to cause a marked
anaemia in vitamin E deficient rats indicating a
possible involvement of this vitamin in the
synthesis of heme protein. It is believed that
Arsenic and lead induced free radical generation
41
Copyright © 2006 C.M.B. Edition
vitamin E, as a scavenger of free radicals, might
be reacting with methyl radicals that might be
formed in the breakdown to provide protection.
Addition of vitamin E may also alleviate arsenic
toxicity. The protective mechanism of vitamin E
could be attributed to its antioxidant property or
its location in the cell membrane and its ability to
stabilize membrane by interacting with
unsaturated fatty acid chain. Flora et al (198)
reported that administration of Vitamin C or
vitamin E when given in combination with
succimer or its monoisoamyl derivative
(MiADMSA) produced profound recoveries in
sub-chronically lead exposed rats. Although the
group suggest that vitamin C was better in
providing clinical recoveries and Vitamin E was
equally efficient in decreasing the lead burden
from the tissues.
Vitamin C (Ascorbic acid)
Vitamin C is a low molecular mass
antioxidant that interacts directly with the
oxidizing radicals (195) and protect the cells
from reactive oxygen species (196). It is well
known that ascorbic acid enhances the absorption
of dietary iron by increasing the solubility of iron
at the alkaline pH of the intestine and by
maintaining the ferrous iron in its reduced
oxidation state (199). Vitamin C scavenges the
aqueous reactive oxygen species (ROS) by very
rapid electron transfer that thus inhibits lipid
peroxidation (199). It acts mainly as an
antioxidant molecule and its beneficial effects
could be attributed to its ability to complex with
lead (188). Vitamin C. Animal studies have
suggested an antagonistic effect of ascorbic acid
on lead absorption and toxicity and ascorbic acid
may even chelate lead as effectively as EDTA.
However, studies in humans have shown some
mixed results. In a study with 78 male workers,
38 received vitamin C and 38 were given placebo
(200). They found no effect of ascorbic acid on
absorption or excretion of lead. However, 47
psychiatric patients receiving ascorbic acid and
zinc showed reduced blood lead concentration
(200). Simon and Hudes investigated the
association between ascorbic acid concentration
and the prevalence of elevated blood lead
concentration in 19, 578 participants ages 6 years
and older in National Health and Nutrition
Examination Survey 1988-94 (NHANES III)
(201). Serum ascorbic acid concentration was
inversely associated with prevalence of elevated
blood lead concentration in children. However,
no relationship between dietary ascorbic acid
intake and blood lead concentration has been
reported. Vitamin C has been shown to be
effective against methyl mercury intoxication
however; no definite conclusion regarding its
beneficial effects could be drawn.
Taurine
Taurine a semi essential amino acid has been
shown to have a role in maintaining calcium
homeostasis, osmoregulation, removal of
hypochlorous acid and stabilizing the membranes
(202,203). The highest concentrations of taurine
occur in developing brain, at which time the
concentrations of other free amino acids tend to
be low (203).
Figure 5. Chemical structures of antioxidants
known for their protective efficacy against arsenic
and lead
Some of the recent data indicate that taurine
can act as the direct antioxidant by scavenging
ROS and/or as an indirect antioxidant by
preventing changes in membrane permeability
due to oxidant injury (202). The zwitterionic
nature of taurine gives it high water solubility
and low lipophilicity. Consequently compared
with carboxylic amino acids, diffusion through
lipophillic membranes is slow for taurine (203).
In the studies conducted by Gurer and Ercal
(165), taurine was shown to have beneficial
effects in lead induced oxidative stress in
Chinese Hamster Ovary (CHO) cells and F344
rats (165). There was an increased cell survival
in taurine treated lead exposed CHO cells while
MDA levels were diminished and GSH levels
FLORA S.J.S. et al.
42
Copyright © 2006 C.M.B. Edition
were increased. Similar effects were found in
RBC and the brains and livers of lead exposed F-
344 rats. In the above study, no chelating effect
of taurine (1.2 g/kg/d) was indicated by any
change in lead concentrations in the blood,
brains, livers and kidneys after taurine treatment.
An antioxidant mechanisms rather than a
chelating activity, seems to underlie this
observed effects of taurine against lead-induced
oxidative stress.
Although a lot of work has been done for the
treatment of lead and arsenic poisoning, still we
are far away from having a safe, specific and
effective chelating agent for the treatment against
these deadly toxic metals. Besides, further
knowledge is needed in several basic research
areas within the field of in vivo chelation of
metals and call for studies on (a) Molecular
mechanism of action of clinically important
chelators, (b) Intracellular and extra cellular
chelation in relation to mobilization of aged
metal deposits and the possible redistribution of
toxic metal to sensitive organs as the brain, (c)
Effect of metal chelators on biokinetics during
continued exposure to metal, especially possible
enhancement or reduction of intestinal metal
uptake, (d) Combined chelation with lipophilic
and hydrophilic chelators, which presently has a
minimal clinical role, (e) Use of antioxidants,
micronutrients or vitamins as complimentary
agents or antagonists (f) Minimization of the
mobilization of essential trace elements during
long-term chelation, and (f) Fetotoxic and
teratogenic effects of chelators.
Acknowledgements – Authors thank Mr. K. Sekhar,
Director of the establishment for his support and for
providing facilities.
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