amelioration of arsenic toxicity by l-ascorbic acid in laboratory · pdf filejournal of...

Journal of Environmental Biology �April, 2007�

Amelioration of arsenic toxicity by L-Ascorbic acid in laboratory rat

Sohini Singh and S.V.S. Rana*

Department of Zoology, Toxicology Laboratory, Ch. Charan Singh University, Meerut-250 004, India

(Received: October 28, 2005 ; Revised received: July 25, 2006 ; Accepted: August 30, 2006)

Abstract: A study, so as to confirm the protective effects of L-ascorbic acid against inorganic arsenic (As2O

3) toxicity was made in male Wistar rats.

Multiphase observations made on iAs concentration in target organs viz. liver and kidney, liver function, histopathological changes, ultrastructural

alterations, lipid peroxidation, oxidative stress and iAs-DNA interaction strongly favoured its ameliorative effects. These effects could mainly be attributed

to its antioxidative property. It offers help in regeneration of GSH and α-tocopherol. The chelation of iAs by ascorbic acid has also been hypothesized.

Inhibition of DNA damage by ascorbic acid in liver and kidney appears to be the most significant part of this study. On the basis of these results, we

conclude that administration of L-ascorbic acid to arsenic affected population may prevent the occurrence of fatal human diseases.

Key words: Arsenic, L-ascorbic acid, Liver, Kidney, Lipid peroxidation and Oxidative stress

*Corresponding author: E-Mail: [email protected], Tel.: 91-121-2774569, Fax: 91-121-2772930

Introduction

Arsenic is a naturally occurring metalloid (atomic number

33), located on group V of the periodic table. Exposure to high

levels of arsenic through drinking water has been recognized for

many decades in some regions of the world, i.e. China, India,

and some countries in Central and South America. Millions of

people are at risk of cancer and other diseases because of chronic

arsenic exposure (NRC, 1999, 2001). Environmental exposure

to arsenic can cause a variety of cancers, most commonly

nonmelanoma skin cancers, and chronic toxicity may manifest

as diffuse symptoms not easily recognizable as chronic heavy

metal toxicity. General adverse health effects associated with

human exposure to arsenicals include cardiovascular diseases,

developmental abnormalities, neurologic and neurobehavioral

disorders, diabetes, hearing loss, fibrosis of the liver and lung,

hematological disorders and blackfoot disease (Abernathy et al.,

1999; Sordo et al., 2001 and Tchounwou et al., 1999). In humans,

arsenic is known to cause cancer of the skin (Rossman et al.,

2004) lung, bladder, liver and kidney (Abernathy et al., 1999;

Kitchin, 2001 and Tchounwou et al., 1999).

Arsenic is methylated by alternating reduction of

pentavalent arsenic to trivalent and addition of a methyl group

from S-adenosylmethionine (Marafante et al., 1985). Glutathione

and possibly other thiols, serve as reducing agents

Fig. 1: Biomethylation is the main mechanism for the metabolism of inorganic arsenic. It involves alternate

steps of two-electron reduction followed by oxidative addition of a methyl group

Journal of Environmental Biology April 2007, 28(2) 377-384 (2007)

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Sohini Singh and S.V.S. Rana

(Delnomdedieu et al., 1994; Styblo et al., 1995). Liver is the most

important site of arsenic methylation (Marafante et al., 1985;

Geubel et al., 1988) but most organs show methylating activity.

The end metabolites are methylarsenic acid (MMA) and dimethyl

arsenic acid (DMAA) (Fig. 1). These compounds are readily

excreted in urine. However, reactive intermediates may be

formed.

Arsenite is known to bind to cellular sulfhydryl, particularly

vicinal ones, accounting for its ability to interfere with energy

generation (Aposhian, 1989). Once in the tissues, arsenic exerts

its toxic effects through several mechanisms, the most significant

of which is, the reversible combination with sulfhydryls groups.

Arsenic also inhibits numerous other cellular enzymes, especially

those involved in cellular glucose uptake, gluconeogenesis, fatty

acid oxidation and product-ion of glutathione through sulfhydryl

group binding

A second major form of toxicity is termed “arsenolysis”.

Pentavalent arsenate can substitute competitively for phosphate

in biochemical reactions, where ADP would normally

phosphorylate into ATP. In the presence of arsenic, ADP arsenate

is the end product and high energy phosphate bonds are not

formed. The unstable ADP-arsenate decomposes spontaneously

and irreversibly resulting in loss of energy by the cell. ROSs are

capable of damaging a wide variety of cellular macromolecules

including DNA, lipids and proteins. Finally, cellular signal

transduction can be altered (e.g. activation of trans factors,

changes of gene expression), cell growth, proliferation and

differentiation can be promoted and apoptosis leading to cell

death or cancer developments can be induced (Yang and Frenkel,

2002; Qian et al., 2003).

The idea that arsenical induced toxicity could be modified

by nutrients was initially proposed in the early 1930’s by Mayer

and Sulzberger (1931), who suggested that adequate levels of

ascorbic acid, in the diet prevented or reduced occurrence of

arsenic induced anaphylaxis. L-ascorbic acid is a primary

defensive nutrient by virtue of its function as a free radical

scavenger. It can react in aqueous media against in vivo

peroxidants, including quenching of singlet oxygen species. It

increases the turn over rate of toxic metals and reduces damage

by scavenging free radicals generated by their metabolism (Hume

et al., 1991). During acute response to different stressors such

as metals and heat shock etc. ascorbic acid is depleted (Parihar

and Dubey, 1995 and Lackner, 1998). Therefore, a study on the

plausible ameliorative effects of L-ascorbic acid against iAs

toxicity in rat was proposed.

Materials and Methods

Chemicals: Arsenic trioxide was purchased from Loba Chemie

(Mumbai). L-ascorbic acid, reduced glutathione (GSH),

nicotinamide adenine dinucleotide (NAD), 5-5, dithio-bis-2-

nitrobenzoic acid, 1-chloro-2, 4-dinitrobenzene, bovine serum

albumin, triton X-100, sodium lauryl sulphate, proteinase-K,

RNAase, were obtained from Sigma Chemical Company (St

Louis, Mo., USA). Thiobarbituric acid (TBA) was purchased from

Wako Chemical Company (Japan). NADPH was obtained from

SRL (Mumbai). All other chemicals or reagents of highest purity

were procured from S. Merck, S. D. Fine Chemicals and

Qualigens (Mumbai).

Model: Male Wistar rats (150 ± 20 g) were procured from the

animal facility of Jamia Hamdard, New Delhi. They were housed

individually in polypropylene cages under standard laboratory

conditions (room temperature 25 ± 5oC; RH = 50 ± 10%). Each

rat was offered food pellets (Golden Feeds, New Delhi) and tap

water ad libitum. All animal treatments and protocols employed

in this study received prior approval of the Institutional Ethical

Committee and met the standards laid down by Govt. of India.

Treatments: Twenty healthy male rats weighing 200 ± 30 g were

selected for present study. After acclimatization to laboratory

conditions, rats were divided into four groups, each containing

five rats. Rats of group A, in addition to food and drinking water

were administered 1.0 ml saline by gavage each alternate day

and treated as controls. Rats of group B were administered

predetermined sublethal dose (4 mg/100 g body weight) (LD50

10 mg/100g body weight) of arsenic trioxide dissolved in saline

through gavage on each alternate day for 30 days as described

earlier (Allen and Rana, 2003). Rats of group C were administered

the same dose of arsenic trioxide in the same concentration and

manner similar to the rats of group B. Moreover, they were

administered with 25 mg/100 g body weight of L-ascorbic acid

simultaneously on each alternate day for thirty days. Rats of group

D were administered L-ascorbic acid only (25 mg/100 g body

weight) as the rats of group C and treated as controls.

Sample preparations: On 31st day rats were starved overnight

and sacrificed next morning by light ether anesthesia. Blood was

collected through cardiac puncture. Serum was separated by

centrifugation at 5000 rpm for 20 min. and processed for the

estimation of serum transaminases and bilirubin. Small pieces

of liver and kidney were carefully removed from each rat and

processed suitably for the estimation of arsenic, glutathione,

microsomal lipid peroxidation, glutathione-S-transferase, isolation

and quantification of DNA, histopathological and EM studies.

Estimation of arsenic in soft tissue, serum and urine: 1.0

gm of wet tissue/1.0 ml serum or urine sample was digested in

10.0 ml of concentrated nitric acid at 80oC for 16 hr. A 2.0 ml aliquot

of the digest was analyzed for inorganic As by hydride generation

at pH 6, using sodium borohydride as the reducing agent. The

analyses were done using atomic absorption spectrophotometer

(Electronic Corporation, India). Absorbance was recorded at 193.7

nm, using a hollow cathode lamp for arsenic.

Determination of serum transaminases: Alanine amino

transaminase (ALT) and aspartate amino transferase (AST)

378


Arsenic toxicity in laboratory rat

activity in serum were determined following the method of

Reitman and Frankel (1957) using a kit procured from Span

Diagnostics (Surat, Gujrat).

Determination of total bilirubin in serum: Total bilirubin in

serum was determined by a commercial kit obtained from Ozone

Biomedicals Pvt. Ltd. (Hyderabad) using the method of Burtis

and Ashwood (1996).

Histopathological study: Small pieces of liver and kidney were

carefully removed from experimental animals and fixed in 10%

buffered neutral formalin. 5 micron thick paraffin sections thus

prepared were stained with hematoxylin and eosin and examined

under research microscopes (Nikon, Japan).

Electron microscopic studies (Transmission electron

microscopy): Very small cubes (1 mm3) of liver and kidney were

immersed in 2.5% glutraldehyde, post fixed in 1.0% osmium

tetraoxide, dehydrated through a graded series of ethanol and

embedded in Epon 812 after several changes of propylene oxide.

Ultra thin sections stained with uranyl acetate and lead citrate

were examined under a Phillips, CMIO Transmission electron

microscope, at AIIMS, New Delhi.

Estimation of reduced glutathione: Glutathione was measured

as acid soluble sulfhydryl levels assayed by the method described

by Ellman (1959). The acid soluble sulfhydryl group generated a

yellow colored complex (5-thio-2-nitrobenzene) with DTNB. Its

absorbance was recorded at 412 nm.

Oxidized glutathione: GSSG was estimated in the liver and kidney

following the method suggested by Ohmori et al. (1986). Peptides

having a c-terminal glycine exhibit a color reaction similar to that

of glycine with acetic anhydride, p-dimethylamino-benzaldehyde

and pyridine. Absorbance was recorded at 458 nm.

Glutathione-S-transferase (E.C.2.5.1.8): Glutathione-S-

transferase was assayed by the method of Habig et al. (1974).

Its activity was determined by the rate of formation of conjugate

between reduced glutathione and 1-chloro 2,4-dinitrobenzene.

The conjugate absorbs strongly at 340 nm wavelength.

Determination of lipid peroxidation: Peroxidized membrane

lipids were estimated by method described by Jordan and

Schenkman (1982). Microsomal pellets were precipitated by

calcium according to the method suggested by Schenkman and

Cinti (1978).

Malondialdehyde and other reactive oxidized products

of membrane lipids, under acidic conditions, react with

thiobarbituric acid and form a pink colored chromogen which is

strongly absorbed at 532 nm wavelangth.

Protein content was determined by the method described

by Lowry and coworkers using bovine serum albumin (Sigma,

USA) as the standard Lowry et al. (1951).

AsIII-DNA interaction: DNA from liver and kidney samples were

eluted using Genelute mammalian genomic DNA miniprep kit

procured from Sigma. The concentration was determined by

spectrophotometric analysis and the absorbance was measured

at 260 nm and 280 nm using a quartz microcuvette (Sambrook

et al., 1989).

Estimation of fragmented DNA: Cells were lysed using tris-

EDTA buffer and Tritox-100. Lysate was centrifuged at 13000xg

and intact and fragmented DNA were separated. The respective

samples were precipitated by trichloroacetic acid. DNA in each

fraction was quantified using diphenylamine reaction. Absorbance

was recorded at 600 nm against a blank (Sellins and Cohen,

1987).

Statistical analyses: Statistical evaluation of results was

simultaneously made employing SPSS software.

Results and Discussion

Urinary excretion of arsenic increased several fold in

arsenic fed male and female rats. Arsenic concentration

decreased in liver, kidney, urine and serum on simultaneous

treatments with ascorbic acid (Table 1, 2). Ascorbic acid has

earlier been reported as a possible chelator of lead with similar

potency as that of EDTA (Bratton et al., 1981). One possibility of

ascorbate protection might be the mobilization of arsenic from

Table - 1: Arsenic concentration (µg/ml) in the serum and urine of arsenic

and ascorbic acid treated rats

Group Treatment Serum Urine

A Control 0.011 ± 0.003 ND

B Arsenic 0.599 ± 0.076* 0.100 ± 0.01*

C Arsenic + 0.136 ± 0.014*� 0.071 ± 0.013*�

ascorbic acid

D Ascorbic acid 0.0126 ± 0.003 NS� 0.020 ± 0.016*�

Results are expressed as mean ± SE (n = 5)NS denotes non significant

* denotes significantly different values from control rats (p<0.05) “t” test� denotes significantly different results from arsenic treated rats (p< 0.05)

‘t’ test

Table -2: Arsenic concentration (µg/gm) in the liver and kidney of arsenic


Group Treatment Liver Kidney

A Control 0.014 ± 0.002 0.032 ± 0.006

B Arsenic 105 ± 13.07* 127 ± 5.41*

C Arsenic + 64.4 ± 7.28*� 71.4 ± 7.39*�

ascorbic acid

D Ascorbic acid 0.008 ± 0.028NS� 0.018 ± 0.006NS�

Results are expressed as mean ± SE (n = 5)

NS denotes non significant


“t” test

379


soft tissues. Another reason might be the reduced

absorption of arsenic from intestine. Ingested arsenic that

accumulates in tissues, binds with sulfhydryl groups (Aposhian

and Aposhian, 1989) and the remaining is excreted in the urine.

Thus it could be envisaged that ascorbic acid might play a

therapeutic role against general arsenic toxicity.

Arsenic is known to produce disturbances in liver

function (Fowler et al., 1977). AST and ALT are reliable

determinants of liver parenchymal injury (Moss et al., 1987).

Activities of both ALT and AST significantly increased in arsenic

treated rats indicating liver dysfunction. In arsenic and ascorbic

acid treated rat, values of ALT and AST declined significantly.

Similarly serum bilirubin increased in arsenic treated rats but

declined in ascorbic acid and arsenic treated rats (Table 3). These

results suggest that ascorbic acid protects against hepato toxicity

of arsenic by improving liver function.

In the present study low levels of GSH were observed in

the liver and kidney of arsenic treated male and female rats. A

moderate increase in oxidized glutathione (GSSG) was, however,

recorded (Table 4 and 5). The intracellular level of GSH has been

inversely correlated with cytotoxicity of arsenic (Ochi et al., 1996).

GSH status improved on ascorbic acid co-treatment. The

improved levels of GSH might protected the sulfydryl groups from

binding with arsenic and promoted the detoxification of arsenic

by modulating arsenic methylation reactions (Buchet and

Lauwerys, 1988). When GSH levels are reduced, inorganic

arsenic becomes more toxic (Shimizu et al., 1998). GSH depletion

causes essentially nontoxic MMAsV to become toxic (Sakurai,

2003). DMAsV induces apoptosis after GSH depletion (Sakurai,

2002), which could allow the survival of damaged cells.

Glutathione-S-transferase (GST) consists of a large family

of GSH utilizing enzymes that play an important role in

detoxication of xenobiotics in mammalian systems. Arsenic

trioxide was found to inhibit glutathione-S-transferase activity

significantly in liver and kidney both (Habig et al., 1974; Lee et

al., 1989 and Wendel, 1980). However, ascorbate co-treatment

restored enzyme activity in male rats. This was considered as an

Table - 4: Reduced glutathione (GSH) (µg/g wet weight) in the liver and kidney of arsenic treated rats

Group Treatment Gender Liver Kidney

A Control Male 0.154 ± 0.060 0.169 ± 0.039

B Arsenic Male 0.126 ± 0.006* 0.105 ± 0.013*

C Arsenic + ascorbic acid Male 0.130 ± 0.010*NS 0.170 ± 0.004NS�

D Ascorbic acid Male 0.139 ± 0.002NS� 0.136 ± 0.006*�


* denotes significantly different values from control rats (p<0.05) “t” test� denotes significantly different results from arsenic treated rats (p< 0.05) “t” test

Table - 5: Oxidized glutathione (GSSG) (µ moles/gm wet weight) in the

liver and kidney of arsenic treated rats


A Control 0.535 ± 0.030 0.429 ± 0.077

B Arsenic 3.168 ± 0.053* 3.644 ± 0.018*

C Arsenic + 1.627 ± 0.114*+ 0.956 ± 0.060*+

ascorbic acid

D Ascorbic acid 0.560 ± 0.023NS+ 0.392 ± 0.033*+


* denotes significantly different values from control rats (p<0.05) “t” test+denotes significantly different results from arsenic treated rats (p< 0.05)

“t” test

Table - 6: Glutathione-S-transferases (GST) (n moles/NADPH/min/mg

protein) in the liver and kidney of arsenic treated rats


A Control 0.865 ± 0.035 0.727 ± 0.015

B Arsenic 0.426 ± 0.021* 0.355 ± 0.008*

C Arsenic +

ascorbic acid 0.677 ± 0.007*� 0.570 ± 0.013*�

D Ascorbic acid 0.806 ± 0.013*� 0.689 ± 0.031*�



“t” test

Table - 3: AST (Karmen Units), ALT (Karmen Units) and bilirubin (mg/dl) in the serum of arsenic treated rats

Group Treatments AST (Karmen Units) ALT (Karmen Units) Bilirubin (mg/dl)

A Control 42.25 ± 1.29 42.0 ± 1.15 17.98 ± 0.53

B Arsenic 120.51 ± 1.48* 136.96 ± 2.19* 40.58 ± 0.921*

C Arsenic + Ascorbic acid 89.71 ± 1.39*+ 98.99 ± 1.057*+ 28.22 ± 1.00*+

D Ascorbic acid only 42.90 ± 0.81NS+ 41.95 ± 0.623NS+ 15.63 ± 0.66NS+

Values are expressed as mean ± SE (n = 5) p<0.05

* denotes values significantly different from control rats.+ Denotes values significantly different from arsenic treated rats

Sohini Singh and S.V.S. Rana380


adaptive response facilitated by ascorbic acid against arsenic

induced stress (Table 6).

Lipid peroxidation has been largely considered as a

molecular mechanism involved in deleterious effects of a variety

of xenobiotics including heavy metals (Sunderman et al., 1985).

Biomembranes and subcellular organelles are the major site of

lipid peroxidation (Halliwell and Gutteridge, 1989). Co-treatments

of ascorbic acid and arsenic inhibited lipid peroxidation in liver

and kidney both. This effect could be attributed to its antioxidative

property. Ascorbic acid is well known to inhibit oxidative damage

to membranes (Li et al., 2001; Nandi et al., 2005) (Table 7).

The induction of oxidative DNA damage by arsenic in

mammalian cells has been paid considerable attention by earlier

workers. (Kessel et al., 2002). There is evidence that both DMAIII

and MMA III exert DNA damaging effect through an intermediate

involving ROS production (Nesnow et al., 2002). Arsenic perturbs

cells in numerous ways i.e. inducing chromosomal abnormalities,

altering DNA repair or DNA methylation patterns and producing

oxidative stress (Hamadeh et al., 2002; Liu and Jan, 2000; Mass

et al., 2001). Present observations on total amount of DNA in

liver and kidney showed that arsenic trioxide significantly

damaged DNA. Results also indicated higher percentage of

fragmented DNA in liver and kidney both after arsenic treatment.

Further, it was found that cotreatments with ascorbic acid reduced

the percentage of fragmented DNA and increased the amount of

total DNA in liver and kidney both (Table 8, 9 and 10). Thus a

protective effect of ascorbic acid on DNA damage could be

envisaged. An earlier study suggested that ascorbic acid inhibited

DNA damage caused by an antitumor drug (Blasiak and Kowalik,

2001).

To gather more evidence to support the protective

behavior of ascorbic acid against arsenic toxicity, histopathological

studies, using both light and electron microscopy were

undertaken. Light microscopical observations showed that arsenic

caused hepatic parenchymal degeneration, hyperplasia and

vascular lesions in male rats. Sinusoids were dilated and found

to be filled with foam cells. Significant protective effects on

different lesions were observed in the liver of rats treated with

arsenic and ascorbic acid. There was no neoplastic formation,

however, mild inflammation of hepatic cells still persisted (Fig. 1,

2). While the ultrastructural study of liver, of arsenic treated rats,

showed inflammed nuclei and increased number of mitochondria

in hepatic cells treated with arsenic and ascorbic acid. The

mitochondria of different shapes and sizes were observed and

several small vacuoles were observed in the matrix of the cell

(Fig. 3 and 4).

Light microscopical observations on kidney of arsenic

treated rats showed glomerulonephritis, proximal tubular necrosis,

epithelial damage and loss of nuclei. In cortex focal tubular

necrosis was observed along with pycnosis and appearance of

Table - 7: Microsomal malondialdehyde (n moles/mg protein) in the

liver and kidney of arsenic treated rats


A Control 0.120 ± 0.004 0.135 ± 0.005

B Arsenic 0.325 ± 0.009* 0.460 ± 0.059*

C Arsenic + 0.211 ± 0.003*� 0.235 ± 0.007*�

ascorbic acid

D Ascorbic acid 0.113 ± 0.012NS� 0.125 ± 0.017NS�



“t” test

Table - 8: Intact and fragmented DNA (µg/ml) in the liver of arsenic and

ascorbic acid treated rats

Group Treatment Total DNA % Fragmentation

A Control 31.59 ± 0.332 23.46 ± 0.478

B Arsenic 12.56 ± 0.253* 34.69 ± 0.445*

C Arsenic + 29.58 ± 0.389*� 25.84 ± 0.345*�

ascorbic acid

D Ascorbic acid 33.22 ± 0.482*� 21.65 ± 0.779*�



“t” test

Table - 9: Intact and fragmented DNA (µg/ml) in the kidney of arsenic


Group Treatment Total DNA % Fragmentation

A Control 26.39±.299 20.76±0.492

B Arsenic 15.48±0.322* 38.20±0.257*

C Arsenic + 31.95±0.425* 26.10±0.697*�

ascorbic acid

D Ascorbic acid 30.71±0.399*� 21.13±0.898NS�



“t” test

Table - 10: DNA (µg/ml) in the liver and kidney of arsenic treated rats


A Control 39.6 ± 0.452 39.2 ± 0.717

B Arsenic 33.4 ± 0.358* 45.60 ± 0.457*

C Arsenic +

ascorbic acid 152.6 ± 5.850*� 55.20 ± 3.23*�

D Ascorbic acid 81.6 ± 4.73*� 35.6 ± 1.807*�



“t” test

Arsenic toxicity in laboratory rat 381


Fig. 2: T.S. of liver of an arsenic fed rat shows parenchymal

degeneration, hyperplasia and vascular lesions (H/E X 400)

Fig. 3: T.S. of liver of rat treated with arsenic and ascorbic acid

shows binucleated cells and focal pycnosis (H/E X400)

Fig. 4: T.E.M. study of liver of arsenic treated rat shows swollen

nuclei and increased number of mitochondria (M) (880X)

Fig. 5: T.E.M. study of liver of rat treated with arsenic and ascorbic acid

shows normal nucleus (N). Several vacuoles are also observed (880X)

Fig. 6: T.S. of kidney of arsenic treated rat shows inflammed

glomerulus, and proximal tubular necrosis (H/E X 400)

Fig. 7: T.S. of kidney of rat treated with arsenic and ascorbic acid

shows mild glomerulonephritis and tubular necrosis (H/E X 400)



Fig. 8: T.E.M. study of kidney of arsenic treated rat shows

mitochondria (M) of different shapes and sizes (880X)

Fig. 9: T.E.M. study of kidney of rat treated with arsenic and

ascorbic acid shows basolateral membrance infoldimgs

tightly associated with mitochondria (M) (1400X)

hyaline like deposits in medulla. Kidney of rat treated with

arsenic and ascorbic acid, showed little improvement in terms of

proximal tubular necrosis and glomerular injury (Fig. 5 and 6).

Ultrastructure of kidney of arsenic treated rats showed, basolateral

infoldings. Number of lysosomes was also found to be increased.

Brush border, the proximal tubule and distal tubules were damaged

(Fig. 7). Ultrastructure of kidney of male rats, treated with arsenic

and ascorbic acid, showed improvements in renal structure.

Proximal tubule was found to be lined with cuboidal cells and

apical brush border. Basolateral membrane infoldings were found

to be tightly associated with mitochondria (Fig. 8).

Ascorbic acid has a few therapeutic characters. It is a

water soluble antioxidant. It predominantly works as a radical

chain terminator. One ascorbate molecle reacts with a peroxyl

radical to yield a hydroperoxide and ascorbyl radical,

subsequently the ascorbyl radical can react with another peroxy-

radical and produce the oxidized ascorbic acid i.e., dihydroscorbic

acid (Combs and Gray, 1998). Thus one molecule of ascorbate

can trap two molecules of peroxyl radicals. Another hypothesis

suggests that ascorbic acid may be involved in the regeneration

or restoration of antioxidant properties of α-tocopherol. The

tocopherol is converted to α-tocopherol quinone (Chow, 1985).

Ascorbate can both chelate and reduce transition metal

ions and the reduced metal ions in turn can reduce oxygen or

H2O

2 to superoxide and hydroxyl radicals, respectively.

AsCH- + Me(n+1)+ → AsC*- + H+ + M n+ (Eq. 1)

H2O

2 + M n+ → OH*. + OH- + M (n+ 1)+ (Eq. 2)

Superoxide and hydroxyl radicals are scavenged by

ascorbate with second order rate constant of 1x105 and 1.1x1010

M-1S-1 respectively. (Carr and Frei, 1999). Therefore, ascorbic

acid forms the first line of antioxidant defense (Frei, 1999; Carr

and Frei, 2000). These properties of ascorbic acid make it a

suitable antidote for arsenic toxicity in rodents and possibly in

human subjects.

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