Article

Ascorbic acid inhibits ferric
nitrilotriacetate induction of
ornithine decarboxylase,
DNA synthesis, oxidative stress,
and hepatotoxicity in rats

Toxicology and Industrial Health
2015, Vol. 31(11) 1008–1014
© The Author(s) 2013
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DOI: 10.1177/0748233713493824
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S Ansar1 and M Iqbal2
Abstract
Ascorbic acid (AA) is a naturally occurring phenolic compound with antioxidant properties used in food,
cosmetics, and pharmaceutical products. In this study, the effect of AA on ferric nitrilotriacetate (Fe-NTA)induced hepatotoxicity in rats has been examined. Fe-NTA alone enhances ornithine decarboxylase activity to
4.5-fold and tritiated thymidine incorporation in DNA to 3.6-fold in livers compared with the corresponding
saline-treated controls. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction
to 3.02- and 1.88-fold, respectively, at a higher dose of 2 mg AA per day per animal, compared with the FeNTA-treated groups. Fe-NTA treatment also enhanced the hepatic microsomal lipid peroxidation to 1.7fold compared to saline-treated controls. These changes were reversed significantly in animals receiving pretreatment of AA. The present data shows that AA can reciprocate the toxic effects of Fe-NTA and can serve as
a potent chemopreventive agent to suppress oxidant-induced tissue injury and hepatotoxicity in rats.
Keywords
Ferric nitrilotriacetate acid, liver, oxidative stress, rats, ascorbic acid

Introduction
Ascorbic Acid (AA) is involved in many biological
and biochemical functions and earlier data has been
published on recommendations for AA intake.
(Levine et al., 1999). Several studies suggest that
AA has protective effects in cell transformation and
tumor promotion (Patel et al., 1998; Zhang et al.,
1999). Ameliorative potential of AA and thiamine
on oxidative stress in liver, kidney, and blood and
of hepatic and renal infliction in arsenic-exposed rats
has earlier been shown by Nandi et al. (2005).
AA suppresses the growth of some tumor cells
resistant to chemotherapy and serves as a potential
antitumor agent (Osmak et al., 1997). The antioxidants like vitamins C and E and b-carotene not only
tap free radicals but also may have some other biological activities, which exert protective effects on
metabolic activation of carcinogens (Van-Poppel and
Van den Berg, 1997). Such chemopreventive agents
like AA may prove to be very effective against chemical carcinogens including some complete carcinogens

like Fe-NTA. These toxic agents produce oxidative
stress in the tissue by generating free radicals that play
a key role in both initiation and promotion stages of
carcinogenesis. Repeated intraperitoneal (i.p.) administration of Fe-NTA produces renal adenocarcinoma
in rodents (Athar and Iqbal, 1998; Toyokuni et al.,
1998).
Iqbal et al. (1995, 1996) have also shown that FeNTA is a potent hepatic and renal tumor promoter
and induces ornithine decarboxylase (ODC) activity,
enhances tritium [3H] uptake, and acts through

1
Department of Clinical Laboratory Sciences, College of Applied
Medical Science, King Saud University, Riyadh, Saudi Arabia
2
Biotechnology Research Institute, Universiti Malaysia Sabah,
Kota Kinabalu Sabah, Malaysia

Corresponding author:
Sabah Ansar, Department of Clinical Laboratory Sciences, College
of Applied Medical Science, King Saud University, Riyadh 43502,
Saudi Arabia.
Email: sansar@ksu.edu.sa

Ansar and Iqbal

generation of oxidative stress by depleting antioxidants
armory of the tissue. Earlier studies have shown kidney
toxicity after subacute administration of Fe-NTA to
rats, formation of 8-hydro guanosine (8-OH-dG) in
DNA isolated from rat kidney cells (Bahnemann et al.,
1998), DNA base modifications in renal chromatin of
rats and formation of 4-hydroxy-2-nonenal-modified
proteins in the kidney treated with Fe-NTA (Toyokuni
et al., 1994a, 1994b). Yamada et al. (1990) also showed
the transformation of rat hepatic epithelial cells by
treatment with Fe-NTA. Role of vitamins and scavengers in prevention of toxicity has been reported earlier
in several studies (Tsou et al., 1996; Zhang et al., 1999;
Osmak et al., 1997). The effect of AA on Fe-NTA
induced hepatic toxicity in rats for protection against
tissue damage has been investigated in this study.

Experimental protocol
Animals and treatment
Male albino rats of Wistar strain (4–6 weeks old)
weighing 125–150 g were used throughout this study.
Animals were housed in an air-conditioned room and
had free access to pellet diet.
For various set of biochemical studies, different
groups of animals were used. For studying the effect
of AA on Fe-NTA-mediated generation of hepatic
oxidative stress and ODC induction, 30 male rats
were divided into 5 groups of 6 rats in each group.
Group I received saline and served as negative control. Group II received an oral treatment of AA
(2 mg per animal perday) and served as negative control, groups IV and V received a dose level of 1 and 2
mg per animal per day in 0.2 ml saline, respectively,
for a period of 1 week administered through the
gavage. Group III received only saline (vehicle of
AA) for 1 week through the gavage. Twenty four
hours after the last treatment of AA or saline, the animals of groups III, IV, and V received an i.p. injection
of Fe-NTA (9 mg Fe per kg body weight). The animals were killed 12 h after the treatment of Fe-NTA
by cervical dislocation within a short span of period
of 1 h. Just before the killing, blood samples of these
animals were collected in different test tubes from
retro-orbital sinus for the estimation of serum transaminases and lactate dehydrogenase activity.
Similarly, animal treatment protocol and the dose
regimen were the same as described above for
studying tritiated [3H] thymidine incorporation in
hepatic DNA. However, 18 h after the treatment of
Fe-NTA, these animals were given an i.p. injection

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of [3H] thymidine (20 mCi per animal). After 2 h
of the administration of [3H] thymidine, these animals were killed by cervical dislocation. Their livers
were quickly removed, cleaned free of extraneous
material, and homogenized in distilled water for
further processing and the separation of DNA.

Materials and methods
Preparation of Fe-NTA. The Fe-NTA solution was prepared by the method of Awai et al. (1979).
Study design. For various sets of biological studies, different groups of animals were used. A total of 60 rats
were used in the study. A total of 30 rats were used for
studying the effect of AA on Fe-NTA-mediated
induction of oxidative stress in liver. Another group
of 30 rats were used for studying the effect of AA
on Fe-NTA-mediated [3H] thymidine incorporation
into hepatic DNA.
Estimation of serum transaminases and lactose
dehydrogenase
Estimation of sGOT and sGPT activity. In a water bath,
0.5 ml of 2 mM a-ketoglutarate was incubated for 5
min at 37 C. Then, 0.1 ml serum was added, and the
volume was adjusted to 1 ml with sodium phosphate
buffer. The reaction mixture was incubated at 37 C for
20 min. The reaction was terminated with an equal volume of trichloroacetic acid (TCA; 10% w/v), stored in
ice, and then centrifuged at 4000g for 5 min. To the
supernatant, 0.5 ml of 2,4 dinitrophenylhydrazine
(DNPH; 1 mM) was added. The reaction mixture was
left for another 30 min at room temperature. Finally,
the color was developed by the addition of 5 ml of
0.4 NaOH N and the product was read at 505 nm.
Estimation of LDH activity. Briefly, the reaction mixture containing 2.7 ml of phosphate buffer (0.1 ml, pH
7.4), 0.01 ml serum, and 0.1 ml of reduced nicotinamide adenine dinucleotide was incubated for 20 min.
To this, 0.1 ml of pyruvate (2.5 mg ml1) was added.
The absorbance was read for 5 min at an interval of
30s.
Preparation of PMS and microsomes. Livers were
quickly removed, perfused immediately with icecold saline (0.85% w/v sodium chloride), and homogenized in chilled phosphate buffer (0.1 M, pH 7.4)
that contained potassium chloride (KCl; 1.17% w/v),
using homogenizer. The homogenate was filtered
through a muslin cloth and was centrifuged at 800g

1010

for 5 min at 4 C in an Eltek refrigerated centrifuge
(model RC 4100 D) to separate the nuclear debris.
The aliquot obtained was centrifuged at 10,500g for
20 min at 4 C to obtain postmitochondrial supernatant
(PMS), which was used as a source of enzymes. A
portion of the PMS was centrifuged in an ultracentrifuge at 105,000 g for 60 min at 4 C. This pellet was
considered to be the microsomal fraction and was suspended in phosphate buffer (0.1 M, pH 7.4) containing KCl (1.17% w/v).
Determination of ODC activity. ODC activity was determined by utilizing 0.4 ml of hepatic 105,000g supernatant fractions per assay tube and measuring the
release of 14CO2 from DL-[1-14C] ornithine. The livers
were homogenized in tris(hydroxymethyl)aminomethane–hydrochloric acid (HCl) buffer (pH 7.5, 50
mM) that contained ethylenediaminetetraacetic acid
(0.1 mM), pyridoxal phosphate (0.1 mM), phenylmethylsulfonyl fluoride (1.0 mM), 2-mercaptoethanol
(1.0 mM), dithiothreitol (0.1 mM), and Tween 80
(0.1% w/v) at 4 C using a polytron homogenizer.
Finally, the central well was transferred to a vial containing 2 ml of ethanol, and 10 ml toluene-based scintillation fluid was added to it, followed by counting
the radioactivity in a liquid scintillation counter. ODC
activity was expressed in picomole of 14CO2 released
per hour per milligram protein.
Lipid peroxidation. The reaction mixture, in a total volume of 1.0 ml, contained 0.58 ml phosphate buffer
(0.1 M, pH 7.4), 0.2 ml of hepatic microsome (10%
w/v), 0.2 ml AA (100 mM), and 0.02 ml ferric chloride (100 mM). The reaction mixture was incubated at
37 C in a shaking water bath for 1 h. The reaction was
stopped by the addition of 1.0 ml TCA (10% w/v).
Following the addition of 1.0 ml thiobarbituric acid
(TBA; 0.67% w/v), all tubes were placed in a boiling
water bath for 20 min. Finally, the tubes were shifted
to an ice bath and centrifuged at 2500g for 10 min.
The amount of malondialdehyde (MDA) formed in
each of the sample was assessed by measuring the
optical density of the supernatant at 535 nm using a
spectrophotometer against a reagent blank. The results
were expressed in nanomoles of MDA formed per
hour per gram of tissue at 37 C using a molar extinction coefficient of 1.563105 M1 cm1.
H2O2 assay. Hepatic microsomes, 2.0 ml (10% w/v)
of each were suspended in 1.0 ml of solutioncontaining phenol red (0.28 nM), horseradish

Toxicology and Industrial Health 31(11)

peroxidase (8.5 U), dextrose (5.5 nM), and phosphate
buffer (0.05 M, pH 7.0) and were incubated at 37 C
for 60 min. The reaction was stopped with the addition of 0.01 ml NaOH (10 N) and then centrifuged
at 800g for 5 min. The absorbance of the supernatant
was recorded at 610 nm against a reagent blank. The
quantity of H2O2 produced was expressed in nanomoles of H2O2 per hour gram tissue based on the standard curve of H2O2-oxidized phenol red.
Effect of AA on Fe-NTA-mediated induction of
protein carbonyl
Determination of protein carbonyl content. Protein
oxidation was measured by an estimation of carbonyl
groups with slight modifications using 2,4-DNPH
reagent. Serum (0.1 ml) was treated with 0.5 ml of
10 mM 2,4-DNPH, dissolved in 2 M HCl as a sample
or with 0.5 ml of 2 M HCl as a control blank. The
reaction mixtures were allowed to stand for 1 h at
room temperature and in dark, with stirring at 15 min
intervals. Next, 0.5 ml of ice-cold 20% TCA was
added, and the sample was left on ice for 15 min. The
tubes were then centrifuged at 11,000 r min1 for 5
min to obtain the protein pellet. The precipitated proteins were subsequently washed three times with 1 ml
of ethanol–ethyl acetate (1:1) to remove unreacted
2,4-DNPH and lipid remnants. Each washing step was
followed by centrifugation at 3000 r min1 for 7 min.
The final protein pellet was dissolved in 0.25 ml of 6
M guanidine hydrochloride and incubated at 37 C for
10 min. The carbonyl content was calculated from
peak absorption (360 nm) using an absorption coefficient (e) of 22,000 M1 cm1
Effect of AA on Fe-NTA-mediated [3H] thymidine incorporation in hepatic DNA. For studying [3H] thymidine
incorporation in hepatic DNA, another 30 rats were
used; animal treatment protocol and the dose regimen
were the same as described above. However, 18 h
after the treatment of Fe-NTA, these animals were
given an i.p. injection of [3H] thymidine (20 mCi per
animal). After 2 h of administration of [3H] thymidine, these animals were killed by cervical dislocation. Livers were quickly removed, cleaned free of
extraneous material, and homogenized in distilled
water for their further processing and the separation
of DNA. The homogenate (10% w/v) was prepared
in ice-cold water. The incorporation of [3H] thymidine in hepatic DNA was performed as described by
Iqbal et al (1995). The precipitate obtained was
washed with cold TCA (5% w/v) and incubated with

Ansar and Iqbal

cold perchloric acid (PCA; 10%) at 4 C overnight.
After incubation, it was centrifuged, and the precipitate was washed with cold PCA (5%). The precipitate
was dissolved in warm PCA (10% w/v) followed by
incubation in a boiling water bath for 30 min and then
filtered through Whatman 50 paper. The filtrate was
counted for [3H] in a liquid scintillation counter
(LKB-Wallace-1410, Pharmacia Biotech, Finland).

Statistical analysis
Values were expressed as mean + SEM. The level of
significance between different groups is based on
Dunnett’s t test followed by analysis of variance
(ANOVA). One-way ANOVA was used to calculate
the statistical significance between various groups.
A value of p < 0.05 was considered to be statistically
significant.

Results
Inhibition of enhanced ODC activity and [3H]
thymidine uptake by AA
An i.p. injection of Fe-NTA induced ODC activity
and DNA synthesis by 450% and 360% in liver,
respectively, of corresponding saline-treated controls.
However, AA (alone)-treated group did not show any
change as compared to the control. Supplementation
with AA to rats prior to the treatment of Fe-NTA
showed a significant inhibition in the induction of
ODC activity and DNA synthesis in both the organs.
At 1 mg per animal per day dose of AA, the inhibition
in ODC activity and DNA synthesis were observed
and the values reached to 370% and 285% as compared to corresponding saline-treated controls. At the
higher dose of AA, the inhibition in ODC activity and
DNA synthesis was by 302% and 188% as compared
to corresponding saline-treated controls (Table 1).

Inhibition of enhanced microsomal lipid
peroxidation and H2O2 generation by AA
Fe-NA induced microsomal lipid peroxidation and
H2O2 generation between 1.7-2.3 folds in rat livers
of corresponding saline-treated controls. However,
marked inhibition by AA was seen in MDA production and H2O2 generation. The inhibition at the higher
dose level of AA (2 mg per animal per day) was
decreased by 125% and 160%, respectively as compared to Fe-NTA treated group. (Figure 1)

1011
Table 1. Modulatory effects of AA (vitamin C) on FeNTA-induced hepatic ODC activity and enhanced DNA
synthesis.a
ODC activity (pmol [3H] Thymidine
of 14CO2 released/ uptake (dpm/mg
Treatment groups
h/mg protein)
DNA)
Saline
Vitamin C (D2)
Fe-NTA
Vitamin C
(D1) þ Fe-NTA
Vit. C (D2) þ
Fe-NTA

137.5 +
139.9 +
620.8 +
509.8 +

38.9
18.6
42.8b
75.2b

416.8 + 36.5b

56.2
61.4
202.8
160.4

+ 8.2
+ 2.9
+ 12.8b
+ 8.5b

106.1 + 11.0b

AA: ascorbic acid; Fe-NTA: ferric nitrilotriacetate; D1: dose 1; D2:
dose 2; SE: standard error; ODC: ornithine decarboxylase.
a
Each value represents mean + SE of six animals. Saline-treated
group served as control for group III. Fe-NTA-treated group
served as control for groups IV and V. Dose regimen and treatment protocol are described in the text. D1 and D2 represent
administration of 1 and 2 mg AA per animal per day, respectively.
b
p < 0.05.

Inhibition of enhanced level of serum
transaminases (sGOT and sGPT) and LDH by AA
Fe-NTA treatment enhanced serum glutamate oxaloacetate transaminase (sGOT), glutamate pyruvate
transaminase (sGPT), and lactate dehydrogenase
(LDH) to 217%, 221%, and 322%, respectively, as
compared to the serum values of the enzymes of
saline-treated control. A significant decrease in their
levels was observed in the animals pretreated
with AA. Their levels were diminished to the values
of 127%, 172%, and 131%, respectively, when compared with the corresponding saline-treated controls
on the group receiving the higher dose of AA
(Figure 2).

Inhibition of enhanced level of protein carbonyl
by AA
The effect of pretreatment of rats with AA on FeNTA-mediated increase in 2,4-DNPH incorporation
into cytosolic proteins, which is a measure of protein
carbonyl, is shown in Figure 3. Fe-NTA alone treatment results in a maximum of 2.6- fold increase in the
2,4-DNPH incorporation into cytosolic proteins in
liver. However, in AA-pretreated animals, there is
an increase in the incorporation to Fe-NTA (alone)treated group. The decrease in the incorporation of
2,4-DNPH into cytosolic protein was dependent on
the dose of AA used. At the higher dose of AA of 2

Toxicology and Industrial Health 31(11)

20

Percentage control

*
*

*

*

*

40

sGOT
sGPT
LDH

300

*

*
*

350

H2O2
LPO

60

*

H2O2 / LPO generation
(nmol H2O2//MDA/h/g tissue)

1012

250
200
150
100
50

0
I

II

III

IV

V

Figure 1. Effect of AA (vitamin C) on Fe-NTA-induced hepatic lipid peroxide and hydrogen peroxide generation. Group I:
Saline; group II: vitamin C (D2); group III: Fe-NTA; group IV:
vitamin C (D1) þ Fe-NTA; group V: vitamin C (D2) þ FeNTA. Saline-treated group served as control for group III.
Fe-NTA-treated group served as control for groups IV and
V. Data represent mean + SE of six animals. Dose regimen
and treatment protocol are described in the text. *p < 0.05,
**p < 0.001. AA: ascorbic acid; D1: dose 1; D2: dose 2; FeNTA: ferric nitrilotriacetate; SE: standard error.

mg per animal per day, the decrease in the incorporation
of 2,4-DNPH into cytosolic protein was about 1.3-fold.

Discussion
AA is known as an important biological antioxidant
and plays a principal role in providing defense against
superoxide anion and other free radicals (Patel et al.,
1998). Treatment with AA alone results in the reversal of oxidative stress without significant decline in
tissue lead burden (Patra et al., 2001). a-Tocopherol
and some plant extracts have also been found to
inhibit N-nitroso compounds formation by destroying
nitrosating agents (Bartsch and Frank, 1996) and
these antioxidants act on different phases of the carcinogenic process usually acting on more than one
target at a time, thereby interrupting the formation
of cancer (Buiatti and Munoz, 1996). AA also acts
as a scavenger of reactive radical species and protects
against human gastric cancer (Drake et al., 1996).
Small doses of AA are required to inhibit the oxidative response of the tissue. As described in earlier
results, Fe-NTA induces ODC activity and DNA
synthesis, both markers of tumor promotion to significant levels to manifest its carcinogenic effect (Iqbal
et al., 1995, 1996; Nomoto et al., 1999).
AA effectively lessens the degree of hepatic damage
by diminishing TBARS formation following Fe-NTA

0

I

II

III

IV

V

Figure 2. Effect of AA (vitamin C) on Fe-NTA-induced hepatic levels of serum transaminases (sGOT and sGPT) and LDH.
Group I: Saline; group II: vitamin C (D2); group III: Fe-NTA;
group IV: vitamin C (D1) þ Fe-NTA; group V: vitamin C
(D2) þ Fe-NTA. Saline-treated group served as control for
group III. Fe-NTA-treated group served as control for groups
IV and V. Dose regimen and treatment protocol are described
in the text.

treatment suggesting that AA acts by intercepting free
radical formation. This close relationship between AA
level, lipid peroxidation, and tissue damage provides a
good evidence that lipid peroxidation and membrane
damage are also involved in the toxicity caused by
Fe-NTA. The present study shows that AA is an effective inhibitor of Fe-NTA-induced ODC activity, DNA
synthesis, and tissue injury. In addition, the protective
effect of vitamin supplementation was assessed on tissue injury caused by Fe-NTA treatment by measuring
serum transaminases, markers of hepatic injury. Both
the doses of AA used in the experiments significantly
reduced a rise in their levels caused by Fe-NTA.
The significance of these findings with respect to
mankind is that supplementation of AA can prevent
possible toxicity in humans resulting from the ingestion of any toxic compound in any form. The vitamins
afford protection either by interfering with absorption
of carcinogen or by increasing their urinary excretion,
thereby, reducing its toxic effect on the target tissues.
This data not only suggest that AA protects against
the Fe-NTA-induced elaboration of oxidative stress
but also protects against the hyperproliferative response of hepatic tissue. AA thus has a unique advantage relative to other remedies for treatment and/or
prevention of toxicity and cancer as its increase intake
could produce measurable benefits.
In summary, present data suggest that AA may be an
effective chemopreventive agent and offer protection
against Fe-NTA-mediated hepatic damage in rats.

Ansar and Iqbal

1013

*

500

*
300
*

Protein carbonyl
(nmol/mg protein)

400

200
100
0
I

II

III

IV

V

Figure 3. Effect of AA (vitamin C) on Fe-NTA-induced hepatic levels of protein carbonyl. Group I: saline; group II: vitamin
C (D2); group III: Fe-NTA; group IV: vitamin C (D1) þ FeNTA; group V: vitamin C (D2) þ Fe-NTA. Saline-treated
group served as control for group III. Fe-NTA-treated group
served as control for groups IV and V Data represent
mean + SE of six animals. Dose regimen and treatment protocol are described in the text. *p < 0.05, **p < 0.001.

Funding
This research project was supported by a grant from the
‘‘Research Center of the Center for Female Scientific and
Medical Colleges’’, Deanship of Scientific Research, King
Saud University.

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