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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
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
Experimental protocol
Animals and treatment
Male albino rats of Wistar strain (46 weeks old)
weighing 125150 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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Statistical analysis
Values were expressed as mean + SEM. The level of
significance between different groups is based on
Dunnetts 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).
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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) Fe-NTA
Vit. C (D2)
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
20
Percentage control
*
*
40
sGOT
sGPT
LDH
300
*
*
350
H2O2
LPO
60
1012
250
200
150
100
50
0
I
II
III
IV
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) Fe-NTA; group V: vitamin C (D2) 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.
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
II
III
IV
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) Fe-NTA; group V: vitamin C
(D2) 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.
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500
*
300
*
Protein carbonyl
(nmol/mg protein)
400
200
100
0
I
II
III
IV
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) FeNTA; group V: vitamin C (D2) 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.
References
Athar M, Iqbal M (1998) Ferric-nitrilotriacetate promotes
N-diethylnitrosamine induced renal tumerigenesis in the
rat: implications for the involvement of oxidative stress.
Carcinogenisis 19(6): 11331139.
Awai M, Narasaki M, Yamanoi Y and Seno S (1979) Induction of diabetes in animals by parentral administration
of ferric nitrilotriacetate. A model of experimental
hemochromatosis. American Journal of Pathology (95):
663672.
Bahnemann R, Leibold E, Kittel B, Jellert W and Jackh R
(1998) Different patterns of kidney toxicity after subacute administration of Na-nitrilotriacetate acid and Fenitrilotriacetic acid to Wistar rats. Toxicological
Sciences 46(1): 166175.
Bartsch H, Frank N (1996) Blocking the endogenous formation of N-nitroso compounds and related carcinogenesis. IARC Scientific Publications 139: 189201.
Buiatti E, Munoz N (1996) Chemoprevention of stomach
cancer. IARC Scientific Publications 136: 3539.
Drake IM, Davis MJ, Mapstone NP, Dixon MF, Schorah
CJ, White KL, et al. (1996) Ascorbic acid may protect
against human gastric cancer by scavenging mucosal
oxygen. Carcinogenesis 17(3): 559562.
1014
somatic mutations in the Tsc2 and VHL tumor suppressor genes. Japanese Journal of Cancer Research 89(8):
814820.
Toyokuni S, Uchida K, Okamato K, Nakakuki YH, Hiai H
and Stadtman ER (1994b) Formation of 4-hydroxy-2nonenal-modified proteins in the renal proximal tubules
of rats treated with renal carcinogen, ferric nitrilotriacetate. Proceedings of the National Academy of Sciences
United States of the America 91: 26162620.
Tsou TC, Chen CL, Liu TY and Yang JL (1996) Induction
of 8-hydroxyguanosine in DNA by chromium (III) plus
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