CURRENT THERAPEUTIC RESEARCH

VOLUME 7 r, NUMBEH 5, OCTOBER 2010

Effects of Vitamin C and Melatonin on Cysteamine-

Induced Duodenal Ulcer in a Cholestatic Rat Model:
A Controlled Experimental Study
Babak Rezvanjoo, DVM]; Samira Rashidi, PharmD2; Abolghasem ]ouyban, PhD\
Seyed Hamed Shirazi Beheshtiha, DVM 4 ; and Morteza Samini, PhDs
'Science & Research Branch, Islamic Azad University, Tehran, Iran; 2Department of
Pharmacology, Faculty of Pharmacy, Pharmaceutical Science Branch, Islamic Azad
University, Tehran, Iran; 3Department of Pharmaceutical and Food Control, Faculty of
Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; "Department of Clinical
Pathology, Faculty of Veterinary Medicine, Islamic Azad University, Karaj Branch,
Karaj, Iran; and 5Department ofPharmacology, Faculty of Specialized Veterinary Science,
Science & Research Branch, Islamic Azad University, Tehran, Iran

ABSTRACT
BACKGROUND: Superoxide dismutase (SOD) is one of the defense mechanisms
against free radicals. Cysteamine is a cytotoxic agent, acting through generation of
reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, and super-
oxide, and may decrease defense activity of SOD against ROS and induce duodenal
ulcer. Melatonin is a suicidal antioxidant that has a protective effect against ROS and
cytoprotecrive effect through inhibition of the decrease in SOD activity.
OBJECTIVES: The primary aim of this study was to assess the effects of pretreat-
ment with vitamin C and melatonin on cysteamine-induced duodenal ulcer. Second-
ary aims were to compare the ulcerogenic effect of cysteamine and the antiulcer effects
of vitamin C and melatonin.
METHODS: This study was performed in male Wistar rats (200-250 g) in 3 groups
of equal size (n = 24): bile duct ligation-induced cholestasis (test), sham, and con-
trol groups. In the test and sham groups, laparotomy was performed under general
anesthesia and the common bile duct was identified; in sham rats, the common bile
duct was left in situ, but in test rats, the common bile duct was isolated and doubly
ligated to induce cholestasis. Animals in each group were also divided into 4 equal
subgroups (n = 6). These subgroups were treated with vitamin C plus cysteamine,
melatonin plus cysteamine, cysteamine alone, and saline, respectively. All animals
were euthanized via overdose of ether anesthesia 24 hours after the last injection of
cysteamine or saline, and 0.5 mL of blood was collected from the heart ventricle. The
duodenum was cut open, washed with saline, fixed, and prepared for calculation of
ulcer index (Szabo method) and histopathologic assessment. SOD activity was mea-
sured using a branded enzyme kit.

Accepted for publicationJune 21, 20 ZO. doi: I O.1016/j.cutrheres.201 0.1 0.001

© 201 0 Elsevier HS Journals, Inc. All righrs reserved. 001l-393X/$ - see front matter

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 B. REZVANJOO ET AL.

RESULTS: In all 3 groups, animals treated with cysteamine had significantly

increased mean (SE) ulcer index (test, 4.00 {0.1O} vs 1.17 {0.30}; sham, 3.83 {0.16} vs
0.50 {0.22}; control, 3.67 {0.21} vs 0 {OJ) and decreased SOD activity (test, 146.41
{2.16} vs 299.83 {1.94} UlmL; sham, 154.75 {2.02} vs 303.08 {0.35} U/mL; control,
157.08 {1.67} vs 314.50 {1.14} UlmL) compared with saline-treated rats (all, P <
0.00l). In the test rats, ulcer index was significantly increased and SOD activity was
significantly decreased compared with the sham and control groups (both, P < 0.001).
Pretreatment with vitamin C and melatonin was associated with attenuation of ulcer
index and increased SOD activity compared with rats treated with cysteamine alone
(P < 0.001). There were no significant differences in ulcer index or SOD activity
between groups administered vitamin C or melatonin.
CONCLUSIONS: In this experimental study, pretreatment with melatonin or
vitamin C in all rats produced significant attenuation of the ulcer index and enhanced
SOD activity. Cysteamine-induced duodenal mucosal damage was greater in cholestatic
rats compared with sham and control rats. (Curr Tber Res Clin Exp. 2010;71:322-330)
© 2010 Elsevier HSJournals, Inc.
KEY WORDS: cysteamine-induced duodenal ulcer, cholestasis, melatonin, super-
oxide dismutase, vitamin C, rats.

INTRODUCTION
It has been reported that melatonin has a protective effect against indomethacin-
induced gastric damage in multiple stress-induced lipid peroxidation.! Gastric mucosal
damage induced by different gastroinvasive agents was found to be significantly
greater (P < 0.00l) in cholestatic bile duct double-ligated rats than in sham rats," and
the frequency of gastrointestinal ulceration was significantly higher (P < 0.01) in the
cholestatic population than in the healthy population.f It has been suggested that
gastric mucosa in cholestatic rats is more vulnerable to NSAIDs compared with-
healthy animals (P < 0.05), which may be due to a variety of different factors, includ-
ing increased free-radical tormation." Melatonin is a powerful antioxidant and a
scavenger of free radicals, such as hydroxyl and peroxyl radicals, that does not undergo
redox cycling (repeated reduction and oxidation). Once oxidized, it cannot be reduced
to its former state, and therefore, it has been referred to as a "suicidal" antioxidant. 5
Melatonin, in different models of in vitro and in vivo oxidative stress, has been found
to protect tissues against oxidant damage induced by various free-radical-generating
agents. 6,7 It has been suggested that melatonin exerts protective and therapeutic
effects against cholestatic liver injury and its associated oxidative stress in bile duct-
ligated rats through its antioxidant actions as well as its anti-inflammatory effects. 8- 12
Vitamin C can reduce and thereby neutralize reactive oxygen species (ROS) such as
hydrogen peroxide. Vitamin C is an antioxidant agent that undergoes redox cycling that
may allow it to act as a pro-oxidant and promote free-radical formation. 13 It has a biologi-
cal role as a reducing agent in a number of hydroxylation reactions and protects essential
substances in the body such as proteins, lipids, carbohydrates, and DNA and RNA from
damage by free radicals. 14 Cysteamine hydrochloride (HCI) has been found to be

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the most potent agent for inducing duodenal ulcer,15,16 and cysteamine-induced duo-
denal ulcer in animals is now used to study the antiulcer activity of drugs. The
cysteamine used in experimental studies has been found to concentrate in the duode-
num.l ' Its ulcerogenic effect may be due to the generation of ROS, the decreasing de-
fense activity of superoxide dismutase (SOD),18,19 and increasing duodenal endothelin-1
concentration, which are all associated with decreased duodenal mucosal blood
flow. l7,18 Oxidative stress, enhanced free-radical levels, and an impaired in-cell anti-
oxidant pool are important factors underlying the pathophysiologic mechanisms in a
variety of diseases. The superoxide radical and SODs (enzymes that catalyze the dis-
mutation of the superoxide radical anion) have been implicated in many disease states,
including inflammatory diseases, diseases of ischemia and reperfusion, neurodegenera-
tive diseases, and cancer. 20
The present study assessed the protective effects of melatonin and vitamin C against
cysteamine-induced duodenal ulcer and their ability to enhance antioxidant enzymatic
defense by measuring ulcer index and SOD activity in a cholestatic rat model.

MATERIALS AND METHODS

EXPERIMENTAL DESIGN
Male adult Wistar rats (n = 72) weighing 200 to 250 g were used in this study. All
animals were provided food and water ad libitum and housed 3 per cage at 22 ± 2°C
with a controlled 12-hour light-dark cycle. The animals were handled in accordance
with the criteria and recommendations of the ethics committee on animal experiments
of the Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Animals were divided into 3 groups of 24 animals each: the bile-duct ligated (test)
group, the sham group, and the control group. All animals were then further divided
into 4 subgroups of 6 animals each that received one of the following: (1) viramin C
(350 mg/kg IP) at 7:30 AM and 11:30 AM plus cysteamine (230 mg/kg SC) at 8:00 AM
and 12:00 PM; (2) melatonin (20 mg/kg IP) at 7:30 AM and 11:30 AM plus cysteamine
(230 mg/kg SC) at 8:00 AM and 12:00 PM; (3) cysreamine (230 mg/kg SC) at 8:00 AM
and 12:00 PM; or (4) saline (1 mLlkg SC) at 8:00 AM and 12:00 PM.

TEST GROUP
laparotomy was performed under general anesthesia induced by intraperitoneal
injection of ketamine (65 mg/kg) and chlorpromazine (20 mg/kg), and the common
bile duct was identified, isolated, and doubly ligated. Then, 7 days after surgery, when
the group had shown overt jaundice, the animals were divided into the 4 subgroups
and treared as described previously. Rats were euthanized via overdose of ether anes-
thesia 24 hours after the last dose of cysteamine. Blood samples were then collected,
duodenums were removed, and ulcer assessment and SOD activity were determined.

SHAM GROUP
laparotomy was performed under general anesthesia induced by injection of keta-
mine (65 mg/kg IP) and chlorpromazine (20 mg/kg IP) and the common bile duct
was identified, manipulated, and left in situ. The abdominal wall was then closed in

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 B . REZVANJOO ET AI..

2 layers. Seven days afrer surge ry, th e ani ma ls were divided into th e 4 subgroups and
treated as described prev iously. Rats were euthanized via overdose of et her anesthesia
24 hours after th e last dose of cysteamine. Blood samples were then collected, d uode-
nums were remo ved , and ulcer assessme nt and SO D activ ity were determined .

CONTROL GROU P
No surge ry was cond ucted in the control gro up. Th e rats were divided into the
4 subgroups and treated as described p reviously. They were then eurhani zed via over-
dose of et her anesthesia 24 hours after the last dose of saline . Blood samples were th en
collected, duodenums were removed, and ulcer assessme nt and SO D activit y were
determined.

ULCER INDEX MEASUREMENT AND HISTOPATHOLOGIC ASSESSMENT

The duodenum (5 ern in length) was removed and th e esoph agus and duodenum
were clamped . Then, after 20 to 30 minutes, th e sto mach and adjacent duodenum
were cut open and washed out with saline. Samples from each g roup were fixed in
10% neutral bu ffered formaldehyde for 24 hours. Ulcers were exami ned by 5 x bin -
ocular magnifier to assess lesions. Ulce rs were scored for intensity using a 4-point
scale (0 = no ulcer ; 1 = superficial m ucosal erosion; 2 = deep ulcer or transmural ulcer;
and 3 = perfo rated or penetrated ulcerj.!" The ulcer index was calculated by using the
following formula I5 . 16 :

where VI was ulc er index, UN was number of ulcers, Us was ulcer score, and U A was
th e ulcer surface area for each duode num .
For histopatho logic assessment, the forma lin-fixed speci mens were embedded in
paraffin, seerio ned (5 prn), and stained wit h hema toxylin and eosin, and th en histo -
chemical sections were evaluated by light microscopy.

SOD ACTIVITY ASSESSMENT

In all 3 groups, 0 .5 mL of blood was drawn from the heart ventricle into a syringe
containi ng EDTA (final EDTA concent ration of 1 mg/rnl.), transfe rred to plasti c ru bes,
cent rifug ed for 10 mi nutes at 3000 rpm, and th en aspi rated off the plasma. T he
red blood cells (RB Cs) were washed 4 times with 3 mL of norm al saline and centri-
fuged for 10 minutes at 3000 rpm after each wash . Then, 2 .0 mL of cold red ist ill ed
wate r was added to the washed , centrifuge d RBCs, mixed and left to sta nd at 4°C for
15 minutes before being subjected to lysis. Th e lysate was diluted with 0.01 mollL
phosphate bu ffer (pH = 7.0) and SOD activity was de termi ned using an enzyme kit
(RAN SO D , Randox Laboratories, Inc., Cru mlin, Uni ted Kingdom). T he method
employs xant hi ne and xanthine oxidase to p roduce superoxide radicals that react with
IN T (2-{4-iodophenyl}-3 -{4-nit rophenyl}-5-p hen yltetrazolium chloride) to form a
red form azan dye. The SOD act ivity was measured photo metrically by the degree of
inhibition of this react ion at 505 nm and 3JOC and expressed as U/mL.

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CURRENT THERAPEUTIC RESEARCH

CHEMICALS

The following chemicals were used in the study: melatonin (Albissola Marina, Savo-
na, Italy), vitamin C (Osvah Pharmaceutical Co., Tehran, Iran), chlorpromazine (Tehran-
Shirni, Tehran, Iran), ketarnine HCl (Pantex, Duizel, the Netherlands), cysteamine HCl
(Merck, Darmstadt, Germany), formaldehyde (Merck), SOD kit (Randox Laboratories),
and EDTA (Merck).

STATISTICAL ANALYSIS

Data were analyzed statistically by t test and expressed as mean (SE). P < 0.05 was
considered statistically significant. Calculations were performed using SPSS version
16 (SPSS Inc., Chicago, Illinois).

RESULTS
TEST GROUP

In animals treated with cysteamine alone in the test group, mean (SE) duodenal
ulcer index (4.00 [0.10]) was significantly increased and SOD activity was signifi-
cantly decreased 046.41 [2.16} U/mL) compared with saline-treated animals
0.17 [0.30} and 299.83 [1.94} U/mL; both, P < 0.001) (Table). Rats pretreated
with vitamin C or melatonin were associated with attenuation in ulcer index (1.83
[0.16} and 1.67 [0.2l}, respectively) and increased SOD activity (234.91 [1.65}
and 236.66 [1.22} U/mL; P < 0.001) compared with rats treated with cysteamine
alone. There were no significant differences in ulcer index or SOD activity between
the groups pretreated with vitamin C or melatonin.

SHAM GROUP

In rats treated with cysteamine alone in the sham group, mean (SE) ulcer index was
3.83 (0.16) and SOD activity was 154.75 (2.02) U/mL (Table). Pretreatment with vi-
tamin C or melatonin was associated with attenuation in ulcer index (1.67 [0.21} and
1.50 [0.22}, respectively) and significantly increased SOD activity (239.50 [1.06}
and 242.2 [0.14} U/mL; both, P < 0.001). There were no significant differences in
ulcer index or SOD activity between the groups pretreated with vitamin C or
melatonin.

CONTROL GROUP

In rats treated with cysteamine alone in the control group, mean (SE) ulcer
index was significantly higher (3.67 [0.2l}) and SOD activity was significant-
ly lower 057.08 [1.67} U/mL) compared with those treated with saline (0 and
314.50 [l.14} U/mL) (Table). Pretreatment with vitamin C or melatonin was associ-
ated with attenuation of the ulcer index (1.50 [0.22} and 1.33 [0.2l}, respectively)
and significantly increased SOD activity (242.08 [1.18} and 246.91 [l.83} U/mL;
both, P < 0.001) compared with rats treated with cysteamine alone. There were no
significant differences in ulcer index or SOD activity between the groups pretreated
with vitamin C or melatonin.

326
 Table. The effects of vitamin C and melatonin pretreatment on ulcer index and activity of superoxide dismutase (SOD) in rats.
Data are mean (SE).

Subgroup
Vitamin C (350 rng/kg)> + Melatonin (20 mg/kg)> + Cysteamine Saline
Cysteamine (230 rng/kg)" Cysteamine (230 mg/kg)" (230 rng/kg)! (1 mLjkg)t
Group (n = 6) (n = 6) (n = 6) (n = 6)
Bile duct ligation (n = 24)
Ulcer index 1.83 (0.16)1' 1.67 (0.21)1' 4.00 (0.10) 1.17 (0.30)
SOD, UjmL 234.91 (1.65)1' 236.66 (1.22)1' 146.41 (2.16) 299.83 (1.94)

Sham (n = 24)
Ulcer index 1.67 (0.21)1' 1.50 (0.22)1' 3.83 (0.16) 0.50 (0.22)
SOD, UjmL 239.50 (1.06)1' 242.2 (0.14)1' 154,75 (2.02) 303,08 (0,35)

Control (n = 24)
Ulcer index 1.50 (0.22)1' 1.33 (0.21)1' 3.67 (0.21) o (0)
SOD, UjmL 242.08 (1.18)1' 246.91 (1.83)1' 157.08 (1.67) 314.50 (1.14)

*Administered at 7:30 AM
and 11:30 AM. 1Il
t Administered at 8:00 AM
and 12:00 PM. ;u
111
l' P < 0.001 versus cysteamine alone,
'~"
...z
o
o
111
-t
!II
N l>
-.I !"
CURRENT THERAPEUTIC RESEARCH

HISTOLOGIC STUDIES

T he m icroscopic observa tion of all rat s treated with sali ne showed normal appea r-
ance of duodenum muco sa, but rat s treated with cysteam ine alone showed ulcer with
a mucosal defect , which penetrated the mus cularis m ucosae and musc ularis properia.
In rats pretreated with vit ami n C or melaton in , prot ecti on aga inst the histopatholog ic
chang es was observed.

DISCUSSION
An adeq ua te balance amo ng th e antioxidant enz yme s is necessary to minim ize the
toxic effects ofROS. 21 SO D is one of the ma in ant ioxidant en zymes found in aerobic
organism s that constitutes a part of the physiologi c defens e against oxidative stress .
It has been suggested th at gast ric mucosal lesions are associa ted with a decrease in
defense activity against ROS and the protective effect of ant ioxidants is related to
their ability to improve oxida ti ve stress in gastri c mucosa.F ROS is known to be
involved in the pathogenesis of mucosal lesions in th e gastro intestinal tract, and free
radicals play an important role in these mucosal lesion s. The role of ROS has been
well est ablished in man y di sord ers. I? The SOD level is estima ted as an index of the
antioxida nt sratus.23 When the ge neratio n of ROS overwhelms the antioxidant de-
fense , lip id peroxid at ion of the cell membrane occurs, wh ich causesd isturbances in
th e cell int eg rity lead ing to cell dam age . The efforts of th e endogenous ant ioxida nt
enzyme s to rem ove th e conti nuo usly ge nerated free radic als ini t ially increase due to
an ind uct ion, but lat er, enzyme depletion takes p lace, resulting in oxid ati ve cell
dam age. Blood concent rat ion of vi ta m in C and other ant ioxi da nt s decreases du e to
th e accelerat ed consum ption in th e blo od. 24
In th e present stu dy, cystea m ine was used as a cyto tox ic ag ent, who se effect
partly depends on th e ge nera t ion of hydro xyl radi cal , and it has been suggested th at
anti oxidants inhibit cysteam ine-ind uced du odenal ulcer. 18 It seems that, in th e
presence of cysteami ne, free-radi cal production overwhelms ant ioxidant defen se, and
th is situat ion causes d isturbances in the cell membrane. In the present stu dy, vit a-
m in C and melatonin were used as antioxid ants, which inhibited cysteamine-
ind uced duodenal ulcer and increased the activit y of SOD in all 3 groups of rat s.
H istologic analysis confirmed the biochemical results . This effect may be du e to
increase in enzymatic antioxidant defenses and protecti on of cell components from
ROS-mediated damage.
limitations of the pr esent stu dy include the open-label design and the small num-
ber of rats used in each gro up . l arge and controlled studi es in humans are need ed to
confirm the results .

CONCLUSIONS
Pretre atment with melaton in and vitamin C in all rat s p rodu ced sig nificant attenua-
ti on of the ulcer index and enha nced SOD acti vit y. The results of thi s experimental
study suggest that cysteami ne-ind uced duodenal mu cosal da mag e was gre at er in
cholestatic rats comp ared with sham and control rat s.

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 B. REZVANJOO ET AL.

ACKNOWLEDGMENTS
This study was conducted as a part of Dr. Rezvanjoo's PhD project. The authors thank
Fariborz Moayer, PhD, for his assistance in the histopathologic assessment. Partial
financial support for this study was provided by the Science & Research Branch and
Pharmaceutical Science Branch, Islamic Azad University, Tehran, Iran. The authors
have indicated they have no other conflicts of interest regarding the content of this
article.
Drs. Rezvanjoo and Rashidi conducted the experimental work. Dr. Jouyban conducted
statistical analysis and assisted in the preparation of the manuscript. Dr. Beheshtiha
assisted with the SOD activity measurement. Dr. Samini was the study guarantor.

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ADDRESS CORRESPONDENCE TO: Morteza Samin i, PhD, Department of

Ph arm acology, Faculty of Specialized Veterinary Science, Islamic Azad Universit y,
Science & Research Bran ch, Tehr an , Iran . E-mail: sam inim @yahoo.com

330