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Advances in Environmental Biology, 7(14) December 2013, Pages: 4660-4666
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Advances in Environmental Biology
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Protective Effects of Hydroalcoholic Extract of Nasturtium officinale R.Br (Watercress)
on Antioxidant Status and DNA Damage in Kidney of Rats Exposed to Sodium Arsenite
1
Felor Zargari, 2Amir Ghorbanihaghjo, 3Hossein Babaei, 4Safar Farajnia, 5Nasim Hayati Roodbari
1
Department of Biology Science and Research branch, Islamic Azad University, Tehran, Iran.
Department of Clinical Biochemistry, Tabriz University of Medical Sciences, Drug Applied Research Center, Tabriz, Iran.
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran School of pharmacy,Tabriz University of Medical
Sciences, Tabriz, Iran.
4
Department of Biotechnology Research Center, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
5
Department of Biology Science and Research branch, Islamic Azad University, Tehran, Iran.
2
3
ARTICLE INFO
Article history:
Received 25 October 2013
Received in revised form 23
January 2014
Accepted 26 January 2014
Available online 20 February 2014
Key words:
Arsenic.
Nasturtium Officinale R.Br,
Kidney, Dna Damage,
Antioxidant Enzymes
ABSTRACT
The present study was carried out to assessment of effects of hydroalcoholic extract of
Nasturtium officinale R.Br (Watercress) on sodium arsenite-induced renal toxicity
, antioxidant status and DNA damage in rat males. Sodium arsenite(SA)[ at dose of 5.5
mg/kg body weight i.p. injection ] exposure led to significantly (p<0.05)increased
serum uric acid, MDA(Malondialdehyde) and decreased serum TAC(total
antioxidant capacity) and antioxidant enzymes in blood and kidney. In addition SA
significantly increased 8-OHdG(Marker of oxidative DNA damage) in serum(p<0.05).
The consumption of plant extract (500 mg/kg body weight via gavage) increased the
activities of antioxidant enzymes and TAC and decreased MDA, uric acid and 8 -OHdG
levels in serum. The findings indicate that the plant extract might have protective
effects on arsenic induced damaged in serum and kidney function.
© 2013 AENSI Publisher All rights reserved.
To Cite This Article: Felor Zargari, Amir Ghorbanihaghjo, Hossein Babaei, Safar Farajnia, Nasim Hayati Roodbari., Protective effects of
hydroalcoholic extract of Nasturtium Officinale R.Br (Watercress) on antioxidant status and DNA damage in kidney of rats exposed to
sodium arsenite. Adv. Environ. Biol., 7(14), 4660-4666, 2013
INTRODUCTION
Arsenic is an element found in soil, water, air and also a potential human carsinogen [21]. Toxic effects of
arsenic attributed to production of reactive oxygen species (ROS) and oxidative stress, this results the alteration
of the antioxidant defense system, increased oxidative stress and cell death [34,9,6]. Liver and kidney are target
organs for arsenic toxicity in rats. Concentration of arsenic was found to vary in these organs [29]. Arsenic
induced nephrotoxicity by disturbing antioxidant defens system, protein oxidation and lipid peroxidation
products [23]. Arsenic toxicity leading to renal functional deterioration [23,20]. Arsenic has high affinity for
sulfydryl groups of protein so combined with –SH groups of proteins in glomerular filtration membrane [41].
Arsenite has a tendency to readily react with the sulfhydryl groups of proteins and this turn inhibit biochemical
pathways and considered to be the biologically active form and the major source to arsenic toxicity [11]. Lipid
peroxidation leading to decreas GFR(glomerular filtration rate) and to increase nitrogenous waste such as urea,
uric acid and creatinine [26]. Also ROS cause deletion and nucleosid modifications. 8-OHdG is formed from
deoxyguanosine (dG) in DNA by hydroxy free radicals. Because of its stability, 8-OHdG is known as one of the
most reliable markers of oxidative DNA damage [16,30]. Defense system against free radical-induced oxidative
stress includes enzymatic antioxidants (superoxid dismutase, catalase, gluthationperoxidase) and non-enzymatic
antioxidants (vit C, vit E, carotenoids, flavonoids) [34]. Many reports evidenced a decrease in the levels of
antioxidants after arsenic exposure [11]. In recent years attention had been paid toward the medicinal
plants[45][46][47]. Phenolic compounds particularly flavonoids have antioxidant activities [13,44]. Querecetin
a flavonoid that has been reported to prevent against oxidative stress, decreased 8-OHdG level and improved
histology change in rat kidney exposed to lead [17]. Nasturtium Officinale R.Br (watercress) from Brassicacea
family is a perennial plant that thrives in cold water and is found in streams and contains vitamin A,C,E,
gluconasturtine and minerals such as iron, phosphorus [27,12]. The brassica variety has been shown to contain
high amounts of phenolic compounds. Intake of vegetables such as cabbage, broccoli, watercress, is associated
with a decreased risk of several cancers [27,12]. Watercress is used as a home remedy for hypertention,
Corresponding Author: Felor Zargari, Department of Biology Science and Research branch, Islamic Azad University,
Tehran, Iran.
Tel: 09143168443 E-mail : [email protected]
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Advances in Environmental Biology, 7(14) December 2013, Pages: 4660-4666
cardiovascular disease and hyperglycemia [43]. Recently, studies have reported the reduction of cancer risk and
alteration of blood antioxidants status in human and animal models [12,32,39,7,4,3]. The objective of this study
was to examine the effects of hydroalcoholic extract of . Nasturtium Officinale R.Br (watercress) on the kidney
functional and determination of the MDA (Malondialdehyde), TAC( total antioxidant capacity) urea,uric acid
and creatinine levels in serum and antioxidant enzymes (SOD,CAT,GPx) and 8-OHdG in serum and kidney .
MATERIALS AND METHODS
Plant material:
Aerial parts of N.Officinale were obtained from Kaleybar, East Azerbaijan Province in Iran in the spring
2012 and identified by Dr.Nazemiyeh (Faculty of Pharmacy, Tabriz University of Medical Science, Iran) (NO.
711Tbz-fph). They were kept in herbariumThen the plant was air-dried and powdered. The powder was kept in
a closed container at 8°c .
Plant Extraction:
Seven hundred grams of the powder was extracted three times with mixture of ethanol:water (80%) at
room temperature overnight.The solvent was completely removed by rotary evaporator at 50°c .The final residue
of extract was used for the investigation.
Animal and experimental groups:
Male Sprague Dawley rats (n=32) weighing 180-200 gr were purchased from the Central Animal House of
Tabriz Medical School, Tabriz, Iran. Animals were housed at 22±2°c and 60± 5% relative humidity with a 12 h
light/dark cycle. They had free access to water and normal diet adlibitum. The experimental protocol was
approved by the medical ethics committee,and all animals received humane care in compliance with the
guidelines of Tabriz Medical University. After the adaptation period (7 days), the rats were divided randomly
into four groups, each containing 8 rats as follows:
Group I: Normal healthy rats
Group II: treated with arsenic (i.p. as sodium arsenite NaAso2 [ SA ] at a dose of 5.5mg/kg body weight per
day)
Group III: treated with hydroalcoholic extract of N.officineal supplementation (via gavage) at a dose of
500mg/Kg body weight
GroupIV: treated with hydroalcoholic extract of N.officineal supplementation (via gavage) at a dose of
500mg/Kg body weight + SA
The rats were killed at the end of the 28th day of treatment, and the liver tissue and serum were examined
with regard to antioxidant/oxidant biochemical parameters.
Preparation of kidney homogenates:
The kidney samples were cut into pieces and homogenized in 1.15%kcl solution. The homogenates were
then centrifuged at 6000 rpm for 5 min at 4°c . The obtained supernatant was used for biochemical analysis. The
protein concentration of each extract was determined [18].
Malondialdehyde assessment:
Malondialdehyde{ (MDA) marker of lipid peroxidation} levels were measured using the tiobarbituric acid
(TAB) [38]. It was expressed as nmol/mg protein in tissue, nmol/ml in serum.
Total Antioxidant Capacity (TAC) assessment:
Total antioxidant capacity was estimated using Randox TAC status test in serum (Randox Laboratories
LTd,United Kingdum) ABTSR(2,2'-Azino- di- [3-ethylbenzthiazoline sulphate]) was incubated with a
peroxidase(metmyoglobin)and H2O2 to produce the radical cation ABTSR.. This has a relatively stable bluegreen color,which is measured at 600nm. Antioxidants in added sample cause suppresion of this color
production to a degree which is propotional to their concentration.
GPx activity assay:
GPX activity kidney tissue (homogenized in 1.15%kcl solution) and serum was measured [22] with a
spectrophotometers at 340 nm. (Ransel, Randox Laboratores Ltd. United Kingdom). The results were expressed
as Unit/mg protein of tissue.
SOD activity assay:
Superoxid dismutase (SOD) activity in the kidney tissue was determined by using xanthine and xanthine
oxidase to generate superoxide radicals ,the results were expressed as Unit /mg protein of tissue [31].
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Advances in Environmental Biology, 7(14) December 2013, Pages: 4660-4666
CAT activity assay:
Catalase (CAT) activity was measured by the method of Aebi [1] and was expressed as CAT/mg protein in
tissue.
Biochemical analysis:
Serum urea,uric acid,and creatinine (for assessment of kidney function) were measured by Abbott
ALCYONTM300 Autoanalyzer, enzymatic methods using Kits (Pars Azmoon, Iran) according to the
manufacture's instruction.
ElISA assay for 8-OhdG:
Serum and kidney tissue(obtained supernatant from hemogeized kidney tissue)) levels of 8-OHdG were
measured by an enzym-linked immunoabsorbant assay (ELISA) using a rat 8-OHdG immunoassay Kit
(Scientific Research center Glory Science Co. Ltd sensitivity = 0.024ng/ml Assay range 0.05-20ng/ml). The
procedures followed the manufacturer's instruction. 40 μL sample and 10 μL of primary antibody were added
to each well of a 8-OHdG coated microtiter plate; then 50 μLstreptavidin-HRP was added and incubated at 37 °c
for 1 h. The antibodies in the sample bound to the coated 8-OHdG were washed by automatic washing method
After Adding chromogen, the solution A, B was incubated for 10 min at 37 °C. Then, stop solution was added to
each well to stop the reaction. Optical density was measured at 450 nm wavelength. The linear regression
equation of the standard curve and the sample's concentration was calculated according to the concentration
standards and the corresponding OD values. The results were expressed in ng/ml.All of the 8- OHdG
measurements were performed within 6 months of postcollection
Statistical Analysis:
The mean value ±SD was determined for each variable in all groups. Data were fed into SPSS, 16.0, and
Mann Whitney U test was used to compare the groups at the significance level p<0.05.
RESULTS AND DISCUSSION
Serum levels of MDA significantly increased in the rats exposed to sodium arsenite (p<0.05), but arsenic
treated groups did not show a significant increase in MDA content of kidney (Table 1,2). A significant
decreasing was obtained after plant extract treatment in serum MDA. Arsenic increased serum MDA and MDA
content of kidney. MDA serves as a reliable marker for the assessment of free radical induced damage to tissues.
Arsenic induced MDA production could be due to the impairment of cells’ natural protective system and could
be related to the GSH depletion [35]. Reduction in MDA content with plant extract treatment may be due to
phenolic compounds, especially flavonoids. Phenolic antioxidants (PhOH) interfere with the oxidation of lipids
and other molecules by the rapid donation of hydrogen atom to radicals [33]. The phenoxy radical intermediates
are relatively stable, so they do not initiate (propagate) further radical reactions. They even act as terminators of
the reaction chain by interacting with other free radicals [33] The reduction of serum MDA indicates that the
plant has a protective effect on arsenic-induced dmage in serum. The Table 1 and 2 shows the levels of
malondialdehyd (MDA) in serum and kidney in the rats treated with plant extract. Based our unpublished data
arsenic decreased the RBC SOD, CAT, GPx activities compared to the control group that may be due to its
detoxification mechanism( Methylation of arsenic by s-adenosylmethionine in the presence of glutathion (GSH),
depletion of GSH and oxidative stress and alteration of antioxidant enzymes and increaes of lipid peroxidation
[25,28]. GSH a tripeptide, plays an important role in maintaining cellular redox status and its level is considered
a significant marker of oxidative stress [11]. There was no significant difference in GPx of kidney when
compared with controls. Only SOD and CAT activity was significantly (p<0.05) declined in rats exposed to
sodium arsenite. Maiti and Chatterjee [19] reported that liver and kidney have different adaptive cellular
protective mechanisms against arsenic exposure (increased lipid peroxidation and decreased SOD and CAT
activity were identified in the rats' kidneys). The decrease in SOD activity may be attributed to enhanced
suoeroxid radical production and the increase in superoxid radicals inhibits catalase activity [39]. Serum levels
of uric acid significantly increased in the rats exposed to sodium arsenite (p<0.05),but there was no significant
increasing in serum urea and Creatinine when compared with control. (Table 1). Serum uric acid decreased
significantly in the rats treated with plant extract (p<0.05). Oxidative damage of kidney leading to functional
deterioration it such as decreasing GFR and retention of nitrogenous waste such as urea ,uric acid and creatinine
[26]. Anetor [2] reported that free radicals causes tubular necrosis ,tubular permeability and decreasing of GFR
,increasing of nitrogenous waste in serum.Yasmin et al [40] also found that nonsignificant changes in the serum
levels of urea,uric acid and creatinine (3mg/kg body weight oral for 15 days). Saxena et al. [26] repoted that
arsenic increased serum levels of uric acid ,urea, creatinine (arsenic trioxide for 7,14 21 days 0.07-1.5 mg/kg
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body weight). Maybe the toxic effect of arsenic is depending on duration, dose and rout of exposure and
chemical form arsenic. Similarly, animal studies indicated that the kidney is not a major target for inorganic
arsenic and at high levels of exposure, mild histological changes in the renal have been noted [15]. Betancourt
[5] reported that levels of DNA damage in well-Nourished and Malnourished rats depending on the structural
and functional characteristics of each tissue so brain and kidney incontrast liver are not susceptible to DNA
damage and each tissue presented different susceptibility to DNA damage. Uric acid derived from the
metabolism of purins and formed by the action of XO (Xanthin Oxidase). Due to effect of arsenic on activity of
XO serum levels of uric acid increased [26] or inhibition of PDH(pyruvate dehydrogenase) that may lead to
decrease production of ATP [24,14] so low ATP lead to increase of uric acid. Watanabe et al [36] reported that
there was significant negative correlation between ATP synthesis and blood uric acid levels. So arsenic due to
increase uric acid causes lipid peroxidation and decrease of total antioxidant capacity(TAC) (Table 1). Also
many reports showed that there was a significant inverse correlation between plasma antioxidant capacity and
arsenic concentration in whole blood [37]. Some of dietary flavonoids inhibit XO(xantin oxidase) and have
superoxide scavenging activity [8]. So in this study N.officinale extract may be contain the flavonoids that
inhibit XO and have superoxide scavenging activity( such as quercetin). Antioxidant effects of flavonoids may
be due to modulating cell signaling,gen regulation and other biological processes and non antioxidant
mechanisms. Other substance such as vitamin C or E and carotenoids (such as β-caroten) in our plant may be
affect plasma total antioxidant capacity.So presence of other substance in N.Officinale apart from the flavonoids
or the other effect of mechanisms in our study may be increased total antioxidant capacity (TAC). A significant
increase was found in serum concentration 8-OHdG after sodium arsenite exposure (p<0.05). Treatment with
plant extract showed a significant decrease(p<0.05) in concentration of 8-OHdG compared to sodium arsenite
exposed rats that suggesting beneficial role of plant extract in the DNA damage (Table 1). ROS can interact with
DNA to produce damage, including single-and double- stranded DNA breaks, deletion and nucleoside
modifications.The interaction of arsenic with glutathione and its related enzymes by changing their redox status
may lead to the alterations of GSH related enzymes, and methyl depletion which could have deleterious effects
on the detoxification processes and other critical cellular processes involving GSH mediated redox regulation
[10]. Studies have shown that watercress is also a good source of lutein and β-caroten [44,12]. β-caroten and
lutein has been shown to have anti-carcinogenic activity [12].
The findings indicate that the plant extract might have protective effects on arsenic induced damaged in
serum and kidney function but further experimentation needs to be done to find the effects of watercress and its
fractions on kidney function by other oxidants or different dose and rout of exposure of sodium arsenite.
Table 1: Results for lipid peroxidation and total antioxidant capacity and urea,uric acid and creatinine and 8-OHdG in the serum of rats fed
with plant extract: group I: normal group , group II (SA group ) , group III( plant extract 500mg/Kgbodyweight), group IV
(SA+plant extract. (500mg/Kg body weight)
Groups I
Groups II
Groups III
Groups IV
Parameters
MDA(nmol/ml)
TAC(mmol/dl)
Urea(mg/dl)
Uric Acid(mg/dl)
Mean±SD
Mean±SD
Mean±SD
Mean±SD
1.74± 0.10
1.95± 0.15*
1.84 ±0.12**
1.7 ±0.24**
0.89±13
0.67±0.23*
1.07±0.33**
1.15±0.37**
38.3±3.2
49.4±10.1 NS1
41.1±6.4 NS2
46.6±4.1 NS2
1.6±0.7
3.20±0.7*
1.91±0.6**
1.89±0.4**
0.52±0.1 NS2
2.93±0.21NS1
0.51±0.4 NS2
3.14±0.14**
Cr(mg/dl)
0.51±0.2
0.55±0.1NS1
8-OHdG(ngr/ml)
3.05±0.2
3.42±0.19*
TAC:total antioxidant capacity, MDA:malondialdehyde, Cr. :creatinine
NS1 :non-significant values against control values
NS2 :non-significant values against arsenic treated rats
*: significantly different values from control rat(group I)
**: significantly different values from arsenic treated rats(groupII)
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Table 2: Results for lipid peroxidation and antioxidant enzymes and 8-OHdG in the kidney of rats fed with plant extract: group I: normal
group, group II (SA group, group III( plant extract 500mg/Kgbodyweight), group IV (SA+plant extract. (500mg/Kg body weight)
Parameters
MDA(nmol/mg protein)
Groups I
Groups II
Groups III
Groups IV
Mean ±SD
0.49±0.26
0.54±0.23
9.05±0.68
5.24±1.62
2.16±0.36
Mean ±SD
0.54±0.19NS1
0.55±0.50NS1
7.94±0.23**
3.31±0.33**
2.23±0.40 NS1
Mean ±SD
0.58±0.00
0.59±0.29
8.27±0.61 NS2
3.95±1.11NS2
1.99±0.40
Mean ±SD
0.50±0.00
0.63±0.06
7.96±0.51 NS2
3.68±0.86 NS2
2.14±0.40 NS2
GPx(U/mg protein)
SOD(U/mg protein)
CAT(U/mg protein)
8-OHdG(ngr/ml)
GPx : Glutathion peroxidase, SOD:superoxid dismutase, CAT: catalase
NS1 :non-significant values against control values
NS2 :non-significant values against arsenic treated rats
*: significantly different values from control rat(group I)
**: significantly different values from arsenic treated rat(groupII)
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