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International Journal of PeptideResearch and Therapeuticsformerly known as "Letters in PeptideScience" ISSN 1573-3149Volume 18Number 3 Int J Pept Res Ther (2012) 18:281-290DOI 10.1007/s10989-012-9300-5
Betaine Elevates Ovarian AntioxidantEnzyme Activities and DemonstratesMethyl Donor Effect in Non-Pregnant Rats
Masoud Alirezaei, Parvin Niknam &Gholamali Jelodar
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Betaine Elevates Ovarian Antioxidant Enzyme Activitiesand Demonstrates Methyl Donor Effect in Non-Pregnant Rats
Masoud Alirezaei • Parvin Niknam •
Gholamali Jelodar
Accepted: 29 April 2012 / Published online: 9 May 2012
� Springer Science+Business Media, LLC 2012
Abstract Chronic alcoholism leads to infertility in male
and female rats, and antioxidant enzymes form the first line
against oxidative stress in organisms. In recent years,
betaine has shown beneficial effects on various tissues, and
this study has attempted to clarify antioxidant and methyl
donor properties of betaine in the rat ovary. For this
purpose, the sexually matured Sprague-Dawley female
rats were divided into Control, Ethanol (EtOH), Betaine,
and Betaine ? EtOH groups. Administration of betaine
in Betaine ? EtOH group significantly increased CAT
activity when compared to the other groups (P \ 0.05).
GPx activity increased significantly in Betaine and Beta-
ine ? EtOH groups as compared to controls (P \ 0.05).
Interestingly, GPx and CAT activities insignificantly
increased (in order compensatory) in EtOH group to sup-
press oxidative stress. In contrast, SOD activity decreased
insignificantly in EtOH group compared to Beta-
ine ? EtOH and control groups (P [ 0.05). TBARS con-
centration (as a lipid peroxidation marker) significantly
increased in ethanol-treated rats as compared to controls,
while total homocysteine concentration significantly
decreased in betaine-treated rats in comparison with EtOH
group. Regarding to oestrous cycles, ethanol-treated ani-
mals had irregular estral cycle and persistent oestrous
phase compared to controls and betaine-treated rats. In
conclusion, these results demonstrate for the first time the
antioxidant and methyl donor properties of betaine in the
rat ovary. Thus, betaine might be used as a potential
therapy in hyperhomocysteinemia and partial infertility
mediated by oxidative stress in females.
Keywords Ovarian antioxidant enzymes � Betaine �Ethanol � Rat
Introduction
Clinical observation and animal experimentation show that
alcohol consumption interferes with reproduction. In this
context, amenorrhea, anovulation, luteal phase dysfunction,
and early menopause have been observed in alcoholic
women (Hugues et al. 1980; Valimaki et al. 1984; Hakim
et al. 1998). Reduced uterine and fallopian tube weight,
reduced estradiol and progesterone levels, irregular oes-
trous cycles, and ovarian failure have been found in etha-
nol-fed rats (Van Thiel et al. 1978; Bo et al. 1982; Krueger
et al. 1982; Hakim et al. 1998). Chronic alcoholism,
whether or not related with liver damage, has often been
associated with reproductive functional imbalances,
including amenorrhea, oligomenorrhea, dysmenorrhea,
partial or total infertility, spontaneous miscarriages (Harlap
and Shiono 1980; Hugues et al. 1980), loss of libido and
early or later menopause onset (Pettersson et al. 1990;
Kinney et al. 2007; Chuffa et al. 2009). Ethanol has the
ability to inhibit FSH production, and interfering with
folliculogenesis and luteogenesis in women (Mello et al.
1993; Chuffa et al. 2009). Ethanol also can exert as a
oxidative agent due to its direct effect on the generation of
reactive oxygen species (ROS) or through its metabolite,
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10989-012-9300-5) contains supplementarymaterial, which is available to authorized users.
M. Alirezaei (&)
Division of Biochemistry, School of Veterinary Medicine,
Lorestan University, P.O. Box 465, Khorram Abad, Iran
e-mail: [email protected]
P. Niknam � G. Jelodar
Department of Physiology, School of Veterinary Medicine,
Shiraz University, 71345 Shiraz, Iran
123
Int J Pept Res Ther (2012) 18:281–290
DOI 10.1007/s10989-012-9300-5
Author's personal copy
acetaldehyde (Dupont et al. 2000; Li et al. 2004). There-
fore, inadequate protection from ROS that are formed in
steroidogenically active granulosa and luteal cells could be
a potential trigger for follicular atresia (Tsai-Turton and
Luderer 2006) and corpus luteum regression (Sugino et al.
1999) in the rat ovary (Kheradmand et al. 2010).
Oxidative stress is defined as an imbalance between
oxidant and antioxidant agents, when levels of ROS and
free radicals overwhelm the body’s antioxidant defense
system (Neamati et al. 2011; Alirezaei et al. 2012b).
Indeed, oxidative stress is a condition in which the ele-
vated levels of ROS damage cells, tissues, and organs
(Agarwal et al. 2005; Neamati et al. 2011). ROS are free
radicals that play a significant role in many of the repro-
ductive physiological processes including; ovulation
(Kheradmand et al. 2010), sperm capacitation and hyper-
activation, as well as, sperm-oocyte fusion (Agarwal et al.
2005). The chronic ethanol intake leads to an increase of
lipid peroxidation products and a reduction of enzymatic
antioxidant defense system including GPx, CAT, and SOD
and non-enzymatic molecules such as glutathione (GSH)
(Kheradmand et al. 2010; Neamati et al. 2011). Antioxi-
dants are the main defense against oxidative stress induced
by free radicals, and therefore, preventional antioxidants
and scavenger antioxidants may be used as a potential
therapy in reproduction (Kheradmand et al. 2010). In this
regard, we have investigated the possibility of utilizing
antioxidants such as oleuropein and betaine in ethanol-
mediated oxidative stress in our recent researches (Ali-
rezaei et al. 2011a; Alirezaei et al. 2011b; Alirezaei et al.
2012b; Alirezaei et al. 2012a).
Homocysteine (Hcy) is a sulphur containing amino acid
which serves as the carbon backbone in methyl group
metabolism through remethylation pathway and as a pre-
cursor for the synthesis of cysteine and GSH via the trans-
sulphuration pathway (Zeisel et al. 2003; Schwahn et al.
2004; Alirezaei et al. 2011a; Alirezaei et al. 2011b; Alirezaei
et al. 2012b). The enzyme 5,10-methylenetetrahydrofolate
reductase (MTHFR; EC 1.5.1.20) catalyses the irreversible
reduction of 5,10-methylenetetrahydrofolate to 5-methyl-
tetrahydrofolate for the remethylation of Hcy to methionine
by methionine synthase (MT; EC 2.1.1.13) (Schwahn et al.
2004; Alirezaei et al. 2010; Alirezaei et al. 2011a; Alirezaei
et al. 2011b). An accessory enzyme, betaine–homocysteine
methyltransferase (BHMT; EC 2.1.1.5), also exist in liver,
kidney, and testis of rats for the remethylation of Hcy to
methionine by the substrate betaine as an alternative methyl
donor (Schwahn et al. 2004; Alirezaei et al. 2010; Alirezaei
et al. 2011a; Alirezaei et al. 2011b). In this context, animal
studies have demonstrated that limiting one remethylation
pathway increases the activity of another pathway to main-
tain hepatic S-adenosyl methionine (SAM) at normal con-
centration (Wallace et al. 2008; Alirezaei et al. 2010;
Alirezaei et al. 2011a; Alirezaei et al. 2011b) (Supplemen-
tary file).
In human medicine, high levels of Hcy are recognized as
an independent risk factor for cardiovascular and neuro-
degenerative diseases (Alirezaei et al. 2010; Alirezaei et al.
2011a; Alirezaei et al. 2011b). High plasma concentration
of Hcy is also associated with an increased risk of neural
tube defects, placental infarcts, abruptio placentae and
eclampsia (Trisolini et al. 2008). Furthermore, research in
the reproduction field has shown that high levels of Hcy are
associated with early embryonic death in mares and rats
(Petrie et al. 2002; Trisolini et al. 2008). In this sense,
we well know that chronic ethanol consumption induces
folate deficiency, subsequently hyperhomocysteinemia and
infertility (Alirezaei et al. 2010; Alirezaei et al. 2011a;
Alirezaei et al. 2011b). Therefore, pregnant women would
be expected to have higher requirement for folate since,
folate is crucial for DNA and RNA biosynthesis (Wallace
et al. 2008; Alirezaei et al. 2011b), and methyl donors such
as vitamin B12, and choline are essential nutrients for fetal
development (Wallace et al. 2008).
Betaine is an important methyl donor in one-carbon
metabolism, mediates the transfer of a methyl group to
homocysteine, forming methionine and dimethylglycine
(Sunden et al. 1997; Millian and Garrow 1998; Slow et al.
2009; Alirezaei et al. 2011b). Betaine supplementation has
proven effective in reduction of hyperhomocysteinemia
and oxidative stress induced by ethanol in our recent
studies (Alirezaei et al. 2010; Alirezaei et al. 2011a;
Alirezaei et al. 2011b). It lowers the elevated plasma
homocysteine concentrations associated with its antioxi-
dant and methyl donor properties in the cerebellum and
testis of rats (Alirezaei et al. 2011a; Alirezaei et al. 2012a).
Betaine feeding is believed to directly enhance homocys-
teine remethylation and, consequently, to increase the
availability of SAM for transmethylation (Alirezaei et al.
2010; Alirezaei et al. 2011a; Alirezaei et al. 2011b). Thus,
this study examined the effects of betaine and ethanol on
the ovarian antioxidant status, plasma homocysteine con-
centration and oestrous cycle in non-pregnant rats.
Materials and Methods
Materials
Betaine (Betafin� 96 %) was obtained from Biochem
Company (Brinkstrasse 55, D-49393 Lohne, Germany).
Alcohol (ethanol 95 %) and thiobarbituric acid (TBA)
were supplied from Merck Chemical Company (KGaA,
Darmstadt, Germany). GPx and SOD kits were obtained
via Randox � Company (Randox, UK). The homocysteine
kit was prepared by Axis� Homocysteine EIA (Axis-Shield
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AS, Germany). All chemicals used were of analytical
grade.
Animals
Twenty-four sexually matured, healthy, colony-bred in
Animal House Center, Shiraz, Iran of Sprague-Dawley
female rats, weighting 190–220 g, were used for the
experiments. The rats were housing in polypropylene
cages, under well-ventilated animal house condition (tem-
perature: 21–24 �C, photoperiod: 12 h natural light/12 h
dark). The rats were given standard pelleted diet and tap
water ad libitum and weight gain and food consumption
was determined at weekly intervals. The experimental
protocol was approved by the recommendations of Animal
Care Committee for the Shiraz University of Medical
Sciences (Shiraz, Iran).
Experimental Design
The animals were divided into 4 groups consisting of six
animals in each group.
Group I: control, received 2 ml normal saline (vehicle).
Group II: EtOH, received 4 g/kg body weight (bw)
ethanol solution in 2 ml vehicle.
Group III: betaine, received 1.5 % (w/w) of the total diet
betaine in 2 ml vehicle.
Group IV: betaine ? EtOH, received betaine, 1.5 % (w/
w) of the total diet, and after 120 min, feeding with
ethanol solution (4 g/kg bw)
Doses of ethanol and betaine were determined according
to the previous studies (Ji and Kaplowitz 2003; Song et al.
2003; Alirezaei et al. 2011a; Alirezaei et al. 2011b; Ali-
rezaei et al. 2012b; Alirezaei et al. 2012a). All the above
treatments were given orally by using stomach tube for 30
consecutive days to cover six regular oestrous cycles. The
treatments were started from oestrous phase only, as the
ovarian antioxidant enzyme activities change markedly
from one phase to another phase of oestrous cycle
(Kheradmand et al. 2010). The treatments were given
orally everyday between 8.00 and 11.00 am for prevention
of circadian rhythm changes among days. The stages of
oestrous cycle were recorded daily by observing vaginal
smears (Kage et al. 2009). On the 31st day, after 24 h of
the last gavage and in fasting state, the rats were sacrificed
upon light diethyl ether anesthesia (Dagenham, UK) by
decapitation. Blood samples were taken via cardiac punc-
ture, whole blood containing EDTA was centrifuged at
3,0009g for 5 min and plasma was prepared in microtubes.
The ovaries were dissected out immediately and carefully
cleaned of adhering, and then ovaries and plasma samples
were stored at -70 �C until analysis.
Tissue Preparation
The ovaries were thawed and manually homogenized using
liquid nitrogen in cold phosphate buffer (0.1 M, pH 7.4,
containing 5 mM EDTA) and debris were removed by
centrifugation at 2,0009g for 5 min (Centrifuge 5415 R;
Rotofix 32A, Germany). Supernatants were recovered and
used for antioxidant enzyme activities, lipid peroxidation
products and protein measurement. Protein content of tis-
sue homogenates was determined using a colorimetric
method of Lowry with bovine serum albumin as a standard
(Lowry et al. 1951).
Measurement of CAT Activity
Tissue catalase activity was assayed using the method as
described previously (Claiborne 1985), was reported by
Kheradmand et al. (2009, 2010). The reaction mixture
(1 ml) consisted of 50 mM potassium phosphate (pH 7.0),
19 mM H2O2, and a 20–50 ll sample. The reaction was
initiated by the addition of H2O2 and absorbance changes
were measured at 240 nm (25 �C) for 30 s by a spectro-
photometer (S2000 UV model; WPA, Cambridge, UK).
The molar extinction coefficient for H2O2 is 43.6 M/cm.
The CAT activity was expressed as the unit that is defined
as lmol of H2O2 consumed per min per milligram of tissue
protein (U/mg protein).
Measurement of GPx Activity
The activity of glutathione peroxidase (GPx) was evaluated
with Randox GPx detection kit according to the manufac-
turer’s instructions, as described previously (Kheradmand
et al. 2010; Alirezaei et al. 2011a; Alirezaei et al. 2012b).
GPx catalyse the oxidation of glutathione (GSH) by
cumene hydroperoxide. In the presence of glutathione
reductase (GR) and NADPH, the oxidised glutathione
(GSSG) is immediately converted to the reduced form with
a concomitant oxidation of NADPH to NADP?. The
decrease in absorbance was measured spectrophotometri-
cally against blank at 340 nm. One unit (U) of GPx was
defined as l lmol of oxidized NADPH per min per milli-
gram of tissue protein. The GPx activity was expressed as
unit per milligram of tissue protein (U/mg protein).
Measurement of SOD Activity
The activity of superoxide dismutase (SOD) was evaluated
with Randox SOD detection kit according to the manu-
facturer’s instructions, as described previously (Kherad-
mand et al. 2009; Kheradmand et al. 2010; Alirezaei et al.
2012b). The role of SOD is to accelerate the dismutation
of the toxic superoxide (O2-) produced during oxidative
Int J Pept Res Ther (2012) 18:281–290 283
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energy processes to hydrogen peroxide and molecular
oxygen. This method employs xanthine and xanthine
oxidase to generate superoxide radicals which react with
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium
chloride (INT) to form a red formazan dye. The SOD
activity is then measured by degree of inhibition of this
reaction. One unit of SOD is that which causes 50 %
inhibition of the rate of reduction of INT under the con-
ditions of the assay. SOD levels were recorded at 505 nm
and through a standard curve and expressed as unit per
milligram of tissue protein (U/mg protein).
Measurement of Lipid Peroxidation
The amount of lipid peroxidation was indicated by the
content of thiobarbituric acid reactive substances (TBARS)
in the ovary. Tissue TBARS determined by following the
production of thiobarbituric acid reactive substances as
described previously (Subbarao et al. 1990), was reported
(Kheradmand et al. 2009; Kheradmand et al. 2010; Ali-
rezaei et al. 2011a). In short, 40 ll of homogenate was
added to 40 ll of 0.9 % NaCl and 40 ll of deionized H2O,
resulting in a total reaction volume of 120 ll. The reaction
was incubated at 37 �C for 20 min and stopped by the
addition of 600 ll of cold 0.8 M hydrochloride acid, con-
taining 12.5 % trichloroacetic acid. Following the addition
of 780 ll of 1 % TBA, the reaction was boiled for 20 min
and then cooled at 4 �C for 1 h. In order to measure the
amount of TBARS produced by the homogenate, the
cooled reaction was spun at 1,5009g in a microcentrifuge
for 20 min and the absorbance of the supernatant was
spectrophotometrically read at 532 nm, using an extinction
coefficient of 1.56 9 105/M cm. The blanks for all of the
TBARS assays contained an additional 40 ll of 0.9 %
NaCl instead of homogenate as just described. TBARS
results were expressed as nanomol per milligram of tissue
protein (nmol/mg protein).
Measurement of tHcy Concentration
Plasma total homocysteine (tHcy), which refers to the sum
of protein-bound, free-oxidized, and reduced species of
homocysteine in plasma, was determined by the Axis�
Homocysteine EIA kit (Golbahar et al. 2005; Karthikeyan
et al. 2007; Alirezaei et al. 2010; Alirezaei et al. 2011a).
The sample volume used was 25 ll. Absorbance was
measured at a wavelength of 450 nm using an ELISA
reader (STAT FAX 2100, USA). All estimations were
performed in duplicate and the intraassay coefficient of
variation was \10 % and the detection limit of the tHcy
assay was 2.0 lM. The tHcy results were expressed as
micromole per liter of plasma (lmol/L).
Statistical Analysis
All results are presented as mean ± (SEM). The statistical
differences were applied among the control and treated rats
by one-way analysis of variance (ANOVA) with Tukey’s
post hoc analysis (Graphpad PRISM version 5; Graphpad
Software Inc., San Diego, CA, USA). Previously, all
variables were tested for normal and homogeneous vari-
ances by Leven’s statistic test. P value of \0.05 was
considered statistically significant.
Results
In order to clarify antioxidant status of the ovary, the
activities of main antioxidant enzymes including CAT,
GPx, SOD, as well as TBARS concentration in the rat
ovarian tissue were measured (Figs. 1, 2, 3, and 4).
Administration of betaine in Betaine ? EtOH group sig-
nificantly increased CAT activity compared to the other
groups (P \ 0.05), and CAT activity was insignificantly
higher in Betaine group as compared to controls and
ethanol-treated rats (P [ 0.05). GPx activity increased
significantly in Betaine and Betaine ? EtOH groups as
compared to controls (P \ 0.05). In fact, when betaine
administered prior to ethanol, it could insignificantly
increase the activity of GPx in comparison with EtOH
group. Interestingly, GPx and CAT activities were insig-
nificantly higher (in order compensatory) in EtOH group as
compared to controls (P [ 0.05). In contrast, SOD activity
decreased insignificantly in EtOH and Betaine groups
compared to Betaine ? EtOH and control groups
(P [ 0.05). Treatment of rats with ethanol significantly
increased lipid peroxidation products (as shown by TBARS
concentration) as compared to controls, while pretreatment
Control EtOH Betaine Betaine+EtOH
0
50
100
150 **
*
CA
T(U
/mg
prot
ein)
Fig. 1 Comparison of catalase (CAT) activity among the control and
treated rats. Values represent mean ± SEM of enzyme activity (unit/
mg protein of ovarian tissue). Asterisk indicates statistical difference
between groups (P \ 0.05)
284 Int J Pept Res Ther (2012) 18:281–290
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of rats with betaine could suppress TBARS concentration
although it was not significant (P [ 0.05).
To evaluate the methyl donor properties of betaine, we
measured plasma total homocysteine (tHcy) concentration.
Treatment of rats with ethanol significantly increased tHcy
concentration in plasma of the EtOH group as compared to
the betaine-treated rats. However, administration of betaine
to the Betaine ? EtOH group could not suppress tHcy
concentration (P [ 0.05; Fig. 5).
The estral cyclicity evaluation of control and treated rats
clearly indicated irregular estral cycles in EtOH group
(Fig. 6). The cycle’s lengths were significantly longer in
ethanol-treated animals than in controls and betaine-treated
groups. Ethanol-treated animals presented a persistent
oestrous phase, with each phase varying from 2 to 4 days.
In contrast, Betaine could return the irregular estral cycles
in Betaine ? EtOH group to normal cycles.
Control EtOH Betaine Betaine+EtOH
0
5
10
15 *
*
GP
x(U
/mg
prot
ein)
Fig. 2 Comparison of glutathione peroxidase (GPx) activity among
the control and treated rats. Values represent mean ± SEM of
enzyme activity (unit/mg protein of ovarian tissue). Asterisk indicates
statistical difference between groups (P \ 0.05)
Control EtOH Betaine Betaine+EtOH
0
20
40
60
80 Not significant
SOD
(U/m
g pr
otei
n)
Fig. 3 Comparison of superoxide dismutase (SOD) activity among
the control and treated rats. Values represent mean ± SEM of
enzyme activity (unit/mg protein of ovarian tissue). There is no
statistical difference among the groups (P [ 0.05)
Control EtOH Betaine Betaine+EtOH
0
20
40
60
80*
Not significant
TB
AR
S(n
mol
/mg
prot
ein)
Fig. 4 Comparison of thiobarbituric acid reactive substances
(TBARS) concentration among the control and treated rats. Values
represent mean ± SEM of TBARS (nanomoles per milligram protein
of ovarian tissue). Asterisk indicates statistical difference between
groups (P \ 0.05)
Control EtOH Betaine Betaine+EtOH
0.0
0.5
1.0
1.5
2.0
2.5
3.0 *T
otal
hom
ocys
tein
e(µ
mol
/L)
Fig. 5 Comparison of plasma total homocysteine (tHcy) concentra-
tion among the control and treated rats. Values represent mean ±
SEM of tHcy (micromoles per liter of plasma). Asterisk indicates
statistical difference between groups (P \ 0.05)
Control EtOH Betaine Betaine+EtOH
0
1
2
3
4
5
* **
Pro
oes
trou
s +
Oes
trou
s ph
ase
(day
)
Fig. 6 Comparison of pro oestrous ? oestrous phase among the
control and treated rats. Values represent mean ± SEM of both
phases (day). Asterisk indicates statistical difference between groups
(P \ 0.05)
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Discussion
This work provides the novel evidence of betaine antioxi-
dant properties in the rat ovary. There are no data on
antioxidant enzyme activities in response to betaine in the
current literature, nor is there information concerning the
role of this antioxidant in prevention of ethanol-induced
oxidative stress in the female rats. The present study also
demonstrates the methyl donor effects of betaine against
hyperhomocysteinemia and partial infertility (as shown by
irregular oestrous cycle) induced by ethanol in non-preg-
nant rats.
The presence of different antioxidant defense systems in
the rat ovary is well known (Kheradmand et al. 2010). It
has been suggested that accumulation of ROS and a
decrease in antioxidant levels are involved in apoptotic cell
death, whereas antioxidants including GPx, CAT, and SOD
can inhibit apoptosis (Rueda et al. 1995; Kheradmand et al.
2010; Kheradmand et al. 2012). The results of the present
investigation indicated that betaine was able to enhance
GPx and CAT activity in the ovary of betaine-treated rats
however; it failed to induce over changes in SOD activity
in betaine group. Indeed, betaine could not considerably
influence SOD activity in which, the SOD activity was
insignificantly higher in betaine-treated rats compared to
EtOH group. The SOD results were similar to our recent
report in testes of rats (Alirezaei et al. 2012a) and consis-
tent with a previous report by Ganesan et al. (2010). In
response to ethanol treatment, the activities of CAT as well
as GPx (in order compensatory) exhibit slight elevation in
ethanol-treated rats than the controls. Therefore, it appears
that in the present study consumption of ethanol (as an
oxidative inducing agent) was able to increase activity of
the antioxidant enzymes such as GPx by the compensatory
mechanism via antioxidant response elements (AREs),
which are located in promoter regions of many of the genes
(Masella et al. 2004). In this regard, previous studies
showed that ethanol could enhance GPx activity in the
kidney (Dinu et al. 2006), cerebellum (Alirezaei et al.
2011a) and testes of rats (Alirezaei et al. 2012b). Herein,
lipid peroxidation process was indicated via markedly
TBARS elevation in ethanol-treated rats and methyl donor
properties of betaine was manifested by significantly tHcy
reduction in betaine group as compared to EtOH group.
Our observation for ovarian antioxidant enzyme activities,
TBARS level and tHcy concentration in the betaine-treated
rats supports the idea that betaine is associated with anti-
oxidant and methyl donor properties through its involve-
ment in homocysteine remethylation and cell membrane
stabilization (Ganesan et al. 2010; Alirezaei et al. 2011a;
Alirezaei et al. 2011b; Alirezaei et al. 2012a).
Antioxidant defense systems generally classified into
enzymatic and non-enzymatic antioxidants (Alirezaei et al.
2011a; Neamati et al. 2011). The antioxidant enzymes
represent a first line of defense against ROS and free rad-
icals by metabolizing them to non-toxic byproducts. The
first enzymatic reaction in the reduction pathway of oxygen
occurs during the dismutation of two molecules of super-
oxide when they are converted to hydrogen peroxide
(H2O2) and diatomic oxygen (Neamati et al. 2011; Rodri-
guez et al. 2004). The enzyme at this step is one of two
isoforms of superoxide dismutase (SOD); CuZnSOD is
present in the cytosol while (MnSOD) is located in the
mitochondrial matrix (Rodriguez et al. 2004). Although
H2O2 is not a radical itself, it is reactive and it is rapidly
converted into the highly reactive hydroxyl radical in the
presence of ferrous ion (Fe2?) via the Fenton reaction
unless it is efficiently removed (Rodriguez et al. 2004;
Kheradmand et al. 2009; Kheradmand et al. 2010; Neamati
et al. 2011; Alirezaei et al. 2012b). Two enzymes partici-
pate in the removal of H2O2 from the cellular environment,
GPx and CAT. The most abundant peroxidase is the glu-
tathione peroxidase (GPx), which is present in both the
cytosol and mitochondria. This enzyme has the transition
metal selenium at its active site and uses reduced gluta-
thione (GSH) as a substrate to transfer electrons to H2O2
(and other peroxides) thereby converting it into two mol-
ecules of water. The second H2O2 metabolizing enzyme is
catalase (CAT); it is present mainly in the peroxisomes,
presents a molecule of ferric ion at its active site and
converts two molecules of H2O2 into one molecule each of
water and diatomic oxygen (Mates 2000; Rodriguez et al.
2004; Kheradmand et al. 2009; Kheradmand et al. 2010;
Neamati et al. 2011; Alirezaei et al. 2012b). Antioxidant
enzymes are regulated by multiple factors. Oxidative status
of the cell is the primary factor regulating gene expression
and activity of these enzymes (Rodriguez et al. 2004). Both
endogenous (Nicotera et al. 1989) and exogenous agents
(Kim et al. 1999; Yoo et al. 1999) act as oxidants and alter
cellular oxidative equilibrium subsequently, antioxidant
enzyme gene expression (Rodriguez et al. 2004). There-
fore, antioxidants play a critical role in limiting the prop-
agation of free radical reactions, which would otherwise
result in extensive lipid peroxidation (Sehirli et al. 2008;
Alirezaei et al. 2011a; Alirezaei et al. 2012b).
As previously mentioned, presence of different antiox-
idant defense systems is well documented in the rat ovary.
Corpus luteum has an antioxidant enzyme to scavenge
ROS: Cu, Zn–SOD (Kheradmand et al. 2010). Decrease in
intracellular SOD activity inhibits progesterone production
by rat luteal cells and results in the loss of luteal function,
which may be mediated by ROS (Sugino et al. 1999).
Furthermore, the role of GPx in maintaining low concen-
trations of hydroperoxides inside the follicle has been
suggested, in which, the mean GPx activity in the follicular
fluid was found to be approximately 70 % of its serum
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activity (Paszkowski et al. 1995). A previous study showed
that the intensity of peroxidation in the Graafian follicle is
much lower than that in serum (Jozwik et al. 1999). This
gradient is the result of the lower rate of initiation of per-
oxidation in the follicular fluid, suggestive of the presence
of efficient antioxidant defense systems in the direct milieu
of the oocyte such as GSH (Tsai-Turton and Luderer 2006)
and GPx (Paszkowski et al. 1995). The intensive metabo-
lism of granulosa cells and the high numbers of macro-
phages and neutrophilic granulocytes in the follicular wall
at ovulation may point to active generation of ROS (Ebisch
et al. 2007). Margolin et al. (1990) observed that ROS are
involved in the loss of sensitivity of granulosa cells to
gonadotrophic hormones and in the loss of steroidogenic
function, both of which are characteristics of follicular
atresia. Inhibiting the ability of a cell to scavenge or
detoxify ROS is another way by which oxidative stress can
induce apoptosis (Kheradmand et al. 2010; Kheradmand
et al. 2012). In the present experiment, betaine treatment
considerably increased GPx activity, as main antioxidant
enzyme, against oxidative damage in the rat ovarian tissue
in Betaine and Betaine ? EtOH groups compared to the
control group. Enhanced level of GPx and CAT activities
in the ovary suggested scavenging of free radicals from the
ovarian tissue following administration of betaine and
prevention of destructive effect of oxidative stress induced
by ethanol in the rat ovaries.
In the present study, a significant elevation in the con-
centration of TBARS was noted in the ovary of the EtOH
group compared to controls (Fig. 4). As indicated in our
results, lipid peroxidation, which functions as a marker of
oxidative injury of cellular membranes (Husain et al. 2001;
Alirezaei et al. 2011b; Alirezaei et al. 2012b) significantly
increased following ethanol treatment. The concentration of
TBARS is a direct evidence of toxic processes caused by free
radicals (Yao et al. 2007; Kheradmand et al. 2010). There-
fore, it can be concluded that betaine preserves the ovarian
cell membranes against oxidative stresses and lipid peroxi-
dation as shown by slightly reduction of TBARS in betaine
group compared to EtOH group. These data support and
extend previous reports about betaine and are in agreement
with our new investigation, in which we demonstrated that
betaine administration increases testicular antioxidant
status, subsequently elevation of sperm motility and con-
centration in rats (Alirezaei et al. 2012a). Likewise, the
antioxidant properties of betaine are consistent with another
recent work, in which we showed that betaine enhances
antioxidant enzyme activities against oxidative stress med-
iated by ethanol in cerebellum of rats (Alirezaei et al. 2011a).
Homocysteine is a potent inhibitor of antioxidant
enzymes in cells at the level of gene expression (Bleich
et al. 2004; Alirezaei et al. 2011a; Alirezaei et al. 2012a).
Likewise, hyperhomocysteinemia is associated with the
production of ROS in endothelial and smooth muscle cells
(Dinu et al. 2006; Alirezaei et al. 2010; Alirezaei et al.
2011a; Alirezaei et al. 2011b). The mechanism of this
oxidative stress returns to auto-oxidation of the highly
reactive thiol group of homocysteines (Forges et al. 2007)
and the formation of intracellular superoxide and peroxyl
radicals with concomitant inhibition of cellular antioxidant
enzymes, such as SOD and GPx (Forges et al. 2007; Ali-
rezaei et al. 2010; Alirezaei et al. 2011a; Alirezaei et al.
2011b). Elevated level of homocysteine has also been
reported to be associated with increased lipid peroxidation
products (Alirezaei et al. 2011a; Alirezaei et al. 2012a).
Herein, it is observed that ethanol feeding significantly
elevated the level of homocysteine in EtOH group and
administration of betaine could suppress homocysteine
concentration in Betaine ? EtOH group. However, it
appears the applied dosage of betaine treatment was not
sufficient to suppress homocysteine accumulation as
markedly (Fig. 5). The toxic accumulation of homocyste-
ine may cause reproductive dysfunction and oxidative
stress within the testis (Alirezaei et al. 2012a; Tremellen
2008), and ovary. The suggestion that chronic ethanol
consumption might interfere with homocysteine remethy-
lation was first raised by Barak and his colleague (Barak
and Beckenhauer 1988), and also with chronic ethanol
consumption in our recent studies (Alirezaei et al. 2010;
Alirezaei et al. 2011a; Alirezaei et al. 2011b; Alirezaei
et al. 2012a). Although, in the present study we were
unable to measure dimethylglycine (DMG) for detection of
BHMT activity in contrast, other studies have shown that
feeding of alcohol or methionine to rats significantly
reduce the activity of methionine synthase followed by an
increase in BHMT activity to maintain adequate tissue
levels of SAM (Barak et al. 1985; Barak and Beckenhauer
1988; Finkelstein 2007; Alirezaei et al. 2010; Alirezaei
et al. 2011a; Alirezaei et al. 2011b). In this sense, betaine
supplementation increased DMG levels in plasma and liver
of rats and specific activity of the liver betaine-metabo-
lizing enzyme (BHMT) increased significantly following
betaine treatment (2 % w/v of the diet) in both Mthfr (?/?)
and Mthfr (±) groups of mice (Schwahn et al. 2004).
Researches with chronic alcoholic women (Hugues et al.
1980) and monkeys (Mello et al. 1983) reported amenor-
rhea followed by low estrogen and LH secretion, endo-
metrial and ovarian atrophy (Chuffa et al. 2009). Ovarian
atrophy also, has been reported in alcoholic rats (Krueger
et al. 1982; Valimaki et al. 1995; Chuffa et al. 2009), and
this was restricted to the ovarian medulla (Chuffa et al.
2009). In the present study, ethanol-treated animals had
irregular estral cycles with large cycles and persistent
oestrous phases, similar to a previous report (Chuffa et al.
2009). It is well-known that follicular atresia in rats occurs
via apoptosis following oxidative stress (Greenfeld et al.
Int J Pept Res Ther (2012) 18:281–290 287
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2007; Chuffa et al. 2009; Kheradmand et al. 2010) and
therefore, ethanol induces oxidative stress results in irreg-
ular cycles. In contrast, betaine administration prevents
oxidative stress in both Betaine and Betaine ? EtOH
groups which indicated regular estral cycles. The prolon-
gation of the oestrous phase observed in ethanol-treated
animals should be associated with the reduction in
b-oestradiol (Chuffa et al. 2009). Although in this research,
the hormone measurement was not applied however, in the
ethanol drinker (UCh) strain rats, interstitial glandular tis-
sue was common and organized in cell isles originated
from thecal layers of atretic follicles, contributing with
b-oestradiol and testosterone replacement (Chuffa et al.
2009). In the human population, betaine is a significant
predictor of tHcy at week 28 and delivery (Wallace et al.
2008). The methyl donor effect of betaine on tHcy con-
centration may also be evident in pregnancy, a time when
methionine and protein turnover are elevated (Rees et al.
2006; Wallace et al. 2008). However, this result may only
be evident when the status of folate is low such as alco-
holism or pregnancy, because in human population, many
of the pregnant women have a serum folate concentration
in the deficient range (Wallace et al. 2008). Therefore, we
think prolonged treatment by betaine or higher doses may
be as a potential therapy in pregnant women however,
further studies are needed to clarify this point.
As above mentioned, this study is the first in vivo
experiment that demonstrates the betaine antioxidant
effects on the rat ovarian tissue. Betaine is believed to play
a significant role in maintaining the structural and func-
tional integrity of cell membranes (Ganesan et al. 2010;
Alirezaei et al. 2011a; Alirezaei et al. 2011b). We con-
cluded, oxidative stress induces irregular cyclicity in rats
and the beneficial effects of betaine are mediated in part by
stimulation of GPx and CAT activities and another part via
suppression of hyperhomocysteinemia (Alirezaei et al.
2011a; Alirezaei et al. 2012a). These results highlight the
importance of betaine in the control of oxidative stress as
our previous report in male rats (Alirezaei et al. 2012a).
Acknowledgment This research was financially supported by
School of Veterinary Medicine-Shiraz University, Shiraz, Iran. We
are most grateful to Saeedeh Ahmadi for the kind technical assistance;
also like to thank M. Shoaei and R. Shirazi (the member and manager
of Aryadalman Company, Tehran, Iran) for providing betaine
(Betafine�).
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