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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: alirezaei_m54@yahoo.com

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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