Biol Trace Elem Res (2014) 160:409–417
DOI 10.1007/s12011-014-0067-8
Combination of Omega-3 Fatty Acids, Lithium, and Aripiprazole
Reduces Oxidative Stress in Brain of Mice with Mania
Pandiyan Arunagiri & Krishnamoorthy Rajeshwaran &
Janakiraman Shanthakumar & Thangavel Tamilselvan &
Elumalai Balamurugan
Received: 15 April 2014 / Accepted: 7 July 2014 / Published online: 18 July 2014
# Springer Science+Business Media New York 2014
Abstract Manic episode in bipolar disorder (BD) was evalu-
ated in the present study with supplementation of omega-3
fatty acids in combination with aripiprazole and lithium on
methylphenidate (MPD)-induced manic mice model.
Administration of MPD 5 mg/kg bw intraperitoneally (i.p.)
caused increase in oxidative stress in mice brain. To retract
this effect, supplementation of omega-3 fatty acids 1.5 ml/kg
(p.o.), aripiprazole 1.5 mg/kg bw (i.p.), and lithium 50 mg/kg
bw (p.o) were given to mice. Omega-3 fatty acids alone and in
combination with aripiprazole- and lithium-treated groups
significantly reduced the levels of superoxide dismutase
(SOD), catalase (CAT), and lipid peroxidation products (thio-
barbituric acid reactive substances) in the brain. MPD treat-
ment significantly decreased the reduced glutathione (GSH)
level and glutathione peroxidase (GPx) activity, and they were
restored by supplementation of omega-3 fatty acids with
aripiprazole and lithium. There is no remarkable difference
in the effect of creatine kinase (CK) activity between MPD-
induced manic model and the treatment groups. Therefore, our
results demonstrate that oxidative stress imbalance and mild
insignificant CK alterations induced by administration of
MPD can be restored back to normal physiological levels
through omega-3 fatty acids combined with lithium and
aripiprazole that attributes to effective prevention against ma-
nia in adult male Swiss albino mice.
Keywords Oxidative stress . Creatine kinase . Mania .
Omega-3 fatty acids . Aripiprazole
P. Arunagiri : K. Rajeshwaran : J. Shanthakumar : T. Tamilselvan :
E. Balamurugan (*)
Department of Biochemistry and Biotechnology, Faculty of Science,
Annamalai University, Annamalainagar, Tamil Nadu 608 002, India
e-mail: balamurugan_au@yahoo.co.in
Introduction
Bipolar disorder (BD) is a severe mental disorder, which is
associated with elevated suicide rates [1]. Mania is the cardi-
nal feature of BD and traditionally has been assessed using
self- and observer-rating scales [2]. Many signs and symptoms
related to BD can be replicated in animal models with dopa-
minergic stimulants, such as amphetamine and cocaine. For
instance, amphetamine administration in rats induces
hyperlocomotion, insomnia, and enhanced sexual drive [3],
which are behavioral alterations that resemble manifestations
of human bipolar mania [4]. In recent years, increased oxida-
tive stress has been implicated in the pathogenesis of numer-
ous diseases including cancer, atherosclerosis, schizophrenia,
and BD [5, 6]. Martins and his colleagues have demonstrated
that methylphenidate (MPD) induces imperative mood and
increased oxidative stress in young rats [7]. Increased neuro-
nal oxidative stress levels generate deleterious effects on
signal transduction, structural plasticity, and cellular resil-
ience, mostly by inducing lipid peroxidation in membranes,
proteins, and genes. Such changes in diverse oxidative stress
parameters have been reported in BD and schizophrenia [5, 6].
Oxidative stress caused by reactive oxygen species (ROS)
occurs as a consequence of imbalance between the production
and inactivation of these species. Important antioxidant en-
zymes include superoxide dismutase (SOD, EC.1.15.1.1),
which catalyzes dismutation of superoxide anion to H2O2,
which is then deactivated to H 2 O by catalase (CAT,
EC.1.11.1.6) and glutathione peroxidase (GPx, EC.1.11.1.9).
Thiobarbituric acid reactive substance (TBARS) levels are
considered a direct index of cell lipid peroxidation and it
was ameliorated by coordinated effects of primary antioxidant
system including SOD, CAT, and GPx. GPx is a selenium (Se)
ion-containing enzyme that is responsible for the reduction of
hydro and organic peroxides in the presence of reduced glu-
tathione (GSH) [8]. Lithium and aripiprazole has been well
410
demonstrated for decreasing the oxidative stress involving
various diverse mechanisms, as it is well documented with
animal model and clinical studies in BD [9, 10]. Some side
effects of antipsychotics limit their long-term use and are
probably associated with oxidative stress and/or energy im-
pairment [11]. Previous reports showed that lipid peroxidation
was diminished by olanzapine and aripiprazole, but not halo-
peridol and clozapine administration in the prefrontal cortex.
On the other hand, haloperidol and clozapine, but not
olanzapine, ultimately cause oxidative stress [12]. Also, Eren
and his colleagues reported the beneficial effects of
lamotrigine, aripiprazole, and escitalopram and clearly
reviewed antiepileptic drugs on oxidative stress molecular
pathways [13, 14]. The brain and other high-energy tissues
are more prone to stress in energy metabolism. In this context,
neuropsychiatry disorders, such as schizophrenia and BD,
have been related to dysfunction in brain metabolism. The
metabolism dysfunction includes mitochondrial impairment
[15], increase in ROS production, and expression of biochem-
ical markers of cellular degeneration [9].
Creatine kinase (CK, EC 2.7.3.2) catalyzes the reversible
transfer of the phosphoryl group from phosphocreatine to
adenosine diphosphate (ADP), regenerating adenosine tri-
phosphate (ATP). It is also known that a decrease in CK
activity is associated with neurodegenerative pathways that
results in neurodegenerative diseases [16], BD, and other
pathological states [17]. Omega-3 fatty acids are essential
for the physiological function of neuronal cell membrane.
Normal function of neuronal cell membrane requires appro-
priate composition of fatty acids in its structure. Decreased
omega-3 fatty acid intake and increased oxidative stress could
contribute to brain docosahexaenoic acid (DHA) depletion
and low blood levels in Alzheimer’s disease (AD) patients
[18]. Omega-3 fatty acid supplementation becomes especially
important because the majority of diets contain a great quan-
tity of omega-6 and insufficient omega-3 fatty acids. Several
epidemiological studies show a protective effect associated
with increased fish consumption on dementia and cognitive
performance [19]. Recent reports from our laboratory have
shown that omega-3 fatty acid supplementation with
aripiprazole and lithium in MPD-induced manic model result-
ed in significant changes in the behavioral activities test
evaluated by forced swim test (FST), actophotometer test,
and open field test (OFT) [20].
However, as far as the reports are concerned, there are no
data regarding the effect of omega-3 fatty acids after MPD
administration related to the CK activity, antioxidant system,
and lipid peroxidation in the brain. The current study was
performed to determine the activity of antioxidant enzymes
including SOD, CAT, GPx, and GSH to assess the scavenging
effects on ROS and MDA level as an indicator of oxidative
damage or lipid peroxidation in mice brain after acute treat-
ment of MPD. There is also lack of data regarding the use of
Arunagiri et al.
these drugs on brain energy metabolism and oxidative stress
impairment in manic model. Taking all these into consider-
ation, in the present study, we had evaluated the effects of
omega-3 fatty acid combined with aripiprazole and lithium in
MPD-induced manic model by analyzing CK activity,
TBARS, SOD, CAT, GPx, and GSH levels in mice brain.
Materials and Methods
Animals and Drug Administration
The male adult Swiss albino mice (weighed 25–30±5 g) were
housed in wire-topped plastic cages, as six animals per cage.
Control and experimental mice received a standard diet of
rodent chow (12–15 g/day) and water ad libitum. All mice
were kept on an alternating 12-h light and 12-h dark cycle. All
experiments were performed at the same time every day and in
the light period (9:00–11:00 A.M.). All experimental proce-
dures were approved, and all the animals were taken care
according to the Institutional Animal Ethical Committee of
Rajah Muthiah Medical College and Hospital, Annamalai
Nagar, Tamil Nadu, India (Reg No. 160/1999/CPCSEA,
Proposal number 933).
After 7 days of acclimatization period, the mice were
randomly assigned to eight groups consisting of six mice per
group. The study group was administered 5 mg/kg/day of
MPD intraperitoneally (i.p.), whereas the control group was
administered distilled water. The dosage of MPD administra-
tion to mice is similar to that of Barbosa et al. [21]. It was
procured from Ipca pharmaceutical company; 1.5 ml of 0.1 %
fish oil (FO) with a homogenous 1 % Tween suspension [22]
contained 120–180 mg eicosapentaennoic acid (EPA)/
docosahexaeonoic acid (DHA), and lithium carbonate
(50 mg/kg bw) given orally [23]; aripiprazole (1.5 mg/kg
bw) [24] was kindly provided by Sun Pharmaceutical,
Karnataka, India, and dissolved in water and administrated
intraperitoneally (i.p.). All other chemicals used in this study
were of analytical grade obtained from HiMedia Laboratories,
Mumbai, India.
The dose of lithium, aripiprazole, and omega-3 fatty acids
were chosen based on previous literature [21–24], and their
combinatorial effects on behavior studies were confirmed
[20].
The experimental design of the current study was as fol-
lows; each of the following groups consists of six animals.
Group I: Control animals
Group II: Control+lithium (50 mg/kg bw)+aripiprazole
(1.5 mg/kg bw)+omega-3 fatty acids (1.5 ml/
kg bw)
Group III: Mania animals (methylphenidate (5 mg/kg b.w))
Group IV: Mania+lithium (50 mg/kg bw)
Omega-3 Fatty Acid with Lithium and Aripiprazole Reduces Mania
Group V: Mania+aripiprazole (1.5 mg/kg bw)
Group VI: Mania+omega-3 fatty acids (1.5 ml/kg bw)
Group VII: Mania+lithium (50 mg/kg bw)+aripiprazole
(1.5 mg/kg bw)
Group VIII: Mania+lithium (50 mg/kg bw)+aripiprazole
(1.5 mg/kg bw)+omega-3 fatty acids (1.5 ml/
kg bw)
Biochemical Assays
Preparation of the Homogenate
After the behavioral analysis, animals were sacrificed by
decapitation immediately (OFT, actophotometer test, FST)
[20]; mice brain tissues were stored at −80 °C until used for
the biochemical analysis. The mice brain tissues were thawed,
weighed, and then placed in chilled 0.1 mol/l Tris–HCl buffer,
pH 7.4. The samples were homogenized using a Potter–
Elvehjem homogenizer (Wheaton Science Products,
Millville, NJ, USA) filled with Teflon pestle to produce
10 % homogenates and used for determining the biochemical
parameters described below.
Estimation of Lipid Peroxidation
The level of lipid peroxidation (TBARS) was determined by
analyzing TBA-reactive substance according to the method of
Niehaus and Samuelsson [25]. The pink-colored chromogen
formed by the reaction of 2-TBA with breakdown products of
lipid peroxidation was measured spectrophotometrically at
532 nm. The values were expressed as nanomoles per milli-
gram of protein.
Determination of SOD and CAT Activities
Superoxide dismutase activity was measured using the
method of Kakkar et al. [26] and calculated using the
percentage of inhibition of formazan formation. One
unit of the enzyme is defined as the amount of enzyme
required for 50 % inhibition of NBT reduction per
minute per milligram protein. CAT was assayed using
the method of Sinha [27]. The reaction mixture
contained 0.1 ml of tissue homogenate, 1 ml of phos-
phate buffer (0.01 mol, pH 7.0), and 0.2 mol H2O2. The
reaction was arrested by the addition of a dichromate
acetic acid reagent, and the chromic acetate formed was
determined spectrophotometrically at 590 nm. The
values are expressed as micromoles of H2O2 utilized
per minute milligram protein.
411
Determination of the Levels/Activities of Glutathione
and Glutathione Peroxidase
Estimation of reduced glutathione (GSH) in the brain tissue
was performed by the method of Ellman [28]. This method is
based on the development of a yellow color, when
dithionitrobenzoic acid is added to compounds containing
sulfhydryl groups. The color developed was read at 412 nm.
Glutathione peroxidase (GPx) activity in the heart tissue was
assayed by the method of Rotruck et al. [29]. A known
amount of enzyme preparation was allowed to react with
hydrogen peroxide and GSH for a specified time period. The
GSH content remaining after the reaction was measured by
Ellman’s reaction.
The Activity of Creatine Kinase in Brain Homogenates
CK activity was measured in brain homogenates pretreated
with 0.625 mmol lauryl maltoside. The reaction mixture
consisted of 60 mmol Tris–HCl, pH 7.5, containing 7 mmol
phosphocreatine, 9 mmol MgSO4, and approximately 0.4–
1.2 μg protein in a final volume of 100 μl. After 15 min of
preincubation at 37 °C, the reaction was started by the addition
of 0.3 μM of ADP plus 0.08 μmol of reduced glutathione. The
reaction was stopped after 10 min by the addition of 1 μmol of
p-hydroxymercuribenzoic acid. The creatine formed was esti-
mated according to the colorimetric method of Hughes [30].
The color was developed by the addition of 100 μl 2 % α-
naphthol and 100 μl 0.05 % diacetyl in a final volume of 1 ml
and read spectrophotometrically after 20 min at 540 nm.
Results were expressed as units per minute × milligram
protein.
Protein Determination
The protein content of the brain tissue homogenate was esti-
mated using the method of Lowry et al. [31]. About 0.5 ml of
brain tissue homogenate was precipitated with 0.5 ml of 10 %
TCA and centrifuged for 10 min, and the precipitate was
dissolved in 1.0 ml of 0.1 N NaOH. About 0.1 ml of aliquot
was taken and made up to 1.0 ml with distilled water. Then,
4.5 ml of alkaline copper reagent was added and allowed to
stand at room temperature for 10 min. After incubation, 0.5 ml
of Folin–Ciocalteu reagent was added, and the blue color
developed was read at 620 nm after 20 min; bovine albumin
was used as standard.
Statistical Analysis
All the values were expressed as mean±SD of six determina-
tions. Statistical analysis of the data was carried out by one-
way ANOVA on Statistical Package for Social Sciences
(SPSS), and the group mean compared by Duncan’s multiple
412
range test (DMRT). A value of P<0.05 was considered sig-
nificant. Student’s t test will be employed whenever necessary.
Results
Effect of Omega-3 Fatty Acid Supplementation with Lithium
and Aripiprazole on Lipid Peroxidation
Figure 1 shows the effect of omega-3 fatty acids, lithium, and
aripiprazole on brain tissue lipid peroxidation. The level of
TBARS in the brain was significantly increased in MPD-
treated mice; after treatment with lithium and aripiprazole,
these levels were significantly decreased. In addition, the
omega-3 fatty acid supplement along with lithium and
aripiprazole reduces the TBARS levels and retracted back to
normal physiological levels. The effect was more pronounced
(P<0.05) when omega-3 fatty acids was supplemented to the
MPD-exposed mice (group VIII).
Effect of Omega-3 Fatty Acid Supplementation with Lithium
and Aripiprazole on the Activity of SOD and CAT
The activities of SOD and CAT in the control and experimen-
tal mice are shown in Figs. 2 and 3. The activities of these
Fig. 1 Effects of omega-3 fatty acid in combination with aripiprazole and
lithium on thiobarbituric acid reactive substances in brain tissue of control
and experimental manic mice. Group I: normal control mice; group II:
mice were treated with lithium (50 mg/kg)+aripiprazole (1.5 mg/kg)+
omega-3 fatty acid (1.5 ml/kg); group III: mice were treated with meth-
ylphenidate (MPD) (5 mg/kg); group IV: mice were treated with MPD
(5 mg/kg)+lithium (50 mg/kg); group V: MPD (5 mg/kg)+aripiprazole-
treated mice (1.5 mg/kg); group VI: mice were treated with MPD
(5 mg/kg)+supplemented with omega-3 fatty acids (1.5 ml/kg); group
VII: mice were treated with MPD (5 mg/kg)+lithium (50 mg/kg)+
aripiprazole (1.5 mg/kg); group VIII: mice were treated with MPD
(5 mg/kg)+lithium (50 mg/kg)+aripiprazole (1.5 mg/kg)+omega-3 fatty
acid (1.5 ml/kg); each column is mean±SD for six mice in each group;
values not sharing a common symbol (*, #) differ significantly with each
other (P<0.05; DMRT)
Arunagiri et al.
Fig. 2 Effects of omega-3 fatty acid in combination with aripiprazole and
lithium on superoxide dismutase in brain tissue of control and MPD-
induced manic mice. U enzyme concentration required to inhibit the
chromogen produced by 50 % in 1 min under standard condition. Group
I: normal control mice; group II: mice were treated with lithium
(50 mg/kg)+aripiprazole (1.5 mg/kg)+omega-3 fatty acid (1.5 ml/kg);
group III: mice were treated with methylphenidate (MPD) (5 mg/kg);
group IV: mice were treated with MPD (5 mg/kg)+lithium (50 mg/kg);
group V: MPD (5 mg/kg)+aripiprazole-treated mice (1.5 mg/kg); group
VI: mice were treated with MPD (5 mg/kg)+supplemented with omega-3
fatty acids (1.5 ml/kg); group VII: mice were treated with MPD
(5 mg/kg)+lithium (50 mg/kg)+aripiprazole (1.5 mg/kg); group VIII:
mice were treated with MPD (5 mg/kg) + lithium (50 mg/kg) +
aripiprazole (1.5 mg/kg)+omega-3 fatty acid (1.5 ml/kg); each column
is mean±SD for six mice in each group; values not sharing a common
symbol (*, #) differ significantly with each other (P<0.05; DMRT)
Fig. 3 Effects of omega-3 fatty acid in combination with aripiprazole and
lithium on catalase in brain tissue of control and MPD-induced manic
mice. U micromoles of H2O2 consumed per minute. Group I: normal
control mice; group II: mice were treated with lithium (50 mg/kg)+
aripiprazole (1.5 mg/kg)+omega-3 fatty acid (1.5 ml/kg); group III: mice
were treated with methylphenidate (MPD) (5 mg/kg); group IV: mice
were treated with MPD (5 mg/kg)+lithium (50 mg/kg); group V: MPD
(5 mg/kg)+aripiprazole-treated mice (1.5 mg/kg); group VI: mice were
treated with MPD (5 mg/kg)+supplemented with omega-3 fatty acids
(1.5 ml/kg); group VII: mice were treated with MPD (5 mg/kg)+lithium
(50 mg/kg)+aripiprazole (1.5 mg/kg); group VIII: mice were treated with
MPD (5 mg/kg)+lithium (50 mg/kg)+aripiprazole (1.5 mg/kg)+omega-
3 fatty acid (1.5 ml/kg); each column is mean±SD for six mice in each
group; values not sharing a common symbol (*, #) differ significantly
with each other (P<0.05; DMRT)
Omega-3 Fatty Acid with Lithium and Aripiprazole Reduces Mania
Fig. 4 Effects of omega-3 fatty acid in combination with aripiprazole and
lithium on glutathione peroxidase in brain tissue of control and MPD-
induced manic mice. U microgram of GSH utilized per minute. Group I:
normal control mice; group II: mice were treated with lithium
(50 mg/kg)+aripiprazole (1.5 mg/kg)+omega-3 fatty acid (1.5 ml/kg);
group III: mice were treated with methylphenidate (MPD) (5 mg/kg);
group IV: mice were treated with MPD (5 mg/kg)+lithium (50 mg/kg);
group V: MPD (5 mg/kg)+aripiprazole-treated mice (1.5 mg/kg); group
enzymes were significantly increased in the mice brain tissues
of the MPD-treated mice (group III) relative to those of the
mice in the control group (group I). Lithium alone-treated
mice and combination of lithium and aripiprazole-treated mice
show decrease in SOD and CAT activity. Omega-3 fatty acid
alone-treated mice had also decreased the SOD and CAT
activity in mice brain. Therefore, the effect of supplementation
of omega-3 fatty acids in lithium and aripiprazole-treated mice
showed statistically significant decrease in both SOD and
Fig. 5 Effects of omega-3 fatty acid in combination with aripiprazole and
lithium on glutathione in brain tissue of control and MPD-induced manic
mice. Group I: normal control mice; group II: mice were treated with
lithium (50 mg/kg)+ aripiprazole (1.5 mg/kg)+ omega-3 fatty acid
(1.5 ml/kg); group III: mice were treated with methylphenidate (MPD)
(5 mg/kg); group IV: mice were treated with MPD (5 mg/kg)+lithium
(50 mg/kg); group V: MPD (5 mg/kg)+ aripiprazole-treated mice
(1.5 mg/kg); group VI: mice were treated with MPD (5 mg/kg)+
413
VI: mice were treated with MPD (5 mg/kg)+supplemented with omega-3
fatty acids (1.5 ml/kg); group VII: mice were treated with MPD
(5 mg/kg)+lithium (50 mg/kg)+aripiprazole (1.5 mg/kg); group VIII:
mice were treated with MPD (5 mg/kg) + lithium (50 mg/kg) +
aripiprazole (1.5 mg/kg)+omega-3 fatty acid (1.5 ml/kg); each column
is mean±SD for six mice in each group; values not sharing a common
symbol (*, #) differ significantly with each other (P<0.05; DMRT)
CAT activity when compared with all the other groups
(P<0.05).
Effect of Omega-3 Fatty Acid Supplementation on GSH Level
and GPx Activity
Figures 4 and 5 depict the activity of GPx and GSH levels, in
the brain homogenates of control and experimental groups of
mice. Acute mania leads to significantly (P<0.05) lowered
supplemented with omega-3 fatty acids (1.5 ml/kg); group VII: mice
were treated with MPD (5 mg/kg)+lithium (50 mg/kg)+aripiprazole
(1.5 mg/kg); group VIII: mice were treated with MPD (5 mg/kg)+lithium
(50 mg/kg)+aripiprazole (1.5 mg/kg)+omega-3 fatty acid (1.5 ml/kg);
each column is mean±SD for six mice in each group; values not sharing a
common symbol (*, #) differ significantly with each other (P<0.05;
DMRT)
414
GPx activity and GSH levels in the brain tissues, i.e., group III
mice as compared to that of group I, whereas GPx activity and
levels of GSH were elevated significantly in the other treat-
ment group. Treatment with omega-3 fatty acid supplementa-
tion combined with aripiprazole and lithium maintained the
balance of GPx and levels of GSH to near-normal physiolog-
ical levels.
Creatine Kinase Activity
Figure 6 depicts CK activity, where treatment with lithium,
aripiprazole alone, and combined treatment with omega-3
fatty acids in MPD-induced manic mice did not show signif-
icant effect in CK activity.
Discussion
Oxidative stress has been proposed to play a significant role in
the pathophysiology of major neuropsychiatric disorders, such
as BD and schizophrenia [6, 7]. Researchers have found
evidence that individuals with BD had increased serum
TBARS in the initial manic episode [11, 12]. Moreover, an
increase in TBARS levels and in superoxide generation in
sub-mitochondrial particles was found in the rat brain after
MPD treatment [32, 33]. There is evidence regarding some
side effects of antipsychotics that it limits their long-term use
and are probably associated to oxidative stress and metabo-
lism impairment [11]. In this context, several studies show that
antipsychotics aripiprazole, olanzapine, clozapine, and
Fig. 6 Effects of omega-3 fatty acid in combination with aripiprazole and
lithium on creatine kinase in brain tissue of control and MPD-induced
manic mice. Values are expressed as units per minute×milligram protein,
for six independent experiments performed in duplicate. Group I: normal
control mice; group II: mice were treated with lithium (50 mg/kg)+
aripiprazole (1.5 mg/kg)+omega-3 fatty acid (1.5 ml/kg); group III: mice
were treated with methylphenidate (MPD) (5 mg/kg); group IV: mice
were treated with MPD (5 mg/kg)+lithium (50 mg/kg); group V: MPD
Arunagiri et al.
especially haloperidol cause oxidative stress. The biochemical
and physiological characteristics of the brain, with high un-
saturated phospholipid content and energy requirement, make
this organ particularly susceptible to free radical-mediated
damage [34].
The present study intends to investigate the role of omega-3
fatty acid supplementation with aripiprazole and lithium to
protect against the increase in lipid peroxidation, one of the
major consequences of free radical-mediated injury, caused by
MPD in the brain of mice. To the best of our knowledge, this is
the first study carried out on the protective effect of omega-3
fatty acids on brain changes linked with oxidative stress and
brain energy metabolism in MPD-induced manic behavior.
We found that lipid peroxidation level in the brain was in-
creased by MPD-induced mania, whereas GPx activity and
GSH levels were decreased. Hence, MPD-induced mania in
the animals was characterized by increased activities of SOD,
CAT, and level of TBARS in whole brain homogenates in
MPD-induced animals. In contrast, previous result shows
aripiprazole, lamotrigine, and escitalopram supplementations
caused a decrease in lipid peroxidation levels, whereas the
values of the antioxidants increased. MPD increases the oxi-
dative stress, when both acute and chronic stress with MPD
results in superoxide production in the brain [32, 33].
Likewise, our results showed that the effect of MPD increased
SOD and hydrogen peroxide in mice. This may be due to
partial blocking of dopamine and norepinephrine transporter
that amplifies the free dopamine in the brain with MPD
treatment. These excessive dopamine levels can induce oxi-
dative stress and inflammation in the brain [35]. A previous
(5 mg/kg)+aripiprazole-treated mice (1.5 mg/kg); group VI: mice were
treated with MPD (5 mg/kg)+supplemented with omega-3 fatty acids
(1.5 ml/kg); group VII: mice were treated with MPD (5 mg/kg)+lithium
(50 mg/kg)+aripiprazole (1.5 mg/kg); group VIII: mice were treated with
MPD (5 mg/kg)+lithium (50 mg/kg)+aripiprazole (1.5 mg/kg)+omega-
3 fatty acid (1.5 ml/kg); each column is mean±SD for six mice in each
group; values not sharing a common symbol (*, #) differ significantly
with each other (P<0.05; DMRT)
Omega-3 Fatty Acid with Lithium and Aripiprazole Reduces Mania
report shows that levels of SOD, CAT, and TBARS were
increased in whole brain homogenates in ouabain-induced as
well as MPD-induced animal model of mania [32, 36].
Similarly, our results also showed that the levels of SOD,
CAT, and TBARS were increased in whole brain homoge-
nates of MPD-induced animals; however lithium,
aripiprazole, and omega-3 fatty acid supplementation reverses
the effect of MPD by lithium acting as a mood stabilizer,
aripiprazole acting as an atypical antipsychotic, and ultimately
omega-3 fatty acids as a supplement. A previous animal
model study suggested that lithium and aripiprazole antago-
nize the amphetamine-induced locomotor stimulation by its
ability to alleviate the drug-induced hyperdopaminergia with-
out draining to hypodopaminergia [37].
We found that the GPx activity and GSH level were
decreased in the brain of MPD-induced manic mice mod-
el. Previous reports clearly demonstrated that agomelatine
(antidepressant agent), selenium (essential dietary trace
element), topiramate (an antiepileptic drug), and also
omega-3 fatty acids relieve the oxidative stress in the cell
lines of the PC12 neurons [38–40]. This animal model,
when measured with lipid peroxidation levels and SOD,
CAT, GPx activity, responded with the reduction of hydro
and organic peroxides in the presence of GSH. In addi-
tion, the observed restraint manic-induced reductions in
GSH level and activity of GPx act as the second line of
antioxidant defense. GSH is a multifunctional intracellular
nonenzymatic thiol antioxidant, and GSH system is very
important in cellular defenses against oxidative stress.
Lithium and aripiprazole are found effectively to reverse
GSH level and GPx activity, which decline in induced
oxidative stress coordinatively by reinstating interrupted
GSH pathways. It is plausible that omega-3 supplementa-
tion with antipsychotic and mood stabilizer treatment may
exert the observed antioxidant effects via restoration of
critical GSH-related processes such as ROS scavenging,
detoxification of electrophilic compounds, modulation of
cellular redox status as well as thiol–disulfide status of
proteins and regulation of cell signaling and repair path-
ways [40, 41]. Similarly, Zhang et al. reported decreased
activity of GPx in patients with schizophrenia [41]. In
contrast, some other studies [5, 8] did not find any differ-
ences in GPx activity between schizophrenic patients and
normal controls.
Our recent study reports that MPD-induced behavioral
alterations were reversed by supplementation of omega-3 fatty
acids in combination with aripiprazole and lithium [20].
Subsequently, the current results also showed that, oxidative
stress was reverted back to redox homeostasis through lithi-
um, aripiprazole, and omega-3 fatty acids reducing the
oxidant/antioxidant imbalances. The fatty acids and mem-
brane antioxidants are structurally intermingled, thereby func-
tioning as a protective “first line of defense” [42]. In the
415
presence of antioxidants, fatty acids with the most unsaturated
bonds like omega-3 fatty acids docosahexaeonoic acid (DHA)
and eicosapentaennoic acid (EPA) rich in fish oil are protected
against oxidative (“free radical”) destruction. Thus, within the
dynamic membrane milieu, DHA and EPA exist in homeo-
static synergy with both their parent phospholipids and the
antioxidants dispersed in the membrane lipid bilayer, provid-
ing “triple cell membrane synergy.” Thus, in addition to
protective antioxidants, supplements that deliver DHA and
EPA bound to phospholipids—such as omega-3 fatty acids
bound to phosphatidylserine and krill oil—will provide as the
building blocks for healthy cell membranes. Also, one of the
convergent functional genomic studies gives us evidence that
omega-3 fatty acid modulation is involved in molecular net-
works when targeted by current psychotropic medications.
Their study also suggests intriguing possible sex differences
for the molecular and behavioral effects of omega-3 fatty
acids, with a more antidepressant-like profile in females and
a more antimanic-like profile in males [43].
Considering the CK activity, a previous study showed that
high-energy phosphate compounds, such as phosphocreatine
and ATP, were decreased in the brain of BD patients [44].
Feier and his colleagues reported that no significant difference
in CK activity was found in lithium- and valproate-treated D-
amphetamine-induced manic model [45]. The CK activity
between MPD-induced and drug-treated mice seems to
be negligible variation without any significant change
between the groups, in which our data corroborates with
earlier studies, suggesting no significant outcome from
the CK activity in the treatment groups. Further studies
are warranted to evaluate whether other enzymes in-
volved in energy metabolism are also affected by those
drugs used in this study.
Sadasivan and his colleagues reported that dopamine inter-
vention has a role in oxidative stress homeostasis [35]. Further
evaluation will be needed for analysis with intervention of
neurotransmitters such as dopamine, norepinephrine, and se-
rotonin (5-HT) as well as molecular (GSK3 and Akt) net-
works targeted by the current psychotic medication in omega-
3 fatty acid supplementation with lithium plus aripiprazole
combined action in animal model of mania. In conclusion, the
present biochemical findings showed that omega-3 fatty acids
in combination with aripiprazole and lithium reversed back
MPD-induced oxidative stress that is evidenced with im-
proved oxidative stress markers. Omega-3 fatty acid supple-
mentation with antipsychotic and mood stabilizer treatment
could possibly illuminate pathophysiological mechanisms and
suggest novel therapeutic approaches in manic treatment in
near future.
Acknowledgments We thank Annamalai University for the financial
assistance in the form of “University Research Fellowship” to Mr. P.
Arunagiri.
416
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