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8/8/2019 Role of an Aminothiazole derivative on ethanol and thermally oxidized sunflower oil induced toxicity
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ROLE OF AN AMINOTHIAZOLE DERIVATIVE ON ETHANOL-AND THERMALLY OXIDIZED SUNFLOWER OIL-INDUCEDTOXICITY
Kode Aruna, Rajagopalan Rukkumani, Penumathsa Sureshvarma,Venugopal P. Menon
D e p a r t m e n t o f B i o c h e m i s t r y , F a c u l t y o f S c i e n c e , A n n a m a l a i U n i v e r s i t y , A n n a m a l a i N a g a r 6 0 8 0 0 2 , T a m i l N a d u ,
I n d i a
Role of an aminothiazole derivative on ethanol- and thermally oxidized sunflower oil-induced toxicity. K. ARUNA, R. RUKKUMANI, P. SURE-SHVARMA, V.P. MENON. Pol. J. Pharmacol., 2004, 56, 233–240.
It is a known fact that ethanol increases lipid levels in humans and ex- perimental animals. In this study, we have investigated the effect of dendro-
doine analogue (DA), DA-[4-amino-5-benzoyl-2-(4-methoxyphenylamino)--thiazole], on alcohol- and thermally oxidized sunflower oil-induced hyper-lipidemia. Ethanol was given to animals at a dose of 5 ml of 20% solutionand thermally oxidized sunflower oil at a level of 15% (15 g oil/100 g feed).Our results showed increased activity of aspartate transaminase (AST), alka-line phosphatase (ALP) and g-glutamyl transferase (GGT) and increased lev-els of cholesterol, triglycerides and phospholipids in the plasma of groupsgiven alcohol, thermally oxidized oil and alcohol + thermally oxidized oilwhen compared with normal control group. The levels of tissue (liver andkidney) cholesterol and triglycerides were increased significantly in groupstreated with alcohol, thermally oxidized oil and alcohol + thermally oxidizedoil when compared with normal control rats. The levels were decreasedwhen DA was given along with alcohol and thermally oxidized oil. The level
of phospholipids decreased significantly in the liver and kidney of rats ad-ministered alcohol, thermally oxidized oil and alcohol + thermally oxidizedoil when compared with normal control rats. The level increased when DAwas administered along with alcohol and thermally oxidized oil. The activityof phospholipase A and C increased significantly in the liver of groups givenalcohol, thermally oxidized oil and alcohol + thermally oxidized oil whencompared with normal control rats, whereas the activity was decreased uponDA treatment. The obtained results indicate that DA can decrease the lipidlevels in alcohol- and thermally oxidized oil-treated rats.
Key words: ethanol, hyperlipidemia, phospholipases, dendrodoine ana-logue, aminothiazole derivative, thermally oxidized sunflower oil, n-6 PUFA
C o p y r i g h t © 2 0 0 4 b y I n s t i t u t e o f P h a r m a c o l o g y
P o l i s h A c a d e m y o f S c i e n c e s
P o l i s h J o u r n a l o f P h a r m a c o l o g y
P o l . J . P h a r m a c o l . , 2 0 0 4 , 5 6 , 2 3 3 2 4 0
I S S N 1 2 3 0 - 6 0 0 2
c o r r e s p o n d e n c e ; e - m a i l : c m r a n a @ s i f y . c o m
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Abbrevations: ALP – alkaline phosphatase, AST
– aspartate transaminase, DA – dendrodoine ana-logue, GGT – γ-glutamyl transferase, HMG CoA –
β –hydroxy-methyl-glutaryl CoA
INTRODUCTION
Ethanol is a powerful inducer of hyperlipidemia
in humans and in animals [6]. Hyperlipidemia oc-
curs when the intracellular redox potential and re-dox sensitive nutrient metabolisms are disturbed by
alcohol [27]. An excessive accumulation of reduc-
ing equivalents favors hepatic lipogenesis, de-
creases hepatic release of lipoproteins, increasesthe mobilization of peripheral fat, enhances the up-
take of circulating lipids and decreases the fatty
acid oxidation and, thus, increases the retention of
lipids in the liver [39].In contrast to earlier studies showing the re-
duced risk of coronary heart diseases due to intake
of polyunsaturated fatty acids (PUFA) [31], current
data on dietary fat indicate that it is not just the presence of PUFA but the type of PUFA that is im-
portant. A high PUFA n-6 content and n-6/n-3 ratio
in dietary fats is considered to be atherogenic [42].
Moreover, heating of oil produces various peroxi-dative changes. During deep fat frying, when the
fat is used repeatedly, oxidative and thermal effects
result in the formation of many volatile and non-volatile products, some of which are potentially
toxic [1]. Ingestion of decomposition products
formed as a result of thermal abuse and oxidation
of frying oils is known to lead to a variety of dis-eases [43].
Aminothiazoles are a group of biologically im-
portant compounds having a wide range of activi-ties, such as anti-tumor, antianoxic and antioxidant[32, 44]. Dendrodoine is a marine alkaloid, which
was isolated from Dendrodoa grossularia [18]. It
possesses a 1,2,4-thiadiazole unit, a rarity among
natural products. Though its synthesis has been re- ported [21], very little biological studies have been
carried out on it and its analogue (Fig. 1).
We undertook the present work, since very little
or no work has been done to study the effect of dendrodoine analogue (DA) on lipid levels in alco-
hol- and thermally oxidized oil-treated animals.
MATERIALS and METHODS
Experimental animals
Male albino Wistar rats 140–160 g bred in theCentral Animal House, Rajah Muthiah Medical
College, were used in this study. The animals were
housed in plastic cages with filter tops under semi-
natural light-dark conditions and at room tempera-ture. The animals were fed on pellet diet (Hindus-
tan Lever Limited, Mumbai) and water was given
ad libitum.
Chemicals
Ethanol was purchased from E. Merck, Darm-
stadt, Germany. DA was synthesized as described
by Rajasekharan et al. [36]. Purity of the compoundwas checked by thin layer chromatography and
structure has been confirmed by FTIR and NMR.
All other chemicals and biochemicals used for the
experiments were of analytical grade.
Experimental design
Dose-response studies were carried out and the
effective dose of DA was found to be 10 mg/kg.
The animals were randomized into the follow-ing groups.
G r o u p 1 c o n t r o l r a t s g i v e n s t a n d a r d p e l l e t d i e t .
G r o u p 2 r a t s g i v e n 2 0 % e t h a n o l .
G r o u p 3 r a t s g i v e n D A ( 1 0 m g / k g ) .
G r o u p 4 r a t s g i v e n t h e r m a l l y o x i d i z e d s u n f l o w e r o i l ( 1 5 % ) .
G r o u p 5 r a t s g i v e n D A ( 1 0 m g / k g ) + t h e r m a l l y o x i d i z e d
s u n f l o w e r o i l ( 1 5 % ) .
234 P o l . J . P h a r m a c o l . , 2 0 0 4 , 5 6 , 2 3 3 2 4 0
K . A r u n a , R . R u k k u m a n i , P . S u r e s h v a r m a , V . P . M e n o n
A) Dendrodoine
B) 4-Amino-5-benzoyl-2-(4-methoxyphenylamino)-thiazole-
NH2
O
C
H
OCH3
N
S N
O
C
H
NCH3
CH3
N
N
NS
Fig. 1. A) Structure of dendrodoine; B) Structure of DA[4-amino–5-benzoyl-2-(4-methoxyphenylamino)-thiazole]
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G r o u p 6 r a t s g i v e n 2 0 % e t h a n o l + t h e r m a l l y o x i d i z e d s u n f l o w e r
o i l ( 1 5 % ) .
G r o u p 7 r a t s g i v e n 2 0 % e t h a n o l + D A ( 1 0 m g / k g ) .
G r o u p 8 r a t s g i v e n 2 0 % e t h a n o l + t h e r m a l l y o x i d i z e d s u n f l o w e r
o i l ( 1 5 % ) + D A ( 1 0 m g / k g ) .
Rats in group 2 were given 20% ethanol 5 ml
each (equivalent to 7.9 g of ethanol/kg) daily using an
intragastric tube for a period of 45 days according to
Rajakrishnan et al. [35]. Rats in group 3 were given
orally DA (10 mg/kg) dissolved in water for 45 days.
Rats in group 4 were given 15% (15 g of oil/100 g of
feed) thermally oxidized sunflower oil for 45 days.
Group 5 received DA (10 mg/kg) and thermally oxi-
dized sunflower oil (15%) for 45 days. Rats in group
6 were given 20% ethanol (5 ml each) and thermally
oxidized sunflower oil (15%) for 45 days. Rats in
group 7 were given 20% ethanol (5 ml each) and DA
(10 mg/kg) for 45 days. Rats in group 8 received ther-
mally oxidized oil (15%) and DA (10 mg/kg) along
with 20% ethanol (5 ml each) for 45 days.
At the end of experimental period (45 days) the
rats were anesthetized (ketamine hydrochloride,
30 mg/kg) and were sacrificed by decapitation after
an overnight fast. Blood was collected in hepar-
inized tubes and plasma was separated for various
assays. Tissues (liver and kidney) were removed,
cleared of blood and collected in ice-cold contain-
ers containing 0.9% NaCl for various assays. The project was approved by the institutional Ethical
Committee.
Biochemical tests
The activity of plasma g-glutamyl transferase
(GGT) (E.C.2.3.2.2) was assayed by the method of
Fiala et al. [13]. The activity of plasma aspartate
transaminase (AST) (E.C.2.6.1.1) was assayed by
the method of Reitman and Frankel [37] and alka-
line phosphatase (ALP) (E.C.3.1.3.1) by the methodof King and Armstrong [25] using reagent kit. Tis-
sue lipids were extracted according to the method
of Folch et al. [14]. Plasma and tissue cholesterol
was estimated by using reagent kit [2]. Triglyc-
erides were estimated by the method of Foster and
Dunn [15] and phospholipids by the method of Zil-
versmit and Davis [48]. Phospholipase A (E.C.
3.1.4.1) was assayed by the method of Rimon and
Shapiro [38] and phospholipase C by the method of
Kleiman and Lands [26].
Statistical analysis
Statistical analysis was carried out using analy-
sis of variance (ANOVA) followed by Duncan’s
multiple range test (DMRT). The level of statistical
significance was set at p < 0.05.
RESULTS
Biochemical findings
Table 1 presents changes in body weight of dif-ferent groups. The average weight gain induced by
alcohol, thermally oxidized oil and ethanol + ther-
mally oxidized oil in rats was significantly reduced
when compared with normal control rats. The ani-
mals showed near normal pattern of weight gainwhen DA was given along with thermally oxidized
oil, alcohol and alcohol + thermally oxidized oil.
Table 2 shows changes in the activity of GGT,AST and ALP in plasma. The activity of GGT, AST
and ALP was increased significantly in groups re-
ceiving thermally oxidized oil, alcohol and alcohol
+ thermally oxidized oil when compared with thecontrol group. The administration of DA resulted in
marked reduction in the activity of liver marker en-
zymes GGT, AST and ALP in the groups given
thermally oxidized oil, alcohol and alcohol + ther-
mally oxidized oil.Table 3 includes changes in the level of plasma
cholesterol, triglycerides and phospholipids. The
levels were increased significantly in the groupstreated with alcohol, thermally oxidized oil and al-
cohol + thermally oxidized oil when compared
with the control group. The levels were decreased
when DA was administered along with thermallyoxidized oil, alcohol and alcohol + thermally oxi-
dized oil.
The changes in the levels of tissue cholesterol,
triglycerides and phospholipids are given in Table
4. The levels of cholesterol and triglycerides wereincreased significantly in the liver and kidney of
the rats given alcohol, thermally oxidized oil and
alcohol + thermally oxidized oil when comparedwith the control group but they decreased after DA
treatment. The level of phospholipids decreased
significantly in the groups administered alcohol,
thermally oxidized oil and alcohol + thermally oxi-dized oil when compared with the control group.
The level was increased after administration of DA
to the above groups.
I S S N 1 2 3 0 - 6 0 0 2 235
P R O T E C T I V E I N F L U E N C E O F A N A M I N O T H I A Z O L E D E R I V A T I V E
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236 P o l . J . P h a r m a c o l . , 2 0 0 4 , 5 6 , 2 3 3 2 4 0
K . A r u n a , R . R u k k u m a n i , P . S u r e s h v a r m a , V . P . M e n o n
T a b l e 2 . C h a n g e s i n t h e a c t i v i t y o f A S T , A L P a n d G G T
G r o u p s A S T ( I U / l ) A L P ( I U / l
)G G T ( I U / l )
1 . C o n t r o l 8 3 . 5 3 ± 3 . 9 2
= ? A C
8 6 . 7 7 ± 3 . 8 0
= ? A C
0 . 5 9 7 ± 0 . 0 2
= ? A C D
2 . A l c o h o l ( A l c ) 1 5 2 . 0 9 ± 8 . 1 1
>
1 4 0 . 4 0 ± 6 . 0 2
>
1 . 2 4 0 ± 0 . 0 7
>
3 . D A 8 0 . 2 7 ± 4 . 6 5
? A C
8 2 . 2 9 ± 4 . 7 8
?
0 . 5 1 9 ± 0 . 0 2
?
4 . T h e r m a l l y o x i d i z e d o i l 1 2 0 . 9 6 ± 1 5 . 8 7
@
1 2 2 . 6 9 ± 8 . 7 6
@ C
0 . 7 1 6 ± 0 . 0 3
@ C
5 . T h e r m a l l y o x i d i z e d o i l + D A 8 7 . 8 1 ± 6 . 0 3
A C D
9 2 . 2 6 ± 6 . 1 1
A C D
0 . 6 1 5 ± 0 . 0 1
A C D
6 . A l c + t h e r m a l l y o x i d i z e d o i l 1 8 1 . 4 5 ± 1 7 . 0 7
B
1 6 2 . 6 3 ± 1 1 . 3 8
B
1 . 4 1 7 ± 0 . 0 8
B
7 . A l c + D A 9 1 . 4 3 ± 6 . 5 0
C D
9 5 . 9 4 ± 5 . 1 0
C D
0 . 6 2 1 ± 0 . 0 5
C D
8 . A l c + t h e r m a l l y o x i d i z e d o i l + D A 9 6 . 2 7 ± 6 . 7 2
D
1 0 0 . 8 3 ± 7 . 1 0
D
0 . 6 3 3 ± 0 . 0 3
D
V a l u e s a r e t h e m e a n ± S D f r o m s i x r a t s i n e a c h g r o u p . V a l u e s n o t s h a r i n g a c o m m o n s u p e r s c r i p t d i f f e r s i g n i f i c a n t l y a t p < 0 . 0 5
( D M R T )
T a b l e 3 . C h a n g e s i n t h e l e v e l o f p l a s m a c h o l e s t e r o l , t r i g l y c e r i d e s a n d p h o s p h o l i p i d s
G r o u p s C h o l e s t e r o l ( m g / d l ) T r i g l y c e r i d e s ( m g / d l ) P h o s p h o l i p i d s ( m g / d l )
1 . C o n t r o l 8 9 . 9 2 ± 3 . 4 1
= ? A
7 6 . 7 4 ± 4 . 7 5
= ? A C
9 7 . 7 4 ± 4 . 8 9
= ? A C
2 . A l c o h o l ( A l c ) 1 2 5 . 4 2 ± 6 . 5 8
>
1 1 6 . 9 2 ± 6 . 8 3
>
1 3 3 . 8 2 ± 6 . 0 9
>
3 . D A 9 1 . 9 1 ± 2 . 9 3
? A
7 2 . 1 6 ± 4 . 7 4
? A
9 4 . 6 2 ± 4 . 8 8
? A C
4 . T h e r m a l l y o x i d i z e d o i l 1 1 1 . 6 6 ± 5 . 2 3
@
1 0 1 . 1 ± 5 . 9 6
@ D
1 2 6 . 1 4 ± 8 . 4 7
@ C
5 . T h e r m a l l y o x i d i z e d o i l + D A 9 5 . 7 6 ± 3 . 7 5
A
8 1 . 8 1 ± 5 . 5 4
A C
1 0 2 . 4 7 ± 4 . 0 1
A C
6 . A l c + t h e r m a l l y o x i d i z e d o i l 1 4 5 . 0 9 ± 7 . 5 1
B
1 3 5 . 0 1 ± 8 . 3 5
B
1 5 6 . 8 4 ± 8 . 5 6
B
7 . A l c + D A 1 0 0 . 5 1 ± 6 . 8 3
C
8 5 . 8 1 ± 3 . 8 9
C
1 0 8 . 9 6 ± 8 . 8 6
C D
8 . A l c + t h e r m a l l y o x i d i z e d o i l + D A 1 1 0 . 2 5 ± 5 . 7 9
D
9 6 . 7 5 ± 4 . 3 5
D
1 1 5 . 2 7 ± 9 . 2 9
D
V a l u e s a r e t h e m e a n ± S D f r o m s i x r a t s i n e a c h g r o u p . V a l u e s n o t s h a r i n g a c o m m o n s u p e r s c r i p t d i f f e r s i g n i f i c a n t l y a t p < 0 . 0 5
( D M R T )
T a b l e 1 . C h a n g e s i n b o d y w e i g h t
G r o u p s I n i t i a l b o d y w e i g h t
( g )
F i n a l b o d y w e i g h t
( g )
C h a n g e i n b o d y w e i g h t
( g )
1 . C o n t r o l 1 3 5 . 2 7 ± 5 . 7 1 2 1 6 . 5 4 ± 1 0 . 1 3 8 1 . 2 7 ± 4 . 2 0
= ? A C
2 . A l c o h o l ( A l c ) 1 4 1 . 3 9 ± 6 . 2 9 1 8 6 . 3 1 ± 7 . 4 5 4 4 . 9 2 ± 2 . 3 8
>
3 . D A 1 3 9 . 6 8 ± 7 . 0 7 2 1 6 . 9 9 ± 8 . 1 2 7 7 . 3 1 ± 3 . 1 7
? A C D
4 . T h e r m a l l y o x i d i z e d o i l 1 5 0 . 2 9 ± 3 . 1 2 2 1 5 . 4 8 ± 9 . 1 0 6 5 . 1 9 ± 2 . 5 7
@
5 . T h e r m a l l y o x i d i z e d o i l + D A 1 4 2 . 2 5 ± 4 . 2 7 2 1 7 . 5 2 ± 5 . 1 3 7 5 . 2 7 ± 3 . 1 5
A C D
6 . A l c + t h e r m a l l y o x i d i z e d o i l 1 4 7 . 7 4 ± 4 . 1 2 2 0 6 . 1 7 ± 7 . 6 7 5 8 . 4 3 ± 1 . 9 1
B
7 . A l c + D A 1 5 0 . 4 6 ± 5 . 0 1 2 2 5 . 4 4 ± 9 . 2 9 7 4 . 9 8 ± 3 . 2 3
C D
8 . A l c + t h e r m a l l y o x i d i z e d o i l + D A 1 4 5 . 8 5 ± 5 . 1 3 2 1 6 . 0 9 ± 7 . 1 2 7 0 . 2 4 ± 3 . 3 5
D
V a l u e s a r e t h e m e a n ± S D f r o m s i x r a t s i n e a c h g r o u p . V a l u e s n o t s h a r i n g a c o m m o n s u p e r s c r i p t d i f f e r s i g n i f i c a n t l y a t p < 0 . 0 5
( D M R T )
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Table 5 gives changes in the activity of phos-
pholipase A and C. Alcohol and thermally oxidized
oil administration significantly increased the activ-
ity of phospholipase A and C in the liver when
compared with the control group. Administration
of DA to the rats treated with thermally oxidized oil
and alcohol resulted in marked reduction in the ac-
tivities of these enzymes.
DISCUSSION
The main pathway of alcohol degradation, alco-
hol dehydrogenase pathway leads to the increased NADH synthesis. This striking redox change inhib-
its TCA cycle, fatty acid oxidation, lipoprotein ex-
port and increases fatty acid uptake [16], thus, pre-
I S S N 1 2 3 0 - 6 0 0 2 237
P R O T E C T I V E I N F L U E N C E O F A N A M I N O T H I A Z O L E D E R I V A T I V E
T a b l e 5 . C h a n g e s i n t h e a c t i v i t y o f p h o s p h o l i p a s e A a n d p h o s p h o l i p a s e C
G r o u p s P h o s p h o l i p a s e A
( m E q o f e s t e r h y d r o l y z e d /
m i n / m g p r o t e i n )
P h o s p h o l i p a s e C
( m m o l e s o f p h o s p h o r y l c h o l i n e f o r m e d /
m i n / m g p r o t e i n )
1 . C o n t r o l 0 . 0 2 9 ± 0 . 0 0 2
= ? A
0 . 7 0 4 ± 0 . 0 4 3
= ? A C
2 . A l c o h o l ( A l c ) 0 . 0 9 3 ± 0 . 0 0 6
>
0 . 8 9 8 ± 0 . 0 4 7
>
3 . D A 0 . 0 2 8 ± 0 . 0 0 2
? A
0 . 6 9 9 ± 0 . 0 3 9
? A C D
4 . T h e r m a l l y o x i d i z e d o i l 0 . 0 6 8 ± 0 . 0 0 3
@
0 . 7 8 2 ± 0 . 0 5 0
@
5 . T h e r m a l l y o x i d i z e d o i l + D A 0 . 0 3 2 ± 0 . 0 0 2
A C
0 . 7 3 0 ± 0 . 0 4 7
A C D
6 . A l c + t h e r m a l l y o x i d i z e d o i l 0 . 1 3 1 ± 0 . 0 0 7
B
1 . 0 3 0 ± 0 . 0 9 4
B
7 . A l c + D A 0 . 0 3 6 ± 0 . 0 0 2
C D
0 . 7 4 3 ± 0 . 0 4 7
C D
8 . A l c + t h e r m a l l y o x i d i z e d o i l + D A 0 . 0 4 1 ± 0 . 0 0 3
D
0 . 7 5 1 ± 0 . 0 4 9
D
V a l u e s a r e t h e m e a n ± S D f r o m s i x r a t s i n e a c h g r o u p . V a l u e s n o t s h a r i n g a c o m m o n s u p e r s c r i p t d i f f e r s i g n i f i c a n t l y a t p < 0 . 0 5
( D M R T )
T a b l e 4 . C h a n g e s i n t h e l e v e l o f t i s s u e c h o l e s t e r o l , t r i g l y c e r i d e s a n d p h o s p h o l i p i d s
G r o u p C h o l e s t e r o l
( m g / 1 0 0 g t i s s u e )
T r i g l y c e r i d e s
( m g / 1 0 0 g t i s s u e )
P h o s p h o l i p i d s
( m g / 1 0 0 g t i s s u e )
L i v e r K i d n e y L i v e r K i d n e y L i v e r K i d n e y
1 . C o n t r o l 3 4 1 . 2 5
± 1 3 . 8 5
= ? A C
3 6 6 . 9 1
± 2 7 . 8 1
= ? A C
3 9 1 . 9 3
± 1 9 . 6 3
= ? A C
4 2 8 . 2
± 2 3 . 5 5
= ? A C
1 8 1 7 . 5 3
± 9 6 . 2 3
= ? A C
1 4 9 3 . 9 4
± 6 7 . 8 0
= ? A C
2 . A l c o h o l ( A l c ) 5 3 8 . 6 5
± 2 9 . 6 7
>
5 6 5 . 0 2
± 2 8 . 3 9
>
6 7 9 . 5 9
± 2 8 . 8 2
>
6 5 4 . 8 9
± 3 2 . 2 1
>
1 4 2 0 . 6 6
± 7 3 . 8 6
>
1 2 0 8 . 1 2
± 5 9 . 4 5
>
3 . D A 3 3 2 . 0 1
± 2 3 . 6 4
? A C
3 7 0 . 5 0
± 2 4 . 3 4
? A
3 8 7 . 2 2
± 2 2 . 1 9
? A C
4 1 9 . 1 8
± 1 9 . 4 1
? A C
1 8 0 0 . 8 3
± 1 2 1 . 7 3
? A C
1 4 8 0 . 7 1
± 7 9 . 8 7
? A C
4 . T h e r m a l l y o x i d i z e d o i l 4 6 7 . 5 0
± 3 5 . 4 7
@
4 7 5 . 7 1
± 2 8 . 4 6
@
5 1 5 . 3 8
± 2 8 . 8 6
@
5 5 0 . 2 9
± 2 5 . 3 7
@
1 5 5 9 . 6 7
± 8 9 . 7 7
@
1 2 6 3 . 6 8
± 8 3 . 3 2
@
5 . T h e r m a l l y o x i d i z e d o i l + D A 3 5 3 . 7 3
± 2 2 . 9 6
A C
3 8 6 . 2 7
± 2 3 . 6 3
A C D
4 1 2 . 7 9
± 2 8 . 1 3
A C
4 4 8 . 2 0
± 1 8 . 5 2
A C
1 7 2 1 . 1 9
± 1 0 6 . 8 2
A C D
1 4 5 2 . 0 3
± 6 8 . 3 6
A C
6 . A l c + t h e r m a l l y o x i d i z e d o i l 6 6 0 . 2 5
± 4 6 . 0 6
B
6 9 6 . 1 8
± 3 6 . 0 1
B
8 2 5 . 9 5
± 4 1 . 2 3
B
9 7 7 . 6 8
± 4 3 . 3 2
B
1 3 0 7 . 8 7
± 8 3 . 9 8
B
1 1 0 6 . 7 7
± 8 5 . 7 6
B
7 . A l c + D A 3 8 1 . 8 4
± 2 7 . 7 0
C D
3 9 5 . 3 2
± 1 9 . 3 1
C D
4 2 1 . 6 7
± 2 6 . 4 9
C
4 6 9 . 8 6
± 2 4 . 1 6
C D
1 7 7 9 . 8 8
± 1 0 5 . 9 9
C D
1 4 3 0 . 0 5
± 5 2 . 0 8
C D
8 . A l c + t h e r m a l l y o x i d i z e d o i l + D A 4 1 0 . 7 8
± 2 1 . 5 9
D
4 1 0 . 3 1
± 2 1 . 3 9
D
4 7 3 . 1 4
± 3 0 . 2 7
D
5 0 3 . 7 5
± 3 1 . 4 3
D
1 7 0 5 . 1 3
± 1 0 1 . 2 7
D
1 3 7 5 . 7 1
± 8 9 . 3 1
D
V a l u e s a r e t h e m e a n ± S D f r o m s i x r a t s i n e a c h g r o u p . V a l u e s n o t s h a r i n g a c o m m o n s u p e r s c r i p t d i f f e r s i g n i f i c a n t l y a t p < 0 . 0 5
( D M R T )
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disposing to fatty liver. In our study, the average
weight gained by rats during experimental period
was significantly reduced in rats given alcohol andthermally oxidized oil compared with the control
rats. Rajakrishnan et al. [34] have also observed a
decrease in weight gain on alcohol treatment. Thedecrease in the body weight could be due to thetoxic effects of ethanol [45]. A better weight gain in
DA-fed groups shows the protective effect of DA.
Damage to the liver after ethanol ingestion is a well-
known phenomenon and the obvious sign of hepaticinjury is the leakage of cellular enzymes into plasma
[7]. We have observed the increased activity of
plasma GGT, AST and ALP in rats administered al-
cohol and thermally oxidized oil. Previous reportsshowed that exposure of hepatocytes to ethanol
perturbed the membrane structure and function,
thereby increasing the leakage of AST [33]. Etha-nol also causes structural and functional changes inthe mitochondria and increases membrane perme-
ability leading to the leakage of mitochondrial en-
zymes into the circulation [11]. Serum GGT is
widely used as a laboratory test for the hepatobili-ary diseases, especially of alcoholic liver disease
and alcohol induced liver damage [28]. It has been
found that susceptibility to alcohol may be related
to the consumption of different types of dietary fat[29]. Induction of CYP 4502E1 by ethanol was
found to be related to the concentration of PUFA in
the diet [3]. The increased activity of GGT, AST
and ALP in thermally oxidized oil and alcohol +thermally oxidized oil fed groups may be due to in-
creased volatile and other toxic agents, which are
produced during thermal oxidation of oil. Previous
reports have shown the increased activity of plasmaAST and ALP in rats fed thermally oxidized oil
[17, 41]. The observed decrease in the activity of
hepatic marker enzymes after DA administration
shows that our drug preserves structural integrity of the liver protecting it from the toxic effects of alco-
hol and thermally oxidized oil.
Marked alterations in lipid metabolism have
been reported in chronic ethanol feeding [10]. Our results showed the increased level of cholesterol
and triglycerides in the plasma, liver and kidney of
the rats fed thermally oxidized oil, alcohol and al-
cohol + thermally oxidized oil when comparedwith normal control group. Alcohol feeding is
known to increase the biosynthesis and decrease
the catabolism of fatty acids and cholesterol result-
ing in their accumulation in liver and causing
hyperlipidemia [24]. The increased cholesterol
level may be due to increased -hydroxy-methyl-
glutaryl CoA (HMG CoA) reductase activity by
ethanol, which is the rate limiting step in choles-
terol biosynthesis [5]. The microsomal induction
resulting from long-term alcohol consumption notonly accelerates the oxidation of ethanol but also
increases the synthesis of triacyl glycerols [40].
Previous studies show that diet rich in PUFA stimu-
lates the production of chylomicrons by the intes-
tine [20], which may be the reason for the increased
level of cholesterol in the rats administered ther-
mally oxidized oil and alcohol + thermally oxi-
dized oil. It has been reported that ingestion of oxi-
dized lipids rich in linoleic acid causes profound al-
terations in membrane composition, fluidity and
function [19]. These alterations are likely to be as-sociated with an enhanced cholesterol turnover, as
indicated by the greater cholesterol excretion ob-
served in the experimental rats. The increased tri-
glyceride levels after oil ingestion may be due to
the increased availability of substrate FFA for es-
terification. The levels of cholesterol and triglyc-
erides were found to be decreased when DA was
administered along with thermally oxidized oil and
alcohol. Decreased cholesterol levels may be due to
decreased absorption from the intestine and de-
creased synthesis or increased catabolism. The de-
creased levels of triglycerides may be due to de-creased free fatty acid synthesis, increased utiliza-
tion or decreased glycerol formation.
Phospholipids, the backbone of all cellular
membranes, are the primary targets of peroxidation
and can be altered by ethanol [46]. Our results
show increased levels of phospholipids in the
plasma and decreased levels in the liver and kidney
of the rats given thermally oxidized oil, alcohol and
alcohol + thermally oxidized oil when compared
with the control group. The decreased concentration
of phospholipids indicates accelerated phospholipid
degradation [9] and can result in modification of the
composition, structure and stability of cellular mem-
branes leading to membrane dysfunction [22]. In
this context, Jaya et al. [23] have reported a decrease
in the phospholipid content in the liver and kidney
of alcohol-fed rats. DA administration to rats treated
with thermally oxidized oil and alcohol brought
down the levels of phospholipids to near normal
showing the ability to repair the cellular membrane
damage caused by ethanol and thermally oxidized
238 P o l . J . P h a r m a c o l . , 2 0 0 4 , 5 6 , 2 3 3 2 4 0
K . A r u n a , R . R u k k u m a n i , P . S u r e s h v a r m a , V . P . M e n o n
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oil and to neutralize their metabolic products
thereby preserving the membrane integrity.
Intracellular phospholipases A are a diversegroup of enzymes with a growing number of mem-
bers. These enzymes hydrolyze membrane phos-
pholipids into fatty acids and lysophospholipids[8]. Lysophospholipids generated by the action of phospholipase A can be further metabolized to po-
tent inflammatory mediators, such as eicosanoids
and platelet-activating factors [12]. Phospholipase
C attacks ester bond in position 3, liberating 1,2-diacylglycerol and a phosphoryl base. Our result
shows increased activity of phospholipase A and C
in alcohol-fed rats compared to control rats, which
agrees with results of Zheng et al. [47] who havefound that chronic exposure to ethanol leads to a
progressive increase in membrane phospholipase
A2
activity. Ethanol ingestion also induces an in-crease in microsomal phospholipase C that corre-lates with an increase in the microsomal CYP 2E1
and a decrease in microsomal 20:4 fatty acids.
These changes are associated with ethanol-induced
liver pathology [30]. Our earlier studies [4] havealso shown increased activity of phospholipases A
and C in the liver of ethanol-administered rats. The
activity of these enzymes was decreased in the
groups given DA along with thermally oxidized oiland alcohol.
Thus, our study shows the protective effect of
DA, an aminothiazole derivative in ethanol and
thermally oxidized sunflower oil-induced toxicity.
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Received: October 16, 2003; in revised form: March 3,2004.
240 P o l . J . P h a r m a c o l . , 2 0 0 4 , 5 6 , 2 3 3 2 4 0
K . A r u n a , R . R u k k u m a n i , P . S u r e s h v a r m a , V . P . M e n o n