Influence of a thiazole derivative on ethanol and thermally oxidized sunflower oil induced oxidative stress

8/8/2019 Influence of a thiazole derivative on ethanol and thermally oxidized sunflower oil induced oxidative stress

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doi: 10.1111/j.1472-8206.2004.00274.x

O R I G I N A L

A R T I C L E

Inuence of a thiazole derivative on ethanoland thermally oxidized sunower oil-inducedoxidative stress

Aruna Kode, Rukkumani Rajagopalan, Suresh Varma Penumathsa,Venugopal P. Menon*Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India

I N T R O D U C T I O N

Ethanol is one of the most widely used and abused drugs.The main pathway for the hepatic oxidation of ethanol toacetaldehyde proceeds via alcohol dehydrogenase. Inaddition to alcohol dehydrogenase pathway, ethanol canalso be reduced by an accessory but inducible micro-somal ethanol oxidizing system [1]. Induction of micro-somal system results in enhanced acetaldehyde

production, which in turn impairs defence systemsduring oxidative stress. The liver cytochrome P450form, CYP 2E1, a major component of the microsomalethanol oxidizing system has been implicated in hepato-toxicity caused by ethanol [2]. This hepatic microsomalcytochrome P450 catalyses the oxidative metabolism of xenobiotics, including ethanol.

Fat is an important dietary component, which affectsboth growth and health. The early classical studies have

indicated that diets containing high level of polyunsat-urated fatty acids (PUFA) are effective in lowering serumcholesterol level [3] and are considered to be benecial inpreventing coronary heart disease [4]. However, studieshave also shown that excessive intake of vegetable oilcontaining PUFA is detrimental to health [5]. It has beensuggested that consumption of diets rich in PUFArenders tissues more susceptible to free radical-mediatedlipid peroxidation, a process which has been implicated

in tissue damage [6].Heating of fats brings about measurable changes intheirphysicalandchemical properties. Duringdeep frying,when the fat is used repeatedly, oxidative and thermaleffects result in the formation of many volatile and non-volatile products, some of which are potentially toxic [7].

Aminothiazoles are a group of biologically importantcompounds having a wide range of activities such asanti-tumour, anti-anoxic and antioxidant properties

Keywordsaminothiazole derivative,antioxidants,dendrodoine analogue,ethanol,lipid peroxidation,thermally oxidizedsunower oil

Received 21 November 2003;revised 11 March 2004;accepted 24 May 2004

*Correspondence and reprints:[email protected]

A B S T R A C T

The present work describes the protective inuence of the dendrodoine analogue (DA)[4-amino-5-benzoyl-2-(4-methoxy phenylamino) thiazole] on thermally oxidizedsunower oil and ethanol-induced oxidative stress. Ethanol was fed to animals at alevel of 20% [(7.9 g/kg body weight (bw)] and thermally oxidized sunower oil at a

level of 15% (15 mL/100 g feed). Hepatotoxicity was assessed by measuring theactivity of plasma aspartate transaminase (AST), alkaline phosphatase (ALP) andc -glutamyl transferase (GGT), which were elevated in thermally oxidized oil, andethanol fed rats when compared with normal control rats. Tissue damage wasassociated with increased lipid peroxidation and disruption in the antioxidant defencemechanism in thermally oxidized oil- and ethanol-fed groups when compared withnormal control group. The activity of liver marker enzymes (AST, ALP and GGT) andthe level of lipid peroxidation decreased when DA was administered along withethanol and thermally oxidized oil. The antioxidant status was near normal inDA-administered groups. Thus we propose that DA exerts antioxidant properties bymodulating the activity of hepatic marker enzymes, level of lipid peroxidation andantioxidant status.

2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 565571 565


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[8,9]. Dendrodoine is a marine alkaloid, which wasisolated from Dendrodoa grossularia [10]. It possesses a1,2,4-thiadiazole unit, a rarity among natural products.Although its synthesis has been reported [11], very littlebiological studies have been carried out on it and itsanalogue ( Figure 1 ). In the present study, we evaluatedthe antioxidant properties of dendrodoine analogue (DA)using ethanol and thermally oxidized oil-induced chan-ges in lipid peroxidation and antioxidant status. Wespeculate that the antioxidant properties of DA may bedue to the presence of an NH 2 group which can reactwith acetaldehyde, the toxic metabolite of ethanol and

also with other free radicals resulting in the formation of Schiffs base, thereby neutralizing the toxic effects of ethanol and thermally oxidized oil ( Figure 2 ).

M AT E R I A L S A N D M E T H O D S

Experimental animalsMale albino rats of the Wistar strain [140160 g bodyweight (bw)] bred in the Central Animal House, Rajah

Muthiah Medical College, were used in this study. Theanimals were fed on pellet diet (Hindustan Lever Limited,Mumbai, India) and water was provided ad libitum.

ChemicalsEthanol was purchased from E.Merck (Darmstadt,Germany). The DA was synthesized as described byRajasekharan et al. [12]. The sun ower oil (Gold winner,Chennai-73, Tamil Nadu, India) was subjected to twofrying cycles of 30 min each at 180 C to producethermally oxidized oil. All other chemicals and biochem-icals used for the experiments were of analytical grade.

Experimental designDose-dependent studies for DA were carried out and theeffective dose was found to be 10 mg/kg bw. Theexperimental period was 45 days.

The animals were randomized into the followinggroups:Group 1 : Control rats fed standard pellet diet;Group 2 : Rats fed 20% ethanol (equivalent to 7.9 gethanol/kg bw) daily using an intragastric tube [13].Group 3 : Rats fed DA (10 mg/kg bw) orally.Group 4 : Rats fed thermally oxidized sunower oil (15%)mixed with diet.Group 5 : Rats fed DA (10 mg/kg bw) + thermallyoxidized sunower oil (15%).Group 6 : Rats fed 20% ethanol + thermally oxidizedsunower oil (15%).

Group 7 : Rats fed 20% ethanol + DA (10 mg/kg bw).Group 8 : Rats fed 20% ethanol + thermally oxidizedsunower oil (15%) + DA (10 mg/kg bw).

At the end of experimental period (45 days), the ratswere killed after an overnight fast by decapitation. Bloodand tissues (liver and kidney) were collected for variousestimations. The institutional ethical committee hasapproved this project (Reg. No. 160/1999/CPSEA).

Biochemical estimationsThe activity of plasma gamma glutamyl transferase(E.C.2.3.2.2) was assayed by the method of Fiala et al.

[14]. The activity of plasma aspartate transaminase(E.C.2.6.1.1) was assayed by the method of Reitman andFrankel [15] and alkaline phosphatase (E.C.3.1.3.1) bythe method of King and Armstrong [16] using a reagentkit. The concentration of thiobarbituric acid-reactivesubstances (TBARS) was estimated by the method of Yagi [17], hydroperoxides by the method of Jiang et al.[18] and the level of free fatty acids (FFA) by the methodof Falholt [19]. Reduced glutathione content in the

O

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Figure 1 (a) Structure of dendrodoine; (b) structure of DA[4-amino-5-benzoyl-2-(4-methoxy phenylamino) thiazole].

Schiffs base+ CH 3CHO

Dendrodoine analogue

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Figure 2 Formation of Schiffs base.

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tissues was determined by the method of Ellman [20].Tissue ascorbic acid was estimated by the method of Roeand Kuether [21] and vitamin E by the method of Bakerand Frank [22]. The activity of glutathione peroxidase(E.C.1.11.1.9) was assayed by the method of Rotrucket al. [23], superoxide dismutase (E.C.1.15.1.1) by themethod of Kakkar et al. [24] and catalase (E.C.1.11.1.6)by the method of Sinha [25].

Statistical analysisStatistical analysis was carried out using analysis of variance ( ANOVA ) followed by Duncans multiple rangetest (DMRT). The level of statistical signicance was setat P 0.05.

R E S U LT S

Biochemical ndingsFigure 3 presents the changes in body weight of differentgroups. The average weight gain by alcohol, thermallyoxidized oil and alcohol + thermally oxidized oil fed ratswas signicantly reduced when compared with normalcontrol rats. The animals showed near normal pattern of weight gain when DA was fed along with thermallyoxidized oil, alcohol and alcohol + thermally oxidized oil.

Figures 4 and 5 represent change in the activity of aspartate transaminase (AST), alkaline phosphatase(ALP) and c -glutamyl transferase (GGT) in plasma. Theactivity of AST, ALP and GGT was increased signicantly

in thermally oxidized oil, alcohol and alcohol + therm-ally oxidized oil fed groups when compared with normalcontrol group. The administration of DA resulted in amarked reduction in the activity of liver marker enzymes

AST, ALP and GGT in thermally oxidized oil, alcohol andalcohol + thermally oxidized oil-fed groups.

Changes in the level of plasma and tissue TBARS,hydroperoxides and FFA are given in Figures 6 , 7 and 8respectively. The level of TBARS, hydroperoxides andFFA was increased signicantly in thermally oxidized oil,alcohol and alcohol + thermally oxidized oil-fed groups

when compared with the normal control group. Whenthe drug was administered along with thermally oxidizedoil, alcohol and alcohol + thermally oxidized oil, thelevel was found to be decreased.

Figures 9 and 10 give changes in the level of reducedglutathione, (GSH) vitamin C and vitamin E in the liverand kidney. The level of GSH, vitamin C and vitamin Edecreased signicantly in the thermally oxidized oil,alcohol and alcohol + thermally oxidized oil-fed groups

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Figure 3 Changes in body weight. Values are mean SD fromsix rats in each group. Values not sharing a common superscriptdiffer signicantly at P 0.05 (DMRT).

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Figure 4 Changes in the activity of AST, ALP. Values aremean SD from six rats in each group. Values not sharing acommon superscript differ signicantly at P 0.05 (DMRT).

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Figure 5 Change in the activity of GGT. Values are mean SDfrom six rats in each group. Values not sharing a commonsuperscript differ signicantly at P 0.05 (DMRT).

Protective inuence of an aminothiazole derivative 567



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when compared with the normal control group. Whenthe drug was administered along with thermally oxidizedoil, alcohol and alcohol + thermally oxidized oil, thelevel of GSH, vitamin C and vitamin E were near normalbut was still less than that in the normal control group.

Figure 11 shows the changes in the activity of superoxide dismutase (SOD), glutathione peroxidase(Gpx) and catalase in the liver. The activity of enzymatic

antioxidants SOD, GPx and catalase in the liver of thermally oxidized oil, alcohol and alcohol + thermallyoxidized oil-fed groups was decreased signicantly whencompared with the normal control group. The activitywas found to be increased signicantly in thermallyoxidized oil + DA-, alcohol + DA- and alcohol + therm-ally oxidized oil + DA-administered groups when com-pared with thermally oxidized oil-, alcohol- andalcohol + thermally oxidized oil-fed groups.

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Figure 6 Changes in the level of plasma and tissue TBARS. Valuesare mean SD from six rats in each group. Values not sharing acommon superscript differ signicantly at P 0.05 (DMRT).

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Figure 8 Change in the level of plasma and tissue free fatty acids.Values are mean SD from six rats in each group. Values notsharing a common superscript differ signicantly at P 0.05(DMRT).

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Figure 7 Changes in the level of plasma and tissue hydroperoxides.Values are mean SD from six rats in each group. Values notsharing a common superscript differ signicantly at P 0.05DMRT).

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Figure 9 Changes in the level of tissue GSH.

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Figure 10 Changes in the level of tissue, vitamin C and vitamin E.Values are mean SD from six rats in each group. Values notsharing a common superscript differ signicantly at P 0.05(DMRT).

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D I S C U S S I O N

Alcohol is a toxic substance when consumed in excessand results in a variety of pathological conditions. In ourstudy, the average weight gain by rats during experi-mental period was signicantly reduced in alcohol,thermally oxidized oil and alcohol + thermally oxidizedoil-fed rats when compared with normal control rats.Rajakrishnan et al. [26] have observed decreased weightgain on alcohol treatment. The weight reduction may be

due to impaired function of the cells and also due toinjuries caused to various organs [27]. A better weightgain after DA administration showed the protective effectof the drug.

Damage to the liver after ethanol ingestion is a wellknown phenomenon and the obvious sign of hepaticinjury is the leakage of cellular enzymes into the plasma[28]. We have observed increased activity of plasmaGGT, AST and ALP in thermally oxidized oil, alcoholand thermally oxidized oil + alcohol-fed rats. Previousreports have shown that exposure of hepatocytes toethanol perturbs the membrane structure and functions

thereby increasing the leakage of AST [29]. Ethanolcauses structural and functional changes in the mito-chondria and increases membrane permeability leadingto the leakage of mitochondrial enzymes into thecirculation [30]. Serum GGT is widely used as alaboratory test for the hepatobiliary diseases especially,of alcoholic liver disease and alcohol induced liverdamage [31]. It has been found that susceptibility toalcohol may be related to the consumption of different

types of dietary fat [32]. Ethanol induction of CYP 4502E1 was found to be related to the concentration of PUFA in the diet [33]. The increased activity of GGT,AST and ALP in thermally oxidized oil- and alco-hol + thermally oxidized oil-fed groups may be due toincreased volatile and other nonvolatile agents, whichare produced during the thermal oxidation of oil.Previous reports have shown increased activity of plasma AST and ALP in rat fed thermally oxidized oil[34,35]. Previous studies in our laboratory [36] havealso showed increased activity of plasma AST and ALP inalcohol + thermally oxidized sunower oil-fed rats. Theobserved decrease in the activity of liver marker enzymesin the plasma after DA administration shows that thedrug can preserve the structural integrity of the liverfrom the toxic effects of alcohol and thermally oxidizedoil.

We have observed signicantly increased levels of lipidperoxidation indices TBARS, hydroperoxides and FFA inplasma, liver and kidney of alcoholic group whencompared with normal control rats. The cytochromeP450 2E1 containing microsomal ethanol oxidizingsystem is strikingly inducible by chronic ethanol con-sumption, which results not only in an increasedproduction of acetaldehyde but also in the generationof substantial amounts of superoxide, as well as hydroxyand other free radicals [37]. Our results also showedincreased levels of TBARS, hydroperoxides and FFA inthermally oxidized oil- and alcohol + thermally oxidized

oil-fed rats when compared with normal control rats.There was a report, which stated that susceptibility tolipid peroxidation is a function of fatty acid unsaturationand increased intake of PUFA increases oxidative tissuedamage [38]. Turpeinen et al. [39] have observed asmall but signicant increase in lipid TBARS andhydroperoxides in vitro after sunower oil diet. Increasedlevel of TBARS and hydroperoxides in thermally oxidizedoil- and alcohol + thermally oxidized oil-fed groups inour study may be due to increased susceptibility of thetissues to the combined effect of toxic metabolites formedfrom the thermally oxidized oil and ethanol.

Increased level of FFA in alcohol fed group may be dueto increased formation of acetate which inturn formsFFA. The increased NADH/NAD + ratio formed duringethanol metabolism also favours fatty acid synthesis[40]. The increased FFA in thermally oxidized oil- andalcohol + thermally oxidized oil-fed groups is due toincreased intake of oil rich in PUFA, which mayeventually increase the level of FFA. The level of TBARS,hydroperoxides and FFA was signicantly reduced on

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Figure 11 Changes in the activities of SOD a , GPx b and catalase c .Values are mean SD from six rats in each group. Values notsharing a common superscript differ signicantly at P > 0.05(DMRT). a Enzyme required for 50% inhibition of NBT reduction/min/mg protein; b Glutathione ( l M ) utilized/min/mg protein;c H2 O2 (l M ) utilized/min/mg protein.




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administration of DA along with thermally oxidized oiland alcohol, which may be either due to scavenging of peroxides and other activated oxygen species formed ordue to the neutralization of free radicals. Previous reportshave shown that aminothiazoles possess antioxidantactivity and inhibit lipid peroxidation [9].

Our results also showed decreased level of nonenzy-matic antioxidants GSH, vitamin C and E in the liver,kidney and decreased activity of enzymic antioxidantsSOD, GPx and catalase in the liver of thermally oxidizedoil-, alcohol- and alcohol + thermally oxidized oil-fedrats when compared with normal control rats. Inductionof CYP 2E1 by ethanol results in enhanced acetaldehydeproduction which in turn impairs the defence systemagainst oxidative stress [41]. The antioxidant status wasfound to be improved upon DA administration indi-cating the antioxidant properties of the drug. Previousreports have shown the in vivo antioxidant activity of 4-aminothiazoles [9].

Hence DA with its antioxidant properties effectivelyprotects the tissues against thermally oxidized oil andalcohol-induced oxidative stress. The antioxidant prop-erties of DA may be due to the presence of amino groupat C 2 , C4 positions and a keto group at C 5 position, whichaccept e-electrons from free radicals and neutralize them.

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Influence of a thiazole derivative on ethanol and thermally oxidized sunflower oil induced oxidative stress