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JOURNAL OF MEDICINAL FOOD J Med Food 8 (2) 2005, 242–245© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition

Expression Pattern of Matrix Metalloproteinases in Alcohol- and Thermally OxidizedSunflower Oil-Induced Toxicity: Protective Role of an Aminothiazole Derivative

Kode Aruna, 1 Rajagopalan Rukkumani, 1 Penumathsa Suresh Varma, 1 and Venugopal P. Menon 1,2

1 Department of Biochemistry and 2 Center for Micronutrient Research, Faculty of Science, AnnamalaiUniversity, Annamalainagar, Tamil Nadu, India

ABSTRACT The matrix metalloproteinases (MMPs) are a family of secreted and membrane-bound zinc endopeptidases.Collectively, these enzymes can degrade all of the components of the extracellular matrix including collagen, fibronectin,laminin, and basement membrane glycoproteins. Regulation in expression and activation of proteinases is one of the most im-portant mechanisms in organ morphogenesis. Fibrosis is a dynamic pathological process with a net accumulation of extra-cellular matrix proteins. In the present communication, we have investigated the changes that occur in the activity of liverMMPs in normal and in pathological conditions. The activity of MMPs was increased in thermally oxidized sunflower oil-and alcohol-treated groups, whereas the activity was decreased in the thermally oxidized oil alcohol-fed group when com-pared with the normal control group. The activity was positively modulated when dendrodoine analogue [4-amino-5-benzoyl-2(4-methoxyphenylamino)thiazole] was administered along with ethanol and thermally oxidized oil, which indicates the pro-tective effect of this drug.

KEY WORDS: • aminothiazole derivative • dendrodoine analogue • ethanol • liver fibrosis • matrix metallopro- teinases • thermally oxidized sunflower oil

INTRODUCTION

T HE INCREASED CONSUMPTION OF ALCOHOL by humans pro-duces a heavy burden on society both socially and eco-

nomically. Chronic consumption of alcohol produces a num-

ber of toxic reactions. This is primarily due to its metaboliteacetaldehyde, which is very unstable and can undergo bothoxidation and reduction. Besides this, acetaldehyde has beenshown to stimulates synthesis of collagen in stellate cells, 1

probably because of an adduct formation between acetalde-hyde and propeptide (an intermediate of collagen synthesis),which results in increased collagen synthesis. 2 Fibrosis, aconsequence of most chronic liver diseases, may be con-sidered to be the result of a disrupted balance between fi-brogenesis and fibrolysis. Progressive fibrosis occurs whenthe rate of matrix synthesis exceeds matrix degradation. 3 Inthe extracellular space, matrix degradation occurs predom-inantly as a consequence of the action of a family of en-zymes called the matrix metalloproteinases (MMPs). Theseare secreted from cells into the extracellular space as proen-zymes, which are then activated by a number of specific,usually cell surface-associated, cleavage mechanisms. 4 The

active enzymes are in turn inhibited by a family of tissueinhibitors of metalloproteinases. 5 By this combination of mechanisms extracellular matrix degradation is closely reg-ulated, which prevents inadvertent tissue damage. 4

The level of dietary fat seems to play a very important rolein ethanol-induced damage to various cellular membranes.Diets with high levels of fat greatly enhance liver steatosisas well as liver membrane damage and liver fibrosis. 6

Aminothiazoles are a group of biologically importantcompounds having a wide range of activities, such as anti-tumor, anti-anoxic, and antioxidant. 7,8 Dendrodoine, a ma-rine alkaloid, was isolated from Dendrodoa grossularia .9 Itpossesses a 1,2,4-thiadiazole unit, a rarity among naturalproducts. Though its synthesis has been reported, 10 few bi-ological studies have been carried out on it and its analogue[4-amino-5-benzoyl-2(4-methoxyphenylamino)thiazole](Fig. 1). The present study was designed to study the effectsof dendroine analogue (DA) on the activity of liver MPPs

in normal and in pathological conditions.

MATERIALS AND METHODS

Experimental animals

Male albino rats Wistar strain (body weight 140–160 g)bred in the Central Animal House, Rajah Muthiah MedicalCollege, Annamalainagar, Tamil Nadu, India, were used inthis study. The animals were housed in plastic cages with

Manuscript received 13 April 2004. Revision accepted 14 July 2004.

Address reprint requests to: Dr. Venugopal P. Menon, Professor and Head, Department of Biochemistry and Center for Micronutrient Research, Annamalai University, Anna-malainagar-608 002, Tamil Nadu, India, E-mail: cmrana@sify.com or biocmr@sify.com

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Statistical analysis

Statistical analysis was carried out using analysis of vari-ance followed by Duncan’s multiple range test. The level of statistical significance was set at P .05.

RESULTS

Figure 2 gives changes in the activity of liver MMPs (Fig.2A) and densitometry of the liver zymogram (Fig. 2B) inthe normal control group and experimental groups giventhermally oxidized oil, alcohol, and thermally oxidized al-cohol thermally oxidized oil given groups. The activityof MMPs was increased in both the thermally oxidized oil-and alcohol-given groups and decreased in the thermally ox-idized alcohol thermally oxidized oil-given group whencompared with the normal control group.

Figure 3 gives changes in the activity of liver MMPs (Fig.3A) and densitometry of the liver zymogram (Fig. 3B) inthe drug control group and experimental groups given ther-mally oxidized oil DA, alcohol DA, and alcoholthermally oxidized oil DA. The activity was decreased inboth the thermally oxidized oil DA- and alcohol DA-

given groups, whereas the activity was found to be increasedin the thermally oxidized oil alcohol DA-given group.

Figure 4 gives changes in the total activity of MMPs (Fig.4A) and densitometry of the multiwell zymogram (Fig. 4B)in various groups. The activity was increased in both thethermally oxidized oil- and alcohol-given groups but de-creased in the thermally oxidized oil alcohol-given groupwhen compared with the normal control group. In the groupsgiven thermally oxidized oil DA and alcohol DA, theactivity of MMPs decreased, whereas the activity was at-tenuated in the alcohol oil DA-given group.

DISCUSSION

MMPs, including collagenase, gelatinase, and stromelysin,have been implicated as being involved in remodeling of connective tissue with degradation of matrix proteins 15 and

244 ARUNA ET AL.

A

B

FIG. 3. Gelatin zymogram ( A) and densitometry of the liver zymo-gram ( B) show the changes in activity of MMPs in liver of DA con-trol rats and experimental groups of rats given thermally oxidized oil

DA, alcohol DA, and alcohol thermally oxidized oil DA. A:Lane (iv), alcohol thermally oxidized oil DA; lane (iii), thermallyoxidized oil DA; lane (ii) alcohol DA; lane (i), DA control.

FIG. 4. Multiwell zymogram ( A) and densitometry of the multiwellzymogram ( B) show the changes in the total activity of MMPs in liverof normal control rats (well and column A) and experimental groupsof rats given alcohol (B), thermally oxidized oil (C), alcohol ther-mally oxidized oil (D), DA control (E), alcohol DA (F), thermallyoxidized oil DA (G), and alcohol thermally oxidized oil DA(H). Well I, blank.

B

A

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are thought to play a crucial role in tissue fibrosis. 16 Our re-sults showed increased activities of MMPs in the liver of both the thermally oxidized sunflower oil- and alcohol-fedgroups of rats when compared with normal control rats. Theincrease in the activities of MMPs in these groups may bedue to the up-regulation of MMPs in the early phase of ex-perimental liver injury. 17 This results in degradation of col-lagen, which may have been synthesized. Chronic ethanolconsumption strikingly enhances the number of hepatic col-lagen-producing activated lipocytes. Both in vivo and invitro ethanol and/or its metabolite acetaldehyde increase col-lagen accumulation and levels of mRNA for collagen. 18 Theactivities of MMPs in both the thermally oxidized oil- andalcohol-fed groups of rats were decreased when DA wasadministered along with thermally oxidized oil and alcohol.

The activities of MMPs were decreased in the alcoholthermally oxidized oil-given group when compared with thenormal control group. Dietary fatty acids play an importantrole in altering some of the deleterious effects of ethanol. 19

A high fat diet rich in linoleic acid correlated positively withthe induction of severe liver pathology. 20 Previous studieshave shown that there is a decrease in the interstitial collage-nase activities in late progressive steps of liver fibrosis. 21 Pre-vious reports have shown that hepatic stellate cell-mediatedactivation of progelatinase-A is significantly induced in thepresence of collagen. 22 This could contribute to further degra-dation of the normal liver matrix, leading to decreased acti-vation of hepatic stellate cells and decreased synthesis of col-lagen. This positive feedback loop would promote progressionof liver fibrosis. 4 Decreased activities of MMPs may be dueto decreased pro-collagenase gene expression and biosynthe-sis and decreased activation of proenzyme/specific inhibitionof native collagenase activity in early stages and reduced ac-tivity in advanced stages of liver fibrosis. 23 The activities of

MMPs were found to be increased in the alcohol thermallyoxidized oil DA-administered group. Thus the activities of MMPs were positively regulated when DA was administeredalong with alcohol and/or thermally oxidized oil, which showsthe protective effect of our drug.

Thus the DA investigated in the present study could ef-fectively protect the liver against ethanol- and thermally ox-idized oil-induced toxicity. We conclude that the NH 2 grouppresent in the DA reacts with acetaldehyde, the toxicmetabolite of ethanol, and also with other free radicals andforms a Schiff’s base, thereby neutralizing the toxic effectsof ethanol and thermally oxidized oil.

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