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 Oxidized

Sunflower Oil-Induced Toxicity: Protective Role of an Aminothiazole Derivative
Kode Aruna,1 Rajagopalan Rukkumani,1 Penumathsa Suresh Varma,1 and Venugopal P. Menon1,2
1Department of Biochemistry and 2Center for Micronutrient Research, Faculty of Science, Annamalai
University, 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 liver
MMPs 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 active enzymes are in turn inhibited by a family of tissue

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 metabolite
acetaldehyde, which is very unstable and can undergo both
oxidation and reduction. Besides this, acetaldehyde has been
shown 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, a
consequence 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 when
the rate of matrix synthesis exceeds matrix degradation.3 In
the extracellular space, matrix degradation occurs predom-
inantly as a consequence of the action of a family of en-
zymes called the matrix metalloproteinases (MMPs). These
are 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
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
inhibitors 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 role
in ethanol-induced damage to various cellular membranes.
Diets with high levels of fat greatly enhance liver steatosis
as well as liver membrane damage and liver fibrosis.6
Aminothiazoles are a group of biologically important
compounds 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 It
possesses a 1,2,4-thiadiazole unit, a rarity among natural
products. 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 effects
of 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 Medical
College, Annamalainagar, Tamil Nadu, India, were used in
this study. The animals were housed in plastic cages with

242
 EXPRESSION OF MATRIX METALLOPROTEINASES 243

A ethanol/kg of body weight) daily using an intragastric tube12;

Group 3, rats given DA (10 mg/kg of body weight); Group
4, rats given thermally oxidized sunflower oil (15%) mixed
with diet; Group 5, rats given 20% ethanol
thermally ox-
idized sunflower oil (15%); Group 6, rats given DA (10
mg/kg of body weight)
thermally oxidized sunflower oil
(15%); Group 7, rats given 20% ethanol
DA (10 mg/kg
of body weight); and Group 8, rats given 20% ethanol

thermally oxidized sunflower oil (15%)
DA (10 mg/kg of
B body weight). The experimental period was 45 days.
At the end of experimental period (45 days) rats were
given anesthesia (ketamine hydrochloride, 30 mg/kg of body
weight) and were sacrificed by decapitation after an
overnight fast. The liver was removed, cleared of blood, and
collected in ice-cold containers containing Tris/HCl buffer
(pH 8.9). The institutional ethical committee approved this
project (registration number 160/1999/CPSEA).
Zymography was performed as described by the method
of Ambili et al.,13 and multiwell zymography was performed
by the method of Ambili and Sudhakaran.14

FIG. 1. Structure of (A) dendrodoine and (B) DA [4-amino-5-ben-

zoyl-2-(4-methoxyphenylamino)thiazole.
A

filter tops under seminatural light-dark conditions and at

room temperature. The animals were fed on pellet diet (Hin-
dustan Lever Ltd., Mumbai, India), and water was given ad
libitum.

Chemicals
Ethanol was purchased from E. Merck (Darmstadt, Ger-
many). Tris, acrylamide, bisacrylamide, gelatin, sodium do-
decyl sulfate, and ammonium persulfate were purchased
form Sigma Chemical Co. (St. Louis, MO). DA was syn-
thesized as described by Rajasekharan et al.11 Purity of the
compound was checked by thin-layer chromatography, and
its structure was confirmed by Fourier transform infrared B
spectroscopy and nuclear magnetic resonance. DA was dis-
solved in 1% dimethyl sulfoxide and was given orally.
Sunflower oil (Gold winner) was purchased from the lo-
cal market in Chidambaram, Tamil Nadu, India. The oil was
subjected to two frying cycles of 30 minutes each at 180°C
to produce thermally oxidized oil. All other chemicals and
biochemicals used for the experiments were of analytical
grade.

Experimental design
Dose-dependent studies of DA were carried out in rats
given alcohol, and the effective dose was found to be 10
mg/kg of body weight.
The animals were randomized into the following groups:
Group 1, control rats given standard pellet diet; Group 2,
rats given 20% ethanol 5 mL each (equivalent to 7.9 g of
FIG. 2. Gelatin zymogram (A) and densitometry of the liver zymo-
gram (B) show the changes in activity of MMPs in liver of normal
control rats and experimental groups of rats given thermally oxidized
oil, alcohol, and alcohol
thermally oxidized oil. A: Lane (i), nor-
mal control; lane (ii), alcohol; lane (iii), thermally oxidized oil; lane
(iv), alcohol
thermally oxidized oil.
244 ARUNA ET AL.

A given groups, whereas the activity was found to be increased

in 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 the
thermally oxidized oil- and alcohol-given groups but de-
creased in the thermally oxidized oil
alcohol-given group
when compared with the normal control group. In the groups
given thermally oxidized oil
DA and alcohol
DA, the
activity of MMPs decreased, whereas the activity was at-
tenuated in the alcohol
oil
DA-given group.

DISCUSSION
MMPs, including collagenase, gelatinase, and stromelysin,
B have been implicated as being involved in remodeling of
connective tissue with degradation of matrix proteins15 and

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), thermally
oxidized oil
DA; lane (ii) alcohol
DA; lane (i), DA control.

Statistical analysis B
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) in
the normal control group and experimental groups given
thermally oxidized oil, alcohol, and thermally oxidized al-
cohol
thermally oxidized oil given groups. The activity
of 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 when
compared with the normal control group.
Figure 3 gives changes in the activity of liver MMPs (Fig. FIG. 4. Multiwell zymogram (A) and densitometry of the multiwell
3A) and densitometry of the liver zymogram (Fig. 3B) in zymogram (B) show the changes in the total activity of MMPs in liver
of normal control rats (well and column A) and experimental groups
the drug control group and experimental groups given ther- of rats given alcohol (B), thermally oxidized oil (C), alcohol
ther-
mally oxidized oil
DA, alcohol
DA, and alcohol
mally oxidized oil (D), DA control (E), alcohol
DA (F), thermally
thermally oxidized oil
DA. The activity was decreased in oxidized oil
DA (G), and alcohol
thermally oxidized oil
DA
both the thermally oxidized oil
DA- and alcohol
DA- (H). Well I, blank.
 EXPRESSION OF MATRIX METALLOPROTEINASES
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-fed
groups of rats when compared with normal control rats. The
increase in the activities of MMPs in these groups may be
due 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 ethanol
consumption strikingly enhances the number of hepatic col-
lagen-producing activated lipocytes. Both in vivo and in
vitro ethanol and/or its metabolite acetaldehyde increase col-
lagen accumulation and levels of mRNA for collagen.18 The
activities of MMPs in both the thermally oxidized oil- and
alcohol-fed groups of rats were decreased when DA was
administered along with thermally oxidized oil and alcohol.
The activities of MMPs were decreased in the alcohol

thermally oxidized oil-given group when compared with the
normal control group. Dietary fatty acids play an important
role in altering some of the deleterious effects of ethanol.19
A high fat diet rich in linoleic acid correlated positively with
the induction of severe liver pathology.20 Previous studies
have 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-mediated
activation of progelatinase-A is significantly induced in the
presence 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 progression
of liver fibrosis.4 Decreased activities of MMPs may be due
to decreased pro-collagenase gene expression and biosynthe-
sis and decreased activation of proenzyme/specific inhibition
of 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
thermally
oxidized oil
DA-administered group. Thus the activities of
MMPs were positively regulated when DA was administered
along with alcohol and/or thermally oxidized oil, which shows
the 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 NH2 group
present in the DA reacts with acetaldehyde, the toxic
metabolite of ethanol, and also with other free radicals and
forms a Schiff’s base, thereby neutralizing the toxic effects
of ethanol and thermally oxidized oil.
REFERENCES
1. Moshage H, Casini A, Lieber CS: Acetaldehyde selectively stim-
ulates collagen production in cultured rat liver fat- storing cells
but not in hepatocytes. Hepatology 1990;12:511–518.
2. Ma X, Svegliati BG, Poniachik J, Baraona E, Lieber CS: Colla-
gen synthesis by liver stellate cells is released from its normal
feedback regulation by acetaldehyde induced modification of the
carboxyl terminal propeptide of procollagen. Alcohol Clin Exp
Res 1997;21:1204–1211.
245
3. Iredale JP: Tissue inhibitors of metalloproteinases in liver fibro-
sis. Int J Biochem Cell Biol 1997;29:43–54.
4. Michael JPA: Metalloproteinases and their inhibitors in liver fibro-
sis. Am J Physiol Gastrointest Liver Physiol 2000;279:245–249.
5. Elke R, Edmund P, Bettina B, Huan N, Peter CH, Stefan RJ,
Siegfried M: TIMP expression in toxic and cholestatic liver in-
jury in rat. J Hepatol 1997;27:535–544.
6. Reitz RC: Dietary fatty acids and alcohol: Effects on cellular
membranes. Alcohol Alcohol 1993;28:59–71.
7. Ohkubo M, Kuno A, Nakanishi I, Takasughi H: Studies on cere-
bral protective agents; synthesis of 2-amino thiazole and 2-thia-
zole carboxamides with antianoxic activity. Chem Pharm Bull
(Tokyo) 1995;43:1497–1504.
8. Uchikawa O: In vivo biological activity of antioxidative amino-
thiazole derivatives. Chem Pharm Bull (Tokyo) 1996;44:2070–
2077.
9. Heitz S, Durgeat M, Guyot M, Brassy C, Bachet B: Nouveau de-
rivé imdolique du thiadiazole-1,2,4 isole d’un tunicier (Den-
drodoa grassularia). Tetrahedron Lett 1980;21:1457–1458.
10. Hogan IT, Sainsbury M: Synthesis of 1,2,4-thiazole derivative,
dendrodoine. Tetrahedron 1984;40:681–682.
11. Rajasekharan KN, Nair KP, Jenardanan GC: Studies on the syn-
thesis of 5-acyl-2,4-diaminothiozoles from amidino thioureas.
Synthesis 1986;1986:353–355.
12. Rajakrishnan V, Viswanathan P, Menon VP: Adaptation of sib-
lings of female rats given ethanol, effect of N-acetyl cysteine.
Amino Acids 1996;12:323–341.
13. Ambili M, Radhakrishna PM, Sudhakaran PR: Characteristics of
60K gelatinase involved in rat mammary gland involution. Indian
J Biochem Biophys 1997;34:347–353.
14. Ambili M, Sudhakaran PR: Assay of matrix metalloprotinases in
substrate impregnated gels in multiwells. Indian J Biochem Bio-
phys 1998;35:317–320.
15. Emonard H, Grimaud JA: Matrix metalloproteinases: A review.
Cell Mol Biol 1996;36:131–153.
16. Bissell DH, Friedman SL, Maher JJ, Roll FJ: Connective tissue
biology and hepatic fibrosis: Report of concern. Hepatology
1990;11:488–498.
17. Knittel J, Mehede M, Grundman A, Saile B, Scharf JG, Ramadori
G: Expression of matrix metalloproteinases and their inhibitors
during hepatic tissue repair in rats. Histochem Cell Biol 2000;113:
443–453.
18. Lieber CS: Susceptibility to alcohol related liver injury. Alcohol
Alcohol 1994;2:315–326.
19. Nanji AA, Sadrzadeh SMH, Dannenberg AJ: Liver microsomal
fatty acid composition in ethanol fed rats: Effect of different di-
etary fats and relationship to liver injury. Alcohol Clin Exp Res
1994;18:1024–1028.
20. Nanji AA, French SW: Dietary linoleic acid is required for de-
velopment of experimental induced alcoholic liver disease. Life
Sci 1989;44:223–227.
21. Lichtinghagen R, Michels D, Haberkoran CI: Matrix metallopro-
teinases (MMP-2, MMP-7), and tissue inhibitors of MMP-1 are
related to the fibroproliferative process in the liver during chronic
hepatitis C. Hepatology 2001;34:239–247.
22. Theret N, Lehti K, Musso O, Clement B: MMP2 activation by
collagen I and Concanavalin A in cultured human heptic stellate
cells. Hepatology 1999;30:462–468.
23. Isao O, Tetsu W, Shigenari H: Reversibility of hepatic fibrosis
from the first report of collagenase in the liver to the possibility
of gene therapy for recovery. Keio J Med 2001;50:58–65.