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Changes in Activities of MMP in Alcohol and Thermally Oxidized

Sunflower Oil-Induced Liver Damage: NAC Antioxidant Therapy
Suresh Varma Penumathsa a; Aruna Kode a; Rukkumani Rajagopalan a; Venugopal P. Menon a
a
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar, India

To cite this Article Penumathsa, Suresh Varma, Kode, Aruna, Rajagopalan, Rukkumani and Menon, Venugopal P.(2006)
'Changes in Activities of MMP in Alcohol and Thermally Oxidized Sunflower Oil-Induced Liver Damage: NAC
Antioxidant Therapy', Toxicology Mechanisms and Methods, 16: 5, 267 — 274
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 Toxicology Mechanisms and Methods, 16: 267–274, 2006
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ISSN: 1537-6524 print / 1537-6516 online
DOI: 10.1080/15376520500194734

Changes in Activities of MMP in Alcohol and Thermally

Oxidized Sunflower Oil-Induced Liver Damage: NAC
Antioxidant Therapy

Suresh Varma Penumathsa, Aruna Kode, Rukkumani Rajagopalan, and

Venugopal P. Menon
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar-608 002, India

fibroblasts. Lieber and colleagues thereafter made an important

Liver fibrosis is the result of imbalance between extracellular observation that acetaldehyde stimulates collagen gene activa-
matrix (ECM) synthesis and breakdown. Ethanol-induced increase
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tion and protein synthesis in hepatic stellate cells (Moshage et al.

in redox state is a sign of major change in hepatic metabolism and
this inhibits tricarboxylic acid cycle activity and, fatty acid oxida-
tion and increases fatty acid uptake, thus predisposing fatty liver.
Fibrotic changes induced by alcohol are provoked by diets rich in
PUFA. Heating of oils rich in PUFA produces toxic volatile and non-
volatile compounds, which aggravate liver damage. Hepatotoxicity
was induced in male Wistar rats by administering alcohol (20%)
and thermally oxidized sunflower oil (
PUFA) (15%). When N-
acetyl cyteine (NAC) (150 mg/kg body weight), an ROS scavenger,
was administered, there was a reversal of liver damage, which was
demonstrated biochemically. Matrix metalloproteinases (MMPs),
being potential biochemical indicators of fibroproliferation, were
estimated in the present study, which were found to be altered in
alcohol,
PUFA, and alcohol +
PUFA. The altered activities of
MMPs in these groups were effectively modulated by treatment
with NAC. Thus, in this study, NAC was found to modulate the
effect of alcohol and
PUFA-induced liver damage.
Keywords NAC, Matrix Metalloproteinases, Alcohol,
PUFA
INTRODUCTION
In alcoholic liver disease, liver fibrosis is considered as a first
step, followed by severe fibrotic changes, which lead eventually
to cirrhosis. Hepatic fibrosis is the result of imbalance between
enhanced matrix synthesis and breakdown of connective tissue
proteins, the net result of which is increased deposition of ECM.
All routes of ethanol oxidation result in formation of acetalde-
hyde. Ethanol as well as its metabolite acetaldehyde have been
involved in the stimulation of collagen synthesis (Niemala et al.
1995). Brenner and Chojkier (1987) were the first to report that
acetaldehyde increased collagen gene transcription in cultured
Received 7 January 2005; accepted 7 July 2005.
Address correspondence to Dr. Venugopal P Menon, Professor
and Head, Department of Biochemistry, Annamalai University,
Annamalainagar – 608 002, Tamil Nadu, India. E-mail:
cdl cmrana@sancharnet.in; biocmr@sify.com; cmrana@sify.com
1990).
The composition of dietary fat is reflected in the composition
of the lipids accumulating in the liver of ethanol-fed animals.
It is also suggested that the more fat in the liver, the higher the
susceptibility to severe damage (Day and James 1998). The pro-
portion of unsaturated fat correlates with the damage. Diets rich
in PUFA have influenced the development of liver injury (Nangi
et al. 1994). Thermal oxidation of oil produces volatile and non-
volatile compounds, leading to variety of diseases (Twefix et al.
1998). Enhanced liver damage has been shown when thermally
oxidized sunflower oil (
PUFA) is supplemented along with
ethanol (Rukkumani et al. 2002; Aruna et al. 2002).
During severe damage, fibrotic changes occur in the liver,
which is characterized by proliferation of hepatic stellate
cells and their transformation into myofibroblasts. Hepatic
myofibroblasts are the source of the overproduction of structural
proteins that constitute liver fibrosis (Friedman 1999). Several
biochemical indicators have been shown as potential markers
of fibroproliferation. Among them, MMPs have been shown
by several groups to correlate more or less closely with the de-
velopment of fibrosis. MMPs are zinc- and calcium-dependent
enzymes that are largely responsible for degrading ECM
components (Mignatti and Rifkin 1993). MMPs digest specific
ECM components during abnormalities and thereby contribute
to matrix equilibrium and structural integrity.
NAC, a reactive oxygen species (ROS) scavenger, is a deriva-
tive of L-cysteine and is present in higher amounts in protein-
rich foods. NAC acts as a source of cysteine and stimulates the
production of GSH, which protects the body. It has antioxidant,
anti-inflammatory, and hypolipidemic properties (Devries and
De Flora 1993). Thiol compounds such as cysteine are known
to increase the survival rate of alcohol-fed rats (Rajakrishnan
et al. 1997) and have been found to be an effective antidote for
acetaldehyde poisoning and an antagonist to hepatotoxic effect
of carbon tetrachloride (Jaya et al. 1994). NAC has also been

267
 268 S. V. PENUMATHSA ET AL.

shown to protect the liver from the adverse effects of several TABLE 1
toxic chemicals (Ben et al. 2000). The purpose of our study is to Fatty acid composition of sunflower oil and heated
find the effect of NAC on the synthesis and activities of MMPs sunflower oil percentage of fatty acid/g oil
during alcohol- and
PUFA-induced toxicity.
Fatty acid Sunflower oil Heated sunflower oil
MATERIALS AND METHODS 9:0 3OH — 0.27 ± 0.02
10:0 1.10 ± 0.08 0.15 ± 0.01
Maintenance of Animals
10:0 2OH — 0.34 ± 0.03
Male albino Wistar rats of body weight ranging from 140
10:0 3OH — 0.15 ± 0.01
to 150 g bred in Central Animal House, Rajah Muthiah Medi-
11:0 — 4.27 ± 0.39
cal College, Annamalai University, were fed on pellet diet (Agro
12:0 5.74 ± 0.53 2.02 ± 0.18
Corp. Pvt. Ltd., Bangalore, India) and water ad libitum. The stan-
13:0 0.76 ± 0.05 —
dard pellet diet comprised 21% proteins, 5% lipids, 4% crude
14:0 1.11 ± 0.06 0.25 ± 0.02
fiber, 8% ash, 1% calcium, 0.6% phosphorous, 3.4% glucose,
15:0 0.49 ± 0.04 0.30 ± 0.02
2% vitamins, and 55% carbohydrates and provided metaboliz-
16:0 17.93 ± 1.54 16.78 ± 1.59
able energy of 3600 kcal/kg. The animals were housed in plastic
16:1 — 0.36 ± 0.03
cages under controlled condition of 12-h light/12-h dark cycle,
17:0 — 0.67 ± 0.05
50% humidity, and 30 ± 3◦ C. The animals used in the present
17:0 cyclo — 0.52 ± 0.04
study were maintained in accordance with the guidelines of the
9.87 ± 0.85 9.92 ± 0.87
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18:0
National Institute of Nutrition, Indian Council of Medical Re-
18:1 60.22 ± 5.74 52.89 ± 4.65
search, Hyderabad, India, and approved by the Animal Ethical
18:1 2OH — 1.82 ± 0.13
Committee, Annamalai University.
19:0 2.17 ± 0.19 0.95 ± 0.05
20:0 0.61 ± 0.04 1.56 ± 0.08
Materials Used 20:1 — 6.78 ± 0.65
1. Ethanol: Absolute ethanol (AR) was obtained from
Hayman Limited, England.
2. Sunflower oil: Sunflower oil marketed by Gold Winner
was purchased from the local market, Chidambaram, NAC : NAC 150 mg/kg body weight (Jaya et al. 1994) dissolved
Tamil Nadu, India. in water.
3. NAC: N-acetyl-L-cysteine was purchased from Sigma
Chemical Company, USA. All animals were maintained on isocalorific diet using glu-
4. Thermally oxidized sunflower oil (
PUFA): Sunflower cose solution (total calories/day: 508 kcal/kg body weight). At
oil was subjected to heating at 180◦ C for 30 minutes, twice the end of experimental period (45 days), the rats were sacrificed
(fatty acid composition is shown in Table 1) (Aruna et al. after an overnight fast by decapitation. The liver was removed,
2002). cleared of blood, and immediately transferred to ice cold con-
tainers containing 0.9% NaCl for various estimation.
All other chemicals used were of analytical grade.

Experimental Design Methods

Normal : Control rats given standard pellet diet
Alcohol : Rats given 20% ethanol [7.9 g/kg body weight]
(Rajakrishnan et al. 1997)

PUFA : Rats given high-fat diet (15%) (thermally oxidized
sunflower oil mixed with diet) (Aruna et al. 2002)
Alcohol +
PUFA : Rats given 20% ethanol orally using an in-
tragastric tube and a high-fat diet (15%) (thermally oxidized
sunflower oil mixed with diet)
Alcohol + NAC : Rats given NAC (150 mg/kg body weight)
dissolved in 20% ethanol

PUFA + NAC : Rats given NAC dissolved in water and high-
fat diet (15%) (thermally oxidized sunflower oil mixed with
diet)
Alcohol +
PUFA + NAC : Rats given NAC dissolved in alco-
hol and high-fat diet (15%) (thermally oxidized sunflower
oil mixed with diet)
1. Multiwell zymography: It was done by the method Ambili
and Sudhakaran (1998).
2. SDS-PAGE zymography: It was done by the method of
Ambili et al. (1997). Gelatin substrate-impregnated SDS-
polyacrylamide gels were used. Samples were separated
according to their molecular weight by electrophoresis.
3. Succinylation: It was done by the method of Vijaykumar
et al. (Baragi et al. 2000) for the quantitative estimation
of MMPs activity.
Statistical Analysis
The data given in the tables are average ± standard deviation
(SD). Data were analyzed statistically by “analysis of variance”
(ANOVA) and groups were compared by Duncan’s Multiple
Range Test (DMRT).
 MMPS IN LIVER DAMAGE
1 brane damage and increase lipid infiltration predisposing fatty
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2 and thus reduce organ fibrosis. Preaux et al. have observed the in-
FIGS. 1 & 2. Multiwell zymogram of Liver.
RESULTS AND DISCUSSION
Figs. 1, 2, and 3 show the total activities of MMP by mul-
tiwell zymography. Figs. 4 to 7 show the individual activities
of MMP by zymography. The overall MMP activities as evi-
denced by multiwell zymography and individual MMP activities
as seen in zymography were increased significantly in alcohol-
and
PUFA-administered rats and decreased significantly in
alcohol +
PUFA-treated rats. Administration of NAC posi-
tively modulated these activities. The changes in the activities
of MMP-2 and MMP-9 by succinylation also correlate with these
results (Figs. 8 and 9).
The increase in the activities of MMPs in alcohol and

PUFA groups may be because of the counterbalancing action
of these enzymes to degrade the excessive collagen deposited
(Fig.10). Ingestion of ethanol induces various processes in-
cluding changes in NAD+ /NADH (Arthur 1995), production
of acetaldehyde protein adducts (Lieber 2000), induction of
269
CYP2El (Tuma et al. 1996), formation of 1-hydroxyethyl free
radicals (Gouillon et al. 2000), and endotoxin-derived activa-
tion of Kupffer cells, which in-turn produce tumor necrosis fac-
tor (Yin et al. 1994). These changes cause severe damage to
the liver. Moreover, acetaldehyde, the main product of ethanol
metabolism, affects hepatic collagen synthesis, both in vivo and
in vitro, leading to increased collagen accumulation, resulting
in fibrosis (Lieber 1993). In
PUFA-treated rats, the increased
intake of PUFA increases the degree of unsaturation of the
biomembrane and makes them more susceptible to lipid peroxi-
dation (Farrel and Jackson 1997). Reports have shown that wide
utilization of fats, which are highly susceptible to oxidation dur-
ing cooking and frying, may alter physiological effects of their
PUFA content and generate lipid peroxides that cause mem-
liver and fibrosis (Jethmalani et al. 1998). Thus, these changes
during alcohol and
PUFA ingestion damage the liver and per-
turb the homeostasis of ECM. Furthermore, cyclooxygenase,
lipoxygenase, and cytochrome P450 monooxygenase pathways
are also activated by PUFA, which may increase liver damage
by enhanced production of free radicals, resulting in fibrosis
(Herrera et al. 2001).
Two possible explanations can be given for the increased
activities of MMPs during alcohol and PUFA ingestion. First,
MMP expression may be induced by increased amount of matrix
proteins with compensatory or homeostatic mechanism against
excessive deposition of matrix components. Second, the in-
creased transforming growth factor (TGF) -β1, which is known
to be increased in chronically diseased liver, may upregulate
MMPs at transcriptional and posttranscriptional levels (Overall
et al. 1991). Previous studies have shown that MMPs are up-
regulated in the early phase of experimental liver injury (Mehde
et al. 1997). The MMPs degrade the basal membrane collagen
creased MMP-2 mRNA expressions during liver injury (Preaux
et al. 1999). Moreover, the hepatic mRNA expression for MMP-
2 and MMP-7 are found to be higher in fibrotic patients than
in normal subjects (Lichtinghagen et al. 1999). Expression of
MMP-2 and MMP-7 increases in the early stages of liver tox-
icity and allows an assessment of the amount of a connective
tissue deposited. Thus, the increase in the activities of MMPs in
our study suggests that alcohol and
PUFA intake might have
caused significant deposition of connective tissue due to liver
damage.
Previous studies suggest that the ROS activate MMP
(Rajagopalan et al. 1996). During alcohol and
PUFA intake
there is an increase in the ROS levels, and this in turn con-
verts inactive zymogen to the active form. Treatment with NAC
brought back the activities of MMPs to near normal. NAC is
widely used as an antioxidant as it scavenges ROS. Previous
studies suggest that scavenging ROS prevent or diminish the
activation of MMP (Devaraj et al. 1996). In the present study,
when NAC was administered, the MMP activities were brought
back to near normal. This may be due to the decreased activation
of MMP zymogen because of decreased ROS levels.
 270 S. V. PENUMATHSA ET AL.
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FIG. 3. Densitometry of Liver Multiwell Zymogram.

FIG. 4. Changes in the activities of Matrix Metalloproteinases in liver.

 MMPS IN LIVER DAMAGE 271

FIG. 5. Densitometry of liver zymogram.

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In our study the activities of MMPs were found to be de-
creased in the alcohol +
PUFA group. Previous reports have
shown that fibrotic changes in rats fed ethanol can be pro-
voked with dietary manipulation (French et al. 1998). A high-fat
ethanol diet has been reported to cause excessive centrilobu-
lar fibrosis within a short period. Previous studies have also
demonstrated that there is a decrease in the interstitial collage-
nase [MMP] activities in late progressive steps of liver fibrosis
(Lichtinghagen et al. 2001). The possible explanation for this
failure of matrix degradation includes decreased procollagenase
gene expression and biosynthesis, decreased activation of proen-
zyme, or specific inhibition of native collagenase. Reports also
show increased collagenase activity in early stages and reduced
activity in advanced stages of liver fibrosis (Isao et al. 2001),
which correlate with our findings.
Administration of NAC improved the activities of MMPs
in this group. This might be because of the hepatoprotective
role of NAC. NAC being an effective antioxidant decreases the
damage to the liver and decreases the extent of fibrosis. Previous
reports have also shown that treatment with NAC modulates the
secretion of MMP-9 during atherosclerosis (Galis et al. 1998).
Thus our results show that NAC is an effective antioxidant
and plays a main role in maintaining the architecture of the
liver. NAC prevents the accumulation of ECM components and

FIG. 6. Changes in the activities of Matrix Metalloproteinases in liver.

 272 S. V. PENUMATHSA ET AL.

FIG. 7. Densitometry of liver zymogram.

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FIG. 8. Changes in the activities of MMP-2.

FIG. 9. Changes in the activities of MMP-9.

 MMPS IN LIVER DAMAGE
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FIG. 10. Mechanism of induction of MMP by Alcohol & PUFA—
Therapeutic role of NAC.
maintains the structural integrity by quenching the ROS pro-
duced during alcohol and heated PUFA intake, thus maintaining
the activities of MMP.
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