Reduction Of Alcohol Intoxication In Experimental Animals By Resveratrol

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Reduction Of Alcohol Intoxication In Experimental Animals By Resveratrol
Parvathy S. Nair & Parvathy R.
S8 Biotechnology & Biochemical Engineering
Mohandas College of Engineering & Technology, Anad

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Abstract
Chronic alcohol consumption induces an increase in oxidative
stress. As polyphenolic compounds are potent
antioxidants, the experiment was aimed to examine whether dietary supplementation of resveratrol (a polyphenol)
may attenuate lipid peroxidation, the major end-point of oxidative
damage, liver problems and alcohol-induced
morality resulting from chronic alcohol administration. Three groups of experimental animals, namely, rat or mice
were used. The first group
served as control. The second and third group was daily injected
with 35% ethanol at 3
g/kg body weight or up to 40%v/v in drinking water. The third group was

supplemented with resveratrol

(5 g/kg) in
standard diet or 10mg/ml in drinking water. Malondialdehyde (MDA), an indicator

of oxidative stress, was
measured in the liver, heart, brain,

and testis. Also, blood levels were determined for transaminase and IL-1. At the
end of a 6 weeks treatment period,

MDA, transaminase and IL-1 levels were significantly increased in the liver,
heart, brain, and testis.

However, when alcohol treated animals were given resveratrol the
increase in MDA,
transaminase and IL-1 levels was significantly reduced to nearly those of control animals. Mortality in the third
group was 22% compared to 78% in the second group. Thus the results obtained shows that resveratrol is

able to
alleviate alcohol-induced liver problems and morality and have
protective effect against oxidative injury.
Keywords: IL-1, MDA, Lipid peroxidation, Oxidative stress, Polyphenols, Transaminase

Introduction
Alcohol Consumption
The average person metabolizes about 10 grams of
alcohol(1 standard drink) per hour. There are many
harmful effects of alcohol consumption. The short
term effects include behavioral or physical
abnormalities. Chronic alcohol consumption causes
adverse effects. Some of the common effects include
alcoholism, malabsorption, chronic pancreatitis, liver
cirrhosis, cancer. Cirrhosis is a condition in which the
liver slowly deteriorates and malfunctions due to
chronic injury. Scar tissue replaces healthy liver
tissue, partially blocking the flow of blood through
the liver.
It also causes damage to central nervous system,
peripheral nervous system. In short, alcohol in
excessive quantities is capable of damaging nearly
every organ and system in the body. It has been
found that intake of resveratrol helps reduce toxic
effects caused by excessive alcohol consumption

Resveratrol (3, 5, 4'-trihydroxy-trans-stilbene) is
a polyphenolic phytoalexin. Phytoalexins are
antimicrobial substances synthesized de novo by
plants that accumulate rapidly at areas of
incompatible pathogen infection. Phytoalexins
produced in plants act as toxins to the attacking
organism. Polyphenols are group of chemical
substances, found in plants, characterized by the
presence of more than one phenol unit.
Resveratrol is present in many plants and fruits,
including red grapes, eucalyptus, spruce,
blueberries, mulberries, peanuts, giant knotweed.
Also red wine contains a lot of it. The longer the
grape juice is fermented with the grape skins the
higher the resveratrol content will be.
Base unit Flavone Class/Polymer (Flavanoid)
Phenolic subcomponent Resorcinol
Eg: Resveratrol
Resveratrol is an antioxidant, posses anticancer
properties and inhibits lipid peroxidation of low-
density lipoprotein and prevents the cytotoxicity of
oxidized LDL. Resveratrol also increases the activity
of some antiretroviral drugs in vitro. They appear to
mimic several of the biochemical effects of calorie
restriction. Possess antioxidant, anticancer, antitoxic
properties, helps in increased lifespan and heart
health.
Experiment
Animals used for the experiment are either mice or
rats. Male Wistar variety were used in case of rats
and male Balb/c variety were used in case of mice,
each weighing about 26g.
The animals were divided into 3 groups of 12 animals
each. They were maintained on a regular 12-hour
light period at a controlled temperature (25 2 C),
with free access to food and water. The mice were
adapted for 2 to 5 days prior to initiation of the xperimental protocol. The diet consisted of 58.5%
carbohydrates, 15.5% proteins, 2.7% fat, 5.5%
minerals, 3.7% fiber and 12% humidity The caloric
contents were 3000 kcal/kg.
Experimental Procedure
First group of animals were taken as control.
In order to induce alcoholic intoxication, second
group was administered pure alcohol by diluting it in
the drinking water. 10% in the first week, 20% in the
second, 30% in the third and 40% in the subsequent
weeks until the end of the study.
Third group was also administered alcohol in the
same manner.In addition, resveratrol was also added
to the drinking water - 10mg in every 1ml. Liquids
and food were changed twice a week and the animals
were monitored daily for general health.
A treatment period of six weeks was given. The
timing of sacrifice for the study of liver damage was
determined from previous trials in which mortality
was seen to be very high after the sixth week. The
animals were sacrificed by decapitation. Few animals
from each group was allowed to live, destined for the
evaluation of mortality were followed until death in
the 7th
week.
The mortality and liver-damage studies were each
repeated on three occasions to confirm the
histological and laboratory alterations (12 mice per
group, for a total of 36 animals per group) and again
on three occasions to confirm the mortality curves
(12 mice per group, for a total of 36 animals per
group).
Animals from each group was taken and tested for
transaminase and IL-1 levels in blood.
MDA(Malondialdehyde) was tested in liver, heart,
brain and testis.
Laboratory Tests
Alcohol in blood was determined by REA assay a
quantitative reagent system for the measurement of
ethanol in murine whole blood.
Transminase (both AST & ALT) was determined on
a computer-controlled biochemical analyzer. It uses a
colored reaction scheme to detect transaminase
enzymatic activity in serum samples.
IL-1 was analyzed using ELIZA kits based on anti-
mouse TNF- monoclonal antibodies.
Extend of MDA production was determined by
TBARS Assay (Thiobarbituiric acid reactive
substances Assay). Thiobarbituric acid reacts with
malondialdehyde to yield a fluorescent product. This
test is indicative of lipid peroxidation in tissues.
Transaminase
Transaminase or Aminotransferase is an enzyme that
catalyzes reaction between amino acid and keto
acid. The reaction involves removing the amino
group from the amino acid, leaving behind an -keto
acid, and transferring it to the reactant -keto acid
and converting it into an amino acid. The reaction is
called Transamination. The presence of elevated
transaminases can be an indicator of liver damage.
Measuring the concentrations of various
transaminases in the blood is important in the
diagnosing and tracking many diseases. In this
experiment Aspartate transaminase (AST) & Alanine
transaminase (ALT) is considered.
Transaminases require the coenzyme pyridoxal-
phosphate, which is converted into pyridoxamine in
the first phase of the reaction, when an amino acid is
converted into a keto acid. Enzyme-bound
pyridoxamine in turn reacts
with pyruvate, oxaloacetate, or alpha-ketoglutarate,
giving alanine, aspartic acid, or glutamic acid,
respectively.
Interlukins
IL-1 refers to Interleukin-1, cytokines secreted by the
immune system. They are a group of three
polypeptides ( IL-1 , IL-1 , interleukin-1 receptor
antagonist (IL-1Ra) ). The term interleukin derives
from (inter-) "as a means of communication", and (-
leukin) "deriving from the fact that many of these
proteins are produced by leukocytes and act on
leukocytes". As the source of secretion suggests, they
play a central role in the regulation of immune and
inflammatory responses.
A peculiar behavior of interleukins is that, low levels
of interleukins helps liver to repair damage while
high levels can cause injury & death of liver cells.
Crystal structure of IL-1 shown in fig.
Lipid Peroxidation
In simple terms, lipid peroxidation is the oxidative
degradation of lipids. It is a process whereby free
radicals "steal" electrons from the lipids in cell
membranes, resulting in cell damage. The reaction
proceeds by a free radical, chain reaction mechanism.
It most often affects polyunsaturated fatty acids,
because they contain multiple double bonds in
between which lie methylene -CH2- groups that
possess especially reactive hydrogens. As with any
radical reaction the reaction consists of three major
steps: initiation, propagation and termination.
Initiation is the step whereby a fatty acid radical is
produced. The initiators in living cells are most
notably reactive oxygen species (ROS), such as OH
which combines with a hydrogen atom to make water
and a fatty acid radical. The fatty acid radical is not a
very stable molecule, so it reacts readily with
molecular oxygen, thereby creating a peroxyl-fatty
acid radical. This too is an unstable species that
reacts with another free fatty acid producing a
different fatty acid radical and a lipid peroxide or a
cyclic peroxide if it had reacted with itself. This cycle
continues as the new fatty acid radical reacts in the
same way. The radical reaction stops when two
radicals react and produce a non-radical species. This
happens only when the concentration of radical
species is high enough for there to be a high
probability of two radicals actually colliding.One
important such antioxidant is vitamin E. Other anti-
oxidants made within the body include the
enzymes superoxide dismutase, catalase,
and peroxidase.
If not terminated fast enough, there will be damage to
the cell membrane, which consists mainly of lipids.
In addition, end products of lipid peroxidation may
be mutagenic and carcinogenic.
For instance, the end product malondialdehyde reacts
with in DNA, deoxyadenosine & deoxyguanosine in
DNA forming DNA adducts to them, primarily M1G.
A DNA adduct is a piece of DNA covalently bonded
to a (cancer-causing) chemical. This has shown to be
the start of a cancerous cell, or carcinogenesis
MALONDIALDEHYDE (MDA)
Fig. showing chemical structure of MDA
Malondialdehyde is an organic compound with the
chemical formula CH2(CHO)2. The structure of this
species is more complex than this formula suggests.
This reactive species occurs naturally and is a marker
for oxidative stress. Reactive oxygen species degrade
polyunsaturated lipids, forming malondialdehyde.
This compound is a reactive aldehyde and is one of
the many reactive electrophile species that cause
toxic stress in cells and form covalent protein adducts
which are referred to as advanced lipoxidation end
products (ALE), in analogy to advanced glycation
end-products(AGE). The production of this aldehyde
is used as a biomarker to measure the level of
oxidative stress in an organism.
Results
The mortality curves were similar in all three test
series. The mice in the alcohol group began to die
after the second week of alcohol intoxication, with a
survival of 22% (4/18) in the seventh week. None of
the mice survived beyond eight weeks. The animals
belonging to the alcohol plus resveratrol group began
to die later (after the fourth week, involving a single
mouse), with a survival of 78% (14/18) in the seventh
week. The control group in turn presented a survival
of 100% (17/18) in the seventh week. Survival was
significantly lower in the alcohol group than in the
rest of groups. The mice subjected to alcohol
intoxication showed a poorer general condition after
the second week, as reflected by decreased activity,
immobility, grouping and coarse hair. There were no
such observations in the other two groups (control
and alcohol plus resveratrol), with no differences
among them. The average food intake among the
control rats was 4.27 0.86 g/day. In the experiment a
significant decrease was observed in food ingestion
and body weight among the alcohol-consuming mice.Fig. shows survival of the different groups of mice
over time (weeks)
In short, alcohol treated animals showed high levels
of transaminase & IL-1 levels in the blood stream.
They also indicated high amounts of MDA in the
liver, heart, brain and testis.
Alcohol treated animals administered with resveratrol
showed transaminase, IL-1 & MDA levels nearly as
low as the control.
Mortality rates as on the 7th
week in the resveratrol
plus ethanol administered animals was 22%
compared to 78% in the alcohol administered group.
Conclusion
From the results we can conclude:
Resveratrol is

able to alleviate alcohol-induced
liver problems and morality. They have
protective effect against oxidative injury.
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