Reduction Of Alcohol Intoxication In Experimental Animals By Resveratrol - Printable Version +- Free Academic Seminars And Projects Reports (https://easyreport.in) +-- Forum: Seminars Topics And Discussions (https://easyreport.in/forumdisplay.php?fid=30) +--- Forum: Engineering Seminars Topics (https://easyreport.in/forumdisplay.php?fid=7) +---- Forum: biotechnology/biomedical engineering seminar topics (https://easyreport.in/forumdisplay.php?fid=62) +---- Thread: Reduction Of Alcohol Intoxication In Experimental Animals By Resveratrol (/showthread.php?tid=1373) |
Reduction Of Alcohol Intoxication In Experimental Animals By Resveratrol - chaitanyaket88 - 08-16-2017 Reduction Of Alcohol Intoxication In Experimental Animals By Resveratrol
Parvathy S. Nair & Parvathy R. S8 Biotechnology & Biochemical Engineering Mohandas College of Engineering & Technology, Anad [attachment=10259] 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. References Borra MT, Smith BC, Denu JM. J Biol Chem, Mechanism of human SIRT1 activation by resveratrol, 280: 17187 17195(2007). Bujanda L, Garc a-Barcina M, Guti rrez-de Juan V, Bidaurrazaga J, de Luco MF, Guti rrez-Stampa M, Larzabal M, Hijona E, Sarasqueta C, Echenique-Elizondo M, Arenas JI. BMC Gastroenterol, Effect of resveratrol on alcohol-induced mortality and liver lesions in mice, 6: 1 9(2008). Kaeberlein M, McDonagh T, Heltweg B, Hixon J, Westman EA, Caldwell SD, Napper A, Curtis R, DiStefano PS, Fields S, Bedalov A, Kennedy BK. J Biol Chem, Substrate-specific activation of sirtuins by resveratrol, 280: 17038 17045(2006). Tilg H, Diehl AM: N Engl J Med, Cytokines in alcoholic and nonalcoholic steatohepatitis, 343:1467-1476(2006). Mart nez J, Moreno JJ: Biochem Pharmacol, Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production, 59:865- 870(2002). Bujanda L: Am J Gastroenterol, The effects of alcohol consumption upon the gastrointestinal tract, 95:3374-3382(2000). Yin M, Gabele E, Wheeler MD, Connor H, Bradford BU, Dikalova A, Rysyn I, Mason R, Thurman RG: Hepatology, Alcohol- induced free radicals in mice: direct toxicants or signaling molecules?, 34:935- 942(2001). Cai, Y. J., Fang, J. G., Yang, L. et al. Biochimica et Biophysica Acta, Inhibition of free radical-induced peroxidation of rat liver microsomes by resveratrol and its analogues, 1637, 31 38(2003). |