Protective Role of Isolated Rat Hepatocytes Against CCL4

Protective Role of Isolated Rat Hepatocytes Against CCL4DISCUSSIONCCl4, Paracetamol and Anti-tubercular drugs cause ROS mediated cellular molest specially in liver, the site of metabolism of these drugs. During the regular physiological functioning the cells/ winds/organs use group O and various nutrients to generate energy. The warrant radicals be also generated in this process as the re make upion intermediates. These free radicals may be very useful because they may promote beneficial oxidative processes. However the higher quantities of much(prenominal) radicals like superoxide anion (O2), NO radical, and hydroxyl ion radical (OH), NOO, etc. may interact with the membrane lipids ahead(p) to lipid per oxidization and attack the DNA resulting DNA marge breaks. The lipid peroxidation also vituperate cell membrane resulting in the leakage of enzymes into the blood stream. Therefore the elevated biochemical levels are treated as biochemical markers of tissue damage. In addit ion the extent of lipid peroxidation is directly proportional to the tissue damage Devasagayam et al., 2003.There are certain inbuilt protective mechanisms, tissue enzymes GSH, SOD, computerized tomography etc. which are involved in the process of combating free radical induce tissue damage. Over powering the inbuilt protective mechanism ascribable to excessive generation of free radicals may fail to destruction of the tissues/organs Tiwari, 2001.Antioxidants are the chemical constituents, which are used for subordinateing the tissue damage by countering the free radicals most of the antioxidants available in the markets are from natural origin e.g. vit-E, vit-C, tocopherol, quercetine, -carotene etc. In addition there are reports that polyphenolic compounds like flavonoids, tannins are useful as antioxidants and organ protectants. Therefore many researchers are attempting to screen the herbs and herbal preparations containing polyphenolic compounds for organ protective propert ies. Mruthunjaya, 2008 in his study reported the presence of very high heart and soul of polyphenols and gallic acid, a well known potent antioxidant Gow- chin et al, 2002 as an important constituent of the roots of a widely grown plant L. inermis. He also reported the antioxidant and in vitro and in vivo hepatoprotective activity of L. inermis root extract against CCl4 induced hepato cyanogenicity in rats. So in the present study L.inermis was selected for investigating the in vivo and in vitro hepatoprotective activity against drugs induced hepato perniciousness.Before screening the test extract for in vivo hepatoprotective activity the extract was interchangejected to the acute ototoxicity studies as per OECD guidelines 425 (limit test). The LIALC was found rubber eraser up to 2000 mg/kg as evident by the absence of mortality in the treated group. Hence, 1/10th (200 mg/kg) and 1/seventh (300 mg/kg) of the safe dose were selected for the in vivo study.In vitro hepatoprotective ScreeningIn the present study, isolated rat hepatocytes were used. The objective of this study was to confirm the protective region of test extracts against CCl4 induced hepatocytes damage.The CCl4 metabolized in (liver) endoplasmic reticulum and mitochondria with the governance of CCl3O (unstable complex radical), by CYT P-450. The nascent oxygen O resulted via lipoperoxidation causes rise in intracellular reactive Fe2+ ions, aldehyde and depletion of GSH, and atomic number 20 sequestration. Oxidative CCl3O, also by direct covalent interaction induces degeneration in Ca2+ sequestration. Failure into sequestration result in change magnitude intercellular Ca2+, assemblage by proteolytic enzymes and cause an increase in Fe2+ ions, which in turn by lipid peroxidation precipitates aldehyde cytotoxicity Zimmerman Hayman, 1976, Agarwal et. al., 1983.Due to this membrane integrity is lost ultimately leading to necrosis. The loss of membrane integrity can be measured by Trypan blue e xclusion assay in which the viable cells exclude the dye whereas assassinated cell take up the dye owing to the alteration in membrane permeability. Due to loss of membrane integrity cytosolic enzymes are leaked into media and the measurement of these cyto blood plasmatic enzymes in the media serves as ideal markers for evaluating the extent of hepatocyte injury and protection offered by extracts. From the results it was apparent that both LIALC and LIAQ protect the hepatocytes against CCl4 damage as evident by the nose candy of cell death and inhibition leakage of cytosolic enzymes. As discussed earlier this strong hepatoprotective activity of LIALC might be callable presence of polyphenols and gallic acid which are potent free radical scavenging agents and antioxidants Gow- chin et al, 2002In vivo hepatoprotective activityParacetamol (N-acetyl-p-aminophenol) is a widely used analgesic and antipyretic drug and is safe when used in therapeutic doses. However over dosage of Parace tamol is known to be hepatotoxic and nephrotoxic in man and in experimental animals Parmar et al., 1995. Paracetamol is a direct hepatotoxin i.e. insobriety is dose dependent and reproducible Tygstrup et al., 1996. Exposure of animals to higher doses produces centrilobular or massive hepatic necrosis followed by congestion and failure. The hepatic necrosis is associated with damage to sub cellular organelle including mitochondria. Thus the drug is used as a typical hepatotoxin to produce hepatic failure experimentally Sing et al., 1999.At lower doses, close to 80% of ingested Paracetamol is eliminated mainly as sulfate and glucoronide conjugates before oxidation and only 5% is oxidized by hepatic CYT P450(CYP2E1) to a highly reactive and toxic electrophile i.e. N-acetyl-p-benzoquineimine (NAPQI). After over dosage of Paracetamol the glucoronidation and sulfation routes become saturated and as a consequence, Paracetamol is increasingly metabolized into NAPQI Remirez et al., 1995. S emiquin 1 radical, one-electron reduction metabolite of NAPQI mediates the cytotoxic effects of NAPQI. Production of these toxic semiquinone radicals is catalyzed by the microsomal CYT P450 reductase. These semiquinone radicals, in turn, can bind directly with cellular macromolecules to produce toxicity or alternatively, the radical can be reoxidized back to their original quinones by donating one electron to molecular oxygen under aerobic conditions. This donation of one electron then generates reduced oxygen radical species and hydroxyl radical. Both semiquinone and oxygen radical are known to be responsible for cytotoxic effects detect with quinones.Also NAPQI is detoxified by glutathione (GSH) to form 3-(GSH-S-yl) acetaminophen. Paracetamol overdose saturates the nontoxic metabolic pathway, i.e. sulfation, glucuronidation, and detoxification of NAPQI by glutathione. The reactive NAPQI may oxidize and arylate cysteinyl thiol group, forming adducts which inhibit the function of c ellular proteins. Adducts formation has been demonstrated for a selenium-binding protein, for microsomal subunit of glutamine. Other mechanism, such as oxidation of pyridine nucleotides and lipid peroxidation, may contribute to cell damage by Paracetamol overdose Tygstrup et al., 1996.Nevertheless at high doses of Paracetamol, NAPQI can alkylate and oxidize intracellular GSH and protein thiol group, which result in the liver GSH pool depletion and the reactive intermediate reacts with other nucleophilic centers of vital molecules in liver cells leading subsequently to hepatotoxicity. In addition Paracetamol is also shown to directly inhibit cellular proliferation, induce oxidative stress, resulting in lipid peroxidation, deplete ATP levels and alter Ca++ homeostasis all of these changes are considered potentially fatal to the cell Sing,. 1999 and Remirez et al., 1995.Anti-tuberculosis drugs act as inducers of hepatic CYT P450 enzymes. For example, Rifampicin is a potent inducer of C YP2D6 and CYP3A4, and Isoniazid induces CYP2E1 Trevor, 2004 and Vuilleumier et al., 2006. The induction of CYT P450 enzymes is known to take part in increased drug disposition and knowledge of multi-drug resistance. Xenobiotics, including anti-tubercular drugs, undergo biotransformation in the liver catalyzed by microsomal enzyme systems Bradford, 2005 Dai et al., 1995and Jeong, 1995. Because of above reasons, Paracetamol and anti-tubercular drugs cause ROS mediated hepatotoxicity and elevate blood serum enzymes levels like SGOT, SGPT, ALP, LDH and Bilirubin content in animals sideline administration of anti-tubercular drugs or toxic dose of Paracetamol. Also these drugs cause depletion of endogenous antioxidant enzymes like GSH, SOD, toss etc and abnormal levels of lipid peroxidation products.The results obtained in the present are in accordance with understanding as discussed above. i.e the serum enzymes levels like SGOT, SGPT, ALP, LDH and Bilirubin contents were significantl y increased, GSH, SOD, cuckoo levels in liver depleted and abnormal levels of lipid peroxidation was observed in Paracetamol and anti-tubercular drugs treated animals.Estimation of serum enzymesSGPT is a cytosolic enzyme primarily present in the liver. The level of SGPT in serum increases due to leakage of this cellular enzyme into plasma by hepatic injury Chenoweth Hake, 1962. Serum levels of SGPT can increase due to damage of the tissues producing acute hepatic necrosis, such as viral hepatitis and acute cholestasis. waterspout liver damage and cirrhosis also can associate with mild to moderate elevation of transaminase Harsh Mohan, 2002. SGOT is a mitochondrial enzyme released from heart, liver, skeletal ponderousness and kidney. Liver toxicity elevated the SGOT level in serum due to the damage to the tissues producing acute necrosis, such as severe viral hepatitis acute cholestasis. Alcoholic liver damage and cirrhosis can also associate with mild to moderate elevation of t ransaminases Harsh Mohan, 2002. In case of toxic liver, ALP levels are very high, which may be due to defective hepatic emptying or by increased production of ALP by hepatic parenchymal or duct cells Harsh Mohan, 2002. LIALC reversed these enzyme levels indicating stabilization of cell membrane by preventing the damage due to free radicals generated by Paracetamol and anti-tubercular drugs.Estimation of bilirubinIn case of toxic liver, bilirubin levels are elevated. Hyperbilirubinemia can result from impaired hepatic uptake of unconjugated bilirubin. Such a situation can occur in generalized liver cell injury. Certain drugs (e.g., Rifampin and Probenecid) interfere with the final uptake of bilirubin by the liver cell and may produce a mild unconjugated hyperbilirubinemia Rubin, 1995. Bilirubin level rises in diseases of hepatocytes, obstruction to biliary excretion into duodenum, in haemolysis and defects of hepatic uptake and conjugation of bilirubin pigment such as in Gilberts disease Harsh mohan, 2002. Significant lapsing of elevated bilirubin level in Paracetamol and anti-tubercular drug treated animals by LIALC indicated the strong hepatoprotective activity of LIALC.Estimation of ProteinA reduction in total serum protein observed in the Paracetamol and anti-tubercular treated control rats may be associated with the decrease in the number of hepatocytes which in turn might result in lessen hepatic capacity to synthesize protein. But, when the LIALC was administered along with Paracetamol or anti-tubercular drugs a significant increase in protein content was observed indicating the hepatoprotection of LIALC.Estimation of endogenous antioxidant enzymes and Lipid PeroxidationCells arouse a number of mechanisms to protect themselves from the toxic effects of the ROS. SOD removes superoxide (O2) by converting it to H2O2, which can be rapidly converted to water by CAT and Glutathione peroxidase. In addition, a large reserve of reduced glutathione is presen t in hepatocytes and red blood cells for detoxification of xenobiotics or free radicals. However, oxidative stress results in toxicity when the rate of which the ROS are generated exceeds the cell capacity for their removal. Lipid peroxidation is an autocatalytic process, which is a common consequence of cell death. This process may cause peroxidative tissue damage in inflammation, cancer and toxicity of xenobiotics and aging. MDA is one of the end products in the lipid peroxidation process. In order to elucidate the protection mechanism of LIALC in Paracetamol and anti- tubercular Drugs induced rat liver, lipid peroxide levels and anti-oxidative enzymes activities were analyzed.GSH is widely distributed in cells. GSH is an intracellular reductant and plays a major role in catalysis, metabolism and transport. It protects cells against free radicals, peroxides and other toxic compounds. GSH is a naturally occurring substance that is abundant in many living creatures. It is well known that a deficiency of GSH within living organisms can lead to tissue disorders and injury. For example, liver injury included by consuming alcohol or by taking drugs like Paracetamol, lung injury by smoking and muscle injury by intense physical activity, all are known to be correlated with low tissue level of GSH.The SOD converts super-oxide radicals (O2_) into H2O2 plus O2, thusly participating in the enzymatic defense against oxygen toxicity. In this study, SOD plays an important role in the elimination of ROS derived from the peroxidative process of xenobiotics in liver tissues.CAT is the key component of the anti-oxidant defense system .Inhibition of this protective mechanisms result in enhanced sensitivity to free radical induced cellular damage. Administration of LIALC increased the activity of catalase in Paracetamol and anti-tubercular drug induced liver damage to prevent the accumulation of excessive free radicals and thus exhibited protection against Paracetamol and anti- tubercular drug.The observed increase of SOD, CAT and GSH activity in liver suggests that the LIALC extract have an efficient protective mechanism in response to ROS. And also, these findings indicate that LIALC may be associated with decreased oxidative stress and free radical mediated tissue damage.In our study, elevation in the levels of end products of lipid peroxidation in liver of rat treated with Paracetamol and anti- tubercular drugs were observed. The increase in MDA level in liver suggests enhanced lipid peroxidation leading to tissue damage and failure of anti-oxidant defense mechanism to prevent formation of excessive free radicals. Treatment with LIALC significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection of LIALC is due to its antioxidant effect.Dept of pharmacology, P.R.R.M college of pharmacy,kadapaPage 1