Screening Models For Hepatoprotective Agents
By - 03/09/2007
Meera Sumanth
Hepatoprotective agents are those compounds, which mitigate the liver injury caused by hepatotoxic agents. 1 Hepatoprotective effects of plant drugs and herbal formulations are studied against chemicals(alcohol, CCl 4 , alcohol-CCl 4 , beta galactosamine, thioacetamide) and drugs(paracetamol, nimusalide, antitubercular drugs like isoniazid, rifampicin etc.) induced hepatotoxicity in rats and mice as they virtually mimic any form of naturally-occurring liver disease.
Hepatotoxicity from drugs and chemicals is the commonest form of iatrogenic disease. Some of the inorganic compounds producing hepatotoxicity are arsenic, phosphorus, copper and iron. The organic agents include certain naturally-occurring plant toxins such as pyrrolizidine alkaloids, mycotoxins and bacterial toxins. The synthetic group of organic compounds is a large number of medicinal agents. In addition, exposure to hepatotoxic compounds may be occupational, environmental or domestic that could be accidental, homicidal or suicidal ingestion. 2 The paper discusses various models used for screening hepatoprotective drugs.
CCl 4 induced hepatotoxicity
Liver injury due to carbontetrachloride in rats was first reported in1936 3 and has been widely and successfully used by many investigators. 4,5 Carbontetrachloride is metabolized by cytochrome P-450 in endoplasmic reticulum and mitochondria with the formation of CCl 3 O - , a reactive oxidative free radical, which initiates lipid peroxidation. 6,7
Administration of a single dose of CCl 4 to a rat produces, within 24 hrs, a centrilobular necrosis and fatty changes. 3 The poison reaches its maximum concentration in the liver within 3 hrs of administration. Thereafter, the level falls and by 24 hrs there is no CCl 4 left in the liver. 8 The development of necrosis is associated with leakage of hepatic enzymes into serum. Dose of CCl 4 : 0.1 to 3 ml/kg I.P.
Galactosamine induced hepatotoxicity
D-Galactosamine induced liver damage has been extensively used as an experimental model. Galactosamine produces diffuse type of liver injury simulating viral hepatitis. It presumably disrupts the synthesis of essential uridylate nucleotides resulting in organelle injury and ultimately cell death. Depletion of those nucleotides would impede the normal synthesis of RNA and consequently would produce a decline in protein synthesis. This mechanism of toxicity brings about an increase in cell membrane permeability leading to enzyme leakage and eventually cell death. The cholestasis caused by galactosamine may be from its damaging effects on bile ducts or ductules or canalicular membrane of hepatocytes Galactosamine decrease the bile flow and it’s content i.e. bile salts, cholic acid and deoxycholic acid. Galactosamine reduces the number of viable hepatocytes as well as rate of oxygen consumption. Dose of D-Galactosamine: 400 mg/kg, I.P. 9
Thioacetamide induced hepatotoxicity
Thioacetamide interferes with the movement of RNA from the nucleus to cytoplasm which may cause membrane injury. A metabolite of thioacetamide (perhaps s-oxide) is responsible for hepatic injury. Thioacetamide reduce the number of viable hepatocytes as well as rate of oxygen consumption. It also decreases the volume of bile and it’s content i.e. bile salts, cholic acid and deoxycholic acid. Dose of thioacetamide:100 mg/kg, S.C. 9
Alcohol induced hepatotoxicity
Liver is among the organs most susceptible to the toxic effects of ethanol. Alcohol consumption is known to cause fatty infiltration, hepatitis and cirrhosis. Fat infiltration is a reversible phenomenon that occurs when alcohol replaces fatty acids in the mitochondria. Hepatitis and cirrhosis may occur because of enhanced lipid peroxidative reaction during the microsomal metabolism of ethanol. It is generally accepted that alcohol can induce in vivo changes in membrane lipid composition and fluidity, which may eventually affect cellular functions. Among the mechanisms responsible for effects of alcohol, an increase in hepatic lipid peroxidation leads to alteration in membrane phospholipid composition. The effects of ethanol have been suggested to be a result of the enhanced generation of oxyfree radicals during its oxidation in liver. The peroxidation of membrane lipids results in loss of membrane structure and integrity. This results in elevated levels of ¡-glutamyl transpeptidase, a membrane bound enzyme in serum. Ethanol inhibits glutathione peroxidase, decrease the activity of catalase, superoxide dismutase, along with increase in levels of glutathione in liver. The decrease in activity of antioxidant enzymes superoxide dismutase, glutathione peroxidase are speculated to be due to the damaging effects of free radicals produced following ethanol exposure or alternatively could be due to a direct effect of acetaldehyde, formed by oxidation of ethanol. 10 Alcohol pre-treatment stimulates the toxicity of CCl 4 due to increased production of toxic reactive metabolites of CCl 4 , namely trichloro-methyl radical by the microsomal mixed function oxidative system. This activated radical binds covalently to the macromolecules and induces peoxidative degradation of membrane lipids of endoplasmic reticulum rich in polyunsaturated fatty acids. This lipid peroxidative degradation of biomembranes is the principle cause of hepatotoxity. 11
Paracetamol induced hepatotoxicity
Paracetamol, a widely used analgesic and antipyretic drug, produces acute liver damage in high doses. Paracetamol administration causes necrosis of the centrilobular hepatocytes characterized by nuclear pyknosis and eosinophilic cytoplasm followed by large excessive hepatic lesion. The covalent binding of N-acetyl-P-benzoquinoneimine, an oxidative product of paracetamol to sulphydryl groups of protein, result in lipid peroxidative degradation of glutathione level and thereby, produces cell necrosis in the liver. Dose of Paracetamol: 1 gm/kg P.O. 11, 4
Antitubercular drugs induced hepatotoxicity
Drug induced hepatotoxicity is a potentially serious adverse effect of the currently used antitubercular therapeutic regimens containing Isoniazid (INH), Rifampicin and Pyrazinamide. Adverse effects of antitubercular therapy are sometimes potentiated by multiple drug regimen. Thus, though INH, Rifampicin and Pyrazinamide each in itself are potentially hepatotoxic, when given in combination, their toxic effect is enhanced. INH is metabolized to monoacetyl hydrazine, which is further metabolized to a toxic product by cytochrome P 450 leading to hepatotoxicity. Patients on concurrent rifampicin therapy have an increased incidence of hepatitis. This has been postulated due to rifampicin-induced cytochrome P 450 enzyme-induction, causing an increased production of the toxic metabolites from acetyl hydrazine (AcHz). Rifampicin also increases the metabolism of INH to isonicotinic acid and hydrazine, both of which are hepatotoxic. The plasma half life of AcHz (metabolite of INH) is shortened by rifampicin and AcHz is quickly converted to its active metabolites by increasing the oxidative elimination rate of AcHz, which is related to the higher incidence of liver necrosis caused by INH and rifampicin in combination. Rifampicin induces hydrolysis pathway of INH metabolism into the hepatotoxic metabolite hydrazine. Pharmacokinetic interactions exist between rifampicin and pyrazinamide in tuberculosis patients, when these drugs are administered concomitantly. Pyrazinamide decrease the blood level of rifampicin by decreasing its bioavailability and increasing its clearance. Pyrazinamide, in combination with INH and rifampicin, appears to be associated with an increased incidence of hepatotoxicity. 12
Models to Evaluate Effect of Drugs on Liver
As liver is a multifunctional organ, a battery of liver function tests are employed to evaluate the effect of drug on liver, which are Non-invasive functional methods:
1. Ascorbic acid content in urine
2. Pentobarbitone induced sleeping time
3. Bromosulphthaline clearance test
Biochemical analysis of blood for
a.SGPT
b.SGOT
c.Alkaline phosphatase
d.Serum bilirubin
e.Total proteins
Morphological test-Wet weight of liver/100 gm body weight
Free radical scavengers
a.Glutathione
b.Lipid peroxidation
c.Superoxide dismutase
d.Catalase
f.Glutathione peroxidase
g.Histopathology of liver
Ascorbic acid content in urine
Measurment of ascorbic acid content of urine is reported as a non-invasive test for screening hepatoprotective agents against CCl 4 induced hepatotoxicity in rats. 13 CCl 4, a pharmacological tool to produce liver damage, reduces the excretion of ascorbic acid in rats.
Hexobarbitone or zoxazolamine induced sleeping time
Toxic liver prolongs duration of sleeping time for pentobarbitone, hexobarbitone, zoxazolamine etc in mice, rats.
Bromosulphthaline clearance test
The liver normally clears bromosulphthalein (BSP), a dye, from the blood. The level of BSP in the blood after intravenous injection of BSP is a sensitive guide to hepatic damage. During the passage of BSP from the plasma to the bile, it undergoes storage, metabolism and excretion by the liver. The abnormal functional effects produced by CCl 4 leads to the retention of BSP in blood. 14
Serum and hepatocyte enzyme
AST i.e. Aspartate Transaminase (SGOT), and ALT i.e. Alanine Transaminase (SGPT), are both sensitive markers of hepatocellular injury. When the liver cell is injured or dies, these proteins can leak through the liver cell membrane into the circulation and serum levels will rise. ALT or SGPT is a cytosolic enzyme primarily present in the liver. Its normal serum level is 10-35 Karmel units/ml. ALT reversibly catalyses amino group from alanine to α-ketoglutarate.
ALT levels are very high in patients of viral hepatitis and hepatic necrosis, 10 to 200 fold higher in patients of post hepatic jaundice, intrahepatic cholestasis and below 10 fold in patients of metastatic carcinoma, cirrhosis and alcoholic hepatitis.
AST or SGOT is a mitochondrial enzyme released from heart, liver, skeletal muscles and kidney. Its normal serum level is 10-40 Karmel units/ml 2 AST reversibly catalyses transfer of amino group from aspartate to α-ketoglutarate.
AST levels are 10 to 200-fold elevated in patients with acute hepatic necrosis, viral hepatitis, CCl 4 and drug induced poisoning.
Alkaline phosphatase
Serum alkaline phosphatase is produced by many tissues, especially bone, liver, intestine and placenta and is excreted in the bile. In the absence of bone disease and pregnancy, an elevated serum alkaline phosphatase levels generally reflect hepatobiliary disease. The mechanism of elevated ALP levels may be due to defective hepatic excretion or by increased production of ALP by hepatic parenchymal or duct cells. Principle involved in estimation of alkaline phosphatase:
ALP hydrolyses substrate P-nitrophenyl phosphate with the formation of P-nitrophenol and liberation of phosphate ion. 15
Serum Bilirubin
Estimation of bilirubin, metabolic product of the break down of heme is one of the better liver function tests. Normally, 0.25 mg/dl of conjugated bilirubin is present in the blood of an adult. Bilirubin level rises in diseases of hepatocytes, obstruction to biliary excretion into duodenum, in hemolysis and defects of hepatic uptake and conjugation of bilirubin treatment such as Gilberts disease. 2
Bilirubin in serum reacts with diazo reagent in the presence of alcohol, after the proteins had been removed by precipitation. 16
Serum Protein
Liver cells synthesize albumin, fibrinogen, prothrombin, alpha-1-antitrypsin, haptoglobin, ceruloplasmin, transferrin, alpha foetoproteins and acute phase reactant proteins. The blood levels of these plasma proteins are decreased in extensive liver damage. 2
Morphological parameters
Morphological parameters like weight of the animals, weight and volume of the liver have also been used to evaluate the protective effect of the drug. Hepatotoxicity causes loss in liver weight/100 gm body weight of rats. 17,18
Hepatocyte Viability and Oxygen Uptake tests
Hepatotoxicants reduce the viability of hepatic cells as assessed by trypan blue exclusion and oxygen uptake tests. 9 In liver, CCl 4 is metabolised to CCl 3 O - by cytochrome P-450 and the reactive oxidative free radical intermediate generated, O - causes further damage. Utilisation of oxygen by hepatocytes gets reduced; therefore the viability of hepatocytes is reduced. 6
Free Radical Scavenging
Free radicals are reactive molecules involved in many physiological processes and human diseases such as cancer, aging, arthritis, Parkinson syndrome, ischaemia, toxin induced reaction, alcoholism, liver injury etc. The damage to hepatic parenchymal cells, leading to hepatic injury, is due to oxidative stress within the cells caused by partially reduced free oxygen (PRFO) species such as O 2 (Superoxide anion), H 2 , O 2 , and OH (hydroxy free radical). The elevation of free radical levels seen during the liver damage is due to enhanced production of free radicals and decreased scavenging potential of the cells. A variety of intrinsic antioxidants (reduced glutathione, superoxide dismutase, glutathione-S-transferase etc.) are present in the organism, which protect them from oxidative stress.
Technically, the estimation of free radicals directly is not possible due to the transient nature of the free radicals. Thus estimations are usually done indirectly by measuring the “Antioxidant defense status” of the liver microsomes. Hepatoprotection by enzymatic free quenching is brought about by elevating the levels of antioxidant enzymes in tissues such as the Superoxide dismutase (SOD), Peroxidase and Catalase.
Figure 1: Mechanism of generation of free radicals.
Free radical generation:
The three partially reduced intermediate species between O 2 to H 2 O are derived from enzymatic and nonenzymatic reaction as under:
1.Superoxide (O 2 - ): superoxide anion O 2 - may be generated by direct autooxidation of O 2 during mitochondrial electron transport reaction. Alternatively O 2 is produced enzymatically by xanthine oxidase and cytochrome P 450 in the mitochondria or cytosol. O 2 so formed is catabolised to produce H 2 O 2 by superoxide dismutase.
2.Hydrogen peroxide (H 2 O 2 ): H 2 O 2 is reduced to water enzymatically by catalase (in the peroxisomes) and glutathione peroxidase GSH (both in the cytosol and mitochondria).
3.Hydroxyl radical: OH - radical is formed by two ways in biologic processes-by radiolysis of water and by reaction of H 2 O 2 with ferrous (Fe ++ ) ions, the latter process is termed as Fenton reaction.
Free radicals may produce membrane damage by the following mechanisms:
1.Lipid peroxidation: Polyunsaturated fatty acids (PUFA) of membrane are attacked repeatedly and severely by oxygen –derived free radicals to yield highly destructive PUFA radicals – lipid hydroperoxy radicals and lipid hydroperoxidation. The lipid peroxidase is decomposed by transition metals such as iron. Lipid peroxidation is propagated to other sites causing widespread membrane damage and destruction of organelles.
2.Oxidation of proteins: Oxygen- derived free radicals cause cell injury by oxidation of protein macromolecules of the cells, cross linking of labile amino acids as well as by fragmentation of polypeptides directly. The end result is degradation of cytosolic neutral proteases and cell destruction.
3.DNA damage: Free radicals cause breaks in the single strands of the nuclear and mitochondrial DNA. This results in cell injury; it may also cause malignant transformation of cells.
4.Cytoskeletal damage: Reactive oxygen species are also known to interact with cytoskeletal elements and interfere in mitochondrial aerobic phosphorylation and thus cause ATP depletion.
Superoxide dismutase (SOD):
SOD is a ubiquitous cellular enzyme, which dismutates superoxide radical to hydrogen peroxide and oxygen. 19 Dismutation is a reaction in which a single reactant is converted into two different products. Superoxide dismutase, one of the chief cellular defense mechanisms, scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen.
Catalase
The hydrogen peroxide formed by superoxide dismutase and other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. 20
Glutathione (GSH)
GSH is a tripeptide of glycine, glutamic acid and cysteine. Glutathione is an important naturally occurring antioxidant as it prevents the hydrogen of sulfydryl group to be abstracted instead of methylene hydrogen of unsaturated lipids. Therefore, levels of glutathione are of critical importance in tissue injury caused by toxic substances. The antioxidant enzymes superoxide dismutase and glutathione form the first line of defense against free radical induced damage, offer protection against free radicals and thereby, maintain low levels of lipid peroxides. 10 The primary biological function of glutathione is to act as a non-enzymatic reducing agent to help keep cysteine thiol side chains in a reduced state on the surface of proteins. Glutathione is also used to prevent oxidative stress in most cells and helps to trap free radicals that can damage DNA and RNA.
Lipid peroxidation
Lipid peroxidation can be defined as the oxidative deterioration of lipids. Lipid hydroperoxides are non-radical intermediates derived from unsaturated fatty acids, phospholipids, glycolipids, cholesterol esters and cholesterol itself. These are formed in enzymatic or non-enzymatic reactions involving free radical. 21
Glutathione peroxidase
Glutathione peroxidase (GPx) is an enzyme which catalyzes the reduction of hydroperoxides, including hydrogen peroxides, and functions to protect the cell from peroxidative damage. It is a seleniumcontaining enzyme and reduces H 2 O 2 to H 2 O by oxidizing glutathione (GSH) Rereduction of the oxidized form of glutathione (GSSG) is then catalysed by glutathione reductase
GPx activity can be measured indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione (GSSG) produced upon reduction of an organic hydroperoxide by GPx, is recycled to its reduced state by glutathione reductase and NADPH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the A340 is directly proportional to the GPx activity in the sample. 22
Conclusion
Thus, estimation of ascorbic acid content in urine, bromosulphthaline clearance from blood, SGPT, SGOT, alkaline phosphatase, serum bilirubin, total proteins, wet weight of liver/100 gm body weight, and hepatic lipid peroxidation, superoxide dismutase, glutathione, catalase and glutathione peroxidase are the suitable models for studying hepatoprotective drugs.
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About the Author
Meera Sumanth
Dr Meera Sumanth, M.Pharm., Ph.D. from Dr H S Gour University,Sagar, M.P. has 21 years of academic experience. She has guided 15 M. Pharm.students. She has presented research papersin national and international conferences. Presently, She is Professor and Head, Dept of Pharmacology, Visweswarpura Institute of Pharmaceutical Sciences , Bangalore , India .