The liver is the largest internal organ and plays a vital role in metabolism, detoxification, protein synthesis, and bile production. Hepatotoxicity refers to liver damage caused by chemicals, drugs, or toxins. Types of Hepatotoxicity are hepatocellular injury, cholestatic injury and mixed injury. In Vivo Screening (Animal Models) are : CCl₄-induced Hepatotoxicity Paracetamol-induced Hepatotoxicity Alcohol-induced Injury Thioacetamide-induced Fibrosis D-galactosamine Model

1. SCREENING MODELS FOR
HEPATOPROTECTIVE DRUGS
PREPARED BY: VAISHNAVI J. AWARE
M. PHARM (PHARMACOLOGY)
GUIDED BY: DR. A. V. KULKARNI
ASSIGNMENT SEMINAR FOR 50 MARKS
DR. D. Y. PATIL COLLEGE OF PHARMACY, AKURDI, PUNE 411044
2024-25

2. CONTENT
• LIVER ANATOMY
• HEPATOTOXICITY
• CLASSIFICATION OF HEPATOTOXICITY
• SCREENING MODELS OF HEPATOTOXICITY
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3. LIVER ANATOMY
• The liver is the largest internal organ and plays a vital role in metabolism,
detoxification, protein synthesis, and bile production.
STRUCTURE OF THE LIVER
• Lobes: Right and left lobes (larger) with caudate and quadrate lobes (smaller).
• Hepatic lobule: Functional unit of the liver, consisting of hepatocytes
arranged in plates around a central vein.
• Cells of the liver:
1. Hepatocytes – Main functional cells, responsible for metabolism and
detoxification
2. Kupffer cells – Macrophages involved in immune defense
3. Stellate cells – Store vit. A and contribute to fibrosis
4. Endothelial cells – Lining cells that regulate exchange between blood and
hepatocytes
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4. HEPATOTOXICITY
• Hepatotoxicity refers to liver damage caused by chemicals, drugs,
or toxins.
Causes Of Hepatotoxicity
• Drugs: Acetaminophen, NSAIDs, Antitubercular Drugs (Isoniazid,
Rifampicin).
• Alcohol: Chronic Alcohol Consumption Leads To Cirrhosis.
• Viruses: Hepatitis A, B, C, And E.
• Metabolic Disorders: Wilson’s Disease, Hemochromatosis.
• Herbal & Environmental Toxins: Aflatoxins, Pyrrolizidine
Alkaloids.
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5. Type Features
Hepatocellular Injury Damage to hepatocytes, elevated ALT & AST (e.g., viral hepatitis, drug-induced).
Cholestatic Injury Bile flow obstruction, elevated ALP (e.g., gallstones, primary biliary cholangitis).
Mixed Injury
Features of both hepatocellular and cholestatic damage (e.g., drug-induced liver
injury).
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Types of Hepatotoxicity

6. CLASSIFICATION OF HEPATOPROTECTIVE DRUGS
Natural Hepatoprotective Agents
• Flavonoids: Silymarin (Milk Thistle), Quercetin
• Terpenoids: Andrographolide (Andrographis
Paniculata)
• Polyphenols: Curcumin (Turmeric), Resveratrol
• Alkaloids: Berberine (Berberis Species)
• Glycosides: Glycyrrhizin (Licorice)
Synthetic Hepatoprotective Agents
• Antioxidants: N-acetylcysteine (NAC) For
Acetaminophen Toxicity
• Cytoprotective Agents: Ursodeoxycholic Acid
(UDCA)
• Enzyme Inducers: Phenobarbital For Improving
Liver Detoxification
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7. SCREENING METHODS FOR HEPATOPROTECTIVE
DRUGS
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8. IN VIVO SCREENING (ANIMAL MODELS)
• CCl -induced Hepatotoxicity
₄
• Paracetamol-induced Hepatotoxicity
• Alcohol-induced Injury
• Thioacetamide-induced Fibrosis
• D-galactosamine Model
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9. CCl -induced Hepatotoxicity
₄
• Carbon tetrachloride (CCl ) is one of the most commonly used hepatotoxins to induce liver damage in
₄
experimental studies. It mimics oxidative stress-mediated liver injury, making it ideal for evaluating
hepatoprotective drugs.
Equipments: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser.
Drugs: Liquid paraffin, Test drug, Carbon tetrachloride, 10% formalin, Silymarin.
Animals: Wistar Albino rats
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10. PRINCIPLE OF CCL -INDUCED
₄
HEPATOTOXICITY
• METABOLISM OF CCL IN THE LIVER:
₄
• CCl is metabolized in hepatocytes by cytochrome P450
₄
(CYP2E1) to generate trichloromethyl radical (CCl •).
₃
• This free radical interacts with lipids in hepatocyte
membranes, leading to lipid peroxidation and oxidative
stress.
• Results in cell membrane damage, inflammatory
responses, and hepatic necrosis.
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11. Group Treatment
Normal control Liquid paraffin (3ml/kg)
Inducer control Liquid paraffin (3ml/kg, s.c.) + CCl4 (1ml/kg, s.c.)
Test drug Test drug + CCl4 (1ml/kg, s.c.)
Reference standard Silymarin (100 mg/kg, p.o.) daily + CCl4 (1ml/kg, s.c.)
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12. PROCEDURE
• Weigh the animal and into groups and label them.
• Administer vehicle to normal control and inducer group, test group with test drug and silymarin
to the reference standard group for 1week.
• 60 minutes after the treatment with drugs administer CCl4 (1ml/kg, s.c.) In liquid paraffin.
• On 8th
day withdraw blood from retroorbital plexus and collect it in sterilized centrifuge tubes.
• Allow the blood to coagulate for 30 mins at room temperature and then separate the clear serum
by centrifugation at 2500 rpm for 10mins.
• Estimate the levels of markers in serum AST, ALT, SGPT, SGOT, ALP, TBL and CHL using
serum diagnostic and an autoanalyzer.
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13. • Thereafter, sacrifice the animals under anaesthesia then dissect them and take out liver, wash
with water, dry with filter paper and preserve in 10% formalin solution for histopathological
studies including cell necrosis, fatty change, hyaline degeneration, ballooning degeneration and
infiltration of Kupffer cells and lymphocytes.
• Compare the results of test drug and reference standard group against the results of inducer
control group using suitable statistical analysis method.
• Reduction in levels of various enzymes, as a marker of liver damage in rats of test drug as
compared to induced control group supported by positive results in histopathological
examination indicated hepatoprotective activity of test drug.
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14. Paracetamol-induced Hepatotoxicity
• Paracetamol-induced hepatotoxicity occurs when excessive doses of paracetamol
(acetaminophen) cause liver damage due to the formation of toxic metabolites. It is a major cause
of drug-induced liver injury (DILI), leading to acute liver failure (ALF) in severe cases.
Equipments: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser.
Drugs: Distilled water, Test drug, Paracetamol, 10% formalin, Silymarin.
Animals: Wistar Albino rats
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15. PRINCIPLE OF PARACETAMOL-INDUCED HEPATOTOXICITY
Metabolism in the Liver
•Paracetamol is primarily metabolized by glucuronidation (60%) and
sulfation (35%) pathways for safe elimination.
•A small fraction (5-10%) is converted by cytochrome P450
(CYP2E1) into N-acetyl-p-benzoquinone imine (NAPQI), a highly
reactive toxic metabolite.
Role of Glutathione (GSH)
•Under normal doses, NAPQI is detoxified by glutathione (GSH) and
excreted.
•In overdose, GSH stores are depleted, leading to the accumulation of
NAPQI, which binds to cellular proteins and causes oxidative stress
and hepatocyte necrosis.
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16. Group Treatment
Normal control Distilled water (1ml/kg, p.o.)
Inducer control Distilled water (1ml/kg, p.o.) + Paraetamol (500 mg/kg, p.o.)
Test drug Test drug + Paracetamol (500 mg/kg, p.o.)
Reference standard Silymarin (100 mg/kg, p.o.) daily + Paracetamol (500 mg/kg,
p.o.)
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17. PROCEDURE
• Weigh the animal and into groups and label them.
• Administer vehicle to normal control and inducer group, test group with test drug and silymarin to
the reference standard group for 15 days.
• 60 minutes after the treatment with drugs administer paracetamol (500 mg/kg, p.o.)
• In distilled water.
• On 16th
day withdraw blood from retroorbital plexus and collect it in sterilized centrifuge tubes.
• Allow the blood to coagulate for 30 mins at room temperature and then separate the clear serum
by centrifugation at 2500 rpm for 10mins.
• Estimate the levels of markers in serum AST, ALT, SGPT, SGOT, ALP, TBL and CHL using serum
diagnostic and an autoanalyzer.
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18. • Thereafter, sacrifice the animals under anaesthesia then dissect them and take out liver, wash with water,
dry with filter paper and preserve in 10% formalin solution for histopathological studies.
• Dehydrate the liver samples fixed for 48 hours in 10% formalin by passing in different mixtures of ethyl
alcohol-water. Clean the sample in xylene and embed them in paraffin.
• Prepare 4-5mm thick sections and then stain with hematoxylin and eosin dye for microscopic
observation of cell necrosis, fatty change, hyaline degeneration, ballooning degeneration and infiltration
of Kupffer cells and lymphocytes.
• Compare the results of test drug and reference standard group against the results of inducer control
group using suitable statistical analysis method.
• Reduction in levels of various enzymes, as a marker of liver damage in rats of test drug as compared to
induced control group supported by positive results in histopathological examination indicated
hepatoprotective activity of test drug.
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19. 19
Alcohol-induced hepatotoxicity
Alcohol-induced hepatotoxicity refers to liver damage caused by excessive alcohol consumption, primarily
due to the toxic effects of ethanol and its metabolites on liver cells. The main mechanism involves oxidative
stress, inflammation, and metabolic dysregulation, leading to liver injury, fibrosis, and cirrhosis.
Equipment: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser.
Drugs: Distilled water, Test drug, 30% Alcohol, 10% Formalin, Anaesthetic ether, Silymarin.
Animals: Wistar albino rats (150-200 gm)

20. 20
PRINCIPLE OF ALCOHOL-INDUCED HEPATOTOXICITY
• Alcohol-induced hepatotoxicity occurs due to the toxic effects of
ethanol and its metabolites on liver cells.
• Ethanol is metabolized into acetaldehyde, a highly reactive
compound, which, along with reactive oxygen species (ROS),
leads to oxidative stress, mitochondrial damage, and
inflammation.
• Chronic alcohol consumption disrupts lipid metabolism, causing
fatty liver (steatosis) and activates hepatic stellate cells, leading
to fibrosis and cirrhosis. This progressive liver damage impairs
liver function and may result in liver failure.

21. 21
Thioacetamide induced hepatotoxicity
Thioacetamide (TAA) is a hepatotoxic compound widely used in experimental models to induce liver injury.
Its toxicity is mediated through its metabolic activation in the liver, leading to reactive metabolites, oxidative
stress, mitochondrial dysfunction, inflammation, and fibrosis. TAA primarily affects hepatocytes by disrupting
cellular structures, causing necrosis, apoptosis, and ultimately leading to cirrhosis.
Equipment: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser.
Drugs: Distilled water, Test drug, Thioacetamide, 10% Formalin, Anaesthetic ether, Silymarin.
Animals: Wistar albino rats (150-200 gm)

22. 22
PRINCIPLE OF THIOACETAMIDE INDUCED HEPATOTOXICITY
• Thioacetamide (TAA) causes liver toxicity through its metabolic
activation in the liver. It is converted by cytochrome P450
enzymes (CYP2E1, CYP3A4) into reactive metabolites (TASO
and TASO ), which induce oxidative stress, mitochondrial
₂
dysfunction, and inflammation.
• This leads to hepatocyte necrosis, apoptosis, and activation of
hepatic stellate cells, resulting in fibrosis and cirrhosis. Chronic
exposure to TAA ultimately impairs liver function, leading to liver
failure.

23. 23
D-galactosamine induced hepatotoxicity
D-galactosamine (d-galn) induces hepatotoxicity by disrupting hepatic RNA and protein synthesis,
primarily due to the depletion of uridine triphosphate (UTP). This leads to mitochondrial
dysfunction, oxidative stress, inflammation, hepatocyte apoptosis, and necrosis, resulting in liver
damage similar to viral hepatitis.
Equipment: Centrifuge, Glass capillary, Serum diagnostic kit, Autoanalyser.
Drugs: Distilled water, Test drug, D-galactosamine, 10% Formalin, Anaesthetic ether, Silymarin.
Animals: Wistar albino rats (150-200 gm)

24. 24
PRINCIPLE OF D-GALACTOSAMINE INDUCED HEPATOTOXICITY
• D-galactosamine (d-galn) induces liver toxicity by depleting
uridine triphosphate (UTP), which inhibits RNA and protein
synthesis, leading to mitochondrial dysfunction, ATP depletion,
and oxidative stress.
• This triggers kupffer cell activation, inflammation (tnf-α, IL-6),
and hepatocyte apoptosis/necrosis, resulting in severe liver
injury resembling viral hepatitis.

25. REFERENCES
• Vyawahare N. S., Karathara V. G., Pujari R. R., “ Preclinical Screening Of Drugs”, Nirali Prakashan, Chapter 7, Page No.
7.13 – 7.22.
• Klaassen, C.D. (2019). "Casarett And Doull’s Toxicology: The Basic Science Of Poisons" (9th Ed.). Mcgraw-hill Education.
• Brunton, L.L., Knollmann, B.C., & Hilal-dandan, R. (2017). "Goodman & Gilman’s: The Pharmacological Basis Of
Therapeutics" (13th Ed.).
• Weber, L.W.D., Boll, M., & Stampfl, A. (2003). "Hepatotoxicity And Mechanism Of Action Of Carbon Tetrachloride
(CCl )."
₄ Toxicology, 189(1-2), 1-12.
• Jaeschke, H., Xie, Y., & Mcgill, M.R. (2014). "Acetaminophen-induced Liver Injury: From Animal Models To Humans."
Journal Of Clinical And Translational Research, 1(1), 5-17.
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26. THANK YOU !!!
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