CHAPTER ONE

INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The liver is a vital organ that plays a crucial role in the maintenance of overall health and well-

being. The liver is an essential organ in the human body, located in the right upper quadrant of

the abdomen. It performs a wide range of functions that are vital for the body's proper f

unctioning, including metabolism, detoxification, storage, and secreti88on (Kalra et al., 2022).

The liver is the largest solid organ in the body, and its importance cannot be overstated. The liver

is a complex organ that consists of two lobes, the right and the left, separated by the falciform

ligament. The liver is supplied with blood through the hepatic artery and the portal vein, which

brings blood from the gastrointestinal tract. The liver has a unique blood supply system, which

allows it to filter and detoxify the blood before it goes to the rest of the body. The liver is also

surrounded by a fibrous capsule, and its outer surface has a smooth, shiny appearance. The liver

is composed of different types of cells, including hepatocytes, Kupffer cells, and stellate cells,

which perform different functions of metabolism, detoxification, storage, and secretion. The liver

is responsible for processing and breaking down nutrients such as proteins, carbohydrates, and

fats, which are essential for energy production.

The liver also plays a crucial role in the detoxification process, removing harmful substances

from the body, such as alcohol, drugs, and toxins. The liver stores essential vitamins and

1
minerals, such as iron and Vitamin B12, and secretes bile, which aids in fat digestion. The liver is

susceptible to numerous diseases, ranging from mild to severe, which can significantly impact its

functions. Liver damage can occur as a result of various factors, including viral infection, alcohol

consumption and drug toxicity (Melaram, 2021). Liver damage is a significant global health

concerns with millions of people affected each year and high mortality rates associated with

severe cases or disorders such as cirrhosis and hepatocellular carcinoma (Cheemerla, 2021).

Liver damage is characterized by destruction of liver cells, inflammation, and the accumulation

of fat in the liver. The symptoms of liver damage include fatigue, abdominal pain, jaundice, and

nausea. Various biochemical parameters, such as serum transaminase levels, bilirubin, and

albumin levels, can be used to diagnose and assess the extent of liver tissue can also provide

valuable insights into the severity and extent of liver damage. The pathogenesis of liver damage

is complex and involves various cellular and molecular mechanism, such as oxidative stress,

inflammation, and apoptosis. Oxidative stress, in particular is a key factor in the development of

liver damage and is caused by an imbalance between the production of reactive oxygen species

(ROS) and the antioxidant defense system (Jindal and Pilkhua, 2013). One of the primary causes

of liver damage is excessive alcohol consumption. Alcohol can cause inflammation and damage

to liver cells, leading to alcoholic fatty liver disease, alcoholic hepatitis, and cirrhosis. Other

causes of liver damage include viral hepatitis, non-alcoholic fatty liver disease, autoimmune

hepatitis, and drug-induced liver damage. Certain medications and supplements can also cause

2
liver damage, such as acetaminophen (Tylenol) when taken in high doses, isoniazid (a

medication used to treat tuberculosis), Isoprenaline (a medication used to treat several

cardiovascular disorders) and herbal supplements like kava and comfrey.

Drug-induced liver injury (DILI) is a significant public health problem that has been associated

with significant morbidity and mortality rates. DILI is defined as liver damage caused by the use

of medications or other chemical agents and is a leading cause of acute liver failure (ALF) in the

United States. The incidence of DILI is difficult to estimate due to the lack of a standardized

definition. However, studies suggest that DILI accounts for approximately 10% of all cases of

acute hepatitis, and up to 50% of cases of ALF in the United States (Alempijevic et al., 2017).

DILI also appears to be more common in certain populations, such as women, older individuals,

and those with pre-existing liver disease. DILI varies across different populations and

geographic regions. In the United States, DILI is responsible for approximately 2% of all

hospital admissions, with an estimated incidence of 10-15 cases per 10,000 patient-years (Abid,

2022). The pathogenesis of DILI is complex and not yet fully understood. The liver is

responsible for metabolizing drugs and other xenobiotics, and this process can result in the

formation of preactive metabolites that can be toxic to the liver. These reactive metabolites can

disrupt cellular function and lead to oxidative stress, inflammation, and cell death (Marina et al.,

2021).The severity and type of liver damage caused by DILI can vary depending on the drug and

the individual's susceptibility to liver injury. Some drugs, such as acetaminophen, can cause rapid
3
and severe liver damage, while others, such as statins, may cause more mild and chronic damage

over time. DILI can result from direct toxicity or immune-mediated reactions (Stefan and

Hamilton, 2011).

Direct toxicity occurs when a drug or its metabolites directly damage liver cells. In some cases,

the drug or its metabolites may form reactive intermediates that can bind to cellular

macromolecules, resulting in oxidative stress, inflammation, and cell death. Immune-mediated

reactions are thought to occur when a drug or its metabolites bind to liver proteins and trigger an

immune response. This can result in the recruitment and activation of immune cells, such as T

cells and natural killer cells, leading to liver inflammation and damage. The type and severity of

liver damage caused by DILI can vary depending on the drug, the dose, and individual factors

such as age, sex, and underlying liver disease. In some cases, DILI may result in acute liver

failure, which can be life-threatening and require urgent medical intervention. Several factors

have been identified as potential risk factors for DILI which includes age, sex, genetics,

concurrent use of multiple medications (Nilesh et al., 2022). The diagnosis of DILI can be

challenging due to the lack of a definitive diagnostic test. The diagnosis is usually made based on

a combination of clinical, laboratory, and histological findings. The diagnosis of DILI is typically

considered when there is a history of exposure to a potentially hepatotoxic agent, and there is

evidence of liver injury, such as elevated liver enzymes or bilirubin levels. Other possible causes

of liver injury, such as viral hepatitis or autoimmune liver disease, must be ruled out before
4
making a definitive diagnosis of DILI. Liver biopsy may be used to confirm the diagnosis and

assess the severity of liver damage. The management of DILI depends on the severity of liver

damage and the cause of the injury. Treatment may involve the withdrawal of the offending drug,

supportive care, and the use of medications to manage symptoms and prevent further liver

damage. In severe cases of DILI, such as those leading to ALF, liver transplantation may be

necessary. Therefore, the development of effective preventive and therapeutic strategies for liver

damage is essential.

Paracetamol was first developed in 1878 from phenacetin and became widespread in the 1950s

as an over-the-counter antipyretic and analgesic. Since that time, there have been numerous

studies connecting paracetamol ingestion with liver injury in a dose dependent fashion. These

effects are compounded in the setting of concomitant alcohol abuse, starvation ketosis or

concurrent infections. Hepatocytes metabolize paracetamol via microsomal cytochrome P450

(CYP450) into non-toxic byproducts. This metabolism pathway via CYP450, specifically

cytochrome P450 2E1 (CYP2E1), produces reactive oxygen species (Wendel and Feuerstein,

2018), originally thought to be the ultimate cause of liver injury in paracetamol overdose. After

recent debunking of that long-standing belief, mitochondrial dysfunction has instead been

attributed as the main source of free radicals and oxidative stress in paracetamol hepatotoxicity

(Jaeschke et al., 2017). Mitochondrial dysfunction begins with the formation of drug-protein

adducts between the reactive paracetamol metabolite, N-acetyl-p-benzoquinone imine (NAPQI),

5
and mitochondrial proteins involved in the electron transport chain. Additionally, increased

activity of mitochondrial complex I, a known site of free radical generation, occurs with

paracetamol overdose, and the level of activity was found to correlate with the degree of liver

injury (Du et al., 2016).

1.2 GENERAL OBJECTIVES OF THE STUDY

The aim of this study is to investigate the effect of metformin on liver enzymes in animals

exposed to sub-acute paracetamol-induced toxicity.

1.3 SPECIFIC OBJECTIVES

The specific objective of this study includes;

i. To determine the effect of administration of metformin on alanine transaminase

(ALT) level of in animals exposed to sub-acute paracetamol-induced toxicity.

ii. To determine the effect of administration of metformin on aspartate transaminase

(AST) level of in animals exposed to sub-acute paracetamol-induced toxicity

iii. To determine the effect of administration of metformin on alkaline phosphatase

(ALP) level of in animals exposed to sub-acute paracetamol-induced toxicity

1.4 SCOPE OF THE STUDY

6
The scope of this study entails the potential of metformin to protect against liver damage caused

paracetamol-induced toxicity in adult male wistar rat. The study involves analyzing various

markers of liver function such as liver enzymes and histological changes in liver tissues to

determine the extent of liver damage and the protective effect of Metformin. The study was

carried out in the Department of Pharmacology, Faculty of Basic Medical Sciences, Delta State

University, Abraka between April to May, 2024.

1.5 SIGNIFICANCE OF THE STUDY

This study will provide necessary information about the possible therapeutics effect of

metformin on acetaminophen-induced liver damage. The study explores the potential of

Metformin, a naturally occurring amino acid, to prevent or mitigate the liver damage caused by

acetaminophen. Acetaminophen is known to induce oxidative stress and damage to liver cells,

which can lead to liver dysfunction and disease. If the results of the study show that Metformin

has a protective effect against liver damage, it could lead to the development of new treatments

for liver disease that incorporate metformin as a therapeutic agent. Additionally, the study could

provide insights into the mechanisms by which metformin exerts its protective effects, which

could have implications for other areas of research on oxidative stress and disease.

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 CHAPTER TWO

LITERATURE REVIEW

2.1 The Liver

The liver is the largest organ in the body, contributing about 2% of the total body weight, or

about 1.5 kilograms (3.3 pounds) in the average adult human. The liver is reddish-brown and

shaped approximately like a cone or a wedge. The liver lies mainly in the right upper quadrant of

the abdomen, where it is protected by the thoracic (rib) cage and the diaphragm. The normal liver

lies deep to ribs on the right side and crosses the midline toward the left nipple. The liver

occupies most of the right hypochondrium and upper epigastrium and extends into the left

hypochondrium. The liver moves with the excursions of the diaphragm and is located more

inferiorly when one is erect because of gravity (Moore et al., 2017). The liver has a convex

diaphragmatic surface and a relatively flat or even concave visceral surface which are separated

anteriorly by its sharp inferior border that follows the right costal margin inferior to the

diaphragm. The diaphragmatic surface of the liver is smooth and dome shaped. In contrast to the

smooth diaphragmatic surface, the visceral surface bears multiple fissures and impressions from

contact with other organs.

8
2.1.1 Anatomical Lobes of Liver

The liver is divided into the right and left lobes by attachment of the falciform ligament

anteriorly and superiorly; by the fissures for the ligamentum teres inferiorly and by the fissure

for the ligamentum venosum posteriorly. The right lobe is much larger than the left lobe, and

forms five sixth of the liver. It contributes to all the five surfaces of the liver, and presents the

caudate and quadrate lobes. The caudate lobe is situated on the posterior surface, the quadrate

lobe is situated on the inferior surface, and is rectangular in shape. The left lobe of the liver is

much smaller than the right and it forms only one-sixth of the liver (Moore et al., 2017)

2.1.2 Embryology of the Liver

The liver originates as a component of the embryonic foregut, emerging from endodermal cells.

Its initial formation occurs as the hepatic diverticulum during the fourth week of prenatal

development. This diverticulum takes shape within the peritoneal cavity and becomes anchored

to the abdominal wall through the falciform ligament, a structure derived from the ventral

mesentery. Notably, the umbilical vein traverses the falciform ligament during its journey from

the umbilical cord to the liver. The induction of this diverticulum is thought to result from the

interplay of various signaling pathways, particularly the Wnt/B-catenin pathway and fibroblast

growth factors (FGF), which are secreted by fetal cardiac cells under the influence of the MAPK

pathway (Kalra et al., 2023).

9
Subsequently, the diverticulum undergoes growth and differentiation, ultimately forming the

primordium of either the liver or the gallbladder. As this primordium expands, it develops into

hepatic cords that interconnect around endothelium-lined spaces, giving rise to the initial

structure of hepatic sinusoids (Kalra et al., 2023). The portal vein, originating from both

umbilical and vitelline veins, serves as the central conduit around which hepatic cords organize.

This phenomenon elucidates the primary role of the portal vein in supplying blood to the liver, in

contrast to the hepatic artery. The development of the hepatic artery progresses alongside that of

the biliary tract and continues postnatally (Kalra et al., 2023). Notably, around the sixth week of

development, the liver assumes a role in hematopoiesis, and hepatocytes begin producing bile

during this phase

10
Figure 2.1 structure of the liver (Johns Hopkins Medicine, 2023)

11
2.1.3 Functions of the Liver

Although the liver is a discrete organ, it performs many different interrelating functions. This

becomes especially evident when abnormalities of the liver occur because many liver functions

are disturbed simultaneously. Below are specific functions of the liver.

2.1.3.1 The Liver Plays a Role as a Blood Reservior

This function is primarily attributed to its unique anatomical structure and the dynamic interplay

between its blood vessels. The liver’s capacity to store and release blood as needed is essential

for maintaining overall cardiovascular homeostasis. This response is particularly significant

during changes in blood volume and pressure. Because the liver is an expandable organ, large

quantities of blood can be stored in its blood vessels. Its normal blood volume, including that in

both the hepatic veins and the hepatic sinuses, is about 450 milimeters, or almost 10% of the

body’s total blood volume. When high pressure in the right atrium causes backpressure in the

liver, the liver expands, and 0.5 to 1 liter of extra blood is occasionally stored in the hepatic veins

and sinuses. The storage of extra blood occurs especially in cases of cardiac failure with

pheripheral congestion. Thus, in effect, the liver is a large, expandable, venous organ capable of

acting as a valuable blood reservoir in times of excess blood volume and capable of supplying

extra blood in times of diminished blood volume (Guyton and Hall, 2016).

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2.2.3.2 Carbohydrate Metabolism

The liver's involvement in carbohydrate metabolism is quite intricate. It plays a central role in

regulating blood glucose and preventing dangerous spikes or drops in levels to ensure a steady

supply of energy for the body.

When blood glucose levels are high, usually after a meal, the liver takes up excess glucose and

converts it into glycogen, store it and return to the blood when glucose concentration begins to

fall too low. This is called the glucose buffer function of the liver. Gluconeogenesis in the liver is

important, as it is capable of generating new glucose molecules from non-carbohydrate sources

such as amino acids (from proteins) and glycerol (from fats). Thus, ensures a constant supply of

glucose, especially during periods of fasting or low carbohydrate intake (Bechmann et al.,2012)

(Guyton and Hall, 2016).

2.2.3.3 Protein Metabolism

The liver is a major site of protein synthesis, producing a variety of plasma proteins that are

essential for maintaining various bodily functions. These proteins include albumin, which helps

maintain osmotic pressure in blood vessels, and clotting factors that are important for blood

coagulation amino acid metabolism. The liver is central to amino acid metabolism, it takes up

amino acids from the bloodstream and either incorporates them into newly synthesized proteins

or converts them into other compounds. For instance, the liver plays a role in deamination of
13
amino acids, which removes the amino group and produces ammonia. Ammonia is then

converted into urea through the urea cycle, which is excreted by the kidneys in urea cycle (Berg

et al., 2011).

2.2.3.4 Fat Metabolism

The liver is involved in the process of synthesizing fatty acids and triglycerides from excess

glucose and other substrates. These triglycerides can be stored in the liver itself as well as

exported to adipose tissue (fat cells) for long-term energy storage. The liver is also responsible

for beta-oxidation, which is the breakdown of fatty acids into acetyl-CoA molecules. These

acetyl-CoA molecules can then enter the citric acid cycle (Krebs cycle) to produce energy via

aerobic respiration. During periods of low carbohydrate availability, such as fasting or a low-carb

diet, the liver synthesizes ketone bodies from fatty acids through a process called ketogenesis.

These ketone bodies, including acetone, acetoacetate, and beta-hydroxybutyrate, can serve as

alternative energy sources for various tissues, including the brain. The liver produces very-low-

density lipoproteins (VLDLs), which transport triglycerides synthesized in the liver to other

tissues, particularly adipose tissue. As triglycerides are removed from VLDLs, the particles

become denser and transition into low-density lipoproteins (LDLs), often referred to as "bad

cholesterol."

14
The liver regulates cholesterol levels by both synthesizing cholesterol and removing excess

cholesterol from the body. It synthesizes cholesterol for various functions, including the

production of cell membranes, hormones, and bile acids. The liver also removes excess

cholesterol by excreting it into bile, which is then eliminated through feces (Harvey, 2011).

2.2.3.5 Liver Regeneration

The liver possesses an impressive capacity to recover from significant loss of hepatic tissue

resulting from actions such as partial hepatectomy or acute liver injury, as long as the injury is

not complicated by viral infection or inflammation. In the case of partial hepatectomy, which

involves the removal of up to 70% of the liver, the remaining lobes undergo enlargement to

eventually restore the liver to its initial size. This regenerative process occurs remarkably swiftly,

taking just 5 to 7 days in rat models. Throughout the process of liver regeneration, hepatocytes

are estimated to replicate once or twice, and once the original size and volume are reestablished,

the hepatocytes return to their typical inactive state. The mechanisms governing this rapid liver

regeneration are not fully comprehended, although hepatocyte growth factor (HGF) seems to

play a significant role in promoting liver cell division and expansion. Normally, hepatocytes

(liver cells) are in a quiescent state, but after injury, they transition from quiescence to an active

state. This transition involves the activation of various growth factor pathways, including

hepatocyte growth factor (HGF) and epidermal growth factor (EGF).

15
2.2.3.6 Detoxification Function

The liver is a central organ in the body's detoxification processes. It plays a critical role in

neutralizing and eliminating toxins, drugs, and harmful substances that can be detrimental to

health. This detoxification process involves various enzymatic reactions that convert these

substances into less toxic forms that can be excreted. The liver contains enzymes, such as the

cytochrome P450 family, that are responsible for metabolizing drugs and toxins. These enzymes

catalyze reactions that make the substances more water-soluble, allowing them to be excreted

through urine or bile (Guyton and Hall, 2016).

2.2.3.7 Heat Production

The liver contributes to the body's heat production through various metabolic processes. As the

site of numerous biochemical reactions, the liver generates heat as a byproduct of these reactions.

The energy released during metabolic processes, such as nutrient metabolism and detoxification,

contributes to the overall body heat. Additionally, the liver is involved in regulating body

temperature through its interactions with various hormones and metabolic pathways. For

example, during digestion, the liver's metabolic activities increase, resulting in increased heat

production that helps regulate body temperature (Guyton and Hall, 2016).

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2.2.3.8 Secretion of Bile

The liver is responsible for producing bile, a greenish-yellow fluid that is essential for the

digestion and absorption of fats and fat-soluble vitamins. Bile is synthesized in liver cells, known

as hepatocytes, and is then transported to the gallbladder for storage and concentration. When

needed, the gallbladder releases bile into the small intestine during meals. Bile contains bile

salts, bile pigments (such as bilirubin), cholesterol, phospholipids, and water .Bile salts, the

major component of bile, aid in the emulsification of dietary fats, which breaks them down into

smaller droplets, increasing their surface area for digestion by enzymes called lipases. Bile also

plays a role in the absorption of fat-soluble vitamins (A, D, E, and K) and cholesterol (Boyer,

2013).

2.2.3.9 Storage function: many substances such as glycogen, amino acids, iron, folic acid and

vitamins A, B12 and D are stored in the liver (Guyton and Hall, 2016).

2.1.4 Liver Lobules and cell types

The fundamental structural and functional unit of the liver is the hepatic lobule, which presents

as a cylindrical entity spanning several millimeters in length and measuring between 0.8 to 2

17
millimeters in diameter. In humans, the liver comprises a range of 50,000 to 100,000 individual

lobules (Guyton and Hall, 2016). Remarkably, the liver stands as the sole organ with the

remarkable capacity for regeneration. The liver is composed of numerous divisions referred to as

hepatic lobes, each of which contains multiple smaller units termed hepatic lobules. These

hepatic lobules represent the fundamental building blocks responsible for both the structural and

functional aspects of the liver. These lobules exhibit a honeycomb-like arrangement and consist

of hepatocytes, which are the principal liver cells. In the liver there are approximately 50,000 to

100,000 lobules within the liver. The hepatic lobule can be characterized through distinct

metabolic zones. Zone 1 designates the periportal region, where activities like gluconeogenesis,

cholesterol synthesis, fatty acid beta-oxidation, and urea formation predominate. Zone 3

encompasses the perivenous vicinity surrounding the central vein, where functions such as

glycolysis, bile acid production, glutamine synthesis, and xenobiotic metabolism occur. Zone 2

denotes the intermediate region between zones 1 and 3, with localized roles including iron

regulation and modulation of insulin-like growth factors (Kriyu et al., 2022).

Within the confines of the lobule, three major cell types coexist:

Hepatocytes: Constituting approximately 80% of liver cells, hepatocytes undertake diverse

metabolic functions. These sizeable, polyhedral epithelial cells are key players in liver activities.

18
Kupffer cells: These specialized macrophages inhabit the liver sinusoids and play roles in

immune responses, phagocytosis, and the breakdown of aged red blood cells (Bonnardel et al.,

2019)( Tortorta et al.,2017).

Stellate cells (Ito cells): Originate from a mesenchymal lineage and occupy the perisinusoidal

space of liver lobules. Stellate cells are chiefly engaged in storing vitamin A and generating

component of the extracellular matrix, which contributes to liver structure maintenance.

Additionally, they play a substantial role in liver regeneration (Bonnardel et al., 2019).

19
Figure 2.2 structure of the liver’s functional unit or lobules (wikipedia,2023)

2.1.5 Hepatic Blood Supply and Bile Synthesis

20
The liver experiences a significant blood influx of approximately 1,500 milliliters per minute.

This blood supply is sourced from two distinct pathways. Firstly, oxygenated blood is delivered

through the hepatic artery, a branch arising from the aorta, constituting approximately 25% of the

total supply. Secondly, nutrient-rich blood is transported via the hepatic portal vein, which

conveys blood from the gastrointestinal tract, spleen, and pancreas, accounting for approximately

75% of the total supply.

In terms of bile synthesis, hepatocytes are responsible for its production. The process commences

with the secretion of bile by hepatocytes, followed by its release into canaliculi. Subsequently,

bile travels through narrow ducts and hepatic ducts until it reaches the common hepatic duct.

From this point, the bile is directed either directly into the intestine or into the gallbladder for

storage and concentration, a decision guided by the specific duct. After being secreted into the

duodenum, bile takes part in enterohepatic circulation. During this process, bile fulfills its

function in the bowel, and any un-excreted components are recycled through their conversion

into bile acids by gut bacteria. These newly generated bile acids are then reabsorbed in the ileum

and transported back to the liver for further utilization. (Boyer et al., 2018)

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Figure 2.3 scheme of blood flow in the liver (hotcore.info, 2023)

2.2. Liver Enzymes and Their Significance

Liver enzymes fulfill distinct physiological roles, and their concentrations serves as valuable

biomarkers for evaluating liver function. Among the frequently assessed liver enzymes are

22
alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP),

and gamma-glutamyltransferase (GGT).

2.2.1 Alanine Aminotransferase (ALT)

ALT, primarily localized within hepatic cells, was initially identified as serum glutamic pyruvate

transaminase (SGPT). The typical serum level of ALT ranges from 13-36 U/L for males and 10-

30 U/L for females (vasudevan, 2018). It holds a prominent position as a clinical biomarker for

assessing liver well-being (Nowicki et al., 2020). ALT levels are frequently employed to evaluate

hepatic impairment and demonstrate elevation in conditions such as hepatitis, liver cirrhosis, and

liver necrosis (Nanda et al., 2018). Typically analyzed alongside AST within a liver function

panel, ALT aids in identifying the source of organ damage. Notably, factors like diet, restraint,

and drug administration can influence plasma ALT levels in rodents (Evans, 2009). Notably, ALT

levels are typically higher in males than females, possibly attributed to hormonal disparities

(Moriles et al., 2022) (Wu et al., 2018).

2.2.3 Aspartate Aminotransferase (AST)

AST, previously designated serum glutamate oxaloacetate transaminase (SGOT), is situated in

the cytoplasm of hepatocytes and other tissues, including skeletal muscle. The standard serum

level for AST is 8-20 U/L (Vasudevan, 2018). AST concentration rises in response to bruising,

trauma, necrosis, infection, or neoplasia affecting the liver or muscles. In comparison to ALT,

23
AST is considered a less specific indicator of liver injury, owing to its expression in diverse

tissues such as the brain, myocardial cells, and skeletal muscle cells (Sharon, 2016). Medical

practitioners may use the AST/ALT ratio to differentiate between hepatic and extrahepatic

injuries, with an AST/ALT ratio of 2:1 indicating hepatic injury (Charles, 2022). Some

researchers have suggested that an AST/ALT ratio of ≤0.4 during recovery from severe acute

hepatotoxicity predicts a favorable prognosis for recuperation (Sharon, 2016).

2.2.4 Alkaline Phosphatase (ALP)

Alkaline phosphatase is a multifunctional enzyme capable of hydrolyzing aliphatic, aromatic, or

heterocyclic compounds. Its optimal pH range for enzymatic activity falls between 9 and 10 (Yu

et al., 2019). The liver and bone exhibit higher concentrations of ALP, whereas lower levels are

present in kidney tubules, intestinal epithelium, lung, and placenta. ALP levels vary across

species, typically increasing due to factors such as digestion, cholestasis, or injury to intestinal or

biliary epithelium. Conversely, fasting, hypothyroidism, or pernicious anemia may lead to

decreased ALP levels (Lowe et al., 2022). The established normal serum ALP range is 40-125

U/L.

2.2.5 Gamma-Glutamyltransferase (GGT)

GGT is situated in the liver, pancreas, and luminal brush border of proximal tubular cells. Its

normal serum concentration ranges from 10 to 30 U/L (Vasudevan, 2018). This enzyme

24
facilitates the transfer of gamma-glutamyl groups from peptides like glutathione to acceptors

such as peptides and L-amino acids. As a result, GGT plays a role in amino acid transport across

cellular membranes and leukotriene metabolism. Infections like hepatitis and prostate cancers

can moderately elevate GGT levels. Notably, alcohol-induced liver damage often leads to

substantially heightened GGT levels (Hussain et al., 2019; Cho et al., 2023).

2.2.6 Bilirubin

Bilirubin, a red-yellow pigment, is a byproduct of the normal catabolic breakdown of heme in

vertebrates. This essential process clears waste stemming from abnormal red blood cell

destruction (Braunstein, 2019). Biliverdin is initially produced from heme, followed by

biliverdin reductase catalyzing its conversion into bilirubin. Subsequently, bilirubin undergoes

further breakdown within the body, with its metabolites excreted through bile and urine. Elevated

bilirubin levels can signal specific diseases (Kalakonda et al., 2022). The standard plasma

bilirubin range is 0.2-1.0 mg/dL. Unconjugated bilirubin ranges from 0.2-0.7 mg/dL, while

conjugated bilirubin ranges from 0.1-0.4 mg/dL. Hyperbilirubinemia arises when plasma

bilirubin surpasses 1 mg/dL, with levels between 1 and 2 mg/dL indicative of latent jaundice.

Bilirubin exceeding 2 mg/dL diffuses into tissues, leading to jaundice-associated yellowish

discoloration (Vasudevan, 2018). Total bilirubin measurement encompasses both conjugated and

unconjugated forms.

25
2.3 Other Enzymes Involved in Liver Function

2.3.1 Lactate Dehydrogenase (LDH)

LDH facilitates the conversion of pyruvate to lactate. Normal serum LDH ranges from 100 to

200 U/L. Remarkably, LDH exists at 100 times higher concentration inside red blood cells

compared to plasma, potentially resulting in false-positive tests due to minor hemolysis.

Elevated LDH values are observed in conditions such as hemolytic anemia, hepatocellular

damage, muscular dystrophy, carcinomas, leukemias, and any circumstance involving cell

necrosis (Vasudevan, 2018).

2.4 Prevalence of Drug induced liver damage

Drug-induced liver injury (DILI) and herbal-induced liver injury (HILI) are well-recognized and

symptomatically can mimic both acute and chronic liver diseases. It is reported that there are

over 1000 prescription medications and over 100,000 herbal and dietary supplements available in

the United States (Fontana et al., 2023). The probability of an individual drug causing liver

injury ranges from 1 in 10,000 to 100,000, with some drugs reported as having an incidence of

100 in 100,000 (chlorpromazine, isoniazid) (Garcia et al., 2020). DILI has a worldwide

estimated annual incidence between 14 to 19.1 per 100,000 persons exposed and 30 percent of

26
cases will develop jaundice. The prevalence and cause of DILI varies geographically. DILI

accounts for approximately 10 percent of all cases of acute hepatitis, is the cause of acute

jaundice in 50 percent of patients who present with new jaundice, and accounts for up to one-half

of the cases of acute liver failure in Western countries (Reuben et al., 2010).DILI is also the most

frequently cited reason for withdrawal of medications from the marketplace (up to 32 percent of

drug withdrawals) (Babai et al., 2021).DILI may not be detected prior to drug approval, because

most new drugs are tested in fewer than 3000 people prior to drug approval. As a result, cases of

DILI with an incidence of 1 in 10,000 may be missed. It has been suggested that for every 10

cases of alanine aminotransferase (ALT) elevation (>10 times the upper limit of normal) in a

clinical trial, there will be one case of more severe liver injury that develops once the drug is

widely available. Following publication of the US Food and Drug Administration (FDA)

guidance statement for DILI in drug development, awareness of the issue increased, and drug

withdrawal significantly decreased (Babai et al., 2021).

2.4.1 Risk factors of Drug induced liver damage

1. Race: Some drugs appear to have different toxicities based on race. For example, blacks and

Hispanics may be more susceptible to isoniazid (INH) toxicity. The rate of metabolism is under

the control of P-450 enzymes and can vary from individual to individual (Lynch and Price,

2007).

27
2. Age: Apart from accidental exposure, hepatic drug reactions are rare in children. Elderly

persons are at increased risk of hepatic injury because of decreased clearance, drug-to-drug

interactions, reduced hepatic blood flow, variation in drug binding, and lower hepatic volume. In

addition, poor diet, infections, and multiple hospitalizations are important reasons for drug-

induced hepatotoxicity (Hunt et al., 1997).

3. Sex: Although the reasons are unknown, hepatic drug reactions are more common in females.

4. Alcohol ingestion: Alcoholic persons are susceptible to drug toxicity because alcohol induces

liver injury and cirrhotic changes that alter drug metabolism. Alcohol causes depletion of

glutathione (hepatoprotective) stores that make the person more susceptible to toxicity by drugs.

5. Liver disease: Preexisting liver disease has not been thought to make patients more susceptible

to drug-induced liver injury, but it may be that a diminished liver reserve or the ability to recover

could make the consequences of injury worse. Although the total cytochrome P-450 is reduced in

chronic liver disease, some may be affected more than others. The modification of doses in

persons with liver disease should be based on the knowledge of the specific enzyme involved in

the metabolism. Patients with HIV infection who are co-infected with hepatitis B or C virus are

at increased risk for hepatotoxic effects when treated with antiretroviral therapy. Similarly,

patients with cirrhosis are at increased risk of decompensation by toxic drugs.

28
6. Genetic factors: A unique gene encodes each P-450 protein. Genetic differences in the P-450

enzymes can result in abnormal reactions to drugs, including idiosyncratic reactions.

Debrisoquine is an antiarrhythmic drug that undergoes poor metabolism because of abnormal

expression of P-450-II-D6. This can be identified by polymerase chain reaction amplification of

mutant genes. This has led to the possibility of future detection of persons who can have

abnormal reactions to a drug (Ahmad and Odin, 2007).

Other comorbidities

 Persons with AIDS, persons who are malnourished, and persons who are fasting may

be susceptible to drug reactions because of low glutathione stores.

 Drug formulation

 Long-acting drugs may cause more injury than shorter-acting drugs.

2.4.2 Drug toxicity mechanisms

The classic division of drug reactions is into at least two major groups, drugs that directly affect

the liver and drugs that mediate an immune response, as follows:

Intrinsic or predictable drug reactions: Drugs that fall into this category cause

reproducible injuries in animals, and the injury is dose related. The injury can be due to

the drug itself or to a metabolite. Acetaminophen is a classic example of a known

29
 intrinsic or predictable hepatotoxin at supertherapeutic doses. Another classic example is

carbon tetrachloride.

Idiosyncratic drug reactions: Idiosyncratic drug reactions can be subdivided into those

that are classified as hypersensitivity or immunoallergic and those that are metabolic-

idiosyncratic. Regarding hypersensitivity reactions, phenytoin is a classic, if not common,

cause of hypersensitivity reactions. The response is characterized by fever, rash, and

eosinophilia and is an immune-related response with a typical short latency period of 1-4

weeks. A metabolic-idiosyncratic reaction occurs through an indirect metabolite of the

offending drug. Unlike intrinsic hepatotoxins, the response rate is variable and can occur

within a week or up to one year later. It occurs in a minority of patients taking the drug,

and no clinical manifestations of hypersensitivity are noted. INH toxicity is considered to

fall into this class. Not all drugs fall neatly into one of these categories, and overlapping

mechanisms may occur with some drugs (eg, halothane).

2.5 PARACETAMOL (ACETAMINOPHEN)

Paracetamol (acetaminophen) is one of the world’s most widely used non-prescription medicines

from cradle to grave. It is readily available and inexpensive. As an analgesic, paracetamol is

better tolerated than the non-steroidal anti-inflammatory drugs (NSAIDs) although it may be

somewhat less efficacious. During the 1980s a decline in the use of aspirin due to its association

30
with Reye’s syndrome allowed paracetamol to become the antipyretic and analgesic of choice in

children (Belay et al. 2019) and it is now the standard antipyretic and analgesic in all age groups.

Although a useful and important drug, the dose of paracetamol is inconveniently large and a full

dose of 4 g daily requires a large number of tablets to be taken.

In his Nobel Prize-winning work on the mechanism of action of aspirin and other NSAIDs, Vane

(2019) demonstrated that these drugs inhibit the formation of prostaglandins (PGs), local factors

that are associated with pain, fever and inflammation. However, paracetamol did not appear to

inhibit PG synthesis, despite its actions similar to those of the NSAIDs. The mechanism of the

basic pharmacological effects of paracetamol is only now becoming clear and it is now

recognised to be an inhibitor of PG synthesis in cellular systems under specific conditions and

has an apparent selectivity for one of the cyclooxygenase (COX) enzymes, namely COX-2.

2.5.1 Chemistry and distribution

Paracetamol is a low-molecular-mass compound (Fig. 1). It is an extremely weak acid (pKa 9.7)

and is, therefore, essentially unionised at physiological pH values (Craig 2019). Its partition

coefficient between octanol and water is 3.2 and in the range where passive diffusion through

cell membranes is likely. The binding of paracetamol to plasma proteins is negligible Gazzard et

al. (2016) and with a volume of distribution of about 50 L after intravenous dosage (Prescott et

al., 2019), it is concluded that paracetamol distributes throughout the body without binding to

31
tissues. This lack of binding indicates that the concentrations of paracetamol in experiments in

vitro can be correlated directly with its concentrations in vivo without corrections for tissue

uptake or protein binding. The peak plasma concentrations of paracetamol after a therapeutic

dose (1 g) are approximately 20 mg/L (130 lM) up to 30 mg/L (200 lM) after intravenous

injection or rapid oral absorption, and concentrations of up to 30 mg/L can be considered as

therapeutic. At a dosage of 1 g four times daily, the trough concentrations are of the order of 2

mg/L (13 lM).

Chemically, paracetamol is a phenol and, like many phenols, it is easily oxidised. This

oxidation is central to its postulated mechanism of action as a substrate and an inhibitor of the

peroxidase function of COX-1 and COX-2. Paracetamol is also oxidised by and inhibits other

haem peroxidases, including myeloperoxidase.

Fig. 2.4: Structure of paracetamol

Therapeutic and toxic actions: similarities and differences from NSAIDs

32
Much of the pharmacology and toxicology of paracetamol are similar to those of the non-

selective NSAIDs, such as ibuprofen, ketoprofen and naproxen, and it shows particular similarity

to the selective COX-2 inhibitors, such as celecoxib and etoricoxib (Table 1). On average,

however, paracetamol has weaker analgesic activity than both groups of NSAIDs. A major

difference is that paracetamol, unlike both groups of NSAIDs, has only weak antiinflammatory

activity (Table 1; see Anti-inflammatory effect).

33
Table 1 Summary of pharmacological and clinical activities of paracetamol, selective COX-2

inhibitors and non-selective NSAIDs

Pharmacological activity Paracetamol Selective COX-2 inhibitor Non-

selective NSAID

Analgesia Active Active Active

Antipyresis Active Active Active
Anti-inflammatory Active in mild Active Active
inflammation
Anti-platelet Low activity Inactive Active
Damage to stomach and Low activity Low activity Active
small intestine
Aspirin-induced asthma Weakly active Inactive Active
Blood pressure Variable data Increase Increase
Renal Lesser effects than both Impaired function in Impaired function
NSAID classes stressed kidneys in stressed
kidneys
Increased risk of Inactive Active Active
thrombosis

Both classes of NSAIDs have been associated with increase in blood pressure (Laine et al.

2008), more so in patients treated for hypertension than normotensive individuals (Snowden and

Nelson 2011). The effect of paracetamol has been studied to a lesser extent with inconsistent

results (Sudano et al. 2012; Turtle et al. 2012). A notable result is that paracetamol increased the

risk of hypertension in women although bias due to taking paracetamol for painful conditions is

possible (Curhan et al. 2002). In studies on patients treated with antihypertensives, paracetamol

has little effect on blood pressure and a lesser effect than NSAIDs (Radack et al. 1987;

Pavlicevic et al. 2008; Aljadhey et al. 2012). It is possible that blood pressure increases in some

patient groups and an important recent finding is that paracetamol increases blood pressure by

34
about 3 mmHg in patients with coronary artery disease (Sudano et al. 2010). In clinical practice,

it is reasonable to check for hypertension in patients taking regular paracetamol. Conversely, a

temporary decrease in blood pressure has been noted after intravenous injection of paracetamol

in acutely ill patients (Hersch et al. 2008; de Maat et al. 2010; den Hertog et al. 2012).

Renal prostaglandins and prostacyclin are synthesised by both COX-1 and COX-2. It is now

apparent that NSAIDs have little or no effect on renal function in patients with good renal

function but may cause renal impairment in patients with risk factors. These risk factors include

already impaired kidney function (particularly in elderly patients) (Murray et al. 1995; Whelton

et al. 2000), dehydration, sodium depletion (Colletti et al. 2019; Farquhar et al. 2019) heart

failure, diabetes, liver disease or taking the combination of a diuretic together with inhibitors of

angiotensin converting enzyme (ACE) or angiotensin receptor (Bouvy et al. 2003; Loboz and

Shenfield 2018). In patients with these risk factors, the function of PGs is important in

maintaining renal function and significant inhibition of their synthesis leads to renal impairment.

Although paracetamol has apparent COX-2 inhibitory activity, it is widely regarded as being safe

in patients with risk factors for renal impairment in contrast to patients taking NSAIDs.

Experimental proof of this concept is scanty but NSAIDs decreased GFR to a greater extent than

placebo or paracetamol in a trial involving stressed kidneys (low sodium diet, dehydration and

exercise) (Farquhar et al. 2019), and immediately after surgery in elderly patients (Koppert et al.

2006). Similarly, ibuprofen depressed renal function to a greater extent than paracetamol during
35
surgery on sodium-depleted, anaesthetised dogs (Colletti et al. 2019). Peripheral oedema was

also less common in a clinical trial comparing paracetamol and naproxen (Temple et al. 2006).

The renal safety of paracetamol is also indicated by two studies finding no increase in the risk of

hospitalisation for heart failure (Merlo et al. 2001) and no worsening of renal function in patients

with grades 4–5 kidney disease (Evans et al. 2009). Both are conditions in which NSAIDs are

expected to worsen renal function. Conversely, in an epidemiological study, Fored et al. (2001)

reported that paracetamol exacerbated the development of chronic renal failure, although bias

could not be excluded in this population study.

The effect of paracetamol on infarction is of considerable interest because of its widespread

use. The highly selective COX-2 inhibitor, rofecoxib, was associated with increased myocardial

infarction and its availability was consequently stopped. There is a small tendency for low doses

of the presently available COX-2 selective inhibitors and the non-selective NSAIDs to lead to

myocardial infarction while paracetamol appears safe (Latimer et al. 2009).

2.5 Paracetamol induced Hepatotoxicity

Overdoses of paracetamol are well known to cause major hepatotoxicity which may progress to

death if the dose is sufficient and the patients are not treated adequately with N-acetylcysteine.

The hepatotoxicity of paracetamol overdoses is due to the formation of the oxidised metabolite

of paracetamol and its reaction with glutathione. Glutathione, in its reduced form, maintains the

36
appropriate redox balance in cells and prevents cell death. Centrilobular necrosis occurs because

of depletion of glutathione and also because, after depletion of hepatocellular glutathione, the

oxidised paracetamol metabolite reacts with essential cellular proteins. A variety of factors affect

the hepatotoxicity of paracetamol but, apart from the contentious area of hepatotoxicity of

therapeutic doses of paracetamol, are not reviewed in this communication. Occasional

hepatotoxicity is claimed widely but critical examination of cases shows that most patients

whose toxicity is claimed from therapeutic doses have probably taken overdoses (Prescott 2010a;

Graham et al. 2018). A background presentation to a FDA conference on paracetamol and

hepatotoxicity contained the statement that ‘‘rare cases of acute liver injury have been linked to

amounts lower than 2.5 g/day’’ but there was no comment on the difficulty in assigning

hepatotoxicity to therapeutic doses of paracetamol (FDA Background 2019), as has been noted

from a large USA survey (Larson et al. 2018). Re-challenge of patients with hepatotoxicity from

claimed therapeutic dosage is extremely uncommon. However, three patients with hepatotoxicity

have been rechallenged with a rapid rise in transaminase concentrations in plasma detected

(Graham and Scott 2018). Elevated plasma transaminases are also noted in some young patients

during treatment with therapeutic doses of paracetamol (Watkins et al., 2016), but the high

transaminase levels have declined or reverted to normal over time despite continuing dosage

(Kuffner et al., 2016). An important recent observation is the comparative levels of alanine

aminotransferase in hospitalised patients taking paracetamol. The levels of alanine

37
aminotransferase were similar in older and younger patients despite higher plasma

concentrations of paracetamol in the older patients (Mitchell et al. 2011). A further indication of

the safety of therapeutic doses of paracetamol is that serious hepatotoxicity has never been

recorded in prospective clinical trials on 30,865 patients (Dart and Bailey 2017). Admittedly,

prospective trials are conducted in controlled conditions and patients with complex medical

histories are often excluded. Nevertheless, the absence of any serious hepatotoxicity in clinical

trials is a strong indicator of the safety of paracetamol.

2.6 Metformin and the liver

Metformin is traditionally thought to act on the liver to improve blood glucose levels and several

lines of evidence support this. First, in mice lacking the organic cation transporter 1 (OCT1),

which take up little or no metformin into the liver, metformin was ineffective at improving blood

glucose after high-fat feeding (Zhou et al., 2016). Second, tracer studies in humans show that

metformin lowers hepatic glucose production, with minimal impact on peripheral insulin-

mediated glucose uptake. However, when only placebo-controlled studies were analysed, the

impact of metformin on endogenous glucose production (EGP) was not significant unless

concomitant drug-induced reductions in plasma insulin were used to ‘adjust’ EGP ( Cameron et al.,

2016). Third, as will be summarised here, multiple studies in mouse hepatocytes and transgenic

mice provide evidence for a role of metformin in reducing hepatic gluconeogenesis and/or

insulin sensitivity.
38
2.7.2 Metformin and the mitochondrial control of hepatic gluconeogenesis

Given that gluconeogenesis is an energyintensive process (consuming six ATP equivalents per

molecule of glucose synthesised), hepatocytes need to balance the demand for ATP with supply,

with the latter primarily provided by mitochondria. Metformin accumulates within mitochondria

to concentrations up to 1000-fold higher than in the extracellular medium, because metformin

carries a positive charge and the membrane potentials across the plasma membrane and

mitochondrial inner membrane (positive outside) drive metformin into the cell and subsequently

into the mitochondria (Zhou et al., 2019). The most intensively studied mitochondrial action of

metformin is the inhibition of Complex I of the respiratory chain ( Howell et al., 2017), which

suppresses ATP production. A persistent criticism of this mechanism has been the high

extracellular concentrations (mmol/l) required to observe rapid effects, although lower

concentrations of metformin (50–100 μmol/l) do inhibit Complex I in rat hepatoma (H4IIE) cells

after several hours; this delay was ascribed to the slow uptake of metformin by

mitochondria ,which has recently been observed experimentally. In addition, some studies do not

detect any changes in cellular ADP:ATP ratios after metformin treatment, although they can be

observed with

binding protein (CREB), thus inducing transcription of the genes encoding the gluconeogenic

enzymes PEPCK and G6Pase.

39
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 MATERIALS

Cages, Feed and water, Oral cannula, Syringes, Hand Gloves, Sample bottles, Organ bottles,
Weighing scale, Sensitive weighing balance, pH meter, Spectrophotometer, Centrifuge,
Phosphate buffer, Normal saline, Permanent marker, Dissecting set, Beaker, Food and water
trough, Plastic basket for transporting rats, Disinfectant, Cotton wool, Detergent, Formalin,
Refrigerated centrifuge, Tissue homogenizer, Microplate reader (spetraMAX),
Microscope,Timer, Lancet, Tissue paper, Cotton wool, Test tubes, Test tube racks, Beaker,
Micropipette and Micropipette tips

3.2 DRUGS

 Acetaminophen (paracetamol) 500 mg

 Silymarin 100 mg tablets
 Metformin 500 mg tablets

3.3 ANIMALS

Wistar rats weighing between 145-180 g were procured from the Animal House of the Faculty of

Basic Medical Science, Delta State University Abraka, Nigeria. The animals were acclimatized

for a period of two weeks prior to the study, and were placed on rats feed and clean water ad

libitum. Guidelines were followed in the handling of animals in accordance with the ethical
40
standards of the Institutional Animals Ethics (IAEC), as adopted by the ethical committee of the

Faculty of Basic Medical Science, Delta State University, Abraka, Nigeria. Ethical approval was

obtained from the Research and Ethics Committee of the Faculty of Basic Medical Science,

Delta State University Abraka, Nigeria (RBC/FBMC/DELSU/24/363).

3.4 EXPERIMENTAL DESIGN AND GROUPING

The experimental animals were grouped into four groups of five animals where group 1 served

as normal control received distilled water (10 ml/kg) daily, while groups 2-4 were administered

paracetamol 500 mg/kg daily for 14 days to induce toxicity. Group 2 also received distilled water

whereas groups 3 and 4 were treated with metformin (MET) 500 mg/kg and silymarin (SLY) 100

mg/kg daily respectively for 14 days.

The drugs were administered orally using an oral gastric cannula. At the end of the experimental

period, the rats were anaesthetize using ether and blood samples were collected for biochemical

analyses.

3.5 STATISTICAL ANALYSIS AND DATA PRESENTATION

All data obtained were expressed as mean ± SEM (standard error of mean), and analyzed by one-

way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Analysis was done using

41
GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA). P-values < 0.05 were taken

as significant. Data were presented in tables and graphs.

CHAPTER FOUR
Results
4.1 Effect of Metfomin on body weight of animals exposed to sub-acute paracetamol-
induced toxicity.

The findings in this investigation showed that the body weight of the wistar rats exposed to sub-

acute paracetamol-induced toxicity showed a significant (P < 0.05 ) increase compared with the

normal control. All treatment had a significant change in body weight, when compared to PCM

Control (Fig. 4.1).

Groups Initial weight (g) Final weight (g)

Normal Control 158.20 ± 6.42 175.60 ± 6.84
PCM Control 500 mg/kg 152.21± 4.66 160.51 ± 6.34
MET 500 mg/kg 156.55 ± 4.00 169.24 ± 3.39
Silymarin 100 mg/kg 162.08 ± 8.86 175.50 ± 6.53
Fig. 4.1: Effect of Metfomin on body weight of animals exposed to sub-acute paracetamol-
induced toxicity. * = P < 0.05 when compared with normal control; # = P < 0.05 when compared
with PCM

42
4.2 Effect of Metfomin on Alanine transaminase (ALT) level of animals exposed to sub-
acute paracetamol-induced toxicity.

The PCM exposed group showed significant increase in Alanine transaminase (ALT), when

compared to the normal control on biochemical analysis, however treatment with Metformin

showed a significant decrease in Alanine transaminase (ALT), compared to the control groups

(fig 4.2).

Fig.4.2: Effect of Metfomin on Alanine transaminase (ALT) of animals exposed to sub-acute

paracetamol-induced toxicity.

* #
= P < 0.05 when compared with normal control; = P < 0.05 when compared with PCM
control

43
4.3 Effect of Metfomin on Alkaline phosphatase (ALP) of animals exposed to sub-acute
paracetamol-induced toxicity.

The PCM exposed group showed significant increase in Alkaline phosphatase (ALP), when

compared to the normal control on biochemical analysis, however treatment with Metformin

showed a significant decrease in Alkaline phosphatase (ALP) compared to the control groups

(fig 4.3).

44
Fig.4.3: Effect of Metfomin on Alkaline phosphatase (ALP) of animals exposed to sub-acute
paracetamol-induced toxicity. * = P < 0.05 when compared with normal control; #
= P < 0.05
when compared with PCM control

4.4 Effect of Metfomin on Aspartate transaminase (AST) of animals exposed to sub-acute

paracetamol-induced toxicity.

The PCM exposed group showed significant increase in Aspartate transaminase (AST), when

compared to the normal control on biochemical analysis, however treatment with Metformin

showed a significant decrease in Aspartate transaminase (AST) compared to the control groups

(fig 4.4).

45
Fig.4.3: Effect of Metfomin on Aspartate transaminase (AST) of animals exposed to sub-acute
paracetamol-induced toxicity. * = P < 0.05 when compared with normal control; #
= P < 0.05
when compared with PCM control

CHAPTER FIVE
DISCUSSION, CONCLUSION, RECOMMENDATION
5.1 Discussion
Hepatic injury may lead to inflammation, fibrosis, and necrosis causing liver failure (Schuppan

and Afdhal). Amino acid have been used for medicinal purposes in many regions of the world

(Odukoya 2021). Acetaminophen (N-acetyl-para-aminophenol, paracetamol, APAP) toxicity is

common primarily because the medication is so readily available, and there is a perception that it

is very safe. Acetaminophen is an antipyretic analgesic with a mechanism of action different

from NSAIDs. Its mode of action is not clearly understood, but it appears to inhibit

cyclooxygenase (COX) in the brain selectively. This results in its ability to treat fever and pain.

It may also inhibit prostaglandin synthesis in the central nervous system (CNS). Acetaminophen

directly acts on the hypothalamus producing an antipyretic effect.

Evidence from numerous studies have shown the effect of paracetamol on Liver enzymes;

elevated plasma transaminases are also noted in some young patients during treatment with

therapeutic doses of paracetamol (Watkins et al., 2016), but the high transaminase levels have

declined or reverted to normal over time despite continuing dosage (Kuffner et al., 2016). An

46
important recent observation is the comparative levels of alanine aminotransferase in

hospitalised patients taking paracetamol. The levels of alanine aminotransferase were similar in

older and younger patients despite higher plasma concentrations of paracetamol in the older

patients (Mitchell et al. 2011). A further indication of the safety of therapeutic doses of

paracetamol is that serious hepatotoxicity has never been recorded in prospective clinical trials

on 30,865 patients (Dart and Bailey 2017). Admittedly, prospective trials are conducted in

controlled conditions and patients with complex medical histories are often excluded.

Nevertheless, the absence of any serious hepatotoxicity in clinical trials is a strong indicator of

the safety of paracetamol.

The present study was aimed to evaluate the Metfomin on Liver enzymes of animals exposed to

sub-acute paracetamol-induced toxicity. The liver is a vital organ that plays an essential role in

carrying out various metabolic functions in the body. However, its proper functioning can be

affected by various factors, one of which is the use of drugs and other agents. The sub-acute

paracetamol-induced toxicity leads to oxidative stress and changes in liver function markers,

including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline

phosphatase (ALP) activities. In our study, we observed that Metformin an amino acid prevents

liver damage induced by Acetaminophen.

5.1.1 Effect of metformin on serum alanine aminotransferase (ALT) level in animals

exposed to sub-acute paracetamol-induced toxicity

47
ALT, primarily localized within hepatic cells, was initially identified as serum glutamic pyruvate

transaminase (SGPT). The typical serum level of ALT ranges from 13-36 U/L for males and 10-

30 U/L for females (vasudevan, 2018). It holds a prominent position as a clinical biomarker for

assessing liver well-being (Nowicki et al., 2020). ALT levels are frequently employed to evaluate

hepatic impairment and demonstrate elevation in conditions such as hepatitis, liver cirrhosis, and

liver necrosis (Nanda et al., 2018). Typically analyzed alongside AST within a liver function

panel, ALT aids in identifying the source of organ damage. Notably, factors like diet, restraint,

and drug administration can influence plasma ALT levels in rodents (Evans, 2009). Notably, ALT

levels are typically higher in males than females, possibly attributed to hormonal disparities

(Moriles et al., 2022) (Wu et al., 2018).This study revealed that metformin treated group showed

a significant decrease in serum alanine aminotransferase (ALT) level compared to the control

groups. This is in agreement with the study conducted by Seyed et al., (2016) on the protective

effect of metformin on acetaminophen-induced oxidative stress, inflammation and subsequent

hepatotoxicity in mice, it was shown that metformin protects hepatocytes against acute

acetaminophen toxicity. Metformin is indicated to diminish oxidative stress, proinflammatory

cytokines, and hepatocyte necrosis.

5.1.2 Effect of metformin on serum Aspartate Aminotransferase (AST) level in animals

exposed to sub-acute paracetamol-induced toxicity
AST, previously designated serum glutamate oxaloacetate transaminase (SGOT), is situated in

the cytoplasm of hepatocytes and other tissues, including skeletal muscle. The standard serum
48
level for AST is 8-20 U/L (Vasudevan, 2018). AST concentration rises in response to bruising,

trauma, necrosis, infection, or neoplasia affecting the liver or muscles. Evidence from this study

revealed that metformin treated group showed a significant decrease in serum Aspartate

Aminotransferase (AST) level compared to the control groups. This is in line with the study

conducted by Seyed et al., (2016) on the protective effect of metformin on acetaminophen-

induced oxidative stress, inflammation and subsequent hepatotoxicity in mice, it was shown that

metformin protects hepatocytes against acute acetaminophen toxicity. Metformin is indicated to

diminish oxidative stress, proinflammatory cytokines, and hepatocyte necrosis.

5.1.3 Effect of metformin on serum Alkaline Phosphatase (ALP) level in animals exposed to
sub-acute paracetamol-induced toxicity.
Alkaline phosphatase is a multifunctional enzyme capable of hydrolyzing aliphatic, aromatic, or

heterocyclic compounds. Its optimal pH range for enzymatic activity falls between 9 and 10 (Yu

et al., 2019). The liver and bone exhibit higher concentrations of ALP, whereas lower levels are

present in kidney tubules, intestinal epithelium, lung, and placenta. ALP levels vary across

species, typically increasing due to factors such as digestion, cholestasis, or injury to intestinal or

biliary epithelium. Results from this study showed that that metformin treated group showed a

significant decrease in serum Alkaline Phosphatase (ALP) level compared to the control groups.

This is in line with the study conducted by Seyed et al., (2016) on the protective effect of

metformin on acetaminophen-induced oxidative stress, inflammation and subsequent

hepatotoxicity in mice, it was shown that metformin protects hepatocytes against acute
49
acetaminophen toxicity. Metformin is indicated to diminish oxidative stress, proinflammatory

cytokines, and hepatocyte necrosis.

5.2 Conclusion
The study demonstrated that Metfomin have a significant preventive potential on sub-acute

paracetamol-induced liver damage in male Wistar rats. The antioxidant and anti-inflammatory

properties of Metfomin are likely responsible for its beneficial effects on liver function. The

reduction in serum liver enzyme levels and histopathological improvements observed in the liver

tissues of Metfomin treated rats suggests that they could be a potential therapeutic agent in the

management of liver damage caused by oxidative stress. The Metformin group showed

significant increase in Aspartate transaminase (AST), when compared to the control groups on

biochemical analysis. The Metformin group showed a significant decrease in Alanine

transaminase (ALT), alkaline phosphatase (ALP) and Aspartate transaminase (AST) compared to

the control groups.

5.3 Recommendations
This study investigated the effect of Metfomin on Liver enzymes of animals exposed to sub-

acute paracetamol-induced toxicity Based on the findings, the following recommendations are

made,

1. Further studies should be conducted to investigate the long-term effects of Metfomin on liver

function in rats. The present study showed that treatment with Metfomin mitigated Sub acute
50
paracetamol-induced liver damage in Wistar rats. However, the study did not investigate the

long-term effects of Metfomin on liver antioxidant parameters. Further studies should, therefore,

be conducted to investigate the long-term effects of Metfomin on liver antioxidant profile in rats.

2. The potential of Metfomin on liver enzymes as a preventive measure against liver damage in

humans should be investigated. The findings of this study showed that Metfomin has preventive

potential against sub-acute paracetamol-induced liver damage in rats. However, this study was

conducted in rats, and the results cannot be directly extrapolated to humans. Further studies

should, therefore, be conducted to investigate the potential of Metfomin as preventive measure

against liver damage in humans.

3. The mechanisms through which Metfomin prevents liver damage should be investigated. The

present study did not investigate the mechanisms through which Metformin prevents liver

damage. Further studies should, therefore, be conducted to investigate the mechanisms through

which Metformin prevents liver damage. Such studies could shed light on the underlying

physiological processes involved in Metformin -mediated prevention of liver damage, which

could lead to the development of effective therapies for liver diseases.

4. The effects of Metfomin in combination with other interventions on liver function should be

studied. The present study investigated the effects of Metfomin alone on liver function. However,

in real-world settings, patients may receive multiple interventions for the prevention or treatment

51
of liver diseases. Therefore, it is important to investigate the effects of Metfomin in combination

with other interventions on liver function. Such studies could provide insights into the potential

benefits or interactions of Metfomin with other interventions for liver diseases.

Further studies are warranted to explore the potential preventive effect of Metfomin on liver

damage caused by other agents besides acetaminophen and in other animal models. The findings

of this study could pave the way for the development of novel therapeutic options for liver

damage diseases and provide valuable information for clinicians in the selection of appropriate

treatment for liver patients.

52
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Appendix 1
Groups Initial weight (g) Final weight (g)
Normal Control 158.20 ± 6.42 175.60 ± 6.84
PCM Control 500 mg/kg 152.21± 4.66 160.51 ± 6.34
MET 500 mg/kg 156.55 ± 4.00 169.24 ± 3.39
Silymarin 100 mg/kg 162.08 ± 8.86 175.50 ± 6.53
Fig. 4.1: Effect of Metfomin on body weight of animals exposed to sub-acute paracetamol-
induced toxicity.

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Fig.4.2: Effect of Metfomin and Silymarin on Alanine transaminase (ALT) of animals exposed to
sub-acute paracetamol-induced toxicity.

* #
= P < 0.05 when compared with normal control; = P < 0.05 when compared with PCM
control

58
59
Fig.4.3: Effect of Metfomin and Silymarin on Alkaline phosphatase (ALP) of animals exposed to
sub-acute paracetamol-induced toxicity. * = P < 0.05 when compared with normal control; # = P
< 0.05 when compared with PCM control

60
61
Fig.4.3: Effect of Metfomin and Silymarin on Aspartate transaminase (AST) of animals exposed
to sub-acute paracetamol-induced toxicity. * = P < 0.05 when compared with normal control; # =
P < 0.05 when compared with PCM control

62