ABSTRACT
The effect of ethanol extract of Dennettiatripetala
on rats exposed to carbon tetrachloride was investigated. Ethanol
extract of the plant was prepared using standard procedure. Sets of 30
female wistar albino rats were divided into 6 groups containing five
animals each and were treated orally with increasing doses of ethanol
extract of Dennettiatripetala for two weeks. CCl4 was
diluted with olive oil in a 1:1 ratio and administered once by oral
route at the end of the extract administration. Results from the study
showed non- significant decreases in the levels of catalase and SOD
activities (P>0.05) in the CCl4 group compared to the
control. The extract treatment however produced a higher activity for
the antioxidant enzymes compared to the CCl4treated groups. The results also showed increased levels of MDA concentration (P<0.05) in the CCl4
group whereas extract treated rats showed lower concentrations of MDA.
The overall results suggests that the ethanolic extract of Dennettiatripetala may have moderate hepatoprotective effect in the CCl4 induced rats.
CHAPTER ONE
1.0 INTRODUCTION
Natural plant products and
their derivatives represent more than 50% of all the drugs in clinical
use in the world (Ben-Eric, 2002). Dennettiatripetalaalso known
as pepper fruit tree is a well-known Nigerian spicy medicinal plant.
It is found in the tropical rainforest region of Nigeria and sometimes
in Savanna areas (Okwu et al., 2005). It is locally called
“Nkarika” by the Efiks of Calabar. The young leaves and fruits have
distinctive spicy taste. The mature fruits constitute the main edible
portions. Some communities in parts of Southern Nigeria also utilize
the leaves and roots, in addition to the fruits for medicinal purpose. Dennettiatripetalahas
been found tocontain lots of minerals, vitamins, alkaloids and trace
elements which are of medicinal importance. It was also indicated that
the rich presence of essential oil (oleoresins) determines the aromatic
flavoring, coloring and pungent properties of pepper fruits. (Nwaoguet al., 2007) investigated phytochemical content of Dennettiatripetala
and detected the presence of saponins, flavonoids, tannins and
cyanogenic glycosides. The intake of flavonoids in any fruit and
vegetable tends to decrease cancer risk (Neuhouser, 2004; Grafet al.,
2005). Flavonoid contributes to the color of plants, their fruits and
flowers. The use of medicinal plants in traditional medicine is not
intended in any way to replace modern medical science but rather an aid
in conventional therapy (Ben-Eric, 2002).
Carbon tetrachloride (CCl4)
is an industrial chemical that does not occur naturally. Most of the
carbon tetrachloride produced is used in the production of
chlorofluorocarbons (CFCs) and other chlorinated hydrocarbons. It was
once used widely as a solvent, cleaner and degreaser, both for
industrial and home use. Today, the scientific database on the effects
of haloalkanes is so vast that it is no longer employed for such
purposes although it is used as a model of experimental liver injury
(Weber et al., 2003).
CCl4 is a well-known hepato- and nephrotoxicant (Thrall et al., 2000; Ogeturket al., 2005), and proves highly useful as an experimental model for the study of certain hepatotoxic effects (Muriel et al., 2003; Moreno and Muriel, 2006). CCl4-induced
toxicity, depending on dose and duration of exposure, covers a variety
of effects. At low doses, transient effects prevail, such as loss of Ca2+ homeostasis, lipid peroxidation, release of noxious or beneficial cytokines (Kyung-Hyun et al.,
2006; Muriel, 2007) and apoptotic events followed by regeneration.
Other effects, with higher doses or longer exposure, are more serious
and develop over a long period of time, such as fatty degeneration,
fibrosis, cirrhosis and even cancer (Weber et al., 2003). In addition, acute intoxication with CCl4
at high doses, when the hepatocellular necrosis exceeds the
regenerative capacity of the liver, fatal liver failure will ensue.
Extreme doses of CCl4 result in nonspecific solvent toxicity, including central nervous system depression and respiratory failure and death.
This study aims at investigating the effect of ethanol extract of Dennettiatripetalaon liver and kidney antioxidant enzyme activity and malondialdehyde concentration of rats exposed to CCl4.
1.1 LITERATURE REVIEW
1.1.0 THE LIVER
The liver is the largest organ
of the human body weighing approximately 1500 g, and is located in the
upper right corner of the abdomen on top of the stomach, right kidney
and intestines and beneath the diaphragm. The liver performs more than
500 vital metabolic functions (Naruseet al., 2007). It is
involved in the synthesis of products like glucose derived from
glycogenesis, plasma proteins, clotting factors and urea that are
released into the bloodstream. It regulates blood levels of amino acids.
Liver parenchyma serves as a
storage organ for several products like glycogen, fat and fat soluble
vitamins. It is also involved in the production of a substance called
bile that is excreted to the intestinal tract. Bile aids in the removal
of toxic substances and serves as a filter that separates out harmful
substances from the bloodstream and excretes them (Saukonenet al.,
2006). An excess of chemicals hinders the production of bile thus
leading to the body’s inability to flush out the chemicals through
waste.
Smooth endoplasmic reticulum
of the liver is the principal ‘metabolic clearing house’ for both
endogenous chemicals like cholesterol, steroid hormones, fatty acids and
proteins, and exogenous substances like drugs and alcohol. The central
role played by liver in the clearance and transformation of chemicals
exposes it to toxic injury (Saukonenet al., 2006).
1.1.0.1 FUNCTIONS OF THE LIVER
The liver has three main functions:
storage, metabolism, and biosynthesis. Glucose is converted to glycogen
and stored; when needed for energy, it is converted back to glucose.
Cholesterol uptake also occurs in the liver. Fat, fat-soluble vitamins
and other nutrients are also stored in the liver. Fatty acids are
metabolized and converted to lipids, which are then conjugated with
proteins synthesized in the liver and released into blood stream as
lipoproteins. Numerous functional proteins such as, enzymes and
blood-coagulating factors are also synthesized by the liver. In
addition, the liver, which contains numerous xenobiotic metabolizing
enzymes, is the main site of xenobiotic metabolism (Hogson and Levi,
2004).
1.1.0.2BIOTRANSFORMATION OF HEPATOTOXICANTS
Liver plays a central role in biotransformation and disposal of xenobiotics.
The close association of liver with the
small intestine and the systemic circulation enables it to maximize the
processing of absorbed nutrients and minimize exposure of the body to
toxins and foreign chemicals. The liver may be exposed to large
concentrations of exogenous substances and their metabolites. Metabolism
of exogenous compounds can modulate the properties of hepatotoxicant by
either increasing its toxicity (toxication or metabolic activation) or
decreasing its toxicity (detoxification).
Most of the foreign substances are
lipophilic thus enabling them to cross the membranes of intestinal
cells. They are rendered more hydrophilic by biochemical processes in
the hepatocyte, yielding water-soluble products that are exported into
plasma or bile by transport proteins located on the hepatocyte membrane
and subsequently excreted by the kidney or gastrointestinal tract
(Totsmannet al., 2008).
The hepatic biotransformation involves
Phase I and Phase II reactions. Phase I involves oxidative, reductive,
hydroxylation and demethylation pathways, primarily by way of the
cytochrome P-450 enzyme system located in the endoplasmic reticulum,
which is the most important family of metabolizing enzymes in the liver.
The endoplasmic reticulum also contains a NADPH-dependent mixed
function oxidase system, the flavin-containing monooxygenases, which
oxidizes amines and sulphur compounds.
Phase I reactions often produce toxic
intermediates which are rendered non-toxic by phase II reactions. Phase
II reactions involve the conjugation of chemicals with hydrophilic
moieties such as glucuronide, sulfate or amino acids and lead to the
formation of more water-soluble metabolite which can be excreted easily.
Another Phase II reaction involves glutathione which can covalently
bind to toxic intermediates by glutathione-S- transferase. As a result,
these reactions are usually considered detoxification pathways.
However, this phase can also lead to the formation of unstable
precursors to reactive species that can cause hepatotoxicity.
The activities of enzymes are influenced
by various endogenous factors and exogenous drugs or chemicals (Lee and
Boyer, 2000). Many substances can influence the cytochrome P450 enzyme
mechanism. Such substances can serve either as inhibitors or inducers.
Enzyme inhibitors act immediately by blocking the metabolic activity of
one or several cytochrome P450 enzymes. Enzyme inducers act slowly and
increase cytochrome P450 activity by increasing its synthesis (Lynch and
Price, 2007).
1.2 KIDNEY
The kidneys are bean-shaped
organs that serve several essential regulatory roles in vertebrates.
They remove excess organic molecules from the blood and their best known
function is the removal of waste products of metabolism. They serve
homeostatic functions such as the regulation of electrolytes,
maintenance of acid-base balance, and regulation of blood pressure (via
maintaining the salt and water balance). In producing urine, the kidneys
excrete wastes such as urea and ammonium. They are responsible for the
reabsorption of water, glucose, and amino acids. They also produce
hormones like calcitriol and erythropoietin.
1.2.1 FUNCTIONS OF THE KIDNEY
Many of the kidney’s functions are
accomplished by relatively simple mechanisms of filtration,
reabsorption, and secretion, which take place in the nephron.
Filtration, which takes place at the renal corpuscle, is the process by
which cells and large proteins are filtered from the blood to make an
ultrafiltrate that eventually becomes urine. The kidney generates 180
litres of filtrate a day, while reabsorbing a large percentage allowing
for the generation of only approximately 2 litres of urine. Reabsorption
is the transport of molecules from this ultrafiltrate into the blood.
Secretion is the reverse process, in which molecules are transported in
the opposite direction, from blood to the urine.(Bard et al., 2003).
1.2.1.0 Excretion of wastes
The kidneys excrete a variety of waste
products produced by metabolism into the urine. These include the
nitrogenous wastes urea, from protein catabolism, and uric acid, from
nucleic acid metabolism. The ability of mammals and some birds to
concentrate wastes into a volume of urine much smaller than the volume
of blood from which the wastes were extracted is dependent on an
elaborate countercurrent multiplication mechanism. This requires several
independent nephron characteristics to operate: a tight hairpin
configuration of the tubules, water and ion permeability in the
descending limb of the loop, water impermeability in the ascending loop,
and active ion transport out of most of the ascending limb. In
addition, passive countercurrent exchange by the vessels carrying the
blood supply to the nephron is essential for enabling this function.
1.2.1.1 Reabsorption of the vital nutrients
Glucose at normal plasma levels is completely reabsorbed in the proximal tubule. The mechanism for this is the Na+/glucose
cotransporter. A plasma level of 350mg/dL will fully saturate the
transporters and glucose will be lost in the urine. A plasma glucose
level of approximately 160 is sufficient to allow glucosuria, which is
an important clinical clue to diabetes mellitus.
Amino acids are reabsorbed by sodium
dependent transporters in the proximal tubule. Hartnup disease is a
deficiency of the tryptophan amino acid transporter which results in
pellagra (Le Tao, 2013)
1.2.1.2 Acid-base homeostasis
Two organ systems, the kidneys and
lungs, maintain acid base homeostasis, which is the maintenance of pH
around a relatively stable value. The lungs contribute to acid-base
homeostasis by regulating carbon dioxide (CO2) concentration.
The kidneys have two very important roles in maintaining the acid-base
balance: to reabsorb and regenerate bicarbonate from urine, and to
excrete hydrogen ions and fixed acids (anions of acids) into urine
(Seldinet al., 1989).
1.2.1.3 Osmolality regulation
Any significant rise in plasma
osmolality is detected by the hypothalamus, which communicates directly
with the posterior pituitary gland. An increase in osmolality causes the
gland to secrete antidiuretic hormone (ADH), resulting in water
reabsorption by the kidney and an increase in urine concentration. The
two factors work together to return the plasma osmolality to its normal
levels.
ADH binds to principal cells in the
collecting duct that translocate aquaporins to the membrane, allowing
water to leave the normally impermeable membrane and be reabsorbed into
the body by the vasa recta, thus increasing the plasma volume of the
body.
There are two systems that create a
hyperosmotic medulla and thus increase the body plasma volume: urea
recycling and the ‘single effect’.
Urea is usually excreted as a waste
product from the kidneys. However, when plasma blood volume is low and
ADH is released the aquaporinsthat are opened are also permeable to
urea. This allows urea to leave the collecting duct into the medulla
creating a hyperosmotic solution that attracts water. Urea can then
re-enter the nephron and be excreted or recycled again depending on
whether ADH is still present or not. The ‘single effect’ describes the
fact that the ascending thick limb of the loop of henle is not permeable
to water but is permeable to NaCl. This allows for a countercurrent
exchange system whereby the medulla becomes increasingly concentrated,
but at the same time setting up an osmotic gradient for water to follow
should the aquaporins of the collecting duct be opened by ADH (Vander,
1985).
1.2.1.4 Blood pressure regulation
Although the kidney cannot directly
sense blood, long term regulation of blood pressure predominantly
depends upon the kidney. This primarily occurs through maintenance of
the extracellular fluid compartment, the size of which depends on the
plasma sodium concentration. Renin is the first in the series of
important chemical messengers that make up the renin-angiotensin system.
Changes in rennin ultimately alter the output of this system,
principally the hormones angotensin II and aldostrone. Each hormone acts
via multiple mechanisms, but both increase the kidney’s absorption of
sodium chloride, thereby expanding the extracellular fluid compartment,
and an increase in blood pressure. Conversely, when rennin levels are
low, angiotensin II and aldosterone levels decrease, contracting the
extracellular fluid compartment, and an increase in blood pressure.
Conversely, when rennin levels are low, angiotensin II and aldosterone
levels decrease, contracting the extracellular fluid compartment, and
decreasing blood pressure.
1.2.1.5 Hormone secretion
The kidneys secrete a variety of
hormones, including erythropoietin, and the enzyme rennin.
Erythropoietin is released in response to hypoxia (low levels of oxygen
at tissue level) in the renal circulation. It stimulates erythropoiesis
(production of red blood cells) in the bone marrow. Calcitriol, the
activated form of vitamin D, promotes intestinal absorption of calcium
and the renal reabsorption of phosphate. Part of the
renin-angiotensin-aldosterone system, renin is an enzyme involved in the
regulation of aldosterone levels (Valtin, 1983).
1.3 HEPATOTOXICITY
Hepatotoxicity refers to liver
dysfunction or liver damage that is associated with an overload of drugs
or xenobiotics(Navaroet al., 2006). The chemicals that cause
liver injury are called hepatotoxins or hepatotoxicants. Hepatotoxicants
are exogenous compounds of clinical relevance and may include overdoses
of certain medicinal drugs, industrial chemicals, natural chemicals
like microcystins, herbal remedies and dietary supplements (Willett et al., 2004).
Certain drugs may cause liver
injury when introduced even within the therapeutic ranges.
Hepatotoxicity may result not only from direct toxicity of the primary
compound but also from a reactive metabolite or from an
immunologically-mediated response affecting hepatocytes, biliary
epithelial cells and/or liver vasculature (Saukkonenet al., 2006).
The hepatotoxic response
elicited by a chemical agent depends on the concentration of the
toxicant which may be either parent compound or toxic metabolite,
differential expression of enzymes and concentration gradient of
cofactors in blood across the acinus. Hepatotoxic response is expressed
in the form of characteristic patterns of cytolethality in specific
zones of the acinus.
1.3.1 SYMPTOMS OF HEPATOTOXICITY
Hepatotoxicity related
symptoms may include a jaundice or icterus appearance causing yellowing
of the skin, eyes and mucous membranes due to high level of bilirubin in
the extracellular fluid, pruritus, severe abdominal pain, nausea or
vomiting, weakness, severe fatigue, continuous bleeding, skin rashes,
generalized itching, swelling of the feet and/or legs, abnormal and
rapid weight gain in a short period of time, dark urine and light
colored stool (Bleibelet al., 2007; Chang and Shaino, 2007).
The symptoms of hepatotoxicity can be subdivided into clinical and drug-induced pathological symptoms.
1.3.1.0 CLINICAL MANIFESTATION
The manifestation of drug induced
hepatotoxicity is highly variable, ranging from a symptomatic evaluation
of liver enzymes to fulminant hepatic failure. The injury may suggest a
hepatocellular injury with evaluation of aminotransferases levels as
the predominant symptom or a cholestatic injury, with elevated alkaline
phosphatase levels with or without hyperbiliruminemia being the main
feature.
In addition, drugs that cause
mild amino transferase elevation with subsequent adaptation are
differentiated from those that result in true toxicity that require
discontinuation.
Hepatotoxicity can be induced in the laboratory by exposing laboratory animals to toxic chemicals such as carbon tetrachloride.
1.3.1.1 PATHOLOGICAL MANIFESTATION
Acute hepatocellular damage
Chronic hepatocellular damage
Chronic cholestasis
Vascular lesions / venocclusive disease
Angiosarcoma (Bleibelet al., 2007; Chang and Shaino, 2007).
1.4 CARBON TETRACHLORIDE
Carbon tetrachloride (CCl4)
is an industrial chemical that does not occur naturally. Most of the
carbon tetrachloride produced is used in the production of
chlorofluorocarbons (CFCs) and other chlorinated hydrocarbons. It was
once used widely as a solvent, cleaner and degreaser, both for
industrial and home use. Today, the scientific database on the effects
of haloalkanes is so vast that it is no longer employed for such
purposes although it is used as a model of experimental liver injury
(Weber et al., 2003).
CCl4 is a well-known hepato- and nephrotoxicant (Thrall et al., 2000; Ogeturket al., 2005), and proves highly useful as an experimental model for the study of certain hepatotoxic effects (Muriel et al., 2003; Moreno and Muriel, 2006). CCl4-induced
toxicity, depending on dose and duration of exposure, covers a variety
of effects. At low doses, transient effects prevail, such as loss of Ca2+ homeostasis, lipid peroxidation, release of noxious or beneficial cytokines (Kyung-Hyun et al.,
2006; Muriel, 2007) and apoptotic events followed by regeneration.
Other effects, with higher doses or longer exposure, are more serious
and develop over a long period of time, such as fatty degeneration,
fibrosis, cirrhosis and even cancer (Weber et al., 2003). In addition, acute intoxication with CCl4
at high doses, when the hepatocellular necrosis exceeds the
regenerative capacity of the liver, fatal liver failure will ensue.
Extreme doses of CCl4 result in nonspecific solvent toxicity, including central nervous system depression and respiratory failure and death. CCl4 can be administered orally, intravenously and intraperitonially.
CCl4 metabolism begins with the formation of the trichloromethyl free radical, CCl3
through the action of the mixed function cytochrome P-450 oxygenase
system of the endoplasmic reticulum. This process involves reductive
cleavage of a carbon-chlorine bond. Free radical activation of CCl4
in mitochondria has also been observed and may contribute significantly
to its toxicity. The major cytochrome iso-enzyme to execute
biotransformation of CCl4 is cytochrome P-450 iso-enzyme 2E1 (CYP2E1). This is evidenced by the absence of toxicity in CYP2E1 knockout mice.
In humans, CYP2E1 dominates CCl4
metabolism at environmentally relevant concentrations, but at higher
concentrations other cytochromes, particularly CYP3A, also contribute
importantly (Zangeret al., 2000). The CCl3 radical
reacts with several important biological substances, like fatty acids,
proteins, lipids, nucleic acids and amino acids (Weber et al., 2003). CCl3 also acts by abstracting hydrogen from unsaturated fatty acids to form chloroform