ABSTRACT
Paracetamol (Acetaminiphen) in toxic doses caused electrolyte imbalance, anaemia and oxidative damage, which manifest itself as renal failure and hepatic centrilobular necrosis. This study aimed to investigate the effect of L-arginine on some haematological and biochemical indices of paracetamol- intoxicated Wistar rats. Twenty-five wistar rats with an average weight of 88.4g were randomly allocated into five groups of 5 rats each. Group A (control) fed with feed and water, Group B (L-arginine) administered 60 mg kg body weight, Group C (Paracetamol) administered 1000 mg kg body weight, Group D (L-arginine/paracetamol) administered 60 mg kg and 1000 mg kg body weight and Group E (High dose L-arginine/ paracetamol) administered 120 mg kg b-wt and 1000 mg kg body weight. Treatment was daily, per oral and lasted for 14 consecutive days. Results of biochemical parameters (Total-bilirubin, Creatinine and MDA) showed significant increase (p<0.05) in paracetamol treated groups when compared to the control. However, treatments with L-arginine did not abate their level. Results of K+ and Cl- showed significant (p<0.05) reduction in groups treated with paracetamol when compared with the control. Treatment with L-arginine, for K+, at different doses after paracetamol intoxication was insignificant while Cl- was increased by treating with L-arginine dose dependently. RBC value in all treated groups showed significant (p<0.05) decrease when compared to the control. Hb and PCV values were non-significant (p>0.05) in the paracetamol treated group when compared to the control. L-arginine treated group for PCV was also non-significant (p>0.05) when compared to the control. However, Hb level of L-arginine control group and the treated groups showed significant (p<0.05) increase when compared to the control. WBC level showed significant (p<0.05) increase in paracetamol group when compared to the control. Treatment with L-arginine showed significant (p<0.05) reduction when compared to the control. Glutathione peroxidase (GPx) activities in the Liver and Kidney of paracetamol group were significantly (p<0.05) reduced when compared to the control. Treatment with L-arginine showed significant (p<0.05) increase both in the Liver and Kidney when compared to the control and this confirmed the antioxidant properties of L-arginine. Catalase activity in all treated groups showed significant (p<0.05) decrease both in the Liver and Kidney when compared to the control. AST and ALT activities showed significant (p<0.05) increase in paracetamol group when compared to the control. However, treatment with L-arginine showed significant (p<0.05) decrease in the activity. ALP activity showed significant (p<0.05) reduction in paracetamol group when compared to the control. Treatment with L-arginine also recorded significant decrease when compared to the control. Intoxication with paracetamol at 1000 mg kg body weight showed a reduction in weight gain (12.04 compared to the control (25.46±4.91) and arginine group (24.70±3.95). Histological examination reveals that treatment of paracetamol intoxicated groups with L-arginine at 60 mg kg showed normal hepatic histo-architecture. However, treatment with L-arginine at 120 mg kg presented a coagulative necrosis of the centrilobular and mid-zonal hepatocytes with moderate infiltration of phagocytic mononuclear leukocytes. Kidney section of all treatment groups showed normal renal histo-architecture, with normal renal tubules. Sections of the testis collected from all treatment groups showed normal testicular histo-architecture with normal seminiferous tubules. This study therefore suggests that L-arginine could be used as a therapeutic agent in restoring paracetamol induced haemato, hepato and renal toxicities in rats.
TABLE OF CONTENTS
Title
page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Table
of Contents vi
Abbreviations vii
List
of Tables viii
List
of Figures
ix
List
of Plates
x
Abstract xi
CHAPTER
ONE: INTRODUCTION
1.1
Background of Study 1
1.2
Aim of Study 4
1.3
Objectives 4
1.4
Justification 4
CHAPTER
TWO: LITERATURE REVIEW
2.1
Overview of Paracetamol
(Acetaminophen) 5
2.2
Metabolism of Paracetamol 6
2.3
Mechanism of Action of Paracetamol 10
2.4
Prostaglandin Inhibition 10
2.4.1
Serotoninergic pathway activation 12
2.4.2
Endocannabinoid enhancement 12
2.4.3
Nitric oxide 14
2.5
Mechanisms of Protection of
Paracetamol Toxicity 14
2.5.1
Preventing covalent binding 14
2.5.2
Scavenging of reactive oxygen and
peroxynitrite 15
2.5.3
Mitochondrial energy substrates 16
2.6
History of L-Arginine 16
2.7.
Physical and Chemical Properties of
L-Arginine 17
2.8.
Major Roles of L-Arginine Amino Acid 17
2.9.
Metabolism of L-Arginine 18
2.10.
Arginine Biochemistry 19
2.11.
Arginine Transport 20
2.12.
Mechanism of Action of L-Arginine 21
2.13.
Clinical Pharmacology of L-Arginine 22
2.13.1.
L-arginine and exercise performance 22
2.13.2
L-arginine and vascular senescence 24
2.13.3
L-arginine and asthma 24
2.13.4
L-arginine and vasomotor
function/hypertension 26
2.13.5
L-arginine and cancer 27
2.13.6
L-arginine and congestive heart failure 28
2.13.7
L-arginine and erectile dysfunction 29
2.14.
Safety, Tolerance and Side Effects of
L-arginine 29
2.15.
Liver Function Test 30
2.15.1.
Serum bilirubin 30
2.15.2.
Alanine amino transferase (ALT) 31
2.15.3.
Aspartate amino transferase (AST) 31
2.15.4
Alkaline phosphatase (ALP) 31
2.16 Kidney Function Test 32
2.16.1
Blood urea nitrogen test (BUN) 32
2.16.2
Urea 32
2.16.3
Creatinine 33
2.16.4
Serum electrolytes 33
2.16.5
Sodium 33
2.16.6
Potassium 34
2.16.7
Chloride 34
2.17
Hematological Parameters 35
2.17.1
Red blood cells (RBC) 35
2.17.2
White blood cells (WBC) 35
2.17.3
Blood platelets 36
2.17.4
Packed cell volume (PCV) 36
2.17.5
Hemoglobin (Hb) 36
2.18
Oxidative Stress Markers 36
2.18.1
Glutathione reductase (GRd) 37
2.18.2
Glutathione peroxidase (GPx) 38
2.18.3
Catalase 38
2.18.4
Superoxide dismutase (SOD) 38
CHAPTER
3: MATERIALS AND METHODS
3.1
Materials 39
3.1.1 Equipments
39
3.1.2 Chemicals and reagents 39
3.2 Methods
40
3.2.1 Animals handling and experimental design 40
3.2.2 Blood collection and separation 41
3.2.3 Preparation of Liver and Kidney Homogenate
for AMP-Kinasa activity 41
3.2.4 Body, relative liver, testis and kidney
weight 41
3.2.5 Determination of urea concentration 42
3.2.6 Determination of creatinine concentration
43
3.2.7
Determination of serum potassium ion
concentration 44
3.2.8 Determination of chloride concentration 45
3.2.9
Determination of alanine transferase
(ALT)
46
3.2.10 Determination of aspartate amino transferase
(AST)
46
3.2.11 Determination of total serum protein
47
3.2.12 Determination of total bilirubin
47
3.2.13 Determination of alkaline phosphatase (ALP) 48
3.2.14 Catalase assay 49
3.2.15
Glutathione estimation 50
3.2.16 Estimation of glutathione peroxidase
50
3.2.17 Superoxide dismutase assay (SOD) 51
3.2.18 Determination of lipid peroxidation
(Malondialdehyde)
52
3.2.19
Determination of erythrocyte count 52
3.2.20
Determination of total leucocyte 53
3.2.21
Packed cell volume estimation 55
3.2.22
Determination of hemoglobin 55
3.2.23
Statistical analysis 56
3.2.24 Histological study 56
3.2.25 Tissue preparation 56
3.2.26 Slide examination and photomicrography 57
CHAPTER
4: RESULTS AND DISCUSSION
4.1 Results 58
4.1.1 Effects of L-arginine on body weight of
paracetamol intoxicated
wistar rats 58
4.1.2 Table of the effects of L-arginine on body
weight of paracetamol
intoxicated
wistar rats 59
4.1.3 Effects of L-arginine on organ weight of
paracetamol intoxicated
wistar
rats 60
4.1.4
Table of the effects of L-arginine on
organ weight of experimental
rat groups 61
4.1.5 Effects of L-arginine on kidney function
parameters of paracetamol
intoxicated
wistar rats 62
4.1.6 Table of the effects of L-arginine on kidney
function parameters of
paracetamol intoxicated wistar rats 63
4.1.7 Effects of L-arginine on liver function
markers of paracetamol
intoxicated
wistar rats 64
4.1.8
Table of the effect of L-arginine on
liver function markers of
paracetamol intoxicated wistar rat. 66
4.1.9 Effect
of L-arginine on serum levels of oxidative stress parameters
in hepatic and
renal function of paracetamol intoxicated wistar rat 67
4.1.10 Table of the effect of L-arginine on serum levels of oxidative
stress
Parameters in
hepatic and renal function of paracetamol intoxicated
wistar rat 69
4.1.11 Effect of L-arginine on haematological parameters of paracetamol
intoxicated wistar
rat 70
4.1.12 Table of the effect of L-arginine on haematological parameters of
paracetamol
intoxicated wistar rat 71
4.1.13 Histopathological examination 72
4.1.13.1 Liver photomicrography 72
4.1.13.2 Kidney photomicrography 76
4.1.13.3 Testes photomicrography 81
4.2 Discussion 87
CHAPTER
5: CONCLUSION AND RECOMMENDATION
5.1
Conclusion 94
5.2
Recommendation 94
References 95
Appendix 120
ABBREVIATIONS
NAPQI N-acetyl-p-benzo-quinone
imine
CNS
Central nervous system
NO
Nitric oxide
TAC Total
antioxidant capacity
SOD
Superoxide dismutase
GSH
Glutathione
AST Aspartate
amino transferase
ALT
Alanine amino transferase
ALP
Alkaline phosphatase
GPx
Glutathione peroxidase
GRd
Glutathione reductase
MDA
Malondialdehyde
RBC
Red blood count
WBC
White blood count
PCV
Packed cell volume
Hb
Hemoglobin
NSAID
Non steroidal anti-inflammatory drugs
UGT UDP-glucurosyl
transferase
SULT
Sulfotransferase
ATP
Adenosine triphosphate
AIF Apoptosis-inducing
factor
EndoG
Endonuclease G
NK
Natural killer
NKT Natural
killer T-cells
DAMP
Damage associated molecular pattern
COX
Cyclooxyginase
PGHs Prostaglandin
H2 synthetase
POX
Peroxidase
PG
Prostaglandin
5-HT3 5-hydroxytryptamine
type 3
FAAH
Fatty acid amide hydrolase
AM404
N-arachidonylphenolamine
TNF-a Tumor
necrosis factor-alpha
HGH
Human growth hormone
NOS
Nitric oxide synthase
OAT
Ornithine aminotransferase
ODC
Ornithine decarboxylase
P5C
Pyrroline 5-carboxylate
CAT
Cationic amino acid transporter
iNOS
Inducible nitric oxide synthase
eNOS
Endogenous nitric oxide
synthase
hCAT
human cationic amino acid transporter
SLC7A
Solute carrier family 7 subfamily A
CVD Cadiovascular
diseases
BP
Blood pressure
MDSCs
Myeloid –derived suppressor cells
CHF Congestive
heart failure
ED
Erectile dysfunction
LFT
Liver function test
BUN Blood
urea nitrogen
NKF
Natural kidney foundation
Na
Sodium
K Potassium
Cl
Chloride
EVF
Erythrocyte volume function
Ht
or Hct
Haematocrit
ROS
Reactive oygen species
CAT
Catalase
GSSG
Oxidized glutathione
NADPH
Nucleotide diphosphate
EDTA
Diethylamine
NBT
Nitroblue tetrazolium
H202 Hydrogen
peroxide
CYP450
Cytochrome 450 enzyme
MPT
Mitochondrial membrane permeability transition
GSK3β
Glycogen synthase kinase 3 β
PKCα
Protein kinase C α
MAPKs
Mitogen activated protein kinase
MLK3
Mixed lineage kinase 3
ASK1
Apoptosis signal regulating kinase 1
MKK4
Mitogen-activated protein kinase kinase 4
sab
SH3 binding protein
RIPK1 Receptor interacting protein
kinase-1
JNK c-Junction N-terminal kinase
CAT2 Cationic amino acid transporter 2
SLC7A2 Solute carrier family 7 member 2
hCAT
Human cationic amino acid transporter
NMDA
N-methyl-D-aspartate
LIST OF TABLES
2.1 Classification
of cationic amino acid transporters 21
4.1.2 Effects of L-arginine on body weight of
paracetamol intoxicated
wistar rat 59
4.1.4
Effect of L-arginine on organ weight of
paracetamol intoxicated
wistar rat 61
4.1.6 Effect of L-arginine on kidney function
parameters of paracetamol
intoxicated wistar rat. 63
4.1.8 Effect of L-arginine on liver function
markers of paracetamol
intoxicated wistar rat. 66
4.1.10 Effect of L-arginine on serum levels of
oxidative stress parameters in
hepatic and renal function of
paracetamol intoxicated wistar rat. 69
4.1.12 Effect of L-arginine on hematological
parameters of paracetamol
intoxicated wistar rat. 71
LIST OF FIGURES
2.1. Pathway of the metabolism of paracetamol 9
2.2. Paracetamol metabolisms after an overdose 9
2.3. Role of paracetamol in inhibition of
prostaglandin production 11
2.4.
Conversion of paracetamol to AM404,
and endocannabinoid enhancement 13 2.5. Pathway of L-arginine metabolism 19
2.6. How L-arginine works in the body 26
2.7.
Imbalance between oxidant and antioxidant 37
LIST OF PLATES
1. Photomicrograph
of the liver section of rats in group A (Control group) magx400
72
2. Photomicrograph
of the liver section of rats in group B (Arginine group) magx400 73
3. Photomicrograph
of the liver section of rats in group C (Paracetamol group) magx400 74
4. Photomicrograph
of the liver section of rats in group D (Arg and para group) magx4 75
5. Photomicrograph
of the liver section of rats in group E (High dose Arg
and para group) magx400 76
6. Photomicrograph
of the kidney section of rats in group A (Control group) magx400 77 7. Photomicrograph
of the kidney section of rats in group B (Arginine group) magx400 78
8. Photomicrograph
of the kidney section of rats in group C (Paracetamol group)
magx400 79
9 Photomicrograph
of the kidney section of rats in group D (Arg and Para group)
magx400 80
10. Photomicrograph
of the kidney section of rats in group E (High dose Arg
and Para group) magx400 81
11. Photomicrograph
of the testis section of rats in group A (Control group) magx400 82
12. Photomicrograph
of the testis section of rats in group B (Arginine group) magx400 83
13. Photomicrograph
of the testis section of rats in group C (Paracetamol group)
magx400 84
14 Photomicrograph
of the testis section of rats in group D (Arg and Para group)
magx400 85
15.
Photomicrograph of the testis section of rats in group E (High dose Arg
and
Para group) magx400 86
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND OF STUDY
Paracetamol (acetaminophen), a popular and
commonly used analgesic and antipyretic drugs around the world, was discovered
100 years ago (Ghaffar and Naser, 2014). Due to its availability, incident
of accidental and intentional abuse are numerous. As a result of the high rate
of abuse, paracetamol has been described as one of the most common cause of
liver failure (Larson et al., 2005;
Iyanda and Adeniyi, 2011). Paracetamol is metabolized in the liver via three pathways-glucuronidation,
sulfation and hepatic cytochrome P450 enzyme system. At intoxicated dose, paracetamol causes hepatic
centrilobular necrosis (Iyanda and Adeniyi, 2011), which has been
linked with excessive generation of the highly toxic metabolite N-acetyl-P-benzo-quinone
imine (NAPQI). Paracetamol is oxidatively transformed to N-acetyl-P-benzo-quinone imine (NAPQI) by the Cytochrome P450 enzyme
system particularly the CYP450 2E1 (Hwang et
al., 2007; Oyedepo, 2014). Endogenous glutathione binds to NAPQ1 and
detoxifies it to a non toxic metabolite (Mercapturic acid) which is excreted in
urine. However, at toxic level, hepatic glutathione depletion occurs when NAPQ1
formation exceeds the available supply of glutathione (Oyedepo, 2014). The
undetoxified NAPQ1 eventually binds to cellular macromolecules (Park et al., 2005; Iyanda and
Adeniyi, 2011) like cellular proteins resulting in impairment in mitochondrial
respiration (Yuan and Kaplowitz,
2013), opening of the mitochondrial permeability transition pores (Jaeschke et al., 2012), elevation of the
oxidative stress (Yuan and Kaplowitz, 2013) as well as hepatic necrosis (Vidhya and Mary, 2012). The risk of paracetamol toxicity increases
with malnutrition (Majeed et al.,
2013), application of paracetamol combined with drugs inducing cytochrome P450
(Waring, 2012, Stirnimann et al., 2010).
Paracetamol mechanism of action is dependent
on the inhibition of prostaglandin and other pro-inflammatory chemical
synthesis that takes place in the central nervous system (CNS) which blocks
pain impulse generation. It provides relief from mild to moderate pain and
fever (Vidhya and Mary, 2012; Marta and Jerzy 2014). In addition, paracetamol interferes with nociception
associated with spinal NMDA receptor activation and as well inhibit spinal
nitric oxide (NO) mechanism (Brain, 2008), therefore, the small amount of NO
released by nitroparacetamol appears to have minimal effect on central pain
mechanism, (Deeb., 2006; Brain, 2008).
The molecular pathway of paracetamol is the
subject of extensive investigation. The paracetamol metabolite, NAPQ1, is a
mitochondrial toxin. Mitochondrial toxicity and the generation of reactive
oxygen species (ROS) results in the activation of signaling molecules such as
Receptor interacting protein kinase 1 (RIPK1), Glycogen synthase kinase 3 β (GSK3β), Protein kinase Cα (PKCα), Mixed
lineage kinase 3 (MLK3), Apoptosis signal regulating kinase 1 (ASK1), and c-Junction
N terminal kinase (JNK) (Dara et al.,
2012 and Dara et al., 2017). This
cascade of signaling event ultimately results in the phosphorylation of JNK and
its translocation to mitochondria where it binds to SH3 binding protein 5 (Sab)
and results in the release of a protein phosphatase that inactivates
intermitochondria src (Huo et al.,
2017). However, certain amino acids of interest (such as L-arginine) have shown
close relationship with the important signal molecule nitric oxide (NO), which
plays important roles in many physiological processes in the human body such
as; neurotransmission, vasorelaxation, cytotoxicity and immunity (Egbuonu et al., 2010c).
L-Arginine,
an amino acid found in many foods, such as dairy products, meat, poultry, and
fish. It is traditionally classified as a semi-essential or conditionally
essential amino acid; it is essential in children and non-essential in adults
(Mohamed, 2010). It was first discovered by Schulze in 1886, when it was
isolated from lupin seedlings. It plays a role in several important mechanisms
in the body; ‘in cell division’, L-arginine improves mitochondrial function and
reduces apoptosis of bronchial
epithelial cells after injury induced by allergic airway inflammation (Mabalirajan et al., 2010), it improves
healing of wounds (Mohamed, 2010, Alan et al.,2016), the removal of ammonia from the body, immune
function, and the secretion of important hormones ( Fayh et al., 2007, Zajac et al., 2010 and Davi et
al., 2014). L-Arginine is required for synthesis of proteins and serves as
a precursor for synthesis of creatine, agmatine, urea, polyamines, proline,
glutamate (Morris, 2006). The body also uses arginine to synthesize nitric
oxide, which relaxes the blood vessels (vasodilation) (Egbuonu et al., 2010c). Based on this, L-arginine
has been proposed as a treatment for various heart conditions (Pahlavani et al., 2014).
It
also decreases blood pressure, heart rate and improves cardiac performance in
congestive heart failure, hypertensive and type II diabetic patients (Martina et al., 2008; Vasdev
and Gill, 2008 ; Sara et al.,2016). In healthy humans, L-Arginine
plays a critical role in the regulation of autonomic cardiovascular control in
humans through nitric oxide (NO) synthesis (Ogungbemi et al., 2013).
Evidence suggests that arginine supplementation may be an effective way
to improve endothelial function in individuals with diabetes mellitus (Mohamed,
2010). It also increases vasodilation, thereby elevating blood flow to the
exercising muscles and enhancing metabolic response to exercise (Koppa et al., 2009; Alvares et al., 2012). Some previous
experimental and clinical studies indicated that L-Arginine can improve antioxi
dant status (Lucotti et al., 2006; Tripati and Misra, 2009). In a recent study,
supplementation with 3gr/day L-Arginine increased serum total antioxidant
capacity (TAC) level in patients with prediabetes after 8 weeks (Siavash et al.,2014) Tripati and Misra (2009),
demonstrated that L-Arginine supplementation increased superoxide dismutase
(SOD) activities in patients with ischemic heart disease. Also, Kochar and
Umathe (2009), reported that L-arginine supplementation in diabetic rats
increased GSH and SOD level. However, L-Arginine supplementation improves antioxidant defenses
through L-arginine/nitric oxide pathways in exercised rats. Therefore, since
L-arginine is the only physiologically significant substrate for the synthesis
of nitric oxide (NO), a signaling molecule in cardiovascular system, it is
necessary to increase its availability in the body. Based on this, L- arginine
and paracetamol is studied to establish whether arginine would amilorate effects
induced by paracetamol toxicity.
1.2 AIM
OF STUDY
The aim of this study was to determine the effect of L-arginine on some
haematological and biochemical indices of paracetamol intoxicated Wistar
rats.
1.3 OBJECTIVES
The objectives of this study were to determine the effect of L-arginine
on some haematological and biochemical indices of paracetamol- intoxicated
Wistar rats on the following biochemical parameters:
-Liver fuction parameters including; AST,
ALT, ALP, albumin, total protein, and bilirubin.
-Kidney function parameters including; serum
creatinine, serum urea and electrolytes
-Oxidative stress parameters including;
Superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase
(GRD), catalase, malondialdehyde (MDA).
-Haematological parameters including; RBC,
WBC, Hb, PCV, of paracetamol intoxicated rats.
-Histological changes of some organs viz: liver,
kidney and testis.
1.4 JUSTIFICATION
This study is justified based on the
therapeutic qualities of L-arginine and its ability to synthesize nitric oxide
(NO) (Vasodilator) in relation to its possible influence on oxidative damage
induced by paracetamol intoxication.
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