ABSTRACT
The research aimed at evaluating the biochemical role of vitamin C and E in ciprofloxacin and gentamicin co-administration. Twenty five female wistar rats weighing (85-140) g were used for the study. Rats were divided into five groups (N=5). The animals received the following treatments: Group 1 received only normal feed and water. Group 2-5 rats were co-administered ciprofloxacin (7.14 mg/kg b.w) and gentamicin (1.14 mg/kg b.w). Group 2 served as the positive control while groups 3-4 received vitamin C (100 mg/kg) and Vitamin E (14.29 IU/kg) respectively. Group 5 received 100 mg/kg vitamin C and 14.29 IU/kg vitamin E. All administration lasted for 11 days. Blood and organ samples were collected on the 12th day and analyzed using standard analytical procedures. The result showed that the co-administration of ciprofloxacin and gentamicin caused a significant (p<0.05) reduction in hemoglobin (Hb) level, packed cell volume (PCV) and red blood cell (RBC) count of group 2 rats compared with the normal control. Total white blood cell (TWBC) count was significantly (p<0.05) increased whereas no significant effect was observed on platelet count of group 2 rats when compared with the normal control. Serum urea, creatinine and total bilirubin were significantly (p<0.05) increased in group 2 rats when compared with the normal control. Equally, alanine amino transferase (ALT) and aspartate amino transferase (AST) showed no significant (p>0.05) change while alkaline phosphatase (ALP) activity, serum total protein, albumin and cholesterol significantly (p<0.05) reduced in group 2 rats when compared with the normal control. Superoxide dismutase (SOD) and catalase activities significantly (p<0.05) reduced whereas glutathione (GSH) concentration, serum malondialdehyde (MDA) concentration, C-Reactive protein concentration and creatine kinase (C.K) activity were significantly (p<0.05) increased in group 2 rats when compared with the normal control. Histological examination of vital organs showed diverse lesions in the brain, kidney and heart of group 2 rats whereas no effect was observed on the liver. All vitamin treatments (vitamin C, E and C+E) significantly (p<0.05) increased the Hb level whereas TWBC counts, serum MDA concentration, CRP concentration and C.K activity was significantly (p<0.05) reduced by same treatment when compared with group 2 rats. Additionally, RBC counts and %PCV were significantly increased by only vitamin E and C+E treatments when compared with group 2. Serum urea and creatinine concentration were significantly (p<0.05) reduced in all the vitamin treated groups. Additionally, in all the vitamin treated groups, serum total protein, albumin, total cholesterol, SOD and catalase activity were significantly (p<0.05) increased whereas GSH significantly reduced when compared with group 2 rats. A significant (p<0.05) reduction in total bilirubin was observed only in the vitamin C and E co-administered groups. The observed renal and heart lesions were attenuated in the vitamin treated groups whereas only the administration of vitamin C and C+E respectively restored the histo-architecture of the brain. In conclusion, co-administration of ciprofloxacin and gentamicin caused some adverse effects. These adverse effects were ameliorated by treatment with antioxidants (vitamin C and E).
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables xii
List of Figures xiii
List of Plates xiv
Abstract xv
CHAPTER 1:
INTRODUCTION
1.1 Background
of the Study 1
1.2
Justification of the
Study 5
1.3 Aim
of the Study 5
1.4 Objectives
of the Study 6
CHAPTER 2: LITERATURE
REVIEW
2.1
Antibiotics 7
2.2 Classification
of Antibiotics 8
2.2.1 Beta-lactams 8
2.2.1.1 Penicillins 9
2.2.1.2 Cephalosporin 9
2.2.1.3 Carbapenems 10
2.2.2 Macrolides 10
2.2.3 Tetracycline 11
2.2.4 Quinolones 12
2.2.5 Aminoglycoside 12
2.2.6 Sulphonamides 13
2.2.7 Oxazolidinones 14
2.3 Mode
of Action of Antibiotics 14
2.3.1 Inhibition of cell wall
synthesis 15
2.3.2 Inhibition of nucleic acid
synthesis 16
2.3.3 Inhibition of protein synthesis 16
2.3.4 Blockage of key metabolic pathways 18
2.3.5 Disorganizing
of the cell membrane 18
2.4 Mechanisms of Antibiotic Resistance 19
2.4.1 Antibiotic
inactivation 19
2.4.1.1 Antibiotic inactivation by hydrolysis 19
2.4.1.2 Antibiotic inactivation by group transfer 20
2.4.1.3 Antibiotic inactivation by redox process 20
2.4.2 Complete replacement or by-pass of target
site 20
2.4.3 Resistance to protein synthesis interference 21
2.4.4 Resistance to DNA synthesis interference 22
2.4.5 Ability of resistant bacteria to decrease
permeability of antibiotics to
target
sites 22
2.4.6 Acquisition
and efficient use of efflux pumps 23
2.5 Gentamicin 25
2.5.1 Adverse effects of gentamicin 26
2.5.1.1 Nephrotoxic effect of gentamicin 26
2.5.1.2 Tubular effect of gentamicin 27
2.5.1.3 Glomerular effect of gentamicin 29
2.5.1.4 Vascular effect of gentamicin 30
2.5.1.5 Central role of oxidative stress and
inflammation in gentamicin toxicity 31
2.6 Ciprofloxacin 32
2.6.1 Hepatotoxic effect of ciprofloxacin 33
2.6.2 Cardiotoxicity of ciprofloxacin 34
2.6.3 Neurotoxicity effect of ciprofloxacin 35
2.6.4 Role
of oxidative stress in inflammation 36
2.7 Ameliorative Roles of Antioxidant
Vitamins in Oxidative Stress
(Health
Disorders) 37
2.7.1 Vitamin C 37
2.7.2
Vitamin E (α-tocopherol)
and its antioxidant activity 38
CHAPTER 3: MATERIALS AND METHODS
3.1 Materials 40
3.1.1 Antibiotics 40
3.1.2 Antioxidants 40
3.1.3
Experimental animals 40
3.1.4
Lists of equipment used 41
3.1.5 List of chemicals/reagents used 41
3.2 Methods 42
3.2.1 Collection of antibiotics and vitamins used
for this study 42
3.2.2 Collection of experimental animals 43
3.2.3
Dose selection and
preparation 43
3.3
Experimental Design 44
3.3.1 Animal grouping 44
3.4 Serum Analysis 45
3.4.1 Determination of red blood cell (RBC) 45
3.4.2
Determination of
hemoglobin concentration (Hb) 46
3.4.3
Determination of
percentage packed cell volume (PCV) 46
3.4.4
Determination of white
blood cell counts (WBC) 47
3.4.5
Determination of platelet
counts 47
3.4.6 Determination of total protein concentration 48
3.4.7 Determination of serum albumin concentration 49
3.4.8 Assay of aspartate aminotransferase (AST)
activity 50
3.4.9 Assay of alanine amino transferase (ALT) activity 51
3.4.10 Assay of alkaline phosphatase (ALP) activity 52
3.4.11 Determination of total bilirubin 53
3.4.12 Determination of serum creatinine
concentration 54
3.4.13 Determination of serum urea concentration 54
3.4.14 Assay of serum creatine kinase activity 55
3.4.15 Determination of the concentration of serum
C-reactive protein (CRP) 57
3.4.16 Determination of serum cholesterol
concentration 58
3.4.17 Assay of superoxide dismutase (SOD) activity 58
3.4.18 Assay
for catalase activity 60
3.4.19 Determination
of malondialdehyde concentration 60
3.4.20 Determination
of reduced glutathione concentration 61
3.4.21 Histopathological
examination 62
3.4.21.1 Tissue preparation 62
3.4.21.2 Slide
examination 63
3.5 Statistical Analysis 63
CHAPTER 4: RESULTS AND
DISCUSSION
4.1 Results 64
4.1.1 Effects of vitamin C and E on body weight of
ciprofloxacin and
gentamicin
co-administration in wistar albino rats 64
4.1.2 Effects of vitamin C and E on haematological
parameters of
ciprofloxacin
and gentamicin co-administration in wistar albino rats 66
4.1.3 Effects of vitamin C and E on serum total
protein concentration of
ciprofloxacin
and gentamicin co-administration in wistar albino rats 69
4.1.4 Effects of vitamin C and E on serum albumin
concentration of
ciprofloxacin
and gentamicin co-administration in wistar albino rats 70
4.1.5 Effects of vitamin C and E on aspartate
aminotransferase (AST) activity
of
ciprofloxacin and gentamicin co-administration in wistar albino rats 71
4.1.6 Effects of vitamin C and E on alanine
aminotransferase activity of
ciprofloxacin
and gentamicin co-administration in wistar albino rats 72
4.1.7 Effects of vitamin C and E on alkaline
phosphatase (ALP) activity of
ciprofloxacin
and gentamicin co-administration in wistar albino rats 73
4.1.8
Effects of vitamin C and
E on total bilirubin concentration of
ciprofloxacin
and gentamicin co-administration in wistar albino rats 74
4.1.9
Effects of vitamin C and
E on serum creatinine concentration of
ciprofloxacin
and gentamicin co-administration in wistar albino rats 75
4.1.10 Effects
of vitamin C and E on serum urea concentration of ciprofloxacin
and
gentamicin co-administration in wistar albino rats 76
4.1.11 Effects
of vitamin C and E on creatine kinase activity of ciprofloxacin
and
gentamicin co-administration in wistar albino rats 77
4.1.12 Effects
of vitamin C and E on serum C-Reactive protein (CRP)
concentration
of ciprofloxacin and gentamicin co-administration
in
wistar
albino rats 78
4.1.13 Effects
of vitamin C and E on total cholesterol (T. cholesterol)
concentration
of ciprofloxacin and gentamicin co-administration in
wistar
albino rats 79
4.1.14
Effects of vitamin C and
E on superoxide dismutase (SOD)
activity
of ciprofloxacin and gentamicin co-administration in wistar
albino
rats 80
4.1.15 Effects of vitamin C and E on catalase
activity of ciprofloxacin and gentamicin co-administration in wistar
albino rats 81
4.1.16
Effects of vitamin C and
E on glutathione concentration of ciprofloxacin
and
gentamicin co-administration in wistar albino rats 82
4.1.17 Effects
of vitamin C and E on serum malondialdehyde (MDA)
concentration
of ciprofloxacin and gentamicin co-administration in
wistar
albino rats 83
4.1.18 Histopathology examination 84
4.1.18.1
Photomicrograph showing rats kidney sections 84
4.1.18.2 Photomicrograph showing rats
liver sections 86
4.1.18.3 Photomicrograph showing rats
brain sections 89
4.1.18.4 Photomicrograph showing rats
heart sections 92
4.2 Discussion 95
CHAPTER 5:
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion 108
5.2 Recommendations 108
References 110
Appendices 129
LIST OF TABLES
4.1: Effects
of ciprofloxacin and gentamicin administration and treatment
with vitamins C
and E on body weight of treated rats 64
4.2
Haematological indices of
rats co-administered ciprofloxacin and
gentamicin, treated with vitamin C and E 66
LIST OF FIGURES
2.1 Mechanism
of action of antibiotics 15
2.2 Efflux system 24
2.3 Chemical structure of gentamicin 32
2.4 Chemical structure of ciprofloxacin 37
2.5 Chemical structure of vitamin C 38
2.6 Chemical structure of vitamin E 39
4.1 Total protein concentration of rats
co-administered ciprofloxacin and
gentamicin,
treated with vitamin C and E 69
4.2 Albumin concentration of rats
co-administered ciprofloxacin and
gentamicin,
treated with vitamin C and E 70
4.3
Aspartate
aminotransferase activities of rats co-administered
ciprofloxacin and gentamicin, treated with
vitamin C and E 71
4.4 Alanine
aminotransferase activities of rats co-administered ciprofloxacin 72
4.4
Alkaline
phosphatase (ALP) activities of rats co-administered
ciprofloxacin and gentamicin, treated with
vitamin C and E 73
4.6 Total
bilirubin concentration of rats co-administered ciprofloxacin and
gentamicin, treated
with vitamin C and E 74
4.7 Serum
creatinine concentration of rats co-administered ciprofloxacin and
gentamicin, treated
with vitamin C and E 75
4.8 Serum
urea concentration of rats co-administered ciprofloxacin and
gentamicin,
treated with vitamin C and E 74
4.9 Creatine
kinase activity of rats co-administered ciprofloxacin and
gentamicin,
treated with vitamin C and E 75
4.10 C-Reactive
protein (CRP) concentration of rats co-administered
ciprofloxacin and gentamicin,
treated with vitamin C and E 78
4.11 Total
cholesterol concentration of rats co-administered ciprofloxacin
and gentamicin, treated
with vitamin C and E 79
4.12 Superoxide
dismutase (SOD) activity of rats co-administered
ciprofloxacin and gentamicin,
treated with vitamin C and E 80
4.13 Catalase
activity of rats co-administered ciprofloxacin and gentamicin,
treated with
vitamin C and E 81
4.14 Glutathione
concentration of rats co-administered ciprofloxacin and
gentamicin,
treated with vitamin C and E 82
4.15 Malondialdehyde (MDA) concentration of rats
co-administered
ciprofloxacin
and gentamicin, treated with vitamin C and E 83
LIST
OF PLATES
4.1 Photomicrograph of rat kidney section of
group 1 rats that received
standard
feed and drinking water only 84
4.2 Photomicrograph showing kidney section of
ciprofloxacin and gentamicin
co-administered
rats (group 2) 84
4.3 Photomicrograph showing kidney section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin C (group 3) 85
4.4 Photomicrograph of kidney section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin E (group 4) 85
4.5 Photomicrograph of kidney section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin C and E (group 5) 86
4.6 Photomicrograph of rat liver section of
group 1 rats that received standard
feed
and drinking water only 86
4.7 Photomicrograph showing liver section of
ciprofloxacin and gentamicin
co-administered
rats (group 2) 87
4.8 Photomicrograph showing liver section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin C (group 3) 87
4.9 Photomicrograph of liver section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin E 88
4.10 Photomicrograph of liver section of
ciprofloxacin and gentamicin co-administered rats, treated with vitamin C and E 88
4.11 Photomicrograph of rat brain section of
group 1 rats that received
standard
feed and drinking water only 89
4.12 Photomicrograph showing brain section of
ciprofloxacin and gentamicin
co-administered
rats (group 2) 90
4.13 Photomicrograph showing brain section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin C (group 3) 90
4.14 Photomicrograph of brain section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin E 91
4.15 Photomicrograph of the brain section of
group 5 91
4.16 Photomicrograph of rat heart section of
group 1 rats that received
standard
feed and drinking water only 92
4.17 Photomicrograph showing heart section of
ciprofloxacin and gentamicin
co-administered
rats (group 2) 92
4.18 Photomicrograph showing heart section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin C (group 3) 93
4.19 Photomicrograph of heart section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin E (group 4) 93
4.20 Photomicrograph of heart section of
ciprofloxacin and gentamicin
co-administered
rats, treated with vitamin C and E (group 5) 94
CHAPTER 1
INTRODUCTION
1.1
BACKGROUND OF THE STUDY
Bacterial infections have been the
major cause of diseases throughout the history of human population. The
introduction of antibiotics was anticipated to counter this problem. However,
bacteria have evolved to develop resistance to available antibiotics (Arias and
Murray, 2009). Increasing bacterial infections and mortalities are some
negative attributes of antibiotic resistance (Arias and Murray, 2009).
Antibiotic resistance occurs when a drug loses its ability to inhibit bacterial
growth; the bacteria therefore continuously multiply even in the presence of
therapeutic levels of antibiotics (Toone, 2011). Antibiotic resistance exists
for both gram positive and gram negative bacteria. Because of their resistance
to common antibiotics and absence of new effective alternative, gram negative
bacteria top the priority list. Bacterial infections remains a leading killer
worldwide (Toone, 2011), as antibiotic resistance has continuously plagued the
effective control of this pandemic problem (Toone, 2011). Resistance to available
antibiotics is dominant; some other existing classes are no longer in use for
increasing number of bacteria species (Arias and Murray, 2009). Multi drug
resistant organisms like methicillin-resistant staphylococcus aureus (MRSA), vacomycin-resistant enterococci and certain gram negative
bacilli like Pseudomonas aeruginosa
(Pa), Acinetobacter baumanii (Ab) can
cause severe and lethal human infections. The new antibiotics produced annually
are unable to counter this development of bacteria resistance (Coates et al., 2011). The existing antibiotics
have lost their potency in managing infections. The invading pathogens may
acquire resistant genes enabling them produce enzymes like beta lactamase,
carbepenemase, express efflux system and modify the drug target (Tenover,
2006).
The alarming increase in drug
resistance rate observed in our era has necessitated the development and
evaluation of alternative ways to cope with drug resistance. Specifically, the
renewal of physician’s interest in older and neglected antibiotic agents as
well as the use of combination of antibiotic agents for the treatment of
bacterial infection has been considered as an important armament in the battle
against antibiotics resistance (Falagas et
al., 2008).
Antibiotic
combination therapy has found wide application especially in improving clinical
efficacy in patients where a given therapy is thought to have limitations when
used alone. Some arguments have it that the traditional strategy of antibiotics
discovery is flawed because the drug discovery process is exhausting and
costly. Therefore the introduction of an appreciable number of effective
antibiotics within a short period is almost impossible. In this regard, a
different approach is needed to replenish our armamentarium against resistant
bacteria (Kalan and Wright, 2011). The most promising strategy is to restore
the therapeutic strengths of the existing antibiotics (Kalan and Wright, 2011).
Combination
antibiotic therapy is used in critically ill patients due to the emergence and
wide spread of multidrug resistance organisms (MDR). Multidrug resistance could
be seen as the lack of sensitivity to at least one agent in three or more
antibiotic groups (Magiorakos et al.,
2012). Over time, carbapenem-resistant Enterobacteriaceae (CRE) has emerged as
one of the most notorious groups due to dissemination of Klebsiella pneumoniae carbapenemase (KPC) and other carbapenemase
subtypes like New Delhi metallo-β-lactamase (NDM1), via mobile genetic
elements(Gupta et al., 2011). Dual
coverage for these resistant enzyme producing organisms is by intuition
believed to be better by many physicians. Combinational antibiotic therapy with
different mechanism of action have been applied in the treatment of infections with the goal of producing a wider
spectrum preventing the emergence of drug resistant sub-population and
achieving a synergistic effect (Paul et
al., 2004).
Gentamicin
is an aminoglycoside antibiotic. It is effective against bacterial infections
(Hathorn et al., 2014). However,
their efficacy is affected by toxicity especially nephrotoxicity, which causes
kidney damage (Khoory et al., 1996).
Nephrotoxicity
appears in 10-25% of therapeutic courses in spite of rigorous patient
monitoring (Lopez-Novoa et al.,
2011). There is a strong relationship between the accumulation of
aminoglycosides within the renal cortex and pathogenesis of nephrotoxicity.
Histopathological findings have equally indicated tubular necrosis
(particularly proximal tubule), glomerular narrowing of Bowman’s capsule,
apoptosis (Martinez-Salgado et al.,
2007). Gentamicin equally induces a reduction in renal blood flow (Morales et al., 2002).
Gentamicin
generates reactive oxygen species (ROS) in the kidney (Banday, 2008).
Reactive oxygen species causes injury
and death cells in tissues like kidney, liver and lungs (Whaley and Sowers, 2012).
Ciprofloxacin
is a commonly used antibiotic for the treatment of bacterial infections. It is
a broad spectrum fluoroquinolone active against Pseudomonas aeruginosa-induced respiratory infections, acute or
chronic osteomyelitis or osteochondritis, multi-drug resistant gram negative
bacterial infections mostly in immune-compromised hosts, meningitis and so many
others (Saracoglu et al., 2009). It
can be used alone or in combination with other drugs including aminoglycosides
like gentamicin. Although this combination is not the first choice, it is
clinically used mostly in resistant cases of bacterial infections caused by Pseudomonas aeruginosa (causative agent
of urinary tract infections, skin infections, pneumonia), extended spectrum
beta lactamase infections, Klebsiella pneumonia (Mandeiila
et al., 2007).
Vitamins have
irreeplaceable role in almost all biochemical reactions. They are ideal
antioxidants capable of protecting tissues from oxidative stress (Cadenas
and Cadenas, 2002).
Vitamin E (α-Tocopherol)
is the primary membrane bound, lipid-soluble, chain-breaking antioxidant that protects
cell membranes against lipid peroxidation (Gulec
et al., 2006).
Pre-treatment of vitamin E has been reportedly beneficial in preventing
tissue damage in rats (Gurel et al.,
2005). The ameliorative effect of vitamin E on cypermethrin or
endotoxin-induced oxidative stress in rat tissues is suggestive of its
antioxidant activity (Atessahin et al.,
2005).
Vitamin C (ascorbic acid) supplementation has a remedial benefit
due to its ability to reduce oxidative stress by reacting with superoxide and
hydroxide radicals as well as alkyl, peroxyl and alkoxyl radicals, thereby neutralizing these radicals, thus stopping
the initiation and propagation of chain reaction (Buettner, 1993).
The
co-administration of two or more drugs is believed to be accompanied by a
variety of therapeutic implications ranging from opposition, alteration,
synergism, physical and chemical antagonism (Esimone
et al., 2002). Clinically
important interactions may occur in ciprofloxacin and gentamicin
co-administration leading to drug resistance or systemic overexposure that may
result in tissue toxicity.
The
safety or toxicity potentials of combinatorial administration of ciprofloxacin
and gentamicin have not been evaluated fully, although there have been reported
cases of nephrotoxicity with such administration (Zafar et al., 2013). Thus, there is need to explore further the
safety/toxicity outcomes of this therapy and the biochemical roles of
antioxidant vitamins (C and E) in such administration.
1.3
JUSTIFICATION
OF THE STUDY
In
recent years, increasing bacterial infections and mortality due to antibiotic
resistance have become rampant. Despite the availability of antimicrobial
therapy, only very little has changed over the last decade. This alarming
increase in resistance necessitated the development and evaluation of
alternative ways of curbing bacterial infections; thus, the combination of
antibiotic agents for the treatment of bacterial infections.
Ciprofloxacin
and gentamicin are two antibiotics with potency against bacterial infections.
Although their co-administration has found clinical applications in the
treatment of bacterial infections, their safety has not been fully ascertained.
The research therefore aims at evaluating their safety or toxicity profile and
the biochemical roles of antioxidant vitamins (C and E) in such
co-administration.
1.3 AIM OF THE STUDY
The
study was aimed at evaluating the biochemical roles of vitamin C and E in
ciprofloxacin and gentamicin co-administration in rats.
1.4 OBJECTIVES OF THE STUDY
The
study was designed to achieve the following specific objectives.
i.
To determine the effects
of vitamin C and E on liver function indices (Aspartate aminotransferase,
alanine aminotransferase, alkaline phosphatase, total bilirubin, total protein,
albumin and cholesterol concentration) in ciprofloxacin and gentamicin
co-administered rats.
ii.
To determine the effects
of vitamin C and E on urea and creatinine concentration) in ciprofloxacin and
gentamicin co-administered rats.
iii.
To determine the effects
of vitamin C and E on hematological indices (Packed cell volume, red blood cell
count, white blood cell count, hemoglobin concentration and platelet counts) in
ciprofloxacin and gentamicin co-administered rats.
iv.
To determine the effects
of vitamin C and E on the antioxidant parameters (catalase and superoxide
dismutase activities, glutathione and malondialdehyde concentration) in
ciprofloxacin and gentamicin co-administered rats.
v.
To determine the effects
of vitamin C and E on the concentration of serum C - reactive protein
concentration and creatine kinase activity in ciprofloxacin and gentamicin
co-administered rats.
vi.
To determine the effects
of vitamin C and E on the histological architecture of sections of kidney,
liver, heart and brain of ciprofloxacin and gentamicin co-administered rats
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