EVALUATION OF SOME APOPTOTIC MARKERS IN SEMINAL PLASMA OF RATS FOLLOWING ADMINISTRATION OF CRUDE OIL AND AGERATUM conyzoides

EVALUATION OF SOME APOPTOTIC MARKERS IN SEMINAL PLASMA OF RATS FOLLOWING ADMINISTRATION OF CRUDE OIL AND AGERATUM conyzoides

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ABSTRACT

This study was designed to evaluate some apoptotic markers in seminal plasma of rats following crude oil and Ageratum conyzoides administration. Twenty male Wistar rats were divided into four groups with five rats in each group. Group I rats were orally administered 3ml/kg body weight of normal saline, group II animals were orally administered 748.33mg/kg body weight ethanol leaf extract of A. conyzoides, group III animals were administered 3ml/kg body weight of Nigerian Bonnylight crude oil (NBLCO) and group IV animals were co-administered 748.33mg/kg body weight A. conyzoides and NBLCO for 28 days. NBLCO administration to group III significantly increased the plasma level of caspases 3, 9 and cytochrome c compared with the control (group I) and extract groups (P<0.05). Also, co-administration of A. conyzoides and NBLCO to group IV animals significantly decreased the plasma level of caspases 3, 9 and cytochrome c compared with group III (NBLCO group) (P<0.05). This study showed that A. conyzoides intervention could mitigate apoptotic process indicated by low caspases 3, 9 and cytochrome c concentration.      

TABLE OF CONTENTS

Pages

Title Page    -        -        -        -        -        -        -        -        -        -        i

Certification         -        -        -        -        -        -        -        -        -        -        ii

Dedication  -        -        -        -        -        -        -        -        -        -        iii

Acknowledgements        -        -        -        -        -        -        -        -        iv

Abstract      -        -        -        -        -        -        -        -        -        -        v

Table of Contents -        -        -        -        -        -        -        -        -        vi

List of Tables       -        -        -        -        -        -        -        -        -       

List of Figures

CHAPTER ONE: INTRODUCTION

1.1     Background of the Study        -        -        -        -        -        -        -        1

1.2     Research Question                   -        -        -        -        -        -        -        3

1.3     Objective of the Study   -        -        -        -        -        -        -        3

1.4     Justification of Study     -        -        -        -        -        -        -        4

CHAPTER TWO: REVIEW OF RELATED LITERATURE

2.1     Crude Oil    -        -        -        -        -        -        -        -        5

2.2     Brief History        -        -        -        -        -        -        -        5

2.3     Composition and Formation   -        -        -        -        -        7

2.4     Source of crude oil in the environment       -        -        -        9

2.5     Toxicity of Petroleum/Crude Oil       -        -        -        -        11

2.6     Influences of Prolonged Exposure to Crude Oil   -        -        15

2.7     Human Semen      -        -        -        -        -        -        -        18

2.8     Reactive Oxygen Species (ROS)       -        -        -        -        22

2.9     Apoptosis   -        -        -        -        -        -        -        -        24

2.10   Apoptosis Signal Pathway      -        -        -        -        -        25

2.11   Apoptosis Induction by ROS  -        -        -        -        -        26

2.12   Cytochrome C-Caspase System        -        -        -        -        -        26

2.13   Caspases cascade in apoptosis         -        -        -        -        -        31     

2.14   Ageratum conyzoides      -        -        -        -        -        -        32

2.15   Ethnopharmacology      -        -        -        -        -        -        35

CHAPTER THREE: MATERIALS AND METHODS

3.1     Materials and Apparatus Used         -        -        -        -        -        37

3.2     Crude Petroleum and Chemical Reagents   -        -        -        37

3.3     Collection of Plant Material    -        -        -        -        -        38

3.3.1  Preparation of leave extract    -        -        -        -        -        38

3.3.2  Acute toxicity test          -        -        -        -        -        -        -        39

3.4     Experimental animals    -        -        -        -        -        -        40

3.4.1  Experimental Design and Treatment of Animals -        -        40

3.5     Collection of Blood Sample for Analysis   -        -        -        41

3.8     Statistical Analysis        -        -        -        -        -        -        47

CHAPTER FOUR: RESULTS AND DISCUSSION

4.1     Results        -        -        -        -        -        -        -        -        48

4.1.1  Result of the level of caspases 3 following crude oil

and Ageratum conyzoides        administration in rats    -        -        48

4.1.2  Results of plasma caspase 9 level     -        -        -        -        50

4.1.3  Cytochrome C plasma level    -        -        -        -        -        52

4.2     Discussion  -        -        -        -        -        -        -        -        54

CHAPTER FIVE: SUMMARY, CONCLUSION AND RECOMMENDATIONS

5.1     Summary    -        -        -        -        -        -        -        -        56

5.2     Conclusion -        -        -        -        -        -        -        -        57

5.3     Recommendations         -        -        -        -        -        -        -        57

References  -        -        -        -        -        -        -        -        58


LIST OF TABLES

Table 2.1:   Solubility (in water) of some common components easily

analysed in crude oil      -        -        -        -        -        -        9

Table 2.2:   Sources and Estimates of crude oil or its products released

into the marine environment   -        -        -        -        -        11

Table 4.1:   Comparison between the mean values of the groups     -        54


LIST OF FIGURES

Figure 2.1:            Showing the Diagram of Ageratum conyzoides     -        34

Figure 4.1.1:         Comparing caspases 3 concentration in NBLCO and

extract of Ageratum conyzoides                  -        -        -        49

Figure 4.1.2:         Comparing caspases 9 concentration in BLCO and

extract of Ageratum Conyzoides        -        -        -        51

Figure 4.1.3:         Comparing cytochrome C concentration in NBLCO

and extract of Ageratum conyzoides -        -        -        53


CHAPTER ONE

INTRODUCTION

1.1     Background of the Study

Cytochrome complex or Cyt C is a small hemoprotein found loosely associated with the inner membrane of the mitochondria. It belongs to the cytochrome C family of proteins. Cytochrome C is an essential component of electron transport chain, where it carries one electron. It is capable of undergoing oxidation and reduction but does not bind oxygen. It transfers electrons between complexes III (coenzyme Q-Cyt-C reductase) and IV (Cyt C Oxidase). In humans, cytochrome C is encoded by the CyCS gene (Tafani et al., 2002). Cytochrome C also has an intermediate role in apoptosis, a controlled form of cell death used to kill cells in the process of development or in response to infection or DNA damage (Liu et al., 1896). Cytochrome C binds to cardiolipin in the inner mitochondrial membrane preventing it from being released out of the mitochrondria and initiating apoptosis.

The release of cytochrome C from the mitochrondria is a key initiative step in the apoptotic process. Once in the cytosol, cytochrome C interacts with its adaptor molecule, Apaf – 1 (apoptotic protease activating factor – 1), resulting in the recruitment, processing and activation of caspases. Caspases (Cysteinyl aspartate- specific proteinases) play a central role mostly in the regulation of apoptosis in the seminiferous epithelium. They are expressed as inactive proenzymes and participate in a cascade triggered in response to pro-apoptotic signals.

To date, 14 caspases have been implicated in the human apoptotic pathway cascade.  Caspase activation occurs by self proteolysis and/or results from the actions of other caspases or regulator proteins. The caspases are under the influence of diverse regulators including activators and inhibitors (Reed, 2000). Functionally, there are two types of caspases: initiator caspases (CP 8, CP9, CP10) and effector caspases (CP3, CP6 and CP7). Among the effector caspases, activated caspase 3 is said to be the most important effector caspase. Its activation hallmarks the point of no return in programmed cell death (PCD) signaling (Earnshaw et al., 1999). Caspase 9 can directly activate caspase 3 to induce the cleavage of cellular substrates during apoptosis (Li et al., 1997). One of the most important initiator caspases is caspase 9 (CP9) for mitochondria-related (type 11) apoptosis.

Modern civilization is facing more than hundreds of disorders associated with free radicals and natural antioxidants from non-edible plants are gaining importance to fight these disorders (Fatema, 2013). Herbal medicine has been revealed to have valid utility and about 80% rural population depends on its efficacy for their primary health care. Ageratum conyzoides, a weed commonly called Billy goat-weed, goat weed etc., generally found in cultivated fields and other ecosystems such as pastures, grasslands, wastelands and even forest areas (Batish et al., 2006) is known in herbal or folk medicine as a remedy for diverse ailments in Africa (Almagbow, et al., 2001) and worldwide. Various pharmacological investigations have verified its antibiotic efficacy (Durodola, 1977) and antioxidative effect (Jagetia et al., 2003).

1.2     Research Question

The evaluation of some apoptotic markers in seminal plasma of rats following administration of crude oil and Ageratum conyzoides.

1.3     Objective of the Study

a.       Determination of seminal plasma  levels of Cytochrome C and  caspases 3 and 9

b.       Evaluation of the  antioxidative activity of Ageratum conyzoides on crude oil – induced free radicals animal models.

1.4     Justification of Study

Various pharmacological investigations have verified the anitibiotic efficacy of Ageratum conyzoides (Durodola, 1997), anagelsic effect in rats (Menut et al., 1993), antioxidative effect (Jagetia et al., 2003), hepatoprotective effects (Ita et al., 2009) and as a blood booster (Ita et al., 2007). The whole plant is traditionally used in management of wounds, burns and bacterial diseases (Ming, 1999).





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