ABSTRACT
This study was carried out to investigate the effects of Allium cepa (onion extract) on ciprofloxacin induced testicular toxicity. Twenty five adult male Wistar rats weighing between 150g – 250g assigned randomly to five groups (A-E) were used for this study. The control group (A) received 1 ml of distilled water daily while group B was given 22.86 mg/Kg body weight(b.w) of ciprofloxacin. Group C was administered 22.86 mg/Kg b.w. of ciprofloxacin plus 100 mg/Kg b.w. of onion extract. Group D received the same dose of ciprofloxacin plus 200 mg/Kg b.w. onion extract while group E was given 100 mg/Kg b.w. of onion extract only. The experiment lasted for 25 days to cover two seminiferous cycles in rats and the route of administration was oral. Semen samples from the epididymis were collected at the end of the experimental period and used for the analysis of sperm count, sperm motility and sperm viability. Blood samples were also collected for the determination of haematological parameters and the blood serum was used for the determination of antioxidant biomarkers. The haematological parameters i.e red blood cell count (RBC), heamoglobin count (Hb), Hematocrit (HCT), white blood cell count (WBC) and platelet count (PLT) were studied. Superoxide dismutase (SOD), catalase (CAT) and Malondialdehyde (MDA) were the antioxidant biomarkers investigated. Testicular protein concentration and caspase-3 activity were also determined. The animals were sacrificed and the testes removed and used for histopathological studies, Flourescence microscopy was also employed to study the DNA integrity of the sperm cells following administration of ciprofloxacin and onion extract.
The results obtained revealed that weights of the testes were comparable in all the groups. Allium cepa administration led to a significant (p<0.05) decrease in body weights in a dose-dependent manner. It was also observed that administration of ciprofloxacin significantly
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(p<0.05) decreased sperm count, motility and viability, and SOD activity. Similarly, administration of ciprofloxacin caused a significant increase (p<0.05) in MDA level, testicular apoptosis (as marked by caspase-3 activity level) and DNA fragmentation. Rats given ciprofloxacin and 100 mg/Kgb.w of onion extract showed significant(p<0.05) increase in sperm parameters with less testicular apoptosis and DNA fragmentation compared to the group given only ciprofloxacin. However, administration of 200 mg/Kgb.w of onion extract to ciprofloxacin-given rats potentiated the toxic effect of ciprofloxacin on sperm parameters, testicular apoptosis and DNA fragmentation.
Histological studies showed that ciprofloxacin caused testicular damage characterized by disruption of spermatogenesis. Onion extract at low dose was observed to protect against the damage caused by ciprofloxacin administration. In conclusion, ciprofloxacin induced caspase-dependent apoptosis in the testis and this was ameliorated by the low dose of Allium cepa extract as the high dose of the extract was found to exacerbate the effect of ciprofloxacin, onion extract was also found to be apoptogenic both at low and high doses.
TABLE OF CONTENTS
itle Page…………………………………………………………………………. ii Declaration………………………………………………………………………… iii Certification……………………………………………………………………….. iv Dedication…………………………………………………………………………. v Acknowledgements ……………………………………………………………….. vi Abstract……………………………………………………………………………. viii Table of Contents………………………………………………………………….. ix List of Figures..……………………………………………………………………. xiv List of Tables……………………………………………………………………… xv List of Plates…………………………………………………………………………. xvi List of Abbreviations………..…………………………………………………….. xvii CHAPTER ONE 1.0INTRODUCTION…………………………………………………………… 1 1.1Background…………………………………………………………………… 1 1.2Statement of Research Problem……………………………………………… 3 1.3Justification for the Study…………………………………………………… 3 1.4Significance of the Study ……………………………………………………. 3 1.5Hypothesis …………………………………………………………….……… 3 1.6Aim and objectives of the study…………………………………………….. 4 1.6.1Aim of the Study …………………………………………………………… 4 1.6.2 Objectives of the study…………………………………………………….. 4 CHAPTER TWO 2.0LITERATURE REVIEW……………………………………………………. 5
2.1Spermatogenesis ……………………………………………………………… 5
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2.2 Ciprofloxacin…….…………………………………………………………… 6 2.2.1 Chemistry …………….…………………………………………………….. 7 2.2.2 Anti-microbial activity ……………………………………………………… 7 2.2.3 Spectrum of activities ………………………………………………………. 7 2.2.4 Pharmacokinetics ………………………………………………………………….. 8 2.2.4.1 Absorption ………………………………………………………………… 8 2.2.4.2 Distribution ……………………………………………………………… 8 2.2.4.3 Metabolism ………………………………………………………………………. 9 2.2.4.4 Excretion ………………………………………………………….. ……… 9 2.2.5 Adverse effects……………………………………………………………. 9 2.2.6 Male reproductive toxicity……………………………………………… 10 2.3. Onion (Allium cepa)……………………………………………………… 11 2.3.1 Description……………………………………………………………… 11 2.3.2 Scientific classification……………………………………………………. 12 2.3.3 Chemical composition…………………………………………………… 12 2.3.4 Action and medical uses………………………………………………… 12 2.3.5 Anti-bacterial activity………………………………………………… …….. 13 2.3.6 Anti-fungal activity……………………………………………………… 13 2.3.7 Anti-oxidant activity……………………………………………………… 13 2.3.8 Androgenic activity …………………………………………………………. 14 2.4The Testis……………………………………………………………………. 15 2.4. 1. Gross anatomy of the testis………………………………………… …….. 15 2.4.2 Histology of the testis ………………………………………………..…….. 16 CHAPTER THREE 3.0MATERIALS AND METHODS ……………………………………………. 19
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3.1Materials………………………………………………………………………. 19 3.1.1 Experimental animals……….…..…………………………………………… 19 3.1.2 Drugs and reagents………………………………………………………… 19
3.1.3 Plant materials…………………………………………………………… 19
3.1.4 Equipment……………………………………………………………….. 19
3.2Methods……………….………………………………………………………. 20 3.2.1Preparation of onion extract………………………………………………. 20 3.2.2Experimental procedures………………………………………………….. 20 3.2.3 Tissue preparation.…………………………………………………………… 21 3.2.4 Testicular histology…….…………………………………………………. 21 3.2.5 Histomorphometric Studies………………………………………………… 21 3.2.6 Fluorescence microscopy…………………………………………………. 21 3.2.7 Sperm analysis……………………………………………………………. 22 3.2.7.1 Progressive sperm motility…………………………………………..…. 22 3.2.7.2 Sperm viability (Life/dead ratio)………………………………………… 22 3.2.7.3 Sperm count……………………………………………………………… 22 3.2.8 Haematological analysis …………………………………………………… 23 3.2.9 Biochemical analysis……………………………………………………….. 23 3.2.9.1 Determination of catalase activity………………………………………. 23 3.2.9.2 Determination of superoxide dismutase (SOD) activity……………….. 24 3.2.9.3 Assessment of lipid peroxidation……………………………………….. 24 3.2.9.4 Determination of protein concentration of the testis…………………… 25 3.2.9.5 Determination of caspase 3- activity…………………………………… 25 3.3 Data analysis………………………………………………………………… 25 CHAPTER FOUR
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4.0 RESULTS……………………………………………………………………… 27 4.1 Morphological Studies………………………………………………………… 27 4.1.1 Weights of the body and testes………………………………………………. 27 4.1.2 Sperm Parameters……………………………………………………………. 27 4.2 Haematological Studies………..……………………………………………… 31 4.3 Biochemical Studies…………………………………………………………… 33 4.3.1 Antioxidant biomarkers……………………………………………………… 33 4.3.2 Protein Concentration………………………………………………………… 33 4.3.3 Caspase-3 Activity………………………………………………………… 34 4.4 Histomorphometric studies…………………………………………………… 38 4.5 Light Microscopic studies (H&E)…………………………………………… 40 4.6 Flourescence microscopic studies (Acridine orange staining)……………. 46 CHAPTER FIVE 5.0 DISCUSSION.………………………………………………………………… 52 CHAPTER SIX 6.0 SUMMARY, CONCLUSION AND RECOMMENDATION……………… 60 6.1 Summary…………………………………………………………………….. 60 6.2 Conclusion……………………………………………………………………… 60 6.3 Recommendation…………..…………………………………………………. 61 Reference…………………………………………………………………………… 62 APPENDIX………………………………………………………………………… 70
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background
Spermatogenesis is the sequence of events by which spermatogonia are transformed into mature sperm (Moore and Persaud, 2008). Proliferation and differentiation of the male germ cells and the intratesticular and extratesticular mechanisms of regulation of spermatogenesis can be disturbed at every level (Holstein et al., 2003). Drugs, hormones and their metabolites, different toxic substances or x-radiation may reduce or destroy spermatogenesis (Holstein et al., 2003, Sheiner et al., 2003). Among the drugs that have been reported to disturb spermatogenesis is ciprofloxacin (Grabe et al., 1986). Ciprofloxacin is a synthetic antibacterial agent that has been approved in more than 100 countriesworld-wide (Wolfson and Hooper, 1985). It belongs to the family of fluoroquinolones with a very broad spectrum against microbial pathogens, especially Gram negative infectious diseases. Ciprofloxacin is well absorbed orally and induced its antibacterial action mainly by inhibition of DNA gyrase, which is equivalent to topoisomerase II in mammalian cell (Akasaka et al., 1998:Liu and Wang, 1999). Ciprofloxacin is routinely used by fertility specialists to treat bacterial infections occurring prior to in vitro fertilization treatment, or when high concentrations of leukocytes are present in the semen of their patients (Herbold et al., 2011).
In vivo genotoxicity studies have shown that ciprofloxacin is safe for therapeutic use (Herbold et al., 2011). However, other studies have demonstrated ciprofloxacin to significantly impair both testicular function and structure. Administration of ciprofloxacin
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significantly reduced sperm count, motility and daily sperm production in rats (Grabe et al., 1986). In healthy rats, ciprofloxacin caused obvious histological damage associated with a decrease in testicular volume and sperm concentration (Abd-Allah et al., 2000). Khaki et al., (2008) reported that oral administration of ciprofloxacin resulted in increase in germ cell apoptosis in the testes of male rats. Weyers et al., (2002) reported that there is an increased peroxide radical generation in the testis following ciprofloxacin treatment in mice. This increase induces DNA single-strand breaks and chromosomal aberrations as demonstrated by in vitro genotoxicity studies (Sanchez et al., 2005: Itoh et al., 2006). In addition, previous study showed that caspase-3, which has an important role in apoptosis, could be activated by ciprofloxacin (Olivia et al., 2002: Zhang et al., 2003). Allium cepa (onion) has a beneficial effect on disease treatment worldwide and has been used since ancient times as a medicinal and food source (Khaki et al., 2009). Allium cepa is commonly used in our daily diet and has been source of much interest because of its antithrombotic, hypolipidaemic, hypotensive, diaphroretic, antibiotic, antidiabetic, antiatherogenic and anticancer medicinal properties (Augusti,1996). The biological action of onion is ascribed to organosulfur and phenolic compounds it contains (Khaki et al., 2009). Research has shown that onion contains exogenous and endogenous antioxidants such as selenium, glutathione, vitamins A, B, and C, and flavonoids such as quercetin and isorhamnetin (Markham, 1982). These antioxidants protect DNA and other important molecules from oxidation and damage, which would otherwise induce apoptosis, and could improve sperm health parameters, increasing the rate of fertility in men (Yang et al., 2006). Researchers were able to show the androgenic activity of Allium cepa juice as well as its ability to recover spermatogenesis in Toxoplasma gondii infected rats (Khaki et al., 2009).
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1.2 Statement of research problem
Onion is one of the most important aphrodisiac foods. As an aphrodisiac, it increases libido and strengthens the reproductory organs (Kumar et al., 2010).Allium cepa (Onion) extract has also been reported to improve sperm parameters in rats (Khaki et al., 2009). Despite this documented androgenic activity of onion, little is known about its mechanism of action. As a result, unravelling the mechanisms of action through which onion improves male reproductive function is the problem this work set out to answer.
1.3 Justification of the study
Antibiotics are commonly prescribed for many disease conditions. Fertility specialist clinics also prescribe ciprofloxacin for the treatment of genitourinary infections. This will exacerbate the infertility conditions of the male as studies have demonstrated that ciprofloxacin significantly impairs both testicular structure and function. As such, discovery of drugs that will inhibit the anti-fertility effect of ciprofloxacin without affecting its antibiotic activity is needed. Allium cepa may serve as a potential candidate for this.
1.4 Significance of the study
This work is significant as it seeks to elucidate the mechanism of action of Allium cepa on testicular toxicity due to ciprofloxacin treatment theknowledge of which canprovide a better understanding of its therapeutic potentials particularly as a sperm boosting agent.
1.5 Hypothesis
Allium cepa extract will protect against testicular toxicity due to ciprofloxacin administration in male Wistar rats.
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1.6 Aim and objectives of the study
1.6.1 Aim of the study
The aim of this study is to investigate the effects of the Allium cepaextract on ciprofloxacin-induced testicular toxicity in Wistar rats.
1.6.2Objectives of the study
The objectives of the study are to investigate the effects of
i. Allium cepa extracton sperm apoptosis using acridine orange and fluorescence microscopy.
ii. Allium cepa extract on changes in the testis histology and sperm parameters due to ciprofloxacin treatment.
iii. Allium cepa extract on testicular apoptosis induced by ciprofloxacin using caspase-3 activity assay.
iv. Allium cepaextract on oxidative stress indicators.
v. Allium cepa extract and ciprofloxacin on haematological parameters.
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