AND Ocimum Gratissimum Linn (Lamiaceae) in CYCLOPHOSPHAMIDE INDUCED UROTOXICITY and MYELOSUPPRESSION

PROTECTIVE ROLE OF AQUEOUS LEAF EXTRACT OF Vernonia amygdalina Del. (Asteraceae) AND Ocimum gratissimum Linn (Lamiaceae) IN CYCLOPHOSPHAMIDE INDUCED UROTOXICITY AND MYELOSUPPRESSION

IKEH, CHIBUEZE

PG/MSC/09/51359

A RESEARCH SUBMITTED IN PARTIAL FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE (M. Sc) DEGREE IN PHARMACOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA.

FEBRUARY, 2013.

CERTIFICATION PAGE

PROTECTIVE ROLE OF AQUEOUS LEAF EXTRACT OF Vernonia amygdalina Del. (Asteraceae) AND Ocimum gratissimum Linn (Lamiaceae) IN CYCLOPHOSPHAMIDE INDUCED UROTOXICITY AND MYELOSUPPRESSION

IKEH, CHIBUEZE

PG/MSC/09/51359

A RESEARCH SUBMITTED IN PARTIAL FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE (M. Sc) DEGREE IN PHARMACOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA.

CERTIFIED BY

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DR. A. C. EZIKE PROF P. A. AKAH (PROJECT SUPERVISOR) (PROJECT SUPERVISOR)

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DR. T. C. OKOYE (Ag. HEAD OF DEPARTMENT)

DEDICATION

This work is dedicated to all patients currently suffering from various human malignancies and metastasizing cancers throughout the globe. I believe this work has provided yet another convenient solution to alleviate their suffering.

ACKNOWLEDGEMENT

Without any reservation, I wish to express my warmest appreciation to the Almighty God for

His eternal mercies and guidance throughout the course of this work without whom it wouldn’t have seen the light of the day.

With utmost sincerity, many thanks to my supervisors Dr. (Mrs.) A. C. Ezike and Prof P.A

Akah for their invaluable contributions; May God continue to bless you abundantly.

I will forever be indebted to my God given parents Rev. Emmanuel and Mrs. Abigail Ike whose kindness and foundational education they provided me with has seen me through in the course of life’s journey.

I will not fail to appreciate my lovely wife Mrs. Patricia E. Ikeh for unalloyed contribution in running the biochemical assay with high proficiency as a chemical pathologist.

To my humble self, I say “well done” for also taking the pain to type and edit this work effectively.

ABSTRACT

Cyclophosphamide (CP) is one of the most potent and widely used alkylating anticancer agents. Urotoxicity and myelosuppression is known as the most prevailing dose-limiting toxicity associated with CP. In the present study, the protective potential of Vernonia amygdalina and Ocimum gratissimum aqueous leaf extracts in CP-induced urotoxicity and myelosupression were evaluated using biochemical and histopathological approaches. Sodium -2-macarptoethane sulfonate (MESNA) was used as a positive control. Forty (40) male Sprague-Dawley outbred albino rats weighing between 130 g – 200 g were randomly separated into eight different groups (n=5). Rats in group 1 received only normal saline orally for gavage for ten consecutive days. Animals in group two were injected with CP only on the tenth day intraperitoneally (i.p) at 200 mg/kg body weight. Animals in group 3 were given MESNA (67 mg/kg) and CP (200 mg/kg) i.p on the tenth day at 5 minutes interval. Rats in groups 4 and 5 received two different doses of O. gratissimum orally by gavage at 250 mg/kg and 500 mg/kg respectively for ten consecutive days before administering CP (200 mg/kg) on the tenth day. Rats in group 6 and 7 received different doses of V. amygdalina orally by gavage at 250 mg/kg and 500 mg/kg respectively for ten consecutive days before administering CP (200 mg/kg) on the tenth day. Rats in group (8) received combination of V. amygdalina and O. gratissimum at a dose of 250 mg/kg each for ten consecutive before administering CP (200 mg/kg) on the tenth day. Results showed that the extract of V. amygdalina protected significantly (P < 0.05) the urothelium and the myeloid system as observed in the biochemical and hematological parameters evaluated. This protection is comparable to MESNA, but MESNA protection was not adequate to prevent myelosupression as observed with V. amygdalina. O. gratissimum did not show significant protection of the urothelium and myeloid system. The protective effects of V. amygdalina was further evident through decreased histopathological alteration of the urinary bladder, kidney and liver tissues unlike the CP and O. gratissimum treated groups. The result of the present study revealed that aqueous leaf extract of V. amygdalina has the potential to prevent urotoxicity and myelosuppression induced by CP and thus can be used as therapeutic adjuvant in the management of CP and other oxazaphosphorine toxicities.

TABLE OF CONTENT

Title page i

Certification page ii

Dedication iii

Acknowledgement iv

Abstract v

Table of content vi

Tables and figures vii

CHAPTER ONE 1

1.0 Introduction 1

1.1 Pharmacology of cyclophosphamide 2

1.1.1 Pharmacodynamics/mechanism of action of cyclophosphamide 3

1.1.2 Pharmacokinetic profile of cyclophosphamide 4

1.1.3 Pharmacokinetic variability 9

1.1.4 Therapeutic uses 12

1.1.5 Drug Interaction 14

1.2.0 Mechanism of cyclophosphamide toxicity 15

1.2.1 Outcomes of cyclophosphamide toxicity 16

1.2.2 Pathophysiology and consequences of urotoxic effects of

cyclophosphamide 17

1.2.3 Pathophysiology and consequences of myelosuppressive effects of

cyclophosphamide 20

1.3 Chemoprevention and amelioration of cyclophosphamide -induced toxicities 24

1.3.1 The Role of MESNA (sodium 2-mercaptoethane sulfonate) in

6 amelioration of cyclophosphamide -induced toxicities 27

1.3.2 Mechanism of action of MESNA 27

1.3.3 Other potential uroprotective agents 29

1.3.4. Medicinal plants used in preventing or ameliorating

cyclophosphamide-induced toxicity 29

1.4. Botanical profile of Vernonia amygdalina Del 39

1.4.1 Taxonomy 39

1.4.2 Description 40

1.4.3 Geographical Distribution 40

1.4.4 Ethnomedicinal Uses 40

1.4.5 Documented research findings on V. amygdalina 40

1.5. Botanical profile of Ocimum gratissimum Linn. 45

1.5.1 Taxonomy 45

1.5.2 Description 46

1.5.3 Geographical Distribution 46

1.5.4. Ethnomedicinal uses. 46

1.5.5 Documented research findings on O. gratissimum. 47

1.6 Aim of the Study 48

CHAPTER TWO 49 2.0 Materials and Methods 49

2.1 Animals 49 2.2 Drugs and Chemicals 49

2.3 Preparation of extracts 50

2.4 Induction of cyclophosphamide -induced toxicity 50

2.5 Blood sample collection 51

2.6 Haematological test 52

2.7 Biochemical analysis 52

2.7.1 Analysis of glutathione (GSH) 52

2.7.2 Superoxide dismutase analysis 52

2.7.3 Catalase assay 53

2.7.4 Malondialdehyde (MDA) Assay 53

2.8 Histopathological Examination 53

2.9 Statistical Analysis 53

CHAPTER THREE 54

3.0 Results 54

3.1. Effects of extracts on cyclophosphamide-induced urotoxicity 54

3.1.1 Effects of extract on glutathione 54

3.1.2 Effects of extracts on Superoxide dismutase analysis activity. 54

3.1.3 Effects on catalase activity 54

3.1.4 Effects of extracts lipid peroxidation (LPO) 55

3.2. Effects of extract on cyclophosphamide -induced myelosupression. 55

3.2.1. Effect on total red blood cell count, total Leucocyte count, absolute neutrophil and absolute lymphocyte counts. 55

3.2.2 Effect on platelet count, absolute basophils and eosinophils 56

3.3 Effect on histopathology of tissues 60

CHAPTER 4 66 4.0 Discussion 66

4.1 Conclusion 71

References 72

List of Tables

Table 1: Normal reference values of blood cells in adult humans and male rats 23

Table 2: Some plants documented to have ameliorating properties in cylophosphamide-induced toxicity. 38

Table 3: Phytochemical constituents of V. amygdalina. 42

Table 4: Documented pharmacological properties of V. amygdalina. 43

Table 5: Effects of O. gratissimum and V. amygdalina compared to MESNA on glutathione, superoxide dismutase and catalase activities in cyclophosphamide-induced toxicity. 57

Table 6: Effects of O. gratissimum and V. amygdalina compared to MESNA on lipid peroxidation (LPO) induced by cyclophosphamide 58

Table 7: Effects of O. gratissimum and V. amygdalina on hematological parameters of peripheral blood in cyclophosphamide-induced toxicities. 59

List of figures Figure 1: Chemical structure of Cyclophosphamide 2

Figure 2: Chemical structure of Sodium 2-mercaptoethane sulfonate 27

Figure 3: Vernonia amygdalina in its natural habitat 39

Figure 4: Ocimum gratissimum in its natural habitat 45

Figure 5: Photomicograph of organ sections from control rats given normal saline 62

Figure 6: Photomicrograph of sections of organs from rats given 200mg/kg of

cyclophosphamide 62

Figure 7: Histologic sections of organs from rats treated with 67mg/kg of

MESNA and 200mg/kg of cyclophosphamide 63

Figure 8: Histologic sections of organs from rats treated with 250mg/kg of

O. gratissimum and 200mg/kg of cyclophosphamide. 63

Figure 9: Histologic sections of organs from rats treated with 500mg/kg of

O. gratissimum and 200mg/kg of cyclophosphamide. 64

Fig.10. Photomicrograph of sections of organs from rats treated with

250mg/kg of V. amygdalina and 200mg/kg of O. gratissimum 64

Figure 11: Photomicrograph of sections of organs from rats given 500mg/kg of

V. amygdalina and 200mg/kg of cyclophosphamide 65

Figure 12: Histologic sections of organs from rats treated with 250mg/kg of

V. amygdalina 250mg/kg of O. gratissimum and cyclophosphamide

200mg/kg. 65

CHAPTER ONE

1.0 INTRODUCTION

It is a well-known fact that neoplasms are deleterious and reduce quality of life. Many alkylating cytotoxic agents, which cyclophoshpamide (CP) is a member, have been well documented to be effective in management of many human malignancies in order to improve quality of life and extend patients life span (Philip et al., 1961; Colvin, 1978; Friedman et al.,

1979; Carter and Livingston, 1982). Despite its adverse effects, many clinicians have continued to use CP either alone or in combination with other agents in cancer chemotherapy due to its efficacy.

Sodium-2-mecarptoethane sulfonate (MESNA), a sulfhydryl-containing agent has long been used in detoxifying and ameliorating specifically the urotoxic effects of CP and other oxazaphosphorines. However, its own side effects are equally disturbing and needs to be addressed (Reinhold-Keller et al., 1992).

Natural products have recently gained acceptance and have continued to gain grounds in therapeutics due to their acclaimed efficacy in management of many ailments with little or no side effects when used appropriately. They are also readily available depending on the region and geographical distribution. Many natural products have been widely reported to ameliorate at varying degrees the side effects of oxazophosphorines e.g. cyclophosphamide and ifosphamide (Łukasz and Piotr, 2012).

Many researchers have shown that Vernonia amygdalina Del. and Ocimum gratissimum

Linn. are capable of detoxifying the body owing to their antioxidant properties thereby

11 protecting the essential organs like the liver, kidney, heart, etc. (Owolabi et al., 2008;

Arhoghro et al., 2009; Asuquo et al., 2010).

This study investigated the protective roles V. amygdalina and O. gratissimum in CP-induced urotoxicity and myelosupression in rats.

1.2 Pharmacology of cyclophosphamide

Figure 1: Chemical structure of Cyclophosphamide

Cyclophosphamide, [2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxzaphosphorine-2- oxide] is one of the most effective and widely used cytotoxic antitumor agents.

Cyclophosphamide (CP) and its structural isomer ifosfamide (N, 3-(bis(2-chloroethyl)- tetradydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide, (IFEX or HOLOXAN), belong to oxazaphosphorine DNA alkylating agents widely used in cancer chemotherapy (Gilman,

1963).

Cyclophosphamide (CP) is a cytotoxic alkylating drug with a high therapeutic index and broad spectrum of activity against a variety of cancers (Fleming, 1997; Baumann and Preiss,

2001). It is often employed in the treatment of a variety of human malignancies despite its serious adverse effects which include: myelosuppression, haemorrhagic cystitis, nausea, vomiting, alopaecia, nephrotoxicity, immunotoxicity, mutagenicity, carcinogenicity and teratogenicity (Mirkes 1985; Fleming, 1997).

Cyclophosphamide was found to have antitumor activity in early 1940s (Gilman, 1963) and was later introduced to clinical cancer chemotherapy. Cyclophosphamide is often used in combination with other antineoplastic agents like methotrexate and fluorouracil to achieve higher efficacy in management of wide range of solid tumours and blood disorders (Zhang et al., 2006).

In non-neoplastic disorders, it has been found useful in organ transplantation procedure

(Zinke et al., 1977) and treatment of some auto-immune diseases (Lares et al., 1971) due to its differential immunosuppressive effects (Schwartz and Grindey, 1973). Although the myelosuppressive effects of cyclophosphamide may be useful clinically, it is however completely undesirable in patients receiving cyclophosphamide regimen in management of neoplastic disorders.

1.1.1 Pharmacodynamics/mechanism of action of CP

The mechanisms of action of oxazaphosphorines are similar. Like other alkylating agents, the mechanism of action of alkylating nitrogen mustard of CP is through the covalent bonding of highly reactive alkyl groups with nucleophilic groups of nucleic acid. Cyclophosphamide itself is devoid of alkylating activity and must first undergo metabolic activation catalysed by the hepatic cytochrome P450 (CYP) monoxygenase systems (Boddy and Yule, 2000).

Following metabolic activation, bifunctional alkylating nitrogen mustards of CP is generated, which are capable of reacting with the nitrogen-7 atom of purine bases in DNA, especially when they are flanked by adjacent guanines (Kohn et al., 1987). At alkaline or neutral pH, the nitrogen mustard is converted to chemically reactive carbonium ion through imonium ion. Carbonium ion reacts with the N7 of the guanine residue in DNA to form a covalent linkage. The second arm in phosphoramide mustard can react with a second guanine moiety in an opposite DNA strand or in the same strand to form crosslinks (Springer et al., 1998).

The O6 atom of guanine may also be a target for oxazaphosphorines (Friedman et al., 1999).

The different intramolecular distance between the chloroethyl group in CP mustard results in a different range of cross-linked DNA. Despite a good understanding of nature of the chemical reactions between alkylating species and DNA molecules (Shulman-Roskes et al.,

1998), the mechanisms linking adduct and crosslink formation with tumor cell death are not fully identified. Cyclophosphamide and ifosphamide, as with all other alkylating agents, destroy tumor cells through apoptosis (programmed cell death) initiated by DNA damage, modulation of cell cycle and other antiproliferative effects (O'Connor et al., 1991; Bhatia et al., 1995; Crook et al., 1986). It is generally accepted that the main mechanism that results in cell death is inhibition of DNA replication, as the interlinked strands do not allow separation of the two strands (Schwartz, 1983; Schwartz and Waxman, 2001). Apoptosis is characterized by a cascade-like activation of intracellular cysteineproteases (i.e. caspases).

Caspases pre-exist as zymogens that are activated by proteolytic cleavage by other caspases or by autocatalysis. Distinct caspase cascades are involved in receptor-mediated or chemical- induced apoptosis (Sun et al., 1999). Drug-induced apoptosis is always mediated by the mitochondrial pathway leading to activation of the initiator caspase-9, which in turn activates the effector caspases-3 and caspase-7. Because CP can damage DNA during any phase of the cell cycle and its cytotoxicity is independent of the cell-cycle (Bruce et al., 1966), it is not cell cycle phase specific.

1.1.2 Pharmacokinetic profile of cyclophosphamide

Absorption: Considerable inter-personal variability exists in pharmacokinetics of CP. This is markedly influenced by route of administration, duration of treatment, patient age, coadministration with other drugs, genetic factors, functional status of the liver and the kidneys (Zhang et al., 2006).

As a monohydrate, CP is readily soluble in water, saline or alcohol. Oral administration is convenient and is well absorbed. The peak concentration appears 1hour following oral drug administration. The oral bioavailability is between 85 – 100% (Wagner and Fenneberg,

1984).

A fraction of the drug undergoes first pass metabolism in the liver and gut. At higher doses

(0.7 g/m2), CP has about 87.7% oral bioavailability (Matthias et al., 1984).

Distribution: After oral or intravenous administration, CP is readily distributed throughout the body. About 30% binds to plasma protein (Moore, 1991). Its metabolite 4- hydroxycyclophosphamide (4-OH-CP) has higher plasma protein binding (approximately

67%) (Moore, 1991). CP is not structurally modified by blood plasma. The volume of distribution (Vd) of CP is increased in obese patients, leading to an increased elimination half-life (t1/2) of CP (Powis et al., 1987). Several studies suggested that CP entered into cerebrospinal fluid through blood-brain barrier (BBB) with varying cerebrospinal fluid to plasma ratios from 0.2 to 4% (Neuwelt et al., 1984; Yule et al., 1995; Hommes et al., 1983).

The active metabolites of CP have limited penetration into the brain due to their increased polarity and higher plasma protein binding (Hommes et al., 1983). This may contribute to the lack of neurotoxicity associated with the intravenous administration of CP unlike its structural isomer, ifosphamide which has more extensive distribution with lower plasma protein binding thus making it more neurotoxic than CP (Zhang et al., 2006)

Transport: CP and its active metabolites are extensively bound by erythrocytes, which may serve as transporters of activated CP metabolites (Highley et al., 1996; Momerency et al.,

1996; Dumez et al., 2004). The protoxic metabolite of CP, 4-hydroxy-CP, is trapped intracellular and transported to tumor tissues. There are increasing data on the transport of

CP and its metabolites across cellular membrane. It is highly hydrophilic and do not diffuse readily through the lipid bilayer of cells (Highley et al., 1997). Similarly, the phosphoramide

15 mustard, the cytotoxic but unstable metabolite of CP bears a negative charge with pKa of 4.5

- 4.8 at physiological pH and are thus relatively difficult to pass the cellular membrane

(Zhang et al., 2006). 4-hydroxycyclophosphamide is the corresponding circulating metabolite that enters tumor cells to form ultimate cytotoxic phosphoramide mustard and the byproduct acrolein. It appears that 4-OH-CP and acrolein can readily cross the cell membrane by passive diffusion in vitro. However, active transport cannot be excluded for CP and its metabolites based on their transport studies (Zhang et al., 2006).

Biotransformation/Metabolism. The initial activation of CP is achieved through 4- hydroxylation at C4 of oxazaphosphorine ring by hepatic CYP2B6, CYP3A4 and CYP2C9 to form 4-hydroxycyclophosphamide (4-OH-CP), which enters blood and is transported to tumor cells by erythrocytes (Colvin et al., 1976, Chang et al., 1993; Huang et al., 2000; Chen et al.,2004). The CYP2B6 is the major contributor (a mean of about 45% of total metabolism) for the activation of CP with the highest intrinsic clearance in vitro and in vivo, compared with 25% and 12% for CYP3A4 and CYP2C9, respectively (Roy et al., 1999;

Huang et al., 2000; Chen et al., 2004). Other CYPs including CYP2A6, CYP2C8 and

CYP2C19 also make a minor contribution to CP 4-hydroxylation (Chang et al., 1993). The 4-

OH-CP so formed, is the major circulating metabolite of CP which is in equilibrium with its tautomer aldophosphamide that can decompose by β-elimination to form ultimate cytotoxic phosphoramide mustard (N,N-bis-2-(2-chloroethyl) phosphorodiamidic acid) and an equimolar amount of the byproduct acrolein (a highly electrophilic α,β-unsaturated aldehyde)

(Hohorst et al., 1976; Fenselau et al., 1977; Colvin, 1999).

Acrolein is implicated in urotoxic effect of CP. It is detoxified by conjugation with glutathione (GSH). Alternatively, 4-hydroxycyclophosphamide is detoxified to form O- carboxyethylcyclophosphoramide mustard (CEPM, namely, carboxyphosphamide) primarily by aldehyde dehydrogenase (ALDH1A1) and, to much lesser extent, by ALDH3A1 and

ALDH5A1 (Jarman, 1973; Domeyer and Sladek, 1980; Hipkens et al., 1981; Boddy et al.,

1992). It is also oxidized by alcohol dehydrogenase (ADH) to form non-toxic 4- ketocyclophosphamide but at much lesser degree than CEPM (Hohorst et al., 1971; Lelieveld et al., 1976; Yule et al., 1995). Furthermore, 4-OH-CP undergoes reversible dehydration to form iminocyclophosphamide that is further conjugated with intracellular GSH, giving rise to another non-toxic 4-glutathionylcyclophosphamide (GSCY), a substrate for multidrug resistance associated protein 2 (MRP2) exhibited by oxazaphosphorines (Hohorst et al.,

1971). By contrast, the inactivation pathway of CP also involves minor (about10%) side chain oxidation (N-dechloroethylation) primarily by CYP3A4/3A5 and, to a minor extent, by

CYP2B6 to generate 3-dechloroethyl-Ifosphamide and the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA) (Ren et al., 1997). The liver is the primary organ for the metabolism of CP through which the drug are activated and eliminated, but metabolism may occur in other sites, including the erythrocytes (Dockham et al., 1997), kidneys (Aleksa et al., 2005) and tumor itself (Schwartz et al., 2003). Various CYPs including CYP1A1, 2A6,

2B6, 2C8/9 and 3A4 are present in a variety of tumors, including those from the central nervous system, breast, colon, lung, ovarian, prostate and kidney, but their relative levels compared with normal tissue are less (Murray et al., 1995; Yu et al., 2001; Kivisto et al.,

1995). Since CYP3A4 and CYP2B6 are the major enzymes for CP activation, their intratumoral level may be a useful predictive marker for the efficacy of CP treatment (Zhang et al., 2006).

Excretion: The CP is primarily (70 %) excreted in urine in forms of metabolites and to a less extent, in the feces (Boddy and Yule, 2000). However, only 10-20% is excreted unchanged in the urine (Fasola et al., 1991, Juma et al., 1979) and only 4% is excreted in the bile following CP administration (Dooley et al., 1982). CEPM is the dominant inactive metabolite of CP found in the urine, while 4-keto-CP is only a minor component in patient

17 urine (<1%) (Hadidi et al., 1988). The renal clearance of CP in cancer patients was reported to be 15 – 44 ml/min (Busse et al., 1997).The reported renal clearance of CP metabolites including 4-OH-CP, dechloroethylifosfamide, keto-CP, and CEPM in cancer patients is 60.6

±9.0, 3.2 ± 1.0, 1.3 ± 0.8 and 7.0 ± 4.5 ml/min, respectively (Busse et al., 1997). These values are lower than the glomerular filtration rate (about125 ml/min). During high-dose therapy of CP (100 mg/kg), the amount of CP excreted in urine is correlated with the urine flow, whereas this correlation does not exist during conventional dose therapy (500 mg/m2)

(Busse et al., 1997). However, there was no correlation between the renal excretion of any metabolites of CP and urine flow during either conventional or high dose therapy (Busse and

Kroemer 1997). Urine flow can have a significant effect of the renal clearance of CP that is substantially reabsorbed. The majority of CP elimination is by metabolic transformation with the metabolites recovered from the urine and feces. Thus, liver impairment may have an impact on pharmacokinetics and disposition of CP. However, such liver impairment only leads to less production of aldophosphamide; fewer adverse effects were observed in patients with liver dysfunction (Koren et al., 1992). Thus, it is not recommended to adjust the dosage of CP in patients with liver dysfunction (Zhang et al., 2006). A number of pharmacokinetic studies have been conducted with CP and the pharmacokinetic parameters reported. The t1/2 of CP ranges from 3.2 - 7.6 hours with total body clearance (CL) values of about 2.5 to 4.0

L/h/m2 (Boddy and Yule, 2000).

1.1.3 Pharmacokinetic variability: Large inter-patient variability in clinical response rate and toxicity has been observed in cancer patients treated with CP. This may be explained by differences in the pharmacokinetics of the agent observed in cancer patients (Yule et al.,

1996; Ren et al., 1998). Inter-patient variability is often greater than intra-patient variability.

Pharmacokinetic parameters of the parent drug vary less (coefficient of variation, 10-30%)

18 than those for the elimination parameters (coefficient of variation, 14-64%) (Busse and

Kroemer, 1997).

Inter-individual variations in the metabolism, transport, distribution and disposition of CP can be influenced by a number of factors associated with the drug (e.g. dosage, dosing regimen, route of administration, and drug combination) and patients (e.g. age, gender, renal and hepatic function, and genetic factor).

Dosage: Dosage and dosing regimen are important factors affecting the pharmacokinetics of

CP. There is increased interest in the use of high dose of CP in cancer chemotherapy. To increase chemotherapy efficacy against human cancer, it is desirable to increase dose intensity to the maximum tolerated dose. High dose oxazaphosphorine chemotherapy is assumed to result in improved antitumor activity due to increased generation of cytotoxic mustards (Zhang et al., 2006). Moderate and high dose CP, doxorubicin, and fluorouracil within the standard range result in greater disease-free and overall survival than the low dose regimen (Citron, 2004). Higher doses (>9.0 g/m2) of CP are usually administered intravenously in either 5% dextrose or 0.9% saline. Clinically, the drug is often given in a single dose over a period of up to 1 hour, repeated every 3 to 4 weeks. However, despite the use of myeloid growth factor and sulfhydryl compounds (e.g. MESNA and amifostine), myelosuppression continues to be a dose-limiting toxicity of oxazaphosphorines (Zhang et al., 2006). At higher doses used prior to marrow transplantation, the dose-limited toxicity is cardiac toxicity. Besides cardiac toxicity, hemorrhagic cystitis, water retention and hyponatremia are found in patients receiving high dose CP (Zhang et al., 2006). High-dose regimens have led to concerns over the existence of dose-dependent pharmacokinetics, with an increase in the production of inactive metabolites as the predominant activation pathway of metabolism is saturated.

The degree of renal excretion and inactive metabolite formation was increased at higher dose of CP, associated with a relative decrease in the formation of the active metabolite (Busse et al., 1997; Busse and Kroemer, 1997). Saturation kinetics at doses of 1 and 4 g/m2 of CP has been observed in cancer patients (Chen et al., 1995). Dosing schedule has significant effect on the pharmacokinetics of CP. In pediatric treatment or dose schedule given prior to bone marrow transplantation, the total dose can be fractionated over several days. There is no obvious evidence that benefits can be achieved from the prolonged intravenous infusions of

CP (Mouridsen et al., 1976).

Age: This is an important factor affecting the pharmacokinetics of CP. Elderly patients with non-small cell lung cancer demonstrated a doubled t1/2, because of an increased volume of distribution (Vd) when total body, renal and non-renal clearance remained unaltered (Lind et al., 1989). Pediatric patients are a specific group of individuals, because they have distinct physiological features from adults. The t1/2 of CP has been shown to be shorter than in adults

(Yule et al., 1996). The differences in body surface area will lead to larger variability of CP doses, which makes comparison between pharmacokinetic parameters more difficult. Since

Vd of CP approximates that of body water, the Vd in children is significantly lower than in adults.

Renal function: Since CP is primarily (70%) excreted in urine, renal function may play a role in the pharmacokinetic variability of CP. Early studies by Bramwell et al. (1979) and

Juma et al. (1981) found that alterations in renal function did not significantly alter the pharmacokinetics of CP in patients, and did not result in any clinically-relevant changes in response rate and toxicity. However, a later study by Haubitz et al. (2002) indicated that the clearance of CP was decreased in patients with impaired renal function, thereby resulting in an increased systemic drug exposure. It appears that minor to moderate renal function impairment, insignificantly alters the clearance of CP or its alkylating metabolites and as

20 such there is no necessity to adjust the doses of CP (Bramwell et al. 1979; Juma et al. 1981).

However, terminal renal insufficiency may have a major impact on the renal excretion of CP and its metabolites. In particular, CP was removed by being taken into the dialysate in hemodialysis-dependent patients (Haubitz et al. 2002). Up to 25 % of administered CP dose

(1.2 ± 0.4 g) was recovered in the dialysate and thus removed from the body during the dialysis. The conclusion was that dialysis should not be initiated earlier than 12 hours after

CP infusion, which can prevent the removal of drug in the early distribution phase (Haubitz et al. 2002). Only when the patients with terminal renal insufficiency are overdosed with CP, that the repetitive dialysis can help remove the drug from the body. Therefore, the severity of renal impairment has to be carefully assessed, dosage of CP should be accordingly adjusted in patients with renal dysfunction on hemodialysis and therapeutic drug monitoring should always be conducted in these patients (Zhang et al., 2006).

Liver function: Cyclophosphamide is extensively metabolized by hepatic CYP2B6, 2C9 and

3A4, indicating that liver impairment has a major impact on pharmacokinetics of CP.

However, such liver impairment only leads to less formation of aldophosphamide; fewer adverse effects were observed in patients with liver dysfunction (Koren et al., 1992).

Therefore, it is not recommended to adjust the dosage of CP in patients with liver dysfunction. Although biliary concentrations of CP are comparable to plasma concentrations, only a low fraction of CP (1.8 %) is found in stool (Dooley et al., 1982).

Disease status: The activation of CP was inhibited in tumor-bearing rats compared to healthy control (Sladek et al., 1978). The clearance of CP was found to reduce in children with

Fanconi’s anemia, probably as a result of altered CYP oxidase-reductase cycling (Yule et al.,

1999). Recently, the risk of recurrence of non-Hodgkin’s lymphoma in children is related to inadequate clearance of CP to active metabolites (Yule et al., 2004).

Genetic factor: Importantly, genetic factors may affect the pharmacokinetics of CP. There are increased studies on the role of genetic polymorphisms of genes encoding various proteins involved in the distribution, metabolism, and transport of CP in the pharmacokinetic variability and therapeutic outcomes (Zhang et al., 2006). Theoretically, polymorphisms of

CYP3A4, CYP2B6, CYP2C9, ALDH1A1, ALDH3A1, GSTT1, GSTM1, GSTP1, and MRP2 may play a role in the disposition of CP, thus resulting in wide inter-patient variability in exposure to CP and its active metabolites, with important clinical consequences in cancer chemotherapy (Wormhoudt et al., 1999; Desta et al., 2002; Rodrigues et al., 2002). Since CP undergo extensive CYP-catalyzed metabolism through which it is activated, deactivated and eliminated, drug interactions may arise due to modulation of the pharmacokinetics, in particular, when inhibition or induction of the relevant CYPs is implicated.

1.1.4 Therapeutic uses: CP is the most widely used alkylating agent in the treatment of hematological malignancies and a variety of solid tumors, including leukemia (Demirer et al., 1996, Rao, et al., 2005), breast cancer (Lippman et al.,1986; Levine, et al.,2005), lung cancer (Chrystal et al., 2004; Hobdy 2004), lymphomas (Escalon et al., 2005; Zinzani, 2005;

Kasamon et al 2005), prostate cancer (Nicolini et al., 2004; Hellerstedt et al., 2003;), ovarian cancer (Morgan et al. 2001; Inoue et al., 1995; Nicoletto et al., 2004), and multiple myeloma

(Dimopoulos et al., 2004; Lundin et al., 2003). This agent has similarly been used extensively for the treatment of diffuse proliferative glomerulonephritis in patients with lupus erythematosus affecting the kidneys (Hengstler et al., 1997). Although its role in the treatment of ovarian cancer and small-cell lung cancer is declining, CP continues to be used in treatment of breast cancer as a critical component of the CMF (CP, methotrexate, flourouracil), CEF (CP, epirubicin, and 5-fluorouracil), MVC (mitoxantrone, vinblastine, and

CP) and DDC (docetaxel, doxorubicin and CP) regimen (Levine et al., 2005; Stewart et al.,

2005). Higher doses of CP are used in the treatment prior to bone marrow transplantation for aplastic anemia, leukemia and other malignancies (Demirer et al., 1996, Rao, et al., 2005).

Recently, there is accumulating evidence indicating the action of CP on the immune system.

CP has modulating effects on both humoral and cell-mediated immunity (Zhang et al., 1993;

Brodsky et al., 2002; Lacki et al., 1997) and thus beneficial effects are obtained when used as an immunosuppressive drug. CP also augmented the efficacy of antitumor immune responses in animals and humans by depleting CD4+/CD25+ regulatory T cells and increasing T lymphocyte proliferation and T memory cells (Ikezawa et al 2005; Lutsiak et al., 2005). The immunostimulatory effect of CP is associated with the marked inhibition of inducible nitric oxide synthase (Loeffler et al., 2005). Furthermore, CP kills circulating endothelial progenitors that present as a marker of tumor angiogenesis (Mancuso et al.,

2003), whereas 4-OH-CP readily destroys various hematopoietic progenitors cells including marrow stromal progenitors (Siena et al., 1985). These findings may provide a solid rationale for the use of CP as an immunosuppressive agent in the treatment of autoimmune diseases or an integral component in combination with other immunotherapy in cancer treatment.

1.1.5 Drug Interaction:

A number of drug interactions with CP have been reported in humans. It seems that the underlying mechanism is inhibition of CYP enzymes for the drug interactions with allopurinol (Yule et al., 1996), chloramphenicol (Faber et al., 1975), sulphaphenazole (Faber et al., 1975), chlorpromazine (Yule et al., 2004), fluconazole (Yule et al., 1999), ranitidine

(Alberts et al., 1991), and triethylenethiophosphoramide (THIOTEPA) (Anderson et al.,

1996). Drug interactions have also been reported with dexamethasone (Yule et al., 1996), prednisolone (Faber et al., 1974), phenobarbitone (Jao et al., 1972), and phenytoin (Slattery et al., 1996) due to induction of CP metabolism. Phenytoin induces the N-dechloroethylation of the S-enantiomer of CP to a greater extent than that of the R-enantiomer (Williams et al.,

1999). The clinical significance of these drug interactions is unclear. In addition, an altered toxicity profile of CP was observed when combined with paclitaxel in a schedule-dependent manner (Kennedy et al., 1998), but pharmacokinetic modulation cannot provide an explanation.

On the other hand, CP may alter the pharmacokinetics and pharmacodynamics of co- administered drugs. Cyclophosphamide reduced digoxin absorption has been reported (Rodin and Jonhson,

1988). Triethylenethiophosphoramide inhibited the activation of CP and decreased the efficacy and toxicity of CP (Anderson et al., 1996), because of the mechanism-based inhibition of CYP2B6 (Rae et al., 2002). Triethylenethiophosphoramide is frequently given in conjunction with CP in high-dose chemotherapy regimens in preparative regimens before autologous bone-marrow and peripheral stem-cell transplantation. Therefore, the sequence and schedule of these two drugs should be critically considered and it has been recommended that these two agents should not be combined (Huitema et al., 2000). Furthermore, the clearance of doxorubicin was significantly reduced (50%) when in combination with CP

(Zhang et al., 2006). The combination of CP and doxorubicin also caused a 10% decrease in clearance of etoposide, but this is of little clinical significance (Zhang et al., 2006).

Cyclophosphamide is often combined with doxorubicin and etoposide in the treatment of breast cancer. Therefore, proper therapeutic drug monitoring is needed when CP is used in combination with doxorubicin and etoposide (Zhang et al., 2006).

1.2.0 Mechanism of CP toxicity

Cyclophosphamide is primarily activated in the liver by CYP3A4, CYP2C9 and CYP2B6 followed by erythrocyte- mediated transport of the activated metabolites to the tumor tissue via blood circulation (Zhang et al., 2006). Cyclophosphamide is considered a prodrug as it

24 requires activation by mixed function oxidase enzyme in the liver (hepatic cytochrome P450 monoxygenase system) to yield the main active metabolite, phosphoramide mustard, and the by-product acrolein (Foley et al., 1961; Struck et al., 1975). However, these activated metabolites, phosphoramide mustard and acrolein also gain entry into normal tissues, where they may induce host toxicity. The resultant phosphoramide mustard is a bifunctional alkylator of DNA and the ultimate cytotoxic metabolite of CP (Struck et al., 1975). The alkylation involves generation of the intermediate phosphoramide aziridinium ion through an intramolecular nucleophilic attack (cyclization reaction) of the nitrogen on the β-carbon of a chloroethyl chain (Ludeman, 1999). Cellular thiols (e.g., GSH) and other nucleophiles react rapidly with phosphoramide aziridinium ions, resulting in thioether products (Gamcsik et al.,

1999). Carboxyethylcyclophosphoramide mustard (CEPM) is one of the major chemically stable metabolites of CP, which are easily detected in patient plasma and urine (Joqueviel et al., 1998). However, acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently disrupts the function and causes organ toxicity (Brock et al., 1979; Kehrer and Biswal, 2000). It is detoxified by conjugation with GSH through glutathione-s-transferases (GSTs) in hepatocytes (Gurtoo et al., 1981a) and this may cause intracellular GSH depletion and injuries of the hepatocytes and urothelium (DeLeve et al.,

1996). Reaction of GSH with acrolein is via nucleophilic addition at the β-carbon atom, generating stable thioether compounds for elimination (Ramu et al., 1995).

1.2.1 Outcomes of CP Toxicity

The usual dose-limiting toxicity is myelosuppression (Bramwell et al., 1987). Since CP is a non-specific alkylating agent, severe host toxicity is inevitable. The urological side effects of

CP are major limiting factor for its use (Brock and Pohl, 1986; Honjo et al., 1988). These side effects include transient irritative voiding symptoms including dypsia, hemorrhagic cystitis, bladder fibrosis, necrosis, con-tracture and vesicometral flux (Levine and Richie,

1989). Several reports have also linked cases of lung toxicity to this medication (Patel et al.,

1984). Similarly, several studies have demonstrated that bladder inflammation induced by CP in rats and mice increased transcript and protein expression in the urinary bladder of several cytokines including IL-6 (Malley and Vizzard, 2002). At higher doses used prior to marrow transplantation, the dose-limited toxicity is cardiac toxicity (Peters et al., 1989). Besides cardiac toxicity, hemorrhagic cystitis, water retention and hyponatremia are found in patients receiving high dose CP. Acrolein is the causal agent of hemorrhagic cystitis. Using MESNA

(sodium-2-mercaptoethanesulfonate) can reduce the incidence of hemorrhagic cystitis (Brock and Pohl 1986). A direct effect of CP on the renal tubules leads to excess water retention.

This can be managed by hydration with isotonic fluids (Bode et al., 1980). In women receiving CP, methotrexate and fluorouracil for treatment of breast cancer, severe thromboembolic events have been reported (Pritchard et al., 1997). Elevation of serum level of aminotransferases in patients treated with CP has also been reported. This CP-induced liver injury results from metabolites of CP, especially acrolein and is mainly dose-dependent

(Honjo et al., 1988). Metabolites of CP have been demonstrated to be teratogens and carcinogens in animals. Malformations have been associated with first trimester exposure to

CP (Zemlickis et al. 1993). It is reported that CP as a single chemotherapeutic agent, induces leukemia in human (Haas et al., 1987). The mechanisms are unknown, but this may be associated with the cytogenetic toxicity of CP (Au et al., 1980). Patients with breast cancer and inheritance of a combined gene deletion of GSTM1 and GSTT1 might bear an increased risk to develop a secondary CP-induced hematological neoplasia (Haase et al., 2002).

Cyclophosphamide treatment also leads to lung injury and cardiac toxicity, which are mediated by the reactive oxygen species (ROS) and lipid peroxide formation (Patel, 1987;

Sulkowska et al., 1998). Cyclophosphamide treatment not only induces lipid peroxidation

(LPO) but also suppresses the tissue and serum level of reduced glutathione (GSH),

26 glutathione peroxidase (GP), glutathione reductase, superoxide dismutase (SOD) and catalase

(CAT) activity (Sulkowska et al., 1998; Kaya et al., 1999).

1.2.2 Pathophysiology and consequences of urotoxic effects of CP.

Urotoxicity and myelosuppression are well documented dose-limiting adverse effects of oxazaphosphorines (CP and Ifosphamide) (Zhang et al., 2006). These undesirable effects tend to reduce the therapeutic benefit of these potent agents. Several in vivo and in vitro studies have shown that these toxic effects result, in part, following free radicals generation and lipid peroxidation with significant depletion of several antioxidants (Haque et al., 2003).

Urotoxicity manifests clinically, mainly as hemorrhagic cystitis. Hemorrhagic cystitis (HC) is defined as lower urinary tract symptoms that include inflammation of the bladder, dysuria

(painful urination), and hematuria (blood in urine) (Ratliff et al., 1998). The condition is very discomforting and if left unattended, may further result to anemia and secondary bacterial infection.

The pathophysiological mechanism of urotoxicity development is complex. This is based on oxazaphosphorines-derived metabolites inflammatory response activation. As it has already been mentioned, CP is metabolised by liver microsomal enzymes, with the simultaneous formation of active phosphoramide mustard derivatives particularly 4-hydroxy derivatives

(so-called peroxynitrite) and co-additional acrolein synthesis (Łukasz and Piotr, 2012). Final metabolic compounds together with acrolein are subsequently excreted intact into the urinary bladder and initiate hemorrhagic cystitis (HC). Acrolein is a reactive unsaturated aldehyde that can react with various structures (proteins, DNA nucleophilic sites), causing their cellular depletion of glutathione and other thiol compounds. Moreover, acrolein may also produce other biological effects. It has the ability to activate both nuclear factor –kB (NF-kB) and activator protein-1 (AP- 1), as well as to initiate lipids peroxidation and to exaggerate

27 oxidative stress damaging rapidly-dividing cells such as the epithelial cells lining the urinary bladder and blood immature progenitors resulting in hemorrhagic cystitis and myelosuppression (Korkmaz et al., 2007).

In fact, the proposed pathomechanism of acrolein-induced hemorrhagic cystitis involves several steps. After entering the urothelium, acrolein directly and indirectly increases the reactive oxygen species (ROS) overproduction in the bladder epithelium. Acrolein react with gluthatione to form glutathione adduct – glutathionylpropionaldehyde. This compound interacts with several enzymes, including xanthine oxidase and aldehyde dehydrogenase that produce acroleinyl and superoxide radicals. Toxic acrolein metabolite – glutathione propionaldehyde is co-responsible for the overproduction of free oxygen radicals by urothelium previously damaged by the same acrolein (Adams and Klaidman, 1993; Korkmaz et al., 2007). Moreover, it has also been demonstrated that inflammatory reaction evoked by oxazaphosphorines involves increasing nitric oxide (NO) and its secondary derivatives production, being an important factor of HC pathogenesis (Korkmaz et al., 2007). It is also known that reactive oxygen and nitrogen species may react and give secondary toxic metabolites. When NO and superoxide O2– react especially in equimolar concentrations, peroxynitrite (ONOO-) is formed, that is in pH-dependent equilibrium with peroxynitrous acid (ONOOH). Its hydrolysis gives highly reactive OH– molecule. The evidence of peroxynitrite formation during CP-induced HC in rats was reported by Korkmaz et al., (2007).

Their study revealed the influence of selective NO synthase (NOS) inhibitor – aminoguanidine and peroxynitrite scavenger – ebselen on bladder damage. They revealed that ebselen administration gave similar results to those observed after aminoguanidine – both agents protected the bladder histologically against CP damage and decreased NOS induction. Thus, it was suggested that not only nitric oxide but also peroxynitrite is involved in the pathogenesis of CP induced cystitis. It is regarded that ONOO– together with OH– augment

28 tissue damages (Korkmaz et al., 2005). Higher ONOO– and OH– levels may result in unwanted oxidation and covalently modification of all major biomolecules, leading to destruction of many cellular constituents, with special attention to DNA cellular injury.

Peroxynitrite oxidant modifies tyrosine in various proteins to nitrotyrosine and this nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathophysiological consequences (Beckman et al., 1996; Korkmaz et al., 2007).

Additionally, peroxynitrite causes increase in DNA strand breakage – that triggers the overactivity of polyadenosine-diphosphatase-ribose polymerase (PARP) – a DNA repairing enzyme. As a consequence, the depletion of oxidised nicotinamide-adenine dinucleotide

(NAD) and adenosine triphosphate (ATP) predisposing to cellular necrosis takes place

(Korkmaz et al., 2007).

Other possible causes of hemorrhagic cystitis include: radiation therapy involving the pelvis, adenovirus infection, some bacterial infection implicated in UTI (urinary tract infections), surgical procedures involving the lower urinary tract, inadvertent placement of vaginal chemical on the urethra, etc. (Friberg et al., 2002)

1.2.3 Pathophysiology and consequences of myelosuppressive effects of CP

Myelosuppression or bone marrow suppression is associated with reduction below normal values of all blood cells which are produced primarily in the bone marrow. Briefly, the bone marrow is the thick liquid in the inner compartment of some bones that is involved in the production of white blood cells (leukocytes), red blood cells (erythrocytes), and platelets

(thrombocytes). These cells are constantly produced and mature rapidly in the bone marrow.

Many chemotherpuetic agents including CP have been implicated in short-term damage of the bone marrow which results in abnormally low numbers of leukocytes, erythrocytes and platelets regarded as bone marrow suppression or myelosuppression (Friberg et al., 2002). As

29 mentioned earlier, whereas acrolein remains the culprit that induces urotoxicity, phosphoramide mustard is responsible for the cytotoxic effect of CP (Bernacki et al., 1987).

While this cytotoxic effect CP is useful in achieving its anticancer activity, it is very undesirable for the host blood cells.

Normally as blood cells wear out, they are constantly replaced by the bone marrow. But following chemotherapy, as these cells wear out, they are not replaced as they would be normally, and the blood cell counts begin to drop. The type and dose of the chemotherapy will influence how low the blood cell counts will drop and how long it will take for the bone marrow to recover (Friberg et al., 2002).

Free radical generation is one of the mechanisms by which CP and its derivatives exert their toxic effects in different tissues as well as the bone marrow (Haque et al., 2003). An overall decrease in the reduced glutathione (GSH) content has often been reported in various tissues as a result of CP treatment (Patel et al., 1984; Haque et al., 2003).

Cyclophosphamide-induced immuno-suppression is reported to prompt various types of infection (Angulo et al., 2002). Some of the infectious agents have GSH depleting effects

(Hung and Wang, 2004). Cyclophosphamide treatment may therefore decrease the GSH content itself but the associated secondary infections are likely to cause an additional decrease in the GSH level. For that reason, a patient undergoing CP chemotherapy needs excessive supply of GSH restoring anti-oxidants or compounds that induce GSH production

(Hamrita et al. 2012).

The resultant cytotoxic metabolites and toxic byproducts of CP are detoxified by various aldehyde dehydrogenases (ALDHs) and by conjugation with glutathione (GSH) via GSH S- transferases (GSTs) intracellularly (Zhang et al., 2006). Therefore the role of tissue antioxidants becomes important in the prevention of such peroxidative damage induced by

CP treatment.

In all species each type of blood cell has a different life span. In mammals:

• Leucocytes come in several types that have a wide range of life spans. They are

classified as granulocytes and agranulocytes. Granulocytes show distinctive stained

granules in the cytoplasm under light microscope, e.g. neutrophils, basophils,

eosinophils. Agranulocytes do not show granules e.g. monocytes and lymphocytes

(Mazza 1995). Neutrophils, a type of leucocyte of special importance in fighting

infections live for an average of 6 hours

• Platelets average 10 days

• Erythrocytes average 120 days

The lowest count that blood cell levels fall to after chemotherapy is called the nadir. The nadir for each blood cell type will occur at different times. Usually neutrophils and platelets will reach their nadir within 7 to 14 days. Because erythrocytes live longer, they will typically take a few weeks to reach their nadir (Mazza, 1995).

Functions of Blood Cells: The knowledge of functions of these three categories of blood cells is necessary for good understanding of the consequences of their reduction in normal values.

• Leukocytes help the body to fight off infections.

• Erythrocytes transport oxygen to cells throughout the body for proper nutrient

utilization and energy production.

• Platelets help prevent bleeding by forming plugs to seal up damaged blood vessels.

Therefore the reduction in number below normal values of these cells will definitely result to immune suppression, anemia and tendency to bleed easily with prolonged clotting time

(Mazza 1995).

The normal reference values of all the blood cells in adult humans and male rats (Sprague

Dawley albino breed) are listed in the table below:

Table 1: Normal reference values of blood cells in adult humans and male rats.

Species Erythroc Total Neutrophil Lymphocyte Basophil Eosinophil Monocyte yte count leukocyte count count count count count x103 x106 µl count x103 µl x103 µl x103 µl x103 µl µl x103 µl

Man 4.28-6.74 4 – 10 1.5 – 7.5 1 – 4 < 0.11 < 0.41 0.2 – 8

Rat 5.73 – 11.86- 1.93-3.59 7.13-11.66 < 0.11 < 0.44 1.33-3.22 7.69 16.14

Adapted from (Mazza 1995) and (Ihedioha et al., 2004).

The clinical implication of hematological assays is that, the total leukocyte count (TLC) and differential leukocyte counts (DLC) reflect the systemic status of an animal in relation to its response and adjustment to injurious agents, stress and/or deprivation; the indices are of value in confirming or eliminating a tentative diagnosis, in making prognosis and guiding therapy (Coles, 1986). The TLC and DLC could further provide information on the severity of an injurious agent, the virulence of an infecting organism, the susceptibility of a host, and the nature, severity and duration of a disease process (Ihedioha et al., 2004)

The significance of the pathophysiological changes caused by these oxidants generated by

CP metabolites cannot be over emphasized. However, there are known intracellular antioxidant defense mechanisms against ROS. They consist of superoxide dismutase (SOD) which converts radical superoxide to H2O2, catalase (CAT) and glutathione peroxidase

(GSH-Px), which inactivate H2O2 (Korkmaz et al., 2007). Under physiological conditions, oxidants and antioxidant defense mechanisms maintain a redox equilibrium. An excessive

ROS production and increased NOS activity (both evoked in bladder by acrolein) disturb the 32 normal equilibrium and NO excess can outcompete SOD for O2–, secondarily resulting in a peroxynitrite formation, with subsequent protein and lipids oxidation, DNA damage, PARP activation and finally cellular energy crisis, which has been already mentioned above

(Korkmaz et al., 2007).

In summary, there is no doubt that the pathophysiology of CP-induced hemorrhagic cystitis and myelosuppression is a complex inflammatory process involving several cytokines, free radicals and non-radical molecules that severely damage host normal cells. Thus all these components of a pathophysiological description must be taken into consideration when looking for more effective chemopreventive compounds against the toxicities so presented

(Korkmaz et al., 2007).

1.3 Chemoprevention and amelioration of CP-induced toxicities

It is well known that any kind of antineoplastic chemotherapy is associated with both mild or moderate and serious life-threatening side effects. They develop due to the inability of cytotoxic drugs to differentiate between their target – malignant cells and normal ones

(Łukasz and Piotr 2012). Antineoplastic chemotherapeutics mostly affect fast-dividing cells, such as blood immature progenitors, the epithelial cells lining the mouth, stomach, intestines, respiratory and urinary tracts, skin and mucosal layers resulting in myelosuppression

(anaemia, depression of the immune system, tendency to bleed easily), malnutrition, progressive body weight loss, hair loss, infertility (Łukasz and Piotr, 2012). These antineoplastic agents also produce nausea and vomiting which is often reported by patients as very unpleasant and severe chemotherapeutic adverse effect. Moreover, damage of the specific organs may also occur, with resultant cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity or even encephalopathy (Links and Lewis, 1999).

Taking into consideration the inevitable cytostatics toxicity of CP, numerous researches are being carried out to find methods to abolish, or at least ameliorate the toxicity it presents

(Links and Lewis, 1999; Łukasz and Piotr 2012). General developments include the synthesis of analogues of established cytotoxic agent with improved toxicity profiles of comparable antitumour efficacy, or alternations in schedules of cytotoxic drug administration (Links and

Lewis, 1999). However, the development of cytoprotection is the most pharmacologically attractive method of diminishing the CP toxicity. The theory of cytoprotection phenomenon during antineoplastic chemotherapy is related to the design of chemoprotectants development. Chemoprotectants are defined as compounds providing tissue-specific cytoprotection, without compromising the desired antitumour efficacy and affecting the additional own toxicity that might jeopardise the effects of adequate chemotherapy (Links and Lewis, 1999).

Since most cytotoxic drugs including CP are myelosuppressive, the bone marrow is a major target for chemoprotective agents. Hematopoietic colony-stimulating factors, used in alleviating CP treatment-related myelosuppression and its consequences, may be considered as one of the first chemoprotectants to have been introduced (Links and Lewis, 1999). These agents entered a wide clinical practice enabling recovery of leukocytes. Erythropoietin use that escalates bone marrow erythrocytes restoration is another example in alleviation of special toxicities (Muggia, 1994; Links and Lewis, 1999).

Numerous studies have demonstrated that nucleophilic sulfur containing compounds such as glutathione can antagonize some harmful effects of the alkylating agents (Dorr, 1991; Links and Lewis, 1999). At present, two agents in this class have drawn the attention of the scientists: amifostine and reduced glutathione. In contrast to dexrazoxane, amifostine is the first broad-spectrum chemoprotectant (bone marrow, peripheral nerve, heart and kidney)

(Links and Lewis, 1999). Preclinical studies have revealed that the amifostine administration protected against a variety of antineoplastic chemotherapy-associated toxicities, including cis-platin nephrotoxicity and neurotoxicity, cyclophosphamide and bleomicin – induced pulmonary toxicity and cytotoxicity evoked by doxorubicin (Santini and Giles, 1999). The broad cytoprotective effects of amifostine seem beneficial and, therefore, the use of this chemoprotectant should be encouraged (Links and Lewis, 1999).

The second nucleophilic sulfur compound – glutathione is also being investigated in view of its potential use especially in cis-platin induced nephrotoxicity. However, its role in determining the cytoprotection remains controversial – extracellular glutathione is not normally taken up by cells except those expressing high level of g-glutamyltranspeptidase activity. This compound is still in preclinical studies (Links and Lewis, 1999).

Korkmaz et al., (2007) reported that the influence of selective NO synthase (NOS) inhibitor – aminoguanidine and peroxynitrite scavenger – ebselen protected bladder damage. They revealed that ebselen administration gave similar results to those observed after aminoguanidine – both agents protected the bladder histologically against CP damage and decreased NOS induction.

Dexrazoxane is a cyclic derivative of EDTA acid that provides cardiac protection from anthracyclines primarily by means of a metal-chelating action (especially free iron and iron bound in anthracycline complexes), therefore preventing the formation of cardiotoxic reactive oxygen radicals (Cvetkovic and Scott, 2005).

The haemorrhagic cystitis, which is the adverse effect of oxazaphosphorines chemotherapy, can also be prevented by co-administration of thiol-containing compound – MESNA (sodium

2-mercaptoethane sulfonate). This agent is regarded to be a specific chemoprotectant against acrolein-induced bladder toxicity.

1.3.1 The Role of MESNA (sodium 2-mercaptoethane sulfonate) in amelioration of

CP-induced toxicities

Sodium-2-mercaptoethanesulfonate MESNA is a sulfhydryl-containing compound (a thiol).

It is a clear, colourless solution with empirical formula of C H NaO S , and a molecular 2 5 3 2 weight of 164.18.

Figure 2: Chemical structure of Sodium 2-mercaptoethane sulfonate

It is employed in regional detoxification of urothelium to prevent urotoxicity (haemorrhagic cystitis) induced by metabolites of oxazaphosphorine alkylating agents, such as ifosfamide and cyclophosphamide (Brock, 1980). Sodium-2-mercaptoethanesulfonate entered clinical trials as a systemic uroprotective agent in the late 1970s, becoming the drug of choice for this purpose within a short period of time (Katz et al., 1995).

1.3.2 Mechanism of action of MESNA

After oral or intravenous administration and entering the circulation, MESNA is rapidly oxidised into the inactive dimer, dithio-bis-sodium-2-mercaptoethanesulfonate (DIMESNA) that does not inhibit the antineoplastic action of oxazaphosphorines (Brock et al., 1982).

DIMESNA is then filtered in the kidneys and about 30-50% of glomerularly filtered

DIMESNA undergoes tubular reabsorption and is reduced back to mesna in the renal tubular epithelium by glutathione reductase and thiol transferase (Brock et al., 1982; Souza-Filho et

36 al., 1997). In such a form it is then delivered into the bladder, and the resulting free sulfhydryl groups of MESNA can combine directly with the double acrolein bonds and with other oxazaphosphorine metabolites to create nontoxic compounds. As MESNA activity is restricted to the urinary tract, the systemic action (and non-urological adverse effects of the oxazaphosphorines) is not affected and, therefore, it is possible to apply MESNA and these alkylating agents simultaneously (Morais et al., 1999). At present, MESNA has been widely accepted as chemoprotectant with established clinical position in cyclophosphamide or ifosfamide induced HC. Moreover, it has been shown that it is as effective in bladder inflammatory alternations blockage as dexamethasone (Morais et al., 1999). However, in about 20% of oxazaphosphorines treated patients receiving MESNA and hydration as present standard against HC, the development of cystitis is observed. Hence, there are further attempts to introduce other methods of HC prevention (Łukasz and Piotr, 2012).

Adverse effects, which may occur after administration of MESNA, include gastro-intestinal effects, headache, fatigue, limb pain, depression and hypotension (Reinhold-Keller et al.,

1992). However, it is difficult to distinguish the adverse reactions, which may be due to

MESNA from those caused by concomitantly administered CP (or other cytostatic agents).

Animal studies suggest MESNA to be non-foetotoxic (Siu and Moore, 1998). However, studies on pregnant women have not been carried out. It is recommended that MESNA should not be given to pregnant women or should be given only under compelling circumstances (Łukasz and Piotr, 2012)

It is important to note that MESNA protects only against the urothelial toxic effect of oxazaphosphorines (i.e. ifosfamide or cyclophosphamide) and not their renal and other toxic effects. Thus the additional prophylactic or accompanying measures recommended during treatment with oxazaphosphorines should not be discontinued (Łukasz and Piotr, 2012).

1.3.3 Other potential uroprotective agents

Other Supportive therapies which include stem cells, hematopoietic growth factors e.g. granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony- stimulating factor (GM-CSF), as well as body detoxifying agents such as amifostine, rasburicase, dexrazoxane, and palifermin are usually combined with CP to protect the key organs of the host (Abd-Allah, 2005; Łukasz and Piotr, 2012). Again a number of GSH- inducing compounds have been found to be effective in reducing CP toxicity in animals

(Manesh and Kuttan, 2005).

Moreover, there are also reports that sulphur containing aminoacids – seleno L-methionine

(Ayhanci et al., 2010) and L-cysteine (Roberts et al., 1991) administration in CP-induced animals resulted in GSH elevation. An experimental study has also revealed that the combination of mesna and hyperbaric oxygen supplementation provides almost complete protection against HC, although it is not practical for the majority of patients undergoing antineoplastic chemotherapy (Korkmaz et al., 2001). Thus MESNA still remains the common administered chemoprotective agent against HC during CP or IF treatment.

Recently, CP and other anticancer drugs have been tried in combination with various detoxifying and protective agents with the purpose of reducing or eliminating their adverse toxic effects. Ayurvedic Rasayana, curcumin, protein A, GSH, and sulphur-containing compounds have shown varying degrees of protection against CP toxicity (Rekha et. al.,

2001; Habs and Hebebrand 1984).

1.3.4. Medicinal plants used in preventing or ameliorating CP-induced toxicity

The mechanisms and the pathophysiology of CP toxicity provided elucidating information for acceptance that haemorrhagic cystitis develops as a non-microbial inflammation, with high level of ROS (peroxynitrite) and proinflammatory cytokines and other proteins production (Korkmaz et al., 2007). Thus, based on the HC pathogenesis, there are efforts

38 towards other chemopreventive agents development, targeted at inflammatory background mentioned above. Among them, numerous phytopharmacological derivatives are being studied, with flavonoids and polyphenoles, being of special interest (Ozcan et al., 2005;

Łukasz and Piotr, 2012). The rationales for the investigation of these compounds were the reports about their significant antioxidant and anti-inflammatory properties (Ozcan et al.,

2005).

Flavonoids have been shown to inhibit enzymes responsible for superoxide production

(xanthine oxidase, protein kinase C) as well as other enzymes involved in ROS synthesis and inflammatory process initiation and maintenance (cyclooxygenase, lipooxygenase, microsomal monooxygenase, mitochondrial succinoxidase and NADH oxidase) (Hanasaki et al., 1994; Brown et al., 1998). Some of them also diminish the activity of immunoglobulin proteins of cell adhesion molecule class (CAM), especially intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) as well as integrins

(CD11b or MAC-1) and selectines (E-selectin and P-selectin) families of CAMs (Spelman et al., 2011). Moreover, flavonoids are regarded to be non-enzyme antioxidants reducing molecular damage by oxygen and nitrogen species. Beneficial role of the flavonoids has been confirmed in many various experimental models of inflammation, and, therefore, they are applied in many clinical inflammatory disturbances (Rotelli et al., 2003). Moreover, apart from anti-inflammatory properties, flavonoids also exert hepatoprotective activity in experimental cirrhosis and diabetes by means of insulin release stimulation. These compounds affect the cardiovascular system, preventing endothelial dysfunction by enhancing the vasorelaxant process, exerting the antiatherosclerotic effect (protecting LDL against oxidative stress) and antithrombotic effects. Flavonoids have been shown to be effective inhibitors of platelet adhesion and aggregation because of their ability to maintain proper concentration of endothelial prostacycline and nitric oxide. Consequently, the therapeutic

39 potential of flavonoids is fairly obvious and it should be expected that these agents might be applied in bladder inflammatory disturbances (Tapas et al., 2008).

There are two flavonoids being currently evaluated in cyclophosphamide-induced toxicity in rats: morin and ternatin (Pessoa et al., 2000; Kim et al., 2010). Morin is a pentahydroxyflavone derivative which is isolated from the Moracae family (Chlorophora tinctoria and Prunus dulcis). This flavonoid is reported to have marked cytoprotective properties against oxidative stress by means of its antioxidative and anti-inflammatory abi- lities that may stem from not only its scavenging activity, but also the anti-NF-kB pathways activation (Kim et al., 2010). Morin was also proven to protect cells against radiation- induced oxidative stress, causing reduced Bax, phospho Bcl-2 and active caspases expression together with exertion of anti-apoptotic effects (Zhang et al., 2010). Based on these data, it seems that morin anti-inflammatory and antioxidative properties may have potential therapeutic application in the treatment and prevention of ROS-induced inflammatory processes, including HC. In one study, this compound partly improved haematological parameters and restored to normal the plasma protein level values in CP treated rats.

However, the morin impact on bladder motility and morphology has not yet been studied and, thus, it is preliminary to predict its role in HC chemoprevention and it is necessary to further clarify its potential application (Ląd-Merwid et al., 2011).

The other flavonoid initially investigated as a HC chemoprotectant is ternatin, which is isolated from Egletes viscosa (Compositae) (Pessoa et al., 2000; Łukasz and Piotr, 2012).

This compound is attributed to possess antiproliferative effect together with anti- inflammatory, and antithrombotic properties (Pessoa et al., 2000). Experimental research demonstrated that ternatin inhibited fat accumulation and reduced fat mass by affecting differentiation stages of adipocytes and decreasing lipogenic enzyme activity (Tapas et al.,

2008). Also consistent with these findings, ternatin was demonstrated to diminish triglyceride

40 synthesis, hence, it might provide a new therapeutic approach to treatment of metabolic disorders (Ito et al., 2009). Vieira et al. (2004) demonstrated that ternatin, when combined with classical HC chemoprotectant – MESNA, could ensure excellent protection in both CP and IF – induced HC. These researchers compared the effects after the ternatin replacement of 1 or 2 doses of MESNA to the threefold MESNA administration in oxazaphosphorine- induced bladder damage. They found evidence indicating that a replacement of 2 doses of

MESNA with ternatin was even more efficient in preventing CP-induced HC than the treatment with 3 doses of MESNA and as efficient as 3 MESNA doses in the prevention of

IF-induced HC. This could be explained in light of the anti-inflammatory properties of ternatin. Moreover, what was also interesting is that replacing all MESNA doses with this flavonoid did not prevent both CP and IF – induced HC, suggesting that MESNA seems to be necessary for initial uroprotection via its neutralising effect on urothelial damage initiated by acrolein, while ternatin seemed to inhibit the inflammatory mediators that follow the acrolein action (Vieira et al., 2004). The studies by Vieira et al., (2004), therefore strongly support the potential flavonoid clinical applicability as adjuvant chemoprotective agents in HC after oxazaphosphorines.

The other phytopharmacological derivative that has also been the subject of considerable attention in view of its varied therapeutic potential is a class of constituents together known as withanolides (steroidal lactones with ergostane skeleton) (Davis and Kuttan, 2000). These agents were isolated from Withania somnifera (Solanacae), popularly known as

Ashwagandha or Winter Cherry (Davis and Kuttan, 2000). Since the withanolides have a structural resemblance to the active compounds present in the plant Panax ginseng known as ginsenosides, Withania somnifera is also named as “Indian Ginseng”. The withanolides group consists of alkaloids (withanine, somniferine, somnine, somniferinine, withananine, tropine, choline, cuscohygrine, isopelletierine, anaferine, anahydrine and others),

41 glycowithanoloids (sitoindoside IX and X) and other sitoindosides (Kulkarni and Dhir,

2008). Withaferin A was one of the first isolated members of withanolides family (Xu et al.,

2011). Considered in general terms, withanolides have been described to possess multiple biological properties which include: anti-inflammatory, immunomodulatory, antineoplastic, antistress as well as adaptogenic and cardiovascular effects (Kulkarni and Dhir, 2008). The antioxidant activity of Withania somnifera active principles was displayed as similar to the one demonstrated by flavonoids. Withanolides (especially withaferin A and sitoindosides

VII-X) modulate the oxidative stress, significantly reducing the lipid peroxidation while increasing the antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) activity, thus possessing free radicals scavenging properties (Kaur et al., 2004). Withanolides have also been found to inhibit complement activity, lymphocyte proliferation and delayed- type hypersensitivity (Mirjalili et al., 2009). These findings may explain at least in part the reported anti-inflammatory, immunomodulatory, antistress and anti-aging effects produced by these compounds in experimental studies and observed in clinical conditions

(Bhattacharya et al., 1997; Kaur et al., 2004; Mirjalili et al., 2009). These features are also closely connected to possible antineoplastic activity of withanolides. These compounds may mitigate unregulated cell growth and proliferation via the potent tumour suppressor gene p53 by sustaining its control activity, affecting NF-kB apoptic pathways and tumour neoangiogenesis inhibition (Mohan et al., 2004; Winters, 2006).

Withanolides were also studied in cyclophosphamide-induced urotoxicity in rats (Davis and

Kuttan, 2000). Their research demonstrated that administration of Withania somnifera extract along with cyclophosphamide could normalize bladder morphological pathology. The CP treated rats, after 24 hours of CP application, showed severe bladder inflammatory changes with uroepithelium completely replaced by metaphasic squamos epithelium with high mitotic activity, whereas, 48 hours after CP administration the lining epithelium was completely

42 replaced by necrotic cells and numerous inflammatory cells. In the presence of Withania compounds bladder histology was normalized, showing almost normal architecture.

Moreover, blood urea nitrogen level was significantly reduced in Withania extract-treated group while the glutathione content both in bladder and liver was enhanced. Therefore, according to (Davis and Kuttan, 2000) morphological, histopathological and biochemical analysis showed that Withania somnifera could alleviate the urotoxicity induced by cyclophosphamide and further that withanolides seem to be promising chemoprotectant agents.

Several other natural compounds and herbal extracts have been investigated as modulators of oxazaphosphorines urotoxicity (Matthew and Kuttan, 1997; Haque et al., 2001).

One of them is Juglans regia (Juglandacae) (Haque et al., 2003). The walnut bark is widely used in folk medicine for treatment of venous insufficiency and haemorrhoidal symptoma- tology and for its antidiarrheic, astringent and antihelmintic properties (Yeung-Him-Che,

1985; Brown, 1995). This plant was also shown to have antioxidant, antiproliferative, blood purifying, and depurative activities by exhibiting high phenolic content (Amaral et al., 2004;

Pereira et al., 2007). This plant is considered to be a source of many phytochemicals also decreasing the risk of oxidative stress and macromolecular oxidation, such as: juglone

(naphtoquinone derivative), hydroxycinnamic acids (3-caffeoylquinic, 3-p-coumaroylquinic,

4-p-coumaroylquinic) and flavonoids (quercetin, kaempferol) (Amaral et al., 2004; Pereira et al., 2007). The walnut extract and its immunomulatory effect have also been studied in CP- induced HC in mice. This treatment resulted in protective restoration of decreased antioxidants in CP-treated animals and lowered the lipid peroxidation in the bladder, thus the promising activity of this plant warrants possible clinical investigations (Bhatia et al.,

2006a).

Trigonella foenum-graecum (Leguminosae) has also been studied in bladder toxicity. This plant has already been extensively evaluated as a source of antidiabetic and antihyperlipidemic compounds (Baquer et al., 2011). One of the active constituent of

Trigonella foenum-graecum (Fenugreek) is an aminoacid – 4-hydroxyisoleucine, which has been shown to exhibit insulinotropic activity related to the immolation of the glucose concentration (Baquer et al., 2011). Thus, it might be considered as a novel secretagogue with a potential interest for the treatment of type II diabetes (Baquer et al., 2011). Moreover, this plant contains steroidal saponins, such as diosgenin and flavonoids (witexin and its derivatives) (Hamrita et al., 2012). It has been found that Trigonella extract is characterised to have immunomodulatory property that might be an advantageous alternative to prevent oxazaphosphorine-induced HC (Hamrita et al., 2012). Bhatia et al. (2006b) examined re- storative effect of Trigonella extract on reduced glutathione (GSH), antioxidant enzymes activity and lipid peroxidation level in CP-treated mice, additionally exposed to a GSH reducing agent – L-buthionine-sulfoximine (BSO) which inhibits g-glutamylcysteine synthetase. The researchers revealed that Fenugreek extract pretreatment not only showed protective effect on CP urotoxicity but that it was also effective in protecting the animals treated with the CP and BSO combination, with depleted GSH. There was restoration of other antioxidants and reduction in bladder lipid peroxidation. Thus, anti-inflammatory and immunomodulatory herbal extract like Fenugreek holds great promise in the reducing the adverse effects in cyclophosphamide treated patients (Bhatia et al., 2006b; Bechr et al.,

2012).

Another interesting plant investigated for its possible applicability in urotoxicity amelioration is Ipomoea obscura (Convolvulaceae). This plant has been shown to have analgesic and antimicrobial effects and is topically used in phytomedicine for pain relief, treatment of sunburns, burns, boils, insect bites and small wounds (Hamsa and Kuttan, 2011). It is

44 suggested to be administered to cure stomach ulcers and in neoplastic tumours. Several indolizidine alkaloids were isolated from extract of Ipomoea (ipomine, ipalbidine, ipalbine and ipalbinium) (Ysrael et al., 2003). This plant also contains muricatin and other lipooligosaccharides derivatives – so called resin glycosides (Ysrael et al., 2003). The protective role of Ipomoea obscura against CP-induced uro- and nephrotoxicities in mice was investigated by Hamsa and Kuttan (2011). The toxicities caused by cyclophosphamide were reversed by Ipomoea extract administration as evident from the decrease in blood urea nitrogen, serum creatinine levels, as well as an increase in body weight. Histopathological assessment of urinary bladder indicated that CP-induced tissue damage was significantly reduced in extract-treated animals. Moreover, the level of proinflammatory cytokine – TNF-

α, which was elevated during CP administration, was significantly reduced by extract application. Thus the study demonstrated that Ipomoea obscura can ameliorate CP-induced bladder and renal toxicities by modulating antioxidant status and proinflammatory cytokines levels.

Incidentally, it should be also mentioned, that naturally occurring sulphur compounds are also regarded to prevent bladder damage after oxazaphosphorine administration. S- allylcysteine – an organosulphur compound of garlic (Allium sativum; Amaryllidaceae) extract regulates the thiol status of the cells and scavenges free radicals. Because depletion of thiols along with an excessive oxidative stress is implicated in CP/IF-induced HC, S- allylcysteine is another phytopharmaceutical agent with potential anti-urotoxicity action. It was also reported that S-allylcysteine showed protection in bladder tissue histology and also improved the decreased activities of antioxidant enzymes in mice with CP-evoked HC

(Bhatia et al., 2008). This compound increased GSH level. Although S-allylcysteine treatment did not ensure full recovery, the marked improvement in bladder histology and antioxidants suggest that it has a significant protective effect on CP-induced urotoxicity

(Bhatia et al., 2008). Similar results were obtained by Manesh and Kuttan (2002), who studied diallyl sulphide and diallyl disulphide in the same experimental model.

In summary, there is lots of naturally occurring phytopharmacological agents which bring large hope on successes of new chemoprotectant agents providing a great promise in the reduction of oxazaphosphorines-evoked haemorrhagic cystitis.

Table 2: Some plants documented to have ameliorating properties in cylophosphamide- induced toxicity. S/N Plant (family) Phytoconstituent Type of toxicity preventented

1 Chlorophora tinctoria flavonoids Urotoxicity

2 Prunus dulcis flavonoids Urotoxicity (Moracae) 3 Egletes viscosa flavonoids Urotoxicity (Compositae) 4 Withania somnifera (Solanacae) withanolides Urotoxicity

5 Juglans regia (Juglandacae) phenoles Urotoxicity flavonoids 6 Trigonella foenum-graecum steroidal saponins Urotoxicity (Leguminosae) flavonoids

7 Ipomoea obscura (Convolvulaceae) indolizidine alkaloids Urotoxicity lipooligosaccharides derivatives– resin glycosides 8 Allium sativum organosulphur compounds Urotoxicity (Amaryllidaceae) 9 Phyllantus amarus - Immunosuppression 10 Sorghum bicolor - Neurotoxicity 11. Vernonia cinerea L. - Immunosuppression and oxidative stress 12 Cardiospermum halicacabum - Immunosuppression and oxidative stress 13 Biophytum sensitivum - Immunosuppression 14 Andrographis paniculata - Immunosuppression 15 Cassia ocidentalis L. - Mutagenicity and immunosuppression 16 Emblica officinalis Gaertn. - Immunosuppression 17 Moringa oleifera Lam. - Immunosuppression 18 Sphaeranthus indicus - Humoral immunity 19 Phyllantus niruri - Urotoxicity

Adapted from Łukasz and Piotr, (2012).

1.4. Botanical profile of Vernonia amygdalina Del.

1.4.1 Taxonomy

Kingdom: Plantae

Phylum: Angiosperm Class: Eudicots Order: Asterales Family: Asteraceae Genius: Vernonia Species: V. amygdalina

Figure 3: Vernonia amygdalina in its natural habitat

In many parts of Africa the plant is variously known as ‘Grawa’ in Amharic, ‘Ewuro’ in

Yoruba, ‘Etidot’ in Ibibio, ‘Onugbu’ in Igbo, ‘Ityuna’ in Tiv, ‘Oriwo’ in Edo and ‘Chusar- doki’ in Hausa (Egedigwe, 2010).

1.4.2 Description

Vernonia amygdalina Del. (family of Asteraceae) is a valuable medicinal plant that is widespread in East and West Africa (Ainslie, 1973; Burkill, 1985). It is a perennial shrub of

2-5m in height that grows throughout tropical Africa. It has a rough bark with dense black straits, and elliptic leaves that are about 6 mm in length. The leaves are green and have a characteristic bitter taste which makes it to be commonly referred to as bitter leaves (Singha,

1966).

1.4.3 Geographical Distribution

In many parts of West Africa, the plant has been domesticated (Igile et al., 1994). V. amygdalina is drought tolerant (though it grows better in a humid environment). It thrives on a range of ecological zones and is used as a hedge plant in some communities (Bonsi et al.,

1995).

1.4.4 Ethnomedicinal Uses

The roots and the leaves are used in ethnomedicine to treat fever, hiccups, kidney problems, and stomach discomfort (Burkill, 1985; Hamowia and Saffaf, 1994). The stem and root divested of the bark are used as chew-sticks in many West Africa countries like Cameroon,

Ghana, and Nigeria. V. amygdalina leaves are one of the most widely leaf vegetables (ndole or bitter leaf) consumed by Nigerians and Cameroonians during special occasions such as marriages, baptisms, Christmas, and birthday.

1.4.5 Documented research findings on V. amygdalina

Pharmacological studies have shown that the leaf extracts of V. amygdalina have both hypoglycaemic and hypolipidaemic properties in experimental animals and so could justify their use in managing diabetes mellitus by local healers (Akah and Okafor, 1992). It has been reported to have anticancer activity (Izevbigie, 2003), anti-bacteria, antimalaria, and anti- parasitic activities (Tadesse, 1993). The beneficial use of V. amygdalina in animal nutrition in Nigeria has been well documented (Onwuka et al., 1989; Aregheore et al., 1998). It was reported that V. amygdalina leaf extract enhanced the prophylactic and therapeutic efficacy of chloroquine against Plasmodium berghei malaria in mice (Iwalokun, 2008). Published studies indicate that V. amygdalina have medicinal properties effective against many diseases other than breast cancer. The molecular mechanisms under which this compound exerts its therapeutic effect in cancer cells have also been reported (Izevbigie, 2003; Izevbigie et al.,

2004). Reports indicate that extracts from planpoiuts are able to inhibit and even reverse

carbon tetrachloride-induced hepatotoxicity in mice and rats (Ijeh et al., 1996; Babalola et

al., 2001).

V. amygdalina Del. has been shown to contain significant quantities of lipids (Ejoh et al.,

2007; Eleyinmi et al., 2008), proteins with high essential amino acid score (Igile et al., 1994;

Udensi et al., 2002; Ejoh et al.,2007; Eleyinmi et al., 2008) that compare favorably with

values reported for Telfairia occidentalis and Talinum triangulare (Ijeh et al., 1996),

carbohydrates (Ejoh et al.,2007) and fiber (Udensi et al., 2002; Ejoh et al., 2007; Eleyinmi et

al., 2008). The plant has also been shown to contain appreciable quantities of ascorbic acid

and caroteinoids (Udensi et al., 2002; Ejoh et al., 2007). Calcium, iron, potassium,

phosphorous, manganese, copper and cobalt have also been found in significant quantities in

V. amygdalina (Bonsi et al., 1995; Ejoh et al., 2007; Eleyinmi et al., 2008).

Table 3: Phytochemical constituents of V. amygdalina.

S/N Phytoconstituent Reference 1 Stigmastane-type saponins – e.g. Ohigashi et al., (1991), Jisaka et al. Vernoniosides A1, A2, A3, A4, B1, B2, (1992), Kamperdick et al. (1992) and B3, C, D, E Jisaka et al. (1993a)

2 Steroidal saponins Ohigashi et al., (1991), Jisaka et al. (1992), Jisaka et al. (1993a), Igile et al. (1994) and Igile et al. (1995) 3 Sesquiterpene lactones – e.g. Kupchan et al. (1969), Jisaka et al. Vernolide, vernodalol, vernolepin, (1992) Jisaka et al. (1993b), Koshmizu et vernodalin, vernomygdin, al. (1994) and Erasto et al., 2006 Hydroxyvernolide 4 Flavonoids Luteolin, luteolin 7-O-β- Igile et al. (1994), Udensi et al. (2002); - glucoroniside, luteolin 7- Farombi, (2003) and Tona et al. (2004). O-β-glucoside 5 Terpenes, coumarins, Wall et al. (1998) and Tona et al.( 2004) phenolic acids, lignans, xanthones, anthraquinones 6 Edoties (peptides) Izevbigie (2003)

Adapted from (Ijeh and Ejike, 2011).

Table 4: Documented pharmacological properties of V. amygdalina.

S/N Property Type of extract References 1 Antibacterial Methanol (60%) Akinpelu (1999) Crude extract Ijeh et al. (1996) Ethanol Erasto et al. (2006) Jisaka et al. (1993b) 2 Antiplasmodial/antimalarial Aqueous Njan et al. (2008) 51

Aqueous Iwalokun. (2008) Ethanol Abosi and Raseroka (2003) - Masaba (2000) 3 Amoebicidal Moundipa et al. (2005) Huffman et al. (1996) 4 Antifungal Aqueous Alabi et al. (2005) Ethanol Erasto et al. (2006) Wedge et al. (2000) 5 Antileishmanial Chloroform and Tadesse et al. (1993) methanol - Huffman et al. (1996) 6 Antischistosomial Ogboli et al. (2000)

7 Wound management Crude extract Giday et al. (2003) 8 Anti-Cancer/Tumor Oyugi et al. (2009) Chloroformic Kupchan et al. (1969) Aqueous Yedjou et al. (2008) Aqueous Gresham et al. (2008) Aqueous Izevbigie (2003) (2004) Aqueous Howard et al. (2006) Aqueous and ethanol of Khalafalla et al., (2009) root cultures Jisaka et al., (1993b) 9 Venereal disease management Kambizi and Afolayin (2001) 10 Antioxidant Aqueous and ethanolic Owolabi et al., (2008) Methanol (30%) Igile et al., (1994) Aqueous Nwanjo, (2005) Methanol Adaramoye et al. (2008) - Iwalokun et al., (2006) 11 Hypoglycemic/Antidiabetic Ethanol Ekpo et al. (2007) Aqueous Osinubi (1996) Aqueous Nwanjo and Nwokoro, (2004) Aqueous Akah and Okafor (1992) Aqueous Uhuegbu and Ogbechi, (2004) Crude extract Atangwho et al. (2007) 12 Oxytocic Aqueous Kamatenesi-Mugisha (2004) Aqueous Kamatenesi-Mugisha et al. (2005)

13 Hepatoprotection Aqueous Arhoghro et al. (2009) Diet incorporation Ijeh and Obidoa (2004) - Babalola et al. (2001) Iwalokun et al. (2006) 14 Nephroprotection Ethanol Atangwho et al. (2007) Ethanol (80%) Atangwho et al. (2009a) 15 Serum lipid modulation Diet incorporation Egedigwe (2010) Diet incorporation Ugwu et al. (2010) Methanol Adaramoye et al. (2008) Ethanol Ekpo et al. (2007) Aqueous Nwanjo (2005) - Ezekwe and Obidoa (2001) 16 Gastric secretion Aqueous Owu et al. (2008) 17 Analgesic Aqueous Njan et al. (2008) 18 Anti-fertility Root extract Steen-Kamp (2003) - Desta (1994) 19 Insecticidal Dust Kabeh and Jalingo (2007) Oil Asawalam and Hassanali (2006)

20 Phytotoxic Alabi et al. (2005)

Adapted from (Ijeh and Ejike, 2011).

1.5. Botanical profile of Ocimum gratissimum Linn.

1.5.1 Taxonomy

Kingdom: Plantae Phylum: Angiosperm Class: Eudicots Order: Lamiales Family: Lamiaceae Genius: Ocimum Species: O. gratissimum

Figure 4: Ocimum gratissimum in its natural habitat

It is popular among various ethnic groups in Nigeria. It is known as “ajasin” in Yoruba,

“ebavbokho” in Bini, “aaid dya ta gida” in Hausa, “nchanwu” in Igbo, “froukena” in Ijaw and “oran” in Urhobo (Iwu, 1993).

1.5.2 Description

Ocimum gratissimum (linn) also known as Clove Basil, African Basil, and in Hawaii as Wild

Basil belongs to the group of plants known as spices. The plant is an erect small plumb with many barnacles usually not more than 1 m high (Vierra and Simon, 2000). It is usually propagated by seed or stem cutting (Iwu, 1993).

1.5.3 Geographical Distribution

The plant is found throughout the tropics and subtropics and its greatest variability occurs in tropical Africa and India (Aruna and Sivaramakrishina, 1990). O. gratissimum (linn) is a plant commonly found around village huts and gardens. It is mostly a weed of wastelands and road sides. It thrives better in wet and fertile soil while growing but will tolerate drought after flowering (Swabirk, 1997). The plant is wide spread in deciduous forest and savannah and is usually cultivated for its medicinal uses. In South East Asia, it is cultivated as a home garden crop but it is grown on a commercial scale in Vietnam.

1.5.4. Ethnomedicinal uses.

It is used for a variety of purposes. In culinary, it is used in salads, soups, pastas, vinegars and jellies in many parts of the world. The Thai people are popularly known to use it in food flavouring.

In traditional medicine in coastal area of Nigeria, the leaves have been used as a general tonic, treatment of epilepsy and diarrhoea as well as conjunctivitis by instilling directly into the eyes (Effraim et al., 2003). The flowers and leaves of this plant are rich in essential oils and therefore used in preparation of teas and infusion (Rabelo et al., 2003). The leaf oil when mixed with alcohol is applied as a lotion for skin infections, and taken internally for bronchitis. In savannah areas, decoction of the leaf is used in the treatment of mental illness

(Akinmoladun et al., 2007). O. gratissimum is used by Igbos in the South Eastern Nigeria in management of umbilical cord to prevent infection (Ijeh et al., 2005). The dried leaves are snuffed to alleviate headaches and fever among other uses (Iwu, 1993).

1.5.5 Documented research findings on O. gratissimum.

Phytopharmacological studies have shown that essential oils of O. gratissimum exhibited antimicrobial, insect repellant and anthelmintic activities (Sofowara, 1982). Its antioxidant

55 activity has also been reported (Oboh, 2008). The extract of O. gratissimum equally exhibited antimicrobial effect and when fed to rabbits, causes weight loss and suppresses the heamopoietic system (Effraim et al., 2000). There is also report that the leaf extracts are able to inhibit or even reverse carbon tetrachloride induced liver damage in rats (Arhoghro et al.,

2009a). Several reports have shown that leaf extract of O. gratissimum exhibited hypoglycemic activity (Aguiyi et al., 2000; Mohammed et al. 2007). Obianime et al., (2011) also reported that the serum levels of the hepatic enzymes on prolonged administration of O. gratissimum in mice were not significantly altered. This suggests that hepatic function in mice is not adversely altered.

Phytochemical screening of leaf extract of O. gratissimum revealed the presence of alkaloids, saponins, anthraquinone, flavonoids and tannin (Obianime et al 2011). Interestingly, saponins and terpene glycoside which O. gratissimum contains in aboundance have been shown to enhance recuperative ability of the body (Obianime et al 2011). Effraim et al.

(2003) showed from histopathological studies that O. gratissimum is capable of protecting the liver.

1.6 Aim of the Study

The aim of the present study is:

1. To scientifically confirm whether or not the aqueous extracts of V. amygdalina and/or

O. gratissimum is capable of protecting the renal system from cyclophosphamide-

induced urotoxicity and myelosupression.

2. To ascertain the degree of the protection compared to that provided by a well-

documented sulfhydryl-containing compound MESNA (sodium-2-mecarptoethane

sulfonate).

CHAPTER TWO

2.0 MATERIALS AND METHODS

2.1 Animals

Forty (40) outbred mature Sprague-Dawley albino male rats (Rattus norvegicus) (130 g –200 g) were used. They were procured from the animal house unit of the department of

Pharmacology, College of Health Sciences, Niger Delta University, Wilberforce Island,

Bayelsa state and housed in a well ventilated room at the same environment at room temperature of about 29 0C ± 3 using standard rat cages. The animals were allowed to acclimatize for two (2) weeks and fed with standard rat chow (pelletized vital feed) and water provided ad libitum. They were placed under 12:12 hr light dark cycle. The entire

57 experimental protocols were performed in accordance with the Institutional Animal Ethical

Committee (IAEC) in line with the directions of the committee for the purpose of control and supervision of experiments on animals (CPCSEA) in the Niger Delta University, Wilberforce

Island, Bayelsa state.

2.2 Drugs and Chemicals

Cyclophosphamide (CYCLOXAN® 500mg) and Sodium – 2 – Mecarptoethane sulfonate

(MESNA) (BIOCHEM Pharmaceutical Industries Ltd.,Mumbai, India), catalase, Supraoxide dismutase and glutathione commercial kits (Sigma Aldrich St Louis, USA) were used for biochemical assay. All other reagents such as normal saline, thiobarbituric acid, adrenaline,

Trichloroacetate and HCl used were of standard analytical grade.

2.3 Preparation of extracts

Leaves of V. amygdalina and O. gratissimum were sourced from Abakpa Nike Enugu, and were identified at the Department of Pharmacognosy, Faculty of Pharmacy, Niger Delta

University, Bayelsa state, Nigeria.

A known weight of freshly collected V. amygdalina (552.5 g) or O. gratissimum (702.2 g) was macerating for 12 hours in distilled water and blended using electric blender, after which it was filtered properly. The filtrate was lyophilized using Yorco™ Lyophylizer (PVT

Industries, China) to yield dry 23.87 g (4.32 %) of V. amygdalina and 15.45 g (2.2 %) O. gratissimum extracts respectively.

2.4 Induction of CP-induced toxicity

Cyclophosphamide (CYCLOXAN 500 mg) and MESNA were suspended in normal saline before administering to the test animals at the dose of 200 mg/kg and 67 mg/kg body weight 58 respectively. The dose of MESNA was 1/3rd (33.5%) the dose of CP used. (Berrigan et al.,

1982.)

The CP and MESNA were given intraperitoneally (i.p) whereas extracts of V. amygdalina

(VA) and O. gratissimum (OG) were given orally (per os) using gavage. The same dose schedule of CP (200 mg/kg body wt) was given to all the groups that received CP. The animals were divided into eight (8) equal groups of five (5) rats each, (n=5).

Group 1 which represents naïve rats received normal saline only orally for ten consecutive days. Group 2 which represents control rats received a single i.p administration of CP on the tenth day after oral gavage of normal saline for ten consecutive days.

Group 3 received normal saline for ten consecutive days before a single i.p dose of MESNA at 67 mg/kg (i.e. 33.5% of CP) on the tenth day after which single dose of CP was administered within five minutes interval on the same day (MESNA+CP).

Group 4 received 250 mg/kg body wt of OG orally for ten consecutive days followed by i.p administration of CP on the tenth day (250 mg/kg OG+CP).

Group 5 received 500 mg/kg body wt of O. gratissimum orally for ten consecutive days followed by i.p administration of CP on the tenth day (500 mg/kg OG+CP).

Group 6 received 250 mg/kg V. amygdalina orally for ten consecutive days followed by i.p administration of CP on the tenth day (250 mg/kg VA+CP).

Group 7 received 500 mg/kg of V. amygdalina orally for ten consecutive days followed by i.p administration of CP on the tenth day (500 mg/kg VA+CP).

Group 8 received 250 mg/kg each of O. gratissimum and V. amygdalina orally for ten consecutive days followed by i.p administration of CP on the tenth day (250 mg/kg OG+250 mg/kg VA+CP).

The dosing procedure was performed in such a way that the entire rats in all the groups could be sacrificed on the 11th day. The CP and MESNA dose schedule was based on our assay of

59 various doses which corresponds with that reported by Berrigan et al. (1982). The doses schedule for the extracts was based on our pilot study.

2.5 Blood sample collection

Prior to death on the 11th day, blood sample was collected through the ophthalmic venous plexus located in the orbital sinus of the rats with the aid of micro-capillary pipette (Stone,

1954), as modified by Rilley (1960). About 1ml of blood collected from each of the rats was introduced into a clean labeled sample bottle containing 1mg Na-EDTA powder and mixed mildly to avoid clotting.

2.6 Haematological test

To determine the level of myelosuppression, the blood sample, immediately upon collection was analyzed using an automated dialyzer machine. Model No: Sysmex kx-21n 3 – part differential analyzer (Sysmex Corporation, Kobe, Japan).

2.7 Biochemical analysis

After the experimental period which lasted for ten days, animals were sacrificed by means of cervical dislocation on the eleventh day (i.e. within 24hr). The urinary bladder was harvested for onward biochemical investigation with some parts preserved using 10% buffered formalin for histopathological studies. The liver and the kidney were also removed for histopathological studies.

2.7.1 Analysis of glutathione (GSH)

The glutathione content of the urinary bladder homogenate was estimated according to the method described by Sedlak and Lindsay (1968). To 1 ml of the sample suspension (1 mg protein/ml), 1 ml of 10 % TCA (Trichloroacetate) containing 1 mM EDTA was added. The

60 protein precipitate was separated by high speed centrifugation at 2500 rpm for 10 min. About

1 ml of supernatant was treated with 0.5ml of Ellmans reagent and 3ml of phosphate buffer

(0.2, pH7.4). The absorbance was read at 412 nm using spectrophotometer.

2.7.2 Superoxide dismutase analysis

A method originally described by Misra and Fridovich (1989) as reported by Magwere et al.,

(1997) was employed. The homogenate was supplemented with 2.5 ml of carbonate buffer, followed by equilibration at room temperature; 0.3ml of 0.3 nM adrenaline solution was then added to the reference and the test solution, followed by mixing and reading of absorbance at

420 nm.

2.7.3 Catalase assay

Activity of catalase in urinary bladder was determined according to procedure of Sinha

(1972). This method is based on the reduction of dichromate in acetic acid to chromic acetate when heated in the presence of H2O2, with the formation of perchromic acid as an unstable intermediate. The chromic acetate so produced is measured. Absorbance was read at 480 nm within 30-60 seconds against distilled water.

2.7.4 Malondialdehyde (MDA) Assay

The MDA assay method of Hunter et al., (1963) as modified by Gutteridge and Wilkins

(1982) was adopted. Malondialdehyde (MDA), a product of lipid peroxidation, when heated with 2-thiobarbituric acid (TBA) under acid conditions forms a pink colored product which has a maximum absorbance of 532 nm. The urinary bladder homogenate was supplemented with I g of TBA in 100 ml of 0.2 % NaOH and 3 ml of glacial acetic acid, thoroughly mixed and incubated in boiling water bath for 15 minutes, then allowed to cool after which they were centrifuged. Absorbance was read at 532 nm and the results expressed as nanomoles

MDA/mg wet tissue.

2.8 Histopathological Examination

Small portions of urinary bladder, kidney and liver tissues were fixed in 10 % buffered formalin, processed and embedded in paraffin wax. Sections of about 5 μm were made and stained with haematoxylin and eosin (H & E) for examination by light microscopy.

2.9 Statistical Analysis

Data obtained were analyzed using One-way Analysis of Variance (ANOVA) in SPSS version 15.0 and subjected to LSD and Duncan post-hoc test. Significant difference between means of treated and control were accepted at P<0.05. Data were expressed as Mean ±SEM.

CHAPTER THREE

3.0 RESULTS

3.1. Effects of extracts on CP-induced urotoxicity

3.1.1 Effects of extract on glutathione

The CP (200 mg/kg body wt.) treated animals showed a significant decrease in GSH content in the urinary bladder (P<0.05) compared to naive group, (Table 5). Animals that received

250 mg/kg and 500 mg/kg of O. gratissimum showed non-significant (P>0.05) increase in

GSH values when compared with the CP treated animals and with a significant reduction

(P<0.05) in relation with naive group and those that received either MESNA, VA 250 mg/kg and 500 mg/kg or VA 250 mg/kg + OG 250 mg/kg +CP. The GSH values for animals that received MESNA+CP, 250 mg/kg VA+CP, 500 mg/kg VA+CP and 250 mg/kg VA+ 250 mg/kg OG+CP were significantly higher (P<0.05) than those of CP alone.

3.1.2 Effects of extracts on SOD Activity.

Supraoxide dismutase activity was significantly reduced (P<0.05) in CP treated animals when compared with naïve (control) animals (Table 5). 250 mg/kg OG+CP and 500 mg/kg 62

OG+CP animals (groups 4 and 5) showed no significant (P>0.05) increase in relation to animals that received CP alone. Animals that were given MESNA+CP, 250 mg/kg VA+CP,

500 mg/kg VA+CP, and 250 mg/kg VA+ 250 mg/kg OG+CP showed no significant (P>0.05) changes in SOD activity but were significantly higher (P<0.05) than CP treated group.

3.1.3 Effects on catalase activity

Catalase activity in the urinary bladder homogenate of CP-treated animals was significantly lower (P<0.05) than naïve (control) animals that received only normal saline, (Table 5).

Groups that received 250 mg/kg OG+CP and 500 mg/kg OG+CP presented significant increase (P<0.05) in catalase activity when compared with animals that received CP alone.

Conversely, 250 mg/kg OG+CP and 500 mg/kg OG+CP animals showed a significant decline (P<0.05) in catalase activity when compared with animals that were given

MESNA+CP, 250 mg/kg VA+CP, 500 mg/kg VA+CP, 250 mg/kg VA+ 250 mg/kg OG+CP.

3.1.4 Effects of extracts lipid peroxidation (LPO)

Lipid peroxidation (LPO) was measured as the amount of thiobabituric acid reactive substances (TBARS) in the urinary bladder. The results were expressed as malondialdehyde

(MDA) formed using molar extinction coefficient of 1.56x105M/cm. Table 6 presented CP- treated animals showing significantly high levels of LPO values (P<0.05) compared to naive

(control). Animals treated with 250 mg/kg OG+CP and 500 mg/kg OG+CP showed similar

LPO which was not significantly different (P>0.05) from those that received only CP.

However, MESNA+CP, 250 mg/kg VA+CP, 500 mg/kg VA+CP and 250 mg/kg VA+ 250 mg/kg OG+CP animals showed LPO values that were similar but significantly lower

(P<0.05) than CP treated animals.

3.2. Effects of extract on CP-induced myelosupression.

3.2.1. Effect on total RBC count, total Leucocyte count, absolute neutrophil and absolute lymphocyte counts. The CP-treated rats depicted values of total RBCs count, leukocyte count, absolute neutrophil and lymphocyte counts that were significantly lower (P<0.05) than those of naïve (control) rats (Table 7). Animals that received 250 mg/kg OG+CP, 500 mg/kg OG+CP and

MESNA+CP had similar values but were significantly higher (P<0.05) than the CP treated animals. However, these values were significantly lower (P<0.05) than the control group.

Values for 250 mg/kg VA+CP, 500 mg/kg VA+CP, 250 mg/kg VA+250 mg/kg OG+CP groups were similar.

3.2.2 Effect on platelet count, absolute basophils and eosinophils

Animals that received only CP had significantly low (P<0.05) values of platelet than those of control animals (Table 7). Platelet count for animals given 250 mg/kg OG+CP and 500 mg/kg OG+CP were similar but significantly higher (P<0.05) than those that received only

CP. However, these values were significantly lower (P<0.05) compared to naïve (control) group. The groups that received MESNA+CP, 250 mg/kg VA+CP, 500 mg/kg VA+CP, 250 mg/kg VA+ 250 mg/kg OG+CP indicated similar platelet counts significantly higher

(P<0.05) than the 250 mg/kg OG+CP and 500 mg/kg OG+CP groups.

The absolute basophils and eosinophils result depicted no significant change (P>0.05) for all the groups, (Table 7).

Table 5: Effects of O. gratissimum and V. amygdalina compared to MESNA on GSH, SOD and catalase activities in CP-induced toxicity.

Treatment Dose mg/kg Reduced glutathion & antioxidant enzymes

GSH (nMGSH/mg) SODµM/mg Catalase tissue nM/mg tissue Naïve Control 0.2ml/kg 5.28±0.45*a 9.79±0.32* a 14.92±0.33* a (Normal saline) CP Alone control 200 1.00±0.10 b 2.42±0.31 b 3.01±0.31 b

MESNA 67 4.77±0.38* a 9.49±0.65* a 13.67±0.51* a

OG 250 2.71±0.28 c 4.27±0.38 c 7.68±0. .50 *c

OG 500 2.51±0.32 c 4.54±0.55 c 7.79±0.66 *c

VA 250 5.16±0.29 *a 10.26±0.33* a 15.27±0.54* a

VA 500 5.78±0.46* a 10.38±0.58* a 15.31±0.81* a

VA+OG 250+250 5.45±0.41* a 10.55±0.68* a 15.47±1.04* a

One-way ANOVA followed by post-hoc LSD and Duncan was adopted. LSD= Means in the same column with asterisks (*) indicate significant different P<0.05, compared to CP treated animals. Duncan= Means in the same column with different superscript letter(s) indicates significant difference, P<0.05 comparing all the groups. Key: - CP: cyclophosphamide; MESNA: sodium-2-mecaptorethane sulphonate; OG: O. gratissimum; VA: V. amygdalina. Data are Mean ±SEM (n = 5). 65

Table 6: Effects of O. gratissimum and V. amygdalina compared to MESNA on lipid peroxidation (LPO) induced by CP. Treatment Dose mg/kg LPO nM MDA/mg wet tissue Naïve (Normal saline) 0.2ml/kg 25.04±0.61* a

CP Alone (control) 200 50.39±3.52 b

MESNA 67 37.43±3.62 a b

OG 250 48.51±4.26 b

OG 500 46.60±6.59 b

VA 250 22.69±0.88* a

VA 500 21.00±1.14* a

VA+OG 250 20.31±1.75* a

Table 7: Effects of O. gratissimum and V. amygdalina on hematological parameters of peripheral blood in CP-induced toxicities. Treatment Dose (mg/kg) HEMATOLOICAL PARAMETERS

Total RBC Total WBC Differential Leukocyte count (x103µl) 3 count count (x10 µl) Neutrophil Lymphocyte Platelet Basophil Eosinophil (x106µl)

Naïve (Normal 0.2ml/kg 9.57±0.24*a 12.02±0.76*a 2.86±0.22*a 8.38±0.37*a 803.00±62.32*a 0.05±0.00* a 0.25±0.03* a saline) b b b b a a CP Alone (control) 200 3.49±0.62 3.17±0.27 0.17±0.07 1.50±0.24 b 221.20±71.44 0.03±0.00* 0.23±0.02*

a c c bc a a a MESNA 67 6.97±0.45* 6.94±0.57* 1.37±.33 5.72±0.91*ac 725.60±46.39* 0.04±0.00* 0.24±0.04*

OG 250 6.22±0.52b c 5.35±0.58b c 1.21±0.16 bc 4.10±0.86b c 431.40±29.39 b 0.03±0.01* a 0.25±0.04* a

OG 500 7.27±0.63*a c 5.10±0.80b c 1.28±0.25 bc 4.16±0.63 c 429.60±31.72 b 0.03±0.01* a 0.24±0.06* a

a a a a a a VA 250 9.27±0.48 *a 11.42±0.59 * 2.60±0.32 * 7.72±0.42* 787.20±56.17 * 0.03±0.01* 0.29±0.07*

a a a a a a VA 500 9.86±0.72 *a 11.74±0.70* 2.42±0.34 * 8.58±0.60* 817.20±44.09 * 0.04±0.00* 0.23±0.02*

a a a a a a VA+ OG 250+250 9.81±0.53 * 10.29±0.61 * 2.70±0.43 * 8.92±0.47 *a 813.80±38.38 * 0.03±0.01* 0.28±0.04*

Data are Mean ±SEM (n = 5). One-way ANOVA followed by post-hoc LSD and Duncan was adopted. LSD= Means in the same column with asterisks (*) indicate significant different P<0.05, compared to CP treated animals. Duncan= Means in the same column with different superscript letter(s) indicates significant difference, P<0.01 comparing all the groups.

3.3 Effect on histopathology of tissues

Urinary Bladder: The photomicrograph showed an intact epithelium and lamina propria of urinary bladder of the control animal (fig. 5A) unlike those of test animals that received only CP (fig. 6A). The urinary bladder of the animals that received only CP showed active congestion of the capillaries with polymorphonuclear cell infilteration and oedema of lamina propria. Histological sections of urinary bladder of animals that received 250 mg/kg OG+CP (fig. 8A) and 500 mg/kg OG+CP (fig.

9A) indicated oedema and vascular congestion with erosions of transitional epithelium respectively and are comparable to those that received CP alone.

On the other hand, photomicrograph of urinary bladder of animals that received

MESNA+CP (fig. 7A), 250 mg/kg VA+CP (fig. 10A), 500 mg/kg VA+CP (fig.

11A), 250 mg/kg VA+ 250 mg/kg OG+CP (fig. 12A) were presented with intact epithelium with no observable histological change comparable to normal group that received only saline without any treatment.

Kidney: The photomicrograph showed intact renal corpuscles of the kidney and tubules of the control animal (fig. 5B) unlike those of test animals that received only

CP (fig. 6B). The kidney of the animals that received only CP in presented with mild cellularity and congestion of the glomerulus. Histological sections of kidney of animals that received 250 mg/kg OG+CP (fig. 8B), 500 mg/kg OG+CP (fig. 9B) and

MESNA+CP (fig. 7B) also indicated mild congestion of the glomerulus which is comparable to those that received CP alone. However, photomicrograph of kidney of animals that received 250 mg/kg VA+CP (fig. 10B), 500 mg/kg VA+CP (fig. 11B),

250 mg/kg VA+ 250 mg/kg OG+CP (fig. 12B) depicted intact tubules and

lxix glomerular tufts with no observable histological change comparable to normal group that received only saline.

Liver: Histopathological sections of the liver of the control animal showed normal plates of hepatocytes separated by sinusoids (fig. 5C) unlike those of test animals that received only CP (fig. 6C). The liver tissue of the animals that received only CP in presented with apoptotic cells. Liver photomicrographs of animals that received

250 mg/kg OG+CP (fig. 8C) and 500 mg/kg OG+CP (fig. 9C) indicated no remarkable histological change and cellular infiltration of kupffer cells respectively.

On the other hand, photomicrograph of liver of animals that received MESNA+CP

(fig. 7C), 250 mg/kg VA+CP (fig. 10C), 500mg/kg VA+CP (fig. 11C) and 250 mg/kg VA+ 250 mg/kg OG+CP (fig. 12C) were presented with normal plates of hepatocytes with no observable histological change comparable to normal group that received only saline without any treatment.

lxx

E R

LP CV

A B C Figure 5: Photomicograph of organ sections from control rats given normal saline showing: A- urinary bladder having intact epithelium (E) and lamina propria (LP); B – the kidney showing normal renal corpuscules (R) and tubules (dark arrow); C – the liver showing the central vein (CV) and normal plates of hepatocytes separated by the sinusoids (arrow).

G E

A B C Figure 6: Photomicrograph of sections of organs from rats given 200mg/kg of CP showing: A- urinary bladder having active congestion of the capillaries (K) with polymorphonuclear cells (arrow) and oedema of the lamina propria (E); B- Kidney showing mild congestion of the glomerulus(G), C- Liver showing the central vein(CV) and apoptotic cells (arrow). H&E ×400

lxxi

CV G LP

A B C Figure 7: Histologic sections of organs from rats treated with 67mg/kg of MESNA and 200mg/kg of CP showing: A- urinary bladder with transitional epithelium (arrow) and lamina propria(LP), B- kidney showing mild cellularity of the glomerulus(G); C- liver showing central vein(CV) and normal hepatocytes. H&E ×400.

A B C Figure 8: Histologic sections of organs from rats treated with 250mg/kg of O. gratissimum and 200mg/kg of CP showing: A- Oedema (E) and vascular congestion (VC) of the urinary

lxxii

bladder; B- kidney having mild congestion of the glomerulus, C-liver with no remarkable histologic change. H&E ×400.

CV MC TE

A B C Figure 9: Histologic sections of organs from rats treated with 500mg/kg of OG and 200mg/kg of CP showing: A- bladder with erosion of the transitional epithelium (TE), B- kidney with mild congestion(MC) of the glomerulus;C- liver with central vein (CV) and hypercelularity of kupfer cells (arrow). H&E ×400

lxxiii

MF T PA LP

A B C Fig.10. Photomicrograph of sections of organs from rats treated with 250mg/kg of VA and 200mg/kg of CP showing: A- urinary bladder with transitional epithelium having mucosal folds (MF) and lamina propria (LP) with no observable histologic change, B- kidney with normal tubules and glomerular tufts (T); C-liver showing the portal area (PA). H&E ×400.

lxxiv

G LP

A B C Figure 11: Photomicrograph of sections of organs from rats given 500mg/kg of VA and 200mg/kg of CP showing: A- the urinary bladder with intact transitional epithelium (T) and lamina propria (LP), B- the kidney with no observable histologic changes (see the glomerulus (G) and renal tubules ‘arrow’), C- liver with decreased nuclear chromatin density of the hepatocytes (arrows).H&E ×400

lxxv

TE G

LP CV

A B C Figure 12: Histologic sections of organs from rats treated with 250mg/kg of VA, 250mg/kg of OG and CP 200mg/kg showing: A- urinary bladder showing normal stretched transitional epithelium(TE) and lamina propria (LP), B- kidney showing mild congestion of the glomerulus (G) and interstitium (arrows); C- Liver showing the central vein (CV) and normal plates of hepatocytes (arrows). H&E ×400.

CHAPTER 4 4.0 Discussion

The main objective of the present study was to explore the possible protective and ameliorative efficacy of V. amagdalina and O. gratissimum in prevention of the undesirable cyclophosphamide-induced urotoxicity and myelosupression in vivo.

lxxvi

The application of natural products as alternative medicine has been encouraged especially because they are relatively cheap and with minimal side effects when compared to modern medicine (Hu et al. 2003). In addition, they could significantly contribute to the improvement of human health regarding cure and the prevention of various diseases affecting man. Studies have shown that oxidative stress contributes a great deal in pathophysiology of many human disease processes (Farber, 1990;

Wiseman and Halliwell, 1996).

Demands have continued to increase concerning the scientific proof and justification of the use of herbs for medicinal purpose. Consequently, attempt was made to investigate the protective role of aqueous leaf extracts of V. amygdalina and O. gratissimum against CP-induced urotoxicity and myelosuppression, using biochemical, histological and haematological parameters.

In this context there are body of evidence that oxidative stress and lipid peroxidation, could in part, be one of the ways in which CP induces its toxicity. The kidney and urinary bladder are actively involved in the excretion of toxic metabolites of CP. The tendency of accumulation of toxic metabolite of CP in urinary bladder is high and is likely to cause more harm to it (Beyer-Boon et al., 1978).

Although there are numerous reports on the various uses of V. amygdalina and O. gratissimum (Owolabi et al., 2008; Arhoghro et al., 2009; Asuquo et al., 2010; Ijeh and Ejike, 2011), this is the first study demonstrating that V. amygdalina and O. gratissimum have the potential to prevent urotoxicity and myelosuppression induced by CP.

lxxvii

In this study, CP caused a significant reduction on levels of all the antioxidants assayed using the bladder homogenate. Similar findings were reported by Hamrita et al., (2012). There was elevation of lipid peroxidation with attendant myelosuppression. Cooper et al., (1986) reported that the oxidative products of CP, phosphoramide mustard and acrolein, responsible for the induction of lipid peroxidation and reactive oxygen species (ROS) generation, resulted in inflammation, thus disturbing the overall redox cycling of the bladder.

The CP-induced depletion of GSH is essentially mediated by the interaction of its reactive metabolite, acrolein with GSH (Kehrer and Biswal, 2000). It is also reported that cysteine which is one of the amino acid constituents of GSH interacts with acrolein in a similar way as GSH itself (Kehrer and Biswal, 2000). Consequently, a number of antioxidants and sulfhydryl (-SH) containing compounds such as sodium-

2-mecarptoethane sulphonate (MESNA), amifostine, disulphiram, L-cysteine, N- acetylcysteine, S-carboxy-methyl –L – cysteine, D-penicillamine, GSH, glutathione esters etc. have been reported to protect the animals from toxic effects of CP, (Habs et al., 1984), (Brock , 1980). Although several antioxidants have been reported in clinical and experimental studies to offer protection to the urinary bladder (Brock,

1980; Habs et al., 1984), yet more attention has been given to the possible roles of dietary antioxidants in protecting the bladder and the myeloid system against CP- induced urotoxicity and myelosuppression possibly because they are cheaper and tend to have minimal side effects (Łukasz and Piotr, 2012). Reports on various plant extracts and their phytochemical constituents including flavonoids which are

lxxviii naturally occurring antioxidants that possess pharmacological actions and therapeutic application are still accumulating (Arhoghro et al., 2012).

Results of antioxidants protection indicated that V. amygdalina offered as much protection as MESNA but much more potent than O. gratissimum which is lower in efficacy than MESNA even at a higher dose. This confirmed that V. amygdalina possesses antioxidant properties as reported by Owolabi et al., (2008). Regarding prevention of lipid peroxidation (LPO), V. amygdalina prevented LPO better than both MESNA and O. gratissimum, irrespective of the doses. V. amygdalina counteracted the CP-induced oxidative stress as evidenced by the increased GSH,

SOD and catalase with decreased LPO levels. The urinary bladder and myeloid system were protected. The value for total RBCs, total leucocyte, platelets as well as absolute neutrophils, and lymphocytes for animals given VA+CP and VA+OG+CP were within normal range compared to MESNA+CP and OG+CP groups which were diminished as similarly reported by Ihedioha et al., (2004).

MESNA on the other hand was able to prevent CP-induced oxidative stress which in turn protected the urinary bladder but not sufficient to protect the myeloid system

(Berrigan et. al., 1982). Although O. gratissimum 250 mg/kg and 500 mg/kg were not able to protect animals against the CP-induced toxicities, it did not interfere adversely with the activities of V. amygdalina as observed in the group that concomitantly received VA+OG+CP.

lxxix

In addition, reports have shown that myelosupression is one of the undesirable side effects induced by CP which is also quite disturbing especially in cancer patients

(Angulo et al., 2002). Myelosuppression is often a pathological condition characterized by reduction in number, below normal values, of erythrocytes, leucocytes especially neutrophils, and plateletes. The consequence is often undesirable because the important cells that play key role in host immunity, oxygen transport and blood clotting would be grossly diminished resulting in anaemia, high disease susceptibility, and blood clotting failure leading to haemorrhage ( Mazza,

1995).

In terms of haematological parameters evaluated, MESNA did not protect the myeloid system from CP-induced myelosuppression. All the blood cells, except platelets, basophils and eosinophils were significantly (P<0.05) diminished below normal values. The animals given MESNA+CP showed high levels of leucopenia, neutropenia and lymphocytopenia. This appears to be in agreement with the report of Berrigan et. al., (1982), who reported that MESNA lacked the ability to protect from CP-induced myelosuppression.

V. amygdalina effectively protected the myeloid system from CP-induced myelosuppression. This may be through another mechanism probably by prevention of lipid peroxidation and nutrient supplementation as earlier reported (Ijeh and Ejike,

2011). Findings on examination of the histopathological slides further authenticates these results as par intact architectural integrity of urinary bladder, kidney and liver tissues of the animals that received VA+CP and VA+OG+CP compared to those of

MESNA+CP and OG+CP which showed active congestion of the capillaries with

lxxx polymorphonuclear cell infilteration and oedema of lamina propria. This is a histological evidence of cystitis within 24 hr as reported by Gray et al., (1986).

The findings of this work revealed that O. gratissimum did not protect the bladder and the bone marrow from CP-induced urotoxicity and myelosuppression. Although there was some level of protection in terms of biochemical and hematological values, it was not sufficient to protect the animals from heamaturia that was observed during clinical examination as well as inflammation of the urinary bladder and suppression of the myeloid system.

V. amygdalina has been widely reported to contain high levels of nutrients including proteins with high essential amino acid score (Igile et al., 1994; Udensi et al., 2002;

Ejoh et al., 2007; Eleyinmi et al., 2008), carbohydrates (Ejoh et al.,2007) and fiber

(Udensi et al., 2002; Ejoh et al., 2007; Eleyinmi et al., 2008). The plant has also been shown to contain appreciable quantities of ascorbic acid and caroteinoids

(Udensi et al., 2002; Ejoh et al., 2007). Calcium, iron, potassium, phosphorous, manganese, copper and cobalt have also been found in significant quantities in V. amygdalina (Bonsi et al., 1995; Ejoh et al., 2007;Eleyinmi et al., 2008). In addition the phytochemical screening of aqueous leaf extract of V. amygdalina has shown that it also contains, flavonoids, saponins and sesquiterpene lactones (Kupchan et al.

(1969); Jisaka et al., 1992)

Flavoniods have been widely reported to posses antioxidant activity and are effective scavengers of reactive oxygen species (Robak and Grygleuski, 1988). The molecular mechanism of V. amygdalina activity against the CP-induced urotoxicity might have been directly related to its ability to scavenge ROS attributable to flavonoids (Robak

lxxxi and Grygleuski, 1988). Similarly, prevention of myelosuppression may in part, be linked to the essential nutrients it contains which is not present in MESNA. In spite of several studies on important usefulness of V. amygdalina, none has focused on uroprotective effects and myeloid system protective efficacy of V. amygdalina on

CP-induced toxicities.

4.1 CONCLUSION

The present study investigated for the first time the potential detoxifying efficacy of

V. amygdalina which is comparable to MESNA focusing on its increased antioxidants activity, reduced LPO and myeloid system protection.

The findings established that V. amygdalina is endowed with properties of a detoxifier and adjuvant therapeutic agent for cancer patients. Again considering the fact that MESNA is relatively more expensive and has its own side effects, V. amygdalina may be an alternative replacement.

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