# EFFECTS OF METHANOL LEAF EXTRACT OF MORINGA OLEIFERA ON NEUROBEHAVIOURAL AND OXIDATIVE STRESS CHANGES INDUCED BY SUBCHRONIC EXPOSURE TO CHLORPYRIFOS IN MALE WISTAR RATS

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EFFECTS OF METHANOL LEAF EXTRACT OF MORINGA OLEIFERA ON NEUROBEHAVIOURAL AND OXIDATIVE STRESS CHANGES INDUCED BY SUBCHRONIC EXPOSURE TO CHLORPYRIFOS IN MALE WISTAR RATS

CHAPTER ONE

INTRODUCTION

1.1 Background

Pesticides are used in agriculture as plant protection agents for boosting food production (Abhilash and Singh, 2009) and in public health (Matthews et al., 2011). Organophosphate (OP) compounds are used widely as pesticides in agriculture industry (Mulchandani et al., 2001; Oates et al., 2014), and as household insecticides (Lovasi et al., 2011). OP insecticides inhibit actetylcholinesterase (AChE) activity by binding to the active site of the enzyme, preventing the break-down of acetylcholine in the nervous system of insects and other pests (Kwong, 2002; Marks et al., 2010). This leads to the accumulation acetylcholine (ACh) in nerve endings, and therefore continuous stimulation of cholinergic receptors (Timofeeva et al., 2008), resulting in tetany, eventually paralysis and death (Ogueji et al., 2013). Animal and human studies have linked OP exposure to sensorimotor and cognitive impairments (Ambali et al., 2010b, 2012a, b; Rauh et al., 2012; Lopez-Granero et al., 2013; Kaur et al., 2014). Similarly, epidemiological studies have linked acute and chronic OP exposure to affective disorders (Da Vies, 1995), resulting in depression and suicide in humans (London et al., 2005, 2012). The wide-spread exposure of humans and animals to OP pesticides have raised increasing concern about their toxicity (El-Hossary et al., 2009; Kim et al., 2014).

Chlorpyrifos (CPF) is an extensively used OP insecticide with many domestic and agricultural pest control applications. It is one of the most widely used organophosphorothionate pesticide (Tirelli et al., 2007). Neurotoxicity is the main manifestation of CPF exposure in animals and man (Sanchez-Santed et al., 2004), and the primary toxic mechanism is AChE inhibition (Kwong, 2002). CPF is also a developmental neurotoxicant, affecting developing foetus (Saunders et al., 2012) and its low doses disrupt brain development and cognitive function (Slotkin and Seidler, 2005). CPF-induced damage extends beyond cholinergic pathways to include other neurotransmitter systems, notably the monoamines, norepinephrine, dopamine (Xu et al., 2012), and serotonin (5HT) (Raines et al., 2001). Recent studies show that 5HT systems are especially sensitive as CPF affects 5HT receptors, the presynaptic 5HT transporter, and 5HT-mediated signal transduction during a discrete critical gestational window (Aldridge et al., 2003). Foetal or neonatal exposure to CPF leads to abnormalities of brain cell development, synaptic function, and behaviour (Dam et al., 2000).

Although inhibition of AChE activity is the primary mechanism of CPF toxicity, studies have shown that toxicity also occurs at doses that do not inhibit AChE activity or long after its restoration (Haviland et al., 2010; Goodman et al., 2013). Therefore, other non-AChE mechanisms have been implicated in CPF toxicity. The generation of reactive oxygen species (ROS), and, hence, induction of oxidative stress has been identified as one of the major non-AChE mechanism of CPF toxicity (Slotkin et al., 2005; Tuzmen et al., 2007; Verma et al., 2007; Mansour and Mossa 2009; Ambali and Ayo 2011; Ambali et al., 2011 a, b, c; Lee et al., 2014; Ventura et al., 2015).

Oxidative stress is associated with increased generation of ROS, exerting toxic effects on cells (Misra et al., 2009). It results in increased lipoperoxidative damage to cell membranes. Under normal circumstances, the body is equipped with agents (antioxidants) that combat the menace of oxidative stress. Antioxidants are agents which play an important role in inhibiting and scavenging free radicals, thus providing protection to tissues against damaging effects of free radicals (Beam et al., 2014). However, oxidative stress results when the body antioxidant reserve system cannot cope with the level of oxidants, as has been previously reported in CPF poisoning (Ambali et al., 2010a, b; Mansour and Mossa, 2010). In this type of situation, exogenous supplementation with antioxidants becomes imperative. Several studies in humans and animals have shown that various antioxidants including, those derived from plants (Ambali et al., 2012a, b) mitigated CPF-evoked adverse health effects.

Moringa oleifera Lam (MO) (Moringaceae), commonly known as Drumstick is a highly valued plant, and a small or medium sized tree of about 10 m high. M. oleifera is a multipurpose tree considered to have its origin in the northwest region of India, south of the Himalayan mountains (Ganesan et al., 2014) with high nutritional values. It is distributed in many countries of the tropics and sub-tropics (Anwar et al., 2007; Razis et al., 2014). It is cultivated for use as a vegetable for spice, cooking and cosmetic oil, and as a medicinal plant (Ajayi et al., 2014). Different parts of this plant contain a profile of important minerals, and are a good source of protein, vitamins, β-carotene, amino acids and various phenolics (Anwar et al., 2007). It contains various chemical constituents such as alkaloids, tannins, flavonoids, carbohydrates, amino acids and glycosides (Belay and Sisay 2014). Leaf extracts of M. oleifera have antioxidant properties, the free-radical scavenging effect of which is comparable with that of many classical antioxidants (Kumar and Pari, 2003; Siddhuraju and Becker, 2003; Sreelatha and Padma, 2009; Ratshilivha et al., 2014).

1.2 Statement of Research Problem

In an effort to feed the world’s rapidly growing population, governments have through various means encouraged increased agricultural production. To this effect, there has been an increase in the application of agricultural inputs such as pesticides, to increase productivity in most countries (Ramaswamy and Sanders, 1992). Pesticide use has  therefore increased over the years, with attendant surge in agricultural productivity and domestic pest control (Poswal and Akpa, 1991). This has however come with its attendant adverse health consequences as they are often not target specific and cause adverse effects in exposed non-target animals and man (Kumar and Singh, 2014).

Chlorpyrifos (CPF) is a broad spectrum, highly effective OP pesticide that is widely used worldwide. Its increasing use has increased the propensity of its neurotoxic effects (El-Hossary et al., 2009), especially in children. This concern has led to the banning of CPF for home and garden use in the United States of America and some European countries (Renner, 2004). However, CPF remains on the shelves of agricultural shops and plazas, and is harnessed for such use in many developing countries, including Nigeria (Asogwa and Dongo, 2009). Many people are exposed to small doses of CPF when applied outdoors to fields, and when used as termiticides (Farahat et al., 2010). The use of CPF has been shown to leave high level of its residues in the soils and several vegetables in some parts of Northern Nigeria (Akan et al., 2013). Factors promoting exposure including eating and drinking during spraying operations, failure to use protective clothing, improper storage and disposal of insecticides, are common among farmers and pesticide applicators in Nigeria (Sosan and Akingbohungbe, 2009; Surajudeen et al., 2014). Thus, pesticide applicators with inadequate protective gears, and farmers and livestock are exposed to sub-lethal and apparently sub-toxic doses of CPF by aerosol and direct contact (Cattani et al., 2001; Quandt et al., 2006). CPF residues that have entered into the food web of the ecosystem in plant parts, soils and water bodies (Akan et al., 2013) therefore constitute a source of hazard to man and animals. CPF induces oxidative stress at low doses and this plays an essential role in its pathophysiology (Slotkin et al., 2001; Renner, 2007), including its effects on a wide variety of neurotransmitter systems. The 5-HT system is especially affected, leading to long-lasting changes in 5-HT-related emotional behaviours such as anxiety and depression (Chen et al., 2011).

Recent studies suggest a link between pesticide exposure and anxiety, depression and suicide (Lee et al., 2007). A study in Hordaland, Norway showed that among other workers, farmers showed a significantly higher level of anxiety and depression than non-farmers. In the same study, full-time farmers showed significantly higher anxiety and depression levels than part-time farmers (Sanne et al., 2004). In a similar vein, studies in Sri Lanka have shown that suicide rates have more than halved since 1995, when the government placed restrictions on the import and sales of WHO Class I toxicity pesticides (Roberts et al., 2003). Adverse effects of chronic CPF exposure such as strained breathing, insomnia, and loss of memory have been observed in several Nigerian farmers (Sosan and Akingbohungbe, 2009; Sosan et al., 2010).

1.3 Justification

There has been a steep increase in the prevalence of neurodegenerative diseases in recent times, which may have been partly associated with an increase in the use of environmental chemicals, including pesticide (Zaganas et al., 2013). Incidences of neurodegenerative diseases and cognitive deficits, including decline in intelligent quotient (IQ) have been shown to be positively correlated with exposure to pesticides (Hayden et al., 2010; Parrón et al., 2011), including OP insecticides (Kamel and Hoppin, 2004; Wang et al., 2014).

Chlorpyrifos is the most commonly used OP insecticide throughout the world (De Felice et al., 2014) and its ability to induce neurobehavioural and cognitive deficits, and predispose to neurodegenerative diseases have been reported (Lee et al., 2007; Jiang et al., 2010). It has been shown to affect motor coordination, locomotor efficiency, motor strength, learning and short-term memory (Ambali et al., 2010c) and 5-HT systems (Slotkin and Seidler, 2005) in animal models. Cognitive deficit, anxiety and depression have also been reported in individuals exposed to CPF (Lee et al., 2007). Induction of oxidative stress is one of the mechanisms implicated in CPF-induced neurotoxicity (Ambali et al., 2010a, b, c; 2012a, b, c) and several antioxidant vitamins and other classical antioxidants have been shown by several workers to ameliorate CPF toxicity (Yu et al., 2008; Mansour and Mossa 2009; Ambali et al., 2010a, b, c; 2011b, c; 2012b, c).

Moringa oleifera, a plant with high flavonoid constituents has been shown to have antioxidant properties (Siddhuraju and Becker, 2003). Although its antioxidant activity has been succinctly elucidated, the possible protective role of M. oleifera in CPF toxicity is yet to be proven. There is the need to add to the available potent antioxidants that have been proven to mitigate against CPF-induced neurobehavioural changes. This will be especially beneficial when the source of the antioxidant is from a natural plant that is abundant in tropical environment. Recent investigations are directed towards natural antioxidants, originated from plants due to concepts of safe therapeutics (Sreelatha and Padma, 2009).

1.4 General Aim of the Study

To evaluate the effect of methanol extract of M. oleifera leaves on CPF-induced neurobehavioural changes.

1.5 Objectives of the Study

The specific objectives of this study were to evaluate:

1. Qualitatively, the phytochemical constituents, and quantitatively, the flavonoids, vitamins A, C and E contents of the oleifera extract.

1. The effect of oleifera extract on some neurobehavioural and cognitive changes induced by subchronic CPF exposure in Wistar rats.

1. The effect of oleifera extract on the brain oxidative status and AChE activity of Wistar rats exposed subchronically to CPF.

1. The effect of oleifera on brain histopathological changes following subchronic exposure of Wistar rats to CPF.

1. The dose-dependency effect of oleifera in the mitigation of neurobehavioural, cognitive, oxidative and histopathological changes in Wistar rats exposed subchronically to CPF.

1.6 Research Hypothesis (H0)

• Moringa oleifera extract does not have any ameliorative effect on neurobehavioural, oxidative and histopathological changes induced by subchronic CPF exposure in Wistar rats.

EFFECTS OF METHANOL LEAF EXTRACT OF MORINGA OLEIFERA ON NEUROBEHAVIOURAL AND OXIDATIVE STRESS CHANGES INDUCED BY SUBCHRONIC EXPOSURE TO CHLORPYRIFOS IN MALE WISTAR RATS