EFFECT OF AQUEOUS EXTRACT OF COWPEA (Vigna unguiculata) ON VISUOSPATIAL LEARNING AND MEMORY IN ACUTE LEAD-INDUCED NEUROTOXICITY IN MICE
ABSTRACT
Learning is the act of acquiring new or modifying and re-inforcing existing knowledge, while Memory is relatively the permanent storage of the learned information. Exposure to lead affect brain regions such as hippocampus that are involved in learning and memory. Succimer drug or meso 2,3 – Dimercaptosuccinic acid (DMSA) is a metal chelator which is used as an antidote to lead toxicity. This study aimed at assessing the effect of cowpea (Vigna uinguiculata (L) walp) on learning and memory in acute lead-induced neuro toxicity in mice using Morris water and Barnes mazes. In this study 50 mice (18-22g, aged 6-8 weeks) were used. The animals were divided into two main groups of 25 mice each of the two memory assessment paradigms. Each paradigm has 5 mice allotted to 5 sub-Groups. Distilled water 10 ml/kg, succimmer 20 mg/kg, 250, 500 and 1000 mg/kg Vigna unguiculata aqueous extract were administered orally. Lead acetate solution at 120 mg/kg was also administered orally using canular to induce acute lead toxicity on the first day. The result was not statistically significant in the acquisition sessions and the probe trials for both the Morris water and Barnes mazes when compared to control. At the end of the study, it was concluded that Vigna unguiculata at the doses administered has no effect on learning and memory in acute lead induced neurotoxicity in mice, but that does not mean it lacks total therapeutic benefit. It was recommended that Co-administration of cowpea and succimmer might be of a better therapeutic benefit.
CHAPTER ONE
Introduction
Lead is a poisonous metal, which exist in both organic (Tetraethyl lead) and inorganic (lead acetate and lead chloride) forms in the environment (Shalan et al., 2005). The main sources are medicines, paintings, pipes, ammunition. And more recently, it is found in alloys for welding storage materials for chemical reagents (Garaza et al., 2006). Exposure to lead mostly occurs through the respiratory and gastrointestinal systems. Lead is conjugated by the liver and passed to the kidney, where it is excreted out in urine and the rest accumulates in various body organs. This affects many biological activities at the molecular, cellular and intercellular levels, which may result in morphological alterations that can remain even after lead level has fallen (Flora et al., 2006; Ibrahim et al., 2012).
Lead poisoning or lead intoxication is defined as exposure to high levels of lead typically associated with severe health effects. Poisoning is a pattern of symptoms that occur with toxic effects from mild to high levels of exposure; toxicity is a wider spectrum of effects, including subclinical ones (those that do not cause symptoms) (Guidotfi and Ragain, 2007). The amount of lead in the blood and tissues, as well as the time course of exposure, determines toxicity. Lead poisoning may be acute (from intense exposure of short duration) or chronic (from repeat low-level exposure over a prolonged period), but the chronic is much more common (Rossi, 2008).
Diagnosis and treatment of lead exposure are based on blood lead level measured in micrograms of lead per deciliter of blood (μg/dL). A blood lead level of 10 μg/dL or above is a cause for concern; however, lead may impair development and have harmful health effects even at lower levels, and there is no known safe exposure level (Barbosa, et al., 2005). Authorities such as the American Academy of Paediatrics defined lead poisoning as blood lead levels higher than 10 μg/dL (Regan and Turne, 2009). Poisoning by organic lead compounds has symptoms predominantly in the central nervous system, such as insomnia, delirium, cognitive deficits, tremor, hallucinations, and convulsions (Karri, et al., 2008).
Acute Lead Toxicity
In acute poisoning, typical neurological signs are pain, muscle weakness, paraesthesia, and symptoms associated with encephalitis such as headache, fever, fatique or weakness, confusion, agitation or hallucinations, seizures, loss of sensation or paralysis in certain areas of the face or body, double vision, perception of foul smells, such as burned meat or rotten eggs, Problems with speech or hearing, loss of consciousness, and so on. Other lead acute symptoms include: Abdominal pain, nausea, vomiting, diarrhoea, and constipation. Lead's effects on the mouth include astringency and a metallic taste. Gastrointestinal problems, such as constipation, diarrhoea, poor appetite, or weight loss, are common in acute poisoning. Absorption of large amounts of lead over a short time can cause shock (insufficient fluid in the circulatory system) due to loss of water from the gastrointestinal tract. Haemolysis (the rupture of red blood cells) due to acute poisoning can cause anaemia and haemoglobin in the urine. Damage to kidneys can cause changes in urination such as decreased urine output. People who survive acute poisoning often go on to display symptoms of chronic poisoning (Pearce, 2007).
Chronic Lead Toxicity
Chronic poisoning usually presents with symptoms affecting multiple systems, but is associated with three main types of symptoms: gastrointestinal, neuromuscular, and neurological. Central nervous system and neuromuscular symptoms usually result from
intense exposure, while gastrointestinal symptoms usually result from exposure over longer periods. Signs of chronic exposure include loss of short-term memory or concentration, depression, nausea, abdominal pain, loss of coordination, and numbness and tingling in the extremities. Fatigue, problems with sleep, headaches, stupor, slurred speech, and anaemia are also found in chronic lead poisoning (karri et al, 2008).
Figure 1.1: Lead Structure (Adopted from Stewart et al., 2006).
Mechanism of Lead Toxicity
Tetraethyl lead, still used as an additive in some fuels, can be absorbed through the skin. Exposure occurs through inhalation, ingestion or occasionally skin contact. Lead may be taken in through direct contact with mouth, nose, and eyes (mucous membranes), and through breaks in the skin. Tetraethyllead, which was a gasoline additive and is still used in fuels such as aviation fuel, passes through the skin; however inorganic lead found in paint, food, and most lead-containing consumer products is only minimally absorbed through the skin (Samarghandian,2013).
The main sources of absorption of inorganic lead are from ingestion and inhalation. In adults, about 35–40% of inhaled lead dust is deposited in the lungs, and about 95% of that goes into the bloodstream. Of ingested inorganic lead, about 15% is absorbed, but this percentage is higher in children, pregnant women, and people with deficiencies of calcium, zinc, or iron. The main body compartments that store lead are the blood, soft tissues, and bone; the half-life of lead in these tissues is measured in weeks for blood, months for soft tissues, and years for bone (Flora et al., 2012).
Lead has no known physiologically relevant role in the body, and its harmful effects are myriad. Lead and other heavy metals create reactive radicals which damage cell structures including DNA and cell membranes. Lead also interferes with DNA transcription, enzymes that help in the synthesis of vitamin D, and enzymes that maintain the integrity of the cell membrane. Anaemia may result when the cell membranes of red blood cells become more fragile as the result of damage to their membranes. Lead interferes with metabolism of bones and teeth and alters the permeability of blood vessels and collagen synthesis. Lead may also be harmful to the developing immune system, causing production of excessive inflammatory proteins; this mechanism may mean that lead exposure is a risk factor for asthma in children. Lead exposure has also been associated with a decrease in activity of immune cells such as polymorphonuclear leukocytes. Lead also interferes with the normal metabolism of calcium in cells and causes it to build up within them (Flora et al., 2012).
Lead Neurotoxicity
Lead Neurotoxicity is a term used to describe neurophysiological changes caused by exposure to toxic agents. Such exposure can result in neurocognitive symptoms and/or psychiatric disturbances. Common toxic `agents include heavy metals, drugs, organophosphates, bacterial, and animal neurotoxins. Among heavy metal exposures, lead exposure is one of the most common exposures that can lead to significant neuropsychological and functional decline in humans. In the brain exposure of animals to lead caused cerebellar oedema, cerebral satellitosis and encephalomalacia (El-Neweshy and El-Sayed, 2011).
Impairments in cortex, hippocampus and cerebellum were also reported. Lead intoxication in humans can be seen from the recent report of high number of children fatalities in Zamfara Nigeria, an estimated 400 children died. Laboratory testing later confirmed high levels of lead in the blood of the surviving children (MSF, 2012), commonly associated neuropsychological difficulties involve in lead poisoning are: intelligence, learning, memory, executive functioning, attention, processing speed, language, visuospatial skills, motor skills, etc. (Samarghandian, 2013).
1.4.1 Mitigative effects of some chemical agents on lead induced neuro toxicity
In the light of lead associated brain toxicity; researches have also reported cases of lead induced brain toxicities that were mitigated by some chemical agents. One of these researches is the role of exogenous hydrogen peroxide (H2O2) in inducing mouse tolerance to lead exposure. Administration of lead was found to significantly (p<0.05) inhibit SOD and CAT activities in the brain. Application of 1.2 micro grams H2O2 per kg body weight efficiently decrease lead induced injury as revealed by decreased growth suppression, increased antioxidative enzyme activity, reduced lipid peroxidation and protection of nuclear DNA integrity (Li et al., 2010). Reckziegel et al., (2011) reported the protective effect of garlic in lead induced brain damage.
Effect of Lead on Different Regions of the Brain
Cerebral cortex
The prefrontal cortex is a likely site of damage responsible for behavioural impairment induced by lead. Indeed, prefrontal cortical damage results clinically in perseveration, inability to inhibit appropriate behavioural response, and increased distractibility. All clinical hallmarks of lead-induced impairment in both monkeys and humans. It has been suggested that basal forebrain and the primary visual cortex may also be damaged by lead. Morphological changes were observed in areas V1 and V2 of the occipital cortex of monkeys following moderate level exposure (Flora et al.,2012).
Hippocampus
Morphologic changes were observed in the rat hippocampus following low level exposure to lead during lactation, and at blood lead levels (BPb) of 20 mg/dl. Namely, a significant increase in the size and numerical density of the mossy fibres, the granule cell layer and the commissural–associational area of the dentate molecular layer were reported. This was related to the high zinc content in the hippocampus. The opposite effect i.e. decreased density of cell layers was observed at much higher (BPb) level of 250 mg/dl., which might be irrelevant to our discussion on low-level exposure. However, this latter finding suggests a bimodal effect of lead on the developing hippocampus, and this type of dose– response curve is consistent with those described for some behavioural outcomes in experimental animals (Flora et al., 2006).
A recent report has demonstrated long-lasting decrease in the density of cholinergic innervation of the hippocampus as the result of perinatal low-level lead exposure. The loss of septohippocampal cholinergic projection neurons in neonate animals resulted in a deficit in hippocampal cholinergic innervation that persisted into young adulthood. This may account for persistent cognitive impairments associated with early Pb exposure (Reckziegel et al., 2011).
Cerebellum
Lead-induced inhibition of postnatal structuring of the rat cerebellum was indicated by an impaired developmental time course of desialylation of the D2-CAM-N-CAM protein. N- CAM, the neural cell adhesion molecule, regulates neuronal fiber outgrowth and synapse formation. This phenomenon was observed at PbB of 20–30 mg/dl, and may contribute to impairment in fine motor skills. Its possible clinical correlate in human is postural disequilibrium, as was described in a clinical study of 6-year-old children with PbB of 10–14 mg/dl measured during their first 5 years of life (Bhattacharya et al., 1993).
Statement of the Research Problem
Lead poisoning has been a recurrent problem in society for many centuries, and its deleterious effects on central nervous system (CNS) are known as lead encephalopathy or lead neuropathy (Flora et al., 2006). Some of the major effects of lead poisoning are neurobehavioral impairments, hyperactivity, alterations in brain structure learning and cognitive deficits in children have been observed even with low blood lead levels (10- 20µg/dl) (Needleman, 2004). Although no general hypothesis is known for the mechanism to explain what cellular events underlie the behavioural and cognitive dysfunction of lead, the detrimental effects of lead have warranted interest in this area (karri et al, 2008).
One of the reasons for the deleterious effects on lead is its ability to strongly bind to sulfhydryl groups of proteins and to mimic or compete with calcium which is one of the major component of cowpea (Vigina unguiculata) (flora et al., 2006). Chronic lead toxicity continues to be a leading environmental health issue especially for children (Mushak, 1992). Recent studies have shown that the toxicity of heavy metals such as lead is a problem for ecological, evolutionary and environmental activities in Nigeria (Nagajyoti et al., 2008).
The procedures required in treating lead toxic patients are tedious. The need to seclude patients, use chelating agents or drugs to treat the patients are all not very effective and not easily applicable to the lead patients. Also the problems of affordability, acceptability and compliance to the drugs intake are all other issues to contend with. Therefore, the need to find easier alternative means in alleviating the sufferings of these patients in our environment. Cowpea is found to be rich in protein content, fibres and vitamins, and have many health benefits which led to a number of researches in this area (Kundua et al., 2008). But the question is whether Cowpea (Vigna unguiculata) play a role in learning and memory in lead induced neurotoxicity?
Justification
Chelation therapy is the only available medical counter measure to treat lead or heavy metal toxicity. The thiol and amino carboxylic acid metal chelators have been used for the prevention as well as the raphy for lead toxicity (saxena and flora, 2004). The goal of chelation is to enhance lead elimination before irreversible changes occur, calcium disodium EDTA (CaNa2 EDTA) and 2,3 – dimercaprol have been used conventionally for the treatment of lead intoxication, however, the clinical use of these clelating agents has been under debate (flora et al., 2012). However, the water soluble analog measo - 2, 3 – dimercaptosuccinic acid (DMSA) was found to be an effective chelator without adverse health effects (Jones, 1994). Clinical human and animal studies have shown that succimmer reduces lead levels in blood and other soft tissues (Smith, 2000). However, its hydrophilic properties have hampered its effectiveness in removing lead from brain and skeleton. Also the problems of dosage regimen compliance or treatment protocol and expensive nature of the drugs are other compounding issues (Cremin et al., 1999).
Studies have shown that the increasing influx of heavy metals in to water bodies from industrial, agricultural and domestic activities is of global concern because of their well- documented negative effects on human and ecosystem (Nakao et al., 2010). This study intends to determine the role of Cowpea (Vigna unguiculata) in cognitive deficits of acute lead induced neuro toxicity, if found to play a role in ameliorating the cognitive deficits of lead toxicity it will reduce the cost of purchasing expensive drugs for treating lead toxicity. This will solve the problems of lack of compliance, acceptability and adverse reactions of the existing drug. Vigna unguiculata is bound free and has high bioavailability, and is a common food grown and consumed in Nigeria.
Hypothesis
Cowpea (Vigna unguiculata) has no effect on Visuospatial Learning and Memory in acute lead induced neurotoxicity in mice.
Aim and Objectives
Aim
The aim of this study is to evaluate the effect of Cowpea (Vigna unguiculata (L). Walp). on visuospatial learning and Memory in acute Lead induced neuro cognitive deficits in mice.
Objectives
The specific objectives are:
i. To evaluate the effect of Cowpea (Vigna unguiculata (L). Walp) on visuospatial learning and memory in acute lead-induced neurotoxicity in mice using Morris water as a wet maze.
To assess the effect of Cowpea (Vigna unguiculata (L).Walp) on visuospatial learning and memory paradigm in acute lead-induced neurotoxicity in mice using Barnes as a dry maze.
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