Role of Mercury Toxicity on Memory and Calcium (Ca2+) Level Determination Using NAA-1 in the Brain Tissues of Adult Wistar Rats

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A.A Sadeeq; A.O Ibegbu; J. A Timbuak; M, Tanko; S.P Akpulu; H.R Bello; S.A Musa
Department of Human Anatomy; Faculty of Medicine. Ahmadu Bello University, Zaria. Nigeria

L. H. Adamu: Department of Anatomy; Faculty of Medicine. Bayero University, Kano. Nigeria 

I.S El-Ladan: Department of Anatomy; Faculty of Medicine. Umaru Musa Yar’addua University, Katsina Nigeria

H.O Kwanashie: Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences. Ahmadu Bello University, Zaria. Nigeria.

All correspondence to: Abubakar Sadeeq Adamu; aasadeeq@abu.edu.ng

ABSTRACT
Mercury is a wide spread environmental pollutant that occurs naturally and has been reported to cause some health problems worldwide. Calcium is an essential neurotrace elements that is required for nerve cells to release neurotransmitters. The present work study the effects of mercury toxicity on memory and level of calcium (ca2+) using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats. Twenty four (24) adults Wistar of both sexes were used for the studies and randomly divided into four groups of six rats per group. Group 1 was the Control and received normal saline, while Groups 2, 3 and 4 received 12.45mg/kg, 28.9mg/kg and 49.8mg/kg body weight of mercuric Chloride respectively for twenty one (21) days through oral route. Animals were trained for spatial learning and memory using Morris water maze method and latency time to find the flat form was recorded. The animals were anesthetized and humanely sacrificed using chloroform. The brain was fixed in Bouin’s fluid and processed histologically using H and E stain. Moreover, some of the tissues were crushed by using mortar and pestle after oven drying at 100oC for short-live irradiation method which was exposed to characteristic Gamma-ray to reveal the presence of Ca2+ ion. The photomicrograph reveals that, there was distortion of the pyramidal cells, congestion, necrosis and sparse distribution of hippocampal cells. The spatial memory test showed a decreased in latency time among the control group while the treated groups showed an increased in latency time that was statistically significant (P=0.05). NAA-1 shows an increased level of calcium concentration among mercury treated animals (P=0.01) when compared to the control animals in group I. It was concluded from this studies that; mercuric chloride exposure has effects on the cyto-archetecture of the hippocampus and alters the level of neurotrace element (calcium ion) which in turn affects spatial learning and memory in adults Wistar rats.

Keywords: NAA-1, short-live analysis, Mercury, memory, calcium ion.

INTRODUCTION
Mercury, a heavy metal is a highly deleterious environmental pollutant that can lead to many health problems in the world (WHO, 2003). Man in his environment is exposed to much potential hazards by heavy metals via bioaccumulation and biodegradation which are transferred to man via food chain due to anthropogenic activities (Wang et al., 2007). Mercury can exist either as elemental, organic and inorganic mercury (Burger et al., 2011). Sources of Mercuric compounds are mostly from Industrial sources, gas, fumes, battery disposals, broken mercury thermometer and coal combustion (Akagi, 1995; Bjomberg et al., 2011). Natural source of Mercury such as Mercury chloride that is found in higher densities in rocks and volcanic activities can give half off of the mercury present in nature (Park, 2000; Booth, 2005). There are many routes of exposure to mercury which include: Oral exposure, inhalational exposure and dermal exposure (Vupputuri et al., 2005: Berlin, 2006). Mercury and its compounds have been shown to also have effects on the growth, weight, renal system, liver, enzymes, memory  and psychological disturbances to mention but a few (Kosan et al., 2001; Rao, 2001; Valera et al., 2008;). Signs and symptoms of mercury poisoning include; Irritability, excitability, restlessness of the skin and eyes, headache, dizziness, difficulty in breathing and frequent urination (ATDRS, 2011). Mercury has no known nutritional or biomedical importance but has various applications and uses such as preservation, employed by pharmaceutical company, agriculture and in cosmetic production (W.H.O, 2005). Calcium is especially important element for signal transduction in cells. For this reason, it is sometimes called a second messenger (Khachaturiazn et al., 1989). Coupled with the fact that calcium is necessary for neurons to release neurotransmitters in the brain, calcium plays an important role in the areas of the brain responsible for storing and retrieving memory (Khachaturiazn et al., 1989). The present work, study the effects of mercury toxicity on memory and level of ca2+ using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats.

MATERIALS AND METHOD
Experimental Animals
Twenty four Wistar rats of average weight of 210g was used for this study and were acclimatized for three weeks in the animal house of the Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria. The animals were fed with Grower’s mesh brand of the Animal feed which was prepared in pellet form to reduce spillages. The animals were fed 3 times daily while clean water was provided in plastic drinking bottle and animals were allowed to feed and drink ad-bilitum. The animals were divided into four (4) groups with six animals per group.
Chemical substances
Mercuric chloride (May and Bakers limited, Dagenham England) with batch number XN202 was obtained from Steve Moore chemicals limited Samaru, Zaria. Kaduna State, Nigeria.
Administration of Mercury Chloride
The LD50 of Mercuric chloride was adopted from the Manufacturer (as 166mg/kg body weight. The Concentration of Mercury chloride used was determined using 30%, 15% and 7.5% of the LD50 per kg body weight according to the manufacturer’s instruction. Animals in Group I served as the Control and were given normal saline, while animals in Group II, III and IV were orally administered with mercuric chloride at 12.45 mg/kg, 24.9 mg/kg and 49.8 mg/kg body weight respectively of the LD50 per kg body weight. The administration of mercuric chloride lasted for 21 days through oral route.

Neurobehavioral test
Spatial learning and memory test using Morris water maze
Morris water maze test was used to develop and test spatial learning and memory in animals according to the methods of Morris (1981) which was further developed by Liu et al (2011). Animals were placed in a circular pool of clear transparent water which was partition into four quadrants. According to this method, a platform was submerged beneath the surface of the maze pool; the animal task is to find the hidden platform. The animal starting point was changed from time to time so as to build a cohesive spatial representation of the pool in order to find the platform and the latency time was recorded both during the training and experimental periods.
Animal Sacrifices
After the administration, the animals were humanely sacrificed and incision was made through the skin and muscle of the skull. The skull was opened through a mid-sagittal incision and the brain was removed and fixed in Bouin’s fluid. The tissues were routinely processed and stained using haematoxylin and eosin. And some tissue were processed for short-lived irradiation for neurotrace elements detection.

Tissues Preparation for Neuro-trace Element Analysis Method
Trace element analysis of the brain tissue was carried out with the aid of Nuclear Research Reactor (NNIR-1) using NAA-1 method at the Center for Energy Research and Training Ahmadu Bello University, Zaria, Kaduna, Nigeria according to IAEA (2004). The brain tissue samples were Oven dried at 100oC using Memmert DINI ovum-280 model-KA. After successful drying, tissues were crushed in Agate mortar and piston at zero contaminant level in order to have the tissues in powdered form which were then put in sealed polyethene bag and weighed using Metter AE 240 Electronic Digital balance to prepare tissue for nuclear irradiation. In NAA-1 short-lived irradiation method used, each sample was irradiated for 5 minutes and exposed to a semi-conductor detector for characteristic gamma-rays detection for 10 minutes followed by another 10 minutes for 3 hours after the first count. This is to allow safe handling of the radioactive nuclei formed (Akaho and Nyako, 2002).The short lived analysis revealed the presence of neurotrace element calcium (Ca2+). The samples were counted for short –lived irradiation using Maestro software attached to an Analog to Digital Converter (ADC) card and Multi-channel Analyzer card (MCA) according to method of Jonah (2005).

Statistical analysis
Data obtained was expressed as Mean ± SEM (Standard error of Mean). One Way Analysis of Variance (ANOVA) was used to compare the Means between and within the groups. P-value less than 0.05 was considered statistically significant. Statistical analysis was performed using EZanalyze v3.0 and a post hoc test of Bonferroni was applied. Chart was produce using Microsoft(R) Excel 2007 for windows. The Trace elements present in the tissue were analyzed using WIN SPAN 2004 software for peak analysis and efficient calibration and calculation of elements detected.

RESULTS
The effect of mercury chloride exposure on Morris water maze test.
The results on mercuric chloride exposure on spatial learning and memory test, showed a decreased in the meantime taken for the animals to complete Morris water maze task among the control group throughout the period of administration as shown in Table 1. While animals in group II and III had an increased in latency time to find an escape route during Morris water maze activity, though the increase is between weeks 3 and weeks 2 respectively which was significant (P=0.05). And animals in group IV showed an increased that was statistically significant (P=0.01) throughout the period of administration as shown in table 1 below.

Table 1: Effect of mercury chloride ingestion on spatial learning and memory using Morris water maze test

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the hippocampus of group III animals, with degeneration of the pyramidal cell layer, loss of some of the pyramidal cells and clumping of pyramidal cell nuclei. Group IV animals show disorientation of the pyramidal cell layer and degeneration of some pyramidal cells with the pyramidal cells appearing to be smaller than normal as shown in Plate D.

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Short-lived analysis for calcium (Ca2+) level in the brain
The results of Neuro-trace element analyses indicates the presence of Calcium (ca2+) in the brain tissues of the animals which showed a significant increase (P =0.01) in concentration of Calcium in Group III and IV animals when compared to the control. While Groups II animals showed an increased in calcium concentration that is not significant as shown in figure 1

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DISCUSSION
The pyramidal cells in the hippocampus manifested some changes like necrosis degeneration and loss of neuronal cell fiber compared to the control group which could be as a result of the exposure of mercuric chloride. This implies that the activity of the hippocampus in memory formation and learning will be impaired and the role of the hippocampus that involved storage and retrieval of information will also be lost. The findings in this study agree with the studies of Wolf et al., (2003), who reported that rats exposed to high concentration of mercury vapor, showed neurodegenerative changes in the hippocampus which was responsible for memory deficit in such animals.
Result from the present study shows that, there was an increased in time taken by the experimental rats to find the hidden platform in Morris water maze test which was significant. Conversely, the pyramidal cell layer of the hippocampus appears to be damaged with dead cells, and vacuolated spaces and distortion in the general morphology of the pyramidal cells. These alterations can consequently result to memory impairments which could be as a result of neuronal degeneration. The destruction of the pyramidal cells implies that activity from the brain region that projects into the pyramidal layer of the hippocampus will also be lost such as memory and learning ability (Wolf, 2009; Quirino, 2012). Mutter (2010) had reported that short term occupational exposure to high levels of mercury induced slight cognitive deficits. A memory deficit among animals exposed to methyl mercury was not significant in latency time or swim length between the different groups of animals according to Olson (2005).
The findings of the present study revealed a high level of calcium in the brain due to mercury intoxication. Increased in calcium level among groups that receive medium and high doses of mercuric chloride implies that, the role of calcium as a second messenger in signal transmission between cells will be loss, which is required for nerve cells to release neurotransmitters in the central nervous system. This finding agrees with Bandtlowc (1993) which explained that excess calcium at the surface of nerve cells causes them to ‘fire’ spontaneously sending messages around the brain for no purpose except confusion and cause a ‘short circuit’ allowing messages sent to one destination to get misrouted to another which causes memory loss.

CONCLUSION
It was concluded from the present study that oral administration of mercuric chloride has effects on spatial learning and memory and can cause histopathological assault in the hippocampus like necrosis, loss of nerve fibers of the pyramidal cells and neuronal clumping. The short-lived analysis for neurotrace elements revealed the presence of calcium in the brain which was high in concentration due to mercury exposure.

ACKNOWLEDGMENT
I appreciate the support of Mal. Shehu Shika of Center for Energy Research and Training. Ahmadu Bello University Zaria, Nigeria for a bench space in his Laboratory and Mal Ado Garba of A. B.U for his financial support.

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