Neurobehavioral and Body Weight Assessment in mercury exposed Rats and the role of Ascorbic Acid (Vitamin C)

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Animoku Abdulrazaq A., Augustine Ibegbu O., Maliki Joseph Simpa and Iliyasu Musa O.
Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria-Nigeria
Samuel Bolaji Mesole, Uthman Ademola Yusuf
Department of Human Anatomy, University of Gitwe-Rwanda
All Correspondence to: Animoku AA email: animokuaa@gmail.com

ABSTRACT

Mercury is a heavy metal of known toxicity, exerting its adverse effects on the health of humans and animals through air, soil, water and food. Aim: The present study was aimed at investigating the role of ascorbic acid on mercury induced spatial learning, memory and body weight impairments in Wistar rats. Methods: Twenty five (25) Adult Wistar rats with average weight 185 g were randomly divided into five groups of five rats per group (n=5); The animals in Group I (Control) were administered with normal saline, Group II was given 49.8mg/kg body weight of mercuric chloride. Animals in Groups III and IV were given 49.8mg/kg body weight of mercuric chloride and distilled water, and 49.8mg/kg body weight of mercuric chloride and 595mg/kg body weight of ascorbic acid respectively while Group V animals were administered with 49.8mg/kg body weight of mercuric chloride and 1,190mg/kg body weight of ascorbic acid. All administration was carried out orally for a period of three to six weeks. Results: The results from Morris water maze test showed significant increase (p<0.05) in mean time taken by the rats to locate the hidden platform in mercury treated groups compared to rats in the Control and Ascorbic acid treated groups, suggestive of neurological toxicity of mercury to learning and memory loss. The results also revealed significant decrease (p<0.05) in weight gain by mercury intoxicated rats when compared to weights gain by rats treated with ascorbic acid and rats in the Control group. Conclusion: The administration of ascorbic acid remarkably ameliorated HgCl2 induced changes in body weights, learning and memory impairments in Wistar rats.
Key words: Memory, Mercuric Chloride, Ascorbic acid, Body Weight, Wistar rats

INTRODUCTION

Mercury is a heavy metal of known toxicity, noted for inducing public health disasters (Berlin, 2007) through bioaccumulation and biomagnifications which has been transferred to man via air, water and food chain (Burger et al., 2011). Human toxicity varies with the form of mercury, the dose and the rate of exposure. The target organ for inhaled mercury vapor is primarily the brain. It is a potential factor in brain damage (Ibegbu et al., 2014), mental impairment, behavioral anomalies (Farina et al., 2011), neuromuscular weaknesses, hearing problems, impaired cognitive functions and coma (Liuji et al., 2002; Verina et al., 2007; Flora et al., 2007). However, the toxicity of mercury can also result from vapor inhalation and ingestion or absorption through the skin. Burning of fossil fuels, batteries disposal and burning mercury painted wood are all sources of mercury exposure (WHO, 2005). Mercury is capable of inducing a wide range of clinical presentations while, nervous, digestive and renal systems are most affected in mercury exposure (Bernhort, 2012). Mercuric salts can be absorbed through the skin of animals (Altmann et al., 2008) and through the skin of humans via mercuric ointments (Berlin et al., 2007). Metabolism of mercuric chloride is similar for humans and laboratory mammals. Once absorbed, mercury chloride enters an oxidation– reduction cycle (Agency for Toxic Substances and Disease Registry, 1999). The urine and feces are the main excretory pathways of mercury compounds in humans (WHO, 2005). In Nigeria, Tilapia fishes from Lagos Lagoon and the use of “Kohl” a traditional cosmetic had been reported to predispose people to mercury toxicity (Fodeke, 1979; Onyeike et al., 2002). Symptoms of mercury poisoning involves depression, headache, dizziness, itching, burning, shedding of the skin, irritability, excitability, restlessness, irrational outburst of temper, profuse sweating, tachycardia, frequent urination, increased salivation, and hypertension (Grant and Lipman, 2009; ATDRS, 2011).
Ascorbic acid is an essential nutrient for humans and some other animal species. In living organisms ascorbate acts as an antioxidant by protecting the body against oxidative stress (Padayatty et al., 2003; Ibegbu et al., 2014). Vitamin C functions as an antioxidant that scavenges free radicals (Padayatty et al., 2003), prevents scurvy (WHO, 2001), pneumonia (Hemila and Louhiala, 2007) and may be useful in lowering the incidence of gout (Choi, et al., 2009). Antioxidants are substances that nullify the effect of free radical by either inhibiting the initial production or inhibiting the preparative phase of free radicals (Sujatha et al., 2011). Examples of antioxidants are Ascorbic acid (Vitamin C), Vitamin E and Vitamin A (Vasudevan and Sreekumari, 2007). These anti-oxides are generally regarded as primary first-line protective agent that nullifies free radicals by donating a single electron to yield dehydro-ascorbic acid (UKFSA, 2007; Gemma et al., 2010).
The aim of the study was to determine the role of ascorbic acid on mercury induced spatial learning, memory and body weight impairments in Wistar rats.

MATERIALS AND METHODS
Experimental Animals
Twenty five (25) Adult Wistar rats of average weight 185g were used for this study and were acclimatized for 3 weeks to enable the rats adapt to laboratory conditions and were kept in the animal house of the Department of Human Anatomy, Ahmadu Bello Univrsity, Zaria. After acclimatization, the rats were divided into five groups of five rats per group for the experiment.

Experimental Chemicals
Mercuric Chloride manufactured by May and Bakers limited, Dagenham England with batch number X-N202 as used for this study. Ascorbic acid manufactured by Sam Pharmaceuticals Limited, Ilorin, Nigeria with batch number S42238 was used for this study. The mercury chloride used for the experiment was of the approved Laboratory grade chemical by Standard Organization of Nigeria, marketed and sold in Nigeria while the Ascorbic acid tablets was approved by National Agency for Food and Drug Administration and Control to be marketed and used in Nigeria.

Experimental Protocol
The animals in Group I (control) were administered with normal saline from 1st to 42nd day, Group II was given 49.8mg/kg body weight of mercuric chloride (HgCl2) from 1st to 21st day, Group III animals were given 49.8mg/kg body weight of HgCl2 from 1st to 21st day and distilled water from 22nd to 42nd day, corresponding to 30% of LD50 of HgCl2 (ATSDR, 2011). Animals in Group IV were administered with 49.8mg/kg body weight of HgCl2 from 1st to 21st day and 595mg/kg body weight of ascorbic acid from 22nd to 42nd day, while Group V rats were administered with 49.8mg/kg body weight of HgCl2 from 1st to 21st day and 1,190mg/kg body weight of ascorbic acid from 22nd to 42nd day; only corresponding to 5% and 10% of LD50 of ascorbic acid respectively (MSDS, 2008) as shown in Table (1).The administration was by oral route daily and lasted for 3-6 weeks, while animal feed and water were allowed ad libitum. 

Animal Sacrifice
After the administration, the animals were weighed and anaesthetized by inhalation of chloroform in the sacrificing chamber. Incision was made through the skin and muscle of the skull. The skull was opened through a mid sagittal incision and brain tissues were removed and fixed in Bouin’s fluid.

RESULTS
BODY WEIGHT ASSESSMENT OF THE ANIMALS
The results of body weight assessment showed that there was progressive increase in the mean body weights of the animals in Groups I-V throughout the duration of the experiment. However, the degree of weight gain by the animals in Groups II and III decreased significantly (p<0.05) when compared to the weights gain by animals in Groups I (Control), IV and V as shown in Table 2, Figure 1 and Figure 2. *p<0.05 indicates significant difference compared to Group I (Normal saline).
g = mean weight in grams. SEM: Standard Error of Mean
*a indicates significant difference between Group V and Group II.
*b indicates significant difference between Group V and Group III.
*c indicates significant difference between Group IV and Group II
*d indicates significant difference between Group IV and Group III.
GI= Control, GII=HgCl2 (alone),
GIII =HgCl2 and Distilled H2O,
GIV =HgCl2 and Vit.C595mg/kg,
GV=HgCl2 and Vit.C1,190mg/kg.

Figure 2: The % body weight change of animals throughout the experiment.
GI= Control, GII=HgCl2 (alone), GIII=HgCl2 and Distilled H2O,
GIV=HgCl2 and Vit.C595mg/kg, GV=HgCl2 and Vit.C1,190mg/kg.

*p<0.05 indicates significant difference compared to Group I (Control).
*a indicates significant difference between Group V and Group II.
*b indicates significant difference between Group V and Group III.
*c indicates significant difference between Group IV and Group II
*d indicates significant difference between Group IV and Group III.

SPATIAL LEARNING AND MEMORY ASSESSMENT IN RATS USING MORRIS WATER MAZE NAVIGATION TEST
The results on spatial learning and memory using Morris water maze navigation test showed neither significant increase nor decrease in the mean time taken by the animals in Groups I to V to complete Morris water maze task at the end of the training period and week 1. However, significant increase (p<0.05) in the mean time were observed in Group II at the end of Week 2 but, in Groups II, III, IV and V at the end of Week 3, in Groups III and IV at Week 4 as well as in Group III at the end of Week 5 when compared to the Control Group. This significant increase (p<0.05) in the mean time was only observed in Group III at Week 6 when compared to Groups I (Control), IV and V as shown in Table 3 and Figure 3.

Figure 3: Mean latencies in seconds of all the Groups in Morris water maze test for spatial learning and memory.
GI= Control, GII=HgCl2 (alone) GIII =HgCl2 and Distilled H2O,
GIV =HgCl2 and Vit.C595mg/kg, GV=HgCl2 and Vit.C1,190mg/kg.

DISCUSSION

BODY WEIGHT ASSESSMENT OF ANIMALS
The present study clearly showed progressive increase in the mean body weights of the animals throughout the period of administration of mercuric chloride and ascorbic acid. However, the degree of weight gain by the animals treated with mercuric chloride alone throughout the experiment was less than that of the weight gain by animals treated with ascorbic acids for 3 weeks and animals in the Control Group. This difference in decreased body weight gain was significant (p<0.05) and this could be attributed to the effect of mercuric chloride that might have slowed down the degree of weight gain by the animals exposed to mercury. This agrees with the findings of Mohammad, (2009) who reported that the body weight of mercuric chloride treated rats was less than their control significantly during the 8 weeks of exposure. The present study is however, in contrast to the study of Hans, (2006) who reported a decreased relative body weight in rats fed diets containing mercuric chloride equivalent to 20mg mercury/kg body weight per day for 4 weeks and Thomas et al., (2001) who observed that exposure of rats, rabbits and dogs to metallic mercury vapour for 7h/day, 5 days/week for 72-83 weeks, resulted significantly in weight decrease. In the present study the degree of weight gain by animals treated with mercuric chloride and ascorbic acid were significantly higher when compared to the weight gain by animals treated with mercuric chloride alone, suggesting the ameliorative effect of ascorbic acid on mercury induced changes on the body weight of the animals. This is related to the findings of Ibegbu et al., (2014) who reported that ascorbic acid was able to ameliorate the toxic effect of mercury on body weights of animals treated with ascorbic acid and mercury.

NEUROBEHAVIORAL FINDINGS
The present study showed significant increase (p<0.05) in the mean time taken by the experimental animals to locate the hidden platform in Morris water maze test for memory and learning during the weeks of mercuric chloride administration. This could be associated with memory loss which could be as a result of neuronal degeneration, distortion in the general morphology of the hippocampus. Hence, activities such as memory and learning abilities from the brain region that projects into the pyramidal layer and CA3 region of the hippocampus Animoku et al., 2016) could be lost (Wolf et al., 2009; Quirino et al., 2012). However, Mutter et al., (2010), had earlier reported that short term occupational exposure to high levels of mercury induced slight cognitive deficits and that, mercury has no effect on memory as observed from cognitive test for memory, which conversely disagrees with the present study. The results of the present study showed significant decrease (p<0.05) in the mean time taken by the animals to locate the hidden platform in Morris water maze test for spatial learning and memory during the weeks of ascorbic acid administration as compared to mercury induced groups. Thus, the study has revealed the ameliorative potential of ascorbic acid on spatial learning and memory impairment in animals intoxicated with mercuric chloride.

CONCLUSION
The findings from the present study justify the ameliorative potential of ascorbic acid against mercury induced body weight changes, learning and memory impairments in rats. Hence populations exposed to mercury poison should consume foods rich in ascorbic acid (Vitamin C) along with other antioxidants to alleviate mercury toxicity.

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