THE EFFECT OF ASCORBIC ACID ON MERCURY-INDUCED CHANGES ON THE HISTOMORPHOLOGY OF THE CEREBELLUM OF ADULT WISTAR RATS

Aim: The present study was aimed at evaluating the effect of ascorbic acid on mercury-induced changes on the cerebellar cortex of adult Wistar rats. Methods: Thirty adult Wistar rats of average weight of 200g were randomly divided into 6 groups of 5 rats each. The animals in Group 1 (control) were administered with distilled water, Groups 2 and 3 were administered with 52mg/kg and 26.25mg/kg body weight of HgCl2 respectively while Groups 4 and 5 were administered with 52mg/kg of HgCl2 and 5mg/kg of ascorbic acid and 26.25gm/kg of HgCl2 and 5mg/kg of ascorbic acid respectively, while Group 6 was administered with 5mg/kg of ascorbic acid. The administration was through the oral route, daily for 3 weeks and the animals were humanely sacrificed. Thereafter, blood and tissue samples were harvested for analysis. Results: The result of the biochemical parameters showed a significant increase (P<0.05) in the mean SOD and LPO values after the administration of mercuric chloride and ascorbic acid. Histological observation of the cerebellar cortex showed normal histo-morphology in Groups 1 and 6 while, the cerebellum in Groups 2, 3, 4 and 5 showed some degenerative, necrotic and cellular changes. Conclusion: Ascorbic acid administration has shown to ameliorate the induced degenerative changes in the cerebellum caused by mercuric chloride toxicity in Wistar rats.


INTRODUCTION
Mercury occurs in the environment as a result of natural processes such as degassing from earth crust, emissions from volcanoes, evaporation from water bodies and anthropogenic processes from coal-fires, power stations, residential heating systems and waste incinerators (Cox, 1997;Burger and Gochfeld, 2011).Mercury can also occur as a result of mining of mercury, gold, copper, zinc, lead and silver (ATSDAR, 1999).There is a growing appreciation of the effects that exposure to heavy metals may cause in the body and in particular, the brain and the nervous system.This is because some of these metals can cross the blood brain barrier and accumulate in the brain and cause damages (Langford and Ferner, 1999;Valko et al., 2005).
Animals and humans interact with their environment on a daily basis and as such are exposed to a broad spectrum of chemicals and heavy metals present in the environment through food, air and water (Burger et al., 2011).The ancient Greek used mercury in ointments while the ancient Egyptians and Romans used it in cosmetics but in China, mercury was thought to prolong life, heal fractures and maintain general good health (Clarkson, 1989;Brian. and Fred, 1995).There are many reported cases of mercury food poisoning in Sweden, Mexico, USA and the Minamata Bay incidence that led to the poisoning of over 800 people.Toxicity of mercury can result from vapor inhalation, ingestion or absorption through the skin.Nervous, digestive and renal systems are most commonly affected in mercury exposure while children and pregnant women are most vulnerable to mercury exposure (De Bont et al., 1986;European Commission, 2005).Fishes, mostly tilapia, from Lagos Lagoon of Nigeria are characterized with relatively high level of mercury concentration and this can be attributed to industrial effluents into the Lagoon (Fodeke, 1979).The level of mercury in fishes from Niger Delta area of Nigeria is less than 10mg/kg (Kakulu and Osibanjo, 1986).In Nigeria, not much work has been done to investigate the relative bio-accumulation potential of mercury on local aquatic species (Oyewo, 1998).Some of the symptoms of mercury poisoning are irritability, excitability, restlessness, irrational outburst of temper, depression, headache and dizziness amongst others (Grant and Lipman, 2009).Other symptoms of mercury poisoning include itching, burning, pain, pink cheeks, swelling of fingertips and toes and shedding of the skin.A person suffering from mercury poisoning may experience profuse sweating, tachycardia, increased salivation, and hypertension (Lucky, 1987;Langford and Ferner, 1999).Affected children may show red cheeks, nose and lips, loss of hair, teeth, and nails, transient rashes, muscle weakness, kidney dysfunction, memory impairment and insomnia (Horowitz et al., 2002;Liuji et al., 2002).Ascorbic acid is a natural antioxidant that prevents the production of free radicals induced by oxidative damage to lipids and lipoproteins in various compartments of cells and tissues (Padayatty et al., 2003;WHO, 2003).Antioxides have been shown to react with superoxides (Nishikimi, 1975, WHO, 2004), hydroxyl radicals (McGregor and Biesalski, 2006) and singlet oxygen (Moreira et al., 2010).Ascorbic acid is an anti-oxidative substance that may protect the cells against the effects of free radicals which are molecules produced when the body breaks down foods or by exposure to tobacco smoke and radiation (Chihuailafet al., 2002).These anti-oxides are regarded as firstline protective agents that nullify free radicals by donating a single electron to yield dehydroascorbic acid (Valkoet al., 2005;Gemma et al., 2010).The aim of the present study was to evaluate the effect of ascorbic acid on mercuric chloride-induced changes on the cerebellar cortex of adult Wistar rats.

Experimental Animals
Thirty adult Wistar rats of average weight of 200g were used for this study and were acclimatized for three weeks and kept in the Animal house of the Department of Human Anatomy, Faculty of Medicine Ahmadu Bello University Zaria.The rats were then divided into six groups of five rats per Group for the experiment.

Experimental Chemicals
Twenty grams of mercuric chloride manufactured by May and Bakers Chemical Laboratory Limited Dagenham England while Vitamin C tablets manufactured by Jopan Pharmaceuticals Ltd. were purchased and used for the experiment.

Animal Experimentation
Twenty Wistar rats were divided into 6 Groups of 5 animals each.Group 1 (Control) was administered with distilled water, Group 2 was given 52.5mg/kg body weight of mercuric chloride (Hg), Group 3 animals were administered with 26.25mg/kg body weight of Hg, corresponding to 25% and 12.5% of LD 50 of Hg respectively (Lucky, 1987).Animals in Group 4 were given 52.5mg/kg body weight of Hg and 5mg/kg body weight of ascorbic acid, while Group 5 animals were administered with 26.25mg/kg body weight of Hg and 5mg/kg body weight of ascorbic acid and Group 6 rats were administered with 5mg/kg body weight of ascorbic acid only corresponding to 10% of LD 50 of ascorbic acid (UKFSA, 2007).The administration was by oral route daily and lasted for 3 weeks while animal feed and drinking water was allowed ad libitum.

Animals Sacrifice
After the administration, the animals were weighed, humanely sacrificed and incision was made through the skin and muscle of the skull.The skull was opened through amid sagittal incision and the cerebellum was removed and fixed in Bouin's fluid.The tissues were routinely processed and stained using haematoxylin and eosin and crystal violet.

Estimation of Oxidative Parameters Determination of Catalase Activity
Catalase activity was determined using the method described by Sinha, (1972) and the absorbance was read at 570 nm.Standard cure was made by plotting the absorbance obtained at various levels of the assay.The catalase activity was obtained from the graph of the standard curve.

Determination of superoxide dismutase (SOD) activity
Superoxide Dismutase (SOD) activity was determined by a method described by Fridovich, (1989).Absorbance was measured every 30 seconds up for a total of 150 seconds at 480 nm from where the SOD activity was calculated.

Assessment of lipid peroxidation
Lipid peroxidation as evidenced by the formation of TBARS was measured by the method of Niehaus and Samuelson (1968).The absorbance of the pink supernatant was measured against a reference blank using a spectrophotometer at 535nm.

Assay of reduced Glutathione concentration
Reduced glutathione (GSH) concentration measurements were done according to the method of Ellman (1959) as described by Rajagopalan et al. (2004), and the absorbance was read at 412 nm.

Statistical Analysis
All data were presented as mean ± SD.For establishing significant differences, data were analyzed by one-way analysis of variance (ANOVA), followed by Tukeypost hoc test.Values were considered statistically significant if P value was p ≤ 0.05.

Physical Observation of the animals
The result of physical observation of the animals showed that rats in Group 1 were very active while Group 2 animals showed less activity, gnawing and restlessness with watery feces.The animals in Group 3 showed gnawing and restlessness, characterized by watery feces while, Group 4 animals exhibited restlessness.The result of physical observation showed little gnawing in Group 5 animals with more activity when compared to Group 2 and Group 4 while Group 6 animals showed no changes in their physical activity as in Group one.

Oxidative Parameters
The result of the analysis of oxidative stress markers namely catalase, SOD, glutathione reductase and lipid peroxidation following administration of mercuric chloride and ascorbic acid showed increase and decrease in some parameters as shown in Table 1.The result showed a significant decrease (P<0.05) in catalase, SOD and glutathione reductase in Groups 2, 3 and 4 when compared with the Control, and a significant increase (P<0.05) in lipid peroxidase in Groups 2 and 3, and a significant decrease (P<0.05) in Groups 5 and 6 when compared with the Control as shown in table 1.
Table1: Effect of ascorbic acid on mercuric chloride-induced changes on the oxidative markers in experimental animals

Histological Observation
The histological observation of the cerebellar cortex of animals in Group 1, showed normal histomorphology of the cerebellar cortical cells and layers as shown in Figure 1

DISCUSSION
The present study showed degeneration and necrosis of Purkinje cells in the Purkinje cell layers of the cerebellum of adult Wistar rats induced with different concentration of mercuric chloride.All layers and cells of the cerebellum of the Control group showed no histological changes.The findings from the present study agree with the findings which reported that many heavy metals such as mercury, lead, cadmium and other organic compounds have the capacity to damage the nervous system (Maurice et al., 1972;Verina et al., 2007).These changes may be transient but permanent abnormalities may be induced only by sustained exposure of these chemicals in an excessive quantity (Wolf et al., 2009).Recent studies have shown that the most sensitive elements of the cerebellar cortex to these chemicals are the Purkinje cells, which react to these noxious substances by undergoing degeneration and as such disappear from their relative positions in the Purkinje cell layer (Jomova Volko, 2011;Farina et al., 2013).It has been shown that cerebellar dysfunction may occur in association with exposure to a wide variety of toxins including heavy metals such as mercury, lead, thallium, manganese, drugs and solvents (Woodside et al., 2005;Burger et al., 2013).These toxins may adversely affect the cerebellum directly or as part of a more generalized brain effects (Jomova et al., 2011).Findings from other studies, show that the cerebellar neurodegeneration will lead to lesions of the cerebellum giving rise to signs and symptoms generally called cerebellar syndromes which include muscular hypotonia, intentional tremor, nystagmus, scanning speech and ataxic gait (Fine et al., 2002;Gemma et al., 2007;Wolf et al., 2009).The present study has shown that ascorbic acid has an ameliorative effect on the cerebellum of the experimental animals induced toxicity of the mercury chloride.Administration of ascorbic acid has shown some improvements in the prevention of degeneration of brain cells when compared with rats exposed to only mercuric chloride.From the present observation, it has been shown that there is a direct relationship between the concentration of mercuric chloride, ascorbic acid and the level of neurological damage in the cerebellum.The present study showed that the ascorbic acid administration reduced the damage done to the cerebellum was in agreement with the findings that showed that natural agents rich in antioxidants are involved in the improvement in enzymatic activity and hence in the reduction of oxidative stress (Ahamed and Siddiqui, 2007;Farina et al., 2013).This may play a significant role in the reversion of mercury toxicity by forming inert complexes and inhibiting their toxicity on the dopaminergic neurons (Flora et al., 2007;Jomovaand Volko, 2011).The present study showed a significant increase in the mean SOD levels in the experimental groups and a significant decrease in the LPO mean values when compared to the Control.Theincrease and decrease in the mean values of SOD and LPO could be related to the ameliorative effects of the antioxidative activities of ascorbic acid on mercury induced toxicity in the experimental animals.It has been shown that heavy metals such as mercury, lead and thallium reduce antioxidative enzymes such as SOD, CAT, GLU and Lipid peroxidase (LPO) (Woodside et al., 2005;Farina et al., 2013).The present study agrees with the findings that showed that oxidative stress was an important component of the mechanism of toxicity by heavy metals (Gutierrez et al., 2006).It has been shown that acute exposure to mercury increases LPO and decreases SOD levels in experimental animals.The decrease in the activity of antioxidative enzymes such as superoxide dismutase level and the elevation of Lipid peroxidation, suggest the formation of free radicals and the participation of free radical induced oxidative cell injury in mediating the toxic effect of mercury (Jomva et al., 2010).There is reduction in the antioxidant defense system by SOD in mercury toxicity leading to the disruption of pro-antioxidant balance in the body.The increase in Lipid peroxidation might be due to peroxidation of unsaturated fatty acids within the cells in the body.Thus, increased lipid peroxidation is suggestive of progressive increase in cellular deformity, increase in membrane permeability and rigidity, and disruption of structural and functional integrity of cell organelles (Gemma et al., 2007).

CONCLUSION
Mercury exposure has induced degeneration in the cerebellar features of the adult Wistar rats and ascorbic acid has shown to significantly protect the neurotoxicity induced by mercuric chloride administration.As such, people exposed to mercury poison should consume food rich in ascorbic acid along with other antioxidants.
while the cerebellum of the animals in Group 2 showed degeneration and necrosis of the Purkinje cells of the Purkinje cell layer of the cerebellar cortex as shown in Figure 2. The cerebellum of animals in Group 3 showed separation of the Purkinje cells due to degeneration of Purkinje cells, degenerated Stallate cells in the cerebellum as shown in Figure 3 while the cerebellar cortices of animals in Group 4 showed few degeneration of Purkinje cells of the cerebellum with clumping and congestion of cells as shown in Figure 4.The cerebellar cortices of animals in Group 5 showed displacement of Purkinje cell nuclei, separation of Purkinje cells due to the degeneration of Purkinje cells and the granular and molecular layers of the cerebellum were not affected as shown in Figure 5, while the results of the observation of the cerebellar cortices of animals in Group 6showednormal arrangement of the layers and cells of the cerebellum as shown in Figure 6.