The Role of Ascorbic Acid on Mercury Induced Molecular Changes and DNA Alteration in Rats.

Animoku Abdulrazaq A.1,5, Mesole Samuel Bolaji2, Okpanachi Omachonu Alfred2,

Yusuf Uthman Ademola3 Shember Pascal Bemseer4

1Department of Anatomy, College of Health Sciences, Kogi State University, Anyigba-Nigeria

2Department of Clinical Medicine, School of Medicine, Eden University-Zambia

3Department of Human Anatomy, School of Medicine, Mulungushi University-Zambia

4Department of Pharmacology and Therapeutics, College of Health Sciences, Benue State University, Makurdi-Nigeria

5Department of Anatomy, Ahmadu Bello University Zaria-Nigeria

All Corresponding to: Animoku Abdulrazaq, animokuaa@gmail.com

ABSTRACT

Background Mercury is a heavy metal contaminant of known toxicity with potential for global mobilization following its give off through air, soil, water and food Aim: The objective of this work was to examine the molecular changes in the DNA caused by a heavy metal pollutant mercuric chloride (HgCl2) in Wistar rats and the role of ascorbic acid administration. Methods: Twenty five adult Wistar rats (average weight 185 g) were randomly divided into five groups of five rats per group; a control group administered normal saline, mercuric chloride (HgCl2; 49.8 mg/kg), HgCl2 with distilled water, HgCl2 with low dose ascorbic acid (595mg/kg) and HgCl2 with high dose ascorbic acid (1,190 mg/kg) groups. All administration was carried out orally for a period of three to six weeks and the animals were humanely sacrificed. Results: The results from molecular studies showed that the level of migration of the genomic DNA were fastest in the mercury treated groups when compared to ascorbic acid and the control groups as characterized on agarose gel electrophoresis. While, significant decrease (p<0.05) in the DNA area and height were observed in mercury treated groups as compared to ascorbic acid and the control groups. Conclusion: The findings from this study showed that ingestion of mercuric chloride has potential deleterious effects on genomic DNA of Wistar rats. However, ascorbic acid administration demonstrate possible ameliorative potential against mercury induced molecular changes

Key words: Mercuric Chloride, Ascorbic acid, DNA, Agarose gel electrophoresis, Wistar rats

INTRODUCTION

Mercury intoxication has been a public health problem for many decades (Burger et al., 2011). Consideration of the role of environmental factors in determining the susceptibility to mercury has recently been renewed by evidence from epidemiological studies (Wang, et al., 2007). Populations are exposed to mercury through the consumption of fishes and sea foods (European Commision, 2005), dental amalgam, mining of silver, gold in industries (WHO, 2007). Mercury readily crosses the blood-brain barrier due to limited lipid solubility and can result in neurological symptoms; seizures, mental retardation, language disorders, vision, hearing and memory loss (WHO, 2003, 2007; Animoku et al., 2018). It is a potential factor in brain damage (Ibegbu et al., 2014), mental impairment, behavioral anomalies (Farina et al., 2011), but little information concerning the potential genotoxicity of mercury compounds (Barregard et al., 1991). However, heavy metals like mercury and lead had been associated with chromosomal aberrations, elongation of X-chromosome, pulverization and birth defects (Valverde et al., 2002), cell death by apoptosis and DNA fragmentation (Hassan et al., 2010). Mercuric salts can be absorbed through the skin of animals (Altmann et al., 2008), Mercury compounds rapidly enter the bloodstream (Berlin et al., 2007), and the level of mercury in the blood is higher after exposure to low dose than a high dose (Berlin et al., 2007). Elimination of mercury is via urine, feaces (WHO 2005), expired air, sweat, and saliva (Booth and Zeller, 2005). In Nigeria, fishes from Lagos Lagoon and the use of “Kohl” a traditional cosmetic had been reported as an agent of mercury toxicity (Onyeike et al., 2002). Presenting symptoms include headache, itching, dizziness, burning, excitability, irritability, restlessness, increased salivation, profuse sweating and frequent urination (Grant and Lipman, 2009; ATDRS, 2011).

Ascorbic acid is an essential nutrient for humans and some other animal species. Vitamin C is an antioxidant that protects our cells against free radicals (Padayatty et al., 2003), pneumonia (Hemila and Louhiala, 2007), prevents scurvy (WHO, 2001) and lowering the incidence of gout (Choi et al., 2009). Ascorbic acid is a well known non-enzymatic antioxidant that can break the chain of lipid peroxidation in cell membranes and scavenges free-radicals such as superoxide ion, hydrogen peroxide, nitric oxide, hydroxyl, alkoxyl and alkyl-peroxyl, collectively called reactive oxygen species (ROS) (Kacuk et al., 2003). Vitamin C is found in high concentration in immune cells and is consumed quickly during infections (Preedy et al., 2010). Ascorbic acid is absorbed in the body by both active transport and simple diffusion (Savini, et al., 2007). Examples of antioxidants are Ascorbic acid (Vitamin C), Vitamin E and Vitamin A (Vasudevan and Sreekumari, 2007). Sources of vitamin C are fruits, vegetables, liver, nutritional supplement, tablets, drinks (Wilson, 2005) and animal products (United Kingdom Food Standard Agency, 2007). Scurvy results from lack of vitamin C, it leads to the formation of brown spot on the skin, spongy gums, and bleeding from all mucous membranes (Stanner et al., 2004).

The aim of the study was to determine the role of ascorbic acid on mercury induced molecular changes and DNA alteration in Wistar rats.

MATERIALS AND METHODS

Twenty five (25) Adult Male Wistar rats of average weight 185g were used for this study. After acclimatization in the Animal House of the Department of Human Anatomy, Ahmadu Bello University, Zaria, the animals were grouped into five groups of five animals each (n = 5). Mercuric chloride (X-N202, May and Bakers, England) was utilized at LD50 of 166 mg/kg body weight as adopted from ATSDR (2011).While; the LD50 of ascorbic acid (S42238, Sam Pharmaceuticals, Nigeria) was adopted from MSDS (2008) as 11,900 mg/kg body weight. The mercury chloride was 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. Before the commencement of the study, ethical approval was sort and obtained from the Ahmadu Bello University Zaria Ethical and Animal Use Committee, Faculty of Veterinary Medicine with reference Number ABU/ FVM/ EAUC/ 2015/12. The animals were dosed as follows: control group was administered with normal saline, group II with 30% mercuric chloride (HgCl2, 49.8 mg/kg) only, group III received HgCl2 with distilled water only, group IV received HgCl2 with 5% low dose ascorbic acid (595 mg/kg), while group V received HgCl2 with 10% high dose ascorbic acid (1,190 mg/kg). However, administrations of distilled water and ascorbic acid from weeks 3-6 were done in order to observe for any possible natural recovery and possible ameliorative potentials of ascorbic acid respectively (Table 1). The administration was by oral route daily and lasted for 3-6 weeks, while animal feed and water were allowed ad libitum.

MOLECULAR STUDIES

Brain tissues were collected in eppendorf tubes containing genomic lysis buffer and were taken to Avian Influenza Laboratory, Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria for Molecular analysis. The tissues were homogenized, kept in water bath overnight and were washed twice through centrifugation set at 14,000 rpm with 200µl of DNA pre-wash buffer, 200µl DNA wash buffer and DNA elution buffer respectively so as to extract the DNA. This protocol was according to the manufacturer’s instruction (Zymo research; the epigenetics company USA). 1% agarose was added to 100 µl Tris Acetic Acid (TAE) buffer and was dissolved in micro wave oven for 5 minutes and allowed to cool to about 45 ˚C; this was followed by the addition of 1µl ethidium bromide to the gel and was mixed thoroughly and gently poured into the casting tray containing the combs and allowed to solidify. Running buffer (Tris Acetic Acid EDTA) was poured into the tank while 20 μl of the DNA sample was mixed with 5 μl DNA loading dye and was loaded into the wells. The DNA sample was run for 40 minutes at 75 mV while the Gel was observed in gel documentation system and the DNA was documented.

STATISTICAL ANALYSIS

All the results were analyzed using the Statistical package for Social Scientist (SPSS version 21) and the results were expressed as Mean ± SEM. The Statistical significance between means were analyzed using one-way analysis of variance (ANOVA) followed by post HOC test; Turkey’s multiple comparison test to test for statistically significant difference between control and experimental groups. A p-value < 0.05 was considered statistically significant.

RESULTS:

MOLECULAR STUDIES

The results from molecular studies as observed from the Genomic DNA showed that the level of migration of the genomic DNA on agarose gel electrophoresis of the animals in Groups II and III appeared fastest characterized by lighter DNA bands when compared to Groups I (Control), IV and V as shown in Plate I.

MEASUREMENT OF GENOMIC DNA AREA, WIDTH, HEIGHT, PERIMETER AND LENGTH OF MIGRATION OF DNA FROM WELL

The measurement of Genomic DNA area, width, height, perimeter and length of migration of DNA showed significant decrease (p<0.05) in the measured areas and heights of the genomic DNA in Groups II and III when compared to Group one (Control) and Group V, while, measurement of the length of migration of genomic DNA revealed significant increase (p<0.05) in Group II when compared to Groups I (Control), III, IV and V respectively as shown in Table 2.

DISCUSSION

The present study was on whether or not mercuric chloride induced toxicity was accompanied by changes in the Genomic DNA and the role of ascorbic acid. The study revealed that the level of migration of the Genomic DNA as characterized on agarose gel electrophoresis were fastest in Groups treated with mercury only, suggesting lesser molecular weights of the DNA molecules which could be related to the effect of mercury on the Genomic DNA. The study showed significant increase (p<0.05) in the mean Genomic DNA length measured from the well in Groups exposed to mercury only when compared to ascorbic acid treated groups and animals in the Control Group. This study agrees with the work of Hasan et al., (2010) who reported that liver cell DNA of mercury treated fishes appeared further away from the well when compared to the position of the Control Group and this change in position was related to cell death by apoptosis accompanied by DNA fragmentation. In another study, it was reported that DNA damage in mercury exposed individuals suggests that mercury overload induces an imbalance in the redox cycle (Hasan et al., 2013). The present study revealed significant decrease (p<0.05) in height and area of the Genomic DNA of animals exposed to mercury only when compared to animals treated with ascorbic acid and animals in the Control Group suggesting the toxic effects of mercury and the possible ameliorative effects of ascorbic acid against mercury induced DNA damage. However, further study on Polymerase chain reaction (PCR), DNA sequencing and comet assay (Tice et al., 2000) could further reveal any alteration in the Genomic DNA sequence or if any mutation might have occurred following mercury exposure and if this alteration could possibly be ameliorated by ascorbic acid, since it was suggestive that ascorbic acid can protect against DNA damage (Cail et al., 2001; Diatrich et al., 2003).

CONCLUSION

The findings from the present study justify the ameliorative potential of ascorbic acid against mercury induced molecular changes and DNA alteration; hence the consumption of ascorbic acid should be encouraged to populations exposed to increased risk of mercury poisoning.

Conflict of Interest

None declared.

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