International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(8), 76-81, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 76 Protective Effects of Vitamin C on Haematological Parameters in Intoxicated Wistar Rats with Cadmium, Mercury and Combined Cadmium and MercuryHounkpatin A.S.Y., Johnson R.C., Guédénon P. 1, Domingo E., AlimbaC.G., Boko M.1 and Edorh P.A.1,4Interfaculty Centre of Training and Research in Environment for Sustainable Development (CIFRED), Laboratory of Toxicology and Environmental Health, University of Abomey-Calavi (UAC), 01 BP 1463, Cotonou, BENIN Department of Geography and Planning, Processing Laboratory of Geographic Information and Planning Analysis, University of Abomey-Calavi (UAC), BP 939, Porto-Novo, BENIN Department of Cell Biology and Genetics, Faculty of Science, university of Lagos, NIGERIA Departement of Biochemistry and Cellular Biology, University of Abomey-Calavi (UAC), 01 BP 526, Cotonou, BENINAvailable online at: www.isca.in Received 21st October 2012, revised 31th October 2012, accepted 3rd November 2012Abstract Cadmium and mercury are reported as heavy metals that induce blood disorders and immunological effects. This study was performed to determine the haematological toxicity of cadmium, mercury and evaluated the protective antioxidant role of vitamin C. For this purpose, 65 rats were divided into 13 lots of 5 animals, grouped into 2 groups of 6 lots and one control group. Two different doses of each metal and their combination were administered orally for 28 consecutive days to 6 lots of 1 group. The first two lots (A, B) respectively were exposed to cadmium chloride, two other lots (C, D) were respectively received mercury chloride and the last two lots of this group (E, F) were respectively treated with the combination of these two metals. The second group of 6 lots (Ac, Bc, Cc, Dc, Ec, Fc) respectively have received over the previous doses of metals, a daily dose of Vitamin C during the same period. The control group (G) received the same volume of distilled water. At the end of exposure, the body weight of rats was weighed and whole blood was collected by retro-orbital sinus for analysis of haematological parameters. The results of this study showed a significant decrease (P 0.05) on white blood cell, red blood cell, hemoglobin concentration, mean corpuscular, hemoglobin concentration with high concentrations of mercury and the combination of high concentrations of cadmium and mercury. However, co-administration of mercury, cadmium and mercury and vitamin C had a protective effect on the potential harmful metals. Keywords: Cadmium, mercury, haematology, antioxidant, vitamin C, rat.. Introduction The increase in the pollution of our day is a major and global problem. This is due to the use of toxic chemicals or xenobiotic substances or by certain synthetic compounds such as heavy metallic compounds. Of these compounds, heavy metals, non-biodegradable induced potential effects at low doses. Discharged by industries, agriculture and urban communities, heavy metals reach the environment. Metallic compounds on land and water pose potential health hazard not only to livestock and wild life but also to fishes, birds, mammals and even to human beings. Also did not help in the metabolism, cadmium and mercury persist in the environment and are the most toxic metals to humans. They have a strong ability to accumulate in the food chain, including halieutics products such as fish5 and also in snails. This situation exposes consumers to food poisoning. In acute, cadmium as well as in chronic cytotixicity and carcinogenesis, activation of endonucleases, generation of reactive free radical such as reactive oxygen species (ROS)7,8and signal transduction pathways involving apoptosis play important roles. Cadmium accelerates lipid peroxidation by stimulating the peroxidation chain reaction in the target organs, resulting in the generation of ROS and consequently the induction of cytotoxicity10. Cadmium causes anemia11,12 and induces immunological effects13,14. The classic symptoms of mercury contamination are carcinogenicity and/or damage to kidney function, visual, metabolic, reproductive, immunological and neurological15-20. Haematological and immunological effects induced by cadmium and mercury were verified by Guédénon et al.21. Studies have shown that vitamin C supplementation has varied effects on induced toxicity22-28. Ascorbic acid has been found to interact with several elements in such a way as to render them less available for animals29. He also demonstrated the protective effect of vitamin C on the toxicity of mercury29. Grosicki30 and Akhere et al.31 reported a decrease in the carcass cadmium burden and the cadmium contents in the liver, kidney, testicles and muscles of cadmium exposed rats given water supplemented vitamin C for 28 days. Within this framework we proposed to explore the protective effect of vitamin C on blood disorders in rats exposed to cadmium, mercury and their combination. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 76-81, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 77 Material and MethodsBiological Material: The animal material was composed of 65 male albino Wistar rats aged from 6 to 8 weeks and weighted about 108±25g. These rats obtained at the Animal Breeding Unit of the University of Lagos, Nigeria were acclimated for two weeks before the experiments. They were placed in designed sterile polypropylene cages in a room whose temperature borders 25-30°C with relative humidity of 60°C±5%. The cages were illuminated with a sequence of 12 hours with light and 12 hours into dark. Animals had free access to water and standard rodent laboratory chow (Ladokun feed Nigeria®) ad libitum, in the animal "Botanical and Zoological Garden" in UNILAG (University of Lagos), of Nigeria where experiments were conducted. Chemicals and Preparation of the Various Solutions: The chemicals tests used for the experiment were cadmium chloride and chloride anhydrous of mercury. The powdered mercuric chloride (HgCl = 271.50; minimum assay: 98%) and chloride cadmium (CdCl = 183.32; minimum assay: 99%) were purchased from "General Purpose Reagent BDH Chemicals Ltd. Poolo England". Vitamin C (100 mg tablet), registered under MAFDAC REG. No. 04-1453, manufactured by 'Emzor Pharmaceutical Industries Ltd., Plot 3C Block A, was obtained "Outpatient Pharmacy Department of the Lagos University Teaching Hospital, Lagos, Nigeria" in Nigeria. The expiry date was scheduled for 02/01/2014. The vitamin C tablets not easily soluble were returned in the form of a fine powder prior to their solution was shaken vigorously before each oral administration. Concentrations were prepared for the experiment (0.25 mg/kg, 2.5 mg/kg) for cadmium chloride; (0.12 mg/kg, 1.2 mg/kg) for mercuric chloride and 150 mg/kg ascorbic acid. The different solution concentrations were based on different daily doses, the average weight of each lot and the daily volume administered to rats (1 mL). Distribution of rats and testing: After two weeks of acclimatization, 65 rats were randomly divided into 13 groups of 5 pooled into 2 groups of 6 lots and one control group. Cadmium, mercury and their combination were administered by gavage (by stomach tube) for 28 consecutive days following the method of Alimba32 and Awodele 22. Six groups of rats received the first group daily doses of cadmium, mercury and their combination in a final volume of 1 ml of water. The first two batches (A, B) were exposed respectively to cadmium chloride (0.25 mg/kg, 2.5 mg/kg), two other batches (C, D) are respectively received mercury chloride ( 0.12 mg/kg, 1.2 mg/kg) and the last two batches of this group (E, F) were respectively treated with the combination of these two metals (0.25 mg/kg Cd + 0.12 mg/kg Hg) (2.5 mg/kg Cd +1.2 mg/kg Hg). The second group of 6 lots (AC, , C, D, E, F) have respectively received in addition to these previous doses of metals (Cd, Hg, Cd + Hg), a daily dose of 150 mg/kg vitamin C during the same period. The control group (G) received only the same volume of distilled water. These different doses of cadmium respectively correspond to a dose producing significant results: 0.25 mg/kg33 and 10 times this concentration (2.5 mg/kg). As for mercury, 1/10 (1.2 mg/kg) and 1/100 (0.12 mg/kg) of the LD 5034 were used. Blood Collection and Haematological Analysis: After 28 days of exposure, rats were fasting overnight. They were weighed before the collection of blood and sacrifice and this to determine the final body weight. All samples were taken between 7 am and 9 am to avoid variations due to circadian rhythm. Whole blood was obtained from a puncture of the retro-orbital sinus by the conventional method35. This methodology is to slowly introduce the tip of the microhaematocrit (70 ml heparinized micro-capillary tubes haematocrit) in the medial angle of the eye. Progression through the tissue was facilitated by slightly turning to the pipette. The vessel wall was very fragile and when we reached the venous plexus, blood spurts and periorbital space rises by capillarity into the pipette that was put into tubes with anticoagulant for hematology. Blood samples collected in EDTA anticoagulant tubes (ethylene diamine tetra-acetic acid 8.5%) was quickly returned by mixing with anticoagulant in the tube. All blood samples were labeled and immediately conveyed to the laboratory for analysis. Haematological parameters were analyzed: white blood cell count (WBC), red blood cells (RBC), hemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT) and the number of lymphocytes (LYM). All haematological parameters were analyzed in the "Haematology Unit, Lagos state University Teaching Hospital (ULTH)" using the automated method with the automatic analyzer “Haematology auto analyzer Sysmex KX-21N”.Statistical Study: Results are expressed as mean ± esm1 of n experiments (where n represents the number of animals used). The differences between the treated and control rats were evaluated using the Students t-test p (T � t) = 0.05. The software used was Microsoft Excel 2010 and XL Stat 2011. The differences were statistically significant if the value of p was less than 0.05 and not significant if the value of p was greater than 0.05. Results and DiscussionHaematological parameters analyzed were: Red Blood Cell (X10L RBC), White blood cell (X10L WBC), hemoglobin concentration (HGB) (g/dL HGB), haematocrit (% HCT), Mean Corpuscular Volume (fL MCV), Mean Corpusculaire Hemoglobin (pg MCH), Mean Corpuscular Hemoglobin Concentration (g/dL MCHC) Platelets (X10L PLT) and lymphocytes (% LYM). The results are presented as mean values and standard deviation of haematological parameters in the blood of rats exposed to cadmium, mercury and cadmium and mercury combination at various concentrations and intoxicated rats treated with vitamin C in table-1. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 76-81, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 78 Table-1 Mean values and SD of hematological parameters of rats exposed to Cd, Hg and treated with vitamin C Concentration Parameters control Cd Hg Cd+Hg G A C E LowWBC 13,95±0,21 a 13,55±1,76 a 10,17± 2, 32 a 8,47±1,50 * b RBC 8,46±0,72 a 7,45±1,76 a 7,36±0,49 a 7,18±0,69 a HGB 14,40±0,35 a 13,60±2,40 a 13,47±1,57 a 13,32±1,12 a HCT 47, 450±0,07 a 44,95±8,83 a 46,47±5,20 a 45,07±4,84 a MCV 60,30±1,98 a 61,00±2,54 a 63,00±4,86 a 62,87±5,15 a MCH 18,20±0,42 a 18,50±1,13 a 18,25±1,40 a 18,60±1,10 a MCHC 31,30±0,84 a 30,30±0,56 a 29,00±1,07 a 29,60±0,89 a PLT 624,50±16,26 a 965,00±158,39 a 768,50±163,59 a 742,50±94,60 a LYM 69,60±10,46 a 69,00±10,46 a 69,87±11,96 a 69,80±6,29 a G B D F HighWBC 13,95±0,21 a 12,50±1, 20 a 9,85±0,49 * b 8,15± 0,49 * b RBC 8,46±0,72 a 7,78±0,66 a 5,59±1,02 * b 5,46±0,78 * b HGB 14,40±0, 35 a 14,47±0,96 a 10,37±1,97 * b 9,65±0,77 * b HCT 47,450±0,07 a 48,00±3,57 a 37,55±8,74 a 35,85±4,73 a MCV 60,30±1,98 a 61,00±1,91 a 66,62±5,86 a 60,50±0,72 a MCH 18,20±0,42 a 18,60±0,58 a 18,52±0,48 a 18,45±0,77 MCHC 31,30±0,84 a 30,20±0,29 a 27,35±1,39 * b 27,00±0,70 * b PLT 624,50±16,26 a 901,50±241,41 a 822,50±378,70 a 841,00±427,09 a LYM 69,60±10,46 a 71,32±11,02 a 65,75±5,47 a 65,30± 4,38 a Concentration Parameters control Cd + Vit C Hg + Vit C Cd+Hg+ Vit C G Ac Cc Ec LowWBC 13,95±0,21 a 24,40±5, 39 a 13, 95± 2, 76 a 14,05±5,20 a RBC 8,46±0,72 a 7,72±1,07 a 7,49± 0,30 a 7,305±0,78 a HGB 14,40±0,35 a 13,55±1,52 a 13,32±0,44 a 12,72±0,69 a HCT 47, 450±0,07 a 47,50±6,02 a 43,67±0,80 a 43, 95±3,65 a MCV 60,30±1,98 a 61,70±3,22 a 58,32±1,99 a 69,30±2,13 a MCH 18,20±0,42 a 17,60±0,67 a 17,80±0,45 a 17,50±1,27 a MCHC 31,30±0,84 a 28,57±1,49 a 30,50±0,78 a 29,02±1,38 a PLT 624,50±16,26 a 1078,75±180,28 a 874,75±46,42 a 1254,50±262,67 a LYM 69,60±10,46 a 54,82±14,15 a 55,32±13,98 a 52,30±15,39 a G Bc Dc Fc HighWBC 13,95±0,21 a 14,75±1,93 a 14,70±4,18 a 10,70±2, 34 a RBC 8,46±0,72 a 7,45±1,26 a 8,10±0,70 a 8,02±0,12 a HGB 14,40±0, 35 a 14,17±0,35 a 14,93±0,68 a 14,25±0,45 a HCT 47,450±0,07 a 46,12±7,58 a 47,00±3,12 a 46,30±1,49 a MCV 60,30±1,98 a 61,50±1,05 a 58,03±1,30 a 60,30±2,13 a MCH 18,20±0,42 a 21,22±5,27 a 18,46±0,65 a 17,77±0,34 a MCHC 31,30±0,84 a 30,85±1,27 a 31,80±0,72 a 30,77±0,56 a PLT 624,50±16,26 a 734,00±248,17 a 1014,33±110,18 * b 907,25±120,40 * b LYM 69,60±10,46 a 49,35±7,86 a 60,86±14,23 a 57,75±9,24 a The difference between the value of blood parameters of the experiment groups and that of the negative control at 0, 05. The averages followed by the same letter are not statistically different; a and b (significativity enters negative control, lots intoxicated at metals and their correspondent intoxicated and treated with vitamin C; a: not significant and b: significant) Interpretation of Results: Count of Red Blood Cell: In Wistar rats, erythrocyte count has made important and significant variations ranging from 8.46±0.72 in animals of Lot G (control) to 5.59±1.02 and 5.46±0.78 in those of Lot D and F (p 0.05). The red blood cell count was significantly higher (8.10±0.70 and 8.02±0.12) in rats and lots Fc Dc when compared to that of rats of Lot D and F but lower than that of rats of Lot G (p 0.05). Count of white blood cells: The lowest values of 9.85±0.49, 8.47±1.5, 8.15±0.49 for leukocytes was encountered respectively in rats lots D, E, F against 14.70±4.18, 14.05±5.20 and 10.70±2,34, 13.95±0.21 for animals in Dc, Ec, Fc and G (p 0.05). As for platelets, the number has increased in all rats intoxicated (A, B, C, D, E, F) compared to control (G) and more increase with the application of vitamin C (Ac, Bc, Cc, Dc, Ec, Fc). Hemoglobin: Hemoglobin concentration was significantly lower in rats of group D and E (10.37±1.97 and 9.65±0.77) respectively when compared to (14.40±0.35, 14.93±0.68, 14.25±0.45) than rats of lots G, Dc and Fc (p 0.05). International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 76-81, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 79 Haematocrit : The haematocrit values in Wistar rats ranged from 47.45±0.07 in animals in the G (37.55±8.74 and 35.85±4.73), respectively, in those of group D and F through intermediate values (47.00±3.12 and 46.30±1.49) rats lots Dc and Fc. Mean corpuscular hemoglobin concentration: The average concentrations corpuscular hemoglobin concentration (MCHC) were fluctuating values with the lowest (27.35±1.39 and 27.00±0.70) respectively in the rats of groups D and F compared to those of other groups (p 0.05). The results of this study found that some parameters vary widely depending on the batches of Wistar rats intoxicated with cadmium, mercury and their combination and treated with vitamin C. This is for example the case of the erythrocyte: indeed animals of lots D and F, it reached values 5.59.10/uL and 5.46.10L against 8.46.10/uL animals of Lot G (control) with intermediate values in animals treated with Fc, Dc and vitamin C. The number of red blood cells obtained from animals in the D and F is very low compared to the results of Boukerche et al.36, where these authors found 8.45.10/uL as the number of red blood cells in healthy Wistar rats. It is likely that the high dose of mercury and the combination of high doses of cadmium and mercury do reduce the number of red blood cells causing anemia in Wistar rats intoxicated. These results have been shown by other authors37,38 conducted on mice poisoned with lead. These authors argue that the number of red blood cells decreased. The red blood cell count not intoxicated rats (control) is stable and varies little from one subject to another. Even if this stability seems characteristic of healthy Wistar rats, the number of red blood cells obtained from rats of Lot G in this study is almost similar to 8.45.10/uL obtained by Boukerche et al.36 but higher value 7.1.10/uL proposed Lahouel et al.39. At the rats that were addicted to high doses of mercury and the combination of high doses of cadmium and mercury treated with vitamin C, erythrocyte regeneration was remarkable in batches (Dc and Fc). Similar results were observed by Lahouel et al.39 in Wistar rats intoxicated with paracetamol and treated with propolis, and by Veena et al.37 in mice intoxicated with lead nitrate and treated with ethanoic extract extra coriander. Regarding hemoglobin and haematocrit, a similar trend was obtained with the lowest levels in the rats of group D and F. The values of these haematological parameters were increased due to treatment with vitamin C. Everything seems to confirm the conclusion Friot and Calvet40 who argue that the factors that affect the general condition of the animal such as nutritional status, fluid balance and status influence pathological haematocrit. Regarding the white blood cells, changes were observed mainly in rats lots D and F in which the number was lower. These low values were increased in rats lots Dc and Fc treated with vitamin C would be related to the toxic action of mercury and cadmium and mercury combination can induce leucopenia and thrombocytopenia in cases of severe liver dysfunction41. The significant decrease in hemoglobin (10.37±1.97 and 9.65±0.77 g/dl) in rats of group D and F, associated with a decrease in MCHC (27.35±1.39 and 27.00±0.70 g/l) indicate a tendency to macrocytosis and hypochromia hematopoiesis in the liver which occurs efficiently. This decrease in blood cells has been corrected in rats and lots Dc Fc due to the favorable action of vitamin C used as a dietary supplement. Vitamin C has antioxidant potential which was confirmed by Szeto et al.42 which states that the high amount of vitamin C in pineapple comus contributes to over 30% of its potential antioxidant, which promotes its favorable action on the liver in the regulation of hematopoiesis. This decrease in hemoglobin was also found by Bersenyi et al.43in rabbits poisoned by lead, by Sinha et al.44 in mice exposed to cadmium chloride, for Ognjanoviet al.45 in rats exposed to cadmium chloride. This result deferred of Smaoui et al.46 which exposed rats to the exhaust gas which had induced erythropoiesis by hypoxia. Mercury and the combination of high concentrations of cadmium and mercury could inhibit heme synthesis of red blood cells and cause anemia signs described by Bottomley and Muller-Eberhard47. These signs are offset by the beneficial effect of vitamin C. However, red blood cells are low in heme, where the decline in MCHC, already indicated by our results in rats of Lot D and F. We also found that the rate Red blood cell, White blood cell, Hemoglobin, Haematocrit, the mean corpuscular hemoglobin concentration were not statistically different in control rats (G) and those addicted and treated (lots Dc and Fc). This could be explained by the antioxidant role of vitamin C. It has the ability to capture and deactivate free radicals. According to Siess et al.48, it acts by preventing the binding of free radicals on DNA by activation of detoxification and protection of the capillary walls as noticed Kawabata et al.49. Awodele et al.22, reported that the administration of vitamin C (8 mg/kg) corrected some of the potential harmful rifampicin of the deoxyribonucleic acid (DNA) in mice. This is due to the small dose lower than that used in this study (150 mg/kg). Ognjanoviet al.45 have also shown that as our of results pretreatment with vitamin E and C showed a protective role on the toxic effects of cadmium on haematological values, lipid peroxide. Similar results were obtained by Fox et al.50, which showed the protective effect of vitamin C on anemia induced by heavy metals in rats. ConclusionThese findings suggest that cadmium and mercury present in the environment and in particular in foodstuffs of first necessity cause of haematological disturbances in the blood. However, co-administration of (cadmium and mercury) and antioxidant (vitamin C) has protective effect hématotoxic due to cadmium and mercury. It could be concluded from the present study that vitamin C has potent antioxidant activity against cadmium and International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 76-81, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 80 mercury sensitive Haematological. It may then be recommended its use as a dietary supplement to rid the body of these xenobiotics that affect the health of the population. The consumption of rich foods in vitamin C is also highly recommended. References1.Jagadeessan G. and Pillai S.S., Hepatoprotective Effect of Taurine against Mercury Induced Toxicity in Rat, J. Environ. Biol., 28, 753-756 (2007)2.Akinyeye A.J. and Okorie T.G., Heavy Metal Studies of Industrial Effluent on Alaro Stream Sediment, I. Res. J. Biological Sci., 1(6), 5-9 (2012)3.Miquel G., Les Effets des Métaux Lourds sur l’Environnement et la Santé, Rapport d’Office Parlementaire d’évaluation des choix scientifiques et technologiques, Sénat (261), Assemblée Nationale (1979), Paris, France, 360p (2001)4.Picot A. and Proust N., Mercury and Its Compounds: from Speciation to Toxicity, Actualit. Chim., 16-24 (1998) 5.Hounkpatin A.S.Y., Edorh P.A., Salifou S., Gnandi K., Koumolou L., Agbandji L., Aissi K.A., Gouissi M. and Boko M., Assessment of Exposure Risk to Lead and Cadmium Via Fish Consumption in the Lacusrian Village of Ganvié in Benin Republic, J. Environ. Chem. Ecotoxicol., 4 (1), 1-10 (2012)6.Edorh A.P., Agonkpahoun E., Gnandi K., Guédénon P., Koumolou L., Amoussou C., Ayedoun A., Boko M., Gbeassor M., Rihn H. and Creppy E., An Assessment of the Contamination of Achatina achatina by Toxic Metals in Okpara Village, Int. J. Biol. Chem. Sci., 3 (6)1428 -1436 (2009)7.Nath K.A., Croatt A.J., Likely S., Berhrens T.W. and Warden D., Renal Oxidant Injury and Oxidant Response Induced by Mercury, J. Kidney Int., 50, 1032-1043 (1996)8.Hultberg B., Andersson A. and Isalsson A., Alterations of Thiol Metabolism in Human Cell lines Induced by Low Amounts of Copper, Mercury, of Cadmium Ions, Toxicol., 126, 203-212 (1998)9.Habeebu S.S., Liu J., Liu Y. and Klaassen C.D., Succeptibility of MT-null Mice to Chronic CdCl- Induced Nephrotoxicity Indicates that Renal Injury is not Mediated by the CdMT Complex, Toxicol. Sci., 46, 197- 203 (1998)10.Oteiza P.I., Adonaylo V.N. and Keen C.L., Cadmium Induced Testes Oxidative Damage in Rats can be Influenced by Dietary Zinc Intak, Toxicol., 137, 13-22 (1999)11.Horiguchi H., Anemia Induced by Cadmium Intoxication, Nihon Eiseigaku Zasshi. Japanese J. of Hygien. 62 (3), 888-904 (2007)12.Kosti M.M., Ognjanovi B., Dimitrijevi S., Ziki R.V., ZSCtajn A., Rosi G.L. and Zivkovi R.V., Cadmium-Induced Changes of Antioxidant and Metabolic Status in Red Blood Cells of Rats: in vivo effects, European J., Haematol., 51 (2), 86-92 (1993)13.Blakley B.R., The Effect of Cadmium Chloride on the Immune Response in Mice, Can. J. Comp. Med., 49 (1), 104-108 (1985)14.Müller S., Gillert K.E., Krause C., Jautzke G., Gross U. and Diamantstein T., Effects of Cadmium on the Immune System of Mice, Experientia., 35(7), 909-910 (1979)15.Mergler D., Anderson H.A., Chan L.H.M., Mahaffey K.R., Murray M., Sakamoto M. and Stern A.H., Methylmercury Exposure and Health Effects in Humans, Worldwide Concern. Ambio., 36(1), 3-11, (2007) 16.Mathiessen T., Ellingsen D.G. and Kjuus H., Neuropsychological Effects Associated with Exposure to Mercury Vapor Among Former Chloralkali Workers, Scan. J. Work. Environ. Health., 25 (4), 342-350 (1999)17.Barregard L., Thomassen Y., Schutz A., Marklund S.L., Levels of Selenium and Antioxidative Enzymes Following Occupational Exposure to Inorganic Mercury, Sci. Total. Environ., 99 (1-2), 37-47 (1990)18.Sallsten G. and Barregard L., Urinary Excretion of Mercury, Copper and Zinc in Subjects Exposed to Mercury Vapour, Biometal., 10 (4), 357-361 (1997) 19.Elghany N.A., Stopford W., Bunn W.B. and Fleming L.E., Occupational Exposure to Inorganic Mercury Vapour and Reproductive Outcomes, Occupational. Med. Oxford., 47(6), 333-336 (1997) 20.Boffetta P., Merler E. and Vainio H., Carcinogenicity of Mercury and Mercury Compounds, Scand. J. Work. Environ. Health., 19(1), 1-7 (1993)21.Guédénon P., Edorh P. A., Hounkpatin A.S.Y., Alimba C.G., Ogunkanmi A., Nwokejiegbe E.G., Deguenon Y., Gbeassor M. and Creppy E.E., Haematological Study of Clarias gariepinus Exposed to Chronic and Subchronic Doses of Cadmium, Mercury and Combined Cadmium and Mercury, Sci. Nat., (2), 2-19 (2012b)22.Awodele O., Olayemi S.O., Alimba C.G., Egbejogu C. and Akintonwa A., Protective Effect of Vitamin C and or Vitamin E on Micronuclei Induction by Rifampicin in Mice, Tanzania. J. Health. Res., 12 (2), 2 (2010)23.Ddique S., Hasan Y., Tanveer T. and Afzal M., Antigenotoxic Effects of Ascorbic Acid against Megesterol Acetate-induced Genotoxicity in Mice, Human. Experiment. Toxico., 24, 121-127 (2005)24.Liopiz N., Francesc P., Ela C., Lui A., Anna A., Cinta B. and Salvado J.M., Antigenotoxic Effect of Grape Seed Procyanidin Extract in Fao Cells Submitted to Oxidative stress, J. Agric. and Food. Chemistry., 52, 1083-1087 (2004) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 76-81, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 81 25.Kaya B., Antigenotoxic Effect of Ascorbic Acid on Mutagenic dose of three Alkylating Agents, Turkish. J. Biology., 27, 241-246 (2003) 26.Aly F.A.E. and Donya S., In Vivo Antimutagenic Effect of Vitamin C and E against Rifampicin Induced Chromosome Aberration in Mouse Bone Marrow, Mut. Res., 518, 1-7 (2002)27.Netke S.P., Roomi M.W., Tsao C. and Niedzwiecki A., Ascorbic Acid Protects Guinea pigs from Acute Aflatoxin Toxicity, Toxicol. Appl. Pharm., 143, 429-435 (1997) 28.Khan P.K. and Sinha, S.P., Antimutagenic Efficacy of Higher Doses of Vitamin C, Mut. Res., 298, 157-161 (1993)29.Hill C.H., Interactions of Vitamin C with Lead and Mercury, Ann. N. Y. Acad. Sci., 355, 262-266 (1950)30.Grosicki A., Influence of Vitamin C on Cadmium Absorption and Distribution in Rats, J. Trace. Elem. Med. Biol., 18 (2), 183-187 (2004)31.Akhere A., Omonkhua F.O. and Obi P., Biochemical Evaluation of the Effects of Vitamin C in Rats Exposed to Sub-chronic Low Doses of Cadmium, Int. J. Toxicol., 1559-3916 (2008) 32.Alimba C.G., Bakare A.A. and Latunji C.A., Municipal Landfill Leachates Induced Chromosome Aberrations in Rat Bone Marrow, African. J. Biotech., , 2053-2057 (2006)33.Ganesh C.J. and Satish K.A., Cadmium Chloride Induces Dose Dependent Increases in the Frequency of Micronuclei in Mouse Bone Marrow, Sci. Mut. Res., 306, 85-90 (1994)34.Bharat B.P., Atish R., Soumik A. and Shelley B., Induction of Oxidative Stress by Non-lethal Dose of Mercury in Rat Liver, Possible Relationships Between Apoptosis and Necrosis, J. Environ. Boil., 31, 413-416 (2010)35.Boussarie D., Hématologie des Rongeurs et Lagomorphes de Compagnie, Bull. Acad. Vét., 72, 209-216 (1999)36.Boukerche S., Aouacheri W. and Saka S., Les Effets Toxiques des Nitrates : Etude Biologique chez l’Homme et chez l’Animal, Ann. Biol. Clin., 65(4), 38 (2007)37.Veena S., Leena K., Arti S., Shweta L. and Sharma S.H., Ameliorating Effect of Coriandrum sativum Extracts on Hematological and Immunological Variables in an Animal Model of Lead Intoxication, J. Pharm. Allied. Health. Sci., , 16-29 (2011)38.Lavicoli I, Carelli G., Stanek E.J., Castellino N. and Calabrese E.J., Effects of Low Doses of Dietary Lead on Red Blood Cell Production in Male and Female Mice, Toxicol. Lett., 137, 193-199 (2003)39.Lahouel M., Boulkour S., Segueni N. and Fillastre J.P., Effet Protecteur des Flavonoïdes contre la Toxicité de la Vinblastine, Cyclophosphamide et du Paracétamol par Inhibition de la Peroxydation Lipidique et Augmentation du Glutathion Hépatique, J. pat. Bio., 10, 314-322 (2004)40.Friot D. and Calvet I.I., Biochimie et Elevage au Sénégal, Rev. Elev. Méd. Vét., 20, 75-98 (1973). 41.Lee W.M., Acetaminophen and the US Acute Liver Failure Study Group: Lowering the Risks of Hepatic Failure, Hepatology., 40, 6-9 (2004)42.Szeto Y.T., Tomlinson B. and Benzie I.F., Total Antioxydant and Ascorbic Acid content of Fresh Fruits and Vegetables: Implications for Dietary Planning and Food Preservation, J. Nutr., 87, 55-59 (2001)43.Bersényi A., Fekete S.G., Szocs Z. and Berta E., Effect of Ingested Heavy Metals (Cd, Pb and Hg) on Haematology and Serum Biochemistry in Rabbits, Acta. Vet. Hung., 51(3), 297-304 (2003)44.Sinha M., Manna P. and Sil P.C., Taurine Protects the Antioxidant Defense System in the Erythrocytes of Cadmium Treated Mice, B. M .B. report., 41(9), 657-663 (2008)45.Ognjanovi B.I., Pavlovi S.Z., Maleti S.D., Ziki R.V., Stajn A.S., Radojici R.M., Saici Z.S. and Petrovi V.M., Protective Influence of Vitamin E on Antioxidant Defense System in the Blood of Rats Treated with Cadmium, Physiol. Res., 52(5), 563-570 (2003)46.Smaoui M., Yhorbel F., Boujelbene M., Makni-Ayadi F. and El Féki A., Impact de l’Exposition Chronique au Gaz d’Echappement d’Origine Automobile sur certains Biomarqueurs touchant la Fonction Hormonale Sexuelle Mâle, la Fonction Rénale et l’Hémogramme chez le Rat, Pollut. Atmosph.,167(2), 439-449 (2000)47.Bottomley S.S. and Muller-Eberhard V., Pathophysiology of the Heme Synthesis, Pemin. Hematol., 25, 282-303 (1998)48.Siess M.H., Le Bon A.M., Canivenc-Lavier M.C. and Susch M., Mechanisms Involved in the Chemoprevention of Flavonoids, Biofactor., 12(4), 193-199 (2000)49.Kawabata T.T., Chapman M.Y., Kim D.H., Stevens W.D. and Holsapple M.P., Mechanisms of In VitroImmunosuppression by Hepatocyte-generated Cyclophosphamide Metabolites and 4-Hydroxycyclophosphamide, Biochem. Pharmacol., 40(5), 927 (1990) 50.Fox M.R.S., Harland B.F., Schertel M.E. and Weeks C.E., Effect of Ascorbic Acid on Cadmium Toxicity in the Young Coturnix, J. Nutr., 101, 1295-1305 (1971)