Antimutagenic Effect of Selenium and Vitamins Against the Genotoxicity Induced by Cobalt Chloride in Mice

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1 2006 The Japan Mendel Society Cytologia 71(3): , 2006 Antimutagenic Effect of Selenium and Vitamins Against the Genotoxicity Induced by Cobalt Chloride in Mice Nagwa H. A. Hassan 1, Maha A. Fahmy 2, *, Ayman A. Farghaly 2 and Entesar E. S. Hassan 2 1 Faculty of science, Ain Shams University, Cairo, Egypt 2 Department of Genetics and Cytology, National Research Center, Dokki, Cairo, Egypt Received January 26, 2006; accepted March 1, 2006 Summary The genotoxic effects of cobalt chloride (CoCl 2 ) were studied in mice using different mutagenic end points in vivo. The possible protection provided by the antioxidant selenium and vitamins (ACE) against CoCl 2 genotoxicity was also assessed. The results indicate that CoCl 2 has genotoxic effects on mice somatic and germ cells. It induced a significant increase in the frequency of chromosomal aberrations in mouse bone-marrow and spermatocytes after single and repeated oral treatments. The induction of morphological sperm abnormalities was also confirmed in the present study. The supplemental selenium and vitamins (ACE) to reduce the genotoxic effect of CoCl 2 was proved. Key words Cobalt chloride, Genotoxicity, Selenium, Vitamins (ACE), Mice. The contamination of our environment with metal compounds has expanded to include the general public health. Metal mutagenesis has only been little studied, although many of them are reported to be carcinogenic (IARC 1990, Carlisle et al. 2000). Mutagenesis by CoCl 2 is of potential interest in view of human exposure to this metal. It is used in many applications: e.g. invisible ink, humidity indicator, glass and porcelain painting, production of vitamin B 12, fertilizer and food additive (Dose 1993, Zenorola et al. 1994). The effect of CoCl 2 in inducing chromosomal aberrations was observed in vivo on mice after single oral administration with different doses (1/10, 1/20, 1/40 LD 50 ) for 6, 12, 18 and 24 h (Palit et al. 1991). It also, induced an increase in the number of dividing cells and chromosomal aberrations in male rats after oral administration for 21 days with 15 mg kg 1 b.wt. (Sanyal et al. 1980). The mutagenic potential of cobalt and its compounds was evaluated by IARC (1991). In this evaluation cobalt (II) compounds were reported to induce DNA damage, DNA protein cross links, gene mutation, sister chromatid exchanges, and aneuploidy in vitro studies, on animal and human cells. According to many authors who studied the action of vitamins in vivo, the treatment protocols that yielded the best results in terms of reduction of chromosome damage were those which vitamin A, C or E was administered as pre-treatment or in simultaneous with clastogenic agent (Ciaccio et al. 1993, Lacava and Luna 1994, Khan and Sinha 1996). The antimutagenic and anticarcinogenic properties of selenium and vitamins A, C and E were reported by many authors (Johnson and Kligman 1992, Schrauzer 2000, Dusinska et al. 2003). In the present investigation the antimutagenic effect of selenium and vitamins (A, C, E) was tested against the mutagenicity induced by CoCl 2 in mice somatic and germ cells. * Corresponding author, Maha_Sadki@yahoo.com

2 214 Nagwa H. A. Hassan et al. Cytologia 71(3) Materials and methods Animals Mice were obtained from the animal house of the National Research Center. The experiments were carried out on male Swiss mice 9 12 weeks old, weighting gm. Food and water were provided ad libitum. Chemical agents CoCl 2 is produced by BDH laboratory supplies, poole, BH/51 TD, England. Selenium-ACE mixture (selenium 100 mg, VA IU, VC 90 mg, VE 30 mg) was purchased from Wassen International Ltd, England. Dosage and treatment The LD 50 for CoCl 2 was found to be 250 mg kg 1 b.wt. Four doses of CoCl , 31.25, and 125 mg kg 1 b.wt. (1/16, 1/8, 1/4, 1/2, LD 50 ) were tested. Another groups of mice received the last 3 doses of CoCl 2 simultaneously with 50 mg selenium-ace kg 1 b.wt. Samples were taken 24 h after single oral treatment. For repeated treatment, mice were orally treated with successive daily doses 15.62, and mg CoCl 2 kg 1 b.wt. for 1, 2 and 3 weeks. Another groups of mice were treated with the same doses of CoCl 2 plus 50 mg selenium-ace kg 1 b.wt. at the same time of treatment. In all experiments an untreated group acted as negative control and cyclophosphamid at a dose of 20 mg kg 1 b.wt. (i.p.) was used as positive control. The third control group was treated orally with 50 mg selenium ACE kg 1 b.wt. Slide preparation and scoring 1. Chromosome aberrations Chromosomes from bone-marrow cells were prepared following the method of Yosida and Amano (1965). Spermatocyte metaphases at diakinase metaphase I were prepared according to Evans et al. (1964). Slides were stained with 7% Giemsa in phosphate buffer PH 6.8. At least well spread metaphases were analyzed for each animal scoring different types of aberrations. 2. Sperm abnormalities For sperm abnormalities, smears were prepared and staining with Eosin Y using the method of Wyrobek and Bruce (1978). Three doses (15.62, and mg CoCl 2 kg 1 b.wt.) were administered for 5 days and mice were sacrificed 30 days after the last dose. For each dose, about 5000 sperms were assessed for morphological abnormalities. The significance of the experimental from control data was calculated using t-test. The resulting data between groups were compared using Fisher s least significance differences and Duncan s multiple range test (Harter 1960). Results 1. Chromosome aberrations Tables 1 and 2 present chromosomal aberrations induced in bone-marrow and spermatocyte cells respectively after a single and repeated oral treatments with different doses of CoCl 2. The results show that the tested doses of CoCl 2 induced a statistically significant increase in the percentage of chromosomal aberrations. Such percentage was found to be dose and time dependent. The results in Tables 1 and 2 also demonstrate that the percentage of aberrations in bone-mar-

3 2006 Effect of Selenium and Vitamins Against 215 Table 1. Percentage and types of aberrations induced in mouse bone-marrow cells after single and repeated oral treatment with different doses of CoCl 2 and CoCl 2 with selenium ACE (continued) No Abnormal metaphases Number of metaphases with Including gaps Excluding gaps Fragments Chromatid gap Mean % S.E Mean % S.E and/or break Deletion RT Endomitosis Polyploidy Single treatment I. Control (non-treated) * ** * ** ** ** ** IV. Selenium ACE CoCl 2 at * ** ** ** ** Repeated treatment A1 week treatment I. Control (non-treated) ** ** ** ** ** ** IV. Selenium ACE CoCl 2 at ** ** ** ** B2 weeks treatment I. Control (non-treated) ** ** ** **

4 216 Nagwa H. A. Hassan et al. Cytologia 71(3) Table 1. continued No Abnormal metaphases Number of metaphases with Including gaps Excluding gaps Fragments Chromatid gap Mean % S.E Mean % S.E and/or break Deletion RT Endomitosis Polyploidy ** ** IV. Selenium ACE CoCl 2 at ** * ** ** ** ** C3 weeks treatment I. Control (non-treated) ** ** ** ** ** ** IV. Selenium ACE CoCl 2 at ** ** ** ** ** ** Cyclophosphamide (positive control) ** ** metaphase examined in 5 mice/treatment. * significant (p 0.05), ** highly significant (p 0.01) (t-test). RT Robertsonian translocation

5 2006 Effect of Selenium and Vitamins Against 217 Table 2. Percentage and types of aberrations induced in mouse spermatocytes after single and repeated treatment with different doses of CoCl 2 and CoCl 2 with selenium ACE (continued) Abnormal metaphases Number of metaphases with No Mean % S.E X-Y Autosomal X-Y Fragment univalent univalent Autosomal and/or brea Gap in X Chain (IV) univalent Single treatment I. Control (non-treated) * ** ** ** IV. Selenium ACE CoCl 2 at ** ** ** Repeated treatment A-1 week treatment I. Control (non-treated) ** ** ** IV Selenium ACE CoCl 2 at ** ** B2 weeks treatment I. Control (non-treated) ** **

6 218 Nagwa H. A. Hassan et al. Cytologia 71(3) Table 2. continued Abnormal metaphases Number of metaphases with No Mean % S.E X-Y Autosomal X-Y Fragment univalent univalent Autosomal and/or break Gap in X Chain (IV) univalent ** III Selenium ACE IV. Selenium ACE (50) CoCl 2 at ** ** ** C3 weeks treatment I. Control (non-treated) ** ** ** IV. Selenium ACE (50) CoCl 2 at ** ** ** Cyclophosphamide (positive control) ** metaphase examined in 5 mice/treatment. * significant (p 0.05), ** highly significant (p 0.01) (t-test).

b j) types of sperm abnormalitied from mice treated with different doses of CoCl 2. b) Without hook, c, d, e) amorphous head, f) big head, g) small head, h) trianglar, i, j) coil tail.

7 2006 Effect of Selenium and Vitamins Against 219 a b a b c d e c Fig. 1. Y X d Y X CIV Metaphases from mouse treated with different doses of CoCl 2 showing a) break, b) fragment, in mouse bone-marrow cells, c) fragment, d) chain (CIV), in mouse spermatocytes Fig. 2. f g h i j a) Normal sperm with a definite head with a marked hook from a non-treated mouse. b j) types of sperm abnormalitied from mice treated with different doses of CoCl 2. b) Without hook, c, d, e) amorphous head, f) big head, g) small head, h) trianglar, i, j) coil tail row and spermatocyte was significantly reduced in all groups of mice treated with selenium-ace at 50 mg kg 1 b.wt. in the same time of administration of CoCl 2 at the tested dose levels. Different types of aberrations in bone-marrow cells (Table 1 and Fig. 1a, b) and spermatocyte cells (Table 2 and Fig. 1 c, d) were also recorded. 2. Sperm abnormalities The effect of different doses of CoCl 2 (15.62, and mg CoCl 2 kg 1 b.wt.) on the induction of sperm abnormalities in male mice are summarized in Table 3. The results show that the 3 tested doses of CoCl 2 induced an increase in the percentage of sperm abnormalities, which show positive correlation with the dose. Such percentage was found to be statistically significant after treatment with the 3 tested doses. Simultaneous treatment of 50 mg selenium-ace kg 1 b.wt. with the tested doses of CoCl 2 reduced the percentage of sperm abnormalities. Various morphological sperm abnormalities were also recorded (Table 3 and Fig. 2). Discussion The results of the present work demonstrated that a single oral treatment with different doses of CoCl 2 induced a significant percentage of aberrations in mouse bone-marrow cells and spermatocytes with a dose-related relationship. A linear increase in the percentage of chromosomal aberrations which was directly related to the concentration, and also to the period of administration of CoCl 2 on mice bone-marrow cells in vivo was reported by Palit et al. (1991). However the repeated dose treatment showed incidence of toxicity on the tested animals and the percentage of aberrations was higher than a single dose treatment. Such phenomenon was observed by Sanyal et al. (1980) who found that repeated oral treatment of CoCl 2 at a rate of 15 mg kg 1 b.wt. up to 21 days produced an increase in the number of dividing cells and chromosomal aberrations in male rats. This phenomenon was explained on the basis that the repeated dose treatment with metals may result in its accumulation in the tissues beyond the capacity to be discharged through the natural physiological mechanisms. This observation agrees with Alexandersson (1988) who reported that following acute occupational cobalt inhalation the urinary elimination is

8 220 Nagwa H. A. Hassan et al. Cytologia 71(3) Table 3. Percentage and types of sperm abnormalities induced in mouse after oral treatment with different doses of CoCl 2 and CoCl 2 with selenium ACE Abnormal sperms No Mean % S.E Amorphous Without hook Triangle I. Control (non-treated) * ** ** IV. Selenium ACE (50) CoCl 2 at * ** V. Cyclophosphamide (positive control) * No of head sperms with Banana Small Big Forked Coiled tail I. Control (non-treated) IV. Selenium ACE (50) CoCl 2 at V. Cyclophosphamide (positive control) sperms examined in 5 mice/treatment. * significant (p 0.05), ** highly significant (p 0.01) (t-test). rapid for 24 h followed by a slower excretion phase lasting several weeks. Farah (1983) observed that i.p. injection of CoCl 2 in Syrian hamsters caused aneuploidy in bone-marrow cells and testes. An increased mutagenic effect of CoCl 2 was evidenced by the induction of micronuclei in mice treated in vivo (Suzuki et al. 1993) and in cultured human lymphocytes (Capomazza and Botta 1991). The present study was undertaken further to validate the genotoxicity of CoCl 2 by studying the effect on morphological sperm abnormalities. The present results indicate that CoCl 2 induced significant sperm abnormalities after treatment with all tested doses of CoCl 2 which induced both head and tail abnormalities. The head shape abnormalities reflect changes in the DNA content (Wyrobek and Bruce 1978). Tail deformities was reported to reduce fertility in human and animals (Topham 1983). Such results are coincide with the results obtained by other authors who reported that CoCl 2 induced hypoxic testicular damage in rats, decreased sperm motility, the percentage of motile forms and epididymal sperm concentration (Mollenhauer et al. 1985, Pedigo et al. 1988). Pedigo and Vernon (1993) observed reversible infertility in male mice exposed to 400 ppm CoCl 2 for 10 weeks. Regarding the direct genotoxic mechanisms, Co (II) induces the formation of reactive oxygen

9 2006 Effect of Selenium and Vitamins Against 221 species (ROS) when combined with hydrogen peroxide in cell free system. These ROS were suggested to give different kinds of site-specific DNA damage, 8-hydroxyl-2-deoxyguanosine formation is included in the DNA damage (Lloyd et al. 1997). Cobalt ions were also shown to substitute for zinc in protein-zinc finger domins which control the transcription of several genes. This substitution is suggested to generate free radicals close to DNA, causing DNA damage (Sarkar 1995). In the present study, the drug, selenium-ace was used as chemoprotective agent. The results showed that selenium-ace was succeeded for minimizing the genotoxicity of CoCl 2 in the different tissues of the mouse but not to the control levels. The protective effect of selenium-ace against the mutagenic effect of chemical agents was also proved by other authors (Stewart et al. 1996, Abdulla and Gruber 2000, Dusinska et al. 2003). Selenium-ACE act as antioxidant which can help to protect the body from damaging effects of free radicals. Also, it has an important role in regulating the immune system (Atroshi et al. 1995, Fomenko et al. 1997, Wright et al. 2004). The same authors also reported that selenium-ace appeared to be more effective for the maintenance of health and the prevention of diseases than either of selenium and vitamins A, C, E alone. In conclusion, the present results indicate that the administration of antioxidants, seleniumvitamins ACE can be considered a promising approach toward inhibiting the genetic damage by chemical pollutants. References Abdulla, M. and Gruber, P Role of diet modification in cancer prevention. Biofactors, 12: Alexandersson, R Blood and urinary concentrations as estimators of cobalt exposure. Arch. Environ. Health. 43: Atroshi, F., Rizzo, A., Biese, I., Salonen, M., Lindberg, L. and Saloniemi, H Effets of feeding T-2 toxin and deoxynivalenol on DNA and GSH contents of brain and spleen of rats supplemented with vitamin E and C and selenium combination. J. Animal phys. Animal Nut. 74: Capomazza, C. and Botta, A Cobalt chloride induces micronuclei in human lymphocytes. Med. Sci. Res. 19: 219. Carlisle, D., Pritchard, D., Singh, J., Owens, B., Blankenship, L., Orenstein, J. and Patierno, S Apoptosis and P53 induction in human lung fibroblasts exposed to chromium VI: Effect of ascorbate and tocopherol. Toxicol. Sci. 55: Ciaccio, M., Velenza, M., Tesoriere, L., Bongiorno, A., Albiero, R. and Livera, M Vitamin A inhibits doxorubicin induced membrane lipid peroxidation in rat tissues in vivo. Arch. Biochem. Biophys. 302: Dose, Dictionary of Substances and Their Effects. Vol. 2. Royal Society of Chemistry, Cambridge. Dusinska, M, Kazimirova, A., Barancokova, M., Beno, M., Smolkova, B., Horska, A., Raslova, K., Wsolova, L. and Collins, A Nutritional supplementation with antioxidants decreases chromosomal damage in humans. Mutagen. 18: Evans, E., Breckon, G. and Ford, C An air drying method for meiotic preparation from mammalian testes. Cytogenetics 3: Farah, S. B Carcirogenicity and Genotoxicity of Lead, Bryllium and Other Metals. In: Chang, L. W. (ed.) Toxicology of Metals. CRC Press. pp Fomenko, L. A., Bezlepkina, T. A., Anoshkin, A. N. and Gaziev, A. I Antioxidant vitamin diet decreases the rate of chromosomal damage and gene mutation in irradiated mice, Biology B, Russian Acad. Sci. 24: Harter, H. L Critical values for Duncan s new multiple range test. Biometrics 16: IARC IARC Monographs on the Evaluation of Carcinogenic Risks to Human. Vol. 49. Chromium, Nickel and Welding. World Health Organization, Lyon. pp International Agency for Research on Cancer, IARC Monographs on the Evaluation of Carcinogenic Risks of Chemicals to Humans. Vol. 23, Some Metals and Metallic Compounds. IARC, Lyon, France. Johnson, K. and Kligman, E. W Preventive nutrition disease-specific dietary intervention for older adults. Geriatrics. 47: Khan, P. K. and Sinha, S. P Ameliorating effect of vitamin C on murine sperm toxicity induced by three pesticides (endosulfan, phosphamidon and mancozeb). Mutagen. 11: Lacava, Z. G. and Lura, H The anticlastogenic effect of tocopherol in peritoneal macrophages of benznidazole-treated and ovariectomized mice. Mut. Res. 305:

10 222 Nagwa H. A. Hassan et al. Cytologia 71(3) Lloyed, D. R., Phillips, D. H. and Carmichael, P. L Generation of putative in tras trand cross-links and strand breaks in DNA by transition metal ion-mediated oxygen radicel attack. Chem. Res. Toxicol. 10: Mollenhauer, H. H., Corrier, D. E., Clark, D. E., Hare, M. F. and Elissalde, M. H Effects of dietary cobalt on testicular structure. Virchows Archiv B Cell Pathol. 49: Palit, S., Sharma, A. and Talukder, G Chromosomal aberrations induced by cobaltous chloride in mice in vivo. Biol. Trace Elem. Res. 29: Pedigo, N. G., George, W. J. and Anderson, M. B Effects of acute and chronic exposure to cobalt on male reproduction in mice. Reprod. Toxicol. 2: and Vernon, M. W Embryonic losses after 10-week administration of cobalt to male mice. Reprod. Toxicol. 7: Sanyal, R., Giri, A. K., Talukder, G. and Sharma, A Effects of cobalt and nickel on mammalian cellular systems. Biol. Bull. Indian. 2: Sarkar, B Metal replacement in DNA-binding zinc finger proteins and its relevance to mutagenicity and carcinogenicity through free radical generation. Nut. 11: Schrauzer, G. N Anticarcinogenic effects of selenium. Cell Mol. Life Sci. 57: Stewart, M. S., Cameron, G. S. and Pence, B. C Antioxidant nutrients protect against UVB-induced oxidative damage to DNA mouse keratinocytes in culture. J. Inv. Dermat. 106: Suzuki, Y., Shimizu, H., Nagae, H., Fukumoto, M., Okonogi, H. and Kadokura, M Micronucleus test and erythropoiesis-effect of cobalt on the induction of micronuclei by mutagens. Environ. Mol. Mutagen. 22: 101. Topham, J. C Chemically induced changes in sperm in animals and humans. Chem. Mutagen. 8: Wright, M. E., Mayne, S. T., Stolzenberg-Solomon, R. Z., Li, Z. H., Pietines, P., Taylor, P. R., Virtamo, J. and Albanes, D Development of a comprehensive dietary antioxidant index and application to lung cancer risk in a cohort of male smokers. Amer. J. of Epidemol. 160: Wyrobek, A. J. and Bruce, W. R The Induction of Sperm-Shape Abnormalities in Mice and Humans. In: Hallaender, A. and De Serres, F. J. (eds.) Chemical Mutagens: Principles and Methods for their Detection. Vol. 5. Plenum, New York. pp Yosida, T. H. and Amano, K Autosomal polymorphism in laboratory bred and wild Norway rats, Rattus norvegicus. Misima Chromosoma 16: Zenorola, P., Bisceglia, M. and Lomuto, M Ashy dermatosis associated with cobalt allergy. Contact Dermatitis 31: 53.

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