Abstract
Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium that are found in cereals and agricultural products. ZEN has been implicated in mycotoxicosis in farm animals and in humans. The toxic effects of ZEN are well known, but the ability of an alkaline Comet assay to assess ZEN-induced oxidative DNA damage in Chang liver cells has not been established. The first aim of this study was to evaluate the Comet assay for the determination of cytotoxicity and extent of DNA damage induced by ZEN toxin, and the second aim was to investigate the ability of N-acetylcysteine amide (NACA) to protect cells from ZEN-induced toxicity. In the Comet assay, DNA damage was assessed by quantifying the tail extent moment (TEM; arbitrary unit) and tail length (TL; arbitrary unit), which are used as indicators of DNA strand breaks in SCGE. The cytotoxic effects of ZEN in Chang liver cells were mediated by inhibition of cell proliferation and induction of oxidative DNA damage. Increasing the concentration of ZEN increased the extent of DNA damage. The extent of DNA migration, and percentage of cells with tails were significantly increased in a concentration-dependent manner following treatment with ZEN toxin (p < 0.05). Treatment with a low concentration of ZEN toxin (25 μM) induced a relatively low level of DNA damage, compared to treatment of cells with a high concentration of ZEN toxin (250 μM). Oxidative DNA damage appeared to be a key determinant of ZEN-induced toxicity in Chang liver cells. Significant reductions in cytolethality and oxidative DNA damage were observed when cells were pre-treated with NACA prior to exposure to any concentration of ZEN. Our data suggest that ZEN induces DNA damage in Chang liver cells, and that the antioxidant activity of NACA may contribute to the reduction of ZEN-induced DNA damage and cytotoxicity via elimination of oxidative stress.
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References
Wood, G.E. (1992) Mycotoxins in foods and feeds in the United States. J. Anim. Sci., 70, 3941–3949.
Muller, H.M., Reimann, J., Schumacher, U. and Schwadorf, K. (1998) Natural occurrence of Fusarium toxins in oats harvested during five years in an area of southwest Germany. Food Addit. Contam., 15, 801–806.
Scudamore, K.A. and Patel, S. (2000) Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom. Food Addit. Contam., 17, 407–416.
Felix D’Mello, J.P., Duffus, C.M. and Duffus, J.H. (1991) Toxic substances in crop plants. The Royal Society of Chemistry, Cambridge, pp. 226–257.
Kiessling, K.H. and Pettersson, H. (1978) Metabolism of zearalenone in rat liver. Acta Pharmacol. Toxicol., 43, 285–290.
Marroufi, K., Chekir, L., Creppy, E.E., Ellouz, F. and Bacha, H. (1996) Zearalenone induces modifications of haematological and biochemical parameters in rats. Toxicon, 34, 535–540.
Obremski, K., Zielonka, L., Zaluska, G., Zwierzchowski, W., Pirus, K. and Gajecki, M. (1999) The influence low doses of zearalenone on liver enzyme activities in gilts. In: proceeding of the X conference “Microscopy Fungi-plant pathogens and their metabolites” pp. 66.
Conková, E., Laciaková, A., Pástorová, B., Seidel, H. and Kovác, G. (2001) The effect of zearalenone on some enzymatic parameters in rabbits. Toxicol. Lett., 121, 145–149.
Abid-Essefi, S., Quanes, Z., Hassen, W., Baudrimont, I., Creppy, E. and Bacha, H. (2004) Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in culture cells exposed to zeararenolne. Toxicol. In Vitro, 18, 467–474.
Hassen, W., El Golli, E., Baudrimont, I., Mobio, A.T., Ladjimi, M.M., Creppy, E.E. and Bacha, H. (2005) Cytotoxicity and Hsp70 induction in HepG2 cells in response to zearalenone and cytoprotection by sub-lethal heat shock. Toxicol., 207, 293–301.
Ouanes, Z., Abid, S., Ayed, I., Anane, R., Mobio, T., Creppy, E.E. and Bacha, H. (2003) Induction of micronuclei by zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of vitamin E. Mutat. Res., 538, 63–70.
Ouanes, Z., Ayed-Boussema, I., Baati, T., Creppy, E.E. and Bacha, H. (2005) Zearalenone induces chromosome aberrations in bone marrow: preventive effect of 17β estradiol, progesterone and vitamin E. Mutat. Res., 565, 139–149.
Abbès, S., Quanes, Z., ben Salah-Abbès, J., Houau, Z., Queslati, R., Bacha, H. and Othman, O. (2006a) The protective effect of hydrated sodium calcium aluminosilicate against hematological, biochemical and pathological changes induced by zearalenone in mice. Toxicon, 47, 567–574.
Abbès, S., Salah-Abbès, J.B., Quanes, Z., Houau, Z., Othman, O., Bacha, H., Addel-Wahhab, M.A. and Queslati, R. (2006b) Preventive role of phyllosilicate clay on the immunological and biochemical toxicity of zearenolone in Balb C/ mice. Int. ImmunoPharmacol., 6, 1251–1258.
Abbès, S., Quanes, Z., Salah-Abbes, J.B., Addel-Wahhab, M.A., Queslati, R. and Bacha, H. (2007) Preventive role of aluminoslicate clay against induction of micronuclei and chromosome aberrations in bone marrow cells of Balb/c mice treated with zearalenone. Mutat. Res., 631, 85–92.
Ayed-Boussema, I., Ouanes, Z., Bacha, H. and Abid, A. (2007) Toxicities induced in cultured cell exposed to zearaleone: apoptosis or mutagenesis? J. Biochem. Mol. Toxicol., 21, 136–144.
Tice, R.R., Agurell, E., Anderson, D., Burlison, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C. and Sasaki, Y.F. (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen., 35, 206–221.
Singh, N.P., McCoy, M.T., Tice, R.R. and Schneider, E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184–191.
Collins, A., Dusinská, M., Franklin, M., Somorovaská, M., Petrovská, S., Duthi, L., Fillion, L., Panayiotidis, M., Raslová, K. and Vaughan, N. (1997) Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ. Mol. Mutagen., 30, 139–146.
Dixon, D.R., Pruski, A.M., Dixon, L.R. and Jha, A.N. (2002) Marine invertebrate eco-genotoxicology: a methological overview. Mutagenesis, 17, 495–507.
Lee, R.F. and Steinert, S. (2003) Use of the single cell gel electrophoresis/Comet assay for detecting DNA damage in aqatic (marine and freshwater) animals. Mutat. Res., 544, 43–64.
Gedik, C.M., Ewen, S.W. and Collins, A.R. (1992) Single cell gel electrophoresis applied to the analysis of UV-C damage and it repair in human cells. Int. J. Radiat. Biol., 62, 313–320.
Jojte, J., Zmyœlony, M., Palus, J., Dziubaltowska, E. and Rajkowska, E. (2001) Protective effect of melatonin against in vitro iron ions and 7 mT 50 Hz magnetic field-induced DNA damage in rat lymphocytes. Mutat. Res., 483, 57–64.
Mohankumar, M.N., Janani, S., Prabhu, B.K., Kumar, P.R. and Jeevanram, R.K. (2002) DNA damage and intergrity of UV-induced DNA repair in lymphocytes of smokers analyzed by the comet assay. Mutat. Res., 520, 179–187.
Lupetti, A., Paulusma-Annema, A., Senesi, S., Campa, M., Van Dissel, J.T. and Nibbering, P.H. (2002) Reactive oxygen species and internal thiols in the candidacidal activity of a N-terminal peptide of human lactoferrin. Antimicrob. Agents Chemother., 46, 1634–1639.
Cotgreave, I.A. (1997) N-acetylcysteine: Pharmacological considerations and experimental and clinical applications. Adv. Pharmacother., 38, 205–227.
Offen, D., Gilgun-Sherki, Y., Barhum, Y., Benhar, M., Grinberg, L., Reich, R., Mealmed, E. and Atlas, D. (2004) A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis. J. Neurochem., 89, 1241–1251.
Grinberg, L., Fibach, E., Amer, J. Atlas, D. (2005) N-acetyl-cysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress. Free Radical Biol. Med., 38, 136–145.
Shi, R., Huang, C.C., Aronstam, R.S., Ercal, N., Martin, A. and Huang, Y.W. (2009) N-acetylcysrein amide decrease oxi-dative stress but not cell death induced by doxorubicin in H9c2 cardiomyocytes. BMC Pharmacol., 9, 7.
Carmichael, J., de Graff, W.G., Gadza, A.F., Minna, J.D. and Mitchell, J.B. (1987) Evaluation of a tetrazolium-based semiautomatic colorimetric assay: assessment of chemosensitivity testing. Cancer Res., 47, 936–942.
Bottalico, A. (1998) Fusarium diseases of cereal: Species complex and related mycotoxin profiles in Europe. J. Plant Pathol., 80, 85–103.
Placinta, C.M., D’Mello, J.P.F. and Macdonald, A.M.C. (1999) A review of world contamination of cerael grains and animal feed with Fusarium mycotoxins. Anim. Feed Sci. Technol., 78, 21–37.
Creppy, E.E. (2002) Update of survey, reguration and toxic effects of mycotoxina in Europe. Toxicol. Lett., 127, 19–28.
Sáenz de Rodríguez, C.A., Bougovanni, A.M. and Conde de Borrego, L. (1985) An epidemic of precocious development in Puerto Rican children. J. Pediatr., 107, 393–396.
Tomaszewski, J., Miturski, R., Semezuk, A., Koarski, J. and Jakowicki, J. (1998) [Tissue zearalenone concentration in normal hyperplasic and neoplastic human endometrium]. Ginekol. Pol., 69, 363–366.
Chang, R.S. (1954) Continous subcultivation of epithelial-like cells from normal human tissue. Proc. Soc. Exp. Biol. Med., 87, 440–443.
Jukes, T.H. (1976) Letter: Evolutionary changes in insulin. Nat., 259, 250.
Abid-Essefi, S., Baudrimont, I., Hassen, W., Quanes, Z., Mobio, T.A., Anane, R., Creppy, E.E. and Bacha, H. (2003) DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by vitamin E. Toxicol., 192, 237–248.
El Golli, E., Hassen, E., Bouslimi, W., Bouaziz, A., Ladjimi, M.M. and Bacha, H. (2006) Induction of Hsp 70 in Vero cells in response to mycotoxins cytoprotection by sub-lethal heat shock and by vitamin E. Toxicol. Lett., 166, 122–130.
Hassen, W., Ayed-Boussema, I., Oscoz, A.A., Lopez Ade, C. and Bacha, H. (2007) The role of oxidative stress in zerale-none-meditated toxicity in Hep G2 cells: Oxidative DNA damage, gluthatione depletion and stress proteins induction. Toxicol., 232, 294–302.
Lioi, M.B., Santoro, A., Barbieri, R., Salzano, S. and Ursini, M.V. (2004) Ochratoxin A and zeralenone: a comparative study on genotoxic effects and cell death induced in bovine lymphocytes. Mutat. Res., 557, 19–27.
Zourgui, L., Golli, E.E., Bouaziz, C., Bacha, H. and Hassen, W. (2008) Cactus (opuntia ficus-indica) caladodes prevent oxidative damage induced by the mycotoxin zearalenone in Balb/C mice. Food Chem. Toxicol., 46, 1817–1824.
Zourgui, L., Ayed-Boussema, I., Ayed, Y., Bacha, H. and Hassen, W. (2009) The antigenotoxic activities of Cactus (opuntia ficus-indica) caladodes against the mycotoxin zearalenone in Balb/C mice.: Preventive of micronuclei, chromosome aberrations and DNA fragmentation. Food Chem. Toxicol., 47, 662–667.
Phillips, T.D. (1999) Dietary clay in the chemoprevention of aflatoxin induced disease. Toxicol. Sci., 52, 118–126.
Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B. and Gustafsson, J.A. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinol., 139, 4252–4263.
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Kang, C., Lee, H., Yoo, YS. et al. Evaluation of Oxidative DNA Damage Using an Alkaline Single Cell Gel Electrophoresis (SCGE) Comet Assay, and the Protective Effects of N-Acetylcysteine Amide on Zearalenone-induced Cytotoxicity in Chang Liver Cells. Toxicol Res. 29, 43–52 (2013). https://doi.org/10.5487/TR.2013.29.1.043
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DOI: https://doi.org/10.5487/TR.2013.29.1.043