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Protective Effect of Melatonin and Catalase in Bovine Neutrophil-induced Model of Mammary Cell Damage1

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      Abstract

      The effect of several antioxidants and a proteinase inhibitor on bovine neutrophil-induced mammary epithelial cell damage was investigated using an in vitro model of co-culturing bovine neutrophils and MAC-T cells, a mammary epithelial cell line. Epithelial cell damages were evaluated by measuring lactate dehydrogenase activity in culture media and by morphological observations of cells after acridine orange staining. Activation of neutrophils with Escherichia coli lipopolysaccharide and phorbol 12-myristate 13-acetate caused superoxide and gelatinase release in media. Activated neutrophils damaged the epithelial cells, as demonstrated by an increase in lactate dehydrogenase release and the observation of morphological changes. The addition of melatonin or catalase reduced neutrophil-induced cytotoxicity in a dose-dependent manner, whereas superoxide dismutase and aprotinin had no effect on cytotoxicity. Melatonin has been reported to scavenge hydroxyl radical and peroxynitrite, whereas catalase and superoxide dismutase scavenge hydrogen peroxide and superoxide, respectively. Our results suggest that hydroxyl radicals released by activated bovine neutrophils cause damage to mammary epithelial cells and that antioxidants may be useful to protect the mammary tissue during bovine mastitis.

      Key words

      Abbreviation key:

      DMEM (Dulbecco's Modified Eagle Medium), FBS (fetal bovine serum), LDH (lactate dehydrogenase), LPS (lipopolysaccharide), OD (optical density), PMA (phorbol 12-myristate 13-acetate), ROS (reactive oxygen species), SOD (superoxide dismutase)

      References

        • Babior B.M.
        • Knipes R.S.
        • Curnutte J.T.
        The production by leukocytes of superoxide, a potential bactericidal agent.
        J. Clin. Invest. 1973; 52: 714-744
        • Beckman J.S.
        • Beckman T.W.
        • Chen J.
        • Marshall P.A.
        • Freeman B.A.
        Apparent hydroxyl radical production by peroxynitrite: Implication for endothelial injury from nitric oxide and superoxide.
        Proc. Natl. Acad. Sci. USA. 1990; 87: 1620-1624
        • Borjigin J.
        • Li X.
        • Snyder S.H.
        The pineal gland and melatonin: molecular and pharmacologic regulation.
        Annu. Rev. Pharmacol. Toxicol. 1999; 39: 53-65
        • Borm P.J.A.
        • Knaapen A.M.
        • Schins R.P.F.
        • Godschalk R.W.L.
        • Van Schooten F.J.
        Neutrophils amplify the formation of DNA adducts by benzo[a]pyrene in lung target cells.
        Environ. Health Persp. 1997; 105: 1089-1093
        • Bouchard L.
        • Blais S.
        • Desrosiers C.
        • Zhao X.
        • Lacasse P.
        Nitric oxide production during endotoxin-induced mastitis in the cow.
        J. Dairy Sci. 1999; 82: 2574-2581
        • Boulanger V.
        • Bouchard L.
        • Zhao X.
        • Lacasse P.
        Induction of nitric oxide production by bovine mammary epithelial cells and blood leukocytes.
        J. Dairy Sci. 2001; 84: 1430-1437
        • Capuco A.V.
        • Paape M.J.
        • Nickerson S.C.
        In vitro study of polymorphonuclear leukocyte damage to mammary tissue of lactating cows.
        Am. J. Vet. Res. 1986; 47: 663-668
        • Carlson G.P.
        • Kaneko J.J.
        Isolation of leukocytes from bovine peripheral blood.
        Proc. Soc. Exp. Biol. Med. 1973; 142: 853-856
        • Cuzzocrea S.
        • Zingarelli B.
        • Gilad E.
        • Hake P.
        • Salzman A.L.
        • Szabò C.
        Protective effect of melatonin in carrageenan-induced models of local inflammation: relationship to its inhibitory effect on nitric oxide production and its peroxynitrite scavenging activity.
        J. Pineal Res. 1997; 23: 106-116
        • Cuzzocrea S.
        • Zingarelli B.
        • Hake P.
        • Salzman A.L.
        • Szabò C.
        Antiinflammatory effects of mercaptoethylguanidine, a combined inhibitor of nitric oxide synthase and peroxynitrite scavenger, in carrageenan-induced models of inflammation.
        Free Rad. Biol. Med. 1998; 24: 450-459
        • Cuzzocrea S.
        • Caputi A.P.
        Protective effect of melatonin on zymosan-induced cellular damage.
        Biol. Signals Recept. 1999; 8: 136-142
        • Desrosiers C.
        • Bouchard L.
        • Lacasse P.
        Milk alteration and tissue damages in mastitis: is plasmin the only protease involved?.
        in: Proceedings of the International Dairy Federation Symposiumon Immunology of RuminantMammary Gland, Stressa, Italy2000: 231-239
        • Dore M.
        • Slauson D.O.
        • Neilsen N.R.
        Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists.
        Am. J. Vet. Res. 1991; 52: 375-380
        • Gilad E.
        • Cuzzocrea S.
        • Zingarelli B.
        • Salzman A.L.
        • Szabò C.
        Melatonin is a scavenger of peroxynitrite.
        Life Sci. 1997; 60: 169-174
        • Ginsburg I.
        Could synergistic interactions among reactive oxygen species, proteinases, membrane-perforating enzymes, hydrolases, microbial hemolysins and cytokines be the main cause of tissue damage in infectious and inflammatory conditions?.
        Med. Hypotheses. 1998; 51: 337-346
        • Guigui B.
        • Rosenbaum J.
        • Preaux A.-M.
        • Martin N.
        • Zafrani E.S.
        • Dhumeaux D.
        • Mevier P.
        Toxicity of phorbol myristate acetate-stimulated polymorphonuclear neutrophils against rat hepathocytes. Demonstration and mechanisms. Lab.
        Invest. 1988; 59: 831-837
        • Haanen C.
        • Vermes I.
        Apoptosis and inflammation.
        Mediators Inflamm. 1995; 4: 5-15
        • Hachiya O.
        • Takeda Y.
        • Miyata H.
        • Watanabe H.
        • Yamashita T.
        • Sendo F.
        Inhibition by bacterial lypopolysaccharide of spontaneous and TNF-alpha-induced human neutrophil apoptosis in vitro.
        Microbiol. Immunol. 1995; 39: 715-723
        • Huynh H.T.
        • Robitaille G.
        • Turner J.D.
        Establishment of bovine mammary epithelial cells (MAC-T): An In vitro model for bovine lactation.
        Exp. Cell. Res. 1991; 197: 191-199
        • Jaeschke H.
        • Smith C.W.
        Mechanisms of neutrophil-induced parenchymal cell injury.
        J. Leuk. Biol. 1997; 61: 647-653
        • Kausalya S.
        • Nath J.
        Interactive role of nitric oxide and superoxide anion in neutrophil-mediated endothelial cell injury.
        J. Leuk. Biol. 1998; 64: 185-191
        • Knaapen A.M.
        • Seiler F.
        • Schilderman P.A.E.L.
        • Nehls P.
        • Bruch J.
        • Schins R.P.F.
        • Borm P.J.A.
        Neutrophils cause oxidative DNA damage in alveolar epihelial cells.
        Free Rad. Biol. Med. 1999; 27: 234-240
        • Lacasse P.
        • Lucy-Hulbert J.
        • Blais S.
        Somatic cell production of the free radical nitric oxide during mastitis.
        Livest. Prod. Sci. 1997; 50 (Abstr.): 168
        • Ledbetter T.K.
        • Paape M.J.
        • Douglass L.W.
        Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells.
        Am. J. Vet. Res. 2001; 62: 286-293
        • Li X.
        • Zhao X.
        • Ma S.
        Secretion of 92 kDa gelatinase (MMP-9) by bovine neutrophils.
        Vet. Immunol. Immunopathol. 1999; 67: 247-258
        • Marshall K.A.
        • Reiter R.J.
        • Poeggeler B.
        • Aruoma O.I.
        • Halliwell B.
        Evaluation of the antioxidant activity of melatonin in vitro.
        Free Rad. Biol. Med. 1996; 21: 307-315
        • Okamoto T.
        • Akaike T.
        • Nagano T.
        • Miyajima S.
        • Suga M.
        • Ando M.
        • Ichimori K.
        • Maeda H.
        Activation of human neutrophil procollagenase by nitrogen dioxide and peroxynitrite: A novel mechanism for procollagenase activation involving nitric oxide.
        Arch. Biochem. Biophys. 1997; 342: 261-274
        • Oliver S.P.
        • Calvinho L.F.
        Influence of inflammation on mammary gland metabolism and milk composition.
        J. Anim. Sci. 1995; 73: 18-33
        • Owen C.A.
        • Campbell E.J.
        The cell biology of leukocyte-mediated proteolysis.
        J. Leuk. Biol. 1999; 65: 137-150
        • Paape M.J.
        • Shafer-Weaver K.
        • Capuco A.V.
        • Van Oostveldt K.
        • Burvenich C.
        Immune surveillance of mammary tissue by phagocytic cells.
        Adv. Exp. Med. Biol. 2000; 480: 259-277
        • Poch B.
        • Gansauge F.
        • Rau B.
        • Wittel U.
        • Gansauge S.
        • Nüssler A.K.
        • Schoenberg M.
        • Beger H.G.
        The role of polymorphonuclear leukocytes and oxygen-derived free radicals in experimental acute pancreatitis: mediators of local destruction and activators of inflammation.
        FEBS Lett. 1999; 461: 268-272
        • Rajala-Schultz P.J.
        • Gröhn Y.T.
        • McCulloch C.E.
        • Guard C.L.
        Effects of clinical mastitis on milk yield in dairy cows.
        J. Dairy Sci. 1999; 82: 1213-1220
        • Reiter R.J.
        • Melchiorri D.
        • Sewerynek E.
        • Poeggeler B.
        • Barlow-Walden L.
        • Chuang J.
        • Ortiz G.G.
        • Acuna-Castro-Vieio D.
        A reviewof the evidence supporting melatonin's role as an antioxidant.
        J. Pineal Res. 1995; 18: 1-11
      1. SAS User's Guide: Statistics, Version 5 Edition. 1985. SAS Inst. Inc., Cary, NC.

        • Smith J.A.
        Neutrophils, host defense, and inflammation: a double-edged sword.
        J. Leuk. Biol. 1994; 56: 672-686
        • Sweeney J.F.
        • Nguyen P.K.
        • Omann G.M.
        • Hinshaw D.B.
        Lipopolysaccharide protects polymorphonuclear leukocytes from apoptosis via tyrosine phosphorylation-dependent signal transduction pathways.
        J. Surg. Res. 1998; 74: 64-70
        • Weiss S.J.
        Tissue destruction by neutrophils.
        N. Engl. J. Med. 1989; 320: 365-376