Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells.
OBJECTIVE
To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers.


METHODS
Polymorphonuclear neutrophils from 3 lactating cows.


METHODS
Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots.


RESULTS
Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner.


CONCLUSIONS
Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.
Ledbetter
T K
TK
Department of Animal and Avian Sciences, University of Maryland, College Park 20742, USA.
Paape
M J
MJ
Douglass
L W
LW
D000814:Aniline Compounds / Q000633:toxicity; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015153:Blotting, Western / Q000662:veterinary; D002417:Cattle / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D005260:Female; D016166:Free Radical Scavengers / Q000633:toxicity; D006639:Histidine / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008070:Lipopolysaccharides / Q000633:toxicity; D008321:Mammary Glands, Animal / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008981:Molsidomine / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000633:toxicity; D018375:Neutrophil Activation / Q000276:immunology; D009504:Neutrophils / Q000276:immunology*; D009566:Nitrates / Q000493:pharmacokinetics / Q000633:toxicity*; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D016877:Oxidants / Q000493:pharmacokinetics / Q000633:toxicity*; D009195:Peroxidase / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D019323:omega-N-Methylarginine / Q000633:toxicity
D000814:Aniline Compounds; D000975:Antioxidants; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D009566:Nitrates; D016877:Oxidants; C039642:peroxynitric acid; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D006639:Histidine; C034343:4-aminobenzhydrazide; C002385:linsidomine; D008981:Molsidomine; D007770:L-Lactate Dehydrogenase; D009195:Peroxidase; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D013755:Tetradecanoylphorbol Acetate