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Antioxidants to Prevent Bovine Neutrophil-Induced Mammary Epithelial Cell Damage^*

¹ Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada J1M 1Z3
² Department of Animal Science, McGill University, Ste-Anne de Bellevue, Québec, Canada H9X 3V9
³ Department of Nuclear Medicine and Radiobiology, Faculty of Medicine, Universitéde Sherbrooke, Québec, Canada, J1H 5N4

Corresponding author: Pierre Lacasse; e-mail: lacassep{at}agr.gc.ca.

Activated neutrophils are able to produce a large quantity of bactericidal molecules such as reactive oxygen species that have been associated with tissue damage in several inflammation models. The protective effects of antioxidants in a context of neutrophil-induced damage to mammary epithelial cells were first evaluated in vitro using a coculture model of activated bovine neutrophils and a bovine mammary epithelial cell line (MAC-T cells). Cell damage was determined by quantifying the release of lactate dehydrogenase by MAC-T cells in culture medium. Morphological observation of cells stained with acridine orange was used to visualize the extent of cell damage. When incubated with neutrophils activated by lipopolysaccharides and phorbol 12-myristate 13-acetate, MAC-T cells released large amounts of lactate dehydrogenase indicating significant cell damage. The addition of dimethylthiourea or bathocuproine disulfonic acid did not reduce the damage whereas catechin, deferoxamine or glutathione ethyl ester significantly reduced neutrophil-induced cytotoxicity in a dose-dependent manner. The effect of deferoxamine, an iron chelator, on the growth of Escherichia coli and the ability of bovine neutrophils to phagocytose these bacteria were then assessed in vitro. Our data showed that deferoxamine did not interfere with the phagocytic activity of neutrophils but inhibited growth of the bacteria. Overall, our results suggest that antioxidants may be effective tools for protecting mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

Key Words: mastitis • inflammation • antioxidant • reactive oxygen species

Abbreviation key: BDA = bathocuproine disulfonic acid, CAT = catechin, DFO = deferoxamine, DMEM = Dulbecco’s modified Eagle medium, DMTU = dimethylthiourea, FBS = fetal bovine serum, GEE = glutathione ethyl ester, GSH = glutathione, LDH = lactate dehydrogenase, PMA = phorbol 12-myristate 13-acetate, PMN = polymorphonuclear neutrophils, ROS = reactive oxygen species

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