title: Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro.
abstract: Despite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA laddering were used as apoptosis markers. Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P<0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P<0.001). Activation with PMA induced a loss of viable neutrophils after 3h, which was greater with AOPP-BSA incubation (P<0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred. The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability.
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Agripolis, 35020 Legnaro (PD), Italy.
authors.forename: Milena
authors.initials: M
authors.lastname: Bordignon
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Agripolis, 35020 Legnaro (PD), Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Da Dalt
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Agripolis, 35020 Legnaro (PD), Italy.
authors.forename: Lieta
authors.initials: L
authors.lastname: Marinelli
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padova, Agripolis, 35020 Legnaro (PD), Italy. Electronic address: gianfranco.gabai@unipd.it.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gabai
mesh_terms: D062107:Advanced Oxidation Protein Products / Q000378:metabolism*; D000818:Animals; D019540:Area Under Curve; D002417:Cattle; D003470:Culture Media; D005609:Free Radicals / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D012710:Serum Albumin, Bovine; D013755:Tetradecanoylphorbol Acetate
chemical_list: D062107:Advanced Oxidation Protein Products; D003470:Culture Media; D005609:Free Radicals; D012710:Serum Albumin, Bovine; D013755:Tetradecanoylphorbol Acetate
keywords: Advanced oxidation protein products; Bovine; Neutrophil; Oxidative stress; Protein oxidation