In vitro effects of very low levels of aflatoxin B1 on free radicals production and bactericidal activity of bovine blood neutrophils

As one of the most potent and hazardous feed/food-originated mycotoxins, aflatoxin (AF) B1 is regarded as a potent immunosuppressor in dairy cows. Neutrophils (PMN), as key effector cells against pathogens, have a high potential to kill engulfed microbes. To investigate the in vitro effects of very low doses of AFB1 on blood PMN functions, we examined the effects of biologically relevant concentrations of AFB1 on the phagocytosis and non-phagocytosis dependent luminol, representative of mainly intracellular free radicals, and isoluminol, representative of mainly extracellular free radicals, chemiluminescence (CL), necrosis and apoptosis of PMN. Isolated blood PMN from healthy dairy cows (n = 12) were exposed to 0, 0.01, 0.05 and 0.5 ng/ml of AFB1 for 0.5 and 18 h depending on the assay. Further, blood PMN of healthy dairy cows (n = 8) were exposed to 0.5 ng/ml of AFB1 for 3 h and myeloperoxidase (MPO) activity, superoxide anion (O2−) production, phagocytosis and killing activities against Staphylococcus (S.) aureus and Escherichia (E.) coli, were examined. Though the effect of extremely low doses of AFB1 were less pronounced, at 0.5 ng/ml the production of free radicals was greatly enhanced, especially extracellularly. In contrast to isoluminol CL, the AFB1-treated PMN showed a remarkably impaired phagocytosis-depended luminol CL. PMN necrosis and apoptosis were not affected by AFB1. MPO activity, O2− production, phagocytosis rates and killing of E. coli and S. aureus by AFB1-treated PMN were significantly lower than those of non-treated ones. Our results show the extracellularly pro-oxidant and antiphagocytic properties of very low doses of AFB1 for bovine PMN. The scope of the suppressive effects of the in vitro AFB1 levels on cellular innate immune functions should be considered for high yielding dairy cows.