Naturally Occurring Level of Aflatoxin B 1 Injures Human, Canine and Bovine Leukocytes Through ATP Depletion and Caspase Activation

Aflatoxin (AF) B is a potent hepatotoxic, mutagenic, teratogenic mycotoxin and may cause immune suppression/dysregulation in humans and animals. Toxic effects of AFB on key mammalian immune cells (ie, leukocytes) needs to be mechanistically elucidated. In this study, along with the determination of AFB 's LC for certain leukocytes, we analyzed the effect of naturally occurring levels of AFB on apoptosis/necrosis of neutrophils, lymphocytes, and monocytes from healthy young humans (20- to 25-year-old male), dogs (1- to 2-year-old Persian/herd breed), and cattle (1- to 2-year-old cattle). Leukocytes were incubated for approximately 24 hours with naturally occurring levels of AFB (10 ng/mL). Intracellular adenosine triphosphate (ATP) depletion and caspase-3/7 activity were then determined by luciferase-dependent bioluminescence (BL). Furthermore, the necrotic leukocytes were measured using propidium iodide (PI)-related flow cytometry. A significant decrease (24%-45%, 33.2% ± 2.7%) in intracellular ATP content was observed in AFB -treated neutrophils, lymphocytes, and monocytes in all studied mammals. Also, with such a low level (10 ng/mL) of AFB , BL-based caspase-3/7 activity (BL intensity) in all 3 tested mammalian leukocyte lineages was noticeably increased (∼>2-fold). Flow cytometry-based PI staining (for viability assay) of the AFB -treated leukocytes showed slightly/insignificantly more increase of necrotic (PI ) neutrophils, lymphocytes, and monocytes in human, dogs, and cattle. Even though in vitro LC s for AFB (∼20,000-40,000 ng/mL) were approximately 2,000 to 4,000 times higher than background, these studies demonstrate leukocytes from human and farm/companion animals are sensitive to naturally occurring levels of AFB . The observed in vitro ATP depletion and caspase activation in AFB -exposed leukocytes can partially explain the underlying mechanisms of AFB -induced immune disorders in mammals.