Naturally Occurring Level of Aflatoxin B1 Injures Human, Canine and Bovine Leukocytes Through ATP Depletion and Caspase Activation.

Aflatoxin (AF) B₁ is a potent hepatotoxic, mutagenic, teratogenic mycotoxin and may cause immune suppression/dysregulation in humans and animals. Toxic effects of AFB₁ on key mammalian immune cells (ie, leukocytes) needs to be mechanistically elucidated. In this study, along with the determination of AFB₁'s LC₅₀ for certain leukocytes, we analyzed the effect of naturally occurring levels of AFB₁ 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 AFB₁ (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 AFB₁-treated neutrophils, lymphocytes, and monocytes in all studied mammals. Also, with such a low level (10 ng/mL) of AFB₁, 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 AFB₁-treated leukocytes showed slightly/insignificantly more increase of necrotic (PI⁺) neutrophils, lymphocytes, and monocytes in human, dogs, and cattle. Even though in vitro LC₅₀s for AFB_1' (∼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 AFB₁. The observed in vitro ATP depletion and caspase activation in AFB₁-exposed leukocytes can partially explain the underlying mechanisms of AFB₁-induced immune disorders in mammals. [ABSTRACT FROM AUTHOR]