Bioluminescence-based detection of astrocytes apoptosis and ATP depletion induced by biologically relevant level aflatoxin B1

Abstract

Although brain accumulation of aflatoxin B1 (AFB1) suggests potential impact on brain cells, including astrocytes, there still exists a scarcity of research on this issue within the literature. This research investigates the apoptosis effect of AFB1 on primary mouse astrocytes. To this aim, a MTT colorimetric assay on astrocytes was performed to measure the toxicity/LC50 of various concentrations (0-320,000 nM) of AFB1 for 24 h. Further, the astrocytes were exposed to concentrations of 8, 16 and 32 nM of AFB1 for 24, 48 and 72 h. Concentration of intracellular ATP) and caspase-3/7 activity was then determined by luciferase-dependent bioluminescence. Furthermore, the percentage of apoptotic cells was obtained using flow cytometry (annexin V+/propidium iodide (PI)−; cytochromec release from mitochondria, a hallmark of cell damage, was carried out by Western blot as well. MTT assay at post-exposure hours (PEH) 24 revealed that the LC50 of AFB1 was ~80,000 nM. Though at PEH 48 only 32 nM of AFB1 resulted in a significant diminished intracellular ATP content, at PEH 72 both 8 and 32 nM of AFB1 led to a significant ATP depletion in astrocytes. Similar patterns of changes were observed in bioluminescence intensity of AFB1-treated astrocytes. Flow cytometry-based annexin V and PI staining of astrocytes at PEH 24, 48 and 72 showed that 32 nM of AFB1 significantly and time dependently increased the percentage of apoptotic astrocytes (annexin V+/PI−). With 32 nM of AFB1, caspase-3/7 activity in astrocytes was increased ~4-fold at PEH 72. A remarkable release of cytochromec was only detected in astrocytes exposed to 32 nM AFB1 for PEH 72. The results indicated that a biologically relevant level of AFB1 (32 nM) induces apoptosis in astrocytes through ATP depletion and caspases activation.