Neurotoxic implications of gliotoxin and ochratoxin A in SH-SY5Y cells: ROS-induced apoptosis and genotoxicity

Abstract

Gliotoxin (GTX) and ochratoxin A (OTA) are naturally produced toxins by fungi and are known for their potential health risks. With the aim of shed some light on the mechanisms by which GTX, OTA, and their combination exert toxicity at neuronal level, the following in vitro studies were conducted in SH-SY5Y cells: a) intracellular ROS monitorization by the H2-DCFDA assay b) study of the expression of pro-apoptotic genes Bcl2, Casp-3, and Bax by RT-qPCR c) study of the apoptotic-necrotic progression of SH-SY5Y cells by flow cytometry; d) study of the genotoxic potential through the in vitro micronucleus (MN) assay also by flow cytometry following OECD TG 487 guidelines. ROS production was increased when cells were exposed to mycotoxins at all scenarios tested highlighting the effects of GTX. Regarding gene expression, increases of Bax and Casp-3 genes at 1.3- and 3- folds respectively were observed when cells were exposed to GTX at 0.75 μM, with a more prominent increase after exposure to the binary combination [GTX + OTA] at [0.2 + 0.1] µM, increasing 3 and 5-folds more, respectively when compared to the control. MN formation increased a 30 % compared to control when exposed to GTX at 0.4 μM, 43 % for OTA at 0.8 μM, with the highest increase observed when cells were exposed to the combination [GTX + OTA] at [0.2 + 1.5] μM, obtaining a 65 % more MN formation. Based on the results obtained, we can conclude that for the proposed scenarios of exposure to GTX, OTA, and their combination, genotoxic effects together with oxidative effects at neuronal level in SH-SY5Y cell line, were found to play a key role in their mechanisms of toxic action.