SY5Y CELLS

AFLATOXIN B1 AND OCHRATOXIN A DISRUPT NEURONAL DIFFERENTIATION IN

SH-SY5Y CELLS

M. Alonso-garrido, M. Frangiamone, C. Carfagnini, G. Font, L. Manyes Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Ave. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain manuel.alonso-garrido@uv.es

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are well known to human health as the most toxic secondary metabolites produced by fungi. They are a risk to public health due to their occurrence in food and feed, being contaminated wheat one of the main concerns in food safety. On the other side, it has been shown that both fermented whey and pumpkin extract can interact with these molecules significantly reducing their occurrence. They can cross the blood brain barrier via paracellular and could trigger different molecular responses associated to a wide range of neurodegenerative pathologies. Four different types of bread (bread (B); pumpkin (1%) bread (PB); fermented whey (1%) bread (FWB); fermented whey (1%) and pumpkin bread (FWPB) (1%)) were elaborated with the same concentrations of mycotoxins. This study is focused in the alteration of neuronal cell differentiation by these different bread extracts previous simulated stomach digestion, using SHSY5Y cells in vitro. Cell cycle by flow cytometry and dopamine and β-tubulin III by immunofluorescence were analyzed as markers of neuronal differentiation. For both experiments, cells were cultivated for seven days and treated with the bread extracts at 2% (AFB1 60 nM; OTA 240 nM; AFB1 (60 nM) + OTA (240 nM)) and retinoic acid (0.1%). For cell cycle analysis 20000 cells per well were seed in 48-well plates and for dopamine and β-tubulin III detection 300000 cells per well were inoculated in 6-well plates. Flow cytometry analysis reported significantly less differentiation for B-OTA, FW-OTA, FW-AFB1-OTA, FWPB-AFB1, FWPB-OTA, FWPB-AFB1OTA and significantly higher differentiation rate for PB-AFB1 and FWB-AFB1 respect to their correspondent controls. Immunofluorescence showed lower significant concentrations of β-tubulin III and dopamine for all treatments versus their control. In conclusion, even low concentrations of mycotoxins can change neuronal differentiation in vitro. Interaction between pumpkin, fermented whey and mycotoxins needs further research.

Acknowledgements: Spanish Ministry of Science and Innovation project (PID2019-108070RB-I00ALI) and PhD grant (BES-2017-081328).

Keywords: whey, pumpkin, flow cytometry, immunofluorescence, in vitro

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