From conventional to advanced cell culture systems to evaluate single and combined cytotoxic effects of mycotoxins: towards an in vivo-like risk assessment

Mycotoxins are low-molecular-weight secondary metabolites of filamentous fungi, mainly produced by species belonging to the genera Aspergillus, Fusarium, and Penicillium. As widespread natural contaminants, mycotoxins can be found in numerous food commodities, both of plant and animal origin. Mycotoxin exposure has been associated to chronic and acute effects on human and animal health. For these reasons, their presence in foods and feeds has been considered by different regulatory agencies, and great efforts are being made to harmonise legislation worldwide. Moreover, increasing evidence proves that humans are exposed to more than one mycotoxin at once through diet. The interaction between two or more mycotoxins can both enhance or decrease the toxic effect of each component of the mixture tested alone. Therefore, the real risk scenario for humans due to concomitant mycotoxins exposure may be different from that predicted based on their individual toxicities. In this context, our study aims to evaluate single and combined cytotoxic effects induced by mycotoxins exposure comparing conventional and advanced cell culture models. For this purpose, two tumour cell lines, human neuroblastoma (SH-SY5Y) and human breast cancer epithelial cells (MDA-MB-231), were cultured both in conventional monolayer and in a 3D spheroid model. Spheroids were generated from single-cell suspensions obtained from trypsinised monolayers, diluted to the appropriate cell seeding densities into ultra-low attachment (ULA) 96-well round bottom plates. Once optimised the protocol for the generation of reproducible and size-appropriated bio-relevant spheroids, the effects of the mycotoxins sterigmatocystin (STE), ochratoxin A (OTA) and patulin (PAT) exposure on spheroid viability were investigated after 1, 2, and 3 days using ATP assay. The results obtained in the spheroids were then compared with those obtained in 2D monolayer cultures, highlighting a statistically significant different sensitivity between the two culture systems. Since cytotoxicity measured on a 3D model reflects more accurately in vivo-like cell behaviour, we proceeded to further evaluate cytotoxic effects induced by mycotoxins co-exposure using the sole spheroid model. The isobologram analysis, which plots the dose–effect curves for each compound and its combinations in multiple diluted concentrations, was used to determine the type of interaction occurred when STE, OTA and PAT were mixed in binary and tertiary combinations. Finally, to improve the knowledge level of complex biological phenomena occurring in humans, a microfluidic platform for culturing cells was used to evaluate the effects induced by continuous mycotoxins co-exposure. In particular, the spheroids were seeded into a micro-bioreactor that allows simultaneous exposure to combined mycotoxins with finely controlled conditions. In conclusion, this study highlights the pivotal role played by cell culture models in the toxicological risk assessment of mycotoxins and how the use of advanced culture system improves the understanding of such complex biological mechanisms. Indeed, our findings first evidence the limits of conventional monolayer cultures compared to advanced systems and provide novel and more accurate evaluations of mycotoxins-induced toxic effects. This information enriches the currently limited knowledge of the multiple toxins’ co-exposure. Finally, the proposed use of a microfluidic device for screening of combined toxins effects builds upon the novelty of our approach. Overall, our study fits into the scenario of a new alternative in vitro toxicology, where it is of paramount importance to optimise advanced culture models to better mimic the physiological environment and reduce in vivo follow-up, in compliance with the European Union recommendations.