Transport of enniatin B and enniatin B1 across the blood-brain barrier and hints for neurotoxic effects in cerebral cells

Enniatins are common contaminants of food and feed and belong to the group of the "emerging" mycotoxins, which are produced by various Fusarium species. Although a wide range of toxic effects, like Antibacterial, Antifungal, insecticidal and cytotoxic properties, have been described in vitro, so far, no cases of mycotoxicosis connected to enniatins in vivo are reported. Among this group of mycotoxins, enniatin B and enniatin B1 are the most prevalent compounds and therefore are present in the human diet. Enniatins can reach systemic circulation, thus, the investigation of possible neurotoxic effects is of importance. Different cerebral cells were used to address effects on cell death having an impact on the blood-brain barrier. The influence of enniatin B and enniatin B1 on cellular viability was examined via Cell Counting kit-8 assay (CCK-8) in three different cell types of the blood-brain barrier: porcine brain capillary endothelial cells (PBCEC), human brain microvascular endothelial cells (HBMEC) and human astrocytoma cells (CCF-STTG1). CCF-STTG1 cells were more sensitive to enniatin B (IC50 = 8.9 μM) and enniatin B1 (IC50 = 4.4 μM) than both endothelial cell types. In CCF-STTG1 cells, Caspase-3 activation and Lactate Dehydrogenase (LDH) release were evaluated. Both compounds did not induce any LDH release and only enniatin B increased Caspase-3 activity as a marker for Apoptosis. The transport kinetics of enniatin B and enniatin B1 across the blood-brain barrier in vitro were evaluated using PBCEC, cultivated on Transwell® filter inserts. Analysis of the apical and the basolateral compartment by high performance liquid chromatography-mass spectrometry revealed high influx rates for enniatin B and enniatin B1. Thus, both compounds can reach the brain parenchyma where neurotoxic effects cannot be ruled out.