In vitro hepatic metabolism and associated binding of enniatin B

Enniatin B (ENNB), a secondary metabolite produced by various Fusarium species, frequently contaminates cereals, which constitutes the main dietary source of human exposure. Critical toxicodynamic and toxicokinetic data gaps currently impede a robust and accurate risk assessment. The primary objective of this study was to characterize the in vitro toxicokinetic profile of ENNB. We investigated its clearance using human and mouse liver microsomes (HLM and MLM), as well as 2D and 3D HepaRG cell models. To facilitate reliable in vitro-in vivo extrapolation (IVIVE), we also determined key parameters: plasma protein binding, binding to microsomes and HepaRG cells, CYP450 inhibition, and the identification of HepaRG metabolites. Binding studies revealed a very high binding of ENNB to human plasma proteins and a high binding to inactivated human liver microsomes and HepaRG cells. The predicted in vivo hepatic clearance (cl_H,blood) of ENNB, calculated using the in vitro results from MLM, HLM, and HepaRG 2D indicated a low hepatic first pass effect. Interestingly, no observable disappearance of ENNB was found in the HepaRG 3D model. Our findings on ENNB-mediated CYP inhibition in HLM, in combination with literature results, suggest a potential for CYP3A4/5-related auto-inhibition. Finally, we successfully performed a putative identification of 13 Phase I metabolites using the human HepaRG cell line. In conclusion, the low hepatic first pass effect could imply a high oral bioavailability in vivo if intestinal barrier passage is significant, as predicted elsewhere. However, this could be counteracted by transport limited hepatic clearance that should be further investigated and a pre-systemic first-pass effect already demonstrated in vitro.