Enniatins: new insights on biotransformation and mechanisms of action

Abstract

Enniatins are cyclic hexadepsipeptides produced mainly by Fusarium species, which are predominantly found in cereal grains and their products. The most common enniatins occurring in grain are enniatin B, B1, A and A1. Because of their frequent occurrence in feed and food grains, enniatins are considered as of potential concern for animal and human health. Previous studies have demonstrated that these compounds are bioactive in vitro and cytotoxic at low concentrations. Recently, the Toxinology Research Group at the Norwegian Veterinary Institute has demonstrated that lysosomal destabilization could be an early event in enniatin -induced cell death in human epithelial colorectal adenocarcinoma cells, Caco-2 cells as well as in murine macrophages, RAW264.7 cells. In this context, the proposed project aims to clarify the biotransformation pathway of enniatin B1 (ENN B1) and get improved insight into the reported mechanism of the lysosomal destabilization induced by enniatin B. The 6-month project will involve a range of techniques and experimental set-ups, including the production and isolation of the newly discovered hepatic metabolite of ENN B1 (MX) using pig liver microsomes, purification of the unknown metabolite (MX) by using semi-preparative HPLC and structural elucidation and precise quantitative determination of purified MX by Nuclear Magnetic Resonance (NMR) spectroscopy. Changes in pH in lysosomes and cytosol after exposure to ENN B1 and MX will also be analyzed by flow cytometry or by using pH sensitive fluorescent probes. Finally, the role of the lysosome-associated membrane proteins 1 (LAMP-1) and 2 (LAMP-2) in lysosomal cell death will be studied by using LAMP1/2 double-deficient cells (mouse embryotic fibroblasts). (AU)