The Transport Manner And Molecular Mechanisms Of P-glycoprotein Induction Of Deoxynivalenol

Deoxynivalenol(DON),belonging to type B trichothenece,is one of the most abundant mycotoxins and posing various toxicological effects to animals and humans.It is also known as vomitoxin because it can induce vomiting in pigs.To date,the underlying mechanisms respecting the transporting manners of DON in mammalian cells remain unclear.To uncover the transporting mechanism of DON,parallel artificial membrane permeability assay(PAMPA)and Transwell model were conducted to determine the transport manners.To screen the transporters related to DON uptake and efflux in mammalian cells,various transporters inhibitors of ABC family,Solute carrier family(SLC)family combined with Caco-2,HepG2 and MDCK cells were used.The results of PAMPA and Transwell models indicate a carrier-mediated transport manner with regards to DON transport in mammalian cells,instead of a free diffusion manner.Moreover,the uptake of DON in mammalian cells exhibit a time-and dosage-dependent manner.Organic anion transporting peptides(OATPs),Organic anion transporters(OATs)and Organic cation transporters(OCTs)were determined as major transporters respecting DON uptake in mammalian cells.P-glycoprotein(P-gp)is the major transporter associated with DON efflux in intestinal cells,hepatocytes and kidney cells.Thus,a carrier-mediated and energy-dependent uptake and efflux manner of DON were proposed.Moreover,P-gp was selected as the major efflux transporter to investigate the induction mechanisms upon DON exposure.The mRNA level of P-gp in Broiler Liver was upregulated after exposure of DON.To date,it still remains unclear whether mRNA and protein level of P-gp can be upregulated in the stress of DON in mammalian cells.P-gp is an important protein located in cell membrane that pumps a lot foreign substances out of cell.We investigated whether DON upregulates the expression and efflux activity of P-glycoprotein and uncover the related signal pathways.Caco-2 and HepG2 cells were treated with DON and changes of P-gp expression and Rhodamine 123 efflux were determined.To identify the related signaling pathways regarding P-gp induction,proteins inhibitors and knockdown via shRNA combined with lentivirus transfection were conducted.DON upregulates the expression and activity of P-gp in a timeand concentration-dependent manner in HepG2 cells.The upregulation of P-gp was strongly inhibited by NF-?B inhibitors and p65 knockdown indicating the key function of NF-?B.NF-?B was activated through AKT phosphorylation.Moreover,the upstream signal pathway of P-gp induction is MAPK due to ribotoxic stress of DON,and JNKs is the key protein of MAPKs with regards to P-gp induction.In summary,DON induces P-gp expression and activity via the NF-?B and AKT pathways through MAPK activation.In return,the upregulation of P-gp dramatically declines the cytotoxicity of DON or other P-gp substrates towards mammalian cells.