Development of siRNA against the CYP1A1 gene for trap of endogenous Ah-receptor ligand

Abstract: The aryl hydrocarbon receptor (Ah-receptor) is a member of the bHLH-PAS protein family. The Ah-receptor is a ligand dependent transcription factor, which activates a wide range of genes, most notably the xenobiotica metabolising genes, CYP1A1 and CYP1A2. The biological function of the Ah-receptor is still unknown and an endogenous ligand has yet not been identified. A possible Ah-receptor ligand is 6-formylindolo[3,2-b]carbazole (FICZ). FICZ has a high affinity for the Ah-receptor and is rapidly metabolised by CYP1A1, CYP1A2 and aldehydeoxidase (AOX). To try to trap FICZ or other possible endogenous Ah-receptor ligands, the metabolising enzymes CYP1A1, CYP1A2 and AOX were blocked. This was achieved through chemical blockage of CYP1A1 and CYP1A2 by ellepticin and through silencing with siRNA directed against CYP1A1 and CYP1A2. Successful blockage would be seen as an increase in Ah-receptor dependent XRE-luciferase activity. Chemical blockage of AOX with tungstate did not affect FICZ-dependent XRE-luciferase activation which could indicate that HepG2 cells lack AOX. The chemical blockage of CYP1A1 and CYP1A2 with ellepticin modified the XRE-luciferase response, but did not completely block Ah-receptor activation. In addition it is possible that ellepticin is a ligand for the Ah-receptor. The blockage of CYP1A1 by siRNA was successful; a silencing of CYP1A1 mRNA by at least 50 percent was detected. However due to lack of time it was not tested if the blockage of CYP1A1 and CYP1A2 was sufficient to trap Ah-receptor ligands.