HNF4 as the Primal Target of TCE Toxicity in the Developing Heart

The hypothesis to be tested was that HNF4a is a target of TCE.  Bioinformatic analysis had suggested that this transcription factor was the most highly-linked molecule to the genes altered by TCE in a chick heart model.  The study section had requested that we provide experimental data to back this up.  Two approaches were taken.  First we used cell culture with human HepG2 and mouse epicardial cells and selected a panel of 11 genes that were altered by TCE in mice and chicks.  We then tested two molecules identified as an antagonist (Bi 6015) and an antagonist (Benflorex).  Both reagents appeared to be antagonists to the selected marker genes.  We then performed a similar series of experiments giving TCE (10ppb), Benflorex (20 µM, Bi 6015 (20µM) or a combination of TCE and Benflorex in drinking water to pregnant mice.  Three liters for each treatment were collected on day 11 of pregnancy and RNA was collected and pooled for heads, hearts and bodies from the embryos of each liter.  Analysis is still being performed but qPCR of the first set of litters shows that each HNF4a antagonist and TCE exposure alone produces a similar inhibition of each of the 4 best markers and that the markers are most highly expressed in the embryonic heart.  Thus the data argue that HNF4a inhibition and TCE exposure similarly alter these markers that are useful indicators of TCE exposure.  We will be using this data for a submission to NIEHS for the Feb. 5, 2015 deadline.