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P1008
In vitroToxicity Screening by Transcription Factor Activity Profiling
Presenter Keith Houck, US EPA, USA
Additional Authors: David Dix, Sergei Romanov, Alexander Medvedev, Maria Gambarian, Natalia Poltoratskaya, Matt Moeser, Liubov Medvedeva, Mikhail Gambarian, Luda Diatchenko, and Sergei Makarov
Assessing large numbers of compounds with in vitro assays for toxicity is a daunting endeavor due to the myriad potential molecular targets and pathways involved in toxicity. Use of microarrays for mRNA expression profiling provides broad coverage of effects, but the data generated remains a challenge for associations with toxic effects, particularly with in vitro systems. An alternative approach is to focus analysis of test compound effects on key transcription factors controlling gene expression. These regulatory proteins modulate gene expression response to environmental perturbations, and are many fewer in number than the total number of expressed genes. To efficiently characterize the activity of 320 chemicals with known toxicities, we used a novel, high-content reporter gene system termed Factorial™. This method afforded multiplexed assay endpoints in transiently transfected HepG2 cells. Two reporter gene assays, one consisting of 52 cis-acting response elements and the other containing 28 trans-acting systems, were run on all 320 chemicals. The results showed robust, reproducible profiles of activity that correlated with known mechanisms of toxicity for many of the chemicals. Transcription factor responses linked to toxicity pathways that were affected included  estrogen receptor, peroxisome proliferator receptor alpha, retinoic acid receptor, aryl hydrocarbon receptor, Nrf2/ARE, and p53. The results obtained through the Factorial assays have been incorporated into a larger ToxCast™ dataset being used to develop predictive signatures of potential for toxicity. A systematic, global analysis of effects on key regulatory proteins offers great promise for screening for adverse effects of drugs and environmental chemicals. This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy.