Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

Abstract

Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the concentration- and time-response of the 320 ToxCast chemicals for changes in expression of genes regulated by nuclear receptors. Fourteen gene targets were monitored in quantitative nuclease protection assays: six representative cytochromes P-450, four hepatic transporters, three Phase II conjugating enzymes, and one endogenous metabolism gene involved in cholesterol synthesis. These gene targets are sentinels of five major signaling pathways: AhR, CAR, PXR, FXR, and PPARα. Besides gene expression, the relative potency and efficacy for these chemicals to modulate cellular health and enzymatic activity were assessed. Results demonstrated that the culture system was an effective model of chemical-induced responses by prototypical inducers such as phenobarbital and rifampicin. Gene expression results identified various ToxCast chemicals that were potent or efficacious inducers of one or more of the 14 genes, and by inference the 5 nuclear receptor signaling pathways. Significant relative risk associations with rodent in vivo chronic toxicity effects are reported for the five major receptor pathways. These gene expression data are being incorporated into the larger ToxCast predictive modeling effort.

Acknowledgments

Supported by EPA CR83323601-DESE cooperative training agreement. The U.S. Environmental Protection Agency through its Office of Research and Development collaborated in the research described in this article, which has been subjected to agency review and approved for publication. Reference to commercial products or services does not constitute or imply endorsement or recommendation by the U.S. government. We acknowledge the help of Drs. John Wambaugh (U.S. EPA/ORD/NCCT), Howard Bondell (North Carolina State University), and Bruce Seligman (High Throughput Genomics) for their contributions to the analysis of these data. Stephen Ferguson, Kimberly Freeman, Edward LeCluyse, Andrew Beam, and Adam Farmer worked for CellzDirect/Invitrogen corporation (a part of Life Technologies), where human hepatocyte culture and qNPA assays are developed and performed for commercial purposes. All other authors have no conflicts of interest to declare.