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Final Progress Reports: University of California-San Diego: Toxicogenomic Analysis of Nuclear Xenobiotic Receptors

Superfund Research Program

Toxicogenomic Analysis of Nuclear Xenobiotic Receptors

Project Leader: Ronald M. Evans (Salk Institute for Biological Studies)
Grant Number: P42ES010337
Funding Period: 2005-2017
View this project in the NIH Research Portfolio Online Reporting Tools (RePORT)

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Final Progress Reports

Year:   2016  2009 

In addition to verifying the strong hits from previous HTP chemical library screening for PXR/CAR/ERα activators, researchers are in the process of collecting and screening more compounds in the CERCLA toxicant list for intrinsic PXR/CAR/ERα activity. Researchers have optimized the assays and increased the sensitivity in order to make them suitable for screening of toxins from actual environmental samples. With the collaboration of the UCSD SRP Project 4 (Dr. Robert Tukey) and the Outreach Core, the researchers tested a number of soil and water samples from the US/Mexico border using the Superfund standard chemicals as positive controls and evaluated the possibility of using the PXR/CAR/ERα bioassay to detect real environmental pollution. Researchers plan to test more such samples when they become available. This study will provide useful information to predict environmental health risks.

In the previous study, researchers have screened ERα activators in CV-1 cells using nearly 5000 structurally diverse compounds and found that ~5-10% of these chemicals including a number of chemicals in CERCLA Priority List of Hazardous Substances such as benzapyrene, fluoranthene can activate ERα but not ERß. In addition, in the ERα antagonist screen using a number of xenobiotics, researchers found that arsenite (As3+) and puromycin have inhibitory effects on ERα but not ERß, though the mechanism remains unclear. Researchers will perform a larger scale screening for ERα antagonists. Among the ERα activators in the CERCLA toxicants list, such as benzapyrene, fluoranthene, benzanthracene, 3-methylcholanthrene (3-MC) and 4-aminobiphenyl (4-ABP), it was found that with the exception of 4-ABP, most of the ERα activators activate multiple nuclear receptors. In order to look at the effects of ERα liver response to xenobiotics, researchers injected 4-ABP into animal, with estrodiol (E2) and a phytoestrogen daidzein as controls and looked at liver drug metabolism gene expression profile. Researchers found that a set of drug metabolism genes were up-regulated in all three treatments, such as Nqo1, Gsta1, Gstm3, Cyp2b10, Por and Pdk4. In Myles Brown's ChIP-Seq study of ERα in human breast cancer cells, ERα was shown to have binding sites in NQO1 and POR, which also indicates the regulation of ERα on drug metabolism genes. This result suggested that the activation of ERα by xenobiotics is able to induce liver drug metabolism thus has properties of a true xenobiotic receptor. In order to examine whether the up-regulation of these genes is directly or indirectly caused by ERα activation, researchers cloned Nqo1, Gsta1, Gstm3, Cyp2b13 gene promoter regions to look at the ERα transactivation on these genes upon the xenobiotic stimulation. In addition, researchers collected the promoter regions of nearly 120 drug metabolism genes constructed in luciferase reporter plasmids and plan to screen the responsiveness of these gene promoters on ERα activation by environmental toxins. The researchers are also performing direct binding assays of ERα with selected xenobiotics and in vivo studies to examine the drug clearance pathway mediated by ERαxenobiotic activation.

Using SMRTRID mutant mice, researchers previously studied the dynamic interaction between CAR and SMRT. In the last reports, the in vitro and in vivo data indicated that SMRT regulates drug metabolism mediated by CAR. Pharmacokinetics study using HPLC/MASS spectrometry method showed that the ketamine degradation and the formation of its metabolite norketamine are much more rapid in mutant mice liver cells, consistent with the observation that CYP2B10 gene expression levels are much higher in the mutant liver cells. Using other selected drugs, for an example, a PXR agonist midazolam, researchers have been studying the regulation of hepatic xenobiotic metabolism by SMRT through other nuclear receptors. They also observed the direct binding of SMRT with PXR using GST pull-down assays and that SMRT suppress PXR activity in cell-based luciferase reporter assays. Researchers will perform a gene expression microarray to examine the overall regulation of SMRT on the hepatic drug metabolism. These results may reveal SMRT as a new key regulator of the hepatic drug metabolism system.

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