Transactivation Assays to Assess Canine and Rodent Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) Activation.

The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important. Screening for NR activation by New Molecular Entities (NMEs) using cell-based transactivation assays is the singular high throughput method currently available for identifying the activation of a particular NR. Moreover, screening for species-specific NR activation can minimize the use of animals in drug development and toxicology studies. With this in mind, we have developed in vitro transactivation assays to identify compounds that activate canine and rat PXR and CAR3. We found differences in specificity for canine and rat PXR, with the best activator for canine PXR being 10 μM SR12813 (60.1 ± 3.1-fold) and for rat PXR, 10 μM dexamethasone (60.9 ± 8.4 fold). Of the 19 test agents examined, 10 and 9 significantly activated rat and canine PXR at varying degrees, respectively. In contrast, 5 compounds exhibited statistically significant activation of rat CAR3 and 4 activated the canine receptor. For canine CAR3, 50 μM artemisinin proved to be the best activator (7.3 ± 1.8 and 10.5 ± 2.2 fold) while clotrimazole (10 μM) was the primary activator of the rat variant (13.7 ± 0.8 and 26.9 ± 1.3 fold). Results from these studies demonstrated that cell-based transactivation assays can detect species-specific activators and revealed that PXR was activated by at least twice as many compounds as was CAR3, suggesting that there are many more agonists for PXR than CAR

Effects of various compounds on transactivation of rat CAR3.

<p>HepG2 cells were transfected with rat CAR3 or empty expression vector and the CYP2B6 promoter and enhancer linked to a luciferase reporter vector (A) or luciferase reporter vector with CYP3A4 proximal and distal promoter regions (B) before seeding in 96-well plates as described in Materials and Methods. Cells were treated with the same panel of chemicals for 48 h before detection of fluorescence and luminescence. All luminescence values were normalized for cell viability. Data represents the mean ± SE from three independent experiments in triplicate expressed as fold activation above vehicle control treated cells. All data is normalized against fold activation values from transactivation assays with an empty expression vector. An asterisk denotes significant difference from vehicle controls at a level of p < 0.01. (C-D), Representative clotrimazole dose-response curves for rCAR3/2B6 (C) and rCAR3/3A4 (D). Cells in triplicate wells were treated with 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 20 μM clotrimazole (C) or 0.1, 0.5, 1, 5, 10 and 20 μM clotrimazole (D) for 48 h before luminescence was detected and normalized against cell viability. Results are expressed as fold activation above 0.1% DMSO treated cells. Data represent the mean ± SE from three independent experiments triplicate. EC₅₀ and E_MAX values were calculated using nonlinear regression of a typical log dose-response curve.</p

Rat PXR transactivation by 19 compounds in a stable cell line.

<p>A rat PXR stable cell line over-expressing rat PXR and a luciferase reporter vector containing the CYP3A4 proximal and distal enhancer regions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164642#pone.0164642.ref014" target="_blank">14</a>] was used to evaluate activation of rat PXR by various compounds (A). rPXR cells were seeded in 96-well plates and treated with the 19 compounds for 48 h before fluorescence (cell viability) and luminescence (transcriptional activation) detection. All luminescence values were normalized for cell viability. The data represent the mean ± SE from three independent experiments in triplicate expressed as fold activation above vehicle control treated cells. An asterisk denotes compounds exhibiting significant difference from their respective vehicle control at a level of p < 0.01. (C-D) Dose-response curves for two rat PXR positive controls, Dexamethasone (B) and Pregnenolone 16α- carbonitrile (C). Concentrations were 0.1, 0.5, 1, 5, 10 and 20 μM for Dexamethasone (B) and 0.01, 0.05, 0.1, 0.5, 1 and 5 μM for Pregnenolone 16α- carbonitrile (C). Results are expressed as fold activation above 0.1% DMSO treated cells. Data represents the mean ± SE from three independent experiments in triplicate. EC₅₀ and E_MAX values were calculated using nonlinear regression of typical log dose-response curves.</p

Effects of various compounds on transactivation of canine PXR.

<p>HepG2 cells were transfected with canine PXR or empty expression vector and luciferase reporter construct harboring the CYP3A4 proximal and distal promoter regions and seeded in 96-well assay plates as described in Materials and Methods. (A) Cells were treated with panel of 19 compounds for 48 h before detection of fluorescence for cell viability and luminescence for transcriptional activation. All luminescence values were normalized for cell viability. Data for each compound and concentration represents the mean ± SE from three independent experiments in triplicate expressed as fold activation above vehicle control treated cells. All data is normalized against fold activation values from transactivation assays with an empty expression vector. An asterisk denotes compounds exhibiting significant difference from their respective vehicle control at a level of p < 0.01. (B) The graph represents a SR12812 dose-response curve for canine PXR. Cells in triplicate wells were treated with 0.1, 1, 2.5, 5, 10, 20 and 30 μM SR12813 for 48h before luminescence was detected and normalized against cell viability (fluorescence values). Results are expressed as fold activation above 0.1% DMSO treated cells. Data represents the mean ± SE from three independent experiments in triplicate. EC₅₀ and E_MAX values were calculated using nonlinear regression of a typical log dose-response curve.</p

Transactivation of canine CAR3 by 19 different compounds.

<p>HepG2 cells were transfected with the canine CAR3 or empty expression vector and the CYP2B6 promoter and enhancer linked to a luciferase reporter vector (A) or luciferase reporter vector with CYP3A4 proximal and distal promoter regions (B) before seeding in 96-well plates as described in Materials and Methods. Cells were treated with the panel of 19 chemicals for 48 h before detection of fluorescence for cell viability and luminescence for transcriptional activation. All luminescence values were normalized for cell viability. Data for each test compound and concentration represents the mean ± SE from three independent experiments in triplicate expressed as fold activation above vehicle control treated cells. All data is normalized against fold activation values from transactivation assays with an empty expression vector. An asterisk denotes compounds exhibiting significant difference from their respective vehicle control at a level of p < 0.01. (C) A representative artemisinin dose-response curve for the cCAR3/2B6. Cells in triplicate wells were treated with 0.5, 1, 2.5, 5, 10, 20, 50, 75 and 100 μM artemisinin for 48 h before luminescence was detected and normalized against cell viability (fluorescence values). Results are expressed as fold activation above 0.1% DMSO treated cells. Data represents the mean ± SE from three independent experiments in triplicate. EC₅₀ and E_MAX values were calculated using nonlinear regression of a typical log dose-response curve.</p