Identification of New Human Pregnane X Receptor Ligands among Pesticides Using a Stable Reporter Cell System

Abstract

Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

24,25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation

“…Although we cannot say with certainty that this could indeed be the case, the application of rifampicilin (5M), a widely recognised reference molecule for PXR binding [49], was also capable of eliciting a very similar response to that afforded by ketoconazole (data not shown).…”

A consecutive three alanine residue insertion mutant of human CAR: a novel CAR ligand screening system in HepG2 cells

“…Although a cultured cell-based screening system for SXR ligand had been established (Lemaire et al, 2004(Lemaire et al, , 2006Noracharttiyapot et al, 2006), no similar system has been established for CAR ligand. CAR is localized in the cytoplasmic compartment in a ternary complex with Hsp90 via CAR cytoplasmic retention protein (CCRP) in the mouse liver and in primary hepatocytes Yoshinari et al, 2003).…”

Assessment of Human Pregnane X Receptor Involvement in Pesticide-Mediated Activation of CYP3A4 Gene

“…There is a possibility of the existence of a chemical modulating the CYP3A4 expression through hPXR activation. Although pesticides have been reported to activate PXR or CAR (Wyde et al, 2003;Jacobs et al, 2005;Lemaire et al, 2006), pesticide-mediated activations of CYP3A4 gene are not well assessed.…”