Identification of new human PXR ligands among pesticides using a stable reporter cell system

1 These authors contributed equally to the study. ABSTRACT Pregnane X receptor (hPXR, 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 hPXR activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG 5 LN, 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 HG 5 LN and stably expressed hPXR ligand binding domain (LBD) fused to GAL4 DNA binding domain (DBD). The HG 5 LN cells were used as a control to detect non-specific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: i) herbicides: pretilachlor, metolachlor and alachlor chloracetanilides, oxadiazon oxyconazole, and isoproturon urea; ii) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles and imazalil triazole; and iii) 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 (CYP3A4) expression in a primary culture of human hepatocytes. HGPXR, with HG 5 LN 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.