Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system.
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.
Lemaire
Géraldine
G
INSERM, U540, F-34090 Montpellier, France.
Mnif
Wissem
W
Pascussi
Jean-Marc
JM
Pillon
Arnaud
A
Rabenoelina
Fanja
F
Fenet
Hélène
H
Gomez
Elena
E
Casellas
Claude
C
Nicolas
Jean-Claude
JC
Cavaillès
Vincent
V
Duchesne
Marie-Josèphe
MJ
Balaguer
Patrick
P
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010575:Pesticides / Q000633:toxicity*; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists* / Q000378:metabolism; D011987:Receptors, Steroid / Q000819:agonists* / Q000378:metabolism
D008024:Ligands; C000625852:NR1I2 protein, human; C000625853:Nr1i2 protein, mouse; D010575:Pesticides; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human