Affinity purification using recombinant PXR as a tool to characterize environmental ligands.
Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis.
IRCM, Institut de Recherche en Cancerologie de Montpellier, CRLC Val d'Aurelle Paul Lamarque, INSERM, U896 34298 Montpellier, France; Université Montpellier 1, 34298 Montpellier, France; UMR 5569 Hydrosciences, Université Montpellier I, 34060 Montpellier, France.
Sonia
S
Dagnino
Virginie
V
Bellet
Marina
M
Grimaldi
Anne
A
Riu
Sélim
S
Aït-Aïssa
Vincent
V
Cavaillès
Hélène
H
Fenet
Patrick
P
Balaguer
D002460:Cell Line; D045744:Cell Line, Tumor; D047628:Estrogen Receptor alpha / Q000378:metabolism; D019015:Geologic Sediments; D006801:Humans; D008024:Ligands; D000077297:Pregnane X Receptor; D011944:Receptors, Androgen / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D047628:Estrogen Receptor alpha; D008024:Ligands; D000077297:Pregnane X Receptor; D011944:Receptors, Androgen; D018336:Receptors, Aryl Hydrocarbon; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D014874:Water Pollutants, Chemical