Use of comprehensive screening methods to detect selective human CAR activators

Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers

“…CAR activation is associated with the increased expression of a number of cell cycle regulators, including Cyclin D1, Mdm2, cMyc, Gadd45β and others (Blanco-Bose et al, 2008;Columbano et al, 2005;Huang et al, 2005;Ledda-Columbano et al, 2003;Yamamoto and Negishi, 2008;Yamamoto et al, 2010). DDT isomers and metabolites activate rodent and human CAR and target gene CYP2B (Kanno and Inouye, 2010;Kiyosawa et al, 2008;Küblbeck et al, 2011;Wyde et al, 2003). Thus, the DDT isomer mixture may elicit complex liver responses through both the ERα and CAR nuclear receptors.…”

Improved assays for xenosensor activation based on reverse transfection

“…This cell line expresses human AhR endogenously. The CMV promoter-driven expression plasmids for GAL4-LBD fusions of human CAR and PXR, all luciferase reporter genes and the b-galactosidase control reporter have been previously described (Küblbeck et al, 2008(Küblbeck et al, , 2011a.…”

“…The conventional calcium phosphate-mediated transfection was done essentially as described before (Küblbeck et al, 2008(Küblbeck et al, , 2011a. After transfection for four hours, the medium was replaced with fresh DMEM complemented with 1% antibiotics/2 mM L-glutamine/5% delipidated FBS and including either DMSO, positive controls for xenosensors (10 lM OME for AhR; 10 lM FL81 for CAR; 10 lM RIF for PXR) or test chemicals at indicated concentrations (Table 1).…”

“…The optimized assay conditions are summarized in Table 2. Next, three-day assay uniformity tests were performed according to the NIH guidelines by adapting the 96-well NIH protocols to a 48-well plate format (Küblbeck et al, 2011a). The strong positive controls (OME, FL81 and RIF) and moderate activators (BNF, clotrimazole and RU486) for xenosensors displayed reproducible activation.…”

“…Recently, we have optimized and formally validated transient transfection assays for all three human xenosensors AhR, CAR, and PXR (Küblbeck et al, 2011a). This assay requires separate stages for DNA/carrier complex formation, transfection of DNA into cells, removal of the DNA/carrier-containing medium and exposure to test chemicals.…”