Background: The constitutive androstane receptor (CAR, NR1I3) plays a key role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes. Results: The expression of CAR mRNA throughout the circadian rhythm is reported for the first time in phase with the clock gene Bmal1 and in antiphase with the clock-controlled gene Rev-erbα mRNAs, with a peak at Zeitgeber time (ZT) 20 and a trough at ZT8, and a peak/trough ratio of 2.0. The diurnal difference in CAR mRNA expression might underlie the 1.7-fold difference in the magnitude of the PB-dependent induction of CYP2B1/2 mRNA. Conclusion: The circadian oscillation of xenosensor gene CAR mRNA expression is partially responsible for chronopharmacokinetics and chronopharmacology in disease.

The expression of the CAR gene is lost in most cultured cell lines, and exogenously expressed CAR accumulates spontaneously in the nuclear compartment, where CAR is constitutively active. Therefore, the assessment of CAR-dependent transcriptional activation has to be evaluated using either animal models or primary cultured hepatocytes. Furthermore, due to the species-specific response of CAR to individual compounds, the results obtained with animal models are not directly applicable to human cases. We established a novel screening system for hCAR ligands (including agonists and inverse agonists) by expressing GAL4/DBD-fused hCAR/LBD, in which three consecutive alanine residues were inserted between helix 11 and helix 12 of LBD, and a commercially obtained plasmid consisting of the firefly luciferase gene downstream of the GAL4 responsive element in a cultured cell line. Androstenol (Andro) and clotrimazole (CLT), which are both inverse agonists of hCAR, were classified as an antagonist and weak agonist, respectively, in our novel assay system. Among DDT and its metabolites DDE and DDD, only DDT worked as an agonist of the hCAR mutant, although all of them were enrolled as agonists of SXR. Using this system, the screening for hCAR ligands can be conducted without sacrificing animals.