Cytochrome P4503A (CYP3A) enzymes involve the metabolism of two thirds of drugs and other xenobiotics. Induction of CYP3A enzymes by many compounds is known as an important contributing factor to many failures of therapy or severe toxicity. CYP3A induction is featured by marked species difference, structural diversity of the inducers, and inter-individual variation. Analyses of CYP3A promoters locate three cis-response elements likely involved in CYP3A induction. A reporter gene construct containing one of the elements can be transactivated by an orphan receptor designated the pregnane X receptor (PXR), and the differential activation of mouse and human PXRs by several compounds largely reflects the species difference observed in vivo. The central hypothesis of the proposed studies is that PXR plays a determinant role in CYP3A induction and multiplicity/polymorphism, along with inducibility of PXR, are responsible for the species difference, inducer diversity and individual variation.
The specific aims of this project are: (1) to determine the multiplicity/polymorphism of PXR in humans; (2) to determine the essentiality of PXR in the CYP3A induction; (3) to determine the synergistic effects of PXR inducers on PXR activator-mediated CYP3A induction; and (4) to determine important residues of PXRs in conferring CYP3A induction by rifampicin (RIF) and pregnenolone 16alpha-carbonitrile (PCN). As part of the studies to determine the molecular basis for the existence of multiple forms and polymorphic variants of PXRs in humans, a cDNA-trapping method will be used to screen cDNA libraries from hepatic and extrahepatic tissues. Transient cotransfection experiments with a CYP3A reporter will be conducted to determine the activation profile of each PXR. To determine the essentiality of PXR in CYP3A induction, PXR antisense constructs will be tested for their ability to block CYP3A induction; and PXR chimeras with a ligand binding domain from another species will be tested for their ability to modulate CYP3A expression in response to species-selective activators. To determine the synergistic effects of PXR inducers on PXR activator-mediated CYP3A induction, rats and hepatocytes will be treated with PXR inducers, PXR activators, or in combination; induction of CYP3A will be determined by Northern and Western blot analyses. Site-directed mutagenesis experiments will be conducted to determine functionally important residues of PXRs in conferring CYP3A induction by RIF and PCN. Significant progress has been made toward the proposed objective. Full-length cDNAs encoding multiple forms of rodent and human PXRs have been isolated. Several compounds are found to drastically increase rPXR-1 mRNA levels. These results support our hypothesis that multiplicity and polymorphism along with inducibility of PXR are responsible for species difference, inducer diversity and individual variation featured by CYP3A induction.
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