Abstract
CYP3A4 is the predominant isoform of CYP3A in humans and is responsible for the metabolism of a wide range of drugs and endogenous compounds (Dresser et al., 2000 & Guengerich et al., 1998). CYP3A4 has a very broad substrate specificity and accounts for up to 60% of the cytochrome P450 enzyme content in human liver and 70% of P450s in enterocytes (Slaughter and Edwards, 1995 & Westlind et al., 1999). Expression of the CYP3A4 gene is markedly induced both in vivo and in primary hepatocytes in response to treatment with a variety of compounds; many of the most efficacious inducers of CYP3A4 expression are commonly used drugs such as the glucocorticoid dexamethasone, the antibiotic rifampicin, and the antimycotic clotrimazole (Lehmann et al., 1998). The inducibility of CYP3A4 expression levels coupled with the broad substrate specificity of the CYP3 A4 protein represents a basis for drug interactions in patients undergoing combination drug therapy or through environmental exposures. Although a wide interindividual variation exists in levels of expression of CYP3A4, as assessed by direct analysis of liver microsomes (Shimada and Guengerich 1989) and through the use of in vivo probe drugs (Lown et al., 1995), there is limited evidence for a functional polymorphism that involves this P450. The initial aim of my research was to study the molecular mechanisms of induction of CYP3A4 gene by a number of steroids and xenobiotics. Specifically, the role of the antiglucocorticoid RU-486, the human glucocorticoid receptor and the human pregnane X receptor in the steroid and xenobiotic induction of CYP3A4 was established by the use of an in vitro CYP3A4 promoter-reporter gene system in HepG2 cells. Approximately lkb of the regulatory region of the CYP3A4 gene (-1087 to -57bp) was previously isolated by PCR and engineered into a reporter plasmid pCMV-cSPAP to create a construct in which expression of the reporter protein Secretory Placental Alkaline Phosphatase (SPAP) was regulated by the CYP3A4 region. This plasmid was transiently transfected into human liver hepatoma cell line, HepG2, in order to study the effects of xenobiotics and steroids on the regulation of the CYP3 A4 gene. The second aim of my project was to isolate approximately 308 bp of the 5' upstream regulatory region of the CYP3 A4 gene which was previously shown to have base pair changes in either the HNF-5 or the estrogen response elements. Sequence confirmation of the isolated regions followed by cloning into a secretory placental alkaline phosphatase (SEAP) expression vector. The functional effect of these mutations on the transactivation of CYP3A4 gene by steroids and xenobiotics has been accomplished. The results presented in this work show that regulation of CYP3 A4 gene is a complex process and that hGR and hPXR are both involved in the transcriptional regulation of CYP3A4 gene by steroids and xenobiotics, which was proved by changes in the rank order inducer potencies of compounds from the basal to the receptor augmented system. Hence, the likelihood exists that there is mutual competition for these two receptors between endobiotics and xenobiotics, which may decrease the therapeutic effects of many administered xenobiotics. RU-486 (1uM) induced the CYP3A4 reporter gene expression and blocked the hGR-mediated induction of CYP3A4 gene by steroids including DEX. The functional HNF-5 RE and ERE mutations identified within the CYP3A4 promoter provide an insight into the possible interindividual variation in CYP3A activity seen in the human population and their mixed response to xenobiotic treatment. They also provide useful tools in our understanding of the molecular mechanism of CYP3A4 transcriptional gene activation.