The marked inter-individual differences in drug oxidations observed in humans is often-times associated with particular subfamilies of P450 enzymes. Among the hepatic P450s to exhibit wide variations in substrate oxidations are those in the CYP2C subfamily. Factors contributing to this phenomenon include induction from exposure to certain xenobiotics. Investigations in our laboratory during the previous funding period noted that the antibiotic, rifampicin (RIF) induces proteins and mRNAs corresponding to CYP2C8, CYP2C9, and CYP2C19 in isolated human hepatocytes. These studies gave rise to the hypothesis that xenobiotic response elements (XRE) exist in the 5'-flanking regions of the CYP2C genes. Studies proposed in specific aim 1 are designed to isolate, clone, and sequence the 5'-upstream region of the human CYP2C19 gene. We hypothesize that isolation of this region will provide information regarding xenobiotics that affect expression of this P450. Moreover, isolation of this regulatory region of the CYP2C19 gene is a prerequisite for the experiments planned in specific aim 2. Experiments described in specific aim 2 will test the hypothesis that RIF enhances expression of the human CYP2C P450 genes. Mechanisms governing RIF- mediated induction of CYP2C8, CYP2C9, and CYP2C19 will be explored with our primary focus on distinguishing DNA elements within each CYP2C gene that are responsive to the chemical agent, RIF. In addition, investigation regarding CYP2C11 in rat hepatocytes have indicated that dexamethasone (DEX) enhances expression of this CYP2C enzyme which occurs via the glucocorticoid receptor (GR). Furthermore, RIF has recently been shown to activate the GR. These studies have directed us to hypothesize that the CYP2C enzymes in humans may also be induced by DEX through the GR. Interestingly, both CYP2C8 and CYP2C9 genes possess several GREs. Thus, we will examine whether DEX enhances human CYP2C expression and if glucocorticoid regulation is via the same response element as that identified for RIF. Finally, the function of the response elements will be tested in situ in specific aim 3. For these studies, cultured human hepatocytes will be treated with RIF and/or DEX and CYP2C mRNA and protein levels measured. In addition, catalytic activities representative of each CYP2C enzyme will be assessed to determine whether enhanced expression of the CYP2C genes ultimately leads to an increase in drug metabolism. In this manner, the function of the CYP2C enhancer elements can be verified in intact liver cells. Taken together, studies planned in this renewal application will extend previous investigation s on the human CYP2C enzymes and further examine causes for their inter-individual variability. Understanding molecular events associated with altered gene expression due to xenobiotic exposure can lead to appropriate mechanisms for identifying individuals with reduced or enhanced capacity to metabolize certain drugs.
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