| Metabolic processes prevalent in the liver are important for the elimination of many endogenous and exogenous substrates in the body. For example, the protein product of the UGT1A1 gene is the primary enzyme responsible for the metabolism of bilirubin, and also glucuronidates SN-38, which can exert severe gastrointestinal toxicities in the unconjugated form. UGT1A1-deficient individuals exhibit disease states such as Gilbert's Syndrome or Crigler-Najjar Syndrome, characterized by hyperbilirubinemia. Phenobarbital therapy has been used clinically to treat these conditions because this drug can induce bilirubin glucuronidation. Because of the physiological significance of this gene, UGT1A1 genetic polymorphisms have been studied extensively. However, the mechanism(s) of UGT1A1 regulation by inducing agents has received little attention, despite the potential clinical implications. The objective of this dissertation research was to characterize UGT1A1 expression and regulation by prototypical inducers and nuclear receptor activators utilizing primary cultures of human hepatocytes. Various experimental approaches were undertaken to accomplish this goal including Western immunoblotting, Real-Time PCR (TagmanRTM), enzymatic activity assays and gene reporter assays. Interindividual differences in UGT1A1-induced expression by prototypical inducers were observed. Results from preliminary experiments indicated that an individual's responsiveness to an inducer could be dependent on inducer concentration. Further analyses were performed employing pharmacodynamic modeling to obtain parameters related to the potencies and magnitudes of UGT1A1 regulation by different compounds. Correlation analyses also were performed between UGT1A1 induced expression and UGT1A1 basal expression or nuclear receptor expression. The 3-methylcholanthrene-elicited UGT1A1 response was related directly to UGT1A1 basal levels, whereas UGT1A1 responses by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) and rifampicin exposure did not correlate well with UGT1A1 basal expression. The induction potential and metabolism of the bioflavonoid, chrysin, in human hepatocytes agreed with results observed in clinical studies, in contrast to results obtained in HepG2 cells. The studies performed in this dissertation comprise the most extensive characterization of UGT1A1 expression and regulation by xenobiotics to date. Collectively, data suggest that primary human hepatocytes can be used to assess UGT1A1 regulation and establish the utility of this model system for examining drug-drug interactions mediated by the UGT1A1 gene. |
