Expression of uridinediphosphate glucuronosyltransferase genes: Focus on alternative splicing and transcriptional regulatory mechanisms that contribute to interindividual differences in drug and carcinogen metabolism.

摘要

There is a complex interplay between genetic and environmental factors that determine inter-individual differences in disease disposition and therapeutic response. Drug metabolism pathways have been a major focus of pharmacogenetic studies because inter-individual differences in the expression and activity of these enzymes may cause clinically significant effects on the kinetic properties of various drugs. Because of their role in the metabolism of chemical toxins and carcinogens, genetic differences in drug metabolizing enzymes are also associated with risk of diseases such as cancer. There are many factors governing interindividual variation in drug-metabolizing enzymes including SNPs, epigenetics, alternative splicing events, transcriptional regulation, and post-translational modifications.;UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism and excretion of endogenous and xenobiotic compounds including drugs and carcinogens. UGT enzymes mediate the phase II conjugation of glucuronic acid to their substrates, thereby increasing substrate polarity and facilitating their excretion. Variations in UGT genes are associated with altered drug metabolism and cancer risk. Some of the genetic factors underlying these associations have been discovered, but often there is wide variability in phenotype within a given genotype. The liver is the organ most commonly associated with metabolism, and most UGTs are expressed in the human liver.;A better understanding of the inter-individual variability and relative abundance of UGT gene expression in different tissues is important as this helps determine the physiological relevance of each UGT enzyme. While many previous studies have used qualitative reverse transcription polymerase chain reaction (RT-PCR) for determining which UGT genes are expressed in different tissues, some quantitative analysis of UGT expression has been performed In studies described in this thesis dissertation, real-time PCR was used to quantify the expression of 16 UGT enzymes in multiple specimens of various normal human tissues including lung, liver, larynx, brain, tongue, floor of mouth, tonsil, esophagus, endometrium, and pancreas.;In the liver, there was a high degree of correlation between the expression levels of many UGT enzymes within the same individual, suggesting a common mechanism of transcriptional regulation. The hepatic expression of UGTs is known to be transcriptionally regulated by ligand-activated and liver-enriched transcription factors (LETFs). The hepatic transcriptional regulation of several UGTs has been partially described, with UGT2B10 a notable exception. UGT2B10 exhibits glucuronidation activity against pharmacological substrates (olanzapine), toxins (nicotine), and carcinogens (NNAL), suggesting that inter-individual variability in the expression of this gene may affect both drug metabolism and cancer risk.;In addition to LETFs, UGTs are also regulated by ligand-activated transcription factors. In particular, UGT genes are known to be regulated by antioxidant response elements (AREs). Phase II enzyme inducers such as L-sulforaphane (SFN) have been shown to induce transcription of UGTs through the nuclear factor-erythroid 2-related factor 2 (nrf2)/ARE pathway.;Another potential regulator of inter-individual differences in glucuronidation is alternative splicing. An alternative exon 5 in the common region of the UGT1A gene cluster leads to the expression of 18 additional mRNA species from this locus. The alternative splice isoforms have a dominant-negative effect on the wild type (WT) isoforms in vitro. We hypothesized that inter-individual variation in the relative abundance of WT and splice variant expression affects glucuronidation capacity in human liver, which could carry important pharmacogenetic implications. In these studies, it was determined that UGT1A splice variants, on average, represent less than 7% of the total UGT1A transcript profile in human liver for all hepatic UGT1A species, with relatively low inter-individual variability in expression between different individuals. A consistent pattern was observed in several extrahepatic tissues as well. UGT1A WT and splice variant expression were both correlated with glucuronidation activity. Sequence alignment of the UGT1A alternative exon 5 with the primate-specific alu transposable element revealed that it is a recent evolutionary event, and it displays a low inclusion rate characteristic of similar exons. Alu-derived exons are usually neutral or only slightly deleterious because the novel, alternatively spliced product represents only a small percentage of the total mRNA species. For this reason, the proteins they encode have often been characterized as nonfunctional, evolutionary intermediates.;This dissertation research has contributed to the understanding of mechanisms underlying UGT gene expression. Studies examining the inter-individual expression patterns of UGT enzymes in human tissues, an analysis of the importance of UGT1A alternative splice variants and how they affect glucuronidation capacity in human liver, and an assessment of the transcriptional regulation of the UGT2B10 promoter by LETFs and SFN were performed in this work. Together these data have improved our knowledge of how inter-individual differences in the expression of metabolizing enzymes are manifested and how these differences may potentially play a role in drug and carcinogen metabolism, personalized medicine, and cancer risk assessment. (Abstract shortened by UMI.).