Lau San Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Title Page --- p.1 / List of Thesis Committee --- p.2 / Declaration Page --- p.3 / Acknowledgements --- p.4 / Table of Contents --- p.5 / Abstract --- p.10 / 論文撰要 --- p.12 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Drug Metabolism --- p.14 / Chapter 1.2 --- Glucuronidation --- p.16 / Chapter 1.3 --- UDP-glucuronosyltransferase (UGTs) / Chapter 1.3.1 --- Nomenclature --- p.18 / Chapter 1.3.2 --- Tissue Distributions of UGTs --- p.20 / Chapter 1.3.3 --- Genetics --- p.26 / Chapter 1.3.4 --- Evolution of UGTs --- p.28 / Chapter 1.4 --- UDP-glucuronosyltransferase related Human Diseases --- p.33 / Chapter 1.4.1 --- Hyperbilirubinemia --- p.33 / Chapter 1.4.2 --- Cancer --- p.37 / Chapter 1.5 --- Rattus norvrgicus UDP-glucuronosyltransferase 1A8 --- p.38 / Chapter 1.6 --- Aims of the Project --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 1. --- Rat liver mRNA Extraction --- p.43 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.43 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.43 / Chapter 4. --- Construction of bacterial expression vector --- p.43 / Chapter 5. --- Expression of recombinant protein in E.coli --- p.44 / Chapter 6. --- Purification of protein with Ni column --- p.44 / Chapter 7. --- Purification of protein with gel filtration column --- p.44 / Chapter 8. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 9. --- Concentration and Desalting of protein --- p.45 / Chapter 10. --- Enzyme activity of glucuronidation --- p.45 / Chapter 11. --- Near UV and far UV circular dichroism (CD) spectroscopy --- p.45 / Chapter 12. --- Fluorescent properties studies --- p.45 / Chapter 13. --- Western Blotting --- p.46 / Chapter 14. --- 3D modeling of UGT1A8 and interactions with ligands --- p.46 / Chapter 2.2 --- Methods / Chapter 1. --- Rat liver mRNA extraction --- p.46 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.47 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.48 / Chapter 4. --- Cloning of UGT1A8 PCR product into expression vector pRSet B --- p.49 / Chapter 5. --- Confirmation of the presence of insert in the plasmid --- p.51 / Chapter 6. --- Sequence checking for UGT1A8 gene in the pRSet B vector --- p.52 / Chapter 7. --- Expression of recombinant protein in E.coli JM109(DE3) cell strain --- p.52 / Chapter 8. --- Purification of recombinant protein by Ni-column --- p.53 / Chapter 9. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.53 / Chapter 10. --- Recombinant protein purification by gel filtration column --- p.54 / Chapter 11. --- Concentration or Desalting of Purified Protein --- p.54 / Chapter 12. --- Determination of Protein Concentration --- p.55 / Chapter 13. --- Far- UV Circular dichroism spectroscopy --- p.55 / Chapter 14. --- Intrinsic Fluorescence Studies of Proteins --- p.57 / Chapter 15. --- Chemical denaturation stability studies --- p.58 / Chapter 16. --- Glucuronidation protein activity assay --- p.59 / Chapter 17. --- Mutagenesis --- p.60 / Chapter 18. --- Western Blotting for the presence of protein --- p.61 / Chapter 19. --- Protein Modeling with Insight II / Chapter 19.1 --- Construction of substrate 1-napthol structure --- p.62 / Chapter 19.2 --- Obtaining UDP-glucuronic acid in PDB file --- p.63 / Chapter 19.3 --- Obtaining rat UGT1A8 model structure in PDB file --- p.63 / Chapter 19.4 --- Optimization of rat UGT1A8 structure --- p.63 / Chapter 19.5 --- Docking studies of interaction between ligands and protein / Chapter 19.5.1 --- Setting up a Grid --- p.66 / Chapter 19.5.2 --- Docking of 1-napthol to UGT1A8 --- p.67 / Chapter 19.5.3 --- Docking of UDP-glucuronic acid in the complex of UGT1A8 and1- napthol --- p.68 / Chapter 19.5.4 --- Definition of Subsets --- p.68 / Chapter Chapter 3 --- Results --- p.70 / Figure 3.1 The extracted RNA from rat liver tissue --- p.76 / Figure 3.2 DNA gel of PCR amplified gene product --- p.77 / Figure 3.3 Colony PCR of UGT1 A8-pRSetB transformed DH5 a bacteria --- p.78 / Figure 3.4 The alignment of amplified gene sequence with the rat UGT1A8 sequence on NCBI database --- p.79 / Figure 3.5 SDS-PAGE of cell lysates with different expression temperature and time duration --- p.82 / Figure 3.6 SDS-PAGE of bacterial cell lysates --- p.83 / Figure 3.7 SDS-PAGE of Ni-column eluted protein --- p.84 / Figure 3.8 Elution Profile of Gel Filtration Chromatography --- p.85 / Figure 3.9 SDS-PAGE analysis of UGT1A8 fractions from Ni-column and gel filtration column --- p.86 / Figure 3.10 Sequence Alignment of UGTs in the rat UGT1A family and 2D structure prediction of UGT1A8 --- p.88 / Figure 3.11 Circular Dichroism (CD) measurements on rat UGT1A8 --- p.89 / Figure 3.12 Western Blotting of UGT1A8 wild-type and mutant proteins --- p.91 / Table 3.1 The specific activity of wild-type and mutated proteins --- p.92 / Figure 3.13 Fluorescence spectrum of wild type and two charged-residue mutants ofUGTlA --- p.93 / Figure 3.14 Fluorescence spectrum of wild type and Trp mutants of UGT1A8 --- p.94 / Figure 3.15 Chemical denaturation of wild type and Trp-mutated UGT1A8 proteins --- p.95 / Figure 3.16 Resolved Stern-Volmer plot of UGT1A8 on acrylamide quenching --- p.96 / Figure 3.17 The 3D modeling structure of rat UGT1A8 --- p.97 / Figure 3.18 Modeling simulated the interaction between UDP-glucuronic acid and UGT1A8 --- p.98 / "Figure 3.19 Modeling simulated the interaction between UDP-glucuronic acid, 1-napthol and UGT1A8" --- p.99 / Chapter Chapter 4 --- Discussion / Chapter 1. --- Successful Expression of Rat UGT1A8 --- p.100 / Chapter 2. --- The recombinant rat UGT1A8 protein was properly folded and enzymatic functioning --- p.102 / Chapter 3. --- Purified recombinant rat UGT1A8 protein contained well-ordered structure --- p.103 / Chapter 4. --- "Relative positions of Trp38, Trp64, Trp98 and Trp208 in the protein" --- p.105 / Chapter 5. --- Contribution of Trp residues in the folding and stability of the protein --- p.106 / Chapter 6. --- Probing of substrate coupling region by mutagenesis --- p.108 / Chapter 7. --- Interaction studies of substrates and UDP-glucuronic acid with UGT1A8 by computer modeling and docking simulation --- p.109 / Chapter Chapter 5 --- Conclusion --- p.111 / Chapter Chapter 6 --- References --- p.113

University of Macau / Institute of Chinese Medical Sciences

Thesis (MSc)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Dissertation (PhD)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (doctoral)--Rijksuniversiteit te Groningen.

Leung Hau Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Thesis Committee --- p.in / Acknowledgement --- p.II / Abstract --- p.III / 摘要 --- p.V / Table of Contents --- p.VII / List of Figures --- p.X / List of Tables --- p.XIII / Appendix Abbreviations --- p.XIV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Breast Cancer --- p.1 / Chapter 1.2 --- Development of Breast Cancer --- p.2 / Chapter 1.3 --- Risk Factors of Breast Cancer --- p.3 / Chapter 1.3.1 --- Age --- p.3 / Chapter 1.3.2 --- Genetic Factors --- p.4 / Chapter 1.3.3 --- Hormonal Factors --- p.5 / Chapter 1.3.4 --- Lifestyles --- p.6 / Chapter 1.4 --- Drug Metabolism --- p.6 / Chapter 1.5 --- UGT1A1 --- p.7 / Chapter 1.5.1 --- UDP-glucuronosyltransferase --- p.7 / Chapter 1.5.2 --- UGT1A1 --- p.9 / Chapter 1.6 --- Cytochrome P450 I Enzyme Family --- p.10 / Chapter 1.6.1 --- CYP450 subfamily --- p.10 / Chapter 1.6.2 --- CYP1A1 --- p.11 / Chapter 1.6.3 --- CYP1B1 --- p.12 / Chapter 1.7 --- Reasons why UGT1A1 is being studied --- p.13 / Chapter 1.8 --- Outline of this Study --- p.14 / Chapter 1.8.1 --- Effects of Over-expressing UDP-Glucuronpsyltransferase and Cytochrome P450 1A1 Against Xenobiotic Assault in Breast Cancer Cells --- p.14 / Chapter 1.8.2 --- Effects of Genistein and Resveratrol on Phase I and II Enzymes in a Non-cancerous Breast Cell Line --- p.15 / Chapter 1.8.3 --- Effects of UGT1A1 on Cancer Drug Treatment --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.16 / Chapter 2.1 --- Chemicals --- p.16 / Chapter 2.2 --- Cell Culture --- p.16 / Chapter 2.2.1 --- Maintenance --- p.16 / Chapter 2.2.2 --- Preparation of Cell Stock --- p.17 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.17 / Chapter 2.3 --- Cloning and Transfection --- p.18 / Chapter 2.3.1 --- Isolation of RNA from cells and cDNA synthesis --- p.18 / Chapter 2.3.2 --- Amplification of UGTlAl --- p.20 / Chapter 2.3.3 --- Separation and Purification of DNA from Agarose Gel --- p.21 / Chapter 2.3.4 --- Restriction Digestion --- p.22 / Chapter 2.3.5 --- Ligation of DNA Fragment and Vector --- p.22 / Chapter 2.3.6 --- Transformation of DH5a --- p.23 / Chapter 2.3.7 --- Small Scale Plasmid Purification (Miniprep) --- p.24 / Chapter 2.3.8 --- Large Scale Plasmid Purification (Maxiprep) --- p.25 / Chapter 2.3.9 --- Stable Transfection into MCF-7 cells with LipofectAMINE PLUS reagent --- p.26 / Chapter 2.4 --- Analytical Procedures --- p.27 / Chapter 2.4.1 --- Western Blot Analysis --- p.27 / Chapter 2.4.2 --- Measurement of cell proliferation (MTT assay) --- p.28 / Chapter 2.4.3 --- Measurement of DMBA-DNA Adduct Formation --- p.28 / Chapter 2.4.4 --- Comet Assay --- p.29 / Chapter 2.4.5 --- Relative Quantitative Real Time PCR --- p.30 / Chapter 2.4.5.1 --- Real Time PCR Using TaqMan Probe --- p.30 / Chapter 2.4.5.2 --- Statistical Analysis of 2-ΔΔCT Comparative Gene Expression --- p.31 / Chapter 2.4.6 --- Flow Cytometry --- p.31 / Chapter 2.4.7 --- EROD Activity in Intact Cells --- p.31 / Chapter 2.4.8 --- High Performance Liquid Chromatography --- p.32 / Chapter 2.5 --- Statistical Analysis --- p.34 / Chapter Chapter 3 --- Effects of Over-Expressing UDP-GIucuronosyltransferase and Cytochrome P450 1A1 Against Xenobiotic Assault in Breast Cancer Cells --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Results --- p.38 / Chapter 3.2.1 --- Effectiveness of Transfection --- p.38 / Chapter 3.2.2 --- Cell Proliferation Experiments --- p.41 / Chapter 3.2.3 --- Regulation of Estrogen Receptor (ER) Expression --- p.43 / Chapter 3.2.4 --- Formation of DMBA-DNA adduct formation --- p.45 / Chapter 3.2.5 --- Single Cell Gel Electrophoresis (Comet Assay) of DMBA-induced DNA Damage in MCF-7UGT1A1 cells --- p.46 / Chapter 3.2.6 --- HPLC for Estradiol-glucuronidation Analysis --- p.49 / Chapter 3.2.7 --- Single Cell Gel Electrophoresis (Comet Assay) of DMBA or E2-induced DNA Damage in MCF-7cyp1A1 cells --- p.51 / Chapter 3.3 --- Discussion --- p.56 / Chapter Chapter 4 --- Effects of Genistein and Resveratrol on Phase I and II Enzymes in a Non-Cancerous Breast Cell Line --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Results --- p.66 / Chapter 4.2.1 --- "Genistein and Resveratrol Reduced DMBA-induced UGT1A1, CYP1A1 and CYP1B1 Expression" --- p.66 / Chapter 4.2.2 --- Genistein and Resveratrol Reduced the Formation of DMBA-DNA Adduct in MCF-10A Cells --- p.73 / Chapter 4.2.3 --- Genistein and Resveratrol Reduced the Single Strand DNA Damage Generated by DMBA in MCF-10A Cells --- p.76 / Chapter 4.2.4 --- Genistein and Resveratrol Reduced DMBA-induced EROD Activities --- p.81 / Chapter 4.3 --- Discussion --- p.84 / Chapter Chapter 5 --- Effects of Ugtlal on Cancer Drug Treatment --- p.89 / Chapter 5.1 --- Introduction --- p.89 / Chapter 5.2 --- Results --- p.93 / Chapter 5.2.1 --- Cell Proliferation Experiment --- p.93 / Chapter 5.2.2 --- "Expression of Bcl-2 and Bax proteins in Paclitaxel- or VCR-treated MCF-7, MCF-7control and MCF-7UGt1A1 cells" --- p.98 / Chapter 5.2.3 --- Flow Cytometric Analysis of Cell Cycle Phase Distributionin Paclitaxel- or VCR-treated MCF-7 cells --- p.103 / Chapter 5.3 --- Discussion --- p.110 / Chapter Chapter 6 --- Summary --- p.114 / Bibliography --- p.116

The UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that glucuronidate small, lipophilic molecules, thereby altering their biological activity and excretion. In humans, important examples of UGT substrates include molecules of both endogenous and xenobiotic origin; thus, UGTs are considered essential contributors to homeostatic regulation and an important defence mechanism against chemical insult. In keeping with both roles, UGTs are most strongly expressed in the liver, a predominant organ involved in detoxification. Rates of glucuronidation in humans are neither uniform among individuals, nor constant in an individual over time. Genetic determinants and non-endogenous signals are both known to influence the expression of UGTs, which in turn may affect the efficacy of certain pharmaceutical treatments or alter long-term risk of developing disease. Thus, this thesis focuses on the transcriptional regulation of UGT genes in humans, particularly on mechanisms that are likely to be relevant to their expression and variation in the liver. Two major approaches were used: firstly, extensive studies of several UGT promoters were performed to identify and characterise transcriptional elements that are important for UGT expression; and secondly, important hepatic transcription factors were investigated as potential regulators of UGT genes. UGT1A3, UGT1A4 and UGT1A5 are a subset of highly related, but independently regulated, genes of the human UGT1 subfamily. UGT1A3 and UGT1A4 are expressed in the liver, whereas UGT1A5 is not. The presented analysis of the UGT1A3, UGT1A4 and UGT1A5 proximal promoters demonstrates that a hepatocyte nuclear factor (HNF)1-binding site common to all three promoters is important for UGT1A3 and UGT1A4 promoter activity in vitro, but is insufficient to drive UGT1A5 expression. Two additional elements required for the maximal activity of the UGT1A3 promoter were also identified that may distinguish this gene from UGT1A4. UGT1A3 was investigated further, focusing on mechanisms that may contribute to interindividual variation in UGT1A3 expression. Polymorphisms in the UGT1A3 proximal promoter were identified and their functional consequences tested. Known variants of HNF1alpha were also tested for altered activity towards the UGT1A3 gene. UGT1A9 is the only hepatic member of the UGT1A7-1A10 subgroup of UGT1 enzymes. Previous work had identified HNF1-binding sites in all four genes, and HNF4alpha as an UGT1A9-specific regulator. The work presented herein extends these findings to show that HNF1 factors and HNF4alpha synergistically regulate UGT1A9, and that HNF4alpha is not the only transcription factor responsible for the unique presence of UGT1A9 in the liver. Liver-enriched transcription factors screened as potential UGT regulators were chosen from the HNF1, HNF4, HNF6, FoxA and C/EBP protein families. Functional interactions newly identified by this work were HNF4alpha with UGT1A1 and UGT1A6, HNF6 with UGT1A4 and UGT2B11, FoxA1 and FoxA3 with UGT2B11, UGT2B15 and UGT2B28 and C/EBPalpha with UGT2B17. Observations were also made regarding different patterns of interaction between each UGT and the transcription factors tested, particularly HNF1alpha.

Based on reactions with two flavanones, three flavonols, and five flavones the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC-MS/MS fragmentation patterns of [Co(II)(flavonoid – H)(4,7-diphenyl-1,10-phenanthroline)2]+ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones, flavonols and flavones. The UGT1A enzyme selectivities are affected by the presence of a hydroxyl group at the 3, 6, 4’, or 3’ positions as well as by the presence of a methoxy at the 3’ position. The UGT2B enzymes show poor to no reactivity with the flavonols or flavones. This result implies that the greater planarity of the flavonols and flavones compared to structure of flavanones inhibits interaction with the UGT2 enzymes. For baicalein and scutellarein, three of the UGT1A isozymes (1A8, 1A9, and 1A10) resulted in the formation of 6-O glucuronides, enabling the fragmentation rules for the metal complexation/MS/MS strategy to be expanded. / text

Flavonoids are polyphenolics compounds that constitute a major group of plant specialized metabolites, biosynthesized via the phenylpropanoid/polymalonate pathways. The resulting specialized metabolites can be due to decoration of flavonoid compounds with sugars, usually glucose, by the action of regiospecific UDP-glycosyltransferase (UGT) enzymes. In some cases, glycosylation can involve enzymatic attachment of other sugar moieties, such as glucuronic acid, galactose, rhamnose or arabinose. These modifications facilitate or impact the bioactivity, stability, solubility, bioavailability and taste of the resulting flavonoid metabolites. The present work shows the limitations of utilizing mammalian UDP-glucuronosyltransferases (UGATs) for flavonoid glucuronidation, and then proceeds to investigate plant UG(A)T candidates from the model legume Medicago truncatula for glucuronidating brain-targeted flavonoid metabolites that have shown potential in neurological protection. We identified and characterized several UG(A)T candidates from M. truncatula which efficiently glycosylate various flavonoids compounds with different/multiple regiospecificities. Biochemical characterization identified one enzyme, UGT84F9, that efficiently glucuronidates a range of flavonoid compounds in vitro. In addition, examination of the ugt84f9 gene knock-out mutation in M. truncatula indicates that UGT84F9 is the major UG(A)T enzyme that is necessary and sufficient for attaching glucuronic acid to flavonoid aglycones, particularly flavones, in this species. Finally, the identified UG(A)T candidates were analyzed via homology modeling and site-directed mutagenesis towards increasing the repertoire of UG(A)Ts applicable for synthesis of flavonoid glucuronides with potential human health benefits in neurological protection.

Polychlorinated biphenyls (PCBs) decrease thyroid function in rats and mice by inducing activity of a liver enzyme, uridine diphosphate-glucuronosyltransferase (UDP-GT), thereby increasing thyroxine (T4) clearance. This loss of T4 can lead to hypothyroidism. In this study, an assay was validated for measuring UDP-GT activity toward T4 in Japanese quail (Coturnix japonica). Then UDP-GT induction by Aroclor 1254 was evaluated in quail, and quail and mice were compared in their responses to Aroclor 1254. In Experiment 1, Japanese quail and Balb/c mice were dosed orally with vehicle or Aroclor 1254 (250 or 500 mg/kg) and sacrificed five days later. In Experiment 2, Japanese quail were dosed orally with vehicle or Aroclor 1254 (500 mg/kg) and sacrificed either five or 21 days later. Total liver UDP-GT capacity increased with Aroclor 1254 exposure in all treatment groups of both species. Enzyme induction led to a trend to decreased plasma T4 concentrations at both doses and exposure times in quail and significantly decreased plasma T4 concentrations at both doses in mice. PCBs altered thyroid function in quail, but they did not become hypothyroid. This was in contrast to mice, which did become hypothyroid. It is unclear how PCBs affect the hypothalamic-pituitary-thyroid (HPT) axis in quail, and activation of the HPT axis appears to be inhibited in mice. Overall, quail showed a lesser response than mice to equivalent doses of Aroclor 1254, so it appears that birds may be less vulnerable to PCBs than mammals. / Master of Science