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Browsing by Subject "UGT"

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  • Kallionpää, Roope (2014)
    Estrogens are female sex hormones that have genotoxic and proliferation-enhancing effects in cells. Life-time exposure to estrogens is linked to the risk of several cancers. Estrone is only a weak agonist of estrogen receptor but it serves as a precursor for biosynthesis of 17β-estradiol, 16α-hydroxyestrone and catechol estrogens. While 16α-hydroxyestrone has relatively weak affinity for estrogen receptor, it has prolonged effect due to covalent binding to the receptor. UDP-glucuronosyltransferases (UGTs) are phase II metabolic enzymes that conjugate estrogens with glucuronic acid to render them more watersoluble. Polymorphisms in UGT genes have been linked to excretion of steroids and risk of some cancers. Generally, subfamily UGT1A enzymes conjugate the 3-hydroxyls of estrogens, while the activity of subfamily UGT2B is directed towards 16- and 17-hydroxyls. Previous results on estrone glucuronidation are incomplete and conflicting, while glucuronidation of 16α-hydroxyestrone has not been systematically studied. The aim of this study was to identify UGTs active in the glucuronidation of estrone and 16α-hydroxyestrone and to further examine the glucuronidation kinetics of the active UGTs. Also the effects of bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) and mutations of UGT1A10F90 and UGT1A10F93 on glucuronidation activity were examined. Activity assays were conducted using recombinant enzymes as well as human liver and intestinal microsomes. Resulting glucuronides were analyzed using high performance liquid chromatography and quantified based on their UV absorbance. UGT1A3, UGT1A10 and UGT2A1 showed the highest activity toward estrone glucuronidation, while UGT1A10, UGT2A1 and UGT2B7 were the most efficient UGTs conjugating 16α-hydroxyestrone. UGT1A10 had the highest Vmax in the glucuronidation of both substrates, although it conjugated estrone at a higher rate than 16α-hydroxyestrone. UGT1A10F93 was shown to have a role in the different glucuronidation activities of UGT1A10 toward estrone and 16α-hydroxyestrone. Affinity of 16α-hydroxyestrone was highest for UGT2B7, while UGT2B17 conjugated 16α-hydroxyestrone relatively slowly. The results confirm earlier observations of the preference of UGT2B7 for α-configured hydroxyls while UGT2B17 favors β-configuration. UGT2A1 showed no strict regioselectivity but had a relatively weak affinity for both substrates. DMSO was found to decrease UGT activity. However, its presence is necessary to solubilize lipophilic substrates. DMSO concentration has to be kept constant to produce comparable data for, for example, kinetic studies. BSA was found to alter especially the kinetics of UGT2A1. BSA also seemed to have solubility-enhancing effect.
  • Hirvisaari, Laura (2012)
    Estradiol is a female sex hormone which is metabolized to two different catechol estradiols. 2-hydroxyestradiol (2-OHE2) is normally the major catechol estradiol metabolite but breast cancer patients have increased amounts of genotoxic 4-hydroxyestradiol (4-OHE2) and it arises to predominant metabolite with these patients. These catechol estradiols can form reactive quinones that can bind to DNA and lead to mutations and finally cause cancer. Catechol-O-methyl transferase can add methyl groups and UDP-glucuronosyl transferase (UGT) glucuronic acid groups to catechol estradiols. These phase II enzymes play important role in the inactivation of catechol estradiols because only non-conjugated catechol estradiols can be oxidized to quinones. The aim of this study was to find out which human UGTs catalyze glucuronidation of 2-OHE2 or 4-OHE2, how many different glucuronides are formed and in which part of the substrate glucuronic acid is added. To answer these questions chromatography methods for 2-OHE2 and 4-OHE2 glucuronides were developed using HPLC. Eleven UGT-enzymes glucuronidate 2-OHE2. UGTs 1A1, 1A7 and 1A10 form two different glucuronides and UGTs 1A3, 1A8, 1A9, 2A1, 2A2, 2A3, 2B7 and 2B15 form only the second glucuronide. It was possible to detect three different glucuronides for 4-OHE2 but the amount of the first glucuronide was under quantification limit. UGT1A10 catalyzed the formation of the second glucuronide and UGTs 1A7, 1A8, 1A9, 2B7 and 2B15 catalyzed the formation of the last glucuronide. One aim of the study was to find out which part of the substrate is glucuronidated but this aim was not achieved because suitable standards were not available.