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Browsing by Subject "16α-hydroxyestrone"

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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.