Določanje in vrednotenje encimske kinetike sulfatiranja bisfenola A in vitro

Pokovec, Eva (author), Peterlin Mašič, Lucija (mentor), Trontelj, Jurij (comentor)

Bisfenol A (BPA) je kemična spojina z zelo široko uporabo, zaradi česar ljudje pogosto pridejo v stik z njim. Kot monomer se uporablja pri izdelavi polikarbonatne plastike in epoksi smol. Polikarbonatno plastiko najdemo v plastenkah, vsebnikih za hrano ter medicinskih in športnih pripomočkih; epoksi smole pa se uporabljajo predvsem za premaze pločevink. Zaradi svojega endokrinega delovanja BPA spada med motilce endokrinega sistema in ima za človeški organizem toksične učinke. S svojim delovanjem lahko vpliva na nastanek debelosti, razvoj sladkorne bolezni in srčno-žilnih bolezni. Zelo pomembno in najbolj raziskano je estrogeno delovanje BPA, zaradi česar ga uvrščamo med t. i. ksenoestrogene. Zaradi toksičnih učinkov spojine je pomembno, da se BPA učinkovito izloča iz človeškega telesa, kar se doseže z metabolizmom. Glavna pot metabolizma BPA je glukuronidacija z uridindifosfoglukuronozil-transferazami (UGT), obenem pa poteka tudi sulfatiranje s sulfotransferazami (SULT). O glukuronidaciji BPA je bilo izvedenih že mnogo študij, manj pa je znanega o sulfatiranju, kar smo raziskovali v tej magistrski nalogi. Sulfatiranje BPA smo preiskovali z modelom in vitro s citosolnimi frakcijami človeških jeter, kar nam je omogočilo, da smo bisfenol A res le sulfatirali in hkrati niso potekale še druge encimske reakcije. Vzorce smo analizirali z metodo LC-MS/MS in z umeritveno krivuljo določili koncentracijo in hitrost nastajanja produkta – bisfenol A-monosulfata. Encimska kinetika je sledila Michaelis-Mentenovim predpostavkam in enačbi, s katero smo nato izračunali parametre encimske reakcije. Da smo lahko reakcijo sulfatiranja ustrezno umestili v metabolizem bisfenola A, smo v študijah poiskali parametre glukuronidiranja BPA in jih medsebojno primerjali. Ugotovili smo, da imata obe reakciji podobno Michaelis-Mentenovo konstanto, vendar pa ima reakcija glukuronidiranja višjo maksimalno hitrost, kar se tudi sklada s podatki iz literature in z in vivo opaženim dejstvom, da nastane mnogo več glukuronidov BPA kot sulfatov.

• Language: Slovenian
• Keywords: bisfenol A, sulfatiranje, sulfotransferaze, motilci endokrinega sistema
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-107516
• Publication date in RUL: 24.04.2019

Secondary language

• Language: English
• Title: Determination and evaluation of the kinetics of bisphenol A sulfation in vitro

Abstract

Bisphenol A (BPA) is a chemical compound widely used in consumer products. As a monomer it is a part of polycarbonate and epoxy resins. Polycarbonates can be found in bottles, food containers, sports and medical equipment; while epoxy resins are used for the coating of food cans. Due to its long history of usage, BPA can be also found in the environment. BPA is considered as an endocrine disruptor with many toxic effects to the human organism. Because of its endocrine actions it is probably involved in the development of obesity, type II diabetes and cardiovascular diseases among others. The estrogenic effects of BPA are the best known and evaluated in many researches. Due to its toxicity, the metabolism and elimination of BPA are very important. BPA is mainly metabolised by glucuronidation with UDP-glucuronosyltransferases (UGTs) and a minor pathway of sulfation with sulfotransferases (SULTs). We studied the sulfation of BPA in vitro with human liver cytosol fractions. With this model we were able to study the sulfation separately from other enzymatic reactions. The samples were then analyzed with LC-MS/MS. We calculated the concentration and velocity of the product – BPA-monosulfate – with the calibration curve. The enzyme kinetics corresponded with the Michaelis-Menten equation with which we also calculated the parameters of the reaction. For the evaluation of the sulfation of BPA as a metabolic reaction we compared the results with the parameters of the glucuronidation of BPA. We came to the conclusion that both reactions have a similar Michaelis-Menten constant but the maximal velocity of the two reactions was higher for glucuronidation. The conclusions were consistent with literature and in vivo facts that there are more BPA-glucuronides than sulfates.

• Keywords: bisphenol A, sulfation, sulfotransferases, endocrine disruptors