Vpliv izbranih bisfenolov na aktivnost glukokortikoidnih receptorjev, izraženih v celicah MDA-kb2

Beganović, Amela (author), Sollner Dolenc, Marija (mentor), Zore, Taja (comentor)

Bisfenoli so spojine, ki se v velikih količinah uporabljajo pri izdelavi plastike. Vsakodnevno smo izpostavljeni nizkim koncentracijam bisfenolov, saj se ti nahajajo v izdelkih za vsakodnevno uporabo, v hrani, vodi in tudi zraku. Najbolj zastopan bisfenol je bisfenol A (BPA), ki je precej poznan tudi laični populaciji, saj je znan motilec endokrinega sistema in posledično že skoraj deset let prepovedan v otroških plastenkah. Kot alternativa pri izdelavi plastike in ostalih proizvodov so se zato pojavili različni analogi BPA, ki so manj raziskani, a imajo zaradi podobne strukture verjetno podobne učinke na endokrini sistem. Njihovi učinki na estrogenski in androgeni receptor so najbolj preučevani, naloga naše raziskave pa je bila ugotoviti, kakšen vpliv imajo na glukokortikoidni receptor (GR). S pomočjo celične linije MDA-kb2 smo ugotavljali vpliv sedmih bisfenolov na GR. Najprej smo izbranim analogom določili najnižjo citotoksično ali najvišjo necitotoksično koncentracijo s testom citotoksičnosti z resazurinom. Najnižja citotoksična koncentracija je za bisfenol E (BPE) znašala 150 µM pri testiranju na agonistično delovanje, za bisfenol P (BPP) 25 µM tako pri testiranju na agonistično kot tudi na antagonistično delovanje in za tetrametil bisfenol F (TMBPF) 25 µM pri testiranju na agonistično in 50 µM pri testiranju na antagonistično delovanje. Bisfenol AP (BPAP) in bisfenol B (BPB) sta se obarjala v mediju pri koncentracijah, višjih od 50 µM, zato sta njuni najvišji testirani koncentraciji bili 25 µM za BPAP ter 50 µM za BPB in se nista izkazali kot citotoksični. Najvišja testirana koncentracija za 2,4-bisfenol S (2,4-BPS) in bisfenol F (BPF) je bila 50 µM, pri kateri spojini nista bili citotoksični. Nadalje smo ugotavljali vpliv bisfenolov na GR s pomočjo luciferaznega testa. Rezultati testiranja so pokazali, da spojina BPE deluje agonistično na GR (EC50 = 13,65 µM), spojine BPAP (IC50 = 15,75 µM), BPB (IC50 = 24,55 µM) in BPP (IC50 = 13,04 µM) pa delujejo antagonistično na GR. Z dodatnim testom agonističnega delovanja na GR s pomočjo mifepristona (RU-486) smo potrdili domnevo, da je BPE povzročil zvišanje aktivnosti luciferaze preko GR, z dodatnim testom inhibicije luciferaze pa da so BPAP, BPB in BPP povzročili znižanje aktivnosti luciferaze preko GR. Rezultati raziskovalnega dela nakazujejo na to, da imajo nekateri bisfenoli lahko vpliv na glukokortikoidni sistem. Da bi popolnoma toksikološko ovrednotili te spojine, pa bi potrebovali še več in vivo, in vitro ter epidemioloških raziskav.

• Language: Slovenian
• Keywords: motilci endokrinega sistema, glukokortikoidni receptor, bisfenol A, analogi bisfenola A, celična linija MDA-kb2
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-120213
• Publication date in RUL: 17.09.2020

Secondary language

• Language: English
• Title: Influence of selected bisphenols on the activity of glucocorticoid receptors expressed in MDA-kb2 cells

Abstract

Bisphenols are compounds that are used in big quantities for manufacturing of plastics. We are exposed to low concentrations of bisphenols daily because they can be found in everyday products, food, water and air. Most common bisphenol is bisphenol A (BPA) which is recognised even in the lay population because it is a known endocrine disruptor and has subsequently been prohibited in baby bottles for the past 10 years. As an alternative, many other bisphenol analogues have come into use but because of their similar chemical structure they are expected to have similar effects on the endocrine system. Their activity on estrogen and androgen receptors is the most studied, but the goal of our research was to determine their effect on glucocorticoid receptors (GR). The activity of seven bisphenols on the GR was assessed on the MDA-kb2 cell line. We performed a cell viability assay with resazurin to determine the lowest cytotoxic or highest non-cytotoxic concentration for each compound. Lowest cytotoxic concentration for bisphenol E (BPE) was 150 µM at testing for agonistic activity, for bisphenol P (BPP) it was 25 µM both at testing for agonistic and antagonistic activity and for tetramethyl bisphenol F (TMBPF) it was 25 µM at testing for agonistic activity and 50 µM at testing for antagonistic activity. Bisphenol AP (BPAP) and bisphenol B (BPB) had a low solubility in the test medium at concentrations higher than 50 µM so the highest concentrations we could test for these two compounds were 25 µM for BPAP and 50 µM for BPB, which were not found to be cytotoxic at either type of testing. For 2,4-bisphenol S (2,4-BPS) and bisphenol F (BPF) the highest tested concentration of 50 µM was not cytotoxic at both types of testing. We assessed glucocorticoid activity of bisphenols with luciferase assay. Only BPE showed agonistic activity on GR (EC50 = 13,65 µM). BPAP (IC50 = 15,75 µM), BPB (IC50 = 24,55 µM) and BPP (IC50 = 13,04 µM) showed antagonistic activity on GR. With an additional test of agonistic activity with mifepristone (RU-486) we confirmed that BPE caused an elevation in luciferase activity through agonistic activity on GR. With an additional test of inhibition of enzyme luciferase we showed that bisphenols that exhibited antagonistic activity lowered luciferase activity not by inhibiting the enzyme luciferase but through activity on GR. The results from our research imply that some bisphenol analogues could have an impact on the glucocorticoid system. More in vivo, in vitro and epidemiological research is needed to fully determine the toxicological profile of these compounds.

• Keywords: endocrine disruptors, glucocorticoid receptor, bisphenol A, bisphenol A analogues, cell line MDA-kb2