Vrednotenje estrogenih učinkov izbranih bisfenolov in izoflavonoidov na celični liniji ERα-HeLa-9903

Flisar, Alja (author), Peterlin Mašič, Lucija (mentor), Gramec Skledar, Darja (co-mentor)

Motilci endokrinega sistema so snovi, ki vplivajo na njegovo homeostazo. V zadnjih 50-ih letih je bila zabeležena porast pojavnosti in razširjenosti zdravstvenih težav, povezanih z motnjami endokrinega sistema, ki so: različne vrste hormonsko-odvisnih rakov, sladkorna bolezen, debelost, metabolni sindrom, zaostanek v razvoju organizma in zmanjšana plodnost. Ker predstavljajo za človeka glavni vir motilcev endokrinega sistema različne snovi, prisotne v hrani, med katere prištevamo med drugim tudi naravne spojine, izoflavonoide, ter sintezne bisfenole, smo se v magistrski nalogi osredotočili predvsem na njih. Namen magistrske naloge je bil s pomočjo testa in vitro, ki temelji na poročevalskem genu, vgrajenem v človeško celično linijo epitelijskih celic raka materničnega vratu, določiti agonistično estrogeno delovanje izbranih izoflavonoidov in bisfenolov. Uporabljena človeška celična linija HeLa-9903 je imela stabilno vstavljena gen za človeški estrogeni receptor alfa in genski konstrukt za encim luciferazo, ki po vezavi agonista na omenjeni receptor omogočata nastanek luminiscentnega signala, katerega jakost omogoča merjenje aktivnosti preučevanih spojin. Preverjali smo tudi živost reporterskih celic s testom citotoksičnosti. Pri tistih preskušanih spojinah, ki so v testih delovale pozitivno in pri katerih smo sumili na prekomerno oz. super-indukcijo gena za luciferazo, smo dodatno izvedli še kompetitivni test, s katerim smo preverili, ali je bil luminiscenčni signal dejansko posledica sproženja omenjenega gena po vezavi določene spojine na človeški estrogeni receptor alfa, ali pa je ta morda delovala na detekcijski sistem preko kakih drugih mehanizmov. Rezultati pri pozitivno delujočih spojinah so pokazali, da je bilo njihovo estrogeno delovanje koncentracijsko odvisno. Vrednosti koncentracij spojin, ki so povzročile agonistični odziv, enak 10% odziva na pozitivno kontrolo (PC10), in tistih, ki so izzvale agonistični odziv, enak 50% odziva na pozitivno kontrolo (PC50), so bile v nM oz. nizkem µM območju, tako v primeru uporabe izoflavonoidov kot bisfenolov. Večina preskušanih izoflavonoidov je pri koncentracijah, >1 μM, povzročila super-indukcijo gena za luciferazo. S kompetitivnimi testi smo za nekatere izoflavonoide dokazali, da so bili odzivi, ki so jih izzvali, posledica njihovega delovanja tako na človeški estrogeni receptor alfa kot preko nekega drugega, še neznanega mehanizma. Kot najmočnejši oz. najučinkovitejši agonisti med preskušanimi izoflavonoidi so se izkazali genistein, formononetin, ononin in prunetin, sledili pa so jim biokanin-A in daidzein ter R,S-ekvol in S-ekvol. Še šibkejša agonista sta bila kalikozin in glicitin, kladrin pa je bil najšibkejši, saj je bil malo nad mejo agonističnega učinka. Puerarin ni deloval agonistično. Vsi preskušani bisfenoli so se izkazali za močne agoniste. Po vrsti so si glede agonističnega delovanja sledili: bisfenol F, bisfenol A in bisfenol AF, s podobnima učinkoma, ter bisfenol S z najmanjšo učinkovitostjo. Zaradi super-indukcije, ki jo je izzvala večina izoflavonoidov, bodo potrebne nadaljnje raziskave glede njihove specifične estrogene aktivnosti. Za to pa bo nujno potrebno razviti nove metode in vitro oz. in vivo, s katerimi bomo lahko pridobili zanesljivejše rezultate.

• Language: Slovenian
• Keywords: motilci endokrinega sistema, test s poročevalskim genom, človeška celična linija HeLa-9903, človeški estrogeni receptor alfa, genski konstrukt za encim luciferazo
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121706
• Publication date in RUL: 23.10.2020

Secondary language

• Language: English
• Title: Estrogenic activity evaluation of selected bisphenols and isoflavonoids on ERα-HeLa-9903 cell line

Abstract

Endocrine disrupting chemicals are substances that affect its homeostasis. In the last 50 years, there have been recordings about growth of incidence and prevalence of health issues due to disruption of endocrine system, including different kinds of hormone-dependent cancers, diabetes, obesity, metabolic syndrome, reduced development of the organism and reduced fertility. Because the main source of endocrine disrupting chemicals for a human represent different substances in food, among which we also count natural compounds, isoflavonoids, and synthetic bisphenols, we have focused in the master's work mainly on them. The purpose of the master's work was to evaluate agonistic estrogenic activity of selected isoflavonoids and bisphenols on epithelial human cervical cancer cell line, with the help of stably transfected transactivation assay in vitro. The used HeLa-9903 human cell line was stably transfected with gene for human estrogen receptor alpha and enzyme luciferase gene construct. After the agonist binds on mentioned receptor, the two produce the luminous signal and its potency enables us to measure the activity of testing substances. We also verified the reporter cells' viability with the citotoxic assay. For substances that caused supra-maximal luciferase activity, we also performed competition assay. With its help we verified, if the luminous signal was really due to gene's induction after binding of selected substance on human estrogen receptor alpha, or it may have induced the detecting system through some other mechanisms. The results among positive-working substances have shown that their estrogenic activity was concentration-dependent. The values of the concentrations, corresponding to 10% (PC10) and 50% (PC50) of the positive control, were in nM or low µM range in testing both isoflavonoids and bisphenols. The majority of tested isoflavonoids caused supra-maximal luciferase activity at concentrations, >1 μM. With competition assays for some of the isoflavonoids we have proven, that they induced responses by their activation of the human estrogen receptor alpha and through some other, yet not known mechanism. Genistein, formononetin, ononin and prunetine proved to be the strongest or the most effective agonists among tested isoflavonoids. They were followed by biochanin-A and daidzein, as well as R,S-equol and S-equol. Even more weaker agonists were calycosin and glycitin, while the weakest one, cladrin, was little above the border of agonistic activity. Puerarin didn't have agonistic activity. All tested bisphenols were proven to be strong agonists. Comparing their agonistic activity, bisphenol F was the most effective agonist, followed by bisphenol A and bisphenol AF, with similar effects, and bisphenol S with the lowest effectiveness. Because of the supra-maximal luciferase activity, which was caused by the majority of isoflavonoids, further researches about their specific estrogenic activity will be necessary. In this case, the development of new methods in vitro and/or in vivo, that would give us more reliable results, will be inevitable.

• Keywords: endocrine disrupting chemicals, transactivation assay, HeLa-9903 human cell line, human estrogen receptor alpha, enzyme luciferase gene construct