Ugotavljanje delovanja izbranih anksiolitikov na estrogenske receptorje v celicah hER[alfa]-HeLa-9903 : magistrski študijski program Laboratorijska biomedicina

Zore, Taja (author), Sollner Dolenc, Marija (mentor)

V današnjem času se pojavlja vse več dokazov, da številne spojine, ki so nekoč veljale za varne, motijo delovanje endokrinega sistema in delujejo kot hormonski motilci. Med temi spojinami so tudi zdravilne učinkovine, ki se vsakodnevno uporabljajo za zdravljenje v humani medicini ali veterini. Te zdravilne učinkovine in njihovi metaboliti pa ne predstavljajo samo problem za bolnike in živali, ki ta zdravila prejemajo, ampak tudi za vsa ostala živa bitja. Zaradi povečane uporabe farmacevtskih učinkovin se namreč povečuje tudi njihova prisotnost v okolju, kjer smo jim pa lahko izpostavljeni vsi. Zato smo se tudi odločili, da bomo preverjali možen vpliv izbranih zdravilnih učinkovin na endokrino delovanje. Na podlagi napovedi, ki smo jo pridobili s pomočjo računalniškega programa Endocrine Disruptome, smo se odločili za vrednotenje učinka štirih anksiolitikov – klonazepama, lorazepama, sertralina in mianserina – na estrogenski receptor. Program je namreč za te štiri izbrane anksiolitike napovedal veliko verjetnost vezave oz. agonističnega/antagonističnega delovanja na estrogenski receptor α (sertralinijev klorid) ali β (klonazepam, lorazepam in mianserinijev klorid). Klonazepam se uporablja za zdravljenje epilepsij, preostali trije pa se uporabljajo za zdravljenje raznih duševnih motenj, kot so depresija in anksioznost. V raziskavi smo za ugotavljanje učinkov izbranih spojin na ERα uporabili celično linijo hERα-HeLa-9903, ki izraža funkcionalni estrogenski receptor α. V prvem delu testiranja smo za vse spojine izvedli test viabilnosti, s katerim smo določili njihovo največjo necitotoksično koncentracijo za to vrsto celic. Klonazepam in mianserinijev klorid sta se izkazala za necitotoksična že pri najvišji testirani koncentraciji (100 μM), za lorazepam in sertralinijev klorid pa smo morali poiskati ustrezni nižji koncentraciji. Prva necitotoksična koncentracija za sertralinijev klorid je znašala 25 μM, za lorazepam pa 10 μM. V drugem delu testiranja spojin smo izvedli presejalni luciferazni test, s pomočjo katerega smo določali agonističen/antagonističen učinek preiskovanih spojin na ERα. Rezultati agonističnega testa so pokazali, da nobena izmed preiskovanih spojin ni agonist za ERα, saj pri nobeni nismo zaznali dovolj velikega povečanja luciferazne aktivnosti. Rezultati antagonističnega testiranja pa so pokazali, da je prišlo do statistično značilnega znižanja aktivnosti luciferaze samo pri višjih koncentracijah (50–100 μM) klonazepama. Znižanje pri teh koncentracijah je bilo večje, kot je meja za pozitiven rezultat v protokolu izvedbe testa. Na podlagi tega lahko sklepamo, da je klonazepam pri visokih koncentracijah antagonist za ERα, a bi morali prej preveriti še možnost njegovega delovanja direktno na encim luciferazo. Rezultati našega eksperimentalnega dela niso potrdili, da bi preiskovane spojine delovale kot modulatorji estrogenskega receptorja α. Edini, za katerega se je pokazala možnost, da modulira estrogenski receptor α, je klonazepam.

• Language: Slovenian
• Keywords: hormonski motilci estrogenski receptorji klonazepam lorazepam endokrini sistem sertralin celična linija hER[alfa]-HeLa-9903
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [T. Zore]
• Year: 2018
• Number of pages: XVI, 76 str.
• PID: 20.500.12556/RUL-104396
• UDC: 577.175:615.015(043.3)
• COBISS.SI-ID: 4591217
• Publication date in RUL: 12.10.2019

Secondary language

• Language: English
• Title: Determination of the action of selected anxiolytics on estrogen receptors using the cells hER[alpha]-HeLa-9903

Abstract

Nowadays, there is growing evidence that many compounds that were once considered safe, interfere with the endocrine system and act as endocrine disruptors. Among these compounds are also active substances of drugs that are used on a daily basis for the treatment in human or veterinary medicine. These active substances and their metabolites do not only present a problem for the patients and animals, receiving them, but also for all other living creatures. Due to the increased use of pharmaceuticals, their presence in the environment, where everyone can be exposed to them, is also increasing. Therefore, we decided to check the possible effect of the selected active substances on the endocrine activity. Based on the prediction obtained with the computer program Endocrine Disruptome we decided to evaluate the effect of four anxiolytics – clonazepam, lorazepam, sertraline and mianserin – on the estrogen receptor. The program predicted a high probability of binding or agonistic/antagonistic activity on the estrogen receptor α (sertraline hydrochloride) or estrogen receptor β (clonazepam, lorazepam and mianserine hydrochloride). Clonazepam is used for the treatment of epilepsy, while the other three are used for the treatment of various mental disorders, such as depression and anxiety. In the research, the cell line hERα-HeLa-9903 expressing the functional estrogen receptor α was used to determine the effects of the selected compounds on the ERα. First, a cell viability test was performed for all compounds to determine their maximum non-cytotoxic concentration. Clonazepam and mianserin proved to be non-cytotoxic at the highest tested concentration (100 μM), but for lorazepam and sertraline a corresponding lower concentration had to be found. The first non-cytotoxic concentration of sertraline was 25 μM and of lorazepam was 10 μM. In the second part of the compound testing, a screening luciferase test was performed to determine the agonistic/antagonistic effect of the investigated compounds on ERα. The results of the agonist test showed that none of the investigated compounds was an agonist for ERα, since no significant increase in their luciferase activity was observed. The results of antagonistic testing showed a statistically significant decrease in luciferase activity only at higher concentrations (50-100 μM) of clonazepam. The decrease at these concentrations was higher than the limit for a positive result in the test protocol. Based on that, we can conclude that clonazepam is at higher concentrations an antagonist for ERα, but we should first check the possibility of its action directly on the luciferase enzyme. The results of our experiment did not confirm that the investigated compounds act as estrogen receptor α modulators. Only clonazepam showed a possible ability to modulate the estrogen receptor α.

• Keywords: endocrine disruptors estrogen receptors clonazepam lorazepam mianserine hydrochloride sertraline hydrochloride cell line hERα-HeLa-9903