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Določanje učinka zmesi paracetamola, diklofenaka, 4'hidroksidiklofenaka ter resveratrola na tiroidne receptorje, izražene v celicah GH3.TRE-Luc
ID Bergant Loboda, Kaja (Author), ID Sollner Dolenc, Marija (Mentor) More about this mentor... This link opens in a new window

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Abstract
Tiroidni motilci so snovi, ki motijo delovanje ščitničnega sistema. Do sedaj je znanih kar nekaj snovi, ki se vpletajo v ščitnični sistem, malo pa je znano o vplivu zdravilnih učinkovin in njihovih presnovkov na ta sistem. Ker pa smo običajno sočasno izpostavljeni kombinaciji različnih učinkovin naenkrat, smo se odločili, da v naši raziskavi testiramo, kako zmes različnih spojin vpliva na modulacijo tiroidnih receptorjev (TR). Do sedaj je znanih nekaj raziskav, ki opisujejo delovanje nekaterih zmesi na ščitnični sistem, vendar nobena od njih ne vključuje zdravilnih učinkovin. Izbrali smo si kombinacijo spojin, ki so pogosto v uporabi in so zato tudi pogosto prisotne v okolju. Njihova skupna lastnost je protivnetno delovanje. Diklofenak (DIC) spada med nesteroidne antirevmatike (NSAID), 4'-hidroksidiklofenak je njegov presnovek, ki je še vedno farmakološko aktiven. Tudi paracetamol (PAR) in resveratrol (RES) imata šibko protivnetno delovanje, čeprav praviloma izkazujeta vpliv na COX-sistem v višjih odmerkih od terapevtskih. Te zdravilne učinkovine so nam prosto dostopne, saj so na voljo tudi kot zdravila brez recepta, RES pa kot prehransko dopolnilo. Njihova prisotnost v okolju je neizogibna, saj tja pridejo s pomočjo odplak iz gospodinjstev, bolnišnic ali tovarn. V naši raziskavi smo preverili, kako vpliva zmes različnih učinkovin na receptorje TRα in TRβ, izražene v celični liniji GH3.TRE-Luc. Najprej smo izvedli test citotoksičnosti, da smo preverili ali preiskovane spojine vplivajo na živost (viabilnost) celic. RES se je pokazal kot citotoksičen pri koncentracijah 100 μM in 250 μM. Ostale testirane zmesi, brez RES-a, niso bile citotoksične. Zatem smo s pomočjo luciferaznega testa preverili, ali so naše spojine sposobne modulacije TR. Testirali smo kombinacije spojin PAR+DIC, PAR+4-HD, DIC+4-HD in kombinacijo vseh treh spojin PAR+DIC+4-HD. Kot pozitivno kontrolo pri luciferaznem testu smo uporabili bisfenol A (BPA). Za DIC, njegov presnovek in PAR smo na osnovi predhodnih testiranj vedeli, da izkazujejo antagonistično delovanje na TR. Ker smo za RES potrdili le agonistično delovanje (EC50= 0.3956 nM), RES v testiranje različnih kombinacij prej omenjenih spojin nismo vključili. Ostale spojine smo dodali v testirane zmesi v takšnih koncentracijah, kot so znašale njihove vrednosti IC50. Te so znašale za DIC, 4-HD in PAR 4.106 μM, 62.140 μM in 1.811 μM v tem zaporedju. Vrednost IC50 za BPA je znašala 52.7 μM. Aktivnost luciferaze je za 4-HD znašala 0.62-krat, za BPA pa 0.51-krat glede na kontrolo (0.1 % DMSO v PCM-ju). Za zmesi so aktivnosti luciferaze, glede na kontrolo, znašale za PAR+DIC, PAR+4-HD, DIC+4-HD, PAR+DIC+4HD 1.31, 0.54, 0.47, 0.47 v tem zaporedju. Zmesi spojin PAR+4-HD, DIC+4-HD in PAR+DIC+4-HD so delovale antagonistično na TR, medtem ko je zmes PAR+DIC imela agonistično delovanje na TR. Rezultati kažejo na to, da zmes spojin vpliva na modulacijo TR na drugačen način kot posamezna spojina, vendar je težko napovedati, kakšni bodo učinki posamezne zmesi. To pa potrjuje potrebo po upoštevanju možne prisotnosti različnih spojin v organizmih, ko se ocenjuje varnost uporabe spojin, predvsem tistih, ki modulirajo delovanje endokrinega sistema.

Language:Slovenian
Keywords:hormonski motilci paracetamol diklofenak tiroidni receptor celična linija GH3 TRE-Luc resveratrol
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:FFA - Faculty of Pharmacy
Place of publishing:Ljubljana
Publisher:[K. Bergant]
Year:2015
Number of pages:XIII, 71 f.
PID:20.500.12556/RUL-121034 This link opens in a new window
UDC:612.018:615(043.3)
COBISS.SI-ID:3919985 This link opens in a new window
Publication date in RUL:29.09.2020
Views:1017
Downloads:119
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Secondary language

Language:English
Title:Determination of the effects of paracetamol, diclofenac, 4'-hidroxydiclofenac and resveratrol mixtures on thyroid receptors, expressed in cells GH3.TRE-Luc
Abstract:
Thyroid disruptors are substances that interfere with the functioning of the thyroid system. Up to now several substances are known that are interlacing in the thyroid system, but little is known about the effects of medicines and their metabolites on this system. Since we are usually simultaneously exposed to a combination of different medicines at once, we decided that in our study we will test how a mixture of different compounds affects the modulation of thyroid receptors (TR). Until now various research describe the effect of some mixtures on the thyroid system, but none of them includes the healing active substances. We have chosen a combination of compounds, which are often used, and thus are often present in the environment. Their common feature is the anti-inflammatory activity. Diclofenac (DIC) is one of the non-steroidal nonsteroidal anti-inflammatory drugs (NSAIDs), 4'-hidroxyidiclofenac is its metabolite, which is still pharmacologically active. Paracetamol (PAR) and resveratrol (RES) have a weak anti-inflammatory activity, too, although as a rule they show the impact on the COX-system in higher doses than the therapeutic ones. These healing active substances are freely available to us, they are also available as non-prescription medicines and RES is available as a dietary supplement. Their presence in the environment is inevitable, because they come there through the sewage from households, hospitals or factories. In our study, we examined the impact of a mixture of different substances on receptors TRα and TRβ expressed in the cell line GH3.TRE-Luc. First, we performed a cytotoxicity test to check whether the investigated compounds affect the cell survival. RES has proved to be cytotoxic at concentrations of 100 μM and 250 μM. Other tested mixtures, without RES, were not cytotoxic. After that we used the luciferase assay to verify whether our compounds are capable of modulation of TR. We have tested a combination of compounds PAR + DIC, PAR + 4-HD, DIC + 4-HD and the combination of all three compounds PAR + DIC + 4-HD. As a positive control for the luciferase assay, we used bisphenol A (BPA). For DIC, its metabolite and PAR, based on pre-tests, we knew that they show antagonistic effects on TR. We confirmed only the agonist activity (EC50= 0.3956 nM) with RES and therefore we did not include RES in the testing of various combinations of the aforementioned compounds. We added the other compounds in the tested mixtures in their IC50 concentrations. These amounted 4.106 μM, 62.140 μM and 1.811 μM for DIC, 4-HD and PAR, respectively. The IC50 value for BPA was 52.7 μM. The activity of luciferase for 4-HD was 0.62-fold and for BPA 0.51-fold in comparison with the control (0.1% DMSO in the PCM). For mixtures, the luciferase activity in comparison with the control, was 1.31, 0.54, 0.47, 0.47 for PAR+DIC, PAR+4-HD, DIC+4-HD, PAR+DIC+4HD, respectively. Mixtures of compounds PAR+4-HD, DIC+4-HD and PAR+DIC+4-HD acted antagonistically on TR, whereas the mixture of PAR+DIC had an agonist activity on TR. The results show that the mixture of the compounds affects the modulation of TR in a different way as a single compound, but it is difficult to predict what the effects of the individual compounds will be. This confirms the need to take into account the possible presence of different compounds in organisms when assessing the safety of substances, particularly those that modulate the functioning of the endocrine system.

Keywords:endocrine disruptors paracetamol diclofenac resveratrol thyroid receptor GH3 TRE-Luc cells

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