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.
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