In doctoral thesis we focused on the identification of new potential endocrine disrupting compounds, their pleiotropic effects and examined hormonal mixture effects thereof. As target group of tested compounds we chose phenolic analogues of bisphenol A (BPA) from the classes of drugs, pollutants and cosmetic ingredients. At first, we verify the hypothesis of the doctoral thesis whether the modulation of hormonal system depends on the chemical structure of the compounds. Among the phenolic analogues of BPA we identified recognizable structural elements responsible for the interaction with nuclear receptors. Using the freely accessible program package, Endocrine Disruptome, we predicted binding affinity to 12 different nuclear receptors for the selected compounds in silico. In particular, we focused on brominated flame retardants (TBB and TBPH), their metabolites (TBBA and TBMEPH) and aminophenol analogues of BPA (diclofenac (DIC), 4-hydroxydiclofenac (4-
HD), paracetamol (PAR) and piceatannol (PIC)). Next, we tested them in vitro on two cell lines with luciferase reporting gene: on the MDA-kb2 cell line, which expresses the glucocorticoid (GR) and the androgen receptor (AR) and the GH3.TRE-Luc cell line expressing thyroid hormone receptors α and β (TRα and TRβ). For compounds which showed modulation of the glucocorticoid and androgen receptor, we additionaly performed an in vitro confirmatory test for binding. Among the first we have proved their different hormonal effects on estrogen (ER) and earlier mentioned receptors at concentrations, which represent real exposure levels on humans. We have found that the metabolism directly affects the
modulation of the androgen, glucocorticoid and thyroid hormone receptors. For the modulation of the glucocorticoid receptor, the following functional groups are important: the free -COOH group and 2-(ethylhexyl)-oxycarbonyl group attached to the aromatic ring and the presence of three halogen atoms in the ortho position. The results showed that the antiandrogen activity depends on the number of carbonyl groups in the molecule and stilbenic scaffold without additional side chains between two benzene rings. We have found that the binding affinity for the androgen receptor depends on the presence Ph-N-C=O fragment, OH and NH-C=O groups, and the aromatic ring in their chemical structure. Also, the compounds with the brominated 3,5-substituent in the phenyl group possess antithyroid hormone effect. It has been observed, that the Ph-N-Ph fragment, that contains the Cl atom on one of the phenyl rings, is present in the compounds with mixed agonist/antagonist androgen activity. We have found that the endocrine disruptors of the androgen, glucocorticoid, thyroid hormone and estrogen receptors in their structures contain phenolic groups and also OH groups in para position with respect to the –R (in this case, R is –NH-Ph fragment, -NH-C=O fragment, -
C=CH-Ph fragment) attached to a phenolic ring. In the second part of the doctoral thesis, we confirm the hypothesis, that the modulation of the
above-mentioned receptors induced by the compounds depends on the presence of other compounds, and that the phenolic analogues of BPA have pleiotropic effect. In addition, we also focus on their genotoxic and immunomodulatory activities. We also performed in vitro assay on the LCL cell line in order to determine the immunomodulatory effect of the aminophenol analogues of BPA. Results obtained using the LCL cell line indicate that certain aminophenol analogues of BPA have immunomodulatory activity at concentrations found in human blood. We proved that the phenolic analogues of BPA as glucocorticoid receptor mimics of the natural ligand, may be associated with altered immune responsiveness.
Making use of the available quantum-chemical methods, we first outline the mechanism of direct damage to the DNA by the toxic metabolite of the aminophenol analogue of BPA (NAPQI). Paracetamol can be classified as an aminophenol analogue of BPA (another are diclofenac (DIC), 4-hydroxydiclofenac (4-HD) and piceatannol (PIC)). We have found that the aminophenol analogue of BPA (PAR) possesses low genotoxicity, which is possible only in the case of the depleted glutathione, and that quinone imines are less reactive when compared to quinones. We presented a detailed overview of most commonly used compounds or their metabolites containing quinone and quinone imine structure as well as the consequences of exposure to these compounds. The effects of mixtures of compounds selected from the groups of pollutants (propylparaben (PP), butylparaben (BP), diethylhexyl phthalate (DEHP) and tetramethrin (TM)), and cosmetic ingredients (2-methylresorcinol (2MR), avobenzone (AVB) and butylated hydroxyanisole (BHA)) was determined in vitro in the same way as the hormonal effects of the individual compounds, explained above. We tested the mixture effects in two different ways: (i) selected compounds were mixed at concentrations to which humans are exposed (1 μM and 10 nM), (ii) at their EC50 or IC50 concentrations. Based on the obtained results, we
found that the components of tested mixtures interact with each other. We pointed out, that the effects of mixtures are not negligible and that it is necessary to include them into the risk assessment of compounds. Also, it is necessary to specify a strategy for testing mixtures of compounds, which will be based on the relevant toxicological tests (already existing in silico and in vitro methods, new biological tests and Euromix modeling tools for testing mixtures).
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