Studies of endocrine effects of phenoloc [!] analogs of bisphenol A and their mixtures in silico and in vitro

V doktorski disertaciji smo se osredotočili na identifikacijo potencialnih hormonskih motilcev fenolnih analogov bisfenola A (BPA) iz skupin zdravil, onesnažil in kozmetičnih sestavin, na njihovo pleiotropno delovanje ter preučili hormonske učinke njihovih mešanic. Najprej smo preverili zastavljeno hipotezo doktorske disertacije ali je modulacija hormonskega sistema odvisna od kemijske strukture. Med fenolnimi analogi BPA smo identificirali prepoznavne strukturne elemente odgovorne za interakcijo z jedrnimi receptorji.Z metodo in silico smo s pomočjo prosto dostopnega programskega paketa Endocrine Disruptome smo napovedali afiniteto vezave z 12 različnimi jedrnimi receptorji za izbran niz spojin. Predvsem smo se osredotočili na bromirana zaviralca gorenja TBB in TBPH, njunametabolita (TBBA in TBMEPH) ter aminofenolne analoge BPA (diklofenak (DIC), 4- hidroksidiklofenak (4-HD), paracetamol (PAR) in piceatanol (PIC)). Testirali smo jih in vitro na celičnih linijah s poročevalskim genom za luciferazo: MDA-kb2 celični liniji, ki ima izražen glukokortikoidni (GR) kot tudi androgeni receptor (AR), in GH3.TRE-Luc celični liniji z izraženima tiroidnima receptorjema α in β (ΤRα in TRβ). Za spojine, ki so izkazovale glukokortikoidno in androgeno moduliranje, smo izvedli še potrditveni test vezave in vitro. Med prvimi smo za prej omenjene spojine dokazali njihove različne hormonske učinke na estrogenskem (ER), androgenem, glukokortikoidnem in tiroidnem receptorju pri koncentracijah, katerim smo ljudje realno lahko izpostavljeni. Ugotovili smo, da metabolizem neposredno vpliva na modulacijo androgenega, glukokortikoidnega in tiroidnega receptorja. Za modulacijo glukokortikoidnega receptorja so pomembni: prosta karboksilna skupina in 2-(etilheksil)-oksikarbonilna skupina vezani na aromatski obroč ter prisotnost treh halogenih atomov v orto položaju. Rezultati so pokazali, da je antiandrogena aktivnost odvisna od števila karbonilnih skupin v molekuli in stilbenske strukture brez dodatnih stranskih verig med benzenskima skupinama. Ugotovili smo, da afiniteto vezave na androgeni receptor izkazujejo spojine, ki imajo v svoji strukturi Ph-N-C=O fragment, OH in NH-C=O skupini ter aromatski obroč. Na splošno, spojine, ki so bromirane na položaju 3 in 5 na fenilnem obroču glede na osnovni skelet, imajo antitiroidni učinek. Opazili smo, da je Ph-N-Ph fragment, ki vsebuje še atom Cl na enem od fenilnih obročev, prisoten pri spojinah z mešanim agonističnim/antagonističnim delovanjem na androgeni receptor. Ugotovili smo tudi, da hormonski motilci androgenega, glukokortikoidnega, tiroidnega in estrogenega receptorja v svoji strukturi vsebujejo fenolne skupine kot tudi OH skupino v para položaju glede na –R ( v našem primeru R je –NH-Ph fragment, -NH-C=O fragment, -C=CH-Ph fragment) vezane na fenolni obroč ter da so te prav tako odgovorne za hormonsko aktivnost omenjenih spojin. V drugem delu doktorske disertacije smo potrdili hipotezi, da je hormonski učinek proučevanih spojin na zgoraj omenjene receptorje odvisen od hkratne prisotnosti drugih snovi in da imajo fenolni analogi BPA pleiotropni učinek. Poleg modulacije jedrnih receptorjev smo se osredotočili tudi na njihovo genotoksično in imunomodulatorno delovanje. Izvedli smo teste in vitro na LCL celični liniji za aminofenolne analoge BPA v smislu določanja vpliva izbranih spojin na produkcijo citokinov. Rezultati na LCL celični liniji kažejo, da imajo določeni aminofenolni analogi BPA imunomodulatorno delovanje pri koncentracijah, najdenih v človeški plazmi ter da na njega znatno vpliva metabolizem teh spojin. Dokazali smo, da lahko predvsem fenolni analogi BPA kot hormonski motilci glukokortikoidnega receptorja spremenijo imunski odziv izpostavljenih posameznikov. S pomočjo kvantno-kemijskih metod smo prvi predstavili mehanizem direktne poškodbe DNA toksičnega metabolita paracetamola (NAPQI). Paracetamol lahko uvrstimo med aminofenolne analoge BPA (tako kot diklofenak (DIC), 4-hidroksidiklofenak (4-HD) in piceatanol (PIC)). Ugotovili smo, da izkazuje aminofenolni analog BPA paracetamol preko svojega metabolita NAPQI nizko verjetnost genotoksičnosti, ki je možna le v primeru izčrpanih zalog glutationa v celici ter da so kinoniminske strukture manj reaktivne v primerjavi s kinonskimi. Predstavili smo podroben pregled vseh najpogosteje uporabljenih spojin, ki vsebujejo kinonsko in kinoniminsko strukturo ali vsebujejo to skupino njihovi metaboliti, ter posledice izpostavitve tem spojinam. Učinke mešanic izbranih spojin iz skupin onesnažil (propilparaben (PP), butilparaben (BP), dietilheksil ftalat (DEHP) in tetrametrin (TM)) in kozmetičnih sestavin (2-metilrezorcinol (2MR), avobenzon (AVB) in butil-hidroksianizol (BHA)) smo določili na celičnih linijah na enak način kot hormonske učinke posameznih spojin. Testiranja učinka mešanic smo se lotili na dva načina: proučevali smo mešanice izbranih spojin pri koncentracijah izpostavitve človeka (1 μM in 10 nM) ter pri koncentracijah, v katerih spojine izkazujejo 50 % spodbujevalni ali zaviralni učinek na proučevane receptorje (EC50 ali IC50). Glede na dobljene rezultate smo ugotovili, da testirane komponente mešanic interagirajo ena z drugo. Pokazali smo, da učinki mešanic niso zanemarljivi ter da jih je nujno potrebno vklopiti v oceno tveganja hormonskih motilcev na zdravje ljudi in njihovo okolje. Ti rezultati kažejo, da je potrebno natančno določiti strategijo za testiranje mešanic spojin, ki bo temeljila na ustreznih toksikoloških testih (že obstoječe in silico in in vitro metode, novi biološki testi ter Euromix modelna orodja za testiranje mešanic).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)