Endocrine disruptors are defined, according to World Health Organization (2002), as exogenous substances or mixtures that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations (1). Exposure to endocrine disruptors is associated with numerous diseases of our time, such as metabolic syndrome, cardiovascular disease, infertility and cancer. Their use is widespread and it is apparent that they can interfere with biological processes in our body at very low, environmentally relevant concentrations. Bisphenol A, for example, can cause measurable changes in certain cellular functions at concentrations of 1pM-1nM (2). Extensive research was performed in recent years in the area of endocrine disruptors. However, many questions remain open and therefore pose a challenge for the future. In this doctoral thesis, two groups of potential endocrine disruptors were evaluated: structural analogs of bisphenol A (BPA), like bisphenol AF (BPAF), bisphenol S (BPS) and bisphenol F (BPF), which are used as BPA substitutes in the manufacture of plastic and thermal paper and novel brominated flame retardants 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), and bis (2-ethylhexyil) 2,3,4,5-tetrabromophtalate (TBPH). Flame retardants are added to everyday products like electronic devices and furniture, to prevent fires. Selected compounds are extremely interesting from a toxicological point of view, because the general population is exposed daily to low concentrations of these chemicals. However, their impact on human health is still largely unknown. The main goal of this doctoral thesis was the evaluation of endocrine activities of selected compounds and their metabolites on the following endocrine receptors in the body: estrogen (ER), androgen (AR), thyroid (TR), glucocorticoid (GR), pregnane X receptor (PXR), farnesoid X receptor (FXR), and peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ), with particular emphasis placed on the effects of metabolism on endocrine activity. Metabolism can have a major influence on the endocrine activity of compounds. Conjugates (glucuronides, sulphates, ...) are usually without activity on endocrine receptors, while oxidative metabolites can have a significantly higher activity from the parent compound (e.g Ipso metabolite of BPA has 500 times greater estrogen activity than BPA). In the first section, the metabolism of selected bisphenols was evaluated. In an article entitled Differences in glucuronidation of bisphenols F and S between two homologous human UGT enzymes, 1A9 and 1A10, glucuronidation of selected bisphenols, both with microsomal fractions as well as with individual UGT enzymes, was studied. We showed that glucuronidation represents the most important metabolic pathway for selected bisphenols, and we highlighted the importance of non-hepatic tissues in the glucuronidation of bisphenols. Despite the great structural similarity between the selected bisphenols, various UGT enzymes are responsible for their glucuronidation. Liver UGT1A9 was the most important for glucuronidation of BPS, UGT2B15 for BPA, intestinal UGT1A10 isoform for BPF and UGT2A1 for BPAF (3). Kinetics parameters of bisphenols glucuronidation with hepatic and intestinal microsomes, that were previously determined in in vitro studies, were further used in collaboration with researchers of the Swiss Institute of Technology, for the establishment of a pharmacokinetic model. With the model we wanted to evaluate the pharmacokinetics of BPA and its analogs, and consequently compare the impact of replacing BPA with other bisphenols. The results of this work are presented in an article entitled Physiologically based pharmacokinetic (PBPK) modeling of bisphenols BPA, BPS, BPF and BPAF with new experimental metabolic parameters: Comparing the pharmacokinetic behavior of BPA with its substitutes. The built-in physiological pharmacokinetic model is the first that describes the kinetics of structural analogs of BPA, and thus represents an important contribution in the field of pharmacokinetics. With the model we found that the replacement of BPA with structural analogs does not necessarily lower the risk of endocrine activity (4). Furthermore, the oxidative metabolism of bisphenols was studied. We focused mainly on BPS oxidative metabolism and the results are collected in an article entitled Influence of metabolism on endocrine activities of bisphenol S. The major oxidative metabolic pathway of BPS is the ortho hydroxylation of the aromatic ring, catalysed by cytochromes CYP3A4, CYP2C19 and CYP2C9. Hydroxylation at meta position and the formation of ipso metabolites were minor oxidative transformations. In contrast to BPA, reactive metabolites of BPS, that were tracked as glutathione conjugates, were not detected (5). The second part of our research included evaluation of endocrine activities of selected compounds and their metabolites. We used variety of in vitro test systems: reporter assay in GH3.TRE-Luc cell line (thyroid activity), reporter assay in MDA-kb2 cell line (androgen and glucocorticoid activity), YES/YAS assay with Saccharomyces cerevisiae (estrogen and androgen activity) and OECD validated reporter gene assay on Hela9903 cell line (estrogen activity). In collaboration with colleagues from University in Perugia, Italy, the activities of selected compounds on pregnane X receptor (PXR), farnesoid X receptor (FXR), and peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) were evaluated. In the paper entitled Influence of metabolism on endocrine activities of bisphenol S, we showed that in the case of BPS, metabolism works as a protective system that lower the endocrine activity of BPS and also accelerates its elimination from the body. BPS showed weak agonist activity on ER (EC50 = 84 μM). Ortho hydroxylated metabolite showed weaker activity on the ER than BPS (EC50 = 670 μM), while BPS glucuronide was without any activity on ER (5). The endocrine activities of BPA, BPAF and BPAF glucuronide were collected in the article entitled Endocrine activities and adipogenic effects in mouse preadipocytes of bisphenol AF and its main metabolite (sent for publication). BPAF showed more potent activity on tested receptors than BPA. The estrogenic activity of BPAF, determined by an in vitro assay using the Hela9903 cell line, was 10 times higher than the BPA activity (EC50 [BPAF] = 0.15 μM; EC50 [BPA] = 1.12 μM). BPAF was also more potent antagonist on TR (IC50 [BPAF] = 7.6 μM; IC50 [BPA] = 88.2 μM) and AR (IC50 [BPAF] = 2.9 μM; IC50 [BPA] = 5.5 μM). BPAF glucuronide, in contrast, was inactive on ER, TR, AR, GR and FXR receptors. However, weak antagonist activity of BPAF glucuronide on PXR and PPARγ receptors was determined. BPAF glucuronide is therefore not an inactive metabolite, as it was believed for a long time. The results of endocrine activities were supplemented by studying the effect of bisphenols on lipid accumulation. Both, BPAF and BPAF glucuronide, significantly increased lipid accumulation and the expression of different adipogenic markers. We showed that glucuronides can have biological activity, which is an important discovery in this field, and indicates the need for further research. Selected flame retardants and their metabolites showed activities on numerous endocrine receptors in the body. In an article entitled New brominated flame retardants and their metabolites as activators of the pregnane X receptor, the PXR agonist activities of both flame retardants (TBB and TBPH) and their metabolites 2,3,4,5-tetrabromobenzoic acid (TBBA) and the 2-ethylhexyltetrabromobenzoate ester (TBMEPH) were presented. The metabolite TBMEPH was more potent PXR agonist than TBPH. All tested compounds increased the expression of the cytochrome CYP3A4 gene, which can result in different interactions at metabolism level (6). In the article entitled Estrogenic and androgenic activities of TBBA and TBMEPH, metabolites of novel brominated flame retardants, and selected bisphenols, using the XenoScreen XL YES/YAS assay, the activities of selected flame retardants and their metabolites on the ER and AR were determined, using in vitro assay on yeasts. The metabolites of selected flame retardants (TBBA and TBMEPH) were ER and AR antagonists, while parent compounds were without activity on tested receptors. Metabolism can therefore even increase the biological activity of the selected flame retardants (7). Furthermore, using reporter gene assays, activities of selected flame retardants and their metabolites on GR and TR were determined and presented in an article entitled Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays. We found that the selected flame retardants (TBB and TBPH) and their metabolites (TBBA and TBMEPH) act as antagonists on TR, and the starting compounds further act as GR antagonists. Given their widespread use and overall presence in the environment, there is a high concern for their impact on human health, especially in the most vulnerable groups, such as pregnant women and infants (8). In the current doctoral thesis, extensive research on metabolism and endocrine effects of bisphenols and newer brominated flame retardants was conducted. We confirmed the activities of tested compounds on numerous endocrine receptors and thus raise the question about suitability of their use. We found that BPAF glucuronide can also have endocrine activity and other biological effects, like the effect on adipogenesis. We therefore refuted the long-standing belief that glucuronides are only inactive metabolites. The new knowledge about activities of glucuronides should be taken into account when performing risk assessment for selected compounds. The extensive research conducted represent a good starting point for the risk assessment of the selected compounds. Additionally, these results can be valuable for the general population. Raising awareness about the harmful effects of selected compounds on the endocrine system can in the long run lead to lower use of products containing these substances as it was already observed with BPA.
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