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Thyroid modulating activity of UV-B filter octylmethoxycinnamate on vascular system

Publication . Lorigo, Margarida Ferreira; Oliveira, Maria Elisa Cairrão Rodrigues; Granadeiro, Luiza Augusta Tereza Gil Breitenfeld; Antunes, Carla Patrícia Quintaneiro

Every day, an extensive and varied range of emerging contaminants encounter humans. Exposure to these endocrine-disrupting chemicals (EDCs) is not without risks and has even been associated with adverse effects on multiple human systems, including the cardiovascular system. Due to the fundamental role of thyroid hormones (TH) in vascular homeostasis, EDCs capable of disturbing thyroid homeostasis (thyroid disruptive chemicals, TDCs) may increase the risk of cardiovascular disease (CVD). However, the mechanisms underlying thyroid disruption associated with the progression or development of CVD are not fully understood. Endocrine disruption is one of the biggest concerns and, therefore, one of the most extensive research forces. Endocrine disruption comprises a diverse set of target organs, processes, and modes of action (MoA), with multiple interactions and complex actions. Periods of development, such as pregnancy, are susceptible to endocrine disruption and constitute a priority area of research. Pregnant women often use various personal care products containing UV filters. The UV-B filters are designed to protect humans against the biological effects of UV radiation. However, their use has been questioned, and some concerns of toxicity to human health have emerged, demonstrating an EDC role for some UV filters. Octylmethoxycinnamate (OMC) is one of the most used UV-B filters worldwide. However, the OMC is also an EDC that affects human health and the environment, and its use remains under intense controversy. The scientific community has given special importance to the environmental and severe effects on coral reefs and aquatic species, which led to OMC being withdrawn from the market in different countries since 2018. On the contrary, the endocrine-disrupting effects in humans have been neglected despite in vitro studies having proven effects at the estrogenic level and non-consensual effects concerning (anti)thyroid and (anti)androgenic activities. Based on the above, it is worrying that the effects of a UV filter widely used worldwide are practically unknown on human health. Within the scope of my MSc dissertation, we demonstrated that OMC acts as a rapid vasodilator of the human umbilical artery (HUA). Although this vasorelaxation has a short-term beneficial effect on the vascular system, also observed with other EDCs, in the long term, all EDCs studied impaired the main vasorelaxation mechanisms of HUA through different MoA. In the case of OMC, only one study evaluated its short-term effects on human vasculature, and the effects of its long-term exposure on human health were not known until this research. Therefore, the main objective of this doctoral thesis was to understand whether exposure to the OMC UV filter impairs human vascular function in the presence of thyroid disorders. We hypothesize that exposure to OMC has adverse effects on the human vasculature, altering the expression of different receptors involved in thyroid functions. To achieve our objective and answer our research hypothesis, various in silico (molecular docking), ex vivo, in vitro (HUA and human umbilical artery smooth muscle cells, HUASMC), and in vivo (zebrafish) approaches were outlined. The first research work analysed the effects of long-term exposure to OMC on HUA vascular homeostasis. Potential changes in the vascular responses of proteins and channels involved in contractile processes were the focus of the research. The effects of 24- hour exposure to OMC (1, 10, and 50 μmol/L) were evaluated on HUA contractile responses to serotonin (5-HT) and potassium chloride (KCl). The data showed that exposure to OMC altered HUA vascular homeostasis; therefore, its MoA was explored in more detail by studying the activity of cyclic guanosine 3’,5’–monophosphate (cGMP) and Ca2+ channels (two pathways involved in its relaxation and contraction, respectively). Prolonged exposure to OMC impaired the main vasorelaxant mechanism of HUA. Interference in the nitric oxide (NO)/soluble guanyl cyclase (sGC)/cGMP/protein kinase G (PKG) signaling pathway and modulation of L-Type voltage-operated Ca2+ channels, BKCa1.1 potassium channels (α subunit), and PKG were the MoA implicated in the decrease in the contractile response. This work demonstrated that OMC could compromise vascular homeostasis in pregnant women and may have a role in the induction of hypertensive diseases of pregnancy. The HUA is regulated mainly by local mediators (5-HT and histamine, His). Membrane receptors, such as G protein-coupled receptors, GPCR induce the effects of these mediators which, when altered, compromises maternal-foetal health. Elevated levels of 5-HT and His increased HUA reactivity/sensitivity to these vasoactive agents, increasing vascular resistance and inducing preeclampsia (PE) and gestational hypertension. On the other hand, it has been demonstrated that EDCs can activate membrane receptors and alter signal transduction. Based on the above and considering the results obtained in the first study, our second research work deepened knowledge about the effects of prolonged exposure to OMC on vascular homeostasis, evaluating the modulation of receptors involved in the contractile processes of HUA. In this work, the research focused on the activity and expression of different 5-HT and His receptors. Thus, the effects of 24-hour exposure to OMC (1, 10 and 50 μmol/L) were evaluated on the contractile responses of HUA to 5-HT and His, using different specific agonists and antagonists, namely 5-HT2A receptor agonist (AMHT), 5-HT7 receptor agonist (AS19), 5- HT1B/1D receptor agonist (L69), H1 receptor agonist (BHI), H2 receptor agonist (dimaprit) and H2 receptor antagonist (cimetidine). Our results demonstrated that exposure to OMC increased HUA reactivity to the vasoactive agents under study. Consequently, and in line with previous research, our results denote a potential role for OMC in the development of hypertensive diseases of pregnancy, such as PE or gestational hypertension. In the third research work, the effects of short- and long-term exposure to OMC were evaluated on the vasculature of pregnant women with hypothyroidism. Although it has been suggested that OMC is a TDC due to its action on thyroid hormone receptor (TR), its disruptive effects on thyroid pathologies during pregnancy have never been evaluated. Therefore, in this study, we assessed the disruptive vascular effects of OMC in hypothyroidism conditions. Computational simulations using molecular docking allowed the correlation of vascular changes with the action of OMC on TRα. Our results indicated that OMC alters the contractility patterns of HUA contracted with 5-HT, His, and KCl, possibly due to interference with 5-HT and His receptors and/or involvement of Ca2+ channels (as shown in two previous investigations, in the absence of thyroid pathologies). Molecular docking analysis confirmed OMC competition with the endogenous hormone T3 for binding to the active centre of TRα, supporting the observed vascular changes. Taken together, our data demonstrate that exposure to OMC alters the vascular reactivity of pregnant women with thyroid pathologies and, thus, may be related to an increased risk and development of cardiovascular diseases. In the fourth research work, developing zebrafish embryos were used as a model to study the effects of OMC exposure in conditions of thyroid pathologies. This work aimed to unravel whether and how the response to OMC exposure is altered by developmental hypothyroidism and hyperthyroidism simulated conditions in zebrafish developing embryos and impair their normal development. For this purpose, developmental hypothyroidism and hyperthyroidism were induced through exogenous exposure to PTU (propylthiouracil, an anti-thyroid drug) or T3 (triiodothyronine endogenous hormone), respectively. Then, embryos were exposed during 120h to OMC isolated and in binary mixture with PTU or T3. In hypo- or hyperthyroidism developmental conditions, exposure to OMC impaired the embryonic development of zebrafish through the induction of several malformations. Pericardial and yolk sac oedema, yolk sac delayed absorption, noninflation of the swim bladder, notochord curvature, and lack of pigmentation were the most prevalent malformations. Furthermore, exposure to OMC under these conditions impairs the behaviour of larvae. Altered transcription of genes related with the HPT axis found proves the TDC action of the OMC. Our findings highlight a modified OMC response in the presence of thyroid pathologies, with harmful effects in development period more sensitive to endocrine disruption, which is in line with research work III. Lastly, the fifth research work was based on the study of the molecular interactions of the OMC in the HPT axis and their implications for humans and zebrafish. This in silico research aimed to reveal the different modes of interaction of the OMC at different levels of the HPT axis, comparing it to the PTU and T3. Our results demonstrated an action of OMC at different levels on the HPT axis, with different actions depending on the target. Molecular docking analysis revealed favourable binding to the corticotrophin-releasing hormone (CRH) receptor and the TH transporter protein, transthyretin (TTR). Furthermore, competition with T3 (inverse agonist activity) was observed for TRα and TRβ. Overall, the action of OMC appears to lead to a decrease in TH, which may promote hypothyroidism. This investigation proves that the OMC acts as a TDC and offers new insights about its disruptive action on the HPT axis, capable of supporting previous investigations. In summary, this doctoral thesis analysed the effects of prolonged exposure to OMC on human vasculature for the first time. Our mechanistic and integrative in silico, ex vivo, in vitro and in vivo approaches demonstrate that OMC impairs vascular homeostasis in pregnant women with and without thyroid pathologies, acting through different molecular initiating events (MIEs) and key events (Kes). Consequently, exposure to OMC can induce the development of cardiovascular diseases. Complementation with in vivo studies made it possible to prove the action of OMC on the HPT axis since the response to OMC was altered in the presence of both thyroid pathologies. In silico studies compared its thyroid modulatory activity to that of the endogenous hormone T3 and the antithyroid drug PTU, which revealed interference at different levels in the HPT axis. Considering the agonist and antagonist actions as an endocrine disruptor, we conclude that prolonged exposure to OMC triggers pathophysiological mechanisms capable of inducing vascular changes with implications for human thyroid health.

2025-01-21Doctoral thesis

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