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Assessing the pro inflammatory effects of bisphenol compounds using exposure relevant in vitro co culture models
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Advisor(s)
Abstract(s)
A inflamação atingiu proporções epidémicas nos países industrializados, principalmente devido a hábitos pouco saudáveis, má alimentação, poluição ambiental e outros fatores ainda não compreendidos. Quando descontrolada ou prolongada, a inflamação pode tornar-se crónica e contribuir para o desenvolvimento de várias doenças humanas, incluindo doenças autoimunes, doenças intestinais e, nos piores casos, tumorigénese e progressão tumoral.
A exposição a disruptores endócrinos (EDCs) é um dos fatores ambientais que contribuem para a inflamação, e estudos recentes trouxeram o grupo dos bisfenóis (BP) para o centro das atenções científicas. Estes compostos têm sido fortemente associados a várias patologias, incluindo inflamação crónica, e o seu efeito na saúde intestinal humana é um tema de grande interesse na atualidade científica.
Tendo isto em conta, o objetivo deste trabalho foi analisar os efeitos de quatro bisfenóis – BPA, BPS-MAE, BPAP e BPP – no stress da barreira intestinal e nos efeitos pró-inflamatórios associados.
Para tal, foi otimizado e estabelecido um sistema de co-cultura, consistindo num protocolo melhorado de células epiteliais polarizadas Caco-2, semeadas em filtros de inserção PET no compartimento apical, juntamente com macrófagos derivados de monócitos THP-1 no compartimento basolateral. Posteriormente, foram testados os efeitos da exposição aos BPs na integridade da barreira, no stress celular e nas citocinas pró-inflamatórias, numa ampla gama de concentrações (de 100 µM a 0,1 µM).
Experimentalmente, verificámos que o modelo foi capaz de fornecer dados específicos sobre os potenciais efeitos dos BPs na saúde. Em termos de resistência transepitelial e stress epitelial, conseguimos identificar algumas tendências claras que precisam de ser consolidadas com mais réplicas experimentais independentes. Em particular, o BPA foi o indutor menos potente de respostas de stress celular e alterações na polarização epitelial, enquanto os análogos de BP testados mostraram ser mais disruptivos do que o BPA, com o BPP a revelar-se potencialmente o mais perigoso, seguido do BPAP e por último o BPS-MAE.
Para avaliar os efeitos moduladores da inflamação desses compostos, testámos macrófagos, tanto diretamente como em co-cultura, para a expressão do marcador pró-inflamatório IL-1β, utilizando uma abordagem de RT-PCR semi-quantitativa. Uma otimização importante foi a sua pré-ativação com IFN-γ para aumentar a sensibilidade do modelo e permitir maior relevância fisiológica. As nossas observações mostraram que, mais uma vez, os novos BPs induziram efeitos mais severos em comparação com o BPA. O BPP revelou-se o indutor mais potente de inflamação, seguido do BPS-MAE. Ambos apresentaram níveis elevados do marcador IL-1β em todas as concentrações testadas. O BPAP e o BPA produziram efeitos mais atenuados, embora significativos em concentrações mais altas.
Em conclusão, este trabalho forneceu-nos resultados fundamentais sobre estes análogos do BPA e os seus efeitos na saúde intestinal, acrescentando novos conhecimentos sobre a "nova geração" emergente de BPs e os seus potenciais efeitos adversos na saúde.
Inflammation has reached epidemic proportions in industrialized countries, mainly due to unhealthy habits, poor diet, environmental pollution and other factors not yet understood. If uncontrolled or prolonged, inflammation can become chronic and contribute to the development of a number of human diseases, including autoimmune diseases, intestinal diseases and, in the worst cases, tumorigenesis and tumor progression. Exposure to endocrine disrupting chemicals (EDCs) is one environmental factor contributing to inflammation, and recent studies have brought the bisphenol (BP) group of EDCs into the scientific spotlight. They have been strongly linked to various pathologies, including chronic inflammation, and their effect on human gut health is a hot topic in the scientific community. With this in mind, the aim of this work was proposed to analyze the effects of four bisphenols, BPA, BPS-MAE, BPAP and BPP, on intestinal barrier stress and associated pro-inflammatory effects. To achieve this, a co-culture system was optimized and established, consisting of an improved protocol of polarized Caco-2 epithelial cells seeded on PET insert filters in an apical compartment, together with THP-1 derived macrophages in a basolateral compartment. Subsequently, the effects of BPs exposure on barrier integrity, cellular stress and pro-inflammatory cytokine were tested in a wide range of concentrations (from 100 µM to 0.1 µM). Experimentally, we found that the model was capable of delivering BP-specific data on potential health effects. In terms of transepithelial resistance and epithelial stress, we were able to identify some clear trends that need to be consolidated with more independent experimental replicates. In particular, BPA was the least potent inducer of cellular stress responses and changes in epithelial polarization, whereas the BP analogues tested proved to be more disruptive than BPA, with BPP appearing to be the most potentially hazardous, followed by BPAP and then BPS-MAE. To access the inflammation-modulating effects of these compounds, we tested macrophages, either directly or as co-cultured cells, for expression of the pro-inflammatory marker IL-1β using a semiquantitative RT-PCR approach. An important optimization was their priming with IFN-γ to increase the sensitivity of the model and allow for more physiological relevance. Our observations showed that, once again, the BP analogues induced greater effects compared to BPA. BPP appeared to be the more potent inducer of inflammation, followed by BPS-MAE. Both showed elevated levels of the IL-1β marker at all concentrations tested. BPAP and BPA produced more attenuated effects, although significant at higher concentrations. In conclusion, this work has provided us with landmark results on these BPA analogues and their effects on gut health, adding new insights into the 'new generation' of emerging BPs and their potential adverse health effects.
Inflammation has reached epidemic proportions in industrialized countries, mainly due to unhealthy habits, poor diet, environmental pollution and other factors not yet understood. If uncontrolled or prolonged, inflammation can become chronic and contribute to the development of a number of human diseases, including autoimmune diseases, intestinal diseases and, in the worst cases, tumorigenesis and tumor progression. Exposure to endocrine disrupting chemicals (EDCs) is one environmental factor contributing to inflammation, and recent studies have brought the bisphenol (BP) group of EDCs into the scientific spotlight. They have been strongly linked to various pathologies, including chronic inflammation, and their effect on human gut health is a hot topic in the scientific community. With this in mind, the aim of this work was proposed to analyze the effects of four bisphenols, BPA, BPS-MAE, BPAP and BPP, on intestinal barrier stress and associated pro-inflammatory effects. To achieve this, a co-culture system was optimized and established, consisting of an improved protocol of polarized Caco-2 epithelial cells seeded on PET insert filters in an apical compartment, together with THP-1 derived macrophages in a basolateral compartment. Subsequently, the effects of BPs exposure on barrier integrity, cellular stress and pro-inflammatory cytokine were tested in a wide range of concentrations (from 100 µM to 0.1 µM). Experimentally, we found that the model was capable of delivering BP-specific data on potential health effects. In terms of transepithelial resistance and epithelial stress, we were able to identify some clear trends that need to be consolidated with more independent experimental replicates. In particular, BPA was the least potent inducer of cellular stress responses and changes in epithelial polarization, whereas the BP analogues tested proved to be more disruptive than BPA, with BPP appearing to be the most potentially hazardous, followed by BPAP and then BPS-MAE. To access the inflammation-modulating effects of these compounds, we tested macrophages, either directly or as co-cultured cells, for expression of the pro-inflammatory marker IL-1β using a semiquantitative RT-PCR approach. An important optimization was their priming with IFN-γ to increase the sensitivity of the model and allow for more physiological relevance. Our observations showed that, once again, the BP analogues induced greater effects compared to BPA. BPP appeared to be the more potent inducer of inflammation, followed by BPS-MAE. Both showed elevated levels of the IL-1β marker at all concentrations tested. BPAP and BPA produced more attenuated effects, although significant at higher concentrations. In conclusion, this work has provided us with landmark results on these BPA analogues and their effects on gut health, adding new insights into the 'new generation' of emerging BPs and their potential adverse health effects.
Description
Tese de mestrado, Ciências Biofarmacêuticas, 2025, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Bisphenols (BPs) BPA BPAP BPS-MAE BPP Inflammation Intestinal barrier Epithelial cellular stress Teses de mestrado - 2025