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Authors
Gonçalves, Beatriz da Silva
Abstract(s)
As mitocôndrias são um organelo celular cuja principal função passa pela produção de
energia, mas também está envolvida no controlo de diversos processos fisiológicos, como
na homeostase do cálcio citosólico e na produção de espécies reativas de oxigénio
(EROs). A função mitocondrial e a regulação do equilíbrio redox são fundamentais no
controlo celular e vias de sinalização de morte celular. A cadeia respiratória mitocondrial
é a principal fonte de espécies reativas de oxigénio (EROs) na célula, tornando as
mitocôndrias bastante vulneráveis ao stress oxidativo. Em certas doenças associadas
com o stress oxidativo, as defesas endógenas do organismo não conseguem diminuir os
níveis de EROs produzidas pelas células. A administração de antioxidantes exógenos
pode ser benéfica nestes casos, e já foi sugerido que uma dieta rica em polifenóis se
relaciona com a prevenção de certas condições como cancro, diabetes, doenças
cardiovasculares e neurodegenerativas. Os ácidos fenólicos são uma subcategoria dos
polifenóis, e são excelentes antioxidantes exógenos candidatos a serem usados com uma
finalidade terapêutica, no entanto, o seu sucesso terapêutico é limitado ao nível da
farmacocinética e é necessário modificar essas moléculas para que sejam entregues
especificamente na mitocôndria. A estratégia mais eficaz até ao momento é a ligação
covalente de um catião lipofílico (TPP+) à molécula bioativa. De entre os ácidos
fenólicos, podemos destacar os ácidos hidroxibenzóicos, mais especificamente o ácido
gálico, que serviu de base para o desenvolvimento dos antioxidantes direcionados à
mitocôndria MitoBEN2 e MitoBEN4.
Tendo como hipótese que o MitoBEN2 e o MitoBEN4 têm um potencial terapêutico para
poderem ser utilizados em áreas como a cosmética e no tratamento de diversas
patologias, o objetivo deste trabalho passou pela avaliação dos efeitos citotóxicos e
efeitos protetores dos dois antioxidantes, no metabolismo e bioenergética celular e
mitocondrial em diferentes modelos celulares.
Os fibroblastos normais da derme humana (NHDF), as células humanas de
hepatocarcinoma (HepG2) e as células humanas de neuroblastoma (SH-SY5Y) foram os
modelos celulares utilizados para avaliar o perfil de toxicidade dos antioxidantes, assim
como a sua eficácia protetora quando apresentados a diferentes indutores de stress
oxidativo, nomeadamente menadiona, peróxido de hidrogénio e tert-butil
hidroperóxido. A avaliação da toxicidade e da eficácia dos antioxidantes passou pela
realização de ensaios de viabilidade celular, nomeadamente pela determinação da massa celular e atividade metabólica, avaliação dos níveis intracelulares de trifosfato de
adenosina (ATP) e níveis celulares de glutationa reduzida (GSH) e glutationa oxidada
(GSSG).
Os resultados demonstraram que o MitoBEN2 apresentou baixa toxicidade e alta eficácia
de proteção contra o stress oxidativo induzido, enquanto que o MitoBEN4 apresentou
uma maior toxicidade na maioria dos ensaios realizados. O desenvolvimento do
MitoBEN2 e MitoBEN4 pode ser assim promissor como terapia antioxidante
direcionada às mitocôndrias.
Durante o decorrer do estágio, também foram criados protocolos para a avaliação da
respiração celular e função mitocondrial e para a avaliação dos níveis celulares de GSH,
respetivamente com o ensaio MitoXpress Xtra e o ensaio monobromobimano (mBB). No
ensaio MitoXpress Xtra foram usados os modelos celulares HepG2 e NHDF, e no ensaio
mBB foram usados os modelos celulares HepG2, NHDF e SH-SY5Y, com diversas
densidades celulares, de forma a otimizar os ensaios. Os dois ensaios mostraram ser
promissores, no entanto, é necessário continuar a sua otimização, de forma a aumentar
as densidades celulares utilizadas, obtendo assim resultados melhores e fidedignos para
que possam ser utilizados diariamente no laboratório.
Mitochondria are a cellular organelle whose main function is energy production, but it is also involved in the control of various physiological processes, as in cytosolic calcium homeostasis and production of reactive oxygen species (ROS). Mitochondrial function and regulation of redox balance are critical in cell control and cell death signaling pathways. The mitochondrial respiratory chain is the main source of reactive oxygen species (ROS) in the cell, making the mitochondria vulnerable to oxidative stress. In certain diseases associated with oxidative stress, the body's endogenous defenses cannot lower the levels of ROS produced by cells. The administration of exogenous antioxidants may be beneficial in these cases, and it has been suggested that a diet rich in polyphenols is related to the prevention of certain conditions such as cancer, diabetes, cardiovascular and neurodegenerative diseases. Phenolic acids are a subcategory of polyphenols, and are excellent exogenous antioxidants candidates to be used for a therapeutic purpose, however, therapeutic success is limited at the level of pharmacokinetics and it is necessary to modify these molecules so that they are specifically delivered to the mitochondria. The most effective strategy so far is the covalent attachment of a lipophilic cation (TPP+) to the bioactive molecule. Among the phenolic acids, we can highlight hydroxybenzoic acids, more specifically gallic acid, which served as the basis for the development of mitochondria-targeted antioxidants MitoBEN2 and MitoBEN4. Based on the hypothesis that MitoBEN2 and MitoBEN4 have a therapeutic potential to be used in areas such as cosmetics and in the treatment of various pathologies, the objective of this work was to evaluate the cytotoxic and protective effects of the two antioxidants, in cellular and mitochondrial bioenergetics and metabolism in different cell models. Normal human dermal fibroblasts (NHDF), human hepatocarcinoma cells (HepG2) and human neuroblastoma cells (SH-SY5Y) were the cell models used to assess the toxicity profile of antioxidants, as well as their protective efficacy when presented to different oxidative stress inducers, namely menadione, hydrogen peroxide and tert-butyl hydroperoxide. The evaluation of the toxicity and efficacy of antioxidants included cell viability assays, namely the determination of cell mass and metabolic activity, assessment of intracellular levels of adenosine triphosphate (ATP) and cellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG). The results showed that MitoBEN2 had low toxicity and high protective efficacy against induced oxidative stress, while MitoBEN4 had greater toxicity in most of the tests performed. The development of MitoBEN2 and MitoBEN4 may thus hold promise as mitochondrial-targeted antioxidant therapy. During the course of the internship, protocols were also created for the assessment of cell respiration and mitochondrial function and for the assessment of cellular levels of GSH, respectively with the MitoXpress Xtra assay and the monobromobimane (mBB) assay. In the MitoXpress Xtra assay, the HepG2 and NHDF cell models were used, and in the mBB assay, the HepG2, NHDF and SH-SY5Y cell models were used, with different cell densities, in order to optimize the assays. Both assays proved to be promising, however, it is necessary to continue their optimization in order to increase the cell densities used, thus obtaining better and reliable results so that they can be used daily in the laboratory.
Mitochondria are a cellular organelle whose main function is energy production, but it is also involved in the control of various physiological processes, as in cytosolic calcium homeostasis and production of reactive oxygen species (ROS). Mitochondrial function and regulation of redox balance are critical in cell control and cell death signaling pathways. The mitochondrial respiratory chain is the main source of reactive oxygen species (ROS) in the cell, making the mitochondria vulnerable to oxidative stress. In certain diseases associated with oxidative stress, the body's endogenous defenses cannot lower the levels of ROS produced by cells. The administration of exogenous antioxidants may be beneficial in these cases, and it has been suggested that a diet rich in polyphenols is related to the prevention of certain conditions such as cancer, diabetes, cardiovascular and neurodegenerative diseases. Phenolic acids are a subcategory of polyphenols, and are excellent exogenous antioxidants candidates to be used for a therapeutic purpose, however, therapeutic success is limited at the level of pharmacokinetics and it is necessary to modify these molecules so that they are specifically delivered to the mitochondria. The most effective strategy so far is the covalent attachment of a lipophilic cation (TPP+) to the bioactive molecule. Among the phenolic acids, we can highlight hydroxybenzoic acids, more specifically gallic acid, which served as the basis for the development of mitochondria-targeted antioxidants MitoBEN2 and MitoBEN4. Based on the hypothesis that MitoBEN2 and MitoBEN4 have a therapeutic potential to be used in areas such as cosmetics and in the treatment of various pathologies, the objective of this work was to evaluate the cytotoxic and protective effects of the two antioxidants, in cellular and mitochondrial bioenergetics and metabolism in different cell models. Normal human dermal fibroblasts (NHDF), human hepatocarcinoma cells (HepG2) and human neuroblastoma cells (SH-SY5Y) were the cell models used to assess the toxicity profile of antioxidants, as well as their protective efficacy when presented to different oxidative stress inducers, namely menadione, hydrogen peroxide and tert-butyl hydroperoxide. The evaluation of the toxicity and efficacy of antioxidants included cell viability assays, namely the determination of cell mass and metabolic activity, assessment of intracellular levels of adenosine triphosphate (ATP) and cellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG). The results showed that MitoBEN2 had low toxicity and high protective efficacy against induced oxidative stress, while MitoBEN4 had greater toxicity in most of the tests performed. The development of MitoBEN2 and MitoBEN4 may thus hold promise as mitochondrial-targeted antioxidant therapy. During the course of the internship, protocols were also created for the assessment of cell respiration and mitochondrial function and for the assessment of cellular levels of GSH, respectively with the MitoXpress Xtra assay and the monobromobimane (mBB) assay. In the MitoXpress Xtra assay, the HepG2 and NHDF cell models were used, and in the mBB assay, the HepG2, NHDF and SH-SY5Y cell models were used, with different cell densities, in order to optimize the assays. Both assays proved to be promising, however, it is necessary to continue their optimization in order to increase the cell densities used, thus obtaining better and reliable results so that they can be used daily in the laboratory.
Description
Keywords
Ácidos
Fenólicos Antioxidantes Espécies Reativas de Oxigénio Mitocôndrias Stress Oxidativo