Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos
Autor(a) principal: | |
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Data de Publicação: | 2015 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações do UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/11232 |
Resumo: | Selenium (Se) is an essential micronutrient present in selenoproteins on living beins, in the form of the amino acid selenocysteine and selenomethionine. Chemically related to Se, tellurium (Te) has no biological function in mammals, however, organic Te compounds showed up as good antioxidant agents. Although ebselen (Ebs), diphenyl diselenide [(PhSe)2] and diphenyl ditelluride [(PhTe)2] present antioxidant properties via their glutathione peroxidase (GPx) mimetic activity, these compounds exhibit toxic effects at high concentrations due to its property in oxidizing thiols groups. However, the toxicity mechanism of these compounds through the modulation of gene expression was never studied in human cells. Thus, this study aimed to evaluate the cytotoxicity and genotoxicity of organochalcogens in human leukocytes, and evaluate their mechanisms through the production of reactive oxygen species (ROS) and modulation of antioxidant proteins expression, and to evaluate the relative amount of organochalcogens in contact with the cells in our ex vivo exposure model. Trypan s blue exclusion test and comet assay were used to evaluate, respectively, cytotoxicity and genotoxicity induced by the compounds. The fluorescence of dichlorofluorescein (DCFH) and propidium iodide (PI) were measured in leukocytes exposed by flow cytometry. The expression of the genes for the enzymes Catalase, Superoxide Dismutase 1, Glutathione Peroxidase 3, Glutathione Peroxidase 4, Thioredoxin Reductase 1 and Nrf-2 were analyzed. A dichloromethane extraction of the buffer and the pellet of the cells was injected into a GC-MS apparatus to evaluate the amount of compound in contact with cells. The compounds induced a reduction in cell viability only in the concentration of 50 μM, being Ebs the most cytotoxic compound, followed by (PhTe)2 and (PhSe)2, while (PhTe)2 was the only compound able of increasing the apoptotic rate of leukocytes, which happened at all concentrations (10-50 μM). (PhTe)2 increased DNA damage index in all tested concentrations (5-50 μM), while Ebs and (PhSe)2 did it only at 50 μM concentration. Surprisingly, (PhSe)2 was the only compound effective in increasing ROS production in all tested concentrations (10-50 μM), which was accompanied by an increase in the SOD1 expression and a decrease in CAT expression. All compounds were effective in decreasing the expression of GPX3 and NFE2L2 (Ebs > (PhTe)2 > (PhSe)2), and none altered the expression of GPX4 and TRXR1. The compounds were found in higher concentrations in leukocyte pellet extraction than in their buffer. We conclude that the toxicity of the compounds in question is not directly related to their property in inducing production of ROS. |
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2015-05-282015-05-282015-02-06BUENO, Diones Caeran. ORGANOCHALCOGEN TOXICITY AND ITS MECHANISMS THROUGH GENE EXPRESSION IN HUMAN LEUKOCYTES. 2015. 69 f. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2015.http://repositorio.ufsm.br/handle/1/11232Selenium (Se) is an essential micronutrient present in selenoproteins on living beins, in the form of the amino acid selenocysteine and selenomethionine. Chemically related to Se, tellurium (Te) has no biological function in mammals, however, organic Te compounds showed up as good antioxidant agents. Although ebselen (Ebs), diphenyl diselenide [(PhSe)2] and diphenyl ditelluride [(PhTe)2] present antioxidant properties via their glutathione peroxidase (GPx) mimetic activity, these compounds exhibit toxic effects at high concentrations due to its property in oxidizing thiols groups. However, the toxicity mechanism of these compounds through the modulation of gene expression was never studied in human cells. Thus, this study aimed to evaluate the cytotoxicity and genotoxicity of organochalcogens in human leukocytes, and evaluate their mechanisms through the production of reactive oxygen species (ROS) and modulation of antioxidant proteins expression, and to evaluate the relative amount of organochalcogens in contact with the cells in our ex vivo exposure model. Trypan s blue exclusion test and comet assay were used to evaluate, respectively, cytotoxicity and genotoxicity induced by the compounds. The fluorescence of dichlorofluorescein (DCFH) and propidium iodide (PI) were measured in leukocytes exposed by flow cytometry. The expression of the genes for the enzymes Catalase, Superoxide Dismutase 1, Glutathione Peroxidase 3, Glutathione Peroxidase 4, Thioredoxin Reductase 1 and Nrf-2 were analyzed. A dichloromethane extraction of the buffer and the pellet of the cells was injected into a GC-MS apparatus to evaluate the amount of compound in contact with cells. The compounds induced a reduction in cell viability only in the concentration of 50 μM, being Ebs the most cytotoxic compound, followed by (PhTe)2 and (PhSe)2, while (PhTe)2 was the only compound able of increasing the apoptotic rate of leukocytes, which happened at all concentrations (10-50 μM). (PhTe)2 increased DNA damage index in all tested concentrations (5-50 μM), while Ebs and (PhSe)2 did it only at 50 μM concentration. Surprisingly, (PhSe)2 was the only compound effective in increasing ROS production in all tested concentrations (10-50 μM), which was accompanied by an increase in the SOD1 expression and a decrease in CAT expression. All compounds were effective in decreasing the expression of GPX3 and NFE2L2 (Ebs > (PhTe)2 > (PhSe)2), and none altered the expression of GPX4 and TRXR1. The compounds were found in higher concentrations in leukocyte pellet extraction than in their buffer. We conclude that the toxicity of the compounds in question is not directly related to their property in inducing production of ROS.O selênio (Se) é um micronutriente essencial presente nas selenoproteínas dos organismos vivos, na forma dos aminoácidos selenocisteína e selenometionina. Quimicamente relacionado ao Se, o telúrio (Te) não possui nenhuma função biológica nos mamíferos, porém, compostos orgânicos de Te se mostraram bons agentes antioxidantes. Apesar do ebselen (Ebs), disseleneto de difenila [(PhSe)2] e ditelureto de difenila [(PhTe)2] possuírem atividade mimética a enzima glutationa peroxidase (GPx), exibindo propriedades antioxidantes, estes compostos apresentam efeitos tóxicos em altas concentrações, devido a sua capacidade de oxidar grupos tióis. Porém, os mecanismos de toxicidade destes compostos através da modulação da expressão gênica nunca foram estudados em células humanas. Desta forma, este estudo objetivou avaliar a citotoxicidade e a genotoxidade dos organocalcogênios em leucócitos humanos, e avaliar seus mecanismos através da produção de espécies reativas de oxigênio (EROs) e modulação da expressão de proteínas antioxidantes, além de avaliar a quantidade relativa de organocalcogênio em contato com as células em nosso modelo de exposição ex vivo. O teste de exclusão do azul de Trypan e o ensaio cometa foram utilizados para avaliar, respectivamente, a citotoxicidade e a genotoxicidade dos organocalcogênios. A fluorescência da diclorofluoresceína (DCFH) e iodeto de propídeo (IP) foi medida nos leucócitos expostos por citometria de fluxo. A expressão dos genes para as enzimas Catalase, Superóxido Dismutase 1, Glutationa Peroxidase 3, Glutationa Peroxidase 4, Tiorredoxina Redutase 1 e Nrf-2 foram analisados. Uma extração de diclorometano do tampão e do pellet das células foi injetada em um aparelho de GC-MS para avaliar a quantidade de composto em contato com as células. Os organocalcogênios induziram a uma redução da viabilidade celular apenas na concentração de 50 μM, sendo que o efeito maior foi do Ebs, seguido pelo (PhTe)2 e pelo (PhSe)2, enquanto o (PhTe)2 foi o único composto capaz de aumentar a taxa apoptótica dos leucócitos, o que aconteceu em todas as concentrações (10-50 μM). O (PhTe)2 aumentou o índice de dano ao DNA em todas as concentrações testadas (5-50 μM), enquanto o Ebs e o (PhSe)2 o fizeram apenas na concentração de 50 μM. Surpreendentemente, o (PhSe)2 foi o único composto efetivo em aumentar a produção de EROs em todas as concentrações testadas (10-50 μM), o que foi acompanhado por um aumento na expressão da SOD1 e uma diminuição da expressão da CAT. Todos os compostos foram efeitovs em diminuir a expressão da GPX3 e do NFE2L2 (Ebs > (PhTe)2 > (PhSe)2), sendo que nenhum alterou a expressão da GPX4 e da TRXR1. Os organocalcogênios foram encontrados em maior concentração na extração do pellet de leucócitos do que no seu tampão. Concluímos que a toxicidade dos compostos em questão não está diretamente relacionada com a propriedade dos mesmos em produzir EROs.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de Santa MariaPrograma de Pós-Graduação em Ciências Biológicas: Bioquímica ToxicológicaUFSMBRBioquímicaSelênioTelúrioOrganocalcogêniosToxicidadeEbselenDisseleneto de difenilaDitelureto de difenilaExpressão gênicaSeleniumTelluriumOrganochalcogensToxicityEbselenDiphenyl diselenideDiphenyl ditellurideGene expressionCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICAToxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanosOrganochalcogen toxicity and its mechanisms through gene expression in human leukocytesinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisRocha, João Batista Teixeira dahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782281H2Nogueira, Cristina Waynehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728219Y9Farina, Marcelohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4706271Y7http://lattes.cnpq.br/1263838473216543Bueno, Diones Caeran20080000000240050030050030027186f63-94dd-4f0f-8181-7ce1b08350096ef82042-d212-4641-bd80-1d9f407e6ac41e373fcc-89bd-4103-adba-676f6dcec787f3323a2d-3cb4-47f8-b0eb-b11216c8695dinfo:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações do UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMORIGINALBUENO, DIONES CAERAN.pdfapplication/pdf4086708http://repositorio.ufsm.br/bitstream/1/11232/1/BUENO%2c%20DIONES%20CAERAN.pdfc588135406b6276c5f5a62fe31f5a771MD51TEXTBUENO, DIONES CAERAN.pdf.txtBUENO, DIONES CAERAN.pdf.txtExtracted texttext/plain103710http://repositorio.ufsm.br/bitstream/1/11232/2/BUENO%2c%20DIONES%20CAERAN.pdf.txtf9650a3c66e9bf691bc744591140a80fMD52THUMBNAILBUENO, DIONES CAERAN.pdf.jpgBUENO, DIONES CAERAN.pdf.jpgIM Thumbnailimage/jpeg5157http://repositorio.ufsm.br/bitstream/1/11232/3/BUENO%2c%20DIONES%20CAERAN.pdf.jpg58d42b7e2dab1a1df677ee33b6372636MD531/112322021-11-17 17:13:08.034oai:repositorio.ufsm.br:1/11232Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufsm.br/ONGhttps://repositorio.ufsm.br/oai/requestatendimento.sib@ufsm.br||tedebc@gmail.comopendoar:2021-11-17T20:13:08Biblioteca Digital de Teses e Dissertações do UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.por.fl_str_mv |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos |
dc.title.alternative.eng.fl_str_mv |
Organochalcogen toxicity and its mechanisms through gene expression in human leukocytes |
title |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos |
spellingShingle |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos Bueno, Diones Caeran Selênio Telúrio Organocalcogênios Toxicidade Ebselen Disseleneto de difenila Ditelureto de difenila Expressão gênica Selenium Tellurium Organochalcogens Toxicity Ebselen Diphenyl diselenide Diphenyl ditelluride Gene expression CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
title_short |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos |
title_full |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos |
title_fullStr |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos |
title_full_unstemmed |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos |
title_sort |
Toxicidade de organocalcogênios e seus mecanismos através da expressão gênica em leucócitos humanos |
author |
Bueno, Diones Caeran |
author_facet |
Bueno, Diones Caeran |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Rocha, João Batista Teixeira da |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782281H2 |
dc.contributor.referee1.fl_str_mv |
Nogueira, Cristina Wayne |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728219Y9 |
dc.contributor.referee2.fl_str_mv |
Farina, Marcelo |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4706271Y7 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/1263838473216543 |
dc.contributor.author.fl_str_mv |
Bueno, Diones Caeran |
contributor_str_mv |
Rocha, João Batista Teixeira da Nogueira, Cristina Wayne Farina, Marcelo |
dc.subject.por.fl_str_mv |
Selênio Telúrio Organocalcogênios Toxicidade Ebselen Disseleneto de difenila Ditelureto de difenila Expressão gênica |
topic |
Selênio Telúrio Organocalcogênios Toxicidade Ebselen Disseleneto de difenila Ditelureto de difenila Expressão gênica Selenium Tellurium Organochalcogens Toxicity Ebselen Diphenyl diselenide Diphenyl ditelluride Gene expression CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
dc.subject.eng.fl_str_mv |
Selenium Tellurium Organochalcogens Toxicity Ebselen Diphenyl diselenide Diphenyl ditelluride Gene expression |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
description |
Selenium (Se) is an essential micronutrient present in selenoproteins on living beins, in the form of the amino acid selenocysteine and selenomethionine. Chemically related to Se, tellurium (Te) has no biological function in mammals, however, organic Te compounds showed up as good antioxidant agents. Although ebselen (Ebs), diphenyl diselenide [(PhSe)2] and diphenyl ditelluride [(PhTe)2] present antioxidant properties via their glutathione peroxidase (GPx) mimetic activity, these compounds exhibit toxic effects at high concentrations due to its property in oxidizing thiols groups. However, the toxicity mechanism of these compounds through the modulation of gene expression was never studied in human cells. Thus, this study aimed to evaluate the cytotoxicity and genotoxicity of organochalcogens in human leukocytes, and evaluate their mechanisms through the production of reactive oxygen species (ROS) and modulation of antioxidant proteins expression, and to evaluate the relative amount of organochalcogens in contact with the cells in our ex vivo exposure model. Trypan s blue exclusion test and comet assay were used to evaluate, respectively, cytotoxicity and genotoxicity induced by the compounds. The fluorescence of dichlorofluorescein (DCFH) and propidium iodide (PI) were measured in leukocytes exposed by flow cytometry. The expression of the genes for the enzymes Catalase, Superoxide Dismutase 1, Glutathione Peroxidase 3, Glutathione Peroxidase 4, Thioredoxin Reductase 1 and Nrf-2 were analyzed. A dichloromethane extraction of the buffer and the pellet of the cells was injected into a GC-MS apparatus to evaluate the amount of compound in contact with cells. The compounds induced a reduction in cell viability only in the concentration of 50 μM, being Ebs the most cytotoxic compound, followed by (PhTe)2 and (PhSe)2, while (PhTe)2 was the only compound able of increasing the apoptotic rate of leukocytes, which happened at all concentrations (10-50 μM). (PhTe)2 increased DNA damage index in all tested concentrations (5-50 μM), while Ebs and (PhSe)2 did it only at 50 μM concentration. Surprisingly, (PhSe)2 was the only compound effective in increasing ROS production in all tested concentrations (10-50 μM), which was accompanied by an increase in the SOD1 expression and a decrease in CAT expression. All compounds were effective in decreasing the expression of GPX3 and NFE2L2 (Ebs > (PhTe)2 > (PhSe)2), and none altered the expression of GPX4 and TRXR1. The compounds were found in higher concentrations in leukocyte pellet extraction than in their buffer. We conclude that the toxicity of the compounds in question is not directly related to their property in inducing production of ROS. |
publishDate |
2015 |
dc.date.accessioned.fl_str_mv |
2015-05-28 |
dc.date.available.fl_str_mv |
2015-05-28 |
dc.date.issued.fl_str_mv |
2015-02-06 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
BUENO, Diones Caeran. ORGANOCHALCOGEN TOXICITY AND ITS MECHANISMS THROUGH GENE EXPRESSION IN HUMAN LEUKOCYTES. 2015. 69 f. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2015. |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/11232 |
identifier_str_mv |
BUENO, Diones Caeran. ORGANOCHALCOGEN TOXICITY AND ITS MECHANISMS THROUGH GENE EXPRESSION IN HUMAN LEUKOCYTES. 2015. 69 f. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Santa Maria, Santa Maria, 2015. |
url |
http://repositorio.ufsm.br/handle/1/11232 |
dc.language.iso.fl_str_mv |
por |
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por |
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200800000002 |
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400 500 300 500 300 |
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openAccess |
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Universidade Federal de Santa Maria |
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Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica |
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UFSM |
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BR |
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Bioquímica |
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Universidade Federal de Santa Maria |
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