Efeitos de uma mistura ambientalmente relevante de ftalatos sobre células prostáticas humanas
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Data
2022-01-28
Autores
Cavalca, Alexandre Matheus Baesso
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os ftalatos representam um grupo de substâncias utilizadas na indústria que possuem atividade antiandrogênica e são encontrados em diferentes concentrações na urina e no plasma humano. Mais de 8 milhões de toneladas de ftalatos são usadas a cada ano, predominantemente como plastificantes em produtos de cloreto de polivinila (PVC), sendo amplamente utilizados em produtos de consumo diário e descartados, de forma inadequada, no meio ambiente. Ainda, estudos in vivo realizados em nosso laboratório, demonstraram que uma mistura de ftalatos, equivalente a usada neste estudo, desregulou a expressão de genes e miRNAs associados ao desenvolvimento e à vias carcinogênicas prostáticas. Dessa forma, este estudo teve como objetivo principal mimetizar as condições da exposição in vivo, com o intuito de estabelecer um modelo in vitro para avaliação das vias relacionadas à sobrevivência, proliferação e morte celular e biossíntese de miRNAs, utilizando células epiteliais prostáticas normais e tumorais expostas a uma mistura de monoésteres de ftalato, ambientalmente relevante, em proporções semelhantes às encontradas em humanos. As linhagens de células epiteliais prostáticas tumorais (LNCaP) e normais (PNT-2) foram expostas por 24h e 72h à mistura de ftalatos. A proporção dos monoésteres na mistura foi calculada com base na concentração média de ftalatos encontradas na urina e sangue humanos. A dose de exposição (1000μmol/L) foi escolhida com base nos ensaios de viabilidade (IC50) e do MTT, balizada pelas doses usadas da literatura. A quantificação da expressão dos genes foi realizada por qRT-PCR. Os miRNAs estudados foram escolhidos com base nos resultados dos estudos in vivo. Para análise do potencial migratório celular foi realizado o ensaio de migração celular através de insertos transwell. Nossos resultados mostraram que a mistura de ftalatos pode causar aumento em células LNCaP, do turnover celular, do estresse oxidativo, da biossíntese e expressão de miRNAs, podendo assim aumentar o potencial expansivo e migratório celular e modular o comportamento tumoral, tornando-as mais agressivas. No entanto, em células normais estes efeitos são menos acentuados, demonstrando que, a curto prazo, as mesmas são capazes de elaborar mecanismos e respostas mais efetivos contra o insulto, ou ainda, que as células PNT-2 são mais resistentes à exposição na dose empregada.
Phthalates represent a group of substances used in industry that have antiandrogenic activity and are found in different concentrations in human urine and plasma. More than 8 million tons of phthalates are used each year, predominantly as plasticizers in polyvinyl chloride (PVC) products, being widely used in everyday consumer products and inappropriately disposed of in the environment. Furthermore, in vivo studies performed in our laboratory demonstrated that a mixture of phthalates, equivalent to the one used in this study, deregulated the expression of genes and miRNAs associated with developmental and prostatic carcinogenic pathways. Thus, the main objective of this study was to mimic the conditions of exposure in vivo, in order to establish an in vitro model for the evaluation of pathways related to cell survival, proliferation and death and miRNA biosynthesis, using normal and tumoral prostatic epithelial cells. exposed to an environmentally relevant mixture of phthalate monoesters in proportions similar to those found in humans. Prostatic tumor (LNCaP) and normal (PNT-2) epithelial cell lines were exposed for 24h and 72h to the phthalate mixture. The proportion of monoesters in the mixture was calculated based on the average concentration of phthalates found in human urine and blood. The exposure dose (1000μmol/L) was chosen based on feasibility tests (IC50) and MTT, based on the doses used in the literature. Quantification of gene expression was performed by qRT-PCR. The miRNAs studied were chosen based on the results of the in vivo studies. To analyze the cell migration potential, the cell migration assay was performed using transwell inserts. Our results showed that the mixture of phthalates can cause an increase in LNCaP cells, cell turnover, oxidative stress, biosynthesis and expression of miRNAs, thus increasing the cellular expansive and migratory potential and modulating tumor behavior, making them more aggressive. However, in normal cells these effects are less accentuated, demonstrating that, in the short term, they are capable of elaborating more effective mechanisms and responses against the insult, or even that PNT-2 cells are more resistant to exposure at the dose maid.
Phthalates represent a group of substances used in industry that have antiandrogenic activity and are found in different concentrations in human urine and plasma. More than 8 million tons of phthalates are used each year, predominantly as plasticizers in polyvinyl chloride (PVC) products, being widely used in everyday consumer products and inappropriately disposed of in the environment. Furthermore, in vivo studies performed in our laboratory demonstrated that a mixture of phthalates, equivalent to the one used in this study, deregulated the expression of genes and miRNAs associated with developmental and prostatic carcinogenic pathways. Thus, the main objective of this study was to mimic the conditions of exposure in vivo, in order to establish an in vitro model for the evaluation of pathways related to cell survival, proliferation and death and miRNA biosynthesis, using normal and tumoral prostatic epithelial cells. exposed to an environmentally relevant mixture of phthalate monoesters in proportions similar to those found in humans. Prostatic tumor (LNCaP) and normal (PNT-2) epithelial cell lines were exposed for 24h and 72h to the phthalate mixture. The proportion of monoesters in the mixture was calculated based on the average concentration of phthalates found in human urine and blood. The exposure dose (1000μmol/L) was chosen based on feasibility tests (IC50) and MTT, based on the doses used in the literature. Quantification of gene expression was performed by qRT-PCR. The miRNAs studied were chosen based on the results of the in vivo studies. To analyze the cell migration potential, the cell migration assay was performed using transwell inserts. Our results showed that the mixture of phthalates can cause an increase in LNCaP cells, cell turnover, oxidative stress, biosynthesis and expression of miRNAs, thus increasing the cellular expansive and migratory potential and modulating tumor behavior, making them more aggressive. However, in normal cells these effects are less accentuated, demonstrating that, in the short term, they are capable of elaborating more effective mechanisms and responses against the insult, or even that PNT-2 cells are more resistant to exposure at the dose maid.
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Palavras-chave
Próstata, Migração, miRNAs, Ftalatos, Cultura celular, Prostate, Phthalates, Migration, Cell culture