Impacto da exposição ao chumbo (Pb) sobre o perfil de expressão de genes relacionados ao metabolismo do metal em indivíduos ocupacionalmente expostos
Data
2022-01-18
Autores
Araújo, Marília Ladeira 
Orientadores
Barcelos, Gustavo Rafael Mazzaron
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O chumbo (Pb) é um metal versátil utilizado por humanos há mais de 9.000 anos e, sua exposição poderá influenciar no processo de regulação da expressão gênica o qual é composta por diversos eventos complexos, caracterizando-os como um processo biológico essencial. Além disso, a identificação de biomarcadores moleculares que podem preceder a efeitos adversos à saúde relacionados à exposição a toxicantes aumentou significativamente na última década. Diante deste contexto, a expressão de genes relacionados com a toxicocinética do Pb e a expressão de microRNAs (miRNAs) em indivíduos expostos a baixas concentrações de Pb podem gerar novas perspectivas na ampliação do entendimento dos mecanismos associados à toxicidade induzida pelo metal. Deste modo, o objetivo do presente estudo consistiu na avaliação do impacto da exposição ao Pb sobre o perfil de expressão transcricional (RNAm) de genes associados ao metabolismo do metal (VDR, SLC11A2, MT2A, ALAD, GCLM, GSS, GSR, GGT1) e sobre a expressão de dois miRNAs, hsa-miR-148a-5p e hsa-miR-23a-3p, em trabalhadores expostos ao metal, por RT-qPCR. Foram coletadas cerca de 10 mL de sangue e cerca de 30 mL de urina de 85 trabalhadores de fábricas de baterias automotivas no estado do Paraná, após esclarecimento detalhado sobre o projeto e assinatura do termo de consentimento livre e esclarecido (TCLE). Foram incluídos no estudo indivíduos com idade entre 19 a 69 anos, do sexo masculino e que declararam não possuírem doenças conhecidas no momento da coleta. As concentrações de Pb no sangue (Pb-s), no plasma (Pb-p) e na urina (Pb-u) foram determinadas por espectrometria de massas com plasma indutivamente acoplado (ICP-MS). Para as análises das expressões transcricionais dos genes de metabolismo e do miRNA hsa-miR-148a-5p foram realizadas a partir de extrações de sangue, enquanto que a extração do miRNA hsa-miR-23a-3p foi obtida pelo plasma e os ensaios de RT-qPCR foram realizados através do método TaqMan. As médias ± desvio padrão (DP) de Pb-s, Pb-p e Pb-u foram de 19 ± 11 µg/dL, 0,54 ± 0,65 µg/dl, 35,39 ± 40 µg/g creatinina, respectivamente, evidenciando que estes valores encontram-se abaixo do permitido pela legislação brasileira. Correlações de Spearman e as análises de regressões multivariadas apontaram para associações positivas entre Pb-s e a expressão dos genes GSR e SLC11A2 (β=0,053 p=0,045 e β= 0,052 p=0,048, respectivamente). As regressões múltiplas apresentaram associações inversas entre a expressão de hsa- miR-148a-5p e metilação global do DNA (%5-mC DNA), em função do Pb-p (β = -4,1; p =0,050). Ademais, em baixas concentrações de Pb, os miRNAs (hsa-miR-148a-5p e hsa-miR-23a-3p) não impactaram na alteração da expressão de genes alvo. Por fim, os dados obtidos colaboraram para uma melhor compreensão sobre a influência da toxicidade do Pb em baixas concentrações, através da ativação do gene carreador de soluto (responsável pela absorção e distribuição do metal no organismo), assim como uma ativação do papel antioxidante, caracterizando como um possível mecanismo de defesa.
Lead (Pb) is a versatile metal used by humans for over 9,000 years and its exposure may influence the process of gene expression regulation, which is composed of several complex events, characterizing them as an essential biological process. Furthermore, the identification of molecular biomarkers that may precede adverse health effects related to exposure to toxicants has increased significantly in the last decade. In this context, the expression of genes related to Pb toxicokinetics and the expression of microRNAs (miRNAs) in individuals exposed to low concentrations of Pb may generate new perspectives in expanding the understanding of the mechanisms associated with toxicity induced by the metal. Thus, the aim of the present study was to evaluate the impact of exposure to Pb on the transcriptional expression profile (mRNA) of genes associated with metal metabolism (VDR, SLC11A2, MT2A, ALAD, GCLM, GSS, GSR, GGT1) and on the expression of two miRNAs, hsa-miR148a-5p and hsa-miR-23a-3p, in workers exposed to the metal, by RT-qPCR. About 10 mL of blood and about 30 mL of urine were collected from 85 workers at automotive battery factories in the state of Paraná, after detailed clarification about the project and signing of the free and informed consent form (ICF). Individuals aged between 19 and 69 years, male and who declared not having known diseases at the time of collection, were included in the study. Pb concentrations in blood (Pb-s), plasma (Pb-p) and urine (Pb-u) were determined by inductively coupled plasma mass spectrometry (ICP-MS). For the analysis of transcriptional expressions of the metabolism genes and the miRNA hsa-miR-148a-5p were performed from blood extractions, while the extraction of miRNA hsa-miR-23a-3p was obtained by plasma and assays of RT-qPCR were performed using the TaqMan method. The means ± standard deviation (SD) of Pb-s, Pb-p and Pb-u were 19 ± 11 µg/dL, 0.54 ± 0.65 µg/dl, 35.39 ± 40 µg/g creatinine, respectively, showing that these values are below the permitted by Brazilian legislation. Spearman correlations and multivariate regression analyzes pointed to positive associations between Pb-s and the expression of the GSR and SLC11A2 genes (β=0.053 p=0.045 and β= 0.052 p=0.048, respectively). Multiple regressions showed inverse associations between hsa-miR-148a-5p expression and global DNA methylation (%5-mC DNA), as a function of Pb-p (β = -4.1; p =0.050). Furthermore, at low concentrations of Pb, miRNAs (hsa-miR-148a-5p and hsa-miR-23a-3p) did not affect the alteration of target gene expression. Finally, the data obtained contributed to a better understanding of the influence of Pb toxicity at low concentrations, through the activation of the solute carrier gene (responsible for the absorption and distribution of the metal in the body), as well as an activation of the antioxidant role, characterizing as a possible defense mechanism.
Lead (Pb) is a versatile metal used by humans for over 9,000 years and its exposure may influence the process of gene expression regulation, which is composed of several complex events, characterizing them as an essential biological process. Furthermore, the identification of molecular biomarkers that may precede adverse health effects related to exposure to toxicants has increased significantly in the last decade. In this context, the expression of genes related to Pb toxicokinetics and the expression of microRNAs (miRNAs) in individuals exposed to low concentrations of Pb may generate new perspectives in expanding the understanding of the mechanisms associated with toxicity induced by the metal. Thus, the aim of the present study was to evaluate the impact of exposure to Pb on the transcriptional expression profile (mRNA) of genes associated with metal metabolism (VDR, SLC11A2, MT2A, ALAD, GCLM, GSS, GSR, GGT1) and on the expression of two miRNAs, hsa-miR148a-5p and hsa-miR-23a-3p, in workers exposed to the metal, by RT-qPCR. About 10 mL of blood and about 30 mL of urine were collected from 85 workers at automotive battery factories in the state of Paraná, after detailed clarification about the project and signing of the free and informed consent form (ICF). Individuals aged between 19 and 69 years, male and who declared not having known diseases at the time of collection, were included in the study. Pb concentrations in blood (Pb-s), plasma (Pb-p) and urine (Pb-u) were determined by inductively coupled plasma mass spectrometry (ICP-MS). For the analysis of transcriptional expressions of the metabolism genes and the miRNA hsa-miR-148a-5p were performed from blood extractions, while the extraction of miRNA hsa-miR-23a-3p was obtained by plasma and assays of RT-qPCR were performed using the TaqMan method. The means ± standard deviation (SD) of Pb-s, Pb-p and Pb-u were 19 ± 11 µg/dL, 0.54 ± 0.65 µg/dl, 35.39 ± 40 µg/g creatinine, respectively, showing that these values are below the permitted by Brazilian legislation. Spearman correlations and multivariate regression analyzes pointed to positive associations between Pb-s and the expression of the GSR and SLC11A2 genes (β=0.053 p=0.045 and β= 0.052 p=0.048, respectively). Multiple regressions showed inverse associations between hsa-miR-148a-5p expression and global DNA methylation (%5-mC DNA), as a function of Pb-p (β = -4.1; p =0.050). Furthermore, at low concentrations of Pb, miRNAs (hsa-miR-148a-5p and hsa-miR-23a-3p) did not affect the alteration of target gene expression. Finally, the data obtained contributed to a better understanding of the influence of Pb toxicity at low concentrations, through the activation of the solute carrier gene (responsible for the absorption and distribution of the metal in the body), as well as an activation of the antioxidant role, characterizing as a possible defense mechanism.
Descrição
Citação
ARAUJO, Marília Ladeira. Impacto da exposição ao chumbo (Pb) sobre o perfil de expressão de genes relacionados ao metabolismo do metal em indivíduos ocupacionalmente expostos. 2022. 126 f. Tese (Doutorado Interdisciplinar em Ciências da Saúde) - Instituto de Saúde e Sociedade, Universidade Federal de São Paulo, Santos, 2022.