Estudo da neurotoxicidade de células SHSY-5Y expostas ao expostas à cetamina, ao etanol e à associação de ambas as substâncias por meio da análise de fluorescência após marcação com laranja de acridina e iodeto de propídeo
Data
2021-02-08
Autores
Almeida, Rafaela Yolanda Silvino de 
Orientadores
Garcia, Raphael Caio Tamborelli
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O consumo de drogas faz parte da história da humanidade, e o uso recreativo dessas substâncias vem crescendo entre adolescentes, que em festas rave e boates consomem as club drugs em busca de ampliação de sentidos. A cetamina, um exemplo dessa classe de droga de abuso, também é utilizada por profissionais de saúde devido à facilidade de obtenção, com objetivo de aliviar a jornada estressante de trabalho. O etanol, bebida presente em muitos contextos por seu uso legal, também registra elevado consumo no Brasil, podendo ser associado com outras drogas, como a cetamina. O uso dessa associação pode levar à danos irreversíveis ao sistema nervoso central, causando neurodegeneração. Assim, esse estudo avaliou a viabilidade de células SH-SY5Y expostas à cetamina, ao etanol e à associação de ambas as substâncias por meio da análise de fluorescência após marcação com laranja de acridina e iodeto de propídeo, utilizando o contador de células automatizado (LUNA). As células foram mantidas em placas de 24 poços (105 células/poço) e incubadas com concentrações variadas de cetamina (0,001; 0,01; 0,1; 1; 2,5; 5; 7,5 e 10 mM) ou etanol (0,1; 1; 10; 100; 250; 500; 750 e 1000 mM), por 24 e 48 horas, para a realização das curvas concentração-resposta (CCR) das substâncias utilizando o ensaio de viabilidade celular de MTT. Cloreto de potássio (250 mM) foi utilizado como controle positivo de morte celular e o meio de cultura como controle negativo. As curvas foram realizadas para se determinar a maior concentração sem efeito tóxico (NOAEL) e a menor concentração em que há efeito tóxico (LOAEL) para cada substância, ou seja, NOAEL do etanol (ETN = 10 mM) e da cetamina (KTN = 0,1 mM), e LOAEL do etanol (ETL = 100 mM) e da cetamina (KTL = 1 mM). Posteriormente, o efeito decorrente da associação das substâncias, após 24h de exposição, foi estudado por meio dos ensaios de MTT, da quantificação da atividade enzimática da lactato desidrogenase (LDH) e da análise de fluorescência após marcação com laranja de acridina e iodeto de propídeo, para os grupos isolados e associados, a saber: KTN+ETN, KTL+ETN, KTN+ETL e KTL+ETL. Os resultados obtidos no ensaio de MTT, mostraram que houve uma diminuição na viabilidade celular após 24 horas de exposição aos grupos: ETL; KTN + ETL; KTL + ETN (p<0,05 para cada grupo) e KTL + ETL (p <0,0001). O mesmo período de exposição não apresentou alterações na atividade da LDH. Os resultados obtidos no LUNA corroboram os do MTT. Por não apresentar variações na atividade da LDH, indicativo de necrose, sugere-se que os
mecanismos subjacentes à morte celular induzida por KT, ET e KT + ET parecem envolver apoptose e/ou autofagia.
Drug use is part of human history, and recreational use of these substances has been growing among teenagers, who at club parties and raves consume club drugs in order to intensify their senses. Ketamine, an example of club drug, is also used by health professionals due to its easier access, in order to relieve stressful work hours. Ethanol, a beverage present in many contexts for its legal use, also registers high consumption rates in Brazil, and it can be associated with other drugs, such as ketamine. The use of this combination can lead to irreversible damage to the central nervous system, causing neurodegeneration. Thus, this study evaluated the viability of SH-SY5Y cells exposed to ketamine, ethanol and the combination of both substances by fluorescence analysis after staining with acridine orange and propidium iodide, using a cell counter equipment (LUNA). The cells were maintained in 24-well plates (105 cells/well) and incubated with a range of ketamine concentration (0.001; 0.01; 0.1; 1; 2.5; 5; 7.5 and 10 mM) or ethanol (0.1; 1; 10; 100; 250; 500; 750 and 1000 mM) for 24 and 48 hours. Potassium chloride (250 mM) was used as a positive control of cell death and the culture medium as a negative control. Concentration-response curves (CCR) were performed for the substances using MTT cell viability assays for 24 and 48 hours of exposure. The CCRs were performed to determine the no observed adverse effect level (NOAEL) and the lowest observed adverse effect level (LOAEL) for each substance, which is, NOAEL of ethanol (ETN = 10 mM) and ketamine (KTN = 0.1 mM), and LOAEL of ethanol (ETL = 100 mM) and ketamine (KTL = 1 mM). Thus, the combination effect of both substances was studied, after 24h of exposure, by MTT assay, quantification of of lactate dehydrogenase (LDH) activity and fluorescence analysis after staining with acridine orange and propidium iodide, for the following experimental groups: KTN + ETN, KTL + ETN, KTN + ETL and KTL + ETL. The results obtained in the MTT assay, showed that there was a decrease in cell viability after 24 hours of exposure for the following groups: ETL; KTN + ETL; KTL + ETN (p <0.05 for each group) and KTL + ETL (p <0.0001). The same period of exposure showed no changes in LDH activity. The results obtained in LUNA corroborate those observed in MTT assay. Since there is no variation in LDH activity, indicative of necrosis, this study suggests that the mechanisms underlying cell death induced by KT, ET and KT + ET may involve apoptosis and/or autophagy.
Drug use is part of human history, and recreational use of these substances has been growing among teenagers, who at club parties and raves consume club drugs in order to intensify their senses. Ketamine, an example of club drug, is also used by health professionals due to its easier access, in order to relieve stressful work hours. Ethanol, a beverage present in many contexts for its legal use, also registers high consumption rates in Brazil, and it can be associated with other drugs, such as ketamine. The use of this combination can lead to irreversible damage to the central nervous system, causing neurodegeneration. Thus, this study evaluated the viability of SH-SY5Y cells exposed to ketamine, ethanol and the combination of both substances by fluorescence analysis after staining with acridine orange and propidium iodide, using a cell counter equipment (LUNA). The cells were maintained in 24-well plates (105 cells/well) and incubated with a range of ketamine concentration (0.001; 0.01; 0.1; 1; 2.5; 5; 7.5 and 10 mM) or ethanol (0.1; 1; 10; 100; 250; 500; 750 and 1000 mM) for 24 and 48 hours. Potassium chloride (250 mM) was used as a positive control of cell death and the culture medium as a negative control. Concentration-response curves (CCR) were performed for the substances using MTT cell viability assays for 24 and 48 hours of exposure. The CCRs were performed to determine the no observed adverse effect level (NOAEL) and the lowest observed adverse effect level (LOAEL) for each substance, which is, NOAEL of ethanol (ETN = 10 mM) and ketamine (KTN = 0.1 mM), and LOAEL of ethanol (ETL = 100 mM) and ketamine (KTL = 1 mM). Thus, the combination effect of both substances was studied, after 24h of exposure, by MTT assay, quantification of of lactate dehydrogenase (LDH) activity and fluorescence analysis after staining with acridine orange and propidium iodide, for the following experimental groups: KTN + ETN, KTL + ETN, KTN + ETL and KTL + ETL. The results obtained in the MTT assay, showed that there was a decrease in cell viability after 24 hours of exposure for the following groups: ETL; KTN + ETL; KTL + ETN (p <0.05 for each group) and KTL + ETL (p <0.0001). The same period of exposure showed no changes in LDH activity. The results obtained in LUNA corroborate those observed in MTT assay. Since there is no variation in LDH activity, indicative of necrosis, this study suggests that the mechanisms underlying cell death induced by KT, ET and KT + ET may involve apoptosis and/or autophagy.