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Avaliação in vitro do efeito de antioxidantes e inibidores da apoptose/autofagia na nefrotoxicidade induzida por catinonas sintéticas
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A introdução incessante de novas substâncias psicoativas no mercado das drogas recreativas e consequente consumo humano continuam a ser um desafio considerável em termos de saúde pública e para as políticas no que toca às drogas. Dentre as várias classes de drogas emergentes, destacam-se as catinonas sintéticas. Foram já descritos vários efeitos adversos associados ao consumo de catinonas sintéticas, incluindo lesões renais, sendo que os mecanismos subjacentes a essa nefrotoxicidade não estão ainda esclarecidos. Neste trabalho pretendeu-se avaliar o potencial nefrotóxico in vitro de duas catinonas frequentemente consumidas, nomeadamente a metilona e a 3,4-dimetilmetcatinona (3,4-DMMC), em células epiteliais do túbulo proximal humano (HK-2). A viabilidade celular foi avaliada usando o ensaio de redução do MTT e o teste de incorporação do corante vermelho neutro. As alterações morfológicas foram identificadas por microscopia de contraste de fase e de fluorescência utilizando marcação com laranja de acridina. Além disso, com o objetivo de explorar os mecanismos subjacentes à toxicidade observada, foram ainda avaliados os efeitos de antioxidantes (ácido ascórbico e N-acetilcisteína), inibidores da apoptose (Z-VAD e Ac-LETD-CHO) e inibidores da autofagia (3-metiladenina e a wortmanina) na citotoxicidade induzida pelas drogas em estudo. Os resultados obtidos mostraram que ambos os derivados induziram citotoxicidade de modo dependente da concentração e do tempo de exposição, tendo o derivado 3,4-DMMC exibido um maior potencial nefrotóxico. Tanto nas células expostas à metilona como à 3,4-DMMC foi possível observar vacúolos citoplasmáticos, sugerindo a indução de autofagia. Este resultado foi corroborado pela deteção de vesículas acídicas intracelulares após marcação com laranja de acridina. A exposição das células HK-2 às catinonas na presença dos inibidores da autofagia promoveu o aumento da morte celular, evidenciando o papel protetor da autofagia na nefrotoxicidade das catinonas sintéticas. Surpreendentemente, verificou-se igualmente um aumento significativo da morte celular na presença dos inibidores da apoptose, sendo necessários estudos adicionais para elucidar as vias envolvidas. Foi também demonstrado que os agentes antioxidantes estudados potenciaram significativamente a morte celular induzida pela metilona e pela 3,4-DMMC, o que poderá estar relacionado com a inibição da autofagia mediada por espécies reativas de oxigénio e consequente potenciação da morte celular por apoptose. Contudo, serão necessários estudos futuros para verificar esta hipótese.
The incessant introduction of new psychoactive substances in the recreational drug market and consequent human consumption remains a considerable challenge in terms of public health and drug policy. Among the several classes of emerging drugs, the synthetic catinones stood out. Several adverse effects have been reported associated with the use of synthetic catinones, including renal injury but the mechanisms underlying this nephrotoxicity have not yet been elucidated. The aim of this study was to evaluate the nephrotoxic potential, in vitro, of two frequently used synthetic cathinones, namely methylone and 3,4-dimethylmethcathinone (3,4-DMMC), in human proximal tubule epithelial cells (HK-2). Cell viability was assessed using the MTT reduction assay and the neutral red dye incorporation assay. The morphological changes were identified by phase contrast and fluorescence microscopy using acridine orange staining. In addition, the effects of antioxidants (ascorbic acid and n-acetylcysteine), apoptosis inhibitors (Z-VAD and Ac-Letd-CHO) and inhibitors of autophagy (3-methyladenine and Wortmannin) were also evaluated to explore the mechanisms underlying the observed toxicity induced by these drugs. The results showed that both derivatives induced cytotoxicity in a concentration-time dependent manner and 3,4-DMMC exhibited a higher nephrotoxic potential. In the cells exposed to both methylone and 3,4-DMMC it was possible to observe cytoplasmic vacuoles, suggesting the induction of autophagy. This result was confirmed by the detection of intracellular acid vesicular organelles in acridine orange-stained cells. The exposure of HK-2 cells to cathinones in the presence of autophagy inhibitors led to increased cell death, evidencing the protective role of autophagy in the nephrotoxicity of synthetic cathinones. Surprisingly, there was a significant increase in cell death in the presence of apoptosis inhibitors, with further studies being needed to elucidate the pathways involved. It was also demonstrated that the antioxidant agents significantly enhanced cell death induced by methylone and 3,4-DMMC, which may be related to the inhibition of autophagy mediated by reactive oxygen species and consequent enhancement of cell death by apoptosis. However, future studies will be needed to verify this hypothesis.
The incessant introduction of new psychoactive substances in the recreational drug market and consequent human consumption remains a considerable challenge in terms of public health and drug policy. Among the several classes of emerging drugs, the synthetic catinones stood out. Several adverse effects have been reported associated with the use of synthetic catinones, including renal injury but the mechanisms underlying this nephrotoxicity have not yet been elucidated. The aim of this study was to evaluate the nephrotoxic potential, in vitro, of two frequently used synthetic cathinones, namely methylone and 3,4-dimethylmethcathinone (3,4-DMMC), in human proximal tubule epithelial cells (HK-2). Cell viability was assessed using the MTT reduction assay and the neutral red dye incorporation assay. The morphological changes were identified by phase contrast and fluorescence microscopy using acridine orange staining. In addition, the effects of antioxidants (ascorbic acid and n-acetylcysteine), apoptosis inhibitors (Z-VAD and Ac-Letd-CHO) and inhibitors of autophagy (3-methyladenine and Wortmannin) were also evaluated to explore the mechanisms underlying the observed toxicity induced by these drugs. The results showed that both derivatives induced cytotoxicity in a concentration-time dependent manner and 3,4-DMMC exhibited a higher nephrotoxic potential. In the cells exposed to both methylone and 3,4-DMMC it was possible to observe cytoplasmic vacuoles, suggesting the induction of autophagy. This result was confirmed by the detection of intracellular acid vesicular organelles in acridine orange-stained cells. The exposure of HK-2 cells to cathinones in the presence of autophagy inhibitors led to increased cell death, evidencing the protective role of autophagy in the nephrotoxicity of synthetic cathinones. Surprisingly, there was a significant increase in cell death in the presence of apoptosis inhibitors, with further studies being needed to elucidate the pathways involved. It was also demonstrated that the antioxidant agents significantly enhanced cell death induced by methylone and 3,4-DMMC, which may be related to the inhibition of autophagy mediated by reactive oxygen species and consequent enhancement of cell death by apoptosis. However, future studies will be needed to verify this hypothesis.
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Keywords
Catinonas sintéticas Nefrotoxicidade Células HK-2 Morte celular Antioxidantes Autofagia Apoptose Synthetic cathinones Nephrotoxicity HK-2 cells Cell death Antioxidants Autophagy Apoptosis