RESUMO
The development of cell-based fluorescent assays has resulted in an incredible tool for searching new ion channels' modulators with a biophysical and clinical profile. Among all the ion channels, potassium (K+)-permeable channels represent the most diverse and relevant for cell function, making them attractive targets for drug discovery. Some of the cell-based assays for K+ channels take advantage of a thallium-sensitive dye whose fluorescence increased upon the binding of thallium (Tl+), an ion able to move through K+ channels. We optimize the FLIPR Potassium Assay Kit based on thallium influx to measure the Kv10.1 activity.
Assuntos
Tálio , Tálio/metabolismo , Humanos , Corantes Fluorescentes/química , Células HEK293 , Fluorescência , Canais de Potássio Éter-A-Go-GoRESUMO
Thallium (TI) is one of the most toxic heavy metals. Human exposure to Tl occurs through contaminated drinking water and from there to food, a threat to health. Recently, environmental contamination by Tl has been reported in several countries, urging the need for studies to determine the impact of endogenous and exogenous mechanisms preventing thallium toxicity. The cytoprotective effect of metallothionein (MT), a protein with high capacity to chelate metals, at two doses (100 and 600 µg/rat), was tested. Prussian blue (PB) (50 mg/kg) was administered alone or in combination with MT. A dose of Tl (16mg/kg) was injected i.p. to Wistar rats. Antidotes were administered twice daily, starting 24h after Tl injection, for 4 days. Tl concentrations diminished in most organs (p < 0.05) by effect of PB, alone or in combination with MT, whereas MT alone decreased Tl concentrations in testis, spleen, lung and liver. Likewise, brain thallium also diminished (p < 0.05) by effect of PB and MT alone or in combination in most of the regions analyzed (p < 0.05). The greatest diminution of Tl was achieved when the antidotes were combined. Plasma markers of renal damage increased after Tl administration, while PB and MT, either alone or in combination, prevented the raise of those markers. Only MT increased the levels of reduced glutathione (GSH) in the kidney. Finally, increased Nrf2 was observed in liver and kidney, after treatment with MT alone or in combination with PB. Results showed that MT alone or in combination with PB is cytoprotective after thallium exposure.
Assuntos
Metalotioneína , Tálio , Animais , Ferrocianetos , Masculino , Metalotioneína/metabolismo , Estresse Oxidativo , Ratos , Ratos Wistar , Tálio/metabolismo , Tálio/toxicidadeRESUMO
BACKGROUND: Ion channels have been proposed as therapeutic targets for different types of malignancies. One of the most studied ion channels in cancer is the voltage-gated potassium channel ether-à-go-go 1 or Kv10.1. Various studies have shown that Kv10.1 expression induces the proliferation of several cancer cell lines and in vivo tumor models, while blocking or silencing inhibits proliferation. Kv10.1 is a promising target for drug discovery modulators that could be used in cancer treatment. This work aimed to screen for new Kv10.1 channel modulators using a thallium influx-based assay. METHODS: Pharmacological effects of small molecules on Kv10.1 channel activity were studied using a thallium-based fluorescent assay and patch-clamp electrophysiological recordings, both performed in HEK293 stably expressing the human Kv10.1 potassium channel. RESULTS: In thallium-sensitive fluorescent assays, we found that the small molecules loperamide and amitriptyline exert a potent inhibition on the activity of the oncogenic potassium channel Kv10.1. These results were confirmed by electrophysiological recordings, which showed that loperamide and amitriptyline decreased the amplitude of Kv10.1 currents in a dose-dependent manner. Both drugs could be promising tools for further studies. CONCLUSIONS: Thallium-sensitive fluorescent assay represents a reliable methodological tool for the primary screening of different molecules with potential activity on Kv10.1 channels or other K+ channels.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Loperamida/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Relação Dose-Resposta a Droga , Fluorescência , Células HEK293 , Humanos , Loperamida/administração & dosagem , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/administração & dosagem , Reprodutibilidade dos Testes , Tálio/metabolismoRESUMO
Thallium (Tl+) is a ubiquitous natural trace metal considered as the most toxic among heavy metals. The ionic ratio of Tl+ is similar to that of potassium (K+), therefore accounting for the replacement of the latter during enzymatic reactions. The principal organelle damaged after Tl+ exposure is mitochondria. Studies on the mechanisms of Tl+ include intrinsic pathways altered and changes in antiapoptotic and proapoptotic proteins, cytochrome c, and caspases. Oxidative damage pathways increase after Tl+ exposure to produce reactive oxygen species (ROS), changes in physical properties of the cell membrane caused by lipid peroxidation, and concomitant activation of antioxidant mechanisms. These processes are likely to account for the neurotoxic effects of the metal. In humans, Tl+ is absorbed through the skin and mucous membranes and then is widely distributed throughout the body to be accumulated in bones, renal medulla, liver, and the Central Nervous System. Given the growing relevance of Tl+ intoxication, in recent years there is a notorious increase in the number of reports attending Tl+ pollution in different countries. In this sense, the neurological symptoms produced by Tl+ and its neurotoxic effects are gaining attention as they represent a serious health problem all over the world. Through this review, we present an update to general information about Tl+ toxicity, making emphasis on some recent data about Tl+ neurotoxicity, as a field requiring attention at the clinical and preclinical levels.
Assuntos
Intoxicação do Sistema Nervoso por Metais Pesados/etiologia , Tálio/intoxicação , Encéfalo/metabolismo , Intoxicação do Sistema Nervoso por Metais Pesados/metabolismo , Intoxicação do Sistema Nervoso por Metais Pesados/fisiopatologia , Humanos , Mitocôndrias/metabolismo , Tálio/metabolismoRESUMO
Thallium (T1+) is a toxic heavy metal which was accidentally discovered by Sir William Crookes in 1861 by burning the dust from a sulfuric acid industrial plant. He observed a bright green spectral band that quickly disappeared. Crookes named the new element 'Thallium' (after thallos meaning young shoot). In 1862, Lamy described the same spectral line and studied both the physical and chemical properties of this new element (Prick, J.J.G., 1979. Thallium poisoning. In: Vinkrn, P.J., Bruyn, G.W. (Eds.), Intoxication of the Nervous System, Handbook of Clinical Neurology, vol. 36. North-Holland, New York. pp. 239-278).