RESUMO
Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na+/K+-ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca2+-sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca2+ influx into the cytosol and that failing to pump Ca2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca2+ or Mn2+ uptake may be a way of controlling oleandrin toxicity.
Assuntos
Cálcio/metabolismo , Cardenolídeos/química , Glicosídeos Cardíacos/química , Glicosídeos Cardíacos/metabolismo , Manganês/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Cardenolídeos/farmacologia , Glicosídeos Cardíacos/farmacologia , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Citosol/ultraestrutura , Inibidores Enzimáticos/metabolismo , Humanos , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrometria de FluorescênciaRESUMO
One of the most important aspects of the detection of antioxidant compounds is developing a fast screening method. The screening of the overall relative antioxidant capacity (RAC) of several Romanian hydrosoluble plant extracts is the focus of this work. This is important because of the presence of increasing levels of reactive oxygen species (such as H2O2) generates oxidative stress in the human body. The consequences are a large number of medical conditions that can be helped by a larger consumption of plant extracts as food supplements, which do not necessarily contain the specified antioxidant contents. By exploiting the catalytic properties of gold nanoparticles, a specific and sensitive nanoparticle-based label-free electrochemical sensor was developed, where the working parameters were optimized for RAC screening of hydrosoluble plant extracts. First, electrochemical measurements (cyclic voltammetry and amperometry) were used to characterize different nanoparticle-based sensors, revealing the best performance of gold nanoparticle-based sensors, obtaining a RAC of 98% for lavender extracts. The sensing principle is based on the quenching effect of antioxidants for H2O2 amperometric detection, where the decrease in electrical signal suggests an increasing antioxidant capacity. The obtained results were expressed in terms of ascorbic acid and Trolox equivalents in order to be able to correlate our results with classical methods like chemiluminescence and UV-Vis spectrophotometry, where a correlation coefficient of 0.907 was achieved, suggesting a good correlation between electrochemistry and spectrophotometry. Considering these results, the optimized gold nanoparticle-based label-free sensor can be used as a simple, rapid alternative towards classical methods for relative antioxidant capacity detection of hydrosoluble plant extracts.
Assuntos
Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , Extratos Vegetais/química , Antioxidantes , Ouro/química , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/químicaRESUMO
Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn2+ surplus. Using cells expressing the Ca2+-sensitive photoprotein aequorin we found that Mn2+ augmented the Ca2+ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn2+-high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn2+ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn2+ activate TRPY1 in the response to oxidative stress.
Assuntos
Cálcio/metabolismo , Haploinsuficiência , Peróxido de Hidrogênio/toxicidade , Manganês/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Canais de Cátion TRPC/genética , Vacúolos/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Diploide , Haploinsuficiência/efeitos dos fármacos , Heterozigoto , Mutação/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Vacúolos/efeitos dos fármacosRESUMO
Epigallocatechin-3-O-gallate (EGCG), the main green tea component, is intensively studied for its anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer effects. In the present study, a screen on a Saccharomyces cerevisiae gene deletion library was performed to identify conditions under which EGCG had deleterious rather than beneficial effects. Two genes were identified whose deletion resulted in sensitivity to EGCG: FET3 and FTR1, encoding the components of the Fet3/Ftr1 high-affinity iron uptake system, also involved in Cu(I)/Cu(II) balance on the surface of yeast cells. The presence of EGCG in the growth medium induced the production of Cu(I), with deleterious effects on fet3Δ and ftr1Δ cells. Additionally, when combined, physiological surpluses of Cu(II) and EGCG acted in synergy not only against fet3Δ and ftr1Δ, but also against wild type cells, by generating surplus Cu(I) in the growth medium. The results imply that caution should be taken when combining EGCG-rich beverages/nutraceuticals with copper-rich foods.
Assuntos
Catequina/análogos & derivados , Ceruloplasmina/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Chá/química , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Ceruloplasmina/deficiência , Cobre/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Saccharomyces cerevisiae/genética , Chá/metabolismoRESUMO
To respond to metal surpluses, cells have developed intricate ways of defense against the excessive metallic ions. To understand the ways in which cells sense the presence of toxic concentration in the environment, the role of Ca2+ in mediating the cell response to high Cu2+ was investigated in Saccharomyces cerevisiae cells. It was found that the cell exposure to high Cu2+ was accompanied by elevations in cytosolic Ca2+ with patterns that were influenced not only by Cu2+ concentration but also by the oxidative state of the cell. When Ca2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca2+ pool under Cu2+ stress was the vacuolar Ca2+ channel, Yvc1, also activated by the Cch1-mediated Ca2+ influx. Using yeast mutants defective in the Cu2+ transport across the plasma membrane, it was found that the Cu2+-dependent Ca2+ elevation could correlate not only with the accumulated metal, but also with the overall oxidative status. Moreover, it was revealed that Cu2+ and H2O2 acted in synergy to induce Ca2+-mediated responses to external stress.
Assuntos
Sinalização do Cálcio , Cobre/metabolismo , Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Cobre/toxicidade , Citosol/metabolismo , Peróxido de Hidrogênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Lanthanides are a group of non-essential elements with important imaging and therapeutic applications. Although trivalent lanthanide ions (Ln³âº) are used as potent blockers of Ca²âº channels, the systematic studies correlating Ln³âº accumulation and toxicity to Ca²âº channel blocking activity are scarce. In this study, we made use of the eukaryotic model Saccharomyces cerevisiae to investigate the correlation between Ln³âº accumulation, their toxicity and their capacity to block the exogenous stress-induced Ca²âº influx into the cytosol. It was found that the Ln³âº blocked the Ca²âº entry into the yeast cells only when present at concentration high enough to allow rapid binding to cell surface. At lower concentrations, Ln³âº were taken up by the cell, but Ca²âº blockage was no longer achieved. At 1 mM concentration, all ions from the Ln³âº series could block Ca²âº entry into cytosol with the exception of La³âº, and to a lesser extent, Pr³âº and Nd³âº. The plasma membrane Ca²âº-channel Cch1/Mid1 contributed to La³âº and Gd³âº entry into the cells, with a significant preference for La³âº. The results open the possibility to obtain cells loaded with controlled amounts and ratios of Ln³âº.
Assuntos
Cálcio/química , Elementos da Série dos Lantanídeos/química , Saccharomyces cerevisiae/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Íons/farmacologia , Elementos da Série dos Lantanídeos/toxicidade , Modelos Biológicos , Saccharomyces cerevisiae/químicaRESUMO
Blueberries (Vaccinium corymbosum L.) are a rich source of antioxidants and their consumption is believed to contribute to food-related protection against oxidative stress. In the present study, the chemoprotective action of blueberry extracts against cadmium toxicity was investigated using a cadmium-hypersensitive strain of Saccharomyces cerevisiae. Four varieties of blueberries were used in the study, and it was found that the extracts with high content of total anthocyanidins exhibited significant protective effect against the toxicity of cadmium and H2O2. Both the blueberry extracts and pure cyanidin exhibited protective effects against cadmium in a dose-dependent manner, but without significantly interfering with the cadmium accumulation by the yeast cells. The results imply that the blueberry extracts might be a potentially valuable food supplement for individuals exposed to high cadmium.