Percorrer por autor "Barreiros, Sara"
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- Evaluating cytotoxic and genotoxic effects of microcystin using Saccharomyces cerevisiæ as eukaryotic cell modelPublication . Barreiros, Sara; Silva, Maria João; Valério, ElisabeteMicrocystins (MC) are one of the most common hepatoxins, produced by cyanobacteria. These toxins are cyclic peptides that have a high affinity for the Serine / Threonine (PPs) protein phosphatases family, namely PP1 / PP2A, acting as their inhibitors. MCs also induce oxidative stress in cells through the production of reactive oxygen species (ROS). Both effects have been associated to cytotoxic and genotoxic effects of MC in animal cells [1]. The effects of MCs on cells of higher eukaryotic organisms, such as animals and plants, have been extensively studied. However, a complete characterization of the effects of these toxins has not yet been achieved [1]. In this study the cytotoxic and genotoxic effects of MC on a eukaryotic cell model (the yeast Saccharomyces cerevisiæ) were evaluated. The cytotoxic effects were assessed using an MTT assay and the genotoxic effects evaluated using the comet assay and the expression levels of genes involved in DNA repair systems obtained by Real-Time PCR (RT-qPCR). The results obtained will be discussed.
- Evaluation of cytotoxic and genotoxic effects of Microcystin-LR in Saccharomyces cerevisiaePublication . Barreiros, Sara; Valério, Elisabete; Dias, DeodáliaMicrocystins (MC) are hepatotoxins produced by cyanobacteria. Among the MCs, the microcystin-LR (MC-LR), produced by several cyanobacterial species, especially by the species Microcystis aeruginosa, is the most abundant and also the most well studied cyanotoxin. MCs are cyclic peptides which have high affinity for protein phosphatases Serine/Threonine (PPs), namely PP1 and PP2A, thus acting as their inhibitors, especially of the last one. It is from these interactions that a series of events occur which are responsible for the MCs cytotoxic and genotoxic effects on animal cells. It is also known that MCs induce oxidative stress in cells due to the production of reactive oxygen species (ROS), however a complete characterization of the effects of these toxins has not yet been obtained. This project intends to clarify some of the molecular mechanisms of MC-LR toxicity in animal cells using Saccharomyces cerevisiae as an eukaryotic organism model. To evaluate the cytotoxic effects of MC-LR, a cell viability assay was used to determine the functional capacity of the mitochondria, the MTT assay, after exposing the yeasts to different concentrations of MC-LR for 4 hours. Genotoxic effects were evaluated by gene expression studies for genes Rad27, Apn1, Apn2, Ntg1 and Ntg2 (from the BER DNA repair system) and Cdc55 gene which encodes the PP2A phosphatase protein, using the Real-Time qPCR technique. The reference genes used for expression normalization were Alg9 and Taf10. Furthermore, it was attempted to adapt the single cell gel electrophoresis assay (comet assay), conventionally performed on mammalian cells, to Saccharomyces cerevisiae cells, in order to quantify induced DNA breaks. MTT was optimized and successfully used in S. cerevisiae. Apparently, MC-LR is not cytotoxic for Saccharomyces cerevisiae, although these results should be confirmed with other methods that accessed cell viability. Regarding the Comet assay, the results were not conclusive, possibly due to the difficulty in optimizing the method when applied to yeast cells, particularly in the DNA migration on the electric field. However, the first two steps of the YCA protocol were optimized. Concerning the RTqPCR method it was possible to obtain tendencies in the gene expression levels, when compared with the control situation, thus revealing that MC-LR affects differently both BER pathways. Despite the difficulty of reproducing some methods in yeast cells, it appears that microcystin- LR plays a critical role in the toxicity of eukaryotic cells. This work allowed us to contribute with a little more information to a still relative unknown study field.
- New Insights in Saccharomyces cerevisiae Response to the Cyanotoxin Microcystin-LR, Revealed by Proteomics and Gene ExpressionPublication . Valério, Elisabete; Barreiros, Sara; Rodrigues, Sara; Turkina, Maria V.; Vasconcelos, Vitor M.; Campos, AlexandreMicrocystins (MCs) are hepatotoxins produced by some cyanobacteria. They are cyclic peptides that inhibit the serine/threonine protein phosphatases (PPs) PP1 and PP2A, especially PP2A. The inhibition of PP2A triggers a series of molecular events, which are responsible for most MC cytotoxic and genotoxic effects on animal cells. It is also known that MCs induce oxidative stress in cells due to the production of reactive oxygen species (ROS). However, a complete characterization of the toxic effects of MCs is still not accomplished. This study aimed to clarify additional molecular mechanisms involved in MC-LR toxicity, using Saccharomyces cerevisiae as eukaryotic model organism. First, a shotgun proteomic analysis of S. cerevisiae VL3 cells response to 1 nM, 10 nM, 100 nM, and 1 µM MC-LR was undertaken and compared to the control (cells not exposed to MC-LR). This analysis revealed a high number of proteins differentially expressed related with gene translation and DNA replication stress; oxidative stress; cell cycle regulation and carbohydrate metabolism. Inference of genotoxic effects of S. cerevisiae VL3 cells exposed to different concentrations of MC-LR were evaluated by analyzing the expression of genes Apn1, Apn2, Rad27, Ntg1, and Ntg2 (from the Base Excision Repair (BER) DNA repair system) using the Real-Time RT-qPCR technique. These genes displayed alterations after exposure to MC-LR, particularly the Apn1/Apn2/Rad27, pointing out effects of MC-LR in the Base Excision Repair system (BER). Overall, this study supports the role of oxidative stress and DNA replication stress as important molecular mechanisms of MC-LR toxicity. Moreover, this study showed that even at low-concentration, MC-LR can induce significant changes in the yeast proteome and in gene expression.