RESUMO
Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment of oral infections as a minimal intervention clinical approach. Its antimicrobial efficacy was demonstrated in several studies; however, there is a lack of evidence on its cytotoxic effect on mouse fibroblasts (NIH/3T3). The aim of this study was to evaluate the cytotoxicity and apoptotic pathways of methylene blue-mediated aPDT on mouse fibroblasts. Cells were treated with 0.1 or 1.0 mg.L-1 methylene blue (MB), and 0.075 or 7.5 J.cm-² LED at 630 nm. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while cDNA expression for Bax, Bad, Bcl-2, VDAC-1, cytochrome C and Fas-L was assessed by qRT-PCR (1, 3, 6 and 24 h). The differences between groups were detected by Kruskal-Wallis and post-hoc Dunn's tests for MTT and CV assays, and by ANOVA and post-hoc Tukey test for qPCR (P < 0.05). The combination of 1.0 mg.L-1 MB and 7.5 J.cm-² LED significantly reduced the cellular viability, whereas MB and LED alone were innocuous to fibroblasts. MB-mediated aPDT increased the expression of cytochrome C and Fas-L after 3 h, and Bax/Bcl-2, Bad/Bcl-2, and VDAC-1 after 6 h from treatment. Based on these results, MB-mediated aPDT induced cytotoxicity on mouse fibroblasts, with consequent activation of Bcl-2 apoptosis signaling pathways. Further studies are needed to determine the adequate parameters of aPDT to inactivate microorganisms without damaging fibroblasts.
Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Azul de Metileno/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Animais , Apoptose/genética , Sobrevivência Celular , Genes bcl-2 , Técnicas In Vitro , Azul de Metileno/toxicidade , Camundongos , Fármacos Fotossensibilizantes/toxicidadeRESUMO
Neospora caninum is an apicomplexan parasite strongly related to reproductive problems in cattle. The neosporosis control is not well established and several fronts are under development, predominantly based on immune protection, immunomodulation and chemotherapy. The use of anti-malarial drugs as therapeutic sources has, in theory, considerable potential for any apicomplexan. Drugs such as methylene blue (MB) and pyrimethamine (Pyr) represent therapeutic options for malaria; thus, their use for neosporosis should be assessed. In this work, we tested the effects of MB and Pyr on N. caninum proliferation and clearance, using LacZ-tagged tachyzoites. The drugs inhibited at nanomolar dosages and its combination demonstrated an antagonistic interaction in proliferation assays, according to the Chou and Talalay method for drug combination index. However, the drug combination significantly improved the parasite in vitro clearance. The repositioning of well-established drugs opens a short-term strategy to obtain low-cost therapeutics approaches against neosporosis.
Assuntos
Antimaláricos/farmacologia , Coccidiose/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Azul de Metileno/farmacologia , Neospora/efeitos dos fármacos , Pirimetamina/farmacologia , Animais , Antimaláricos/uso terapêutico , Antimaláricos/toxicidade , Chlorocebus aethiops , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/toxicidade , Concentração Inibidora 50 , Azul de Metileno/uso terapêutico , Azul de Metileno/toxicidade , Pirimetamina/uso terapêutico , Pirimetamina/toxicidade , Células Vero/efeitos dos fármacosRESUMO
The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysical properties suitable for photodynamic therapy (PDT)-based cancer treatment. However, the clinical application of MB is limited because of its high hydrophilicity, which hinders its penetration into tumor tissues. Therefore, new methods to improve the entry of MB into the cytoplasm of target cells are necessary. Because MB has a mass of 319 Da, transient pores on the plasma membrane, such as the pore induced by the P2X7 receptor (P2X7R) that allows the passage of molecules up to 900 Da, could be used. Using MTT viability assays, flow cytometry experiments, and fluorescence microscopy, we evaluated the toxicity and phototoxicity of MB and potentiation effects of ATP and MB on cell death processes in the J774 cell line (via a P2X7-associated pore). We observed that treatment with 5 µM MB for 15 min promoted the rate of entry of MB into the cytoplasm to 4.7 %. However, treatment with 5 µM MB and 1 mM ATP for the same amount of time increased this rate to 90.2 %. However, this effect was inhibited by pretreatment with a P2X7 antagonist. We used peritoneal macrophages and a cell line that does not express P2X7R as controls. These cells were more resistant to PDT with MB under the same experimental conditions. Taken together, these results suggest the use of the pore associated with P2X7R as a drug delivery system to increase the passage of hydrophilic drugs into cells that express this receptor, thus facilitating PDT.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fotoquimioterapia/métodos , Receptores Purinérgicos P2X7/uso terapêutico , Trifosfato de Adenosina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Macrófagos/citologia , Azul de Metileno/farmacocinética , Azul de Metileno/toxicidade , Camundongos , PorosidadeRESUMO
Methylene blue (MB) has been widely applied in the clinical area and is currently being used in aquaculture as biocide. Some recent studies have emphasized the importance of understanding the action mechanism and the MB cellular targets. In this sense, zebrafish is considered a relevant model to study the intrinsic pathway of apoptosis as well as the cellular responses involving DNA damage and repair. So, the aim of the present study was to compare MB action mechanisms in a zebrafish cell line, both in the absence (MB alone; dark toxicity) and in the presence of photosynthetically active radiation (MB+PAR; phototoxicity). There was a significant increase of the levels of reactive oxygen and nitrogen species 3 h after MB treatment, whereas this increase was only observed 12 h after treatment with MB+PAR. All treatments with MB resulted in an increase in DNA damage after 3 and 6 h. However, cell death by apoptosis was observed from 6 h after treatment with MB+PAR and 12 h after treatment with MB alone. The expression of genes related to apoptosis was altered after MB and MB+PAR treatment. Therefore, this zebrafish cell line is sensitive to the photodynamic action of MB; MB is able to generate DNA damage and induce apoptosis in this cell line both alone and in the presence of PAR. However, the pathways leading to apoptosis in this model appear to be dependent on the type of MB exposure (in the presence or absence of PAR).
Assuntos
Hepatócitos/efeitos dos fármacos , Hepatócitos/efeitos da radiação , Azul de Metileno/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Linhagem Celular , Dano ao DNA , Expressão Gênica/efeitos dos fármacos , Luz , Peixe-ZebraRESUMO
In this study, methylene blue (MB) removal from an aqueous phase by electrical discharge non-thermal plasma (NTP) over water was investigated using three different feed gases: N(2), Ar, and O(2). The results showed that the dye removal rate was not strongly dependent on the feed gas when the electrical current was kept the same for all gases. The hydrogen peroxide generation in the water varied according to the feed gas (N(2)Assuntos
Corantes/química
, Ferro/química
, Azul de Metileno/química
, Sulfetos/química
, Poluentes Químicos da Água/química
, Animais
, Argônio/química
, Artemia/efeitos dos fármacos
, Catálise
, Corantes/toxicidade
, Peróxido de Hidrogênio/química
, Dose Letal Mediana
, Azul de Metileno/toxicidade
, Nitrogênio/química
, Oxigênio/química
, Eliminação de Resíduos Líquidos/métodos
, Poluentes Químicos da Água/toxicidade
, Purificação da Água/métodos
RESUMO
OBJECTIVES: To investigate whether the major fungal multidrug efflux systems (MESs) affect the efficiency of methylene blue (MB)-mediated antimicrobial photodynamic inactivation (APDI) in pathogenic fungi and test specific inhibitors of these efflux systems to potentiate APDI. METHODS: Candida albicans wild-type and mutants that overexpressed two classes of MESs [ATP-binding cassette (ABC) and major facilitator superfamily (MFS)] were tested for APDI using MB as the photosensitizer with and without addition of MES inhibitors. The uptake and cytoplasm localization of photosensitizer were achieved using laser confocal microscopy. RESULTS: ABC MES overexpression reduced MB accumulation and APDI killing more than MFS MES overexpression. Furthermore, by combining MB APDI with the ABC inhibitor verapamil, fungal killing and MB uptake were potentiated, while by combining MB APDI with the MFS inhibitor INF(271), fungal killing and MB uptake were inhibited. This latter surprising finding may be explained by the hypothesis that the MFS channel can also serve as an uptake mechanism for MB. CONCLUSIONS: The ABC pumps are directly implicated in MB efflux from the cell cytoplasm. Both the influx and efflux of MB may be regulated by MFS systems, and blocking this gate before incubation with MB can decrease the uptake and APDI effects. An ABC inhibitor could be usefully combined with MB APDI for treating C. albicans infections.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/toxicidade , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Azul de Metileno/toxicidade , Fármacos Fotossensibilizantes/toxicidade , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antifúngicos/metabolismo , Humanos , Azul de Metileno/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Confocal , Fármacos Fotossensibilizantes/metabolismoRESUMO
Azospirillum brasilense, a plant-growth-promoting rhizobacterium, is exposed to changes in its abiotic environment, including fluctuations in temperature, salinity, osmolarity, oxygen concentration and nutrient concentration, in the rhizosphere and in the soil. Since extra-cytoplasmic function (ECF) sigma factors play an important role in stress adaptation, we analysed the role of ECF sigma factor (also known as RpoE or σ(E)) in abiotic stress tolerance in A. brasilense. An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slow-growing, and was sensitive to salt, ethanol and methylene blue stress. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis and sensitivity to different stresses. Based on data from reverse transcriptase-PCR, a two-hybrid assay and a pull-down assay, we present evidence that rpoE is cotranscribed with chrR and the proteins synthesized from these two overlapping genes interact with each other. Identification of the transcription start site by 5' rapid amplification of cDNA ends showed that the rpoE-chrR operon was transcribed by two promoters. The proximal promoter was less active than the distal promoter, whose consensus sequence was characteristic of RpoE-dependent promoters found in alphaproteobacteria. Whereas the proximal promoter was RpoE-independent and constitutively expressed, the distal promoter was RpoE-dependent and strongly induced in response to stationary phase and elevated levels of ethanol, salt, heat and methylene blue. This study shows the involvement of RpoE in controlling carotenoid synthesis as well as in tolerance to some abiotic stresses in A. brasilense, which might be critical in the adaptation, survival and proliferation of this rhizobacterium in the soil and rhizosphere under stressful conditions.
Assuntos
Antibacterianos/toxicidade , Azospirillum brasilense/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/biossíntese , Fator sigma/biossíntese , Estresse Fisiológico , Transcrição Gênica , Antibacterianos/metabolismo , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Etanol/metabolismo , Etanol/toxicidade , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Azul de Metileno/metabolismo , Azul de Metileno/toxicidade , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Repressoras/genética , Fator sigma/genética , Cloreto de Sódio/metabolismo , Cloreto de Sódio/toxicidade , Técnicas do Sistema de Duplo-HíbridoRESUMO
Methylene blue (MB) is a phenothiazine with radio and photosensitizing properties and anti-tumoral activity. Our group has shown that MB was capable of inhibiting the in vitro growth of erythroleukemic cells with multidrug resistance (MDR). However, there are no studies comparing the cytotoxicity of this molecule for normal and tumoral cells. In this work, the cytotoxicity of MB was measured by MTT method in erythroleukemic and melanoma lineages, comparing it with that of normal cells:lymphocytes and melanocytes. MB was more cytotoxic for tumoral cells; however, there was no difference between erytroleukemic cells with or without MDR phenotype. Lymphocytes and erythroleukemic cells were much more sensitive to the effects of MB than melanoma cells and melanocytes. The proliferation of phytohemagglutinin-activated lymphocytes was inhibited when 3H-thymidine incorporation to DNA was measured. We tried to analyze whether the cells were dying, via apoptosis or necrosis, using Anexin-V and propidium iodide. Despite higher levels of Anexin-V, it was not possible to distinguish necrosis from apoptosis, as the fluorescence of MB is in the same channel as propidium iodide. The production of hydrogen peroxide was measured by cytometry using dihydrorhodamine 123 (DHR). Despite the erythroleukemic cells and lymphocytes being capable of producing free radicals, there was no relation between the production and the sensitivity of various cells to MB. Our results suggest that MB should be used as a chemotherapeutic agent, because of its preferential cytotoxic effects over tumor cells, considering the fact that MDR cells are also sensitive, and due to its radio and photosensitizing activities.
Assuntos
Antineoplásicos/toxicidade , Células K562/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Azul de Metileno/toxicidade , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/biossíntese , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Humanos , Peróxido de Hidrogênio/metabolismo , Células K562/metabolismo , Células K562/patologia , Leucócitos Mononucleares/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Fito-Hemaglutininas/farmacologiaRESUMO
In this work we describe an experimental model to evaluate the photodynamic toxicity on amphibian embryos, as well as the protective effect of antioxidants against the lethal oxidative stress induced by photosensitization. Bufo arenarum embryos were treated with 10 mg/l methylene blue (MB) in AMPHITOX solution for 72 h and then irradiated with a red laser or white light for variable times. Both light sources affected the survival of MB-treated animals and lethal effects occurred within the initial 12 h post-irradiation. For white light irradiation, the most effective phototoxic condition in our study, the LD10, 50 and 90 at 6 h post-irradiation corresponded to 13.57, 19.87 and 29.10 J/cm2, respectively. To explore the action of antioxidants against the photogenerated oxidative stress, MB-treated embryos were incubated with 1mM glutathione (GSH) or ascorbic acid (AA) during 48 h before irradiation. For GSH and 21.6 J/cm2 irradiation, the survival increased from 20 to 90%, whereas 100% survival was achieved with AA even after 43.2 J/cm2 irradiation. These results indicate that both the lethal photodynamic effect and its prevention by antioxidants can be evaluated by means of a simple toxicity test employing amphibian embryos.
Assuntos
Antioxidantes/uso terapêutico , Embrião não Mamífero , Luz/efeitos adversos , Azul de Metileno/toxicidade , Estresse Oxidativo , Fármacos Fotossensibilizantes/toxicidade , Animais , Ácido Ascórbico/uso terapêutico , Bufo arenarum , Relação Dose-Resposta à Radiação , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/efeitos da radiação , Glutationa/uso terapêutico , Lasers/efeitos adversos , Dose Letal Mediana , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Testes de ToxicidadeRESUMO
Photodynamic action has been advocated as an alternative treatment of tumors but the most common used dyes, hematoporphyrin derivatives, are substrate for P-glycoprotein. This study investigated the MDR-reverting properties of methylene blue (MB) and compared the sensitivity to its photodynamic action (PDA) in five cell lines that either express or do not express the MDR phenotype. MB was able to revert the MDR phenotype and there was no difference in sensitivity to MB-PDA between MDR and non-MDR cells, suggesting that MB has the advantage of being used simultaneously as a MDR reverser and a photodynamic agent.