RESUMO
Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications.
Assuntos
Corantes , Fenotiazinas , Humanos , Fenotiazinas/farmacologia , Cisplatino/farmacologia , Vermelho Neutro , Dimetil Sulfóxido , Azul de MetilenoRESUMO
The fast-emerging and multidrug-resistant Candida auris is the first fungal pathogen to be considered a threat to global public health. Thus, there is a high unmet medical need to develop new therapeutic strategies to control this species. Antimicrobial photodynamic therapy (APDT) is a promising alternative that simultaneously targets and damages numerous microbial biomolecules. Here, we investigated the in vitro and in vivo effects of APDT with four phenothiazinium photosensitizers: (i) methylene blue (MB), (ii) toluidine blue (TBO), and two MB derivatives, (iii) new methylene blue (NMBN) and (iv) the pentacyclic derivative S137, against C. auris. To measure the in vitro efficacy of each PS, minimal inhibitory concentrations (MICs) and survival fraction were determined. Also, the efficiency of APDT was evaluated in vivo with the Galleria mellonella insect model for infection and treatment. Although the C. auris strain used in our study was shown to be resistant to the most-commonly used clinical antifungals, it could not withstand the damages imposed by APDT with any of the four photosensitizers. However, for the in vivo model, only APDT performed with S137 allowed survival of infected G. mellonella larvae. Our results show that structural and chemical properties of the photosensitizers play a major role on the outcomes of in vivo APDT and underscore the need to synthesize and develop novel photosensitizing molecules against multidrug-resistant microorganisms.
Assuntos
Anti-Infecciosos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Azul de Metileno/farmacologia , Candida auris , Antifúngicos/farmacologia , Cloreto de Tolônio , Fotoquimioterapia/métodos , Anti-Infecciosos/farmacologiaRESUMO
The widespread use of conventional chemical antifungal agents has led to worldwide concern regarding the selection of resistant isolates. In this scenario, antimicrobial photodynamic treatment (APDT) has emerged as a promising alternative to overcome this issue. The technique is based on the use of a photosensitizer (PS) and light in the presence of molecular oxygen. Under these conditions, the PS generates reactive oxygen species which damage the biomolecules of the target organism leading to cell death. The great potential of APDT against plant-pathogenic fungi has already been reported both in vitro and in planta, indicating this control measure has the potential to be widely used in crop plants. However, there is a lack of studies on environmental risk with ecotoxicological assessment of PSs used in APDT. Therefore, this study aimed to evaluate the environmental toxicity of four phenothiazinium PSs: i) methylene blue (MB), ii) new methylene blue N (NMBN), iii) toluidine blue O (TBO), and iv) dimethylmethylene blue (DMMB) and also of the commercial antifungal NATIVO®, a mixture of trifloxystrobin and tebuconazole. The experiments were performed with Daphnia similis neonates and zebrafish embryos. Our results showed that the PSs tested had different levels of toxicity, with MB being the less toxic and DMMB being the most. Nonetheless, the environmental toxicity of these PSs were lower when compared to that of NATIVO®. Furthermore, estimates of bioconcentration and of biotransformation half-life indicated that the PSs are environmentally safer than NATIVO®. Taken together, our results show that the toxicity associated with phenothiazinium PSs would not constitute an impediment to their use in APDT. Therefore, APDT is a promising approach to control plant-pathogenic fungi with reduced risk for selecting resistant isolates and lower environmental impacts when compared to commonly used antifungal agents.
Assuntos
TriazóisRESUMO
Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 µM, 11 ± 2 µM, 0.7 ± 0.1 µM, and 0.9 ± 0.2 µM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.
Assuntos
Coccidiostáticos/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Redutase/efeitos dos fármacos , Azul de Metileno/análogos & derivados , Neospora/efeitos dos fármacos , Cloreto de Tolônio/farmacologia , Animais , Citoplasma/enzimologia , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Cinética , Masculino , Azul de Metileno/farmacologia , Camundongos Endogâmicos BALB C , Neospora/enzimologia , Neospora/genética , Neospora/crescimento & desenvolvimentoRESUMO
Antimicrobial photodynamic therapy (aPDT) is a complementary therapeutic modality for periodontal and endodontic diseases, in which Gram-negative bacteria are directly involved. Currently, there are few evidences regarding the effects of aPDT on bacterial components such as lipopolysaccharide (LPS) and it would represent a major step forward in the clinical use of this therapy. In this context, this study aimed to evaluate the efficacy of different photosensitizers (PSs) used in aPDT in LPS inhibition. Four PSs were used in this study: methylene blue (MB), toluidine blue (TBO), new methylene blue (NMB), and curcumin (CUR). Different approaches to evaluate LPS interaction with PSs were used, such as spectrophotometry, Limulus amebocyte lysate (LAL) test, functional assays using mouse macrophages, and an in vivo model of LPS injection. Spectrophotometry showed that LPS decreased the absorbance of all PSs used, indicating interactions between the two species. LAL assay revealed significant differences in LPS concentrations upon pre-incubation with the different PSs. Interestingly, the inflammatory potential of LPS decreased after previous treatment with the four PSs, resulting in decreased secretion of inflammatory cytokines by macrophages. In vivo, pre-incubating curcumin with LPS prevented animals from undergoing septic shock within the established time. Using relevant models to study the inflammatory activity of LPS, we found that all PSs used in this work decreased LPS-induced inflammation, with a more striking effect observed for NMB and curcumin. These data advance the understanding of the mechanisms of LPS inhibition by PSs.
Assuntos
Odontologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Animais , Macrófagos/efeitos dos fármacos , Macrófagos/efeitos da radiação , Camundongos , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêuticoRESUMO
Antimicrobial photodynamic treatment (APDT) has emerged as an effective therapy against pathogenic fungi with both acquired and intrinsic resistance to commonly used antifungal agents. Success of APDT depends on the availability of effective photosensitizers capable of acting on different fungal structures and species. Among the phenothiazinium dyes tested as photoantifungals, new methylene blue N (NMBN) and the novel pentacyclic compound S137 are the most efficient. In the present study we compared the effects of APDT with NMBN and S137 on the survival of Candida albicans and employed a set of fluorescent probes (propidium iodide, FUN-1, JC-1, DHR-123 and DHE) together with confocal microscopy and flow cytometry to evaluate the effects of these two chemically diverse photosensitizers on cell membrane permeability, metabolism and redox status, and mitochondrial activity. Taken together, our results indicate that, due to chemical features resulting in different lipophilicity, NMBN and S137 localize to distinct subcellular structures and hence inactivate C. albicans cells via different mechanisms. S137 localizes mostly to the cell membrane and, upon light exposure, photo-oxidizes membrane lipids. NMBN readily localizes to mitochondria and exerts its photodynamic effects there, which was observed to be a less effective way to achieve cell death at lower light fluences.
Assuntos
Anti-Infecciosos/química , Candida albicans/metabolismo , Azul de Metileno/química , Fármacos Fotossensibilizantes/química , Frações Subcelulares/metabolismo , Anti-Infecciosos/metabolismo , Corantes Fluorescentes/química , Azul de Metileno/metabolismo , Fármacos Fotossensibilizantes/metabolismoRESUMO
Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9-Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019-1.83 µM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9-Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9-Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control.
Assuntos
Coccidiose , Azul de Metileno/análogos & derivados , Neospora/crescimento & desenvolvimento , Animais , Chlorocebus aethiops , Coccidiose/tratamento farmacológico , Coccidiose/metabolismo , Masculino , Azul de Metileno/farmacologia , Camundongos , Células VeroRESUMO
The in vitro susceptibilities of Candida albicans and Candida tropicalis to Antimicrobial Photodynamic Treatment with aluminum phthalocyanine chloride in nanoemulsion (ClAlPc/NE) were investigated. PS concentration- and fluence-dependent cell survival after APDT were compared before and after unbound extracellular PS had been washed out. The PS uptake and its subcellular localization were also determined. Exposure to light in the absence of the PS and treatment with the PS in the absence of light did not kill the fungi. APDT with ClAlPc/NE resulted in a reduction of five orders of magnitude in viability for C. albicans and between four and five orders of magnitude for C. tropicalis. Washing the cells to remove unbound PS before light exposure did not impair fungal inactivation, suggesting that cell photosensitization was mainly carried out by cell bound ClAlPc. The degree of ClAlPc uptake was dependent on its concentration. Internalization of ClAlPc by C. albicans and C. tropicalis was confirmed by confocal fluorescence microscopy that showed the PS does not penetrate the nucleus and instead accumulates in specific regions of the cytoplasm. Our results show that incorporating the water-insoluble ClAlPc into a nanoemulsion leads to an efficient formulation capable of photoinactivating both Candida species.
Assuntos
Candida albicans , Candida tropicalis , Viabilidade Microbiana , Candida albicans/efeitos dos fármacos , Candida albicans/efeitos da radiação , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/efeitos da radiação , Indóis/farmacologia , Compostos Organometálicos/farmacologia , Fármacos Fotossensibilizantes/farmacologiaRESUMO
Fusarium keratoplasticum and Fusarium moniliforme are filamentous fungi common in the environment and cause mycosis in both animals and plants. Human infections include mycetoma, keratitis and onychomycosis, while deeper mycosis occurs in immunocompromised patients. Most of the Fusarium spp. are frequently resistant to treatment with currently used antifungals. The frequent occurrence of antifungal resistance has motivated the study of antimicrobial photodynamic therapy as an alternative treatment for fungal infections. Many studies have investigated the in vitro use of antimicrobial photodynamic therapy to kill fungi, but rarely in animal models of infection. Thus, here we employed the invertebrate wax moth Galleria mellonella to study the in vivo effects of antimicrobial photodynamic therapy with three different phenothiazinium photosensitizers, methylene blue, new methylene blue N and the pentacyclic S137 against infection with microconidia of Fusarium keratoplasticum and Fusarium moniliforme. The effect of antimicrobial photodynamic therapy using these photosensitizers and light-emitting diodes with an emission peak at 635 nm and an integrated irradiance from 570 to 670 nm of 9.8 mW cm-2 was investigated regarding the toxicity, fungal burden, larval survival and cellular immune response. The results from this model indicate that antimicrobial photodynamic therapy with methylene blue, new methylene blue N and S137 is efficient for the treatment of infection with F. keratoplasticum and F. moniliforme. The efficiency can be attributed to the fungal cell damage caused by antimicrobial photodynamic therapy which facilitates the action of the host immune response.
Assuntos
Fusarium/efeitos dos fármacos , Fenotiazinas/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Animais , Antifúngicos/farmacologia , Dipeptídeos/farmacologia , Farmacorresistência Fúngica , Larva/efeitos dos fármacos , Lasers Semicondutores/uso terapêutico , Azul de Metileno/análogos & derivados , Azul de Metileno/farmacologia , Mariposas , Pirimidinas/farmacologiaRESUMO
BACKGROUND: Fungal secondary metabolites are important sources for the discovery of new pharmaceuticals, as exemplified by penicillin, lovastatin and cyclosporine. Searching for secondary metabolites of the fungi Metarhizium spp., we previously identified tyrosine betaine as a major constituent. METHODS: Because of the structural similarity with other inhibitors of neprilysin (NEP), an enzyme explored for the treatment of heart failure, we devised the synthesis of tyrosine betaine and three analogues to be subjected to in vitro NEP inhibition assays and to molecular modeling studies. RESULTS: In spite of the similar binding modes with other NEP inhibitors, these compounds only displayed moderate inhibitory activities (IC50 ranging from 170.0 to 52.9 µM). However, they enclose structural features required to hinder passive blood brain barrier permeation (BBB). CONCLUSIONS: Tyrosine betaine remains as a starting point for the development of NEP inhibitors because of the low probability of BBB permeation and, consequently, of NEP inhibition at the Central Nervous System, which is associated to an increment in the Aß levels and, accordingly, with a higher risk for the onset of Alzheimer's disease.