RESUMEN
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.
Asunto(s)
Colorantes , Fenotiazinas , Humanos , Fenotiazinas/farmacología , Cisplatino/farmacología , Rojo Neutro , Dimetilsulfóxido , Azul de MetilenoRESUMEN
The neutral red uptake (NRU) assay is a cell viability assay that can be used for the assessment of compound-induced cytotoxicity. It is based on the ability of living cells to incorporate neutral red, a weak cationic dye, in lysosomes. The quantification of xenobiotic-induced cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red when compared to cells exposed to corresponding vehicle controls. The NRU assay is mainly used for hazard assessment in in vitro toxicology applications. Hence, this method has been incorporated in regulatory recommendations such as the OECD test guideline TG 432, in which an in vitro 3T3-NRU-phototoxicityassay is described to assess the cytotoxicity of compounds in the presence or absence of UV light.This book chapter describes a detailed protocol to carry out the NRU assay using the human hepatoma cell line HepG2, which is frequently employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetylsalicylic acid is assessed.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Rojo Neutro/metabolismo , Hepatocitos/metabolismo , Línea Celular , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Supervivencia CelularRESUMEN
BACKGROUND: Millions of people are diagnosed with cancer each year, and fighting it puts a heavy financial burden on communities and governments. Numerous advances have been made in the field of cancer; one of the newest methods is using oncolytic viruses. This study aimed to evaluate the effect of oncolytic Newcastle disease virus wild-type strains (NDV-WTS) on the immune system. MATERIAL AND METHODS: Forty mice were divided into four groups (10 animals in each group). The control group received phosphate buffered saline, and experimental group 1 (NDV-WTS 1), experimental group 2 (NDV-WTS 2), and experimental group 3 (NDV-WTS 3) received 10-1, 10-2, and 10-3 titers of Newcastle virus on 0, 14th, and 28th days. On the 31st day, 100 µL of Newcastle virus was injected into the left footpads of animals. After 48 hours, delayed-type hypersensitivity (DTH) reactions were measured. On the 33rd day, peritoneal macrophages were isolated. Then proliferation of the cells was measured by the methyl-thiazolyl-tetrazolium (MTT) test. Neutral red uptake and respiratory burst of peritoneal macrophages were also assessed. Data were analyzed using statistical software SPSS, version 19. RESULTS: The results of the DTH test showed that footpad swelling in control, NDV-WTS 1, NDV-WTS 2, and NDV-WTS 3 groups were 23.5%, 23.5%, 23.6% and 23.6%. No significant differences were seen between the groups in this regard (P > 0.05). A negative nitroblue tetrazolium (NBT) reduction test as an indicator of macrophage's respiratory burst, showed no significant difference between the groups (P > 0.05). The neutral red uptake assay and MTT test showed no significant differences between the groups (P > 0.05). CONCLUSION: The results of this study showed that NDV-WTS in doses of 10-1, 10-2, and 10-3 have no adverse effects on healthy normal cells.
Asunto(s)
Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Ratones , Humanos , Animales , Virus Oncolíticos/fisiología , Virus de la Enfermedad de Newcastle/fisiología , Viroterapia Oncolítica/métodos , Rojo Neutro , Neoplasias/terapia , Inmunidad , Línea Celular TumoralRESUMEN
Commercial immunochromatographic assay (ICA) is a convenient tool for controlling antibiotic abuse. Although great efforts have been made to improve the detection performance and quantitative capabilities, simplify the manufacturing process of commercial ICA is rarely mentioned. Here, a proof-of-principle work are developed to solve the above problem. Inspired by dyestuff chemistry, we developed an instant immune-network label strategy by dynamic protonation capacity of neutral red (NR), achieving a desirable labeling efficiency (ï¼1min), and applying for ICA detection of chloramphenicol (CAP). Benefits from the efficiently protonation of NR, lengthy probe production time and organic reagents can be avoided, displaying excellent strip production efficiency and detection performance. Eventually, this strategy presents a visual limit of detection (vLOD) at 3 ng/mL, cut-off value is 9 ng/mL. The assay recoveries in milk and honey were 74.45-107.15 %, with the total RSD of 1.62-6.90 %. We envision that this strategy raises the possibility of commercializing of laboratory prototype products.
Asunto(s)
Antibacterianos , Cloranfenicol , Cloranfenicol/análisis , Rojo Neutro , Cromatografía de Afinidad/métodos , Límite de Detección , Antibacterianos/análisis , Inmunoensayo/métodosRESUMEN
Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics
Asunto(s)
Esquizofrenia/patología , Antipsicóticos/efectos adversos , Clozapina/análisis , Haloperidol/análisis , Células 3T3 NIH/clasificación , Rojo Neutro/farmacologíaRESUMEN
The phototoxic potential of a number of furocoumarins is well established. On the other hand, studies have shown that bergamottin, a furocoumarin containing a bulky, hydrophobic side chain, has significantly less or is even absent of phototoxicity potential. The OECD Test Guideline 432 3T3/Neutral Red Uptake (NRU) in vitro phototoxicity test has shown to be a highly predictive test for identifying compounds that exhibit no phototoxicological potential. In this study using OECD 432, the established phototoxic furocoumarin 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and psoralen were phototoxic, whereas bergamottin showed no phototoxic potential. When compared to 5-MOP, 8-MOP and psoralen, bergamottin was clearly negative at molar-adjusted concentrations that were more than 9 times higher than those that produced phototoxicity in 8-MOP; nearly 16 times than those for psoralen and more than 36 times higher than those for 5-MOP. These data using in vitro 3T3 NRU Phototoxicity Test (OECD 432) are supportive of earlier studies showing bergamottin does not exhibit phototoxicological properties. The detection and quantification of bergamottin should therefore not contribute to the potential marker furocoumarins for risk management interventions intended to reduce the phototoxicity of natural furocoumarin containing preparations.
Asunto(s)
Dermatitis Fototóxica , Furocumarinas , Humanos , Metoxaleno/toxicidad , Organización para la Cooperación y el Desarrollo Económico , Rayos Ultravioleta , Furocumarinas/toxicidad , Dermatitis Fototóxica/etiología , Rojo NeutroRESUMEN
Cancer and infectious diseases are among the leading causes of death in the world. Despite the diverse array of treatments available, challenges posed by resistance, side effects, high costs, and inaccessibility persist. In the Solanaceae plant family, few studies with Vassobia breviflora species relating to biological activity are known, but promising results have emerged. The phytochemicals present in the ethyl acetate fraction were obtained using ESI-MS-QTOF, and the antioxidants assays 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical capture (ABTS), plasma ferric reduction capacity (FRAP), and total antioxidant capacity (TAC). Cytotoxic activity was evaluated by MTT, Neutral Red, and lactate dehydrogenase (LDH) released. The production of reactive oxygen species, nitric oxide, and purinergic enzymes was also investigated. Antibacterial activity was measured through minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antibiofilm activity, in addition to genotoxicity in plasmid DNA. Five major masses were identified D-glucopyranose II, allyl disulfide, γ-lactones, pharbilignoside, and one mass was not identified. V. breviflora exhibited relevant antioxidant and cytotoxic activity against the HeLa cell line and enhanced expression effect in modulation of purinergic signaling. Antibacterial activities in the assays in 7 ATCC strains and 8 multidrug-resistant clinical isolates were found. V. breviflora blocked biofilm formation in producing bacteria at the highest concentrations tested. However, there was no plasmid DNA cleavage at the concentrations tested. Data demonstrated that V. breviflora exhibited an antioxidant effect through several methods and proved to be a promising therapeutic alternative for use against tumor cells via purinergic signaling and multidrug-resistant microorganisms, presenting an anti-biofilm effect.
Asunto(s)
Antioxidantes , Solanaceae , Acetatos , Antibacterianos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Bacterias , ADN/farmacología , Células HeLa , Humanos , Lactato Deshidrogenasas , Lactonas/farmacología , Pruebas de Sensibilidad Microbiana , Rojo Neutro/farmacología , Óxido Nítrico , Extractos Vegetales/química , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno , Ácidos SulfónicosRESUMEN
The aim of the present study was to investigate the effects of PDT using the photosensitizer 5-aminoulevulinic acid (5-ALA) in oral squamous cell carcinoma (OSCC) behavior, mainly regarding its role on the cancer stem cell (CSC) phenotypes and in maintenance of the stem cell properties. Two OSCC cell lines were used and divided in the groups: Control, 5-ALA, LED 6 J/cm2 and PDT. MTT and Neutral red assays were used to access cellular viability, cell migration was evaluated by the wound healing assay. The stem cell phenotype was analyzed by flow cytometry to evaluate the CD44high/ESAhigh, CD44high/ESAlow and CD44low populations, by the clonogenic and tumor sphere formation assays as well as by RT-qPCR. The presence of Protoporphyrin IX in each CSC fraction was evaluated by flow cytometry. The OSCC cell lines showed a significant decrease in cell viability and migration after PDT. The percentage of CD44high/ESAhigh cells decreased after PDT, which was associated with an increase in the CD44low cells and with a functional decrease in the colony and sphere formation capacity. CD44high/ESAhigh cells showed increased PpIX, which contributed for their greater sensitivity to PDT. INV gene increased significantly after PDT, indicating cellular differentiation. Altogether, our results demonstrate that 5-ALA mediated PDT decreases not only the fraction of oral CSC but also their functional capabilities, inducing their differentiation.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de la Boca , Fotoquimioterapia , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Rojo Neutro/uso terapéutico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Protoporfirinas/metabolismoRESUMEN
Deep eutectic solvents (DES), which have low toxicity and are low cost, biodegradable, and easily synthesized, were used for the extraction of neutral red (NR) dye before its spectrophotometric analysis. DES, containing choline chloride as a hydrogen bond acceptor and phenol as a hydrogen bond donor with a molar ratio of 1:2, was used for the extraction of NR dye from aqueous media. The possible interaction of different DESs with NR was studied using density functional theory (DFT) calculations. Experimentally, a UV-visible spectrophotometer was used for the quantitative analysis. The most important parameters affecting method performance, such as pH, extraction temperature, DES type, its volume, THF volume, sonication time, and centrifugation time, were optimized. The developed method provides exceptional sensitivity in terms of LOD and LOQ, which were 2.2 and 7.3 µg/L respectively. The relative standard deviation was 1.35−1.5% (n = 10), and the pre-concentration factor was 40. The method was found to be linear in the range of 2−300 µg/L (R2 = 0.9967). The method was successfully used for the determination of NR in wastewater samples. Finally, the DES-based method presents operational simplicity, high sensitivity, and rapid determination (<5 min) compared with other analytical procedures.
Asunto(s)
Microextracción en Fase Líquida , Colina , Disolventes Eutécticos Profundos , Límite de Detección , Microextracción en Fase Líquida/métodos , Rojo Neutro , Fenoles , Solventes/química , Aguas ResidualesRESUMEN
Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.
Asunto(s)
Virus de la Fiebre Aftosa , Fiebre Aftosa , Animales , Fiebre Aftosa/diagnóstico , Virus de la Fiebre Aftosa/genética , Técnicas de Diagnóstico Molecular , Rojo Neutro , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transcripción Reversa , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4. PURPOSE: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity. METHODS: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes. RESULTS: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process. CONCLUSION: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.
Asunto(s)
Virus del Dengue , Naranja de Acridina/farmacología , Antivirales/farmacología , Araquidonato 5-Lipooxigenasa/genética , Ácidos Cafeicos , Colorantes/farmacología , Virus del Dengue/fisiología , Masoprocol/farmacología , Rojo Neutro/farmacología , ARN , Resveratrol/farmacología , Serogrupo , Esteroles/farmacología , Proteínas Virales , Replicación ViralRESUMEN
Renewable electricity-based microbial electrosynthesis can upgrade CO2 into value-added chemicals and simultaneously increase the number of biocatalysts by cell growth, helping to achieve sustainable carbon-negative processes. In most studies, the main strategy for improving the MES performance was to enhance H2-based electron uptake by decreasing the overpotential and electrical conductivity of the electrode. Less is known about the electrode-based direct electron uptake for CO2 conversion in MES. In this study, a solid neutral red/Nafion conductive layer was developed on the carbon electrode surface using a feasible dip and dry method. The modified electrode showed higher HER overpotential and lower capacitance but enhanced redox capability and hydrophobicity, which increased direct electron transport to the bacteria rather than hydrogen-based indirect electron delivery. The Neutral red/Nafion-implemented MES showed faster start-up, higher acetate production, and energy efficiency than the non-modified electrode.
Asunto(s)
Dióxido de Carbono , Carbono , Acetatos/metabolismo , Dióxido de Carbono/metabolismo , Fibra de Carbono , Conservación de los Recursos Energéticos , Conductividad Eléctrica , Electrodos , Polímeros de Fluorocarbono , Hidrógeno , Rojo NeutroRESUMEN
In this study, colorimetric indicator films (CIFs) were developed by integrating neutral red covalently immobilized onto TEMPO-oxidized cellulose nanofibrils (NR@TOCNFs) and poly(acrylic acid) (PAA) inside a poly(vinyl alcohol) (PVA) matrix. The successful covalent immobilization of NR onto the TOCNFs was confirmed using attenuated total reflection-Fourier transform infrared, X-ray photoelectron spectroscopy, and thermogravimetric analyses. The CIFs had a visible color change from red to yellow as the pH changed from 2.0 to 10.0. The colorimetric response of CIFs improved as the NR@TOCNF content increased, while it decreased as the PAA level increased. The critical pH ranges for the color change of CIFs were 6-7, 7-8, and 8-9 for 3 %, 5 %, and 7 % PAA, respectively, at 0.3 % NR@TOCNF. The best ammonia sensitivity was found in the indicator films containing 3 % PAA and 0.3 % NR@TOCNF. These results showed that the CIFs could be applied for freshness detection in food packaging.
Asunto(s)
Amoníaco , Celulosa , Embalaje de Alimentos/métodos , Concentración de Iones de Hidrógeno , Rojo NeutroRESUMEN
Reactive oxygen species (ROS) produced by cell metabolism have a duplex role in oxidation and inflammation reactions which involve cell damage or repair responses. Excess ROS production has detrimental effects on the survival of cells. We examined the protective effect of a semi-natural compound NF2 (deacetylepoxyazadiradione), for its protective activity against free radical-mediated stress and inflammatory response to lipopolysaccharide (LPS) using zebrafish larvae. Preliminary antioxidant assays indicated an increase in scavenging of free radicals from NF2 than NF1 (Epoxyazadiradione) in a concentration-dependent manner. Cell cytotoxicity was determined using rat myoblast cell lines (L6), and more than 95 % of cell viability was obtained. Zebrafish developmental toxicity test indicated that NF2 is not toxic even at 150â µM. The percentage of ROS, lipid peroxidation, nitric oxide and apoptosis were reduced significantly in NF2 treated LPS-stressed zebrafish larvae. The reduced number of employed macrophages on NF2 treatment was observed in neutral red dye-marked macrophage localization images. Relative expression of antioxidant genes in zebrafish larvae after treatment with NF2 is significantly increased. The RT-PCR quantification of antioxidant and anti-inflammatory gene expression indicated decreased relative folds of pro-inflammatory cytokines, iNOS and increased relative folds of mitochondrial antioxidant genes (GR, GST and GPx) in LPS stressed zebrafish larvae after treatment with NF2. From the overall obtained results, it can be concluded that NF2 reduced the oxidative stress and inflammatory response by scavenging free radicals caused by LPS.
Asunto(s)
Azadirachta , Animales , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Citocinas/metabolismo , Frutas/metabolismo , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Larva , Limoninas , Lipopolisacáridos/farmacología , Rojo Neutro/farmacología , Óxido Nítrico , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Pez Cebra/metabolismoRESUMEN
Bleeding-related complications following vascular surgeries occur in up to half of the patients-500 000 cases annually in the United States alone. This results in additional procedures, increased mortality rate, and prolonged hospitalization, posing a burden on the healthcare system. Commercially available materials rely, in large, on forming covalent bonds between the tissue and the biomaterial to achieve adhesion. Here, it is shown that a biomaterial based on oxidized alginate and oxidized dextran together with polyamidoamine (PAMAM) dendrimer amine provides simultaneous electrostatic and covalent interactions between the biomaterial and the tissue, maximizing adhesion. This study finds that the material withstands supraphysiological pressures (≈300 mmHg) and prevents bleeding in a rabbit aortic puncture model and in a pig carotid bilateral poly(tetrafluoroethylene) graft model-achieving superior performance to commercially available materials such as Tisseel and BioGlue. Material biocompatibility is validated in comprehensive in vitro and in vivo studies in accordance with the US Food and Drug Administration (FDA) guidelines, including in vitro neutral red uptake test, subcutaneous implantation in rabbits, ames genotoxicity, and guinea pig maximization test. This material has the potential to provide with adequate seal and reduced complications following complex vascular surgeries, including hard-to-seal tissue-graft interfaces.
Asunto(s)
Dendrímeros , Adhesivos Tisulares , Conejos , Cobayas , Animales , Hidrogeles/química , Adhesivo de Tejido de Fibrina , Adhesivos Tisulares/química , Ensayo de Materiales , Dextranos , Rojo Neutro , Alginatos/química , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/uso terapéutico , Anastomosis QuirúrgicaRESUMEN
Natural mineral waters (NMWs) emerge from the earth as springs and their beneficial therapeutic effect has been empirically recognized in different countries. Portugal has diverse NMW resources that are sought for the relief of different afflictions including dermatological complications. However, there is a lack of scientific validation supporting this empiric knowledge. In this study, we aimed to screen the in vitro bioactivity of Portuguese NMWs with different chemical profiles, namely sulfurous/bicarbonate/sodic (SBS), bicarbonate/magnesium, sulfated/calcic, sulfurous/chlorinated/sodic, sulfurous/bicarbonate/fluoridated/sodic, and chlorinated/sodic, focusing on aging-related skin alterations. Mouse skin fibroblasts and macrophages were exposed to culture medium prepared in different NMWs. Cellular viability was evaluated by MTT assay and etoposide-induced senescence was analyzed through the beta-galactosidase staining kit. Wound healing was investigated by the scratch assay, and phototoxicity/photoprotection after UVA irradiation was evaluated using a neutral red solution. ROS production was quantified using the 2'7'-dichlorofluorescin diacetate dye, and the activity of superoxide dismutase (SOD) was analyzed by a commercial kit after lipopolysaccharide exposure. NMWs within the SBS profile demonstrated anti-senescence activity in skin fibroblasts, along with a variable effect on cellular viability. Among the tested NMWs, two decreased cellular senescence and preserved cell viability and were therefore selected for subsequent studies, together with a SBS NMW with therapeutic indications for dermatologic diseases. Overall, the selected NMW promoted wound healing in skin fibroblasts and activated SOD in macrophages, thus suggesting an anti-oxidant effect. None of the NMWs prevented phototoxicity after UV irradiation. Our results shed a light on the anti-aging potential of Portuguese NMW, supporting their putative application in cosmetic or medical products.
Asunto(s)
Aguas Minerales , Envejecimiento de la Piel , Animales , Antioxidantes/farmacología , Bicarbonatos , Células Cultivadas , Etopósido/farmacología , Lipopolisacáridos/farmacología , Magnesio , Ratones , Rojo Neutro/farmacología , Portugal , Especies Reactivas de Oxígeno , Piel , Superóxido Dismutasa , Rayos Ultravioleta , beta-Galactosidasa/farmacologíaRESUMEN
BACKGROUND: Exopolysaccharide, a carbohydrate polymer, is known to possess several biological activities. This approach was designed to clarify the cytotoxic mechanism of Bacillus sonorensis exopolysaccharide (EPS-1) on Huh7, HepG2 and BNL cells besides exploring its influence on the expression of the tumor suppressor protein p53. p53 is the biomarker of the prognosis and occurrence of severe stages of the tumor and activation of both cell-cycle arrest and apoptosis in cancer cells which are the most targeted cellular processes for the therapy of tumor patients. METHODS: The cytotoxic impact of EPS-1 was quantified via neutral red uptake assay and the results were confirmed by a morphology study. The expression level of p53 was analyzed using quantitative real-time PCR. RESULTS: The outcomes of the present study explicated that EPS-1 with IC 50 = 164 and 398 µg ml -1 exhibited an inhibitory influence on Huh7 and HepG2 cells growth after 48 h incubation time respectively. EPS-1 showed no influence on normal BNL cells. Furthermore, the molecular genetic analysis revealed that EPS-1 provoked significant upregulation in the expression level of the p53 gene in the treated Huh7 cell line more than that in HepG2, whereas no significant gene expression was noticed in BNL cells ( P = 0.006, 0.65 and 0.83), respectively. CONCLUSION: The antitumor activity displayed by this compound may be of interest for further studies of its structure-activity relationship. Before application in phase 1 of the clinical study, in-vivo studies would be needed to confirm the results obtained in the hope of finding more active and selective anticancer agents for drug development in the future.
Asunto(s)
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Bacillus , Línea Celular Tumoral , Proliferación Celular , Humanos , Rojo Neutro/farmacología , Rojo Neutro/uso terapéutico , Polímeros/metabolismo , Polímeros/farmacología , Polímeros/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/farmacologíaRESUMEN
Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy.
Asunto(s)
Naranja de Acridina , Catepsina D , Naranja de Acridina/metabolismo , Naranja de Acridina/farmacología , Antraquinonas/farmacología , Apoptosis , Autofagia , Caspasa 3/metabolismo , Catepsina D/metabolismo , Cloroquina/metabolismo , Cloroquina/farmacología , Células HeLa , Humanos , Lisosomas/metabolismo , Rojo Neutro/metabolismo , Rojo Neutro/farmacología , Óxidos/metabolismo , Óxidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Costus spiralis (Jacq.). Roscoe (Costaceae) is traditionally used in Brazil for the treatment of kidney diseases such as pyelonephritis, urethra inflammation, kidney stones, and inflammatory conditions. There are reports of its use by Brazilian Indians since the 17th century when it was known as "pacocatinga." Currently, the use of the Costus species in Brazil is widespread, which was evidenced by the inclusion of the genus in the Brazilian National List of Medicinal Plants of Interest to the Unified Health System (RENISUS). AIM OF THE STUDY: This study aimed to confirm the ethnopharmacological use of Costus spiralis in the treatment of kidney diseases, toxicity study using animal models, and the phytochemistry of the species. MATERIALS AND METHODS: The chemical profile of Costus spiralis leaves extract (CSLE) was obtained for the hydroethanolic extract by ultra-performance liquid chromatography coupled to a mass spectrometer and ultraviolet detector with diode array (UPLC-UV/DAD-ESI-MS). The acute oral toxicity of the extract was predicted using the neutral red uptake cytotoxicity assay. Wistar rats were used in a model in vivo for confirmation of acute oral toxicity (2000 mg/kg p.o. for 14 days.) and determination of the effect on a cisplatin-induced nephrotoxicity model. RESULTS: The analysis by UPLC-UV/DAD-ESI-MS showed that the chemical composition of the extract is mostly di-glycosylated flavones of apigenin. In the extract were identified the flavones vicenin II and schaftoside. The quantification of total flavonoids by spectrometry showed 0.880%. CSLE proved to be safe for acute oral administration (2000 mg/kg) with an IC50 value of 222.9 µg/mL and predicted oral toxic dose of 523.82 µg/mL in a neutral red uptake cytotoxicity assay. The absence of death allows the classification of the extract in class 5 according to OECD 423 guidelines and therefore it can be considered as a high acute safety product, which is highly relevant, considering the wide popular use of the species. In the cisplatin-induced nephrotoxicity model, C. spiralis extract (5, 15, and 30 mg/kg) significantly improved renal function, reversing almost completely the effects on plasma creatinine levels and creatinine clearance (p < 0.001). CONCLUSIONS: This study demonstrates that oral administration of Costus spiralis extract leaves is safe and effective in restoring the renal function in rats in a cisplatin-induced nephrotoxicity. It is suggested that the observed activity is related to the flavonoids present. This hypothesis should be confirmed, and the participation of other secondary metabolites should be investigated in the future.
Asunto(s)
Costus , Flavonas , Animales , Apigenina , Cisplatino/toxicidad , Costus/química , Creatinina , Flavonas/análisis , Flavonoides/análisis , Humanos , Riñón , Rojo Neutro/análisis , Extractos Vegetales/análisis , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Hojas de la Planta/química , Ratas , Ratas WistarRESUMEN
Today, easy availability and efficient detection assays in low-resource settings are mandatory. Therefore, a smartphone-assisted paper-based detection strategy using eosin Y (EY)-embed ZIF-8 nanocomposite for phosphate (Pi), neutral red (NR) and heparin (HP) on site testing was fabricated. After adding the targets in a specific order, the prepared strips produced brilliant color transitions (astonishing colors, ranging from pistachio to bright yellow for Pi, from bright yellow to jacinth for NR and from jacinth to light orange for HP). These variation on tonality can be evaluated by the smartphone-based readout device (Color Assist App) through the ratio change of B/(R + G + B), which exhibits superior performance of on-site detection in wastewater, including high sensitivity (LOD is 0.015, 0.031 and 0.014 mM for Pi, NR and HP, respectively), ultra-fast response time and excellent selectivity. This strips and smartphone dual-mode monitoring strategy may offer a convenient idea to identify Pi, NR and HP, with great prospects for environmental wastewater applications. Additionally, the probe was enabled for bioimaging of Pi, NR and HP in living cells. Finally, the related anti-counterfeiting fluorescent ink of probe was proposed on schedule.