RESUMEN
This paper demonstrates for the first time a simple analytical method for differentiation and quantification of dead/live cancer cells using acridine orange (AO) enabled fluorescence spectroscopic techniques. Based on the differential fluorescence (live cells fluoresce green and dead cells orange) exhibited when intercalated with AO, the live/dead cells can be easily differentiated. The optimal AO concentration for enhanced sensitive differentiation has been optimized as 0.001%. These studies offer a promising application for rapid differentiation and quantification of live/dead cells in the case of cytotoxic treatment/therapies within several minutes.
Asunto(s)
Naranja de Acridina/química , Naranja de Acridina/efectos de la radiación , Neuroblastoma/patología , Animales , Muerte Celular , Diferenciación Celular , Supervivencia Celular , Ratones , Procesos Fotoquímicos , Espectrometría de Fluorescencia , Células Tumorales CultivadasRESUMEN
Development of new, ecologically safe technologies to control insect pest populations is of great importance. Photoactive compounds usually used for photosensitization might be effective as pesticide agents, with low impact on the environment, being non-toxic and not mutagenic. Phosensitizer accumulates within the insect body and, following exposure to visible light, induces lethal photochemical reactions and death. The aim of this study is to evaluate the possible usage of several photosensitizers (acridine orange, aminolevulinic acid, hematoporphyrin dimethyl ether, methylene blue) as photopesticides to control population of polyphagous plant pest Liriomyza bryoniae (Kaltenbach, 1858) (Diptera, Agromyzidae). Fluorescence measurements of intact cooled insects indicate that insect feeding with bait containing HPde and sugar induces remarkable accumulation of this compound in the body of insect. This accumulation is strongly dependent on sex and feeding duration. The highest HPde amount in the body of insect was detected 16 h after feeding, whereas no significant photosensitizer amount was detected in the same insect following 48 h. Following irradiation with visible light results in fast death of L. bryoniae. Of importance to note that survival of insects after feeding and irradiation depends on sex: female insect died much faster than males.
Asunto(s)
Dípteros/efectos de los fármacos , Dípteros/efectos de la radiación , Control de Insectos/métodos , Fármacos Fotosensibilizantes/farmacología , Naranja de Acridina/farmacología , Naranja de Acridina/efectos de la radiación , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/efectos de la radiación , Animales , Conservación de los Recursos Naturales , Dípteros/metabolismo , Conducta Alimentaria/efectos de los fármacos , Femenino , Fluorescencia , Hematoporfirinas/química , Hematoporfirinas/metabolismo , Hematoporfirinas/farmacología , Hematoporfirinas/efectos de la radiación , Luz , Masculino , Azul de Metileno/farmacología , Azul de Metileno/efectos de la radiación , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/efectos de la radiación , Plantas , Caracteres Sexuales , Tasa de SupervivenciaRESUMEN
The study was conducted to clarify the cytocidal effect of combination therapy consisting of administration of acridine orange (AO), which is a photosensitizer, and radiation therapy using in vitro and in vivo mouse osteosarcoma models. The results revealed that AO combined with low-dose X-ray irradiation of about 1-5 Gy had a strong cytocidal effect on the cultured mouse osteosarcoma cells regardless of their chemosensitivity, and that this combination therapy inhibited growth of the in vivo mouse osteosarcoma by induction of tumor necrosis. This effect was inhibited by L-histidine, but not by mannitol. These findings suggested that AO might be excited by X-rays and kill osteosarcoma cells through the release of singlet oxygen, which is toxic to living cells. This mechanism is similar to that of photodynamic therapy with AO.
Asunto(s)
Naranja de Acridina/efectos de la radiación , Naranja de Acridina/uso terapéutico , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/radioterapia , Fotoquimioterapia/métodos , Naranja de Acridina/efectos adversos , Animales , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/secundario , Ratones , Microscopía Fluorescente , Trasplante de Neoplasias , Osteosarcoma/patología , Tasa de Supervivencia , Factores de Tiempo , Células Tumorales Cultivadas , Rayos XRESUMEN
Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Colorantes Fluorescentes/efectos de la radiación , Luz , Microscopía Fluorescente , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/efectos de la radiación , Naranja de Acridina/efectos de la radiación , Animales , Benzo(a)pireno/efectos de la radiación , Células Cultivadas , Colesterol/análogos & derivados , Colesterol/efectos de la radiación , Fibroblastos/metabolismo , Fluoresceína-5-Isotiocianato , Fluoresceínas/efectos de la radiación , Técnica del Anticuerpo Fluorescente , Humanos , Cinética , Hígado/metabolismo , Fotoquímica , Ratas , Tiocianatos/efectos de la radiaciónRESUMEN
As shown by electron paramagnetic resonance, acridine orange induces the formation of peroxide radicals in DNA when dye-DNA mixtures frozen at 77 K are irradiated with visible light. The reaction is oxygen dependent and strongly reduced by the addition of an electron scavenger. Factors of the medium can modulate the reaction: an ionic strength increased up to 0.3 greatly enhances the dye efficiency whereas the presence of phosphate ions has an inhibiting influence. Acridine orange, which is slightly less efficient than proflavine on native DNA, induces an important peroxide radical formation in poly(dG).poly(dC) but has no action on the poly(dA).poly(dT)polymer.