Detection of reactive oxygen species (ROS) by the oxidant-sensing probe 2',7'-dichlorodihydrofluorescein diacetate in the cyanobacterium Anabaena variabilis PCC 7937.
ABSTRACT The generation of reactive oxygen species (ROS) under simulated solar radiation (UV-B: 0.30Wm(-2), UV-A: 25.70Wm(-2) and PAR: 118.06Wm(-2)) was studied in the cyanobacterium Anabaena variabilis PCC 7937 using the oxidant-sensing fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is a nonpolar dye, converted into the polar derivative DCFH by cellular esterases that are nonfluorescent but switched to highly fluorescent DCF when oxidized by intracellular ROS and other peroxides. The images obtained from the fluorescence microscope after 12h of irradiation showed green fluorescence from cells covered with 295, 320 or 395nm cut-off filters, indicating the generation of ROS in all treatments. However, the green/red fluorescence ratio obtained from fluorescence microscopic analysis showed the highest generation of ROS after UV-B radiation in comparison to PAR or UV-A radiation. Production of ROS was also measured by a spectrofluorophotometer and results obtained supported the results of fluorescence microscopy. Low levels of ROS were detected at the start (0h) of the experiment showing that they are generated even during normal metabolism. This study also showed that UV-B radiation causes the fragmentation of the cyanobacterial filaments which could be due to the observed oxidative stress. This is the first report for the detection of intracellular ROS in a cyanobacterium by fluorescence microscopy using DCFH-DA and thereby suggesting the applicability of this method in the study of in vivo generation of ROS.
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ABSTRACT: Reactive oxygen species (ROS) have captured the interest of many researchers in the chemical, biological, and medical fields since they are thought to be associated with various pathological conditions. Fluorescent probes for the detection of ROS are promising tools with which to enhance our understanding of the physiological roles of ROS, because they provide spatial and temporal information about target biomolecules in in vivo cellular systems. ROS probes, designed to detect specific ROS with a high selectivity, would be desirable, since it is now becoming clear that each ROS has its own unique physiological activity. However, dihydro-compounds such as 2',7'-dichlorodihydrofluorescein (DCFH), which have traditionally been used for detecting ROS, tend to react with a wide variety of ROS and are not completely photostable. Some attractive fluorescent probes that exhibit a high degree of selectivity toward specific ROS have recently been reported, and these selective probes are expected to have great potential for elucidating unknown physiological mechanisms associated with their target ROS. This review focuses on the design, detection mechanism, and performance of fluorescent probes for the detection of singlet oxygen ((1)O(2)), hydrogen peroxide (H(2)O(2)), hydroxyl radicals ((.)OH), or superoxide anion (O(2) (-.)), a field in which remarkable progress has been achieved in the last few years.Analytical and Bioanalytical Chemistry 11/2006; 386(3):532-43. · 3.66 Impact Factor
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ABSTRACT: Our studies indicate differential response of growth, photosynthesis, antioxidant enzymes and lipid peroxidation of three cyanobacteria: Nostoc muscorum, Plectonema boryanum and Aphanothece sp. to UV-B stress and the sensitivity to UV-B was maximum in N. muscorum and minimum in Aphanothece sp. Cyanobacteria exhibited varied sensitivity to UV-B radiation (280–315 nm: 0.4 W m− 2) as 30 min of UV-B exposure caused 32, 88 and 95% growth yield after 10th day of treatment in N. muscorum, P. boryanum and Aphanothece sp., respectively. Photosynthetic pigment contents, whole cell oxygen yield, 14C-fixation and PS II activity decreased with increasing doses of UV-B exposure (15 and 60 min), however, the inhibitory effect in N. muscorum was more pronounced than in P. boryanum and a least effect was noticed in Aphanothece sp. Among the photosynthetic pigments, phycocyanin was severely affected by UV-B in N. muscorum followed by P. boryanum and Aphanothece sp. 14C-fixation was found to be more sensitive parameter to UV-B than whole cell photosynthetic oxygen evolution. UV-B exposed spheroplasts exhibited severe damage on PS II activity and little effect on the activity of PS I. Partial restoration of PS II activity by electron donors (NH2OH, MnCl2 and DPC) suggested that UV-B interrupted the electron flow by affecting the component of water oxidation site as well as reaction center of PS II and the extent of damage on PS II was dependent on UV-B exposure time as well as the cyanobacterial strains. Suppression in emission peak (λmax 651 nm) of phycocyanin revealed that UV-B altered the energy transfer efficiency of phycocyanin to PS II reaction center. Furthermore, UV-B with increasing doses enhanced lipid peroxidation and the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). However, the enhancement in SOD activity in N. muscorum (13–25%), P. boryanum (18–34%) and Aphanothece sp. (24–53%) was high as compared with CAT and POD activity.South African Journal of Botany. 01/2009;
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ABSTRACT: Endogenously produced pro-oxidant reactive species are essential to life, being involved in several biological functions. However, when overproduced (e.g. due to exogenous stimulation), or when the levels of antioxidants become severely depleted, these reactive species become highly harmful, causing oxidative stress through the oxidation of biomolecules, leading to cellular damage that may become irreversible and cause cell death. The scientific research in the field of reactive oxygen species (ROS) associated biological functions and/or deleterious effects is continuously requiring new sensitive and specific tools in order to enable a deeper insight on its action mechanisms. However, reactive species present some characteristics that make them difficult to detect, namely their very short lifetime and the variety of antioxidants existing in vivo, capable of capturing these reactive species. It is, therefore, essential to develop methodologies capable of overcoming this type of obstacles. Fluorescent probes are excellent sensors of ROS due to their high sensitivity, simplicity in data collection, and high spatial resolution in microscopic imaging techniques. Hence, the main goal of the present paper is to review the fluorescence methodologies that have been used for detecting ROS in biological and non-biological media.Journal of Biochemical and Biophysical Methods 01/2006; 65(2-3):45-80. · 2.33 Impact Factor