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      Melatonin Enhances Photo-Oxidation of 2′,7′-Dichlorodihydrofluorescein by an Antioxidant Reaction That Renders N1-Acetyl-N2-Formyl-5-Methoxykynuramine (AFMK)

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          Abstract

          The indolamine melatonin (MEL) is described as an antioxidant and a free radical scavenger. However occasionally, the indoleamine has been reported to increase free radicals with insufficient mechanistic explanation. In an attempt to find a reason for those controversial results, a potential mechanism that explains MEL prooxidant activity is investigated. The current controversy about redox detection methods has prompted us to search a possible interaction between MEL and dichlorodihydrofluorescein (DCFH 2), perhaps the most widely fluorescence probe employed for free radicals detection in cellular models. Here, it is demonstrated that melatonin potentiates the photooxidation of DCFH 2 in a cell-free system, increasing the production of its fluorescent metabolite. Indeed, MEL works as an antioxidant scavenging hydroxyl radicals in this system. Thus, this reaction between MEL and DCFH 2 produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a biogenic amine with antioxidant properties too. This reaction is O 2 and light dependent and it is prevented by antioxidants such as N-acetylcysteine or ascorbic acid. Furthermore, when DCFH 2 has been employed to evaluate antioxidant or prooxidant activities of MEL in cellular models it is confirmed that it works as an antioxidant but these results can be modulated by light misleading to a prooxidant conclusion. In conclusion, here is demonstrated that DCFH 2, light and melatonin interact and results obtained using these fluorescence probes in studies with melatonin have to be carefully interpreted.

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          Most cited references48

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          Melatonin as a natural ally against oxidative stress: a physicochemical examination.

          Oxidative stress has been proven to be related to the onset of a large number of health disorders. This chemical stress is triggered by an excess of free radicals, which are generated in cells because of a wide variety of exogenous and endogenous processes. Therefore, finding strategies for efficiently detoxifying free radicals has become a subject of a great interest, from both an academic and practical points of view. Melatonin is a ubiquitous and versatile molecule that exhibits most of the desirable characteristics of a good antioxidant. The amount of data gathered so far regarding the protective action of melatonin against oxidative stress is overwhelming. However, rather little is known concerning the chemical mechanisms involved in this activity. This review summarizes the current progress in understanding the physicochemical insights related to the free radical-scavenging activity of melatonin. Thus far, there is a general agreement that electron transfer and hydrogen transfer are the main mechanisms involved in the reactions of melatonin with free radicals. However, the relative importance of other mechanisms is also analyzed. The chemical nature of the reacting free radical also has an influence on the relative importance of the different mechanisms of these reactions. Therefore, this point has also been discussed in detail in the current review. Based on the available data, it is concluded that melatonin efficiently protects against oxidative stress by a variety of mechanisms. Moreover, it is proposed that even though it has been referred to as the chemical expression of darkness, perhaps it could also be referred to as the chemical light of health. © 2011 John Wiley & Sons A/S.
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            Identification of ROS using oxidized DCFDA and flow-cytometry.

            Cells constantly generate reactive oxygen species (ROS) during aerobic metabolism. The ROS generation plays an important protective and functional role in the immune system. The cell is armed with a powerful antioxidant defense system to combat excessive production of ROS. Oxidative stress occurs in cells when the generation of ROS overwhelms the cells' natural antioxidant defenses. ROS and the oxidative damage are thought to play an important role in many human diseases including cancer, atherosclerosis, other neurodegenerative diseases and diabetes. Thus, establishing their precise role requires the ability to measure ROS accurately and the oxidative damage that they cause. There are many methods for measuring free radical production in cells. The most straightforward techniques use cell permeable fluorescent and chemiluminescent probes. 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most widely used techniques for directly measuring the redox state of a cell. It has several advantages over other techniques developed. It is very easy to use, extremely sensitive to changes in the redox state of a cell, inexpensive and can be used to follow changes in ROS over time.
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              Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader.

              Oxidative stress (OS) has been implicated in various degenerative diseases in aging. In an attempt to quantify OS in a cell model, we examined OS induced by incubating for 30 min with various free radical generators in PC12 cells by using the dichlorofluorescein (DCF) assay, modified for use by a fluorescent microplate reader. The nonfluorescent fluorescin derivatives (dichlorofluorescin, DCFH), after being oxidized by various oxidants, will become DCF and emit fluorescence. By quantifying the fluorescence, we were able to quantify the OS. Our results indicated that the fluorescence varied linearly with increasing concentrations (between 0.1 and 1 mM) of H2O2 and 2,2'-azobios(2-amidinopropane) dihydrochloride (AAPH; a peroxyl radical generator). By contrast, the fluorescence varied as a nonlinear response to increasing concentrations of 3-morpholinosydnonimine hydrochloride (SIN-1; a peroxynitrite generator), sodium nitroprusside (SNP; a nitric oxide generator), and dopamine. Dopamine had a biphasic effect; it decreased the DCF fluorescence, thus acting as an antioxidant, at concentrations <500 microM in cells, but acted as a pro-oxidant by increasing the fluorescence at 1 mM. While SNP was not a strong pro-oxidant, SIN-1 was the most potent pro-oxidant among those tested, inducing a 70 times increase of fluorescence at a concentration of 100 microM compared with control. Collectively, due to its indiscriminate nature to various free radicals, DCF can be very useful in quantifying overall OS in cells, especially when used in conjunction with a fluorescent microplate reader. This method is reliable and efficient for evaluating the potency of pro-oxidants and can be used to evaluate the efficacy of antioxidants against OS in cells.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                2 October 2014
                : 9
                : 10
                : e109257
                Affiliations
                [1 ]Departamento de Morfologia y Biologia Celular, School of Medicine, University of Oviedo, Oviedo, Spain
                [2 ]Instituto Universitario Oncologico del Principado de Asturias (IUOPA), Oviedo, Spain
                [3 ]Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
                Instituto de Química, Universidade de São Paulo, Brazil
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: DH RMS DXT. Performed the experiments: DH ARG. Analyzed the data: JCM DH. Contributed reagents/materials/analysis tools: JCM DH. Wrote the paper: DH JCM RMS.

                Article
                PONE-D-14-23350
                10.1371/journal.pone.0109257
                4183549
                25275380
                342647e7-818c-4301-8652-2007a643bb15
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 26 May 2014
                : 8 September 2014
                Page count
                Pages: 10
                Funding
                This work has been supported by an ISCIII (FISS-09-PS09/02204) grant. ARG acknowledges support from the “Severo Ochoa” fellowship program (FICYT). DH and JCM acknowledge sponsorship from Instituto Universitario Oncologico del Principado de Asturias (IUOPA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Circadian Oscillators
                Cell Biology
                Oxidative Stress
                Chronobiology
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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