Evaluation of the probe dihydrocalcein acetoxymethylester as an indicator of reactive oxygen species formation and comparison with oxidative DNA base modification determined by modified alkaline elution technique.
Reactive oxygen species (ROS) play a predominant role in various diseases and the development of fast and easy methods for the quantification of intracellular ROS represents an important goal. Therefore, the aim of the present study was the evaluation of the fluorogenic probe dihydrocalcein acetoxymethylester (AM) for the detection of intracellular ROS. A flow cytometric method was developed using MCF-7 cells and the kinetics of ester hydrolysis and the cellular distribution and stability of calcein were characterized using calcein AM. Then, MCF-7 cells were challenged with model agents for the generation of singlet oxygen (illumination with visible light), peroxyl and hydroxyl radicals (tert-butylhydroperoxide, tBHP), superoxide anion radicals (potassium dioxide), and the intracellular formation of superoxide anion radicals by redox cycling (menadione) and the formation of calcein was compared with the induction of oxidative DNA base modifications assessed by modified alkaline elution technique. Every model agent significantly induced formamidopyrimidine-DNA glycosylase-sensitive sites (i.e. oxidative DNA base modifications) and most also induced DNA strand breaks. In contrast, exclusively tBHP and illumination with visible light induced the intracellular formation of calcein. In conclusion, though intracellular oxidation of dihydrocalcein represents a fast screening method, it detects a limited spectrum of ROS.
Rohnstock
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University of Karlsruhe, Institute of Applied Biosciences, Section of Food Chemistry and Toxicology, Kaiserstrasse 12, D-76131 Karlsruhe, Germany. Leane.Lehmann@chemie.uni-karlsruhe.de
Lehmann
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D000468:Alkalies; D002460:Cell Line; D045744:Cell Line, Tumor; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000378:metabolism; D013481:Superoxides; D013997:Time Factors; D024483:Vitamin K 3; D020122:tert-Butylhydroperoxide
D000468:Alkalies; D005452:Fluoresceins; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D013481:Superoxides; C039316:potassium superoxide; C085925:calcein AM; D024483:Vitamin K 3; D020122:tert-Butylhydroperoxide