Use and evaluation of OH radicals generated with the new synthesized fluorescent probes in fibroblast cells

Reactive oxygen species (ROS), being pro-oxidant molecules synthesized in the body are essential for life owing to their various functions. Nevertheless, these reactive species can be very harmful increasing the oxidative stress whether produced too much or the levels of counterbalancing antioxidants decrease in the body. In order to determine the levels of ROS, Electron Spin Resonance, Fluorescence, and Luminescence Spectroscopy are often used in practice. Among these methods, Fluorescence Probes are the ones with higher specificity and sensitivity with their excellent sensors to detect ROS?s. They also offer different probes specifically designed for each radical. OH? radical is a free radical among ROS's, with a tendency to bind to lipids, DNA and proteins causing irreversible damage in cells. The most devastating consequences related to excess OH? radicals occur via direct binding to nucleic acids and proteins. This radical being very reactive and short lived, is very difficult to determine. The aim of this study was to identify the most suitable probe for detection of OH ? radical levels. The fluorescence probes commonly used in detection of OH? radical levels 2-[6-(4V-Hydroxy) phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and coumarin-3-carboxylic acid (3-CCA) are compared. Human dermal fibroblast cell line was used in this study. The cells were cultured under normal conditions in Dulbecco's Modified Eagle's Medium (DMEM) including 10% fetal bovine serum (FBS). Following the formation of OH? Radicals with Fenton reaction in the presence of H2O2 (Hidrojen peroxide), DMSO (Dimethyl sulfoxide), ascorbic acid and iron, the newly synthesized HPF and coumarin probes were added and spectrofluorometric measurements were performed in their respective wavelengths. The mean amplitude of fluorescence obtained for HPF was 32.72±7.22 F.I (n=40) and for coumarin was 52.1±4.22 F.I (n=40). As a result, the coumarin probe was found to be significantly more diligent than the HPF in statistical sense (p< 0.05). This experimental study was carried out through 2012-2013 in Dokuz Eylul University School of Medicine Department of Medical Biochemistry. Key Words: 2-[6-(4V-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), coumarin-3-carboxylic acid (3-CCA), fibroblast, fluoresan prob.