Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species

Abstract

We designed and synthesized 2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical ( ⅐ OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite ( ؊ OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H 2 O 2 ), nitric oxide (NO), and superoxide (O 2 . ) by using HPF or APF alone, but ؊ OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2,7-dichlorodihydrofluorescein, and for the first time we could visualize ؊ OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and ؊ OCl in many biological and chemical applications.

Amyloid β induces neuronal cell death through ROS-mediated ASK1 activation

“…We next examined whether Ab induces ROS within cells by using a fluorescence probe, HPF, that can selectively detect highly reactive oxygen species (hROS) such as hydroxyl radical ( K OH) and peroxynitrite (ONOO À ). 31 The fluorescence intensity of PC12 cells was clearly increased after treatment with H 2 O 2 for 30 min (Figure 4d), and this increase was blocked by PG (Figure 4e), MnTBAP and VitE (data not shown). We could clearly visualize Ab-induced production of ROS (Figure 4f).…”

“…Ab [25][26][27][28][29][30][31][32][33][34][35] and Ab 1-42 were purchased from Bachem and American Peptide Company, respectively. Ab [25][26][27][28][29][30][31][32][33][34][35] was dissolved in water at 1 mM and Ab was dissolved in water containing 0.1% NH 3 at 1 mM.…”

“…Ab [25][26][27][28][29][30][31][32][33][34][35] was dissolved in water at 1 mM and Ab was dissolved in water containing 0.1% NH 3 at 1 mM. Dissolved Ab peptides were mixed with the same volume of PBS and incubated at 371C for 3-5 days before use.…”

Dual Functionality of Myeloperoxidase in Rotenone-Exposed Brain-Resident Immune Cells

“…Notably, rotenone exposure significantly increased the fluorescence of APF, a highly sensitive probe that selectively detects hydroxyl radicals and hypochloride anions ( Figure 4A). 36 The formation of hypochloride is catalyzed by MPO from chloride ions and H 2 O 2 . Therefore, we considered the possibility that MPO could be involved in rotenoneinduced activation of microglia and subsequent pathophysiological events.…”

Effects of cadmium chloride on some mitochondria-related activity and gene expression of human MDA-MB231 breast tumor cells

“…in the dark [39], then observed under an Olympus BX50 microscope with 10X objective, equipped with a Cool-SNAP-Pro Colour digital camera (Media Cybernetics, Silver Springs/MD, USA) and images were recorded using Image Pro Plus software (Media Cybernetics). Control images were obtained from cells treated with 100 lM H 2 O 2 .…”