Identification of the Hydroxyl Radical and Other Reactive Oxygen Species in Human Neutrophil Granulocytes Exposed to a Fragment of the Amyloid Beta Peptide

A fragment of the amyloid beta protein, &#103 A(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that &#103 A(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, &#103 A(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by &#103 A(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to &#103 A(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 &#148 &#109 ) - and hydroxyl ( &#148 OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of &#148 OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of &#148 OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to &#103 A(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that &#103 A(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 &#148 &#109 can lead to HOCl and further formation of &#148 OH, which both have a cytotoxic potential.