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

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

Jannike M Andersen, Oddvar Myhre, Halvor Aarnes, Tor Arne Vestad, Frode Fonnum

Free Radic Res. 2003 Mar;37(3):269-79.

PMID: 12688422

Abstract:

A fragment of the amyloid beta protein, betaA(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 betaA(25-35) stimulated formation of ROS in concentration and time dependent manner. The inverted peptide, betaA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by betaA(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 betaA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide (O2*-)- and hydroxyl (*OH)-radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of *OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of *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 betaA(25-35). The phospholipase A2 (PLA2) 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 betaA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA2. Production of O2*- can lead to HOCl and further formation of *OH, which both have a cytotxic potential.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP89560010	7,7-Dimethyl-(5Z,8Z)-eicosadienoic acid		89560-01-0	Price

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