Effects of polychlorinated biphenyls on the neutrophil NADPH oxidase system.

Polychlorinated biphenyls (PCBs) are reported to induce the formation of reactive oxygen species (ROS) in human neutrophil granulocytes through the activation of the NADPH oxidase. The purpose of the present study is to elucidate the cellular mechanisms responsible for the activation of the NADPH oxidase after exposure to PCB. We have previously shown that PCB activates human neutrophil granulocytes through a calcium dependent activation of phospholipase D and/or phospholipase C, followed by the activation of protein kinase C. In the present study, pharmacological characterization of Aroclor (A) 1242-induced respiratory burst in human neutrophils was conducted by the use of enzymatic inhibitors. Pre-incubation with U0126, SB203580, SP600125, cyclosporin A and FK506 attenuated the A 1242-induced respiratory burst, measured by DCF-fluorescence, and luminol-amplified chemiluminescence. Our results show that the Erk1/2 kinases and p38MAPK/JNK are involved in ROS formation in neutrophils exposed to A 1242.

Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation.

This study attempts to provide a critical assessment of three different common approaches to identifying teactive species formed in biological systems: the 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay, and the luminol- and lucigenin-amplified chemiluminescence assays. There have been several contradictory reports about the specificity of these methods. Our results show that DCFH is oxidized to the fluorescent compound 2',7'-dichlorofluorescin (DCF) in human neutrophils exposed to the following compounds: Aroclor (A)1242, hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and FeSO(4). Use of a cell-free DCFH system showed increased formation of DCF by peroxynitrite (ONOO(-)), horseradish peroxidase (HRP) alone, and HRP in combination with H(2)O(2), FeSO(4) alone, and a mixture of FeSO(4) and H(2)O(2). The hydroxyl radical (z.rad;OH) scavenger formate and the iron ion chelator deferoxamine reduced the DCF formation induced by FeSO(4) in combination with H(2)O(2). DCFH was insensitive to NO and H(2)O(2) in the cell-free system. In the presence of neutrophils, the A1242-induced luminol chemiluminescence was decreased by the superoxide dismutase inhibitor diethyldithiocarbamic acid (DDC) and the myeloperoxidase inhibitor salicylhydroxamic acid (SHA). Exposure of the neutrophils to NO, FeSO(4), or H(2)O(2) alone did not have any effect. A1242-induced lucigenin chemiluminescence in the neutrophils was increased slightly by DDC, but was not affected by SHA, NO, FeSO(4), or H(2)O(2). In conclusion, we suggest that the DCF assay is only suitable for measurements of ONOO(-), H(2)O(2) in combination with cellular peroxidases, and z.rad;OH. Luminol is sensitive towards HOCl, while lucigenin is oxidized by O(2)z.rad;(-).

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, 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.