The use of dichlorofluorescin (DCFH) as a measure of reactive oxygen species was studied in aqueous media. Hydrogen peroxide oxidized DCFH to fluorescent dichlorofluorescein (DCF), and the oxidation was amplified by the addition of ferrous iron. Hydrogen peroxide-induced DCF formation in the presence of ferrous iron was completely inhibited by deferoxamine and partially inhibited by ethylenediaminetetraacetic acid, but was augmented by diethylenetriaminepentaacetic acid. Iron-peroxide-induced oxidation of DCFH was partially inhibited by catalase but not by horseradish peroxidase. Nonchelated iron-peroxide oxidation of DCFH was partially inhibited by several hydroxyl radical scavengers, but was independent of the scavenger concentration, and this suggests that free hydroxyl radical is not involved in the oxidation of DCFH in this system. Superoxide anion did not directly oxidize DCFH. Data suggest that H2O2-Fe(2+)-derived oxidant is mainly responsible for the nonenzymatic oxidation of DCFH. In addition, peroxidase alone and oxidants formed during the reduction of H2O2 by peroxidase oxidize DCFH. Since DCFH oxidation may be derived from several reactive intermediates, interpretation of specific reactive oxygen species involved in biological systems should be approached with caution. However, DCFH remains an attractive probe as an overall index of oxidative stress in toxicological phenomena.

The etiology of neurodegenerative disorders is at present unknown. However, many of these disorders are associated with an increase in oxidative and inflammatory events. Although a small percentage of these disorders are familial cases linked to specific genetic defects, most are idiopathic. Thus, environmental factors are thought to play an important role in the onset and progression of such disorders. We have demonstrated that exposure (4 h, 5 days per week for 2 weeks) to concentrated airborne particulate matter increases inflammatory indices in brain of ovalbumin-sensitized BALB/c mice. Animals were divided into three exposure groups: filtered air (control), ultrafine particles, or fine and ultrafine particles. The levels of proinflammatory cytokines interleukin-1 alpha (IL-1alpha) and tumor necrosis factor alpha (TNF-alpha) were increased in brain tissue of mice exposed to particulate matter compared to that of control animals. Levels of the immune-related transcription factor NF-kappaB were also found to be substantially elevated in the brain of exposed groups compared with the control group. These data indicate that components of inhaled particulate matter may trigger a proinflammatory response in nervous tissue that could contribute to the pathophysiology of neurodegenerative diseases.

Support was observed for hypothesized excess neurodegenerative disease associated with a variety of occupations, 60 Hz magnetic fields and welding.

Abstract-The formation of oxygen reactive species in response to oxidative stimuli was measured in rat synaptosomes. Studies employed the non-fluorescent probe 2', 7' -dichlorofluorescin diacetate (DCFH-DA), which after de-esterification is oxidized in the presence of oxygen reactive species to the highly fluorescent 2',7'-dichlorofluorescein (DCF). Oxygen reactive species formation, as measured by DCF fluorescence, was stimulated by ascorbate and/or FeS0 4 , and xanthine/xanthine oxidase under various buffering conditions. These agents all increased DCF formation in Tris, HEPES and phosphate buffer. Ascorbate also stimulated the formation of DCF in a concentration-dependent manner. The presence of Ca 2 + in HEPES buffer did not enhance or diminish the effects of ascorbate/FeS0 4 on DCF formation. Deferoxamine inhibited the ascorbate/FeS0 4 -induced stimulation of DCF formation, but xanthine/xanthine oxidaseinduced stimulation was not affected by pretreatment with superoxide dismutase. Results indicate that DCF fluorescence is a sensitive, quantitative and direct measure of oxygen reactive species formation in synaptosomes, providing a rapid method for investigating early neuronal events that occur during oxidative stress.