Ammonia-induced production of free radicals in primary cultures of rat astrocytes

Elevated levels of ammonia in blood and brain result in derangement of cere bral function. Recently, lipid peroxidation and oxidative stress have been implicated in ammonia neurotoxicity. Because ammonia is primarily detoxifie d in astrocytes, we postulated that pathophysiological concentrations of am monia might induce free radical formation in these cells. To test this hypo thesis, we examined the extent of free radical production in primary cultur es of astrocytes that had been preloaded with the fluorescent dye 5- (and 6 -)carboxy-2 ' ,7 ' -dichlorodihydrofluorescein diacetate (DCFDA). DCFDA flu oresence was found to be increased in a dose-dependent manner when astrocyt es were exposed to 1, 5, and 10 mM NH4Cl. This phenomenon was transitory; i t peaked at 2.5 min after exposure and declined subsequently. By 2 hr after treatment, DCFDA fluorescence was below control level. Addition of catalas e or superoxide dismutase to 5 mM NH4Cl-treated astrocytes reduced free rad ical formation. Pretreatment with 3 mM methionine sulfoximine, an inhibitor of glutamine synthetase, also suppressed free radical formation by 5 mM NH 4Cl. The results of this study suggest that elevated concentrations of ammo nia induce the formation of free radicals in astrocytes and that this proce ss is associated with the synthesis of glutamine. We propose that astrocyte -derived free radicals may be responsible for some of the pathophysiologica l changes associated with hyperammonemic conditions. (C) 2001 Wiley-Liss, I nc.