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

Abstract

Elevated levels of ammonia in blood and brain result in derangement of cerebral function. Recently, lipid peroxidation and oxidative stress have been implicated in ammonia neurotoxicity. Because ammonia is primarily detoxified in astrocytes, we postulated that pathophysiological concentrations of ammonia might induce free radical formation in these cells. To test this hypothesis, we examined the extent of free radical production in primary cultures of astrocytes that had been preloaded with the fluorescent dye 5- (and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCFDA). DCFDA fluorescence was found to be increased in a dose-dependent manner when astrocytes were exposed to 1, 5, and 10 mM NH(4)Cl. This phenomenon was transitory; it peaked at 2.5 min after exposure and declined subsequently. By 2 hr after treatment, DCFDA fluorescence was below control level. Addition of catalase or superoxide dismutase to 5 mM NH(4)Cl-treated astrocytes reduced free radical formation. Pretreatment with 3 mM methionine sulfoximine, an inhibitor of glutamine synthetase, also suppressed free radical formation by 5 mM NH(4)Cl. The results of this study suggest that elevated concentrations of ammonia induce the formation of free radicals in astrocytes and that this process is associated with the synthesis of glutamine. We propose that astrocyte-derived free radicals may be responsible for some of the pathophysiological changes associated with hyperammonemic conditions.

Ammonia induces RNA oxidation in cultured astrocytes and brain in vivo†

“…7 The current study shows that an acute ammonia load, which stimulates cerebral ROS/RNS formation in vitro 5,8,24 and in vivo, 8,9,[25][26][27][28] and can precipitate HE episodes in patients with liver cirrhosis, 7 increases RNA oxidation in cultured rat astrocytes, vital mouse brain slices, and rat brain in vivo. Also, hypo-osmolarity, diazepam, and TNF-␣ trigger RNA oxidation in cultured astrocytes and vital brain slices, respectively.…”

Selective alterations of brain osmolytes in acute liver failure: protective effect of mild hypothermia

“…In favour of this mechanism, pretreatment of the animals with the glutamine synthetase inhibitor methionine sulfoximine (MSO) prior to hepatic desvascularization significantly diminished brain water accumulation [3]. Based upon these findings, preliminary 1H-NMR studies in patients with ALF suggest that monitoring of the α-proton of glutamine could be useful for assessment of encephalopathy in these patients [21]. However, despite several experimental data supporting glutamine as accumulating osmolyte in HE, whether or not glutamine accumulation is a major cause for the development of brain edema in ALF is uncertain Furthermore, a previous study [35] did not reveal any significant correlations between either brain glutamine concentrations or glutamine synthesis with the severity of encephalopathy or occurrence of brain edema in experimental ALF.…”

“…For example, like ammonia, glutamine also induces the mitochondrial permeability transition [27], and mediates the generation of free radicals in cultured astrocytes [21].…”

Brain edema in acute liver failure and chronic liver disease: Similarities and differences

“…There is ample evidence stating that oxidative stress plays a role in the pathogenesis of brain edema in HE. Cultured astrocytes acutely treated with ammonia concentrations (>1 mM) display cell swelling and production of ROS (Murthy et al, 2001), which are attenuated with antioxidant treatments (Jayakumar et al, 2006). This suggests that ammonia-induced astrocyte swelling is linked to the generation of ROS.…”