N-Acetylcysteine Inhibits Kynurenine Aminotransferase II.

The tryptophan metabolite kynurenic acid (KYNA) may play an important role in normal and abnormal cognitive processes, most likely by interfering with α7 nicotinic and NMDA receptor function. KYNA is formed from its immediate precursor kynurenine either by non-enzymatic oxidation or through irreversible transamination by kynurenine aminotransferases. In the mammalian brain, kynurenine aminotransferase II (KAT II) is the principal enzyme responsible for the neosynthesis of rapidly mobilizable KYNA, and therefore constitutes an attractive target for pro-cognitive interventions. N-acetylcysteine (NAC), a brain-penetrant drug with pro-cognitive efficacy in humans, has been proposed to exert its actions by increasing the levels of the anti-oxidant glutathione (GSH) in the brain. We report here that NAC, but not GSH, inhibits KAT II activity in brain tissue homogenates from rats and humans with IC50 values in the high micromolar to low millimolar range. With similar potency, the drug interfered with the de novo formation of KYNA in rat brain slices, and NAC was a competitive inhibitor of recombinant human KAT II (Ki: 450 µM). Furthermore, GSH failed to S-glutathionylate recombinant human KAT II treated with the dithiocarbamate drug disulfiram. Shown by microdialysis in the prefrontal cortex of rats treated with kynurenine (50 mg/kg, i.p.), peripheral administration of NAC (500 mg/kg, i.p., 120 and 60 min before the application of kynurenine) reduced KYNA neosynthesis by ∼50%. Together, these results suggest that NAC exerts its neurobiological effects at least in part by reducing cerebral KYNA formation via KAT II inhibition.

Xanthurenic Acid Formation from 3-Hydroxykynurenine in the Mammalian Brain: Neurochemical Characterization and Physiological Effects.

Xanthurenic acid (XA), formed from 3-hydroxykynurenine (3-HK) in the kynurenine pathway of tryptophan degradation, may modulate glutamatergic neurotransmission by inhibiting the vesicular glutamate transporter and/or activating Group II metabotropic glutamate receptors. Here we examined the molecular and cellular mechanisms by which 3-HK controls the neosynthesis of XA in rat, mouse and human brain, and compared the physiological actions of 3-HK and XA in the rat brain. In tissue homogenates, XA formation from 3-HK was observed in all three species and traced to a major role of kynurenine aminotransferase II (KAT II). Transamination of 3-HK to XA was also demonstrated using human recombinant KAT II. Neosynthesis of XA was significantly increased in the quinolinate-lesioned rat striatum, indicating a non-neuronal localization of the process. Studies using rat cortical slices revealed that newly produced XA is rapidly released into the extracellular compartment, and that XA biosynthesis can be manipulated experimentally in the same way as the production of kynurenic acid from kynurenine (omission of Na+ or glucose, depolarizing conditions, or addition of 2-oxoacids). The synthesis of XA from 3-HK was confirmed in vivo by striatal microdialysis. In slices from the rat hippocampus, both 3-HK and XA reduced the slopes of dentate gyrus field EPSPs. The effect of 3-HK was reduced in the presence of the KAT inhibitor aminooxyacetic acid. Finally, both 3-HK and XA reduced the power of gamma-oscillatory activity recorded from the hippocampal CA3 region. Endogenous XA, newly formed from 3-HK, may therefore play a physiological role in attentional and cognitive processes.

Alterations in kinetic and thermotropic properties of cerebral membrane-bound acetylcholineesterase during thioacetamide-induced hepatic encephalopathy: correlation with membrane lipid changes.

Acetylcholinesterase (AchE) is an important peripheral membrane-bound enzyme, crucial for cholinergic neurotransmission. Changes in AchE activity, kinetic and thermotropic properties were studied in hepatic encephalopathy (HE) associated with acute liver failure induced experimentally by the administration of the hepatotoxin thioacetamide (TAA). Activity of AchE decreased significantly following TAA administration. AchE from cerebral cortex membrane isolates of TAA-treated rats also showed a decrease in Vmax and an increase in Km. Arrhenius plots revealed considerable changes in the thermotropic behavior of AchE from membrane isolates obtained from TAA-treated rats as evident from the decreased transition temperature. A positive correlation was observed between changes in membrane cholesterol (r2=0.987), sphingomyelin (r2=0.99) levels and AchE activity, thus indicating that membrane lipid changes could underlie the observed changes in kinetic and thermotropic properties of membrane-bound AchE during TAA-induced HE.

Fulminant hepatic failure in rats induces oxidative stress differentially in cerebral cortex, cerebellum and pons medulla.

Hepatic Encephalopathy (HE) is one of the most common complications of acute liver diseases and is known to have profound influence on the brain. Most of the studies, available from the literature are pertaining to whole brain homogenates or mitochondria. Since brain is highly heterogeneous with functions localized in specific areas, the present study was aimed to assess the oxidative stress in different regions of brain-cerebral cortex, cerebellum and pons medulla during acute HE. Acute liver failure was induced in 3-month old adult male Wistar rats by intraperitoneal injection of thioacetamide (300 mg/kg body weight for two days), a well known hepatotoxin. Oxidative stress conditions were assessed by free radical production, lipid peroxidation, nitric oxide levels, GSH/GSSG ratio and antioxidant enzyme machinery in three distinct structures of rat braincerebral cortex, cerebellum and pons medulla. Results of the present study indicate a significant increase in malondialdehyde (MDA) levels, reactive oxygen species (ROS), total nitric oxide levels [(NO) estimated by measuring (nitrites + nitrates)] and a decrease in GSH/GSSG ratio in all the regions of brain. There was also a marked decrease in the activity of the antioxidant enzymes-glutathione peroxidase, glutathione reductase and catalase while the super oxide dismutase activity (SOD) increased. However, the present study also revealed that pons medulla and cerebral cortex were more susceptible to oxidative stress than cerebellum. The increased vulnerability to oxidative stress in pons medulla could be due to the increased NO levels and increased activity of SOD and decreased glutathione peroxidase and glutathione reductase activities. In summary, the present study revealed that oxidative stress prevails in different cerebral regions analyzed during thioacetamide-induced acute liver failure with more pronounced effects on pons medulla and cerebral cortex.

Co-administration of C-Phycocyanin ameliorates thioacetamide-induced hepatic encephalopathy in Wistar rats.

Fulminant hepatic failure (FHF) is a condition with a sudden onset of necrosis followed by degeneration of hepatocytes, without any previously established liver disease, generally occurring within hours or days. FHF is associated with a wide spectrum of neuropsychiatric alterations ranging from stupor to coma, culminating in death. In the present study FHF was induced in rats by the administration of thioacetamide (TAA). Oxidative stress is thought to play a prominent role in the pathophysiology of cerebral changes during FHF leading to the assumption that antioxidants might offer protection. Hence, in the present study the protective effect of C-Phycocyanin (C-PC), a natural antioxidant, was evaluated on TAA-induced tissue damage. C-Phycocyanin was administered intraperitoneally twice at 24 h interval (50 mg/kg body weight) along with the hepatotoxin TAA (300 mg/kg body weight). The animals were sacrificed 18 h after the second injection of TAA treatment and various biochemical parameters were analysed in liver, serum and brain tissues. These studies revealed significant prevention of TAA-induced liver damage by C-PC, as evidenced by a) increase in survival rate; b) the prevention of leakage of liver enzymes (AAT and AST) and ammonia into serum; c) increase in prothrombin time and d) liver histopathology. Ultrastructural studies of astrocytes of different regions of brain clearly showed a decrease in edema after C-PC treatment. TAA-induced histopathological lesions in different regions of the brain namely cerebral cortex, cerebellum and pons medulla were significantly reduced by the co-administration of C-PC with TAA. Further C-PC treatment resulted in a) decrease in the levels of tryptophan and markers of lipid peroxidation and b) elevation in the activity levels of catalase, glutathione peroxidase in different regions of brain. These studies reveal the potential of C-PC in ameliorating TAA-induced hepatic encephalopathy by improving antioxidant defenses.

Changes in cerebral membrane lipid composition and fluidity during thioacetamide-induced hepatic encephalopathy.

Lipids are an essential structural and functional component of cellular membranes. Changes in membrane lipid composition are known to affect the activities of many membrane-associated enzymes, endocytosis, exocytosis, membrane fusion and neurotransmitter uptake, and have been implicated in the pathophysiology of many neurodegenerative disorders. In the present study, we investigated changes in the lipid composition of membranes isolated from the cerebral cortex of rats treated with thioacetamide (TAA), a hepatotoxin that induces fulminant hepatic failure (FHF) and thereon hepatic encephalopathy (HE). HE refers to acute neuropsychiatric changes accompanying FHF. The estimation of membrane phospholipids, cholesterol and fatty acid content in cerebral cortex membranes from TAA-treated rats revealed a decrease in cholesterol, phosphatidylserine, sphingomyelin, a monounsaturated fatty acid, namely oleic acid, and the polyunsaturated fatty acids gamma-linolenic acid, decosa hexanoic acid and arachidonic acid compared with controls. Assessment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in the annular membrane fluidity, whereas the global fluidity was unaffected. The level of the thiobarbituric acid reactive species marker for lipid peroxidation also increased in membranes from TAA-treated rats, thereby indicating the prevalence of oxidative stress. Results from the present study demonstrate gross alterations in cerebral cortical membrane lipid composition and fluidity during TAA-induced HE, and their possible implications in the pathogenesis of this condition are also discussed.