Mirian Bonaldi Sgarbi

γ-Hydroxybutyric acid induces oxidative stress in cerebral cortex of young rats

GHB is a naturally occurring compound in the central nervous system (CNS) whose tissue concentration are highly increased during drug abuse and in the inherited deficiency of succinic semialdehyde dehydrogenase (SSADH) activity. SSADH deficiency is a neurometabolic-inherited disorder of the degradation pathway of gamma-aminobutyric acid (GABA). It is biochemically characterized by increased concentrations of gamma-hydroxybutyric acid (GHB) in tissues, cerebrospinal fluid (CSF), blood and urine of affected patients. Clinical manifestations are variable, ranging from mild retardation of mental, motor, and language development to more severe neurological symptoms, such as hypotonia, ataxia and seizures, whose underlying mechanisms are practically unknown. In the present study, the in vitro and in vivo effects of GHB was investigated on some parameters of oxidative stress, such as chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), as well as the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in homogenates from cerebral cortex of 15-day-old Wistar rats. In vitro, GHB significantly increased chemiluminescence and TBA-RS levels, while TRAP and TAR measurements were markedly diminished. In contrast, the activities of the antioxidant enzymes SOD, CAT and GPX were not altered by GHB in vitro. Acute administration of GHB provoked a significant enhance of TBA-RS levels and a decrease of TRAP and TAR measurements. These results indicate that GHB induces oxidative stress by stimulating lipid peroxidation and decreasing the non-enzymatic antioxidant defenses in cerebral cortex of young rats. If these effects also occur in humans, it is possible that they might contribute to the brain damage found in SSADH-deficient patients and possibly in individuals who consume GHB or its prodrug gamma-butyrolactone.

Tyrosine promotes oxidative stress in cerebral cortex of young rats.

Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II, where tyrosine levels are highly elevated in tissues and physiological fluids of affected patients. In tyrosinemia type II, high levels of tyrosine are correlated with eyes, skin and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in the present study, we investigated whether oxidative stress is elicited by l‐tyrosine in cerebral cortex homogenates of 14‐day‐old Wistar rats. The in vitro effect of 0.1–4.0 mM l‐tyrosine was studied on the following oxidative stress parameters: total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), ascorbic acid content, reduced glutathione (GSH) content, spontaneous chemiluminescence, thiobarbituric acid‐reactive substances (TBA‐RS), thiol‐disulfide redox state (SH/SS ratio), protein carbonyl content, formation of DNA‐protein cross‐links, and the activities of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glucose‐6‐phosphate dehydrogenase (G6PDH). TRAP, TAR, ascorbic acid content, SH/SS ratio and CAT activity were significantly diminished, while formation of DNA‐protein cross‐link was significantly enhanced by l‐tyrosine in vitro. In contrast, l‐tyrosine did not affect the other parameters of oxidative stress evaluated. These results indicate that l‐tyrosine decreases enzymatic and non‐enzymatic antioxidant defenses, changes the redox state and stimulates DNA damage in cerebral cortex of young rats in vitro. This suggests that oxidative stress may represent a pathophysiological mechanism in tyrosinemic patients, in which this amino acid accumulates.

Acute administration of 5-oxoproline induces oxidative damage to lipids and proteins and impairs antioxidant defenses in cerebral cortex and cerebellum of young rats

Abstract5-Oxoproline accumulates in glutathione synthetase deficiency, an autossomic recessive inherited disorder clinically characterized by hemolytic anemia, metabolic acidosis, and severe neurological symptoms whose mechanisms are poorly known. In the present study we investigated the effects of acute subcutaneous administration of 5-oxoproline to verify whether oxidative stress is elicited by this metabolite in vivo in cerebral cortex and cerebellum of 14-day-old rats. Our results showed that the acute administration of 5-oxoproline is able to promote both lipid and protein oxidation, to impair brain antioxidant defenses, to alter SH/SS ratio and to enhance hydrogen peroxide content, thus promoting oxidative stress in vivo, a mechanism that may be involved in the neuropathology of gluthatione synthetase deficiency.

N-Acetylaspartic acid promotes oxidative stress in cerebral cortex of rats

N‐Acetylaspartic acid accumulates in Canavan Disease, a severe leukodystrophy characterized by swelling and spongy degeneration of the white matter of the brain. This inherited metabolic disease, caused by deficiency of the enzyme aspartoacylase, is clinically characterized by severe mental retardation, hypotonia and macrocephaly, and also generalized tonic and clonic type seizures in about half of the patients. Considering that the mechanisms of brain damage in this disease remain not fully understood, in the present study we investigated whether oxidative stress is elicited by N‐acetylaspartic acid. The in vitro effect of N‐acetylaspartic acid (10–80 mM) was studied on oxidative stress parameters: total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), chemiluminescence, thiobarbituric acid‐reactive substances (TBA‐RS), reduced glutathione content, sufhydryl content and carbonyl content in the cerebral cortex of 14‐day‐old rats. The effect of the acute administration of N‐acetylaspartic acid (0.1–0.6 mmol/g body weight) was studied on TRAP, TAR, carbonyl content, chemiluminescence and TBA‐RS. TRAP, TAR, reduced glutathione content and sulfhydryl content were significantly reduced, while chemiluminescence, TBA‐RS and carbonyl content were significantly enhanced by N‐acetylaspartic acid in vitro. The enhancement in TBA‐RS promoted by N‐acetylaspartic acid was completely prevented by ascorbic acid plus Trolox, and partially prevented by glutathione and dithiothreitol. The acute administration of N‐acetylaspartic acid also significantly reduced TRAP and TAR, and significantly enhanced carbonyl content, chemiluminescence and TBA‐RS. Our results indicate that N‐acetylaspartic acid promotes oxidative stress by stimulating lipid peroxidation, protein oxidation and by decreasing non‐enzymatic antioxidant defenses in rat brain. This could be another pathophysiological mechanism involved in Canavan Disease.

Citrulline and Ammonia Accumulating in Citrullinemia Reduces Antioxidant Capacity of Rat Brain In Vitro

Citrullinemia is an inborn error of the urea cycle caused by deficient argininosuccinate synthetase, which leads to accumulation of L-citrulline and ammonia in tissues and body fluids. The main symptoms include convulsions, tremor, seizures, coma, and brain edema. The pathophysiology of the neurological signs of citrullinemia remains unclear. In this context, we investigated the in vitro effects of L-citrulline and ammonia in cerebral cortex from 30-day-old rats on oxidative stress parameters, namely thiobarbituric acid-reactive substances (TBA-RS), chemiluminescence, mitochondrial membrane protein thiol content, intracellular content of hydrogen peroxide, total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR) as well as on the activities of the antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase). L-Citrulline significantly diminished TRAP (26%) and TAR (37%), while ammonia decreased TAR (30%). Ammonia increased SOD activity (65%) and L-citrulline did not affect the activities of any antioxidant enzymes. We also observed that L-citrulline and ammonia did not alter lipid peroxidation parameters, levels of hydrogen peroxide, and mitochondrial membrane protein thiol content. Taken together, these results may indicate that L-citrulline and ammonia decreased the antioxidant capacity of the brain, which may reflect a possible involvement of oxidative stress in the neuropathology of citrullinemia.

N-acetylaspartic acid impairs enzymatic antioxidant defenses and enhances hydrogen peroxide concentration in rat brain.

N-Acetylaspartic acid accumulates in Canavan Disease, a severe inherited neurometabolic disease clinically characterized by severe mental retardation, hypotonia, macrocephaly and generalized tonic and clonic type seizures. Considering that the mechanisms of brain damage in this disease remain poorly understood, in the present study we investigated the in vitro and in vivo effects of N-acetylaspartic acid on the activities of catalase, superoxide dismutase and glutathione peroxidase, as well as on hydrogen peroxide concentration in cerebral cortex of 14-day-old rats. Catalase and glutathione peroxidase activities were significantly inhibited, while hydrogen peroxide concentration was significantly enhanced by N-acetylaspartic acid both in vitro and in vivo. In contrast, superoxide dismutase activity was not altered by N-acetylaspartic acid. Our results clearly show that N-acetylaspartic acid impairs the enzymatic antioxidant defenses in rat brain. This could be involved in the pathophysiological mechanisms responsible for the brain damage observed in patients affected by Canavan Disease.