Diabetic encephalopathy-related depression: experimental evidence that insulin and clonazepam restore antioxidant status in rat brain.

There is increasing evidence suggesting that oxidative stress plays an important role in the development of many chronic and degenerative conditions such as diabetic encephalopathy and depression. Considering that diabetic rats and mice present higher depressive-like behaviour when submitted to the forced swimming test and that treatment with insulin and/or clonazepam is able to reverse the behavioural changes of the diabetic rats, the present work investigated the antioxidant status, specifically total antioxidant reactivity and antioxidant potential of insulin and clonazepam, as well as the effect of this drugs upon protein oxidative damage and reactive species formation in cortex, hippocampus and striatum from diabetic rats submitted to forced swimming test. It was verified that longer immobility time in diabetic rats and insulin plus clonazepam treatment reversed this depressive-like behaviour. Moreover, data obtained in this study allowed to demonstrate through different parameters such as protein carbonyl content, 2'7'-dichlorofluorescein oxidation, catalase, superoxide dismutase, glutathione peroxidase assay, total radical-trapping antioxidant potential and total antioxidant reactivity that there is oxidative stress in cortex, hippocampus and striatum from diabetic rats under depressive-like behaviour and highlight the insulin and/or clonazepam effect in these different brain areas, restoring antioxidant status and protein damage.

Glutathione metabolism enzymes in brain and liver of hyperphenylalaninemic rats and the effect of lipoic acid treatment.

Phenylketonuria (PKU) is a disorder caused by a deficiency in phenylalanine hydroxylase activity, which converts phenylalanine (Phe) to tyrosine, leading to hyperphenylalaninemia (HPA) with accumulation of Phe in tissues of patients. The neuropathophysiology mechanism of disease remains unknown. However, recently the involvement of oxidative stress with decreased glutathione levels in PKU has been reported. Intracellular glutathione (GSH) levels may be maintained by the antioxidant action of lipoic acid (LA). The aim of this study was to evaluate the activity of the enzymes involved in the metabolism and function of GSH, such as glutathione peroxidase (GSH-Px), glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), glutamate-cysteine ligase (GCL), glutathione-S-transferase (GST) and GSH content in brain and liver of young rats subjected to a chemically induced model of HPA and the effect of LA for a week. In brain, the administration of Phe reduced the activity of the GSH-Px, GR and G6PD and LA prevented these effects totally or partially. GCL activity was increased by HPA and was not affect by LA antioxidant treatment. GST activity did not differ between groups. GSH content was increased by LA and decreased by HPA treatment in brain samples. Considering the liver, all parameters analyzed were increased in studied HPA animals and LA was able to hinder some effects except for the GCL, GST enzymes and GSH content. These results suggested that HPA model alter the metabolism of GSH in rat brain and liver, which may have an important role in the maintenance of GSH function in PKU although liver is not a directly affected organ in this disease. So, an antioxidant therapy with LA may be useful in the treatment of oxidative stress in HPA.

Role of catalase and superoxide dismutase activities on oxidative stress in the brain of a phenylketonuria animal model and the effect of lipoic acid.

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase which leads to accumulation of phenylalanine and its metabolites in tissues of patients with severe neurological involvement. Recently, many studies in animal models or patients have reported the role of oxidative stress in PKU. In the present work we studied the effect of lipoic acid against oxidative stress in rat brain provoked by an animal model of hyperphenylalaninemia (HPA), induced by repetitive injections of phenylalanine and α-methylphenylalanine (a phenylalanine hydroxylase inhibitor) for 7 days, on some oxidative stress parameters. Lipoic acid prevented alterations on catalase (CAT) and superoxide dismutase (SOD), and the oxidative damage of lipids, proteins, and DNA observed in HPA rats. In addition, lipoic acid diminished reactive species generation compared to HPA group which was positively correlated to SOD/CAT ratio. We also observed that in vitro Phe inhibited CAT activity while phenyllactic and phenylacetic acids stimulated superoxide dismutase activity. These results demonstrate the efficacy of lipoic acid to prevent oxidative stress induced by HPA model in rats. The possible benefits of lipoic acid administration to PKU patients should be considered.

Exercício aeróbico agudo restaura a concentração de triptofano em cérebro de ratos com hiperfenilalaninemia / Acute aerobic exercise restores tryptophan concentration in the brain of rats with hyperphenylalaninemia

INTRODUÇÃO: A fenilcetonúria (PKU) é caracterizada pela deficiência da enzima fenilalanina hidroxilase, causando acúmulo de fenilalanina. O diagnóstico precoce e a subordinação à dieta pobre em fenilalanina são importantes para prevenir os efeitos prejudiciais da hiperfenilalaninemia. Não aderir estritamente à dieta provoca, entre outros efeitos, um desequilíbrio entre os aminoácidos neutros que usam o mesmo transportador da fenilalanina na barreira hematoencefálica, causando, então, a diminuição da entrada de triptofano, o precursor de serotonina no cérebro. Esse neurotransmissor tem sido implicado na regulação dos estados de humor, sendo sua alta produção ligada à fadiga central em indivíduos submetidos a exercício prolongado. O exercício físico aumenta os níveis de triptofano livre no sangue, o que facilita seu influxo no cérebro, podendo, portanto, ser útil nos estados hiperfenilalaninêmicos. OBJETIVO: Avaliar se o exercício aeróbico é capaz de normalizar as concentrações de triptofano no cérebro de ratos com hiperfenilalaninemia. MÉTODOS: Trinta e dois ratos foram separados nos grupos sedentário (Sed) e exercício (Exe), e cada um deles subdividido em controle (SAL) e hiperfenilalaninemia (PKU). A hiperfenilalaninemia foi induzida pela administração de alfa-metilfenilalanina e fenilalanina durante três dias, enquanto os grupos SAL receberam salina. Os grupos Exe realizaram uma sessão de exercício aeróbico com duração de 60min e velocidade de 12m.min-1. RESULTADOS: A concentração de triptofano no cérebro nos grupos PKU foi significativamente menor que nos grupos SAL, tanto Sed como Exe, compatível com a condição hiperfenilalaninêmica. O exercício aumentou a concentração cerebral de triptofano comparada aos animais sedentários. O achado mais interessante foi que a concentração cerebral de triptofano no grupo ExePKU não foi diferente do SedSAL. CONCLUSÃO: Os resultados indicam um importante papel do exercício aeróbico para restaurar a concentração de triptofano no cérebro em ratos hiperfenilalaninêmicos.

INTRODUCTION: Phenylketonuria (PKU) is characterized by deficiency of the enzyme phenylalanine hydroxylase, leading to accumulation of phenylalanine. Early diagnosis and subordination to low-phenylalanine diet are important to prevent the harmful effects of hyperphenylalaninemia. In case the diet is not strictly followed, some possible effects are imbalance in the neutral amino acids that use the same carrier of phenylalanine to cross the blood-brain barrier, causing hence reduction in tryptophan entry, the precursor of serotonin in the brain. This neurotransmitter has been implicated in the regulation of mood states, and its high production is linked to central fatigue in individuals subjected to prolonged exercise. Physical exercise increases free tryptophan levels in the blood, which facilitates its influx in the brain, and therefore, may be useful in hyperphenylalaninemia states. OBJECTIVE: To assess whether aerobic exercise is able to normalize the concentrations of tryptophan in the brain of rats with hyperphenylalaninemia. METHODS: 32 rats were randomly assigned to sedentary (Sed) and exercise (Exe) groups, and then divided into control (HEA) and hyperphenylalaninemia (PKU). Hyperphenylalaninemia was induced by administration of alpha-metylphenylalanine and phenylalanine for three days, while the HEA groups received saline. Exe groups held a session of aerobic exercise lasting 60 minutes and speed of 12 m.min-1. RESULTS: The concentration of tryptophan in the brain of PKU groups was significantly lower than HEA groups (both in Sed and Exe groups), compatible with the condition of hyperphenylalaninemia. The exercise increased brain tryptophan levels comparing to sedentary animals. The most interesting finding was that the brain tryptophan levels of ExePKU group were not different from SedHEA group. CONCLUSION: The results indicate an important role of aerobic exercise to restore the concentration of tryptophan in the brain in hyperphenylalaninemic rats.

Phenylpyruvic acid decreases glucose-6-phosphate dehydrogenase activity in rat brain.

Phenylketonuria is a recessive autosomal disorder that is caused by a deficiency in the activity of phenylalanine-4-hydroxylase, which converts phenylalanine to tyrosine, leading to the accumulation of phenylalanine and its metabolites phenyllactic acid, phenylacetic acid, and phenylpyruvic acid in the blood and tissues of patients. Phenylketonuria is characterized by severe neurological symptoms, but the mechanisms underlying brain damage have not been clarified. Recent studies have shown the involvement of oxidative stress in the neuropathology of hyperphenylalaninemia. Glucose-6-phosphate dehydrogenase plays an important role in antioxidant defense because it is the main source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), providing a reducing power that is essential in protecting cells against oxidative stress. Therefore, the present study investigated the in vitro effect of phenylalanine (0.5, 1, 2.5, and 5 mM) and its metabolites phenyllactic acid, phenylacetic acid, and phenylpyruvic acid (0.2, 0.6, and 1.2 mM) on the activity of enzymes of the pentose phosphate pathway, which is involved in the oxidative phase in rat brain homogenates. 6-Phosphogluconate dehydrogenase activity was not altered by any of the substances tested. Phenylalanine, phenyllactic acid, and phenylacetic acid had no effect on glucose-6-phosphate dehydrogenase activity. Phenylpyruvic acid significantly reduced glucose-6-phosphate dehydrogenase activity without pre-incubation and after 1 h of pre-incubation with the homogenates. The inhibition of glucose-6-phosphate dehydrogenase activity caused by phenylpyruvic acid could elicit an impairment of NADPH production and might eventually alter the cellular redox status. The role of phenylpyruvic acid in the pathophysiological mechanisms of phenylketonuria remains unknown.

Pyruvate and creatine prevent oxidative stress and behavioral alterations caused by phenylalanine administration into hippocampus of rats.

Phenylketonuria is characterized by a variable degree of mental retardation and other neurological features whose mechanisms are not fully understood. In the present study we investigated the effect of intrahippocampal administration of phenylalanine, isolated or associated with pyruvate or creatine, on rat behavior and on oxidative stress. Sixty-day-old male Wistar rats were randomly divided into 6 groups: saline; phenylalanine; pyruvate; creatine; phenylalanine + pyruvate; phenylalanine + creatine. Phenylalanine was administered bilaterally in the hippocampus one hour before training; pyruvate, at the same doses, was administered in the hippocampus one hour before phenylalanine; creatine was administered intraperitoneally twice a day for 5 days before training; controls received saline solution at same volumes than the other substances. Parameters of exploratory behavior and of emotionality were assessed in both training and test sessions in the open field task. Rats receiving phenylalanine did not habituate to the open field along the sessions, indicating deficit of learning/memory, but parameters of emotionality were normal, not interfering in the habituation process. Pyruvate or creatine administration prevented the lack of habituation caused by phenylalanine. Pyruvate and creatine also prevented alterations provoked by phenylalanine on lipid peroxidation, total content of sulfhydryls, total radical-trapping antioxidant potential and total antioxidant reactivity. The results suggest that the behavioral alterations provoked by intra-hippocampal administration of phenylalanine may be caused, at least in part, by oxidative stress and/or energy deficit. If this also occurs in PKU, it is possible that pyruvate and creatine supplementation to the phenylalanine-restricted diet might be beneficial to phenylketonuric patients.

Regular exercise prevents oxidative stress in the brain of hyperphenylalaninemic rats.

Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase, leading to accumulation of phenylalanine and its metabolites. Clinical features of PKU patients include mental retardation, microcephaly, and seizures. Oxidative stress has been found in these patients, and is possibly related to neurophysiopatology of PKU. Regular exercise can leads to adaptation of antioxidant system, improving its capacity to detoxification reactive species. The aim of this study was to verify the effects of regular exercise on oxidative stress parameters in the brain of hyperphenylalaninemic rats. Animals were divided into sedentary (Sed) and exercise (Exe) groups, and subdivided into saline (SAL) and hyperphenylalaninemia (HPA). HPA groups were induced HPA through administration of alpha-methylphenylalanine and phenylalanine for 17 days, while SAL groups (n = 16-20) received saline. Exe groups conducted 2-week aerobic exercise for 20 min/day. At 18th day, animals were killed and the brain was homogenized to determine thiobarbituric acid reactives substances (TBA-RS) content, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. Soleus muscles were collected to determine glycogen content as a marker of oxidative adaptation. Exe groups showed enhanced glycogen content. HPA condition caused an increase in TBA-RS and SOD, and reduces CAT and GPx. Exercise was able to prevent all changes seen in the HPA group, reaching control values, except for SOD activity. No changes were found in the ExeSAL group compared to SedSAL. Hyperphenylalaninemic rats were more responsive to the benefits provided by regular exercise. Physical training may be an interesting strategy to restore the antioxidant system in HPA.

Dehydroepiandrosterone improves hepatic antioxidant reserve and stimulates Akt signaling in young and old rats.

This study examined, in the liver of young and old (3- and 24-month-old, respectively) healthy Wistar rats, the in vivo effect of dehydroepiandrosterone (DHEA) (10mg/kg body weight) administered subcutaneously for 5 weeks. Reduced (GSH) and oxidized (GSSG) glutathione levels, glucose-6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities, hydrogen peroxide concentration, GST and p-Akt/Akt immunocontent ratio were assessed in hepatic tissue. DHEA treatment significantly increased total glutathione content (17%) and GSH (22%) in 3- and 24-month-old treated groups when compared to control groups. The aging factor increased G6PDH (51%) and GPx (22%) activities as well as the hydrogen peroxide concentration (33%), independently of treatment. DHEA treatment increased p-Akt (54%) and p-Akt/Akt ratio (36%) immunocontents in both treated groups. Increased serum levels of alanine aminotransferase (ALT) in aged rats were reduced by DHEA treatment (34%).

Redox imbalance influence in the myocardial Akt activation in aged rats treated with DHEA.

This study examined, in young and old (3 and 24 month-old, respectively) healthy Wistar rats, the in vivo effect of DHEA (10 mg/kg body weight) administered subcutaneously for 5 weeks. Reduced (GSH) and oxidized (GSSG) glutathione levels, glucose-6-phosphate dehydrogenase (G6PDH), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and thioredoxin (Trx) reductase activities, hydrogen peroxide steady-state concentration and Nrf2, GST, Trx-1, Akt and p-Akt expressions were assessed in heart tissue. DHEA treatment significantly increased GST activity in 3 and 24 month-old treated groups. The aging factor diminished hydrogen peroxide concentration and Nrf2 expression, independently of treatment. However, the aging process increased GST, Akt and p-Akt expressions in both 24 month-old groups. The aged group responded differently to DHEA respective to GSSG content, GPx activity and p-Akt concentration. Further studies are needed to form conclusions about the efficacy and safety of DHEA replacement in the elderly, and to better understand DHEA's net effect on oxidative stress parameters and its modulation of signaling cascades.

Neuroprotective role of lipoic acid against acute toxicity of N-acetylaspartic acid.

N-Acetylaspartic acid (NAA) accumulates in Canavan disease, a severe inherited neurometabolic disorder clinically characterized by mental retardation, hypotonia, macrocephaly, and seizures. The mechanisms of brain damage in this disease remain poorly understood. Recent studies developed by our research group showed that NAA induces oxidative stress in vitro and in vivo in cerebral cortex of rats. Lipoic acid is considered as an efficient antioxidant which can easily cross the blood-brain barrier. Considering the absence of specific treatment to Canavan disease, this study evaluates the possible prevention of the oxidative stress promoted by NAA in vivo by the antioxidant lipoic acid to preliminarily evaluate lipoic acid efficacy against pro-oxidative effects of NAA. Fourteen-day-old Wistar rats received an acute administration of 0.6 mmol NAA/g body weight with or without lipoic acid (40 mg/kg body weight). Catalase (CAT), glutathione peroxidase (GPx), and glucose 6-phosphate dehydrogenase activities, hydrogen peroxide content, thiobarbituric acid-reactive substances (TBA-RS), spontaneous chemiluminescence, protein carbonyl content, total antioxidant potential, and DNA-protein cross-links were assayed in the cerebral cortex of rats. CAT, GPx activities, and total antioxidant potential were significantly reduced, while hydrogen peroxide content, TBA-RS, spontaneous chemiluminescence, and protein carbonyl content were significantly enhanced by acute administration of NAA. Those effects were all prevented by lipoic acid pretreatment. Our results clearly show that lipoic acid may protect against the oxidative stress promoted by NAA. This could represent a new therapeutic approach to the patients affected by Canavan disease.

Lipoic acid prevents oxidative stress in vitro and in vivo by an acute hyperphenylalaninemia chemically-induced in rat brain.

Phenylketonuria (PKU) is a recessive autosomal disorder caused by a severe deficiency of phenylalanine-4-hydroxilase activity which leads to the accumulation of L-phenylalanine (Phe) in the tissues and plasma of patients. The main clinical features are retarded development and intellectual impairment. Recent studies have shown that oxidative stress may be involved in neuropathology of hyperphenylalaninemia. Lipoic acid (LA) is considered a potent antioxidant which is well absorbed from diet and can easily cross the blood-brain barrier. We investigated the neuroprotective effects of lipoic acid against oxidative stress caused by Phe in vivo and in vitro. Lipoic acid prevented the inhibition provoked by Phe on the activities of catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. It also prevented Phe alterations on total radical-trapping antioxidant potential, thiobarbituric acid-reactive substances, glutathione concentration and on production of reactive species. It is concluded that lipoic acid may be an efficient antioxidant in the CNS against oxidative stress induced by hyperphenylalaninemia. If the present results are confirmed in PKU patients, it is possible that supplementation of lipoic acid may contribute to the treatment of PKU as an adjuvant therapeutic approach to Phe-restricted dietary treatment and amino acid mixture.

Modulation of antioxidant and detoxification responses mediated by lipoic acid in the fish Corydoras paleatus (Callychthyidae).

Lipoic acid (LA) has been reported as a potential therapeutic agent due its antioxidants proprieties. It was considered its effect in different organs (gills, brain, muscle and liver) of the fish Corydoras paleatus (Callychthyidae). LA (70 mg/kg of body mass) was added to a commercial fish diet, organisms being fed daily (1% body weight). Sixty animals (mean mass: 2.37+/-0.09 g) were placed randomly in aquariums and received (+LA) or not (-LA) lipoic acid enriched diet during four weeks. After, fish were killed and the brain, muscle, gills and liver were dissected. LA treatment reduced significantly (p<0.05) reactive oxygen species concentration in brain and increased (p<0.05) glutamate-cysteine ligase activity in brain and liver of the same experimental group. LA fed organisms showed higher (p<0.05) brain glutathione-S-transferase activity, indicating that LA improves the detoxification and antioxidant capacity face components that waste glutathione in phase II reactions. A conspicuous reduction of protein oxidation was observed in muscle and liver of +LA organisms, indicating that the treatment was also effective in reducing oxidative stress parameters.

Antioxidant responses and reactive oxygen species generation in different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae).

The aim of this study was to analyze the total antioxidant capacity (TOSC), generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) in the different body regions of the estuarine polychaeta Laeonereis acuta (Nereididae) sampled at non-polluted (NOPOL) and polluted (POL) sites from Lagoa dos Patos (Southern Brazil). Organisms collected at POL during summer showed similar (p>0.05) TOSC values along the body, but worms collected at NOPOL presented higher (p<0.05) TOSC values in the posterior (P) region in respect of anterior (A) region and middle (M) region. TOSC in the P region at NOPOL was higher (p<0.05) compared with the same body region of worms at POL. In summer, ROS concentration was higher in A and M regions of worms at POL in respect of the organisms at NOPOL. During winter all the regions showed higher ROS in worms sampled at POL. It was registered absence of season influence on LPO content, but in the P region at NOPOL in summer there were lower LPO levels compared with the others regions (p<0.05). In vitro assays showed that P region, despite a higher basal ROS, presented a higher competence to cope with pro-oxidants compared with A and M regions (p<0.05), corroborating the field results. A lower proteic sulfhydril content was observed in P in respect of the other regions (p<0.05) supporting the idea of a highest oxidant condition in this region. The results indicate that worms collected at the POL site are confronted to higher ROS concentrations, affecting its antioxidant capacity, a result that depends of body regions.