Experimental hyperprolinemia induces mild oxidative stress, metabolic changes, and tissue adaptation in rat liver.

The present study investigated the effects of chronic hyperprolinemia on oxidative and metabolic status in liver and serum of rats. Wistar rats received daily subcutaneous injections of proline from their 6th to 28th day of life. Twelve hours after the last injection the rats were sacrificed and liver and serum were collected. Results showed that hyperprolinemia induced a significant reduction in total antioxidant potential and thiobarbituric acid-reactive substances. The activities of the antioxidant enzymes catalase and superoxide dismutase were significantly increased after chronic proline administration, while glutathione (GSH) peroxidase activity, dichlorofluorescin oxidation, GSH, sulfhydryl, and carbonyl content remained unaltered. Histological analyses of the liver revealed that proline treatment induced changes of the hepatic microarchitecture and increased the number of inflammatory cells and the glycogen content. Biochemical determination also demonstrated an increase in glycogen concentration, as well as a higher synthesis of glycogen in liver of hyperprolinemic rats. Regarding to hepatic metabolism, it was observed an increase on glucose oxidation and a decrease on lipid synthesis from glucose. However, hepatic lipid content and serum glucose levels were not changed. Proline administration did not alter the aminotransferases activities and serum markers of hepatic injury. Our findings suggest that hyperprolinemia alters the liver homeostasis possibly by induction of a mild degree of oxidative stress and metabolic changes. The hepatic alterations caused by proline probably do not implicate in substantial hepatic tissue damage, but rather demonstrate a process of adaptation of this tissue to oxidative stress. However, the biological significance of these findings requires additional investigation.

Chronic hyperhomocysteinemia induces oxidative damage in the rat lung.

Tissue accumulation of homocysteine occurs in classical homocystinuria, a metabolic disease characterized biochemically by cystathionine ß-synthase deficiency. Vascular manifestations such as myocardial infarction, cerebral thrombosis, hepatic steatosis, and pulmonary embolism are common in this disease and poorly understood. In this study, we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress (thiobarbituric acid-reactive substances, protein carbonyl content, 2',7'-dichlorofluorescein fluorescence assay, and total radical-trapping antioxidant potent) and activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in the rat lung. Reduced glutathione content and glucose 6-phosphate dehydrogenase activity, as well as nitrite levels, were also evaluated. Wistar rats received daily subcutaneous injections of Hcy (0.3-0.6 µmol/g body weight) from the 6th to the 28th days-of-age and the control group received saline. One and 12 h after the last injection, rats were killed and the lungs collected. Hyperhomocysteinemia increased lipid peroxidation and oxidative damage to protein, and disrupted antioxidant defenses (enzymatic and non-enzymatic) in the lung of rats, characterizing a reliable oxidative stress. In contrast, this amino acid did not alter nitrite levels. Our findings showed a consistent profile of oxidative stress in the lung of rats, elicited by homocysteine, which could explain, at least in part, the mechanisms involved in the lung damage that is present in some homocystinuric patients.

In vivo neuroprotective effect of L-carnitine against oxidative stress in maple syrup urine disease.

Maple syrup urine disease (MSUD) is an autosomal recessive inborn error of metabolism caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase (BCKAD) leading to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine and valine and their corresponding branched-chain α-keto acids. Affected patients present severe brain dysfunction manifested such as ataxia, seizures, coma, psychomotor delay and mental retardation. The mechanisms of brain damage in this disease remain poorly understood. Recent studies have shown that oxidative stress may be involved in neuropathology of MSUD. L-Carnitine (L-Car) is considered a potential antioxidant through its action against peroxidation as a scavenger of reactive oxygen species and by its stabilizing effect of damage to cell membranes. In this study we evaluate the possible neuroprotective in vivo effects of L-Car against pro-oxidative effects of BCAA in cerebral cortex of rats. L-Car prevented lipoperoxidation, measured by thiobarbituric acid-reactive substances, protein damage, measured by sulfhydryl and protein carbonyl content and alteration on catalase and glutathione peroxidase activity in rat cortex from a chemically-induced model of MSUD. Our data clearly show that L-Car may be an efficient antioxidant, protecting against the oxidative stress promoted by BCAA. If the present results are confirmed in MSUD patients, this could represent an additional therapeutic approach to the patients affected by this disease.

Experimental evidence that phenylalanine provokes oxidative stress in hippocampus and cerebral cortex of developing rats.

High levels of phenylalanine (Phe) are the biochemical hallmark of phenylketonuria (PKU), a neurometabolic disorder clinically characterized by severe mental retardation and other brain abnormalities, including cortical atrophy and microcephaly. Considering that the pathomechanisms leading to brain damage and particularly the marked cognitive impairment in this disease are poorly understood, in the present study we investigated the in vitro effect of Phe, at similar concentrations as to those found in brain of PKU patients, on important parameters of oxidative stress in the hippocampus and cerebral cortex of developing rats. We found that Phe induced in vitro lipid peroxidation (increase of TBA-RS values) and protein oxidative damage (sulfhydryl oxidation) in both cerebral structures. Furthermore, these effects were probably mediated by reactive oxygen species, since the lipid oxidative damage was totally prevented by the free radical scavengers alpha-tocopherol and melatonin, but not by L-NAME, a potent inhibitor of nitric oxide synthase. Accordingly, Phe did not induce nitric oxide synthesis, but significantly decreased the levels of reduced glutathione (GSH), the major brain antioxidant defense, in hippocampus and cerebral cortex supernatants. Phe also reduced the thiol groups of a commercial GSH solution in a cell-free medium. We also found that the major metabolites of Phe catabolism, phenylpyruvate, phenyllactate and phenylacetate also increased TBA-RS levels in cerebral cortex, but to a lesser degree. The data indicate that Phe elicits oxidative stress in the hippocampus, a structure mainly involved with learning/memory, and also in the cerebral cortex, which is severely damaged in PKU patients. It is therefore presumed that this pathomechanism may be involved at least in part in the severe cognitive deficit and in the characteristic cortical atrophy associated with dysmyelination and leukodystrophy observed in this disorder.

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.

Evidence that 2-methylacetoacetate induces oxidative stress in rat brain.

In the present study we investigated the effects of 2-methylacetoacetate (MAA) and 2-methyl-3-hydroxybutyrate (MHB), the major metabolites accumulating in mitochondrial 2-methylacetoacetyl-CoA thiolase (KT) and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies, on important parameters of oxidative stress in cerebral cortex from young rats. We verified that MAA induced lipid peroxidation (increase of thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence values), whereas MHB did not alter these parameters. MAA-induced increase of TBA-RS levels was fully prevented by free radical scavengers, indicating that free radicals were involved in this effect. Furthermore, MAA, but not MHB, significantly induced sulfhydryl oxidation, implying that this organic acid provokes protein oxidative damage. It was also observed that MAA reduced GSH, a naturally-occurring brain antioxidant, whereas MHB did not change this parameter. Furthermore, the decrease of GSH levels caused by MAA was not due to a direct oxidative action, since this organic acid did not alter the sulfhydryl content of a commercial solution of GSH in a cell free medium. Finally, MAA and MHB did not raise nitric oxide production. The data indicate that MAA induces oxidative stress in vitro in cerebral cortex. It is presumed that this pathomechanism may be involved in the brain damage found in patients affected by KT deficiency.

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.

Glycine provokes lipid oxidative damage and reduces the antioxidant defenses in brain cortex of young rats.

Patients affected by nonketotic hyperglycinemia (NKH) usually present severe neurological symptoms and suffer from acute episodes of intractable seizures with leukoencephalopathy. Although excitotoxicity seems to be involved in the brain damage of NKH, the mechanisms underlying the neuropathology of this disease are not fully established. The objective of the present study was to investigate the in vitro effects of glycine (GLY), that accumulate at high concentrations in the brain of patients affected by this disorder, on important parameters of oxidative stress, such as lipid peroxidation (thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence) and the most important non-enzymatic antioxidant defense reduced glutathione (GSH) in cerebral cortex from 30-day-old rats. GLY significantly increased TBA-RS and chemiluminescence values, indicating that this metabolite provokes lipid oxidative damage. Furthermore, the addition of high doses of the antioxidants melatonin, trolox (soluble vitamin E) and GSH fully prevented GLY-induced increase of lipid peroxidation, indicating that free radicals were involved in this effect. GLY also decreased GSH brain concentrations, which was totally blocked by melatonin treatment. Finally, GLY significantly reduced sulfhydryl group content from a commercial GSH solution, but did not oxidize reduced cytochrome C. Our data indicate that oxidative stress elicited in vitro by GLY may possibly contribute at least in part to the pathophysiology of the neurological dysfunction in NKH.

Intracerebroventricular administration of N-acetylaspartic acid impairs antioxidant defenses and promotes protein oxidation in cerebral cortex of rats.

N-acetylaspartic acid (NAA) is the biochemical hallmark of Canavan Disease, an inherited metabolic disease caused by deficiency of aspartoacylase activity. NAA is an immediate precursor for the enzyme-mediated biosynthesis of N-acetylaspartylglutamic acid (NAAG), whose concentration is also increased in urine and cerebrospinal fluid of patients affected by CD. This neurodegenerative disorder is clinically characterized by severe mental retardation, hypotonia and macrocephaly, and generalized tonic and clonic type seizures. Considering that the mechanisms of brain damage in this disease remain not fully understood, in the present study we investigated whether intracerebroventricular administration of NAA or NAAG elicits oxidative stress in cerebral cortex of 30-day-old rats. NAA significantly reduced total radical-trapping antioxidant potential, catalase and glucose 6-phosphate dehydrogenase activities, whereas protein carbonyl content and superoxide dismutase activity were significantly enhanced. Lipid peroxidation indices and glutathione peroxidase activity were not affected by NAA. In contrast, NAAG did not alter any of the oxidative stress parameters tested. Our results indicate that intracerebroventricular administration of NAA impairs antioxidant defenses and induces oxidative damage to proteins, which could be involved in the neurotoxicity of NAA accumulation in CD patients.

Effects of 1,4-butanediol administration on oxidative stress in rat brain: study of the neurotoxicity of gamma-hydroxybutyric acid in vivo.

gamma-Hydroxybutyric acid (GHB) is a naturally occurring compound in the central nervous system (CNS) whose tissue concentration are highly increased in the neurometabolic-inherited deficiency of succinic semialdehyde dehydrogenase (SSADH) activity or due to intoxication. SSADH deficiency is biochemically characterized by increased concentrations of GHB in tissues, cerebrospinal fluid, blood and urine of affected patients. Clinical manifestations are variable and include retardation of mental, motor, and language development along with other neurological symptoms, such as hypotonia, ataxia and seizures, whose underlying mechanisms are practically unknown. The precursor of GHB, 1,4-butanediol (1,4-BD) has been used to study the mechanisms of in vivo GHB neurotoxicity. Therefore, in the present work, the effect of acute administration of 20 or 120 mg/Kg 1,4-BD was investigated on various parameters of oxidative stress, such as spontaneous chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total antioxidant reactivity (TAR), sulfhydryl and protein carbonyl contents, as well as the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in homogenates from cerebral cortex of 14-day-old Wistar rats. Acute administration of 120 mg/Kg 1,4-BD significantly increased spontaneous chemiluminescence and TBA-RS levels, while TAR measurement was markedly diminished, whereas injection of a lower dose (20 mg/Kg) did not change the parameters examined. Other parameters of oxidative stress evaluated were not affected by administration of 1,4-BD. These results indicate that 1,4-BD induces in vivo 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 intoxicated by GHB or its prodrugs (gamma-butyrolactone or 1,4-BD).

Evidence that 3-hydroxy-3-methylglutaric acid promotes lipid and protein oxidative damage and reduces the nonenzymatic antioxidant defenses in rat cerebral cortex.

In the present work we investigated the in vitro effect of 3-hydroxy-3-methylglutarate (HMG) that accumulates in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMGLD) on important parameters of oxidative stress in rat cerebral cortex. It was observed that HMG induced lipid peroxidation by significantly increasing chemiluminescence and levels of thiobarbituric acid-reactive substances (TBA-RS). This effect was prevented by the antioxidants alpha-tocopherol, melatonin, N-acetylcysteine, and superoxide dismutase plus catalase, suggesting that free radicals were involved in the lipid oxidative damage. On the other hand, HMG did not change TBA-RS levels in intact or disrupted mitochondrial preparations, indicating that generation of oxidants by this organic acid was dependent on cytosolic mechanisms. HMG also induced protein oxidative damage in cortical supernatants, which was reflected by increased carbonyl content and sulfhydryl oxidation. Furthermore, HMG significantly reduced the nonenzymatic antioxidant defenses total-radical trapping antioxidant potential, total antioxidant reactivity, and reduced glutathione (GSH) levels in rat cerebral cortex. HMG-induced GSH reduction was totally blocked by melatonin pretreatment. We also verified that the decrease of GSH levels provoked by HMG in cortical supernatants was not due to a direct oxidative effect of this organic acid, because exposition of commercial GSH and purified membrane protein-bound thiol groups to HMG in the absence of cortical supernatants did not decrease the reduced sulfhydryl groups. Finally, the activities of the main antioxidant enzymes were not altered by HMG exposure. Our data indicate that oxidative stress elicited in vitro by HMG may possibly contribute at least in part to the pathophysiology of the brain injury in HMGLD.

Guanidinoacetate decreases antioxidant defenses and total protein sulfhydryl content in striatum of rats.

Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of guanidinoacetate (GAA) and depletion of creatine. Affected patients present epilepsy and mental retardation whose pathogeny is unclear. In the present study we investigated the in vitro and in vivo (intrastriatal administration) effects of GAA on some oxidative stress parameters in rat striatum. Sixty-day-old rats were used for intrastriatal infusion of GAA. For the in vitro studies, 60-day-old Wistar rats were killed by decapitation and the striatum was pre-incubated for 1 h at 37 degrees C in the presence of GAA at final concentrations ranging from 10 to 100 microM. Parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP), antioxidant enzymes (SOD, GPx, and CAT), protein carbonyl and thiol contents were measured. DNA damage was also evaluated. Results showed that GAA administration (in vivo studies) or the addition of 100 microM GAA to assays (in vitro studies) significantly decreased TRAP, SOD activity, and total thiol levels in rat striatum. In contrast, this guanidino compound did not alter protein carbonyl content and the activities of CAT and GPx. DNA damage was not found after intrastriatal administration of GAA. The data indicate that the metabolite accumulating in GAMT deficiency decreases antioxidant capacity and total thiol content in the striatum. It is therefore presumed that this pathomechanism may contribute at least in part to the pathophysiology of the brain injury observed in patients affected by GAMT deficiency.

In vitro evidence for an antioxidant role of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in the brain.

We investigated the in vitro effect of 3-hydroxykynurenine (3HKyn), 3-hydroxyanthranilic acid (3HAA), kynurenine (Kyn) and anthranilic acid (AA) on various parameters of oxidative stress in rat cerebral cortex and in cultured C6 glioma cells. It was demonstrated that 3HKyn and 3HAA significantly reduced the thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence measurements in rat cerebral cortex, indicating that these metabolites prevent lipid peroxidation in the brain. In addition, GSH spontaneous oxidation was significantly prevented by 3HAA, but not by the other kynurenines in cerebral cortex. We also verified that 3HKyn and 3HAA significantly decreased the peroxyl radicals induced by the thermolysis of 2,2'-azo-bis-(2-amidinopropane)-derived peroxyl radicals, and to a higher degree than the classical peroxyl scavenger trolox. 2-Deoxy-d-ribose degradation was also significantly prevented by 3HKyn, implying that this metabolite was able to scavenge hydroxyl radicals. Furthermore, the total antioxidant reactivity of C6 glioma cells was significantly increased when these cells were exposed from 1 to 48h to 3HKyn, being the effect more prominent at shorter incubation times. TBA-RS values in C6 cells were significantly reduced by 3HKyn when exposed from 1 to 6h with this kynurenine. However, C6 cell morphology was not altered by 3HKyn. Finally, we tested whether 3HKyn could prevent the increased free radical production induced by glutaric acid (GA), the major metabolite accumulating in glutaric acidemia type I, by evaluating the isolated and combined effects of these compounds on TBA-RS levels and 2',7'-dihydrodichlorofluorescein (DCFH) oxidation in rat brain. GA provoked a significant increase of TBA-RS values and of DCFH oxidation, effects that were attenuated and fully prevented, respectively, by 3HKyn. The results strongly indicate that 3HKyn and 3HAA behave as antioxidants in cerebral cortex and C6 glioma cells from rats.

Erythrocyte glutathione peroxidase activity and plasma selenium concentration are reduced in maple syrup urine disease patients during treatment.

Maple syrup urine disease (MSUD) is an inherited disorder caused by a deficiency of the branched-chain alpha-keto acid dehydrogenase complex activity. In the present study we evaluated selenium levels in plasma from MSUD patients at diagnosis and under treatment and the activities of glutathione peroxidase, catalase and superoxide dismutase in erythrocytes from treated patients. We verified that MSUD patients present a significant selenium deficiency at diagnosis, which becomes more pronounced during treatment, as well as a decrease of erythrocyte glutathione peroxidase activity during treatment. In contrast, erythrocyte catalase and superoxide dismutase activities were not altered in these patients. Our present results suggest that the reduction of an important antioxidant enzyme activity may be partially involved in the pathomechanisms of this disorder and that plasma selenium levels must be corrected through dietary supplementation in MSUD patients.

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.

Evidence for a synergistic action of glutaric and 3-hydroxyglutaric acids disturbing rat brain energy metabolism.

Glutaric acidemia type I is an inherited metabolic disorder caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase activity leading to accumulation of predominantly glutaric and 3-hydroxyglutaric acids in the brain tissue of the affected patients. Considering that a toxic role was recently postulated for quinolinic acid in the neuropathology of glutaric acidemia type I, in the present work we investigated whether the combination of quinolinic acid with glutaric or 3-hydroxyglutaric acids or the mixture of glutaric plus 3-hydroxyglutaric acids could alter brain energy metabolism. The parameters evaluated in cerebral cortex from young rats were glucose utilization, lactate formation and (14)CO(2) production from labeled glucose and acetate, as well as the activities of pyruvate dehydrogenase and creatine kinase. We first observed that glutaric (5 mM), 3-hydroxyglutaric (1 mM) and quinolinic acids (0.1 microM) per se did not alter these parameters. Similarly, no change of these parameters occurred when combining glutaric with quinolinic acids or 3-hydroxyglutaric with quinolinic acids. In contrast, co-incubation of glutaric plus 3-hydroxyglutaric acids increased glucose utilization, decreased (14)CO(2) generation from glucose, inhibited pyruvate dehydrogenase activity as well as total and mitochondrial creatine kinase activities. The glutaric plus 3-hydroxyglutaric acids-induced inhibitory effects on creatine kinase were prevented by the antioxidants glutathione and catalase plus superoxide dismutase, indicating the participation of reactive oxygen species. Our data indicate a synergic action of glutaric and 3-hydroxyglutaric acids disturbing energy metabolism in cerebral cortex of young rats.

Oxidative stress induction by cis-4-decenoic acid: relevance for MCAD deficiency.

Patients affected by medium-chain acyl-CoA dehydrogenase deficiency (MCADD) suffer from acute episodes of encephalopathy whose underlying mechanisms are poorly known. The present work investigated the in vitro effect of cis-4-decenoic acid (cDA), which accumulates in MCADD, on important parameters of oxidative stress in cerebral cortex of young rats. cDA markedly induced lipid peroxidation, as verified by the increased levels of spontaneous chemiluminescence and thiobarbituric acid-reactive substances. Furthermore, cDA significantly increased carbonyl formation and sulphydryl oxidation, which is indicative of protein oxidative damage, and promoted 2',7'-dihydrodichlorofluorescein oxidation. It was also observed that the non-enzymatic tissue antioxidant defenses were decreased by cDA, whereas the antioxidant enzyme activities catalase, superoxide dismutase and glutathione peroxidase were not altered. Moreover, cDA-induced lipid peroxidation and GSH reduction was totally blocked by free radical scavengers, suggesting that reactive species were involved in these effects. The data indicate that oxidative stress is induced by cDA in rat brain in vitro and that oxidative damage might be involved in the pathophysiology of the encephalopathy in MCADD.

Differential inhibitory effects of methylmalonic acid on respiratory chain complex activities in rat tissues.

Methylmalonic acidemia is an inherited metabolic disorder biochemically characterized by tissue accumulation of methylmalonic acid (MMA) and clinically by progressive neurological deterioration and kidney failure, whose pathophysiology is so far poorly established. Previous studies have shown that MMA inhibits complex II of the respiratory chain in rat cerebral cortex, although no inhibition of complexes I-V was found in bovine heart. Therefore, in the present study we investigated the in vitro effect of 2.5mM MMA on the activity of complexes I-III, II, II-III and IV in striatum, hippocampus, heart, liver and kidney homogenates from young rats. We observed that MMA caused a significant inhibition of complex II activity in striatum and hippocampus (15-20%) at low concentrations of succinate in the medium, but not in the peripheral tissues. We also verified that the inhibitory property of MMA only occurred after exposing brain homogenates for at least 10 min with the acid, suggesting that this inhibition was mediated by indirect mechanisms. Simultaneous preincubation with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) and catalase (CAT) plus superoxide dismutase (SOD) did not prevent MMA-induced inhibition of complex II, suggesting that common reactive oxygen (superoxide, hydrogen peroxide and hydroxyl radical) and nitric (nitric oxide) species were not involved in this effect. In addition, complex II-III (20-35%) was also inhibited by MMA in all tissues tested, and complex I-III only in the kidney (53%) and liver (38%). In contrast, complex IV activity was not changed by MMA in all tissues studied. These results indicate that MMA differentially affects the activity of the respiratory chain pending on the tissues studied, being striatum and hippocampus more vulnerable to its effect. In case our in vitro data are confirmed in vivo in tissues from methylmalonic acidemic patients, it is feasible that that the present findings may be related to the pathophysiology of the tissue damage characteristic of these patients.

Brain zinc chelation by diethyldithiocarbamate increased the behavioral and mitochondrial damages in zebrafish subjected to hypoxia.

The increase in brain levels of chelatable zinc (Zn) in dysfunctions involving oxygen deprivation has stimulated the treatment with Zn chelators, such as diethyldithiocarbamate (DEDTC). However, DEDTC is a redox-active compound and it should be better evaluated during hypoxia. We use the hypoxia model in zebrafish to evaluate DEDTC effects. The exploratory behavior, chelatable Zn content, activities of mitochondrial dehydrogenases, reactive species levels (nitric oxide, superoxide anion, hydroxyl radical scavenger capacity) and cellular antioxidants (sulfhydryl, superoxide dismutase) of zebrafish brain were assessed after recovery, with or without 0.2 mM DEDTC. The increased brain levels of chelatable Zn induced by hypoxia were mitigated by DEDTC. However, the novel tank task indicated that DEDTC did further enhance the exploratory deficit caused by hypoxia. Furthermore, these behavioral impairments caused by DEDTC were more associated with a negative action on mitochondrial activity and brain oxidative balance. Thus, due to apparent pro-oxidant action of DEDTC, our data do not support its use for neuroprotection in neuropathologies involving oxygen deprivation.

Experimental hyperphenylalaninemia provokes oxidative stress in rat brain.

Tissue accumulation of L-phenylalanine (Phe) is the biochemical hallmark of human phenylketonuria (PKU), an inherited metabolic disorder clinically characterized by mental retardation and other neurological features. The mechanisms of brain damage observed in this disorder are poorly understood. In the present study we investigated some oxidative stress parameters in the brain of rats with experimental hyperphenylalaninemia. Chemiluminescence, total radical-trapping antioxidant potential (TRAP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were measured in the brain of the animals. We observed that chemiluminescence is increased and TRAP is reduced in the brain of hyperphenylalaninemic rats. Similar data were obtained in the in vitro experiments using Phe at various concentrations. CAT activity was significantly inhibited by Phe in vitro and in vivo, whereas GSH-Px activity was reduced in vivo but not in vitro and SOD activity was not altered by any treatment. The results indicate that oxidative stress may be involved in the neuropathology of PKU. However, further studies are necessary to confirm and extend our findings to the human condition and also to determine whether an antioxidant therapy may be of benefit to these patients.