Francieli M. Stefanello

Chronic hyperhomocysteinemia alters antioxidant defenses and increases DNA damage in brain and blood of rats: protective effect of folic acid.

We have previously demonstrated that acute hyperhomocysteinemia induces oxidative stress in rat brain. In the present study, we initially investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative damage, namely total radical-trapping antioxidant potential and activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), as well as on DNA damage in parietal cortex and blood of rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of Hcy (0.3-0.6 micromol/g body weight), and/or folic acid (0.011 micromol/g body weight) from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, parietal cortex and total blood was collected. Results showed that chronic homocysteine administration increased DNA damage, evaluated by comet assay, and disrupted antioxidant defenses (enzymatic and non-enzymatic) in parietal cortex and blood/plasma. Folic acid concurrent administration prevented homocysteine effects, possibly by its antioxidant and DNA stability maintenance properties. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in disorders that accumulate homocysteine.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats.

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical‐trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid‐reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia.

Concurrent folate treatment prevents Na+,K+-ATPase activity inhibition and memory impairments caused by chronic hyperhomocysteinemia during rat development.

We investigated the hypothesis that folate administration would prevent hyperhomocysteinemia‐induced memory deficits and Na+,K+‐ATPase activity inhibition. Chronic hyperhomocysteinemia was induced from the 6th to the 28th day of life by subcutaneous injection of homocysteine (0.3–0.6 μmol/g), twice a day; control Wistar rats received the same volume of saline solution (0.9% NaCl). Half of the homocysteine‐ and saline‐treated groups also received intraperitoneal administration of folate (0.011 μmol/g) from the 6th to the 28th day of life. A group of animals was killed 12 h after the last injection, plasma and parietal cortex were collected for biochemical analysis. Another group stayed at Central Animal House until 60th day of life, when the rats were submitted to behavioral testing in water maze or were killed for evaluation of cortical Na+,K+‐ATPase activity. Results showed that hyperhomocysteinemia impaired reference memory for platform location, as assessed by fewer crossings to the platform place and increased latency for the first crossing, when compared to controls. In the working memory task homocysteine‐treated animals also needed more time to find the platform. We also observed that Na+,K+‐ATPase activity was reduced in parietal cortex of hyperhomocysteinemic rats sacrificed 12 h after the last injection of homocysteine (29‐day‐old rats). In contrast, this enzyme was not altered when the rats were sacrificed 31 days after the treatment (60‐day‐old rats). Hyperhomocysteinemic rats treated with folate had all those impairments prevented, an effect probably related to folate antioxidant properties.

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 inxa0vitro and inxa0vivo (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 inxa0vitro studies, 60-day-old Wistar rats were killed by decapitation and the striatum was pre-incubated for 1xa0h at 37°C in the presence of GAA at final concentrations ranging from 10 to 100xa0μM. 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 (inxa0vivo studies) or the addition of 100xa0μM GAA to assays (inxa0vitro 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.

Methionine alters Na+,K+-ATPase activity, lipid peroxidation and nonenzymatic antioxidant defenses in rat hippocampus

In the present study we investigated the effect of methionine exposure of hippocampus homogenates on Na+,K+‐ATPase activity from synaptic plasma membrane of rats. Results showed that methionine significantly decreased this enzyme activity. We also evaluated the effect of incubating glutathione (GSH) and trolox (α‐tocopherol) alone or combined with methionine on Na+,K+‐ATPase activity. The tested antioxidants per se did not alter the enzymatic activity, but prevented the inhibitory action of methionine on Na+,K+‐ATPase activity, indicating that Met inhibitory effect was probably mediated by free radical formation. Besides, we tested the in vitro effect of methionine on some parameters of oxidative stress, namely chemiluminescence, thiobarbituric acid reactive substances (TBARS), total radical‐trapping antioxidant potential (TRAP), as well as on the antioxidant enzyme activities catalase, glutathione peroxidase and superoxide dismutase in rat hippocampus. We observed that methionine significantly increased chemiluminescence and TBARS, decreased TRAP, but did not change the activity of the antioxidant enzymes. These findings suggest that reduction of Na+,K+‐ATPase activity and induction of oxidative stress may be involved in the brain damage observed in human hypermethioninemia.

Arginine Administration Decreases Cerebral Cortex Acetylcholinesterase and Serum Butyrylcholinesterase Probably by Oxidative Stress Induction

In the present study we investigated the action of vitamins E and C on the inhibition of acetylcholinesterase and butyrylcholinesterase activities provoked by arginine in cerebral cortex and serum of 60-day-old rats. Animals were pretreated for 1 week with daily intraperitoneal administration of saline (control) or vitamins E (40 mg/kg) and C (100 mg/kg). Twelve hours after the last injection, animals received one injection of arginine (0.8 μM/g of body weight) or saline. Results showed that acetylcholinesterase and butyrylcholinesterase activities were decreased in the arginine-treated rats. Furthermore, pretreatment with vitamins E and C prevented these effects. The data indicate that the reduction of acetylcholinesterase and butyrylcholinesterase activities caused by arginine was probably mediated by oxidative stress. Assuming the possibility that these effects might also occur in the human condition, our findings may be relevant to explain, at least in part, the neurological dysfunction associated with hyperargininemia and might support a novel therapeutic strategy to slow the progression of neurodegeneration in this disorder.

Evidence that oxidative stress is involved in the inhibitory effect of proline on Na+,K+-ATPase activity in synaptic plasma membrane of rat hippocampus

In the present study, we investigated the effect of Vitamins E and C on the inhibition of Na+,K+‐ATPase activity provoked by proline (Pro) administration in rat hippocampus. Five‐day‐old rats were pretreated for 1 week with daily i.p. administration of saline (control) or Vitamin E (40 mg/kg) and Vitamin C (100 mg/kg). Twelve hours after the last injection, animals received one single injection of Pro (12.8 μmol/g of body weight) or saline and were killed 1 h later. Results showed that Na+,K+‐ATPase activity was decreased in the Pro‐treated rats and that the pretreatment with Vitamins E and C prevented this effect. In another set of experiments, we investigated the in vitro effect of 1.0 mM Pro on Na+,K+‐ATPase activity from synaptic membranes of hippocampus of rats. Pro significantly inhibited (30%) Na+,K+‐ATPase activity. We also evaluated the effect of preincubating glutathione, trolox and Nϖ‐nitro‐l‐arginine methyl ester (l‐NAME) alone or combined with Pro on Na+,K+‐ATPase activity. Tested drugs did not alter Na+,K+‐ATPase activity, but glutathione prevented the inhibitory effect of Pro on this enzyme activity. These results suggest that the in vivo and in vitro inhibitory effect of Pro on Na+,K+‐ATPase activity is probably mediated by free radicals that may be involved in the neurological dysfunction found in hyperprolinemic patients.

Reduction of butyrylcholinesterase activity in rat serum subjected to hyperhomocysteinemia.

In the present study we investigate the effect of homocysteine (Hcy) administration, the main metabolite accumulating in homocystinuria, on butyrylcholinesterase (BuChE) activity in serum of rats. For the acute treatment, 29-day-old Wistar rats received one subcutaneous injection of Hcy (0.6 μmol/g) or saline (control) and were killed 1 h later. For the chronic treatment, Hcy was administered subcutaneously to rats from the 6th to the 28th day of life. Control rats received saline. The rats were killed 12 h after the last injection. In another set of experiments, rats were pretreated for one week with vitamins E and C or saline and 12 h after the last injection received one single injection of Hcy or saline, being killed 1 h later. Serum was used to determine BuChE activity. Our results showed that acute and chronic administration of Hcy significantly decreased BuChE activity. Furthermore, vitamins E and C per se did not alter BuChE activity, but prevented the reduction of this enzyme activity caused by acute administration of Hcy. The data suggest that the inhibitory effect of Hcy on BuChE activity is probably mediated by free radicals, since vitamins E and C administration prevented such effect.

Chemically induced model of hypermethioninemia in rats

In the present study, we developed a chronic chemically induced model of hypermethioninemia in rats. We induced elevated concentrations of methionine in the blood by injecting subcutaneously methionine (1.34-2.68 micromol/g of body weight) to developing animals of various ages. Brain methionine concentrations were approximately 1.25 micromol/g wet tissue ( approximately 1.0mM). We then injected the same doses of methionine to young rats twice a day at 8h intervals from the 6(th) to the 28(th) postpartum day. Controls received saline in the same volumes. The body, brain and hippocampus of rats were weighed after treatment and showed that hypermethioninemic animals had no differences in these parameters, when compared to the control group, suggesting that methionine did not cause malnutrition in the rats. Considering that experimental animal models are useful to understand the pathophysiology of human disease, the present model of hypermethioninemia may contribute to the investigation of the mechanisms of brain damage caused by high tissue methionine levels.

Hypermethioninemia Increases Cerebral Acetylcholinesterase Activity and Impairs Memory in Rats

In the present study we investigated the effect of chronic hypermethioninemia on rat performance in the Morris water maze task, as well as on acetylcholinesterase (AChE) activity in rat cerebral cortex. For chronic treatment, rats received subcutaneous injections of methionine (1.34–2.68xa0μmol/g of body weight), twice a day, from the 6th to the 28th day of age; control rats received the same volume of saline solution. Groups of rats were killed 3xa0h, 12xa0h or 30xa0days after the last injection of methionine to AChE assay and another group was left to recover until the 60th day of life to assess the effect of early methionine administration on reference and working spatial memory of rats. AChE activity was also determined after behavioral task. Results showed that chronic treatment with methionine did not alter reference memory when compared to saline-treated animals. In the working memory task, we observed a significant days effect with significant differences between control and methionine-treated animals. Chronic hypermethioninemia significantly increased AChE activity at 3xa0h, 12xa0h or 30xa0days after the last injection of methionine, as well as before or after behavioral test. The effect of acute hypermethioninemia on AChE was also evaluated. For acute treatment, 29-day-old rats received one single injection of methionine (2.68xa0μmol/g of body weight) or saline and were killed 1, 3 or 12xa0h later. Results showed that acute administration of methionine did not alter cerebral cortex AChE activity. Our findings suggest that chronic experimental hypermethioninemia caused cognitive dysfunction and an increase of AChE activity that might be related, at least in part, to the neurological problems presented by hypermethioninemic patients.