Daniela Delwing

Proline induces oxidative stress in cerebral cortex of rats

In the present study we investigated the in vivo and in vitro effects of proline on some parameters of oxidative stress, such as chemiluminescence, total radical‐trapping antioxidant potential (TRAP) and the activity of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase in rat cerebral cortex. Ten‐day‐old rats received one subcutaneous injection of proline (12.8 μmol/g body weight), while control rats received saline in the same volumes. The animals were killed 1 h after injection, the cerebral cortex was isolated and the assays immediately carried out. For the in vitro studies, homogenates from cerebral cortex of 10‐day‐old untreated rats were incubated for 1 h at 37 °C with various concentrations of proline (3.0 μM–1.0 mM). Results showed that proline‐treated rats presented a decrease of TRAP (30%) and an increase of chemiluminescence (78%). In contrast, the activities of catalase, glutathione peroxidase and superoxide dismutase were not modified by proline acute treatment. Furthermore, the presence of proline in the medium increased chemiluminescence, decreased TRAP and the activity of superoxide dismutase at proline concentrations similar to those observed in tissues of hyperprolinemic patients (0.5–1.0 mM). However, catalase and glutathione peroxidase activities were not affected by the presence of proline in the medium. The results indicate that proline induces oxidative stress in the brain, which may be related, at least in part, to the neurological dysfunction observed in hyperprolinemia.

Proline reduces acetylcholinesterase activity in cerebral cortex of rats.

In the present study we investigated the in vivo and in vitro effect of proline (Pro) on acetylcholinesterase (AChE) activity in rat cerebral cortex. The action of vitamins E and C on the effects produced by Pro was also tested. Twelve-day-old rats received one s.c. injection of Pro (12.8 μmol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. In another set of experiments, 5-day-old rats were pretreated for 1 week with daily i.p. administration of saline (control) or vitamins E (40 mg/kg) and C (100 mg/kg). Twelve hours after the last injection the rats received one s.c. injection of Pro (12.8 μmol/g body weight) or saline (control) and were killed 1 h later. For the in vitro studies, cerebral cortex homogenates of 12-day-old untreated rats were incubated for 1 h with various concentrations of Pro (3.0 μM–1.0 mM) or with 1.0 mM Pro, 1.0 mM trolox, or 1.0 mM Pro plus 1.0 mM trolox. Controls did not contain Pro in the incubation medium. Our results showed that the AChE activity significantly decreased (25%) in rat brain subjected to Pro administration and that the pretreatment with vitamins E and C prevented this effect. Furthermore, Pro (0.5 and 1.0 mM) also inhibits AChE activity in vitro and trolox prevented this effect. The data suggest that the inhibitory effect of Pro on AChE activity is associated with oxidative stress. Although it is difficult to extrapolate our findings to the human condition, our results may be relevant to explain, at least in part, the neurologic dysfunction associated with hyperprolinemia type II.

In vivo and in vitro effects of proline on some parameters of oxidative stress in rat brain.

Hyperprolinemia type II is an autosomal recessive disorder caused by the severe deficiency of delta(1)-pyrroline-5-carboxylate dehydrogenase activity leading to tissue accumulation of proline (Pro). Most patients detected so far show neurological manifestations including epilepsy and mental retardation, whose pathophysiology is not yet fully established. In the present study, we determined the in vivo and in vitro effects of Pro on some parameters of oxidative stress, namely chemiluminescence, total radical-trapping antioxidant potential (TRAP) and the activity of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) from cerebral cortex of 29-day-old Wistar rats. Results showed that acute administration of Pro provoked a significant increase of chemiluminescence and a decrease of TRAP, whereas chronic administration of the metabolite did not alter these parameters. Furthermore, in vitro brain exposure to Pro resulted in increased chemiluminescence and decreased TRAP at Pro concentrations similar to those observed in tissues of hyperprolinemic patients (0.5-1.0 mM). As regards to the antioxidant enzymes, acute injection of Pro significantly decreased CAT activity and did not alter SOD and GSH-Px activities, whereas chronic Pro administration provoked a significant increase of CAT activity, a decrease of GSH-Px activity and did not modify SOD activity. Furthermore, CAT, GSH-Px and SOD activities were not affected by the presence of Pro in the incubation medium. The data indicate that Pro induces oxidative stress in vivo and in vitro, which may be involved in the brain dysfunction observed in hyperprolinemic patients.

α-Tocopherol and ascorbic acid prevent memory deficits provoked by chronic hyperprolinemia in rats

In the present study we investigated the action of alpha-tocopherol and ascorbic acid on the effects elicited by chronic hyperprolinemia on rat performance in the Morris water maze. Rats received subcutaneous injections of proline (experimental group) twice a day, with 10 h-interval, from the 6 to 28th days of age or an equivalent volume of 0.9% saline solution (controls). Half of the proline-treated group also received intraperitoneal administration of alpha-tocopherol (40 mg/kg) and of ascorbic acid (100 mg/kg) from the 6 to 28th days of life. On the 60th day of life, rats were subjected to testing in the water maze. Results show that chronic proline administration provokes impairment on spatial learning in reference memory task, as revealed by the increase of latency in acquisition, in the probe trial and in crossing over the platform location, as well as by the number of crossings, when compared to saline-treated animals. Proline-treated rats also demonstrated a reduced efficiency to find the platform position in the working memory task. Rats chronically treated with proline plus alpha-tocopherol and ascorbic acid had above effects prevented, suggesting the participation of oxidative stress in such effects. Our findings lend support to a novel therapeutic strategy, based on these vitamins, to the cognitive dysfunction associated with hyperprolinemia type II.

Brain Na+,K+-ATPase Inhibition Induced by Arginine Administration Is Prevented by Vitamins E and C

Hyperargininemia is a metabolic disorder caused by deficiency of arginase activity resulting in tissue accumulation of arginine and neurological dysfunction. We have previously demonstrated that arginine induces oxidative stress and decreases Na+,K+-ATPase in rat midbrain. In the present study we investigated the action of vitamins E and C on the inhibition of Na+,K+-ATPase provoked by arginine in the midbrain of 60-day-old rats. Animals were pretreated for 1 week with daily IP administration of saline (control) or vitamins E (40 mg/kg) and C (100 mg/kg). Twelve h after the last injection, animals received one injection of arginine (0.8 μmol/g of body weight) or saline. Chemiluminescence was significantly increased, whereas total antioxidant capacity and Na+,K+-ATPase activity were significantly decreased. Furthermore, treatment with vitamins E and C prevented these effects. If these effects also occur in the human condition, it is possible that antioxidant administration might slow the progression of neurodegeneration in this disorder.

In vitro stimulation of oxidative stress in cerebral cortex of rats by the guanidino compounds accumulating in hyperargininemia

Hyperargininemia is a metabolic disorder biochemically characterized by tissue accumulation of arginine and other guanidino compounds. Convulsions, lethargy and psychomotor delay or cognitive deterioration are predominant clinical features of this disease. Although neurologic symptoms predominate in this disorder, their pathophysiology is still unknown. In the present study we investigated the in vitro effects of arginine, N-acetylarginine, argininic acid and homoarginine on some oxidative stress parameters in rat brain in the hope to identify a possible mechanism for the brain damage in hyperargininemia. Chemiluminescence, total radical-trapping antioxidant potential (TRAP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were measured in the cerebral cortex of rats in the presence of various concentrations of these compounds. The results showed that all guanidino compounds tested significantly increased chemiluminescence and decreased TRAP at concentrations similar to those observed in the tissue of hyperargininemic patients. Furthermore, these compounds inhibited CAT and GSH-Px activities to varying extents, with GSH-Px activity being more susceptible to their action. In turn, argininic acid inhibited all enzyme activities, and its main action was also directed towards GSH-Px. The results suggest that oxidative stress caused by guanidino compounds may be involved in the brain dysfunction amongst other potential pathophysiological mechanisms observed in hyperargininemia.

Ovariectomy increases Na+, K+-ATPase, acetylcholinesterase and catalase in rat hippocampus

In the present work we investigated the effect of ovariectomy on Na+, K+-ATPase and acetylcholinesterase (AChE) activities in rat hippocampus. We also studied some parameters of oxidative stress, namely total radical-trapping antioxidant potential (TRAP), thiobarbituric acid-reactive substances (TBA-RS), as well as the antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. Our hypothesis is that ovariectomy might cause alterations in essential enzyme activities necessary to brain normal functioning and that these chances could be caused by oxidative stress. Female adult Wistar rats were divided into three groups: (1) naive (control); (2) sham-operated; and (3) ovariectomized. Thirty days after ovariectomy rats were sacrificed. Results showed that rats subjected to ovariectomy presented a significant increase in Na+, K+-ATPase, AChE and CAT activities, but did not change the oxidative stress parameters studied when compared to sham or naive rats. Since ovariectomy mimics postmenopausal changes, our findings showing alteration in the activities of brain Na+, K+-ATPase, AChE and CAT may be related to problems in postmenopausal women.

Proline reduces brain cytochrome c oxidase : prevention by antioxidants

In the present study, we initially investigated the in vivo (acute and chronic) and in vitro effects of proline on cytochrome c oxidase (complex IV) activity in rat cerebral cortex to test the hypothesis that proline might alter energy metabolism and that this alteration could be provoked by oxidative stress. The action of α‐tocopherol and ascorbic acid on the effects produced by proline was also evaluated. For acute administration, 29‐ and 60‐day‐old rats received one subcutaneous injection of proline (18.2 μmol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were sacrificed 1 h later. For chronic treatment, proline was injected subcutaneously twice a day at 10 h intervals from the 6th to the 28th day of age. Rats were sacrificed 12 h (29th) or 31 days (60th) after the last injection. Results showed that acute administration of proline significantly diminished the activity of cytochrome c oxidase in the cerebral cortex of 29‐ and 60‐day‐old rats. On the other hand, chronic hyperprolinemia reduced this complex activity only on day 29, but not on the 60th day of life. In another set of experiments, 22‐day‐old rats or 53‐day‐old rats were pretreated for 1 week with daily intraperitoneal administration of α‐tocopherol (40 mg/kg) and ascorbic acid (100 mg/kg) or saline. Twelve hours after the last antioxidant injection, rats received a single injection of proline or saline and were killed 1 h later. In parallel to chronic treatment, rats received a daily intraperitoneal injection of α‐tocopherol and ascorbic acid from the 6th to the 28th day of life and were killed 12 h after the last injection. Results showed that the pretreatment with α‐tocopherol and ascorbic acid before acute proline administration or concomitant to chronic proline administration significantly prevented these effects. We also observed that proline (3.0 μM–1.0 mM) when added to the incubation medium (in vitro studies) did not alter cytochrome c oxidase activity. Data suggest that the inhibitory effect of proline on cytochrome c oxidase activity is possibly associated with oxidative stress and that this parameter may be involved in the brain dysfunction observed in hyperprolinemia.

Protective effect of nitric oxide synthase inhibition or antioxidants on brain oxidative damage caused by intracerebroventricular arginine administration

We have previously demonstrated that acute arginine administration induces oxidative stress and compromises energy metabolism in rat hippocampus. In the present study, we initially investigated the effect of intracerebroventricular infusion of arginine (0.1, 0.5 and 1.5 mM solution) on Na(+),K(+)-ATPase activity and on some parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant parameter (TRAP) in the hippocampus of rats. Results showed that 1.5 mM arginine solution significantly increases TBA-RS and reduces Na(+),K(+)-ATPase activity and TRAP in the rat hippocampus. We also evaluated the influence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), and antioxidants, namely alpha-tocopherol plus ascorbic acid, on the effects elicited by arginine on Na(+),K(+)-ATPase activity, TBA-RS and TRAP. Results showed that treatment with alpha-tocopherol plus ascorbic acid per se did not alter these parameters but prevented these effects. Furthermore, intracerebroventricular infusion of L-NAME prevented the inhibition caused by arginine on Na(+),K(+)-ATPase activity, as well as the increased of TBA-RS. Our findings indicate that intracerebroventricular infusion of arginine induces oxidative stress in rat hippocampus and that the inhibition of Na(+),K(+)-ATPase activity caused by this amino acid was probably mediated by NO and/or its derivatives ONOO(-) and/or other free radicals. Finally, we suggest that the administration of antioxidants should be considered as an adjuvant therapy to specific diets in hyperargininemia.

Protective effect of antioxidants on brain oxidative damage caused by proline administration

We have previously demonstrated that acute and chronic hyperprolinemia induce oxidative stress in cerebral cortex of rats. In the present study, we investigated the action of Vitamins E and C on the oxidative damage elicited by acute and chronic administration of proline (Pro) in rat cerebral cortex. Results showed that treatment with Vitamins E and C prevented the alterations caused by acute and chronic administration of proline on chemiluminescence, total radical-trapping antioxidant potential (TRAP) and on the activities of catalase and glutathione peroxidase. If these effects also occur in the human condition, it is possible that antioxidant administration might serve as a potential adjuvant therapy to avoid the progression of the neuropsychiatric dysfunction observed in hyperprolinemic patients.