Rodrigo Binkowski de Andrade

Homocysteine induces energy imbalance in rat skeletal muscle: is creatine a protector?

Homocystinuria is a neurometabolic disease caused by a severe deficiency of cystathionine beta‐synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction. In this study, we investigated the effect of chronic hyperhomocysteinemia on the cell viability of the mitochondrion, as well as on some parameters of energy metabolism, such as glucose oxidation and activities of pyruvate kinase, citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, respiratory chain complexes and creatine kinase in gastrocnemius rat skeletal muscle. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injections of homocysteine (0.3–0.6 µmol/g body weight) and/or creatine (50 mg/kg body weight) from the 6th to the 28th days of age. The animals were decapitated 12 h after the last injection. Homocysteine decreased the cell viability of the mitochondrion and the activities of pyruvate kinase and creatine kinase. Succinate dehydrogenase was increased other evaluated parameters were not changed by this amino acid. Creatine, when combined with homocysteine, prevented or caused a synergistic effect on some changes provoked by this amino acid. Creatine per se or creatine plus homocysteine altered glucose oxidation. These findings provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function, more studies are needed to elucidate them. Although creatine prevents some alterations caused by homocysteine, it should be used with caution, mainly in healthy individuals because it could change the homeostasis of normal physiological functions. Copyright

Creatine and Pyruvate Prevent the Alterations Caused by Tyrosine on Parameters of Oxidative Stress and Enzyme Activities of Phosphoryltransfer Network in Cerebral Cortex of Wistar Rats

Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II. In this disease caused by tyrosine aminotransferase deficiency, eyes, skin, and central nervous system disturbances are found. In the present study, we investigated the chronic effect of tyrosine methyl ester (TME) and/or creatine plus pyruvate on some parameters of oxidative stress and enzyme activities of phosphoryltransfer network in cerebral cortex homogenates of 21-day-old Wistar. Chronic administration of TME induced oxidative stress and altered the activities of adenylate kinase and mitochondrial and cytosolic creatine kinase. Total sulfhydryls content, GSH content, and GPx activity were significantly diminished, while DCFH oxidation, TBARS content, and SOD activity were significantly enhanced by TME. On the other hand, TME administration decreased the activity of CK from cytosolic and mitochondrial fractions but enhanced AK activity. In contrast, TME did not affect the carbonyl content and PK activity in cerebral cortex of rats. Co-administration of creatine plus pyruvate was effective in the prevention of alterations provoked by TME administration on the oxidative stress and the enzymes of phosphoryltransfer network, except in mitochondrial CK, AK, and SOD activities. These results indicate that chronic administration of TME may stimulate oxidative stress and alter the enzymes of phosphoryltransfer network in cerebral cortex of rats. In case this also occurs in the patients affected by these disorders, it may contribute, along with other mechanisms, to the neurological dysfunction of hypertyrosinemias, and creatine and pyruvate supplementation could be beneficial to the patients.

Pyruvate Kinase Activity and δ-Aminolevulinic Acid Dehydratase Activity as Biomarkers of Toxicity in Workers Exposed to Lead

Lead (Pb2+) is a heavy metal that has long been used by humans for a wide range of technological purposes, which is the main reason for its current widespread distribution. Pb2+ is thought to enter erythrocytes through anion exchange and to remain in the cell by binding to thiol groups. Pyruvate kinase (PK) is a thiol-containing enzyme that plays a key role in erythrocyte cellular energy homeostasis. δ-aminolevulinic acid dehydratase (δ-ALAD) is the second enzyme in the heme biosynthetic pathway and plays a role in the pathogenesis of Pb poisoning. Our primary objective was to investigate the effect of Pb2+ on the activity of the thiolenzymes δ-ALAD and PK and on the concentration of glutathione (GSH), a nonenzymatic antioxidant defense, in erythrocytes from Pb-exposed workers. The study sample comprised 22 male Pb workers and 21 normal volunteers (15 men and 6 women). The Pb-exposed workers were employed in manufacturing and recycling of automotive batteries. Basic red-cell parameters were assayed and total white blood cell counts performed. PK and δ-ALAD activity and blood Pb (BPb) concentrations were determined in all subjects. Pb-exposed individuals had significantly greater BPb levels than controls. Both PK and δ-ALAD activity levels were significantly lower in Pb-exposed individuals than in controls. Pb significantly inhibited PK and δ-ALAD activity in a dose-dependent manner. We found that erythrocyte GSH levels were lower in Pb-exposed individuals than normal volunteers. Pb-exposed individuals had lower values than controls for several red cell parameters (hemoglobin, hematocrit, red blood cell count, mean corpuscular volume). These results suggest that Pb inhibits δ-ALAD and PK activity by interacting with their thiol groups. It is therefore possible that Pb disrupts energy homeostasis and may be linked with decreased glucose metabolism because it affects the heme synthesis pathway in erythrocytes, contributing to the cell dysfunction observed in these in Pb-exposed individuals. These results indicate an apparent dose-effect relationship between PK activity and BPb. PK activity in human erythrocytes can be used for biological monitoring of Pb exposure. Study of the mechanisms by which Pb acts may contribute to greater understanding of the symptoms caused by Pb.

Acute treatment with the organochalcogen 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one produces behavioral changes and inhibition of creatine kinase activity in the brain of rats.

Organotellurium compounds have been synthesized since 1840, but their pharmacological and toxicological properties are still incipient. Therefore, the objective of this study was to verify the effect of acute administration with the organochalcogen 3‐butyl‐1‐phenyl‐2‐(phenyltelluro)oct‐en‐1‐one on the activity of brain creatine kinase (CK), a key enzyme in energy metabolism, and on behaviors in the open field test of 30‐day‐old rats. Animals were treated intraperitoneally with a single dose of the organotellurium (125, 250, or 500 μg/kg body weight) and after 55 min of the drug administration the open field test was carried out. Behavior analyses were performed during 5 min and the number of the squares crossed, number of rearing, number of groomings and number of fecal boli were recorded by an observer. Then, the animals were sacrificed and the cerebral cortex, the hippocampus, and the cerebellum were dissected, and CK activity and sulfhydryl content were measured in the brain. The organotellurium increased the ambulation and rearing behaviors in the open field test at doses of 250 and 500 μg/kg. Moreover, the compound inhibited CK activity and provoked a reduced of thiol content measured by the sulfhydryl assay in all the tissues studied. Therefore, changes in energy homeostasis and motor behavior in rats treated with this organotellurium support the hypotheses that the brain is a potential target to pharmacological and toxicological effects of this compound.

Inhibition of creatine kinase activity by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one in the cerebral cortex and cerebellum of young rats.

In the present study, we investigated the potential in vitro toxicity of the tellurium compound 3‐butyl‐1‐phenyl‐2‐(phenyltelluro)oct‐en‐1‐one on creatine kinase activity in cerebral cortex and cerebellum of 30‐day‐old Wistar rats. First, enriched mitochondrial and cytosolic fractions from the two tissues were pre‐incubated for 30 min in the presence or absence of 1, 5 or 20 µm of organotellurium and the creatine kinase activity was measured. The organochalcogen reduced creatine kinase activity in a concentration‐dependent pattern in the two tissues studied. Furthermore, the enzyme activity was performed after pre‐incubation for 30, 60 or 90 min in the presence of 5 µm of the organotellurium. The compound inhibited creatine kinase activity in a time‐dependent way in the enriched mitochondrial fraction of both tissues, but not in the cytosolic fraction, indicating different mechanisms for the organochalcogen in the mitochondrial and in the cytosolic creatine kinase. Pre‐incubation of tellurium compound with reduced glutathione suggests that creatine kinase activity inhibition might be caused by direct interaction with thiol groups or by oxidative stress. Our findings suggest that creatine kinase inhibition may be one of the mechanisms by which this organotellurium could cause toxicity to the rat brain. Copyright

Kinetic studies on the inhibition of creatine kinase activity by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one in the cerebral cortex of rats

Tellurium has been used as an industrial component of many alloys and in the electronic industry. Organotellurium compounds can cause poisoning which leads to neurotoxic symptoms such as significant impairment of learning, spatial memory and are potentially neurotoxic to human beings. However, the molecular mechanisms of neurotoxicity of organotellurium compounds are not well understood. Considering that creatine kinase plays a key role in energy metabolism of tissues with intermittently high and fluctuating energy requirements, such as nervous tissue, the main objective of this study was to investigate the mechanisms by which 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one inhibit creatine kinase activity, a key enzyme of energy homeostasis, in the cerebral cortex of 30-day-old Wistar rats. For the kinetic studies, the Lineweaver-Burk plot was used to characterize the mechanisms of enzyme inhibition by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one. The results suggested that this compound inhibits creatine kinase activity by two different mechanisms: competition with ADP and oxidation of critical sulfhydryl groups for the functioning of the enzyme. The potential for inhibition of creatine kinase to occur in vivo may contribute to the neurotoxicity observed by this organochaocogen.

Evaluation of Oxidative Stress Parameters and Energy Metabolism in Cerebral Cortex of Rats Subjected to Sarcosine Administration.

Sarcosine is an N-methyl derivative of the amino acid glycine, and its elevation in tissues and physiological fluids of patients with sarcosinemia could reflect a deficient pool size of activated 1-carbon units. Sarcosinemia is a rare inherited metabolic condition associated with mental retardation. In the present study, we investigated the acute effect of sarcosine and/or creatine plus pyruvate on some parameters of oxidative stress and energy metabolism in cerebral cortex homogenates of 21-day-old Wistar rats. Acute administration of sarcosine induced oxidative stress and diminished the activities of adenylate kinase, GAPDH, complex IV, and mitochondrial and cytosolic creatine kinase. On the other hand, succinate dehydrogenase activity was enhanced in cerebral cortex of rats. Moreover, total sulfhydryl content was significantly diminished, while DCFH oxidation, TBARS content, and activities of SOD and GPx were significantly enhanced by acute administration of sarcosine. Co-administration of creatine plus pyruvate was effective in the prevention of alterations provoked by sarcosine administration on the oxidative stress and the enzymes of phosphoryltransfer network. These results indicate that acute administration of sarcosine may stimulate oxidative stress and alter the energy metabolism in cerebral cortex of rats. In case these effects also occur in humans, they may contribute, along with other mechanisms, to the neurological dysfunction of sarcosinemia, and creatine and pyruvate supplementation could be beneficial to the patients.

Assessment of changes in energy metabolism parameters provoked by carbon tetrachloride in Wistar rats and the protective effect of white grape juice

The objective of this study was to evaluate the effect of organic and conventional grape juices consumption on the behavior of rats and their neuroprotective effect on the activity of brain energy metabolism enzymes in different brain areas of adult rats on the experimental model of hepatic encephalopathy. Male Wistar rats (90-days-old) were treated once a day with conventional or organic white grape juice by gavage for 14 days (7 μL/g). On the 15th day the rats received carbon tetrachloride (CCl4) in a single dose of 3.0 mL/kg. Cerebral cortex, hippocampus and cerebellum were dissected to measure the activity of creatine kinase (CK) and pyruvate kinase (PK). No changes in feeding behavior were observed after the treatment with the grapes juices. However, there was an increase in grooming behavior in the open field test provoked by both juices. CCl4 inhibited CK activity in cerebral cortex and hippocampus of the rats and CCl4 also reduced PK activity in all brain structures studied. Furthermore, both white grape juices prevented the decrease in the activity of CK and PK. Therefore, we can suggest that organic and conventional white grape juices could restore the activity of enzymes with a central role in brain energy metabolism.

Toxicological evaluation of chronic exposure to the organochalcogen 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one in male rats

The aim of this study was to evaluate the effect of chronic treatment with the organochalcogen 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one on some behavioral and biochemical parameters in the brain, liver, kidney and serum of 90-day-old male Wistar rats. The animals received the organoselenium at doses of 125, 250 or 500 μg/kg body weight intraperitoneally once daily for 30 days. Results showed that chronic treatment with this compound induced behavioral changes in animals, such as increasing of rearing at dose of 250 μg/kg and increasing of ambulation in all concentrations tested. On the other hand, we did not observe any alterations in the body weight gain of the animals. Moreover, the activity of the enzyme creatine kinase (CK) decreased in the cerebral cortex, cerebellum and kidney and increased in the liver after the chronic treatment with the organoselenium compound at dose of 500 μg/kg. The compound also increased aspartate aminotransferase (AST) and urea levels in serum of rats at 500 μg/kg. Glucose, cholesterol, triglycerides, creatinine, alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels were not changed by the treatment. Our results thus show that chronic administration of 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one is able to significantly change the activity of CK in Wistar rats, resulting in a change in cellular energy homeostasis in these tissues, liver damage and behavioral changes in the animals studied.

Acute exposure to the vinyl chalcogenide 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one induces oxidative stress in different brain area of rats

The mechanisms that lead to the onset of organoselenium intoxication are still poorly understood. Therefore, in the present study, we investigated the effect of acute administration of 3‐methyl‐1‐phenyl‐2‐(phenylseleno)oct‐2‐en‐1‐one on some parameters of oxidative stress and on the activity of creatine kinase (CK) in different brain areas and on the behaviour in the open field test of 90‐day‐old male rats. Animals (n = 10/group) were treated intraperitoneally with a single dose of the organoselenium (125, 250 or 500 µg kg−1), and after 1 h of the drug administration, they were exposed to the open field test, and behaviour parameters were recorded. Immediately after they were euthanized, cerebral cortex, hippocampus and cerebellum were dissected for measurement of thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, catalase (CAT), superoxide dismutase (SOD) and CK activity. Our results showed that the dose of 500 µg kg−1 of the organoselenium increased the locomotion and rearing behaviours in the open field test. Moreover, the organochalcogen enhanced TBARS in the cerebral cortex and cerebellum and increased the oxidation of proteins (carbonyl) only in the cerebral cortex. Sulfhydryl content was reduced in all brain areas, CAT activity enhanced in the hippocampus and reduced in the cerebellum and SOD activity increased in all brain structures. The organoselenium also inhibited CK activity in the cerebral cortex. Therefore, changes in motor behaviour, redox state and energy homeostasis in rats treated acutely with organoselenium support the hypotheses that the brain is a potential target for the organochalcogen action. Copyright