Dihydrodichlorofluorescein Oxidation Research Articles

Low maternal care is associated with increased oxidative stress in the brain of lactating rats

Maternal care is crucial for offspring development and licking/grooming patterns can be induced by sensorial, neuroendocrine, and metabolic variations in the CNS. Important brain functions, such as learning and memory, can be influenced by oxidative stress, which can also modulate pathophysiological processes (e.g., depression, anxiety, and other psychiatric disorders). This study evaluated oxidative stress in the hippocampus (HP), olfactory bulb (OB), and plasma in Low-Licking (LL) and High-Licking (HL) lactating rats through superoxide dismutase (SOD) and catalase (CAT) activities, DNA damage (comet assay), and dihydrodichlorofluorescein (DCF) oxidation assay. Results demonstrate that in the HP of LL, the activities of SOD and CAT were increased compared to HL. In the OB, the activities of SOD and CAT were also increased in LL. The comet assay in the HP showed that LL had higher levels of basal damage and increased levels of DNA breaks than HL. In the OB, LL also had higher levels of DNA damage. In the plasma, no difference was observed in either SOD or CAT activities, but the DCF oxidation assay revealed that LL had higher levels of ROS production than HL. In conclusion, we observed that LL mothers showed evidence of increased oxidative stress when compared to HL, suggesting that variations in maternal behavior might be related to these biochemical parameters.

Brain and muscle redox imbalance elicited by acute ethylmalonic acid administration.

Ethylmalonic acid (EMA) accumulates in tissues and biological fluids of patients affected by short-chain acyl-CoA dehydrogenase deficiency (SCADD) and ethylmalonic encephalopathy, illnesses characterized by neurological and muscular symptoms. Considering that the mechanisms responsible for the brain and skeletal muscle damage in these diseases are poorly known, in the present work we investigated the effects of acute EMA administration on redox status parameters in cerebral cortex and skeletal muscle from 30-day-old rats. Animals received three subcutaneous injections of EMA (6 μmol/g; 90 min interval between injections) and were killed 1 h after the last administration. Control animals received saline in the same volumes. EMA administration significantly increased thiobarbituric acid-reactive substances levels in cerebral cortex and skeletal muscle, indicating increased lipid peroxidation. In addition, carbonyl content was increased in EMA-treated animal skeletal muscle when compared to the saline group. EMA administration also significantly increased 2’,7’-dihydrodichlorofluorescein oxidation and superoxide production (reactive species markers), and decreased glutathione peroxidase activity in cerebral cortex, while glutathione levels were decreased only in skeletal muscle. On the other hand, respiratory chain complex I-III activity was altered by acute EMA administration neither in cerebral cortex nor in skeletal muscle. The present results show that acute EMA administration elicits oxidative stress in rat brain and skeletal muscle, suggesting that oxidative damage may be involved in the pathophysiology of the brain and muscle symptoms found in patients affected by SCADD and ethylmalonic encephalopathy.

Acute renal failure potentiates methylmalonate-induced oxidative stress in brain and kidney of rats

Tissue methylmalonic acid (MMA) accumulation is the biochemical hallmark of methylmalonic acidemia. The disease is clinically characterized by progressive neurological deterioration and kidney failure, whose pathophysiology is still unclear. In the present work we investigated the effects of acute MMA administration on various parameters of oxidative stress in cerebral cortex and kidney of young rats, as well as the influence of acute renal failure on MMA-elicited effects on these parameters. Acute renal failure was induced by gentamicin, an aminoglycoside antibiotic whose utilization over prolonged periods causes nephrotoxicity. The administration of gentamicin alone increased carbonyl content and inhibited superoxide dismutase (SOD) activity in cerebral cortex, as well as increased thiobarbituric acid-reactive substances (TBA-RS) and sulfhydryl levels and diminished glutathione peroxidase activity in kidney. On the other hand, MMA administration increased TBA-RS levels in cerebral cortex and decreased SOD activity in kidney. Furthermore, the simultaneous administration of MMA and gentamicin to the rats provoked an augment in TBA-RS levels and superoxide generation in cerebral cortex and in TBA-RS, carbonyl and sulfhydryl levels in kidney, while diminished SOD activity in both studied tissues. Finally, nitrate/nitrite content, reduced glutathione levels, 2’,7’-dihydrodichlorofluorescein oxidation and catalase activity were not affected by this animal treatment in either tissue. In conclusion, our present data are in line with the hypothesis that MMA acts as a toxin in brain and kidney of rats and suggest that renal injury potentiates the toxicity of MMA on oxidative stress parameters in brain and peripheral tissues.

Administration of Histidine to Female Rats Induces Changes in Oxidative Status in Cortex and Hippocampus of the Offspring

Histidinemia is an inherited metabolic disorder biochemically characterized by high concentrations of histidine in biological fluids. Usually affected patients are asymptomatic although some individuals have mental retardation and speech disorders. Considering the high prevalence of histidinemia and the scarce information on the effects of maternal histidinemia on their progeny, we investigated various parameters of oxidative stress in brain cortex and hippocampus of the offspring from female rats that received histidine (0.5 mg/g of body weight) in the course of pregnancy and lactation. At 21 days of age we found a significant increase of thiobarbituric acid reactive substances (TBARS), 2',7'-dihydrodichlorofluorescein oxidation, superoxide dismutase (SOD) activity, catalase (CAT) activity, total sulfhydryls and glutathione (GSH) content in cerebral cortex and hippocampus. We also verified that at 60 days of age, GSH, SOD and total sulfhydryls returned to normal levels in brain cortex, while the other parameters decreased in the same structure. In the hippocampus, at 60 days of age GSH returned to normal levels, CAT persisted elevated and the other parameters decreased. These results indicate that histidine administration to female rats can induce oxidative stress in the brain from the offspring, which partially recovers 40 days after breastfeeding stopped.

Rapamycin Reduces Reactive Oxygen Species in Cultured Human Corneal Endothelial Cells

Purpose: To investigate the protective effect of rapamycin on oxidative stress-induced cell death of human corneal endothelial cells (HCECs).Methods: HCECs were cultured according to previously published methods. With treatment of 0 mM or 5 mM of tert-butyl hydroperoxide (tBHP) with various concentrations (0, 25 and 50 nM) of rapamycin, reactive oxygen species (ROS) production was measured using an oxidation-sensitive fluorescent probe, 2′7′-dichlorofluorescin diacetate (DCFH-DA, USA) methods. Cell viability was assayed by the method of Cell Counting Kit-8 (CCK-8, Wako). The levels of cellular glutathione were also assessed enzymatically with glutathione reductase by using a commercial glutathione (GSH) assay kit (Cayman Chemical, USA).Results: Rapamycin reduced 2′7′-dihydrodichlorofluorescein oxidation and increased GSH in HCECs. Rapamycin significantly inhibited tBHP-induced ROS production. Cells treated with rapamycin showed higher viability compared to control at 5 mM tBHP. Rapamycin effectively protected HCECs from ROS-induced cell death through increasing intracellular GSH.Conclusion: Our data suggest that rapamycin protects HCECs from oxidative injury-mediated cell death via inhibition of ROS production and enhancement of GSH.

Effect of Cysteamine on Oxidative Stress-induced Cell Death of Human Corneal Endothelial Cells

Purpose: The principal objective of this study was to evaluate the protective effect of cysteamine against the oxidative stress-induced cell death of human corneal endothelial cells.Methods: In this study, human corneal endothelial cells (HCECs) were cultured according to a previously published method. With treatment of 0 mM or 5 mM of tert-butyl hydroperoxide (tBHP) with various concentrations (0–50 mM) of cysteamine, reactive oxygen species (ROS) production was measured using an oxidation-sensitive fluorescent probe, and dichlorofluorescein diacetate (DCFH-DA) methods. Cell viability was assayed via the Cell Counting Kit-8 method. The levels of cellular glutathione were also assessed enzymatically with glutathione reductase using a commercial glutathione assay kit (Cayman Chemical, USA).Results: This study showed that cysteamine reduced 2′,7′-dihydrodichlorofluorescein oxidation and increased glutathione. Cysteamine significantly inhibited tBHP-induced ROS production. Cysteamine-treated cells evidenced higher viability relative to the controls at 5 mM tBHP, and cysteamine also effectively protected HCECs against ROS-induced cell death via an increase in intracellular glutathione.Conclusions: Our data indicate that cysteamine was not toxic at low concentrations and, at high concentrations, protects HCECs against oxidative injury-mediated cell death via the inhibition of ROS production, although cysteamine is toxic in cells at high concentrations without oxidative stress.

Tryptophan administration induces oxidative stress in brain cortex of rats

Despite the significant brain abnormalities, the neurotoxic mechanisms of brain injury in hypertryptophanemia are virtually unknown. In this work, we determined the thiobarbituric acid-reactive substances, 2',7'-dihydrodichlorofluorescein oxidation, reduced glutathione and the activities of catalase, superoxide dismutase and glutathione peroxidase in cerebral cortex from rats loaded with L-tryptophan. High L-tryptophan concentrations, similar to those found in hypertryptophanemic patients were induced by three subcutaneous injections of saline-buffered tryptophan (2 micromol/g body weight) to 30-day-old Wistar rats. The parameters were assessed 1 h after the last injection. It was observed that tryptophan significantly increased thiobarbituric acid-reactive substances, 2',7'-dihydrodichlorofluorescein oxidation and reduced glutathione, whereas it reduced catalase activity. Pre-treatment with taurine (1.6 micromol/g of body weight), or alpha-tocopherol plus ascorbic acid (40 and 100 microg/g body weight, respectively) prevented those effects of tryptophan, reinforcing the hypothesis that tryptophan induces oxidative stress in brain cortex of the rats. Therefore, these findings also occur in human hypertryptophanemia or in other neurodegenerative diseases in which tryptophan accumulates, then oxidative stress may be involved in the mechanisms leading to the brain injury observed in patients affected by these disorders.

Effects of cysteamine on oxidative status in cerebral cortex of rats

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in most tissues. Tissue damage depends on cystine accumulation, but the mechanisms of this damage are still obscure. Cysteamine administration depletes cystine accumulated, increasing survive of affected patients. Studies performed in fibroblasts of cystinotic patients suggest that apoptosis is enhanced in this disease. Considering that oxidative stress is a known apoptosis inducer, our main objective was to investigate a possible antioxidant effect of cysteamine on several parameters of oxidative stress in the brain of young rats. Animals received three subcutaneous injections at 3-h intervals of a buffered solution (pH 7.4) of 10 mg/kg body weight cysteamine and were sacrificed 1 h after the last injection. Cysteamine decreased lipoperoxidation and glutathione peroxidase activity, and increased the carbonyl content of proteins and catalase activity. In vitro studies showed that cysteamine reduced lipoperoxidation, 2',7'-dihydrodichlorofluorescein oxidation, carbonyl content of proteins and catalase activity, and increased glutathione peroxidase activity. These results suggest that cysteamine may act as a scavenger of superoxide free radicals and hydrogen peroxide. Therefore, it is possible that cysteamine may extend life of cystinotic patients acting not only as a cystine depleting drug, but also as a free radical scavenger, reducing cell damage by apoptosis.

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.