Analysis of oxidative stress markers in rat brain: the effect of maternal separation

Adverse events that cause stress during the early stages of life may alter the normal development of the brain and neuroendocrine system and increase the vulnerability of the individual to various disorders. Chronic stress and subsequent releasing of stress mediators can lead to oxidative stress and cell damage. The first aim of this work was to determine selected oxidative stress markers in the cerebral cortex, hippocampus, and cerebellum after the exposure of rats to early life stress. To model the stressful situation, we used maternal separation of the offspring for three hours a day during the first three weeks of life. We choose reduced glutathione, protein carbonyls, lipid peroxides and hydroperoxides as typical markers. These markers were determined in the brains of rats aged 22 days. Any significant changes were found in the levels of the studied markers after maternal separation. Damage to brain cells may also be reflected in behavior. Studies of numerous neuropsychiatric and neurodegenerative diseases have indicated that oxidative stress is a promising candidate for inducing changes at the cellular level. The second aim of this work was to monitor the behavior of rats by the light/dark box test after maternal separation along with administration of N-acetylcysteine (NAC), a drug with...Nepřiznivé události vyvolávající stres během rané fáze života mohou změnit normální vývoj mozku a neuroendokrinního systému a zvýšit zranitelnost jedince vůči různým poruchám. Chronický stres a následné vyplavení mediátorů může vést k oxidativnímu stresu a poškození buněk. Prvním záměrem této práce bylo stanovení vybraných markerů oxidativního stresu v mozkové kůře, hipokampu a mozečku po vystavení potkanů časnému chronickému stresu. Pro modelování stresové situace jsme používali tříhodinovou maternální separaci mláďat během prvních tří týdnů života. Vybrané markery byly koncentrace redukovaného glutathionu, proteinových karbonylů, lipidových peroxidů a hydroperoxidů. Markery jsme stanovili v mozcích potkanů starých 22 dnů. Pomocí použitých metod se nám nepodařilo zjistit žádné signifikantní změny v hladinách sledovaných markerů po maternální separaci. Poškození mozkových buněk se může projevit i v chování. Výzkumy četných neuropsychiatrických a neurodegenerativních onemocnění nastínily, že oxidativní stres je slibným kandidátem pro vyvolání změn na buněčné úrovni. Druhým tématem práce bylo sledování chování potkanů pomocí testu light/dark box po maternální separaci a zároveň po aplikaci N-acetylcysteinu (NAC), látky se známými antioxidačními účinky. Ze získaných výsledků testů z 22. postnatálního...Department of PhysiologyKatedra fyziologiePřírodovědecká fakultaFaculty of Scienc

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.</p

Molecular mechanisms engaged in the development of drug addiction

Drug use is part of the human life from the ancient times. Besides their recreational utilization, sustained misuse of these substances can lead to the development of drug addiction especially in susceptible individuals and thus cause serious health and social problems. The aim of this thesis is to briefly introduce brain structures which are affected by addictive substances, and describe some of the mechanisms and molecules that contribute to addiction. A crucial brain structure which plays a role in drug addiction is the reward system, with dopamine as the main neurotransmitter. After repeated use of drugs, in neurons of this system certain molecules and epigenetic changes are accumulating that promote chronic nature of addiction. Especially important is the highly stable transcription factor ΔFosB, which in cooperation with other molecules promotes relapse even after several months or years of the last drug use. Powered by TCPDF (www.tcpdf.org

Analysis of oxidative stress markers in rat brain: the effect of maternal separation

Adverse events that cause stress during the early stages of life may alter the normal development of the brain and neuroendocrine system and increase the vulnerability of the individual to various disorders. Chronic stress and subsequent releasing of stress mediators can lead to oxidative stress and cell damage. The first aim of this work was to determine selected oxidative stress markers in the cerebral cortex, hippocampus, and cerebellum after the exposure of rats to early life stress. To model the stressful situation, we used maternal separation of the offspring for three hours a day during the first three weeks of life. We choose reduced glutathione, protein carbonyls, lipid peroxides and hydroperoxides as typical markers. These markers were determined in the brains of rats aged 22 days. Any significant changes were found in the levels of the studied markers after maternal separation. Damage to brain cells may also be reflected in behavior. Studies of numerous neuropsychiatric and neurodegenerative diseases have indicated that oxidative stress is a promising candidate for inducing changes at the cellular level. The second aim of this work was to monitor the behavior of rats by the light/dark box test after maternal separation along with administration of N-acetylcysteine (NAC), a drug with..

Molecular mechanisms engaged in the development of drug addiction

Drug use is part of the human life from the ancient times. Besides their recreational utilization, sustained misuse of these substances can lead to the development of drug addiction especially in susceptible individuals and thus cause serious health and social problems. The aim of this thesis is to briefly introduce brain structures which are affected by addictive substances, and describe some of the mechanisms and molecules that contribute to addiction. A crucial brain structure which plays a role in drug addiction is the reward system, with dopamine as the main neurotransmitter. After repeated use of drugs, in neurons of this system certain molecules and epigenetic changes are accumulating that promote chronic nature of addiction. Especially important is the highly stable transcription factor ΔFosB, which in cooperation with other molecules promotes relapse even after several months or years of the last drug use. Powered by TCPDF (www.tcpdf.org

Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics

Abstract Alterations in metabolism are a hallmark of cancer. It is unclear if oxidative phosphorylation (OXPHOS) is necessary for tumour cell survival. In this study, we investigated the effects of severe hypoxia, site-specific inhibition of respiratory chain (RC) components, and uncouplers on necrotic and apoptotic markers in 2D-cultured HepG2 and MCF-7 tumour cells. Comparable respiratory complex activities were observed in both cell lines. However, HepG2 cells exhibited significantly higher oxygen consumption rates (OCR) and respiratory capacity than MCF-7 cells. Significant non-mitochondrial OCR was observed in MCF-7 cells, which was insensitive to acute combined inhibition of complexes I and III. Pre-treatment of either cell line with RC inhibitors for 24–72 h resulted in the complete abolition of respective complex activities and OCRs. This was accompanied by a time-dependent decrease in citrate synthase activity, suggesting mitophagy. High-content automated microscopy recordings revealed that the viability of HepG2 cells was mostly unaffected by any pharmacological treatment or severe hypoxia. In contrast, the viability of MCF-7 cells was strongly affected by inhibition of complex IV (CIV) or complex V (CV), severe hypoxia, and uncoupling. However, it was only moderately affected by inhibition of complexes I, II, and III. Cell death in MCF-7 cells induced by inhibition of complexes II, III, and IV was partially abrogated by aspartate. These findings indicate that OXPHOS activity and viability are not correlated in these cell lines, suggesting that the connection between OXPHOS and cancer cell survival is dependent on the specific cell type and conditions

Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited

The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition