Rotenone exerts similar stimulatory effects on H2O2 production by isolated brain mitochondria from young–adult and old rats

Michelini, Luiz G. B.; Figueira, Tiago Rezende; Siqueira-Santos, Edilerie S.; Castilho, Roger F.

doi:10.1016/j.neulet.2015.01.030

Cited by 7 publications

(3 citation statements)

References 35 publications

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“…Six rats were used for the present study and were euthanized by decapitation [ 16 ]. Rat brain mitochondria were isolated as described by Michelini et al [ 17 ] using digitonin to permeabilize the synaptosomal plasma membrane [ 18 ]. The use of a similar procedure to isolate mitochondria from intact cultured glioma cells did not result in functional mitochondria in sufficient quantity to conduct experiments (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells

et al. 2016

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The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.

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Section: Methodsmentioning

confidence: 99%

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells

et al. 2016

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“…To support the above observations, when HEK-293 LRRK2 WT cells were untreated or treated with ROT, the opposite phenotypic responses to KO cells were observed. Indeed, HEK-293 LRRK2 WT cells exposed to ROT had a significant increase in intracellular ROS via inhibition of mitochondrial complex I [ 29 , 30 , 31 ], as revealed by a high percentage of DCF+ cells and oxidized DJ-1Cys 106 -SO 3 , which is specifically oxidized by H 2 O 2 [ 24 ]. In agreement with others [ 19 , 20 ], we found that ROT/H 2 O 2 activated LRRK2 kinase, as evidenced by a high percentage of p-S 935 -LRRK2.…”

Section: Discussionmentioning

confidence: 99%

LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis

Quintero-Espinosa

Sánchez‐Hernández

Vélez-Pardo

et al. 2023

IJMS

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Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson’s disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 μM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, −58%) and PTEN induced putative kinase 1 (PINK1, −49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.

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“…The pesticide was shown to cause the increased production of intracellular ROS, lipid peroxidation, protein carbonylation, decrease the level of GSH, and induce apoptotic cell death in vitro (Sherer et al, 2003;Testa et al, 2005) and following systemic administration in rodents (Abdel-Salam et al, 2014). Rotenone being highly lipophyllic is capable of readily crossing the blood brain barrier and causes inhibition of mitochondrial complex I or NADH-ubiquinone oxidoreductase, resulting in the increased generation of superoxide (Li et al, 2003) or hydrogen peroxide (Michelini et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

The Anti-obesity Agent d-norpseudoephedrine Protects Against Rotenone-induced Parkinson’s Disease in Mice

2022

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The effect of the ephedrine derivative D-norpseudoephedrine on rotenone-induced Parkinson’s disease in mice was studied. Mice received subcutaneous injections of rotenone (1.5 mg/kg, every other day for two weeks) and were treated with the vehicle, L-dopa (25 mg/kg) or pseudoephedrine at doses of 1.8, 5.4 and 10.8 mg/kg once daily orally. Brain levels of malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide (NO) were determined and histopathological study of the brain was done. Motor testing included stair, wire hanging and wood walking tests. Results indicated that ompared with vehicle controls, rotenone caused significant increases in brain MDA and NO along with GSH depletion. Rotenone impaired neuromuscular strength, and motor balance and coordination. There were also marked decrease in size and number of substantia nigra pigmented cells and deeply stained neurons and karyorrhexis in the cerebral cortex and hippocampus. Treatment with pseudoephedrine reduced brain MDA, NO and increased GSH levels and improved motor performance. Furthermore, pseudoephedrine prevented substantia nigra dopaminergic cell death and neurodegeneration in the cortex and hippocampus brain regions in a dose-dependent manner. These data suggest that pseudoephedrine might prove of benefit adjunctive therapy of Parkinson’s disease.

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Rotenone exerts similar stimulatory effects on H2O2 production by isolated brain mitochondria from young–adult and old rats

Cited by 7 publications

References 35 publications

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells

LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis

The Anti-obesity Agent d-norpseudoephedrine Protects Against Rotenone-induced Parkinson’s Disease in Mice

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