Rotenone

The herbs of Derris trifoliata Lour.

Rotenone is an inhibitor of the mitochondrial complex I electron transport chain with IC50 value of 1.7 - 2.2 μM.

Electron transport chain (ETC) is a series of compounds that transfer electrons from electron donors to electron acceptors and transfer protons (H+ ions) across a membrane. The proton gradient drives ATP synthesis. Complex I is one of the main sites of production of superoxide.

Rotenone is an inhibitor of the mitochondrial complex I electron transport chain. In the transformed cell line HEK 293 and cancer cell lines U87, rotenone (50 μM) induced cell death by 30% and 40% respectively in a dose dependent way, which was mediated by reactive oxygen species (ROS). Also, rotenone significantly induced autophagy formation [1]. In SH-SY5Y cells, rotenone induced cell apoptosis in a caspase-dependent way. Also, rotenone induced phosphorylation of p38 MAP kinase, c-Jun and JNK, which indicated activation of the p38 and JNK pathways [2]. In differentiated SH-SY5Y neuroblastoma cells, rotenone (50 nM) induced cell death by 60% and slowed mitochondrial movement. While rotenone didn’t induce the formation of resembling Lewy bodies [3].

References:
[1].  Chen Y, McMillan-Ward E, Kong J, et al. Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species. J Cell Sci, 2007, 120(Pt 23): 4155-4166.
[2].  Newhouse K, Hsuan SL, Chang SH, et al. Rotenone-induced apoptosis is mediated by p38 and JNK MAP kinases in human dopaminergic SH-SY5Y cells. Toxicol Sci, 2004, 79(1): 137-146.
[3].  Borland MK, Trimmer PA, Rubinstein JD, et al. Chronic, low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson's disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells. Mol Neurodegener, 2008, 3: 21.

GAPDH-knockdown reduce rotenone-induced H9C2 cells death via autophagy and anti-oxidative stress pathway.[Pubmed:25725130]

Toxicol Lett. 2015 May 5;234(3):162-71.

BACKGROUND: GAPDH, well known for its house-keeping functions, has also been shown to be involved in cell injury, apoptosis and death under conditions of stress such as starvation, chemical injury and oxidative stress. This study examines the effect of GAPDH knockdown on cell injury in response to Rotenone. METHODS: GAPDH was knocked down in H9C2 cardiomyoblasts using siRNA prior to exposure to Rotenone (0 nM, 20 nM, 40 nM and 80 nM). Autophagy was detected by western blot for autophagy proteins (Beclin-1, Atg5, LC-3A/B and p62) and MDC staining for acidic substances. Pro-apoptosis protein and flow cytometry were used to assess cell apoptosis and death and intracellular ATP relative concentration was measured. Oxidant stress was assessed by measuring DCFH-DA, TBARS, GSH and SOD. RESULTS: In this study, GAPDH-knockdown enhanced autophagy in Rotenone-induced H9C2 cells, decreased oxidant stress and increased antioxidant pathways; and reduced cell apoptosis and death. Furthermore, GAPDH-knockdown preserved cell energy. CONCLUSION: siRNA-mediated GAPDH knockdown reduced Rotenone-induced H9C2 cell death occurring via autophagy and anti-oxidative stress pathway. This study enriches the understanding of GAPDH pathophysiology role, and provides potential new therapeutic targets for cardiac disease states characterized by oxidative stress.

The molecular mechanism of rotenone-induced alpha-synuclein aggregation: emphasizing the role of the calcium/GSK3beta pathway.[Pubmed:25433145]

Toxicol Lett. 2015 Mar 4;233(2):163-71.

Environmental toxin exposure is associated with the development of Parkinson's disease (PD), and environmental factors can influence the onset of the majority of sporadic PD cases via genetically mediated pathways. Rotenone, a widespread pesticide, induces Parkinsonism and the formation of Lewy bodies in animals; however, the molecular mechanism that underlies alpha-synuclein aggregation remains unclear. Here, we assessed the aggregation of alpha-synuclein in PC12 cells with or without cross-linking following Rotenone exposure via a variety of methods, including western blotting, immunofluorescence and electron microscopy. We demonstrated that Rotenone increased the intracellular calcium levels and induced the aggregation and phosphorylation of alpha-synuclein in a calcium-dependent manner. Aggregated alpha-synuclein is typically degraded by autophagy, and Rotenone impaired this process. The attenuation of autophagy and alpha-synuclein alterations were reversed by scavenging calcium. Calcium regulates the activity of AKT-glycogen synthase kinase 3 (GSK3)beta. We demonstrated that Rotenone attenuated the phosphorylation of AKT and GSK3beta, and the elimination of calcium reversed these phenomena. As a GSK3beta inhibitor, lithium promoted autophagy and decreased the aggregation and phosphorylation of alpha-synuclein. GSK3beta activation through overexpression depressed autophagy and increased the total protein level and phosphorylation of alpha-synuclein. These results suggest that Rotenone-induced alpha-synuclein aggregation is mediated by the calcium/GSK3beta signaling pathway.

JNK inhibition of VMAT2 contributes to rotenone-induced oxidative stress and dopamine neuron death.[Pubmed:25496994]

Toxicology. 2015 Feb 3;328:75-81.

Treatment with Rotenone, both in vitro and in vivo, is widely used to model dopamine neuron death in Parkinson's disease upon exposure to environmental neurotoxicants and pesticides. Mechanisms underlying Rotenone neurotoxicity are still being defined. Our recent studies suggest that Rotenone-induced dopamine neuron death involves microtubule destabilization, which leads to accumulation of cytosolic dopamine and consequently reactive oxygen species (ROS). Furthermore, the c-Jun N-terminal protein kinase (JNK) is required for Rotenone-induced dopamine neuron death. Here we report that the neural specific JNK3 isoform of the JNKs, but not JNK1 or JNK2, is responsible for this neuron death in primary cultured dopamine neurons. Treatment with taxol, a microtubule stabilizing agent, attenuates Rotenone-induced phosphorylation and presumably activation of JNK. This suggests that JNK is activated by microtubule destabilization upon Rotenone exposure. Moreover, Rotenone inhibits VMAT2 activity but not VMAT2 protein levels. Significantly, treatment with SP600125, a pharmacological inhibitor of JNKs, attenuates Rotenone inhibition of VMAT2. Furthermore, decreased VMAT2 activity following in vitro incubation of recombinant JNK3 protein with purified mesencephalic synaptic vesicles suggests that JNK3 can inhibit VMAT2 activity. Together with our previous findings, these results suggest that Rotenone induces dopamine neuron death through a series of sequential events including microtubule destabilization, JNK3 activation, VMAT2 inhibition, accumulation of cytosolic dopamine, and generation of ROS. Our data identify JNK3 as a novel regulator of VMAT2 activity.

Rotenone decreases intracellular aldehyde dehydrogenase activity: implications for the pathogenesis of Parkinson's disease.[Pubmed:25645689]

J Neurochem. 2015 Apr;133(1):14-25.

Repeated systemic administration of the mitochondrial complex I inhibitor Rotenone produces a rodent model of Parkinson's disease (PD). Mechanisms of relatively selective Rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the 'catecholaldehyde hypothesis,' buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether Rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with Rotenone (0-1000 nM, 180 min), without or with F-dopamine (2 muM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, Rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD(+) was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that Rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective Rotenone-induced toxicity in dopaminergic neurons. We report that Rotenone, a mitochondrial complex I inhibitor that produces an animal model of Parkinson's disease, increases intracellular levels of the toxic dopamine metabolite 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), via decreased DOPAL metabolism by aldehyde dehydrogenase (ALDH) and decreased vesicular sequestration of cytoplasmic dopamine by the vesicular monoamine transporter (VMAT). The results provide a novel mechanism for Rotenone-induced toxicity in dopaminergic neurons.

Rotenone inhibits autophagic flux prior to inducing cell death.[Pubmed:23259041]

ACS Chem Neurosci. 2012 Dec 19;3(12):1063-72.

Rotenone, which selectively inhibits mitochondrial complex I, induces oxidative stress, alpha-synuclein accumulation, and dopaminergic neuron death, principal pathological features of Parkinson's disease. The autophagy-lysosome pathway degrades damaged proteins and organelles for the intracellular maintenance of nutrient and energy balance. While it is known that Rotenone causes autophagic vacuole accumulation, the mechanism by which this effect occurs has not been thoroughly investigated. Treatment of differentiated SH-SY5Y cells with Rotenone (10 muM) induced the accumulation of autophagic vacuoles at 6 h and 24 h as indicated by Western blot analysis for microtubule associated protein-light chain 3-II (MAP-LC3-II). Assessment of autophagic flux at these time points indicated that autophagic vacuole accumulation resulted from a decrease in their effective lysosomal degradation, which was substantiated by increased levels of autophagy substrates p62 and alpha-synuclein. Inhibition of lysosomal degradation may be explained by the observed decrease in cellular ATP levels, which in turn may have caused the observed concomitant increase in acidic vesicle pH. The early (6 h) effects of Rotenone on cellular energetics and autophagy-lysosome pathway function preceded the induction of cell death and apoptosis. These findings indicate that the classical mitochondrial toxin Rotenone has a pronounced effect on macroautophagy completion that may contribute to its neurotoxic potential.

Pesticides and impairment of mitochondrial function in relation with the parkinsonian syndrome.[Pubmed:17127363]

Front Biosci. 2007 Jan 1;12:1079-93.

The Parkinsonian syndrome induced by pesticides is associated with the impairment of mitochondrial function. Toxicants that inhibit selectively NADH-dehydrogenase activity, as Rotenone or pyridaben, also show a selective inhibition of O2 uptake and respiratory control in rat brain mitochondria in the presence of NAD-dependent substrates. The IC50 of Rotenone and pyridaben for complex I inhibition were in the range 1.7-2.2 microM. The determination of NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome oxidase activities in rat brain submitochondrial showed again the selective inhibition of Complex I by Rotenone and pyridaben, whereas paraquat produced a non-selective inhibition affecting all the respiratory chain complexes. In rat brain mitochondria, Rotenone and pyridaben markedly decreased mtNOS functional activity with NAD-dependent substrates but not when the substrate was succinate. This observation suggest than mtNOS activity is regulated by the activity of complex I. This regulation and the role of mitochondrial NO diffusion as a signal for mitochondrial biogenesis could have a role in the etiopathology of Parkinson's disease.

NADH oxidase activity of rat cardiac sarcoplasmic reticulum regulates calcium-induced calcium release.[Pubmed:14699012]

Circ Res. 2004 Mar 5;94(4):478-86.

NADH and Ca2+ have important regulatory functions in cardiomyocytes related to excitation-contraction coupling and ATP production. To elucidate elements of these functions, we examined the effect of NADH on sarcoplasmic reticulum (SR) Ca2+ release and the mechanisms of this regulation. Physiological concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2)-mediated Ca2+-induced Ca2+ release (CICR) from SR membranes (IC50=120 micromol/L) and significantly lowered single channel open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca2+ release. Blockers of electron transport prevented the inhibitory effect of NADH on CICR in isolated membranes and permeabilized cells, as well as on the activity of RyR2 channels reconstituted in lipid bilayer. An endogenous NADH oxidase activity from rat heart copurified with SR enriched with RyR2. A significant contribution by mitochondria was excluded as NADH oxidation by SR exhibited >9-fold higher catalytic activity (8.8 micromol/mg protein per minute) in the absence of exogenous mitochondrial complex I (ubiquinone) or complex III (cytochrome c) electron acceptors, but was inhibited by Rotenone and pyridaben (IC50=2 to 3 nmol/L), antimycin A (IC50=13 nmol/L), and diphenyleneiodonium (IC50=28 micromol/L). Cardiac junctional SR treated with [3H](trifluoromethyl)diazirinyl-pyridaben specifically labeled a single 23-kDa PSST-like protein. These data indicate that NADH oxidation is tightly linked to, and essential for, negative regulation of the RyR2 complex and is a likely component of an important physiological negative-feedback mechanism coupling SR Ca2+ fluxes and mitochondrial energy production.

Neurotoxicant-induced animal models of Parkinson's disease: understanding the role of rotenone, maneb and paraquat in neurodegeneration.[Pubmed:15258850]

Cell Tissue Res. 2004 Oct;318(1):225-41.

The etiologic basis of Parkinson's disease (PD), the second most common age-related neurodegenerative disorder, is unknown. Recent epidemiological and experimental studies indicate that exposure to environmental agents, including a number of agricultural chemicals, may contribute to the pathogenesis of this disorder. Animal models are important tools in experimental medical science for studying the pathogenesis and therapeutic intervention strategies of human diseases. Since many human disorders do not arise spontaneously in animals, characteristic functional changes have to be mimicked by neurotoxic agents. Recently, agricultural chemicals, when administrated systemically, have been shown to reproduce specific features of PD in rodents, thus opening new routes for the development of animal models for this disorder. In addition to a brief historical overview of the toxin-induced PD models, this study provides a detailed description of exiting models in which Parkinsonism is initiated via the exposure of animals to such agricultural chemicals as Rotenone, paraquat, and maneb. Suggested neurotoxicity mechanisms of these chemicals are considered, and the major lessons learned from the analysis of pesticide-induced PD models are discussed.

Rotenone is an mitochondrial electron transport chain complex I inhibitor. Rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production.

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