Mitochondria Are a Major Source of Paraquat-induced Reactive Oxygen Species Production in the Brain

Abstract

Paraquat (PQ2+) is a prototypic toxin known to exert injurious effects through oxidative stress and bears a structural similarity to the Parkinson disease toxicant, 1-methyl-4-pheynlpyridinium. The cellular sources of PQ2+-induced reactive oxygen species (ROS) production, specifically in neuronal tissue, remain to be identified. The goal of this study was to determine the involvement of brain mitochondria in PQ2+-induced ROS production. Highly purified rat brain mitochondria were obtained using a Percoll density gradient method. PQ2+-induced hydrogen peroxide (H2O2) production was measured by fluorometric and polarographic methods. The production of H2O2 was evaluated in the presence of inhibitors and modulators of the mitochondrial respiratory chain. The results presented here suggest that in the rat brain, (a) mitochondria are a principal cellular site of PQ2+-induced H2O2 production, (b) PQ2+-induced H2O2 production requires the presence of respiratory substrates, (c) complex III of the electron transport chain is centrally involved in H2O2 production by PQ2+, and (d) the mechanism by which PQ2+ generates H2O2 depends on the mitochondrial inner transmembrane potential. These observations were further confirmed by measuring PQ2+-induced H2O2 production in primary neuronal cells derived from the midbrain. These findings shed light on the mechanism through which mitochondria may contribute to ROS production by other environmental and endogenous redox cycling agents implicated in Parkinson's disease. Paraquat (PQ2+) is a prototypic toxin known to exert injurious effects through oxidative stress and bears a structural similarity to the Parkinson disease toxicant, 1-methyl-4-pheynlpyridinium. The cellular sources of PQ2+-induced reactive oxygen species (ROS) production, specifically in neuronal tissue, remain to be identified. The goal of this study was to determine the involvement of brain mitochondria in PQ2+-induced ROS production. Highly purified rat brain mitochondria were obtained using a Percoll density gradient method. PQ2+-induced hydrogen peroxide (H2O2) production was measured by fluorometric and polarographic methods. The production of H2O2 was evaluated in the presence of inhibitors and modulators of the mitochondrial respiratory chain. The results presented here suggest that in the rat brain, (a) mitochondria are a principal cellular site of PQ2+-induced H2O2 production, (b) PQ2+-induced H2O2 production requires the presence of respiratory substrates, (c) complex III of the electron transport chain is centrally involved in H2O2 production by PQ2+, and (d) the mechanism by which PQ2+ generates H2O2 depends on the mitochondrial inner transmembrane potential. These observations were further confirmed by measuring PQ2+-induced H2O2 production in primary neuronal cells derived from the midbrain. These findings shed light on the mechanism through which mitochondria may contribute to ROS production by other environmental and endogenous redox cycling agents implicated in Parkinson's disease. Parkinson disease (PD) 2The abbreviations used are: PD, Parkinson disease; ROS, reactive oxygen species; PQ2+, paraquat; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; TTFA, 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; ETC, electron transport chain; SOD, superoxide dismutase; HPLC, high pressure liquid chromatography. is an age-related neurodegenerative disorder believed to originate in part via reactive oxygen species (ROS) overproduction, leading to oxidative stress and mitochondrial dysfunction (1Jenner P. Ann. Neurol. 2003; 53: S26-S36Crossref PubMed Scopus (1760) Google Scholar). The disease is characterized pathologically by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Both environmental and genetic factors are implicated in specific damage to the nigrostriatal system, contributing to the motor dysfunctions characteristic of PD (2Fahn S. Ann. N. Y. Acad. Sci. 2003; 991: 1-14Crossref PubMed Scopus (736) Google Scholar, 3Dauer W. Przedborski S. Neuron. 2003; 39: 889-909Abstract Full Text Full Text PDF PubMed Scopus (4294) Google Scholar). However, the mechanisms by which such factors cause neurodegeneration remain relatively unknown. Neurotoxins represent the most accepted tools used to produce experimental models of PD both in vivo and in vitro. The most commonly used agents include 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 6-hydroxydopamine, rotenone, and paraquat (PQ2+) (4Bove J. Prou D. Perier C. Przedborski S. NeuroRx. 2005; 2: 484-494Crossref PubMed Scopus (592) Google Scholar). PQ2+, a member of the widely used bipyridyl herbicides, is a prototypic compound known to exert its toxic effects via oxidative stress. A number of studies have exploited this action of PQ2+ to test response and sensitivity to oxidative damage in various models (5Bus J.S. Gibson J.E. Environ. Health Perspect. 1984; 55: 37-46Crossref PubMed Google Scholar, 6Sato B. Tanaka A. Mori S. Yanabu N. Kitai T. Tokuka A. Inomoto T. Iwata S. Yamaoka Y. Chance B. Biochim. Biophys. Acta. 1995; 1268: 20-26Crossref PubMed Scopus (24) Google Scholar, 7Bonilla E. Medina-Leendertz S. Villalobos V. Molero L. Bohorquez A. Neurochem. Res. 2006; 31: 1425-1432Crossref PubMed Scopus (72) Google Scholar, 8Arkblad E.L. Tuck S. Pestov N.B. Dmitriev R.I. Kostina M.B. Stenvall J. Tranberg M. Rydstrom J. Free Radic. Biol. Med. 2005; 38: 1518-1525Crossref PubMed Scopus (90) Google Scholar, 9Cicchetti F. Lapointe N. Roberge-Tremblay A. Saint-Pierre M. Jimenez L. Ficke B.W. Gross R.E. Neurobiol. Dis. 2005; 20: 360-371Crossref PubMed Scopus (133) Google Scholar, 10Bishop A.L. Rab F.A. Sumner E.R. Avery S.V. Mol. Microbiol. 2007; 63: 507-520Crossref PubMed Scopus (79) Google Scholar). The possibility of PQ2+ as a neurotoxin has arisen from evidence that significant damage to the brain is observed in individuals exposed to lethal doses of the herbicide (11Grant H. Lantos P.L. Parkinson C. Histopathology. 1980; 4: 185-195Crossref PubMed Scopus (94) Google Scholar, 12Hughes J.T. Neurotoxicology. 1988; 9: 243-248PubMed Google Scholar). Furthermore, epidemiological studies have suggested an increased risk for developing PD following exposure to the herbicide (13Liou H.H. Tsai M.C. Chen C.J. Jeng J.S. Chang Y.C. Chen S.Y. Chen R.C. Neurology. 1997; 48: 1583-1588Crossref PubMed Scopus (584) Google Scholar). This has lead to an increased interest in the neurotoxic actions of PQ2+, along with the idea that PQ2+ and other herbicides may act as environmental triggers for PD (14Di Monte D.A. Lancet Neurol. 2003; 2: 531-538Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Therefore, in recent years, PQ2+ has become an increasingly popular model for studying the etiology of PD, alone and also in combination with other environmental toxins (15McCormack A.L. Thiruchelvam M. Manning-Bog A.B. Thiffault C. Langston J.W. Cory-Slechta D.A. Di Monte D.A. Neurobiol. Dis. 2002; 10: 119-127Crossref PubMed Scopus (652) Google Scholar, 16Thiruchelvam M. Richfield E.K. Baggs R.B. Tank A.W. Cory-Slechta D.A. J. Neurosci. 2000; 20: 9207-9214Crossref PubMed Google Scholar). Systemic exposure of rodents to PQ2+ has been shown to reproduce many of the pathological features of PD, including selective degeneration of dopaminergic neurons in the substantia nigra and presence of intracellular α-synuclein deposits (15McCormack A.L. Thiruchelvam M. Manning-Bog A.B. Thiffault C. Langston J.W. Cory-Slechta D.A. Di Monte D.A. Neurobiol. Dis. 2002; 10: 119-127Crossref PubMed Scopus (652) Google Scholar, 17Manning-Bog A.B. McCormack A.L. Li J. Uversky V.N. Fink A.L. Di Monte D.A. J. Biol. Chem. 2002; 277: 1641-1644Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 18Thiruchelvam M. McCormack A. Richfield E.K. Baggs R.B. Tank A.W. Di Monte D.A. Cory-Slechta D.A. Eur. J. Neurosci. 2003; 18: 589-600Crossref PubMed Scopus (230) Google Scholar, 19Peng J. Mao X.O. Stevenson F.F. Hsu M. Andersen J.K. J. Biol. Chem. 2004; 279: 32626-32632Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Although these studies show that agents such as PQ2+ can produce selective neurodegeneration, the pathophysiological mechanisms underlying this damage remain unknown. Interestingly, PQ2+ exhibits a remarkable structural similarity to MPP+, the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Although both of these compounds are believed to cause toxicity via oxidative stress, PQ2+ exerts its deleterious effects on dopamine neurons in a unique manner compared with MPP+ (20Richardson J.R. Quan Y. Sherer T.B. Greenamyre J.T. Miller G.W. Toxicol. Sci. 2005; 88: 193-201Crossref PubMed Scopus (203) Google Scholar). PQ2+ belongs to a broad class of redox cycling compounds known to produce ROS. The proposed mechanism of redox cycling for PQ2+ involves the enzymatic reduction to form its cationic radical (Fig. 1, reaction 1), which then can reduce molecular oxygen (O2) to superoxide radical (O¯·2) while also regenerating the parent compound (reaction 2). O¯·2 is subsequently converted to H2O2 either spontaneously (reaction 3) or through the actions of superoxide dismutase (reaction 4). The rate-limiting reaction in this process is the enzymatic reduction of PQ2+ depicted in reaction 1. The identities of enzymes capable of transferring electrons to PQ2+ in this reaction are not completely defined. It is widely accepted that PQ2+-induced generation of ROS arises from a number of cellular sources. Enzymes capable of initiating the redox cycling process of PQ2+ have been identified in microsomal, plasma membrane, and cytosolic components through the actions of NADPH oxidase (21Wu X.F. Block M.L. Zhang W. Qin L. Wilson B. Zhang W.Q. Veronesi B. Hong J.S. Antioxid. Redox. Signal. 2005; 7: 654-661Crossref PubMed Scopus (133) Google Scholar, 22Purisai M.G. McCormack A.L. Cumine S. Li J. Isla M.Z. Di Monte D.A. Neurobiol. Dis. 2007; 25: 327-331Crossref Scopus (209) Google Scholar), M. L. Chang J.S. Acad. Sci. S. A. PubMed Scopus Google Scholar), and Environ. Health Perspect. PubMed Scopus Google Scholar). However, the of mitochondria in PQ2+ toxicity is unknown. The of this study was to the that mitochondria in PQ2+-induced ROS production in the This was by measuring of H2O2 production using polarographic and fluorometric methods. mitochondrial components capable of in the redox ROS generation by PQ2+ were of was in with of Health were from using Percoll density gradient Res. 2000; PubMed Scopus Google Scholar). brain was in mitochondrial and then in were for The was as the cytosolic and the was to Percoll gradient on for The the of the was with mitochondrial and for The were in of and for to of was measured by using the of of cytosolic and mitochondrial from Percoll were by on and to were with primary for or oxidase were with and for oxidase were with The were using oxidase was in cytosolic and in mitochondrial was in cytosolic purified cellular not of H2O2 was measured using the fluorometric were to a of reaction and of the following or was used as an to determine the of mitochondrial potential. The reaction was with the of PQ2+ The following inhibitors were used in complex A and inhibitors used in these studies were obtained from was by a dismutase and were as of and of H2O2 production in cellular components was measured using an Free with a H2O2 The were in a with in a reaction of was with the of reaction to the the the following were or and cellular of The was to to by the of PQ2+ to the and the of or of the mitochondrial respiratory chain was of the respiratory chain were the of PQ2+-induced H2O2 by were from to for M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). the of PQ2+-induced H2O2 production, cells were in a density of were obtained from were for for H2O2 production via was for in of a also and PQ2+ and inhibitors were the was using a of PQ2+ brain mitochondria were in reaction in the presence of PQ2+, substrates, and inhibitors of the were and for and was The were with of reaction inhibitors The was in of and by the of and for The of PQ2+ in the was by high liquid using a P. J. Toxicol. 25: PubMed Scopus Google with was on a The of with a of PQ2+-induced ROS the of cellular components of the brain are involved in PQ2+-induced ROS production, brain and mitochondrial were for H2O2 production the of for mitochondrial and cytosolic were to purified mitochondrial of from other cellular components the fluorometric of PQ2+-induced H2O2 production in rat brain and mitochondrial the of PQ2+ in the presence of mitochondria an and production of were in and cytosolic the of the to H2O2 have arisen from the possibility that endogenous may with the Biophys. 2004; PubMed Scopus Google Scholar). Therefore, in to results from the high fluorometric to H2O2 production, a polarographic was also in H2O2 production using this polarographic in cellular following the of of H2O2 production obtained from these the are in 1. are compared with the H2O2 production in mitochondria in the presence of and which is as the of substrates, mitochondria H2O2 a that of brain the the of The of the and electron transport chain the of complex or the of complex not have on of H2O2 production from the cytosolic (Fig. 2). the brain and mitochondrial the of increased production in the of and mitochondrial of H2O2 production with were (Fig. of PQ2+-induced H2O2 production in cellular from were for H2O2 production using a fluorometric following the of H2O2 production in mitochondrial with as was and are as a of this endogenous from cytosolic of by from cytosolic of by from cytosolic of by from cytosolic of by from cytosolic of by from of by from cytosolic of by from of by from cytosolic of by from of by from cytosolic of by from of by from cytosolic of by from of by in a of the in PQ2+-induced H2O2 in that mitochondria are a involved in PQ2+-induced ROS production, mitochondrial components implicated in this process were The generation of ROS via the redox cycling action of PQ2+ requires reduction to its cationic radical as an The of the represent for this act to transport electrons and redox in the for test this H2O2 production in brain mitochondria was in the presence of inhibitors of the of H2O2 production by a fluorometric mitochondria by and in the presence of PQ2+ an and in H2O2 production. on H2O2 production completely this process (Fig. a specific of complex of H2O2 by However, with PQ2+ to observed with A alone (Fig. and 4). the polarographic A of PQ2+-induced H2O2 production was to results obtained via (Fig. the of PQ2+ in the presence of and H2O2 was measured a of the the of H2O2 production to the as The of A to complex III the of PQ2+-induced H2O2 production was as a a to a H2O2 production and in the an of complex not the of H2O2 generation in the presence of and as of inhibitors on PQ2+-induced H2O2 production in were with (a) or and H2O2 production was measured in the or presence of were as H2O2 production in mitochondrial with as in the presence of PQ2+ was and are as a of this compared with mitochondria of A was subsequently to determine effects on PQ2+-induced H2O2 production in the presence of inhibitors of of the H2O2 production are in in the presence of and (Fig. or (Fig. These act to electrons the the of complex via or to complex of and III the of PQ2+ a significant of H2O2 this production was of the following PQ2+ in in of H2O2 production were observed with both complex and III by and This was to an that is with that obtained in the of PQ2+ (Fig. 4). also H2O2 generation in mitochondria electrons via to a A or shown in on PQ2+-induced H2O2 production, was to completely this of on PQ2+-induced H2O2 the transport of electrons through the is by of from the to the an gradient that the inner mitochondrial transmembrane ROS production is by the of 2005; PubMed Scopus Google Scholar). the of in PQ2+-induced ROS production, the of a mitochondrial FCCP, was the presence of and (Fig. or (Fig. to mitochondrial was to completely production of H2O2 by of of the components in the mitochondrial inner and either the of the membrane, the or the Therefore, for PQ2+ to with and ROS, be capable of the mitochondrial and the and The of PQ2+ to this was in the following were with PQ2+ experimental and PQ2+ was measured via with as in the of PQ2+ was mitochondria a of compared with the in the 2). to of PQ2+ measured by mitochondria increased of PQ2+, not from the of A or on mitochondrial PQ2+ that these compounds not PQ2+ by rat brain mitochondria mitochondria were in PQ2+ in the presence of and and PQ2+ were measured via are presented as PQ2+ PQ2+ PQ2+ PQ2+ PQ2+ in a of PQ2+-induced H2O2 in mitochondrial mechanisms of H2O2 production are involved in a model of primary obtained from were H2O2 production was via the fluorometric to of polarographic in of with PQ2+ in the presence of complex H2O2 production was measured and as of (Fig. this in H2O2 production was completely in the presence of A compared with also PQ2+-induced H2O2 production to a A. observed in mitochondrial PQ2+-induced H2O2 production. findings from the study the mechanism of PQ2+-induced ROS production in brain mitochondria are a cellular involved in ROS generation by PQ2+ in the rat PQ2+-induced H2O2 production by brain mitochondria is on a electron by and a the of specific inhibitors of the ETC, complex III has been identified as a site of action for PQ2+ in the process of redox cycling to ROS. the mechanism of PQ2+-induced ROS production is on mitochondrial potential. a in PQ2+-induced ROS mechanism of ROS generation by redox cycling of PQ2+ has been widely in both in vivo and in These studies the involvement of mitochondrial T. T. A. N. Y. J. Neurosci. Res. 1995; PubMed Scopus Google Scholar), B. J.R. Toxicol. PubMed Scopus Google Scholar, Toxicol. PubMed Scopus Google Scholar), and cytosolic M. L. Chang J.S. Acad. Sci. S. A. PubMed Scopus Google Scholar, Biophys. 1984; PubMed Scopus Google enzymes in ROS production by studies have suggested the involvement of mitochondria in PQ2+-induced O¯·2 the was suggested that the mitochondrial be a site for PQ2+ reduction M. J. D. T. Microbiol. Google Scholar). in the of completely PQ2+-induced generation of ROS S. S. Y. V. Med. 2002; PubMed Scopus Google Scholar). However, the of mitochondria in has relatively this that the of the rat brain, mitochondria are implicated in PQ2+-induced ROS the of substrates, PQ2+-induced H2O2 production was in the mitochondrial by the and the This can be by the that mitochondria in the following Therefore, mitochondria are to this in Health and 2005; Google Scholar). The brain also which is this H2O2 production in the of This also an in H2O2 production is observed in the following the of The of PQ2+-induced H2O2 in the mitochondria compared with other These suggest that PQ2+-induced H2O2 production is on that mitochondrial The transport of PQ2+ the brain and the mitochondria an its toxic actions as in this It has been that PQ2+ can be the brain by in the and subsequently cells in a manner T. M. N. H. Res. PubMed Scopus Google Scholar). It is that mitochondria may transport for this evidence that mitochondria are to transport PQ2+ both and A and on this a transport of or transmembrane potential. The of and as in this study the of PQ2+-induced ROS production in PQ2+-induced ROS production the the of H2O2 production to the of O¯·2 to H2O2 other mechanisms the oxygen However, to PQ2+-induced ROS production. This that PQ2+ ROS in the which is then converted to H2O2 by mitochondrial such as by H2O2 is then capable of through the mitochondrial the can be by or measured via polarographic and shown of the and of III in PQ2+-induced H2O2 this that are for mitochondria to significant of H2O2 in the presence of the of complex and III in mitochondria by and completely this These suggest that in mitochondria with a ETC, electrons can be to PQ2+ the of these initiating the redox cycling reaction and ROS production. However, in primary complex III by A was to H2O2 production, was observed with complex by This may be as the mitochondria used in this study are in a that mitochondria to a high potential. such in the in a to electron and electron transport that may lead in part to the observed generation of ROS. These electrons may also the of PQ2+ to the redox cycling process involved in H2O2 production. However, in the model of primary mitochondria are a of electron and electron transport are not 2004; PubMed Scopus Google Scholar). Therefore, that may act to reduce PQ2+ via such mechanisms in mitochondria may not be involved in this for the and III involves the by the of and of the is also known as an involved in the mitochondrial mitochondria with and or the of both actions of the and may completely electron the ETC, other are to this such an electrons be to reduce PQ2+, and ROS subsequently be as was observed in this in primary is as the the presence of may also be as a of and capable of the or is to electrons to in the ETC, of and Therefore, in cells is to complex and part of the may a significant in to this in the and of the an for complex not PQ2+-induced H2O2 production in primary The obtained from this in model a for complex III of the in the PQ2+ mechanism of toxic of in PQ2+-induced ROS gradient the mitochondrial and a transmembrane of The B. Google used in this study a high and high of reduction of the redox in the of the on the of a mitochondrial FCCP, results that an is for PQ2+-induced H2O2 production. a high reduction of complex III may be a in the reduction of PQ2+ to its cationic radical capable of redox The of the with the of the of as a the to electrons to PQ2+ and lead to the generation of ROS. in PQ2+-induced ROS of PQ2+-induced ROS generation on the presence of in mitochondria the involvement of the The and or and are to in the and subsequently electrons the the of complex is an in the and is to electrons the of complex The electrons from either of these then the and electrons to complex It is this in the of electrons through complex III that electrons are to be to PQ2+, initiating the process of redox This process is to the production of O¯·2 from complex III that has been characterized L. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A for complex III was by Chen S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google in H2O2 production in mitochondria using for complex and complex and a of on can the following PQ2+-induced H2O2 production in (a) The that in PQ2+-induced H2O2 production in mitochondria that complex may not be the site of PQ2+-induced H2O2 generation in mitochondria complex (b) III a reduction of PQ2+-induced H2O2 production in the presence of complex or substrates, that the site of ROS production is in the respiratory most complex (c) may be to the of of on PQ2+-induced H2O2 production. (d) of PQ2+-induced H2O2 production by complex inhibitors in the presence of complex or may be to reduction of PQ2+ by the complex or by electrons to complex However, results using cells suggest that complex has a compared with complex III as a primary site for H2O2 production by in brain It is to the mechanism for PQ2+ reduction and ROS production the of the complex that A can the electron and and from to A. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), a involvement of these in PQ2+ reduction may be these have is to the site of H2O2 generation PQ2+ However, studies show that a significant of H2O2 by PQ2+ is from which that following reduction of PQ2+ that H2O2 in a site the mitochondria to Biochim. Biophys. Acta. 1995; PubMed Scopus Google and C. A. 2007; PubMed Scopus Google suggest that high of PQ2+ may exert oxidative damage leading to an of This may be to oxidative damage and of the as a of to the site of mitochondrial ROS production by PQ2+, complex This mechanism is by the that mitochondrial are to by a of Y. C. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). It has also been that and of NADPH oxidase a significant in PQ2+-induced dopaminergic degeneration F. Lapointe N. Roberge-Tremblay A. Saint-Pierre M. Jimenez L. Ficke B.W. Gross R.E. Neurobiol. Dis. 2005; 20: 360-371Crossref PubMed Scopus (133) Google Scholar, 22Purisai M.G. McCormack A.L. Cumine S. Li J. Isla M.Z. Di Monte D.A. Neurobiol. Dis. 2007; 25: 327-331Crossref Scopus (209) Google Scholar). These studies suggest that NADPH oxidase may be with However, the of NADPH oxidase is a response following PQ2+ in results a and production of H2O2 by mitochondria exposure to that mitochondrial H2O2 production by PQ2+ in the brain is an that may other cellular such as and NADPH oxidase (Fig. these results the and of PQ2+-induced mitochondrial ROS production. PQ2+ is an increasingly popular model of and cellular via the production of ROS (5Bus J.S. Gibson J.E. Environ. Health Perspect. 1984; 55: 37-46Crossref PubMed Google Scholar, 6Sato B. Tanaka A. Mori S. Yanabu N. Kitai T. Tokuka A. Inomoto T. Iwata S. Yamaoka Y. Chance B. Biochim. Biophys. Acta. 1995; 1268: 20-26Crossref PubMed Scopus (24) Google Scholar, 7Bonilla E. Medina-Leendertz S. Villalobos V. Molero L. Bohorquez A. Neurochem. Res. 2006; 31: 1425-1432Crossref PubMed Scopus (72) Google Scholar, 8Arkblad E.L. Tuck S. Pestov N.B. Dmitriev R.I. Kostina M.B. Stenvall J. Tranberg M. Rydstrom J. Free Radic. Biol. Med. 2005; 38: 1518-1525Crossref PubMed Scopus (90) Google Scholar, 9Cicchetti F. Lapointe N. Roberge-Tremblay A. Saint-Pierre M. Jimenez L. Ficke B.W. Gross R.E. Neurobiol. Dis. 2005; 20: 360-371Crossref PubMed Scopus (133) Google Scholar, 10Bishop A.L. Rab F.A. Sumner E.R. Avery S.V. Mol. Microbiol. 2007; 63: 507-520Crossref PubMed Scopus (79) Google Scholar). These findings may mechanisms of the actions of PQ2+ in Furthermore, lead to the that mechanisms of mitochondrial ROS generation may be by other both environmental and that structural and to