The International Journal of Biochemistry & Cell Biology 40 (2008) 1792–1805

Available online at www.sciencedirect.com

Oxidative stress caused by blocking of mitochondrial Complex IH+ pumping as a link in aging/disease vicious cycle

Andrea Dlaskova 1, Lydie Hlavata 1, Petr Jezek ∗Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic,

Vıdenska 1083, Prague 14220, Czech Republic

Received 12 December 2007; received in revised form 14 January 2008; accepted 14 January 2008Available online 19 January 2008

Abstract

Vulnerability of mitochondrial Complex I to oxidative stress determines an organism’s lifespan, pace of aging, susceptibilityto numerous diseases originating from oxidative stress and certain mitopathies. The mechanisms involved, however, are largelyunknown. We used confocal microscopy and fluorescent probe MitoSOX to monitor superoxide production due to retarded forwardelectron transport in HEPG2 cell mitochondrial Complex I in situ. Matrix-released superoxide production, the un-dismuted surplus(Jm) was low in glucose-cultivated cells, where an uncoupler (FCCP) reduced it to half. Rotenone caused a 5-fold Jm increase(AC50 2 �M), which was attenuated by uncoupling, membrane potential (�Ψm), and �pH-collapse, since addition of FCCP (IC50

55 nM), valinomycin, and nigericin prevented this increase. Jm doubled after cultivation with galactose/glutamine (i.e. at obligatoryoxidative phosphorylation). A hydrophobic amiloride that acts on the ND5 subunit and inhibits Complex I H+ pumping enhancedJm and even countered the FCCP effect (AC50 0.3 �M). Consequently, we have revealed a new principle predicting that Complex Iproduces maximum superoxide only when both electron transport and H+ pumping are retarded. H+ pumping may be attenuated byhigh protonmotive force or inhibited by oxidative stress-related mutations of ND5 (ND2, ND4) subunit. We predict that in a vicious

cycle, when oxidative stress leads to higher fraction of, e.g. mutated ND5 subunits, it will be accelerated more and more. Thus,inhibition of Complex I H+ pumping, which leads to oxidative stress, appears to be a missing link in the theory of mitochondrialaging and in the etiology of diseases related to oxidative stress.© 2008 Elsevier Ltd. All rights reserved.

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Keywords: In situ mitochondrial superoxide production; MitoSOX; ProAging

1. Introduction

Complex I (H+-pumping NADH:quinone oxidore-ductase) is an essential component of the mitochondrialrespiratory chain (Brandt, 2006; Lenaz, Baracca, Fato,

∗ Corresponding author. Fax: +420 296442488.E-mail addresses: dlaskova@biomed.cas.cz (A. Dlaskova),

hlavata@biomed.cas.cz (L. Hlavata), jezek@biomed.cas.cz (P. Jezek).1 These authors contributed equally to the work.

1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.biocel.2008.01.012

ping NADH:quinone oxidoreductase; HEPG2 cells; Oxidative stress;

Genova, & Solaini, 2006; Lenaz, Fato, Formiggini,& Genova, 2007; Yagi & Matsuno-Yagi, 2003), par-ticipating not only in cell respiration, but also incellular/organismal reactive oxygen species homeostasis(Brand et al., 2004; Jezek & Hlavata, 2005), apopto-sis initiation or modulation (Ott, Gogvadze Orrenius,& Zhivotovsky, 2007), and O sensing (Piruat &

2Lopez-Barneo, 2005). This huge 46 subunit mammaliancomplex is vulnerable to oxidative stress, hence it isone of the factors that determines lifespan, pace ofaging, susceptibility to oxidative stress-related diseases,

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A. Dlaskova et al. / The International Journal

nd certain mitopathies (Bai et al., 2004; Bourges etl., 2004; Chomyn & Attardi, 2003; Smigrodzki &han, 2005). This is so because Complex I com-rises 7 of 13 proteins encoded by the mitochondrialenome (membrane arm subunits ND1-6, and ND4L,mong which ND2, ND4, and ND5 act in H+ pump-ng; Gemperli, Schaffitzel, Jakob, & Steuber, 2007;akamaru-Ogiso, Boo Seo, Yagi, & Matsuno-Yagi,003; Nakamaru-Ogiso, Sakamoto, Matsuno- Yagi,yioshi, & Yagi, 2003). Thus, mtDNA mutations in

egions encoding Complex I subunits, induced by con-inuous inevitable mitochondrial superoxide (O2

•−)roduction, might initiate a vicious cycle due to fur-her enhanced O2

•− production brought on by theow impaired function of Complex I or other respi-atory machinery elements (Muller, Lustgarten, Jang,ichardson, & Van Remmen, 2007). The progres-

ive increase in mitochondria-derived oxidative stressay also contribute to numerous diseases includ-

ng atherosclerosis, hypertension, ischemia-reperfusionnjury, inflammation, cystic fibrosis, cancer, type-2 dia-etes, and neurodegenerative diseases (e.g. multipleclerosis, Parkinson’s and Alzheimer’s disease) (Brandt al., 2004; Jezek & Hlavata, 2005). Despite the sugges-ion that mitochondria-derived oxidative stress impactsany disease states and aging, it is not understood whyomplex I-related mtDNA mutations lead to oxidative

tress. Notably, we lack a high-resolution structure ofomplex I, a detailed mechanism of O2

•− productionithin the Complex I, and a detailed pathway of elec-

ron transport and H+ pumping (Brandt, 2006; Brand etl., 2004; Grivennikova & Vinogradov, 2006; Ohnishi &alerno, 2005).

Scant direct evidence exists for the dependence ofitochondrial O2

•− formation in intact cells on the mito-hondrial inner membrane potential (�Ψm) or on thentire protonmotive force (�p =�Ψm +�pH, in mV)Jezek & Hlavata, 2005). There are no indications ofttenuation of in situ mitochondrial O2

•− productiony uncoupling or by other means of �p modulationespite clear demonstrations of such phenomenon in iso-ated mitochondria (Brand et al., 2004; Jezek & Hlavata,005). Rather, when uncouplers like carbonyl cyanide p-trifluoro-methoxy)phenylhydrazone (FCCP) are addedo culture cells, remodeling (De Vos, Allan, Grierson,

Sheetz, 2005) or fragmentation/fission of mitochon-rial network (Benard et al., 2007; Duvezin-Caubet et al.,006; Ishikara, Fujita, Oka, & Mihara, 2006; Lyamzaev

t al., 2004; Pletjushkina et al., 2006), apoptosis (Aronist al., 2003; Dispersyn, Nuydens, Connors, Borgers, &eerts, 1999), or changes in gene expression (Desquiret

t al., 2006; Kuruvilla et al., 2003) are observed.

emistry & Cell Biology 40 (2008) 1792–1805 1793

It is still un-clear whether Complex I derived O2•−

generation is sensitive to�Ψm. Complex I derived O2•−

production due to both forward (Lambert & Brand,2004a) and reverse electron transport diminishes withdecreasing �pH in isolated skeletal muscle mitochon-dria (Lambert & Brand, 2004a, 2004b). The reverseelectron transport is inhibited by rotenone. ComplexIII (ubiquinol-cytochrome c oxidoreductase) can alsoproduce O2

•− as shown in vitro in isolated glutathione-depleted nonphosphorylating rat heart mitochondriarespiring with succinate at state 4. These mitochon-dria exhibited a 55% decrease in H2O2 formation when�Ψm decreased by only 10% (Korshunov, Skulachev,& Starkov, 1997). About 30–43% (Starkov & Fiskum,2003; Starkov et al., 2004) of the levels of state 4H2O2 generation was maintained in the phosphorylat-ing state, i.e. state 3. Thus, H+ backflux to the matrix,ensured either by ATP synthase or by uncoupling, atten-uates O2

•− production. Still, the relative contributions ofComplexes I and III to overall O2

•− formation in mito-chondria is not known (Jezek & Hlavata, 2005; Muller,Liu, & Van Remmen, 2004). Nevertheless, almost 100%of Complex I O2

•− production is released to the matrix(Brand et al., 2004; Muller et al., 2004).

In this work, we used confocal microscopy with themitochondrial superoxide indicator MitoSOX Red toassess excessive matrix O2

•− release in situ. We foundthat Complex I O2

•− production is enhanced only andexclusively when both electron transport and H+ pump-ing are retarded. Since inhibition of Complex I H+

pumping usually results from oxidative stress-inducedmutations in mtDNA encoding subunits ND2, ND4, andND5, the fact that this inhibition produces further oxida-tive stress represents a missing link in a vicious cycle ofaging or oxidative stress-related diseases.

2. Materials and methods

2.1. Cell cultivation

The human hepatocellular carcinoma cell lineHEPG2 (ECACC 85011430) was cultivated at 37 ◦Cin humidified air with 5% CO2 in DMEM (Gibco, cat.no. 11995-065; contains no glucose) supplemented with3 mM glutamine, 5% FCS (Biochrome, cat. no. S0113),10 mM HEPES, 100 IU/ml penicillin, and 100 �g/mlstreptomycin. The added carbon source was either25 mM glucose or 10 mM galactose (with glucose-free

dialyzed FCS, PAA, cat. no. A15-107). The latter isreferred to as oxphos conditions (cells), because cellsrely largely on oxidative phosphorylation (Rossignol etal., 2004). The doubling time for glc and oxphos cells was

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1794 A. Dlaskova et al. / The International Journal

on average 23.5 and 39.4 h, respectively. A stable cellline HEPG2-mRoGFP was selected by Geneticin aftertransfection with a pcDNA vector encoding mRoGFP(obtained from Dr. Rossignol, University of Bordeaux2, France). For confocal microscopy, HEPG2 cells werecultured for 2 days on glass coverslips coated with poly-l-lysine.

2.2. Confocal microscopy

The confocal inverted fluorescent microscope LeicaSP2 AOBS DM IRE2 HC Fluo TCS 1-B (an objective PLAPO 100 × /1.40–0.70 oil) was used with an Argon laser(488 nm/20 mW, 514 nm/20 mW) for excitation of cellsin a thermostable sample chamber set to 37 ◦C, suppliedfrom a CO2 incubator, to mimic cultivation conditions. Apinhole 1 Airy unit was used to set confocal conditions.

2.3. Semiquantitative analysis of Jm using confocalmicroscopy

Loading/incubation of cells with 4.1 �M MitoSOXRed for 15 min at 37 ◦C was done exactly the sameway for each experiment and all reagents were addedthereafter. Excitation was at 514 nm and emission wasdetected between 580 (or 610) and 679 nm. A series ofimages was taken after the addition of a reagent to thecells, typically each 30 s for the next 20 min. Regions ofthe interest corresponding to mitochondria of approxi-mately 15–20 cells per slide were selected using Ellipsesoftware (ViDiTo, Kosice, Slovakia) so that nearly anequal pixel number was analyzed in each image. Changesin integrated fluorescence intensity at these loci werequantified from plots of fluorescence in selected area vs.time. Slopes were determined by linear regressions.

2.4. Response of MitoSOX Red spectra tosuperoxide and/or DNA in vitro and in situ

The fluorescent probe MitoSOX Red, a triphenyl-phosphonium-(TPP)-conjugated dihydroethidine,should indicate O2

•−, in addition to other reactiveoxygen species. In a cell-free system, where O2

•−was generated by xanthine/xanthine oxidase for 30 s, asubstantial increase in both ∼610 and ∼663 nm peaksof MitoSOX emission spectra was observed at 500 nmexcitation (Fig. 1a vs. b), as recorded on a Fluorolog322 (Spex–Jobin–Yvon–Horiba) fluorometer with

double grating monochromators for both excitation andemission. Addition of double-stranded herring DNAincreased fluorescence up to eightfold and the emissionmaximum shifted slightly to 593 nm (a shoulder shifted

emistry & Cell Biology 40 (2008) 1792–1805

to ∼655 nm, Fig. 1a). Both the MitoSOX Red free andDNA-intercalated forms were sensitive to O2

•− as indi-cated by the lack of high emission increase when SODwas present (Fig. 1a). Higher signal intensities withSOD (Fig. 1a) than in the absence of enzyme system(Fig. 1b) reflect its optical interference. Excitation spec-tra of MitoSOX Red without DNA and independently ofthe presence of O2

•− exhibited a maximum at 468 nmwith a small peak at 378 nm, whereas in the presenceof DNA two equally obvious peaks at 510 and 390 nmwere observed (data not shown).

In situ, MitoSOX Red should detect surplus mito-chondrial O2

•− generation released to the matrix (Jm),that is to say, that portion that is not consumed by theMn-superoxide dismutase (MnSOD). O2

•− reacts withthe dihydroethidine moiety, forming a highly fluorescentcomplex of 2-OH-dihydroethidine, whereas ethidium,as a positively charged entity, can arise from spon-taneous oxidation (Georgiou, Papapostolou, Patsoukis,Tsegenidis, & Sideris, 2005; Mukhopadhyay, Rajesh,Yoshihiro, Haslo, & Pachem, 2007; Robinson et al.,2006; Zhao et al., 2003, 2005; Zielonka, Zhao, Xu,& Kalyanaraman, 2005). The positively charged TPPmoiety of MitoSOX Red enables its high accumula-tion in the mitochondrial matrix. Its ability to intercalateinto mtDNA (Fig. 2) allows for a sufficient portion ofMitoSOX Red to be retained in the matrix even in theabsence of �Ψm. Due to the existing order of magni-tude of Jm (pmol O2

•− �l−1 min−1), this “dark” portionis not consumed during the typical 20-min experimentand can still react with O2

•−. Hence, the probe indicatesJm for many more minutes even after the�Ψm collapse(see Predicted MitoSOX Red response). Note, that onceit has interacted with O2

•− and becomes fluorescent, theprobe is not quenched. Therefore, only an increase in flu-orescence with time is expected when unreacted probe isavailable. The probe reacting with O2

•− during the load-ing procedure establishes a certain fluorescence levelenabling confocal imaging (plus a small background ofnon-specifically oxidized probe, e.g. due to incident laserlight); moreover as a significant portion of it intercalatesinto mtDNA, it is not expulsed from the matrix upon col-lapse of �p (�Ψm). Indeed, we did not observe a dropin fluorescence upon FCCP addition.

2.5. Predicted MitoSOX Red response

Consider the external medium compartment, hav-

ing a volume Vext, layered on a coverslip with cellsof the overall total volume Vcell, equal to the volumeof cytoplasm Vcyt plus the volume of mitochondrialmatrix Vm (the volume of the intermembrane space is

A. Dlaskova et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 1792–1805 1795

Fig. 1. Emission spectra of MitoSOX Red. (a) The emission spectra of MitoSOX Red were measured in the presence or absence of superoxide.Emission spectra (2 nm slit width) at excitation at 500 nm (10 nm slit width) of MitoSOX Red (7 �M) in PBS either with (upper spectra) orwithout (lower spectra) dsDNA (43 �g/ml isolated from herring) were recorded after 30 s incubation with xanthine oxidase (0.025 U, giving 25 nmolsuperoxide per min; total 12.5 nmol) and 286 �M xanthine (solid lines) or with 0.028 U of CuZnSOD (dotted lines, absence of superoxide). (b) Thee idase anD was sub∼ A.

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wrfmimp2

mission spectra of MitoSOX Red were measured without xanthine oxNA (upper spectra) were present. Background buffer light scattering663 nm) without DNA and at 593 nm (shoulders ∼655 nm) with DN

eglected). In our case Vext was 1000 or 2000 �l, Vcellas 0.24 �l (for 200,000 HEPG2 cells and assuming an

verage cell volume of 1.2 pl, see Wehner, Lawonn, &inel, 2002), and Vm is 0.024 or 0.12 �l, if one assumesitochondria represent 10 or 50% of cell volume, respec-

ively. Cells have plasma membrane potential �Ψpith a typical magnitude of 60 mV and a �Ψm typi-

ally 120–180 mV. We neglect ethidium formation ando we assume that MitoSOX Red is bearing a singleositive charge on the TPP moiety. Under these con-itions, redistribution of MitoSOX Red between thexternal medium compartment and the matrix will beiven by Eq. (1), derived by Rafael and Nicholls (1984),hile neglecting concentration of MitoSOX in the

ytosol:

ψp +�ψm = (2.303RT/F ) log{AVcytcm/Vmcext}(1)

here R and F are the gas and Faraday constants,espectively, T the absolute temperature, A accountsor the difference in activities in different compart-ents, and c stands for the MitoSOX concentration

n the matrix, cm = nm/Vm, or the external compart-ent, cext = next/Vext, where n is the probe amounts in

icomoles. Using a �Ψp of 60 mV, �Ψm of 180 mV,.303RT/F as 60, and Vcyt/Vm equal to 10 or 2, and A

d xanthine. Only MitoSOX Red (lower spectra) or MitoSOX Red andtracted from all spectra. Emission maxima were at 610 nm (shoulders

equal to 1, then

log{10Vextnm/Vmnext} = 4 (2)

Vextnm/Vmnext = 1000 (3)

nm + next = ntotal (4)

Considering mitochondria as only 10% of the cellvolume (Vm of 0.024 �l) and at a ctotal of 4.1 �M and aVext of 2000 �l, one can calculate nm to be 97 pmol andcm to be 4.04 mM; considering the mitochondria as 50%of the cell volume (Vm of 0.120 �l), then nm would be464 pmol and cm 3.87 mM.

The calculated amount of cm and nm is impor-tant with respect to possible order of magnitude ofmitochondrial O2

•− production. Our quantification ofH2O2 release in isolated rat liver mitochondria gave60 pmol min−1 mg mitochondrial protein−1. If SOD dis-muted 90% of this production, 6 pmol min−1 mg−1

would remain. Assuming 1 mg mitochondrial proteinper 1 �l of Vm matrix volume, we estimated O2

•−production of 0.144 and 0.72 pmol O2

•− min−1 for Vm0.024 and 0.12 �l, respectively. Consequently, it wouldtake 675 and 644 min, respectively, for that amount of

MitoSOX Red residing in the matrix to be convertedby O2

•− into the fluorescent form. When one considersthat in the presence of FCCP or other agents collapsing�Ψm, there would be a three orders of magnitude lower

1796 A. Dlaskova et al. / The International Journal of Bioch

Fig. 2. Localization of MitoSOX in HEPG2 cells. (a–c) Confocalimages of MitoSOX Red in glc HEPG2 cells (a, red) expressingmRoGFP (b, green) and magnified merged images (c); staining for15 min with 4.1 �M MitoSOX. Excitation for MitoSOX was set at514 nm and for mRoGFP at 488 nm. The contrast of the original Tiffimages was enhanced by 30% and of the merged image by 70%. (d–f)Confocal images of MitoSOX Red (d, red) and DNA stain SYTO (e,green) in glc HEPG2 cells and the magnified merged image (f). HEPG2cells were stained with 0.25 �M SYTO for 10 s and subsequently with4.1 �M MitoSOX for 15 min; excitation for SYTO was set at 488 nm.The contrast of the merge Tiff image was enhanced by 40%.

emistry & Cell Biology 40 (2008) 1792–1805

amount of MitoSOX Red in the matrix, one would expecta problem in the inability of such a low probe amount toindicate O2

•− for a sufficiently long time. This obstacleis compensated for by the fact that a significant por-tion of MitoSOX Red is intercalated into mtDNA. With10% of intercalated probe in mtDNA, 9.7 and 46.4 pmolstill remains after the FCCP addition, allowing for thepossibility of observation for the first 67 and 64 min,respectively. With a higher fraction of probe intercalatedor a lower excessive O2

•− release rates, higher observa-tion times would be possible. For high intercalation, theintensity scale for MitoSOX emission is similar in boththe case of a high �Ψm and zero �Ψm.

2.6. Cell in situ �Ψm monitoring

In situ �Ψm was monitored with tetramethylrho-damine ethyl ester (TMRE, Molecular Probes) usingconfocal microscopy. Cells were stained with 10 nMTMRE and excited at 514 nm in time-lapse sequences.Emission was detected between 580 and 650 nm.

2.7. Total cellular SOD, CuZnSOD, and MnSODactivities

Cellular activities of SOD, CuZnSOD, and MnSODwere measured using an SOD assay activity kit (Cay-man) according to the manufacturer’s instructions. Theassay utilizes a tetrazolium salt to detect O2

•− generatedby xanthine oxidase and hypoxanthine. Addition of KCNto the assay inhibits CuZnSOD, resulting in detection ofonly MnSOD activity.

2.8. Quantification of electron transport complexes

The amount of electron transport complexes wasquantified either by running blue native PAGE gels(4–13% gradient separation gel), enabling separation ofdigitonin-solubilized mitochondrial complexes (Wittig,Braun, & Schagger, 2006) or by immunoblotting usingthe Human Total OXPHOS Complexes Detection kit(MitoScience) after separating mitochondrial proteinsby SDS-PAGE. Band quantification was performed bydensitometry using the Sciom Image software beta 4.02Win 1.

3. Results

3.1. Subcellular localization of MitoSOX Red

To verify that the O2•−-sensitive probe MitoSOX

Red localizes to the mitochondria, we have incubated

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A. Dlaskova et al. / The International Journal

uman hepatocellular carcinoma (HEPG2) cells express-ng matrix-targeted redox-sensitive GFP (mRoGFP)ith MitoSOX. Fig. 2a–c shows high, about 99.8%,

o-localization of MitoSOX and mRoGFP signalsmRoGFP taken as 100%) in the confocal plane. Welso co-incubated HEPG2 cells with MitoSOX Rednd SYTO, a stain for both nuclear DNA and mtDNAFig. 2d–f). SYTO only partially (82–87%) co-localizedith the more spatially distributed MitoSOX Red. IfYTO exclusively stained mtDNA, ∼80% emission

ntensity of MitoSOX within the mitochondrial SYTO-ositive region would imply that free MitoSOX was20%, because intercalated MitoSOX has a higher quan-

um yield (Fig. 1). Thus MitoSOX was either intercalatednto mtDNA or free in the matrix aqueous compartment.

.2. HEPG2 cells cultivated with glucose do notndergo significant oxidative stress

We measured in situ rates of surplus O2•− release (i.e.

2•− unconsumed by Mn-superoxide dismutase) into

he mitochondrial matrix (Jm). In HEPG2 cells cultivatedith 25 mM glucose (glc cells) the MitoSOX emission

ignal increased only slightly (Figs. 3, 4, and 5a, noddition), indicating no significant surplus O2

•− pro-uction. This suggests that MnSOD can dismute theajority of O2

•− produced by the respiratory chainnd released to the matrix. The already slow Jm ratesf the glc cells were further decreased by ∼50% uponddition of an uncoupler (FCCP) after probe loadingFigs. 3, 4, and 5a). Oligomycin activated Jm morehan fourfold, while establishing the nonphosphorylatingtate 4.

.3. Uncoupling attenuates rotenone-induced Jm initu

Addition of 20 �M rotenone to glc cells afterhe probe equilibration markedly enhanced JmFigs. 3, 4, and 5a) without inducing cell death. Twentyicromoles of rotenone increased Jm on average

.5-fold in glc cells with an AC50 of 2 �M (Fig. 5a),roviding excessive oxidative stress. IC50s for inhibitionf normal (state 3) cell respiration by rotenone rangedrom 34 to 65 nM (but was ∼3 �M with FCCP, Table 1).bout 13% (20–30% with FCCP) of state 3 respiration

emained at rotenone >1 �M. This reflects the facthat rotenone does not prevent succinate-supported

espiration in situ. TMRE indicated that �Ψm was keptather high with rotenone (data not shown).

When 1 �M FCCP was added prior to rotenone butfter MitoSOX Red loading, the rotenone-induced Jm

emistry & Cell Biology 40 (2008) 1792–1805 1797

increase was substantially prevented. It was reduced onaverage 4.5-fold, frequently down to the basal Jm ofintact glc cells (Figs. 3, 4, and 5a). An apparent IC50was 55 nM, consistent with AC50s (Table 1) for acti-vation of glc cell respiration without rotenone. Due tosufficient MitoSOX intercalation into mtDNA, the �pcollapse by FCCP did not completely deplete the unre-acted probe from the matrix, hence MitoSOX responsesto matrix O2

•− were apparent. This was further sup-ported by an experiment where H2O2 was added afterFCCP, restoring a substantial increase in fluorescence(Fig. 4). Although caused by hydroperoxyl radical, therestoration clearly indicates that sufficient amount ofunreacted probe existed in the matrix to be oxidized evenafter �p collapse.

3.4. Conversion of �pH into �Ψm decreasesrotenone-induced Jm in situ

Nigericin is a K+/H+ antiporter with a strict 1:1 sto-ichiometry that converts the whole �pH component of�p into�Ψm while increasing�Ψm. Addition of 10 nMnigericin to glc cells did not affect magnitudes of Jm(Fig. 5a). Nigericin also prevented the high Jm valuesinduced by rotenone (Fig. 5a), but glc cell respirationwas not significantly affected by up to 1 �M nigericin(Table 1).

3.5. �Ψm collapse decreases rotenone-induced Jm

in situ

Valinomycin-mediated K+ uniport short-circuits�Ψm and converts whole �p into �pH. Valinomycin(10 nM) abolished high rotenone-induced Jm in glc cellsbut also slightly increased basal Jm (Fig. 5a). Glc cellrespiration was activated up to 3.8-fold (1.4-fold withrotenone) with AC50s 3–6 nM, but was inhibited by>1 �M valinomycin, which caused MitoSOX releaseinto the cytoplasm and apoptosis and/or necrosis.

3.6. The Jm pattern in HEPG2 cells relying onoxidative phosphorylation

When HEPG2 cells were cultivated in galac-tose/glutamine and had to rely on oxidative phospho-rylation, oxphos cells (Rossignol et al., 2004), the Jmpattern was identical to that of glc cells under all con-ditions tested, with the exception of FCCP treatment

(Fig. 5a and Table 1). Except for the rotenone, the basalJm values of oxphos cells were on average 1.8-fold highercompared with glc cells, corresponding to their higher�Ψm (Fig. 5b). Addition of 20 �M rotenone led to an

1798 A. Dlaskova et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 1792–1805

Fig. 3. First and last confocal images in a series accessing the rates of fluorescence increases of MitoSOX Red. HEPG2 glc cells were stained with4.1 �M MitoSOX for 15 min and images were taken every 30 s (the first images are displayed in the left panels) up to 20 min (the last images aredisplayed in the right panels). Additions were as follows: no addition (a and b); 20 �M rotenone (c and d); 1 �M FCCP (e and f); 20 �M rotenoneplus 1 �M FCCP (g and h).

A. Dlaskova et al. / The International Journal of Bioch

Fig. 4. Derivation of Jm in mitochondria in situ. HEPG2 glc cells werestained with 4.1 �M MitoSOX Red for 15 min. Images were takenevery 30 s (see Fig. 8) and were processed to produce derived tracesfor the integral intensity of MitoSOX Red fluorescence within mito-coow

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arhhiofl(

FsJ(F((

hondrial matrix vs. time. Jm values were determined from the slopesf the traces. Arrow, 500 �M H2O2 was added during the time coursef glc cells with FCCP to show the ability of MitoSOX to be oxidizedithin mitochondria after their de-energization.

qually high Jm in oxphos cells and glc cells (Fig. 5a).he amount of Complex I in oxphos cells was maximally10% of glu cells (Table 1).

Details of differences between glc and oxphos cellsre summarized in Table 1. Higher oxidative phospho-ylation of oxphos cells was reflected by: (i) ∼2-foldigher average respiration of 106 cells, (ii) ∼30–40 mVigher�Ψm, as indicated by on average 4.2-fold higher

nitial TMRE emission (n = 6), and (iii) higher autoflu-rescence excited at 488 nm reflecting either a higheravoprotein content or higher oxidation of flavoproteinsHuang, Heikal, & Webb, 2002); both are consistent

ig. 5. (a) Relative Jm values of oxphos- (dashed columns) and glucose-cultieries for effects of 10 nM nigericin, “Nig”, and valinomycin, “Val”) for each

m values were calculated from the slopes of the integral intensity vs. time a100%). Where indicated, 1 �M FCCP was added. The t-test yielded significCCP or no additions (only oxphos) vs. rotenone + FCCP; for no additions vs.only oxphos). (b) Ratios of Jm for oxphos- vs. glucose-cultivated HEPG2 celabsolute rates) for oxphos- vs. glucose-cultivation as indicated: *p < 0.1; **p

emistry & Cell Biology 40 (2008) 1792–1805 1799

with higher state 3 respiration. Oxphos cells had higherMnSOD activity (Table 1). TMRE staining and in somecases MitoSOX imaging also indicated a distinct mito-chondrial morphology (Fig. 6) of the oxphos-cultivatedHEPG2 cells characterized by an interconnected net-work of mitochondrial reticulum of thin tubules witha few bulkier spherical mitochondria in contrast to lessfrequent thicker tubules and frequent predominant bulkymitochondria within the reticulum or solitary mitochon-dria observed under glucose cultivation (Fig. 6b–d).

3.7. Inhibition of Complex I H+ pumping enhancessuperoxide production and abolishes itsuncoupler-mediated suppression

Because rotenone-induced O2•− production orig-

inates entirely from Complex I, and as uncouplingmay accelerate residual H+ pumping in rotenone-inhibited Complex I, we further studied the effects ofan H+-pumping inhibitor, hydrophobic amiloride, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), known to bind tothe membrane subunit ND5 (Gemperli et al., 2007;Nakamaru-Ogiso, Boo Seo, et al., 2003; Nakamaru-Ogiso, Sakamoto, et al., 2003). Addition of 1 �M EIPAapproximately doubled the basal Jm in both glc andoxphos cells (Fig. 7) independently of FCCP withoutaffecting respiration, thus directly activating O2

•− pro-duction (not trivially via respiration increase). EIPA did

not prevent, but instead activated by ∼10%, the alreadyhigh rotenone-induced Jm (Fig. 7). Surprisingly, EIPAat 0.5–1 �M (Fig. 7, AC50 < 0.3 �M EIPA) preventedsuppression by FCCP of rotenone-induced Jm in both

vated HEPG2 cells (black columns). Twelve to 20 image series (5–6reagent or combination, such as shown in Fig. 2, were performed, andnd finally normalized to the Jm values obtained for 20 �M rotenoneant differences (p < 0.1) between the calculated relative Jm values forFCCP (only glc), vs. rotenone + FCCP, or vs. rotenone + valinomycin

ls. The t-test yielded significant differences between ensembles of Jm

< 0.01; ***p < 0.001.

1800 A. Dlaskova et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 1792–1805

Table 1Comparison of glucose- and galactose/glutamine-cultivated HEPG2 cellsa

Parameter Glc cells Oxphos cells

Surplus superoxide production released to the matrixJm(state 3)/Jm(state 4) (n = 3) 0.2 ± 0.1 0.3 ± 0.1Rotenone activation (AC50) 2 �M 3 �MPrevention of rotenone activation by FCCP (IC50) 55 nM 70 nM

RespirationState 3 respiration per 106 cells (pmol O2 s−1) (n = 50) 51 ± 17 97 ± 24State 3/state 4 (state 4: n = 6) 4 ± 0.5 5.6 ± 1.6Rotenone inhibition in state 3 (IC50) 68, 64, and 34 nM (13% remaining) 74, 40, and 20 nM (5% remaining)Rotenone inhibition after FCCP (IC50) 3 �M (20–30% remaining) 100 nM (2% remaining)FCCP activation in state 3 (AC50) 25, 57, 144, and 220 nM 26, 38, 65, 265, and 300 nMFCCP activation in state 4 (AC50) 132 and 450 nM 127 and 428 nMFCCP activation after rotenone (AC50) 186 nM 200 nMEIPA inhibition No inhibition even at >1 mM No inhibition even at >1 mMValinomycin activation (AC50 Glc: up to 30 nM,

oxphos: up to 80 nM)3, 5, and 6 nM 2 and 2.5 nM

Valinomycin inhibition (IC50) >500 and >1000 nM >1000 nM and >10 �MNigericin activation up to100 nM <10% activation <3% activationNigericin inhibition (IC50) >1000 and >500 nM >1000 nM and >10 �M

Other parametersTotal SOD activity (U/ml, n = 5) 0.07 ± 0.02 0.16 ± 0.05CuZnSOD activity (U/ml, n = 5) 0.02 ± 0.01 0.05 ± 0.03MnSOD activity (U/ml, n = 5) 0.04 ± 0.02 0.08 ± 0.05Complex I content from BN-PAGE, n = 4, Glc cells 100 ± 9 113 ± 11

been caablishe

set to 100%

aWhen for a reagent, an apparent IC50 or AC50 is mentioned, it hasshown, those originate from repeated estimations. State 4 has been est

glc and oxphos cells, in many cases returning Jm torotenone-induced values.

4. Discussion

Our principal findings on the mechanism of O2•−

formation within the mitochondrial Complex I led usto predict significant consequences for mitochondria-derived oxidative stress. We hypothesize that the missinglink in the mitochondrial theory of aging and etiol-ogy of diseases originating from oxidative stress liesin our finding that inhibition of Complex I H+ pump-ing leads to elevated O2

•− production, and hence tooxidative stress, given also by cascade of downstreamproducts of O2

•− (Brand et al., 2004; Jezek & Hlavata,2005). This link has been clearly demonstrated for theND5 subunit via studies with its inhibitor, the hydropho-bic amiloride derivative EIPA (Gemperli et al., 2007;Nakamaru-Ogiso, Boo Seo, et al., 2003; Nakamaru-Ogiso, Sakamoto, et al., 2003). ND5 participation in H+

pumping is derived from its sequence homology withancient Na+/H+ antiporters which provides the abilityof Escherichia coli ND5 homolog NuoL to pump Na+.Such a Na+ pumping was found to be inhibited by EIPA

lculated from a corresponding Hill plot. When several numbers ared by 1 �g ml−1 oligomycin.

(Gemperli et al., 2007). Moreover, Complex I-related H+

pumping of isolated rat liver mitochondria was recentlyfound to be inhibited by EIPA without inhibiting respi-ration (Dlaskova et al., unpublished).

Our hypothesis explains why severe consequencesmay result from slight initial oxidative modifications.They lead to oxidative damage of mtDNA thereby tomutated subunits ND2, ND4, and ND5, and hence tothe impaired H+ pumping. This provides further oxida-tive stress and further oxidative damage of mtDNA,thus representing ever accelerating amplification by avicious spiral (cycle) leading to aging, diseases, andcell death. Oxidatively damaged subunit proteins maybe replaced by new protein expression at a sufficientturnover, whereas oxidative damage of mtDNA leads tomutated subunits and, at high enough levels, is lethal(Sato, Nakada, & Hyashi, 2006; Smigrodzki & Khan,2005). Indeed, severe diseases such as Leber’s hered-itary optic neuropathy, mitochondrial encephalopathylactic acidosis stroke-like episodes, Leigh syndrome,

and Parkinson’s disease originate from oxidative mod-ifications or inherited mutations of the mtDNA codingregion for the H+ pumping subunit ND5 (Bourges etal., 2004; Chomyn & Attardi, 2003; Parker & Parks,

A. Dlaskova et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 1792–1805 1801

Fig. 6. Morphological differences in the mitochondrial network between glucose- and oxphos-cultivated HEPG2 cells. (a and b) Visualization usingTMRE of oxphos- (a) and glucose-cultivated (b) HEPG2 cells. Cells were incubated with 50 nM TMRE for 10 min, then images were taken. Thefl er photR e (d ant

2cbuphiR

uorescence intensity was actually ∼4-fold higher in (a), since a highed prior to (c and e) and after 20 min incubation with 20 �M rotenon

he same scale.

005; Smigrodzki, Parks, & Parker, 2004). Even if mito-hondria perhaps defend against such oxidative stressy complementation, i.e. fusing into a network or retic-lum (Chan, 2006; Sato et al., 2006), a threshold of

oint of no return likely exists. The vicious cycle mayappen also with oxidatively mutated coding regionsn mtDNA for 22 tRNAs and 12S and 16S ribosomalNAs, which obviously affect expression of the whole

omultiplier gain was used for (b). (c–f) Visualization using MitoSOXd f) for glc (c and d) and oxphos cells (e and f). Image pair (c–f) have

mtDNA, and with oxidatively mutated mitochondria-coded subunits of Complex III (cyt b) and ATP synthase(ATP6/8).

Concerning a mechanism of O •− production within

2Complex I (Brandt, 2006; Brandt, Kerscher, Drose,Zwicker, & Zickermann, 2003; Lambert & Brand,2004a; Yagi & Matsuno-Yagi, 2003), our data suggestthat not only retardation of electron transport (mostly

1802 A. Dlaskova et al. / The International Journal of Bioch

Fig. 7. EIPA elevates Jm and attenuating effect of FCCP on rotenone-mediated induction is prevented by EIPA. Figure illustrates Jm inoxphos- (dashed columns) vs. glucose-cultivated HEPG2 cells (blackcolumns). Three to six image series for each reagent or combina-tion were measured and Jm values were normalized to those obtainedwith 20 �M rotenone (100%). Where indicated, 1 �M EIPA or 1 �MFCCP was present. AC for abolishing FCCP attenuation of rotenone-

50

induced Jm was <0.3 �M EIPA. The t-test yielded significant (p < 0.01)differences between Jm values with EIPA or EIPA + FCCP vs. noaddtions.

pathophysiological) within Complex I must occur, but

that the simultaneous additional retardation of H+ pump-ing is essential for high O2

•− formation. Decreased H+

pumping may result from the pathophysiological impair-ment due to mutated ND2, ND4, and ND5 subunits or is

Fig. 8. Principles for superoxide production within Complex I. The schema illuonly when electron transfer is highly retarded (e.g. by rotenone) and when H+

at high �p (a) or pathophysiologically when H+ pumping is inhibited (c). WWhen substrate oxidation is faster than the Q cycle or electron flow within tsimilarly as a relative retardation of electron flow, which together with highchanges are depicted by zigzag lines.

emistry & Cell Biology 40 (2008) 1792–1805

manifested physiologically as feedback suppression by�p or �Ψm (Fig. 8). Indeed, either rotenone-mediatedelectron transport retardation at Δp ∼0 (uncoupling,Fig. 5a) or the inhibition of H+ pumping itself bythe ND5-bound inhibitor EIPA (Gemperli et al., 2007;Nakamaru-Ogiso, Boo Seo, et al., 2003; Nakamaru-Ogiso, Sakamoto, et al., 2003) led only to intermediateO2

•− formation (Fig. 7), perhaps resulting from fullyreduced flavin as seen for isolated Complex I (Kussmaul& Hirst, 2006). Rotenone-binding in proximity to theQ-site (ubiquinone binding site) highly retards electrontransport throughout the peripheral arm of Complex I(Brandt, 2006; Brandt et al., 2003; Lambert & Brand,2004a; Yagi & Matsuno-Yagi, 2003), which results inthe formation of longer-lived semiquinone species hav-ing a higher probability of reacting with oxygen and thusforming O2

•−. Even with retarded electron transport, ahigh rate of O2

•− formation is reached only when H+

pumping is also attenuated by a high �p (Fig. 8a) orby high�Ψm. When the�p feedback retardation of H+

pumping is released by uncoupling and H+ pumping isre-accelerated, O2

•− is not formed in excess, even ifinhibition by rotenone persists (Fig. 8b). This view isindependently supported by the fact that EIPA, whichdoubled basal Jm and slightly increased the rotenone-induced Jm, strongly overcame the suppressive effect

of FCCP on rotenone-induced O2

•− formation (Fig. 7).Blockage of H+ pumping through EIPA-mediated inhibi-tion (Gemperli et al., 2007; Nakamaru-Ogiso, Boo Seo,et al., 2003; Nakamaru-Ogiso, Sakamoto, et al., 2003)

strates O2•− production by Complex I, which takes place in high rates

pumping also is slowed down. The latter can happen physiologicallyith H+ pumping intact but at �p zero, O2

•− production ceases (b).he remaining respiratory chain, the resulting NADH “overflow” acts�p generates an intermediate O2

•− production (d). Conformational

of Bioch

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A. Dlaskova et al. / The International Journal

as sufficient to act in concert with electron flow blockedt the Q-site by rotenone (Fig. 8c).

We predict that a mild uncoupling causing re-cceleration of electron flow through the respiratoryhain may have an “antioxidant” role in vivo but only inases where the H+ pumping subunits are not extensivelyamaged. We also interpret valinomycin prevention ofhe rotenone-induced Jm as a direct consequence of

collapsed �Ψm on Complex I H+ pumping, i.e. aseleased “feedback pressure” of H+ pumping requiredor high O2

•− formation (Fig. 8). In conclusion, dur-ng aging and pathogenesis, oxidative modifications ofuclear-coded subunits or mutations resulting from pre-ious oxidative stress in mitochondria-coded subunitsf Complex I are serious when occur in residues (ornfluence them) participating either in electron transportathway or in pathway including transfer of conforma-ion changes and H+ pumping. The resulting elevated

2•− formation provides further accelerating oxidative

tress in a vicious cycle especially when the damageccurs in both pathways.

The above schemes are grounded in the structure ofomplex I and reflect a yet unknown H+ pumping mech-nism. The consensus view is that the H+ pumping withinomplex I (stoichiometry 4H+ per 2 e−) is provided by

ong-range conformational changes occurring when anlectron leaves the redox center N2 located on the PSSTubunit on a peripheral arm in proximity to the matrixide of the membrane (Brandt, 2006). Conformationalhanges relay redox changes between the pH-dependentid redox potential of redox center N2 (−150 mV) and

biquinone (QH2/Q, +90 mV), residing at the Q-site, to+ transport across the entire membrane-associated partf the complex (Brandt, 2006). One can speculate thatp or�Ψm slow down these changes, which also delay

edox changes leading to longer-lived semiquinones thateact with oxygen to form O2

•−. These conformationhanges are also blocked by EIPA (Nakamaru-Ogiso,oo Seo, et al., 2003; Nakamaru-Ogiso, Sakamoto, etl., 2003) and thus its effect fits into our interpretation.

Complex I is likely an important player in home-stasis of reactive oxygen species (Kussmaul & Hirst,006), significantly contributing to O2

•− release intohe mitochondrial matrix in vivo. Even if Complex IIIrobably contributed to our measured Jm values (Mullert al., 2004), since only rotenone-sensitive portions arenequivocally attributed to the Complex I, we mayeduce a Complex I role from our comparison of cells

elying on oxidative phosphorylation, which exhibited2-fold Jm compared to glucose-cultivated cells. Thus

he basal Jm in oxphos cells was higher by the same factors their respiration increased (up to twofold), but was still

emistry & Cell Biology 40 (2008) 1792–1805 1803

rather low due to a sufficient MnSOD capacity (MnSODactivity actually rose, Table 1). An increased NADH orNADH/NAD+ ratio (Kussmaul & Hirst, 2006) leading toa high electron flow exceeding output electron flow onComplex I and higher �Ψm are likely causes (Fig. 8d).We speculate that electron transport within Complex I(and maybe also Complex III) in oxphos cells is rel-atively more retarded (Fig. 8d) due to higher substrateoxidation resulting in accumulating NAD(P)H levels andfaster respiration, thereby faster H+ pumping and creat-ing higher �p (confirmed by higher TMRE emission).As a result, the Jm is higher than in glc cells (Fig. 5b),although Complex I content is similar. The substrateload (NADH/NAD+ ratio) in oxphos cells should be highenough that compensation by the increased H+ backflowvia FOATP synthase (Desquiret et al., 2006; Rossignolet al., 2004) and doubled MnSOD content cannot atten-uate such elevated O2

•− formation. In turn, the situationin glucose-cultivated cells (moderate levels of oxidativephosphorylation) resembles the effect of calorie restric-tion (Muller et al., 2007). Thus fast electron flux viathe whole respiratory chain at a high substrate pressureproduces more O2

•− than when slower flux occurs atthe same relative retardation (same oxidation/reductionstates). Moreover, we have shown that uncoupling atten-uates O2

•− production in situ, at least for basal Jm of glccells, and that Jm is >4-fold and >3-fold higher at state4 in glc and oxphos cells, respectively.

We have also demonstrated that MitoSOX Red is auseful indicator of mitochondrial oxidative stress. This isdue to the fact that it principally increases its fluorescenceonly when excessive O2

•− production takes place, i.e.production that cannot be efficiently dismuted by matrixMnSOD. Thus we can predict that in situ mitochondria ofany cells with insufficient MnSOD capacity or any cellswhere excessive O2

•− is produced by the respiratorychain due to disease or aging processes will be indicatedby increased MitoSOX emission with time. This increasewill thus indicate excess O2

•− production especiallywhen a high �Ψm is established. Comparing only fluo-rescence levels (Georgiou et al., 2005; Mukhopadhyayet al., 2007; Robinson et al., 2006; Zhao et al., 2003,2005; Zielonka et al., 2005) could be misleading dueto unknown contribution of non-specific oxidation andextent of intercalation into mtDNA.

Acknowledgements

Supported by grants NR/7917-6 from the Czech Min-istry of Health; IAA500110701 and AV0Z50110509from the Academy of Sciences; 301/05/0221 (to P.J.)and 303/05/P100 (to L.H.) from the Grant Agency of

of Bioch

1804 A. Dlaskova et al. / The International Journal

the Czech Republic (GACR). Help of T. Spacek and J.Santorova with tissue culture cells is gratefully acknowl-edged.

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