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Homocysteine-dependent Alterations in Mitochondrial Gene Expression, Function and Structure

HOMOCYSTEINE AND H2O2 ACT SYNERGISTICALLY TO ENHANCE MITOCHONDRIAL DAMAGE*
      Mitochondrial abnormalities have been identified in hepatocytes of patients with hyperhomocysteinemia and in endothelial cells from the aortas of rats with diet-induced hyperhomocysteinemia. However, the mechanism by which homocysteine affects mitochondria is unknown. In this report, homocysteine-induced expression of the mitochondrial electron transport chain gene, cytochrome coxidase III/ATPase 6,8 (CO3/ATPase 6,8), was identified in a human megakaryocytic cell line DAMI using mRNA differential display. Steady-state mRNA levels of CO3/ATPase 6,8, as well as other mitochondrial transcripts, were increased in DAMI cells by homocysteine in a concentration- and time-dependent manner. Despite an increase in mitochondrial RNA levels and changes in mitochondrial ultrastructure, no effect on either cell growth or mitochondrial respiration rates was observed in DAMI cells exposed to homocysteine at concentrations up to 1 mm. In contrast, 1 mmhomocysteine in the presence of Cu2+, which is known to generate H2O2, significantly decreased mitochondrial RNA levels, caused gross morphological changes in mitochondrial ultrastructure, and inhibited both cell growth and mitochondrial respiration rates. However, precursors of cellular glutathione and preexposure to heat shock blocked the decrease in mitochondrial RNA levels caused by homocysteine and Cu2+. The observations that (i) homocysteine and H2O2, but not H2O2alone, caused a decrease in mitochondrial RNA levels, (ii) intracellular levels of H2O2 were significantly increased in the presence of homocysteine and Cu2+, and (iii) catalase, but not free radical scavengers, prevented a decrease in mitochondrial RNA levels, provide evidence that homocysteine and H2O2 act synergistically to cause mitochondrial damage. Furthermore, our findings suggest that intracellular glutathione and heat shock proteins play a role in protecting mitochondria against the adverse effects elicited by homocysteine and H2O2.
      CO3/ATPase 6
      8, cytochrome c oxidase III + ATPase 6,8
      ND5
      NADH-coenzyme Q oxireductase subunit 5
      HH
      hyperhomocysteinemia
      EC
      endothelial cell
      HSP60
      60-kDa heat shock protein
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      SAM
      S-adenosyl-l-methionine
      SAH
      S-adenosyl-l-homocysteine
      NAC
      N-acetyl-l-cysteine
      PBS
      phosphate-buffered saline
      DCF
      dichlorofluorescin
      GSH
      glutathione.
      Homocysteine is a thiol-containing amino acid that is formed when methionine is converted to cysteine. Once synthesized, homocysteine may either be metabolized to cysteine by the transsulfuration pathway or remethylated to methionine (
      • Mayer E.L.
      • Jacobsen D.W.
      • Robinson K.
      ,
      • Ueland P.M.
      • Refsum H.
      • Brattstrom L.
      ,
      • Welch G.N.
      • Loscalzo J.
      ,
      • McCully K.S.
      ,
      • Loscalzo J.
      ). Deficiencies of the enzymes involved in these pathways (i.e. cystathionine β-synthase and 5,10-methylenetetrahydrofolate reductase) and/or cofactors necessary for the metabolism of homocysteine (i.e., vitamin B6, vitamin B12, or folate) can cause aberrant intracellular processing of homocysteine, leading to hyperhomocysteinemia (HH).1
      The most common hereditary causes of HH result from mutations in the genes encoding cystathionine β-synthase or 5,10-methylenetetrahydrofolate reductase. Patients with severe HH due to cystathionine β-synthase or 5,10-methylenetetrahydrofolate reductase deficiency exhibit a wide range of clinical manifestations, including mental retardation, ectopia lentis, osteoporosis, skeletal abnormalities, and a fatty liver (
      • Mudd S.H.
      • Levy H.L.
      • Skovby F.
      ). The major cause of death in these patients is premature vascular and thrombotic disease (
      • Mudd S.H.
      • Levy H.L.
      • Skovby F.
      ,
      • Mudd S.H.
      • Havlik R.
      • Levy H.L.
      • McKusick V.A.
      • Feinleib M.
      ,
      • Ueland P.M.
      • Refsum H.
      ), and recent evidence suggests that patients with mild HH also have an increased risk of cardiovascular disease (
      • Mayer E.L.
      • Jacobsen D.W.
      • Robinson K.
      ,
      • Ueland P.M.
      • Refsum H.
      • Brattstrom L.
      ,
      • Welch G.N.
      • Loscalzo J.
      ,
      • McCully K.S.
      ,
      • Loscalzo J.
      ,
      • Selhub J.
      • Jacques P.F.
      • Bostom A.G.
      • D'Agostino R.B.
      • Wilson P.W.F.
      • Belanger A.J.
      • O'Leary D.H.
      • Wolf P.A.
      • Schaefer E.J.
      • Rosenberg I.H.
      ,
      • Fortin L.-J.
      • Genest J.
      ).
      Experimental evidence indicates that homocysteine causes cell injury when administered to animals (
      • Harker L.A.
      • Slichter S.J.
      • Scott C.R.
      • Ross R.
      ,
      • Harker L.A.
      • Harlan J.M.
      • Ross R.
      ,
      • Hladovec J.
      ) or when added directly to cultured mammalian cells (
      • Wall R.T.
      • Harlan J.M.
      • Harker L.A.
      • Striker G.E.
      ,
      • Starkebaum G.
      • Harlan J.M.
      ,
      • de Groot P.G.
      • Willems C.
      • Boers G.H.J.
      • Gonsalves M.D.
      • VanAken W.G.
      • Mourik J.A.
      ,
      • Hultberg B.
      • Andersson A.
      • Isaksson A.
      ). It has been suggested that homocysteine-induced cell injury involves oxidative damage because homocysteine is readily oxidized in plasma to form reactive oxygen species, such as H2O2, and cell injury caused by homocysteine is blocked by catalase (
      • Wall R.T.
      • Harlan J.M.
      • Harker L.A.
      • Striker G.E.
      ,
      • Starkebaum G.
      • Harlan J.M.
      ). More recently, we demonstrated that, unlike their response to other oxidative stress conditions, exposure of cultured human vascular endothelial cells (ECs) to homocysteine fails to elicit an oxidative stress response (
      • Outinen P.A.
      • Sood S.K.
      • Liaw P.C.Y.
      • Sarge K.D.
      • Maeda N.
      • Hirsh J.
      • Ribau J.
      • Podor T.J.
      • Weitz J.I.
      • Austin R.C.
      ). Instead, homocysteine causes reductive stress with decreased expression of several antioxidant enzymes and subsequent endoplasmic reticulum dysfunction. Given that homocysteine decreases intracellular glutathione peroxidase activity (
      • Upchurch Jr., G.R.
      • Welch G.N.
      • Fabian A.J.
      • Freedman J.E.
      • Johnson J.L.
      • Keaney Jr., J.F.
      • Loscalzo J.
      ), these studies raise the possibility that homocysteine may enhance the cytotoxic effect of agents or conditions known to generate reactive oxygen species. The purpose of this study was to test this possibility.
      In this investigation, we demonstrate that homocysteine alters mitochondrial gene expression, structure, and function and that severe mitochondrial damage occurs in the presence of homocysteine and H2O2. These studies support a mechanism whereby homocysteine and H2O2 act synergistically to cause mitochondrial damage, thereby promoting cell injury and/or dysfunction.

      EXPERIMENTAL PROCEDURES

       Cell Culture and Treatment Conditions

      The human megakaryocytic cell line DAMI was obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) containing 100 units/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Inc.). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. Homocysteine, homoserine, methionine, cysteine,S-adenosyl-l-methionine (SAM),S-adenosyl-l-homocysteine (SAH),N-acetyl-l-cysteine (NAC),dl-dithiothreitol, cupric sulfate (Cu2+), and catalase (Sigma) were prepared in RPMI 1640 medium, which was filtered and added to cell cultures at the desired concentrations. For heat shock experiments, DAMI cells were heated for 2 h at 42 °C in RPMI medium containing 5% fetal bovine serum. After exposure to heat, cells were allowed to recover at 37 °C for 6 or 18 h and then treated with homocysteine, in the absence or presence of Cu2+. Unless otherwise stated, DAMI cells were used at a concentration of 1 × 106 cells/ml. After incubation for the appropriate time period, cell number and viability were assessed by counting cells in a hemocytometer after suspension in PBS containing 0.5% trypan blue.

       Total RNA Isolation and Purification

      All solutions were prepared with water that had been treated with 0.1% diethyl pyrocarbonate (Sigma) and autoclaved. Following treatment, DAMI cells were washed with PBS and pelleted by centrifugation. Total RNA was isolated from cells using the RNeasy total RNA kit as described by the manufacturer (Qiagen, Chatsworth, CA). For mRNA differential display experiments, contaminating DNA was removed by incubation of the RNA with 2 units of RQ DNase (Promega, Mississauga, Ontario, Canada) and 12 units of RNasin (Promega) for 30 min at 37 °C. After the addition of diethyl pyrocarbonate-treated water to 100 μl, samples were mixed with an equal volume of phenol/chloroform (3:1) and centrifuged, and the aqueous phase was transferred to an RNase-free, 1.7-ml centrifuge tube. After the addition of 10 μl of 3m sodium acetate and 300 μl of absolute ethanol, the RNA was precipitated overnight at −70 °C. RNA was collected by centrifugation at 14,000 × g for 10 min at 4 °C, washed briefly with 85% ethanol, dried, and resuspended in diethyl pyrocarbonate-treated water. RNA samples withA 260/A 280 ratios above 1.6 were stored at −70 °C for further analysis.

       mRNA Differential Display

      mRNA differential display was performed using the RNA map kits (GenHunter, Brookline, MA) as described previously (
      • Outinen P.A.
      • Sood S.K.
      • Liaw P.C.Y.
      • Sarge K.D.
      • Maeda N.
      • Hirsh J.
      • Ribau J.
      • Podor T.J.
      • Weitz J.I.
      • Austin R.C.
      ). Candidate cDNA fragments excised from the display gel were amplified by PCR, purified from 1% agarose gels, and used as probes on Northern blots.

       Cloning and Sequencing of cDNA Fragments

      Amplified cDNA fragments were subcloned into T-ended pBluescript II (KS) (Stratagene), and double-stranded DNA sequencing was done using a modified T7 polymerase system (Sequenase, version 2, U. S. Biochemical Corp.). The cDNA sequences were analyzed and compared for similarity with sequences deposited in the EMBL and GenBankTM DNA data bases.

       Northern Blot Analysis

      Total RNA was fractionated on 2.2m formaldehyde/1.2% agarose gels and transferred overnight onto Zeta-Probe GT nylon membranes (Bio-Rad) in 10× SSC. The RNA was cross-linked to the membrane using a Stratalinker UV cross-linker (PDI Bioscience, Toronto, Ontario, Canada) prior to hybridization. Specific probes were generated by labeling the reamplified or cloned cDNA fragments with [α-32P]dCTP (NEN Life Science Products) using a random primed DNA labeling kit (Boehringer Mannheim). After overnight hybridization at 42 °C, the filters were washed as described previously (
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      ), covered in plastic wrap, and exposed to Kodak X-Omat AP film at −70 °C for 1–3 days. Probes for NADH-coenzyme Q oxireductase subunit 5 (ND5), 12 S and 16 S rRNAs were derived from mouse mitochondrial DNA, whereas HSP60 cDNA was obtained from StressGen (Vancouver, British Columbia, Canada). Hybridization with a partial cDNA fragment to human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Changes in the expression of mRNA transcripts were quantified using the ImageMaster VDS and Analysis Software (Amersham Pharmacia Biotech).

       cDNA Library Screening

      A human EC 5′-STRETCH cDNA library in λgt11 (CLONTECH, Mississauga, Ontario, Canada) was screened using a 32P-labeled dhc-1 cDNA probe. Positive cDNA phage clones were plaque-purified as described previously (
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      ), and the size of the cDNA inserts was determined by PCR using λgt11 forward and reverse primers (synthesized at the Institute for Molecular Biology and Biotechnology, McMaster University). The largest cDNA inserts were excised from the λgt11 vector with EcoRI and subcloned into the EcoRI site of pBluescript II (KS). DNA sequencing and homology searches were done as described above.

       Immunoblot Analysis

      Total cellular proteins were immunoblotted as described previously (
      • Outinen P.A.
      • Sood S.K.
      • Liaw P.C.Y.
      • Sarge K.D.
      • Maeda N.
      • Hirsh J.
      • Ribau J.
      • Podor T.J.
      • Weitz J.I.
      • Austin R.C.
      ) using antibodies directed against either cytochrome c oxidase subunits I, II, IV, Vb (Molecular Probes, Eugene, OR), or HSP60 (StressGen). Protein concentrations were determined using the Bio-Rad DC protein assay kit with bovine IgG as the standard.

       Transmission Electron Microscopy

      Control or treated DAMI cells were pelleted, washed with PBS, and fixed for 2 h in 2% glutaraldehyde in PBS. After overnight fixation in 1% OsO4in PBS, cells were washed, dehydrated in a graded alcohol series, and embedded in Spurr's resin. Thin sections (70 nm) were stained with uranyl acetate and examined using a Jeol 100B electron microscope. A minimum of 10 micrographs were evaluated for each experimental condition.

       Mitochondrial Respiration Rates

      To measure mitochondrial respiration rates in control or homocysteine-treated DAMI cells, steady-state oxygen consumption of whole cells permeabilized with digitonin (0.005%) was quantified at 37 °C using a Clark-type oxygen electrode (Yellow Springs Instrument Co. Inc., Yellow Springs, OH). After treatment, cells were pelleted by centrifugation and resuspended in respiration buffer (0.25 m sucrose, 0.01m MgCl2, 0.02 m HEPES, 0.02m KH2PO4, pH 7.1) at a concentration of 1 × 107 cells/ml. To each 100-μl aliquot of cell suspension was added 1 μl of digitonin diluted in 0.5% dimethyl sulfoxide, and 610 μl of the suspension was then loaded into the microchamber. Trypan blue uptake was used to confirm plasma membrane permeabilization. After measuring baseline respiration rates, 5 mm succinate and 1 mm adenosine diphosphate were added to maximally stimulate mitochondrial respiration rates. Respiration rates were measured in the absence or presence of 1 μm oligomycin, a specific inhibitor of mitochondrial oxidative phosphorylation, so that the increase attributable to mitochondria could be determined. Respiration rates were calculated as percentage of oxygen consumed per min per 1 × 106cells, and air-saturated buffer was considered as 100%.

       Detection of Intracellular H2O2 in DAMI Cells

      Intracellular levels of H2O2 were determined by flow cytometry using the nonfluorescent probe 2′-7′-dichlorofluorescin (DCF) diacetate (Molecular Probes, Eugene, OR). Removal of the acetate groups by intracellular esterases results in the release of dichlorofluorescin, which upon exposure to hydroperoxides, such as H2O2 and lipid peroxides, is hydrolyzed to the fluorescent probe dichlorofluorescein (
      • Cathcart R.
      • Schwiers E.
      • Ames B.N.
      ). DAMI cells (5 × 105 cells/ml) pulsed for 60 min in medium containing 5% fetal bovine serum and 100 μmDCF-diacetate (10 mm stock solution freshly prepared in dimethyl sulfoxide) were pelleted and resuspended in fresh media without DCF-diacetate. Cells were then treated for 60 min in medium containing either Cu2+, homocysteine, homocysteine and Cu2+, or H2O2. A minimum of 5 × 105 cells from each treatment were analyzed to obtain a distribution of population fluorescence at 525 nm for emission and 488 nm for excitation using an EPICS PROFILE II flow cytometer (Coulter, Burlington, Ontario, Canada). Data represent a comparison of treatedversus control samples in which control means were set to 100% in each individual test.

       Determination of Intracellular Glutathione Levels

      Intracellular glutathione (GSH) was measured using the BIOXYTECH GSH-400 colorimetric assay as described by the manufacturer (OXIS International, Portland, OR). Briefly, DAMI cells (1 × 106 cells/ml) were incubated for 4 h in control medium or medium containing either homocysteine or NAC. After washing in Hanks' balanced saline solution, cells were snap-frozen on dry ice, homogenized in ice-cold 2.5% (w/v) metaphosphoric acid (Sigma), and centrifuged at 4 °C to remove any insoluble material. Samples were measured at a final absorbance of 400 nm in a LKB Ultrospec II spectrophotometer (Amersham Pharmacia Biotech), and intracellular GSH levels were determined from a standard curve using reduced GSH (Sigma).

       Determination of Mitochondrial Mass

      Mitochondrial mass was measured by flow cytometry using nonyl acridine orange (Molecular Bioprobes), a fluorescent dye capable of binding mitochondrial cardiolipin in an energy-independent manner. DAMI cells were seeded into 24-well plates at a concentration of 5 × 105cells/ml in RPMI medium containing 5% fetal bovine serum. After a 4-h adjustment period in fresh medium, cells were treated for 18 h in medium containing either Cu2+, homocysteine, or homocysteine and Cu2+. Nonyl acridine orange was then added to a final concentration 0.1 μm for 45 min, and the fluorescence distribution of each treated cell population was evaluated by flow cytometry.

       Statistical Analysis

      Data are presented as the means ± S.E. Significance of differences between control and treated groups was determined by one-way analysis of variance. If significant differences were detected, unpaired Student's t tests were performed. For all analyses, p values <0.05 were considered significant.

      RESULTS

       Effect of Homocysteine ± Cu2+ on Cell Viability and Growth

      Earlier studies have shown that in the presence of Cu2+, homocysteine is readily oxidized in plasma or culture medium to generate H2O2, which can cause cell lysis (
      • Wall R.T.
      • Harlan J.M.
      • Harker L.A.
      • Striker G.E.
      ,
      • Starkebaum G.
      • Harlan J.M.
      ). As illustrated in Table I, exposure of DAMI cells (1 × 106 cells/ml) to 1 mm homocysteine for up to 18 h, in the absence or presence of Cu2+, had no effect on cell viability. However, viability was dependent on initial cell density because DAMI cells seeded at <1 × 105cells/ml were lysed when exposed to homocysteine and Cu2+for 18 h. In contrast, neither Cu2+ nor catalase alone had any significant effect on cell viability.
      Table IEffect of homocysteine, in the absence or presence of Cu2+, on DAMI cell viability
      TreatmentLive cells
      %
      Control93 ± 0.6
      Control + 4 μm Cu2+87 ± 2.6
      1 mmhomocysteine93 ± 1.0
      5 mm homocysteine87 ± 1.9
      1 mm homocysteine + 4 μmCu2+90 ± 1.8
      5 mm homocysteine + 4 μm Cu2+54 ± 4.4
      1 mmhomocysteine + 4 μm Cu2+ + catalase (2500 units/ml)89 ± 0.6
      5 mm homocysteine + 4 μm Cu2+ + catalase (2500 units/ml)80 ± 1.7
      Catalase (2500 units/ml)93 ± 1.5
      DAMI cells (1 × 106 cells/ml) were incubated in control or test medium supplemented with 5% fetal bovine serum at 37 °C for 18 h. Cell viability was assessed by counting cells on a hemocytometer after suspension in PBS containing 0.5% trypan blue stain. The data represent the means ± S.E. from three independent experiments.
      To examine the effect on cell growth, DAMI cells (1 × 105 cells/ml) were exposed to varying concentrations of homocysteine, in the absence or presence of Cu2+, for periods up to 7 days (Fig. 1). Although 1 mm homocysteine had no significant effect on growth rates, 5 mm homocysteine significantly reduced growth (p < 0.01) beyond 2 days. Unlike 1 mmhomocysteine alone, exposure to 1 mm homocysteine and Cu2+ completely blocked cell growth and led to cell lysis by 48 h. In contrast, Cu2+ alone had no significant effect on cell growth rates (data not shown).
      Figure thumbnail gr1
      Figure 1Effect of homocysteine ± Cu2+ on DAMI cell growth. DAMI cells, initially seeded at 1 × 105 cells/ml, were grown in the absence (•) or presence of either 1 (▪) or 5 (▴) mm homocysteine or 1 mm homocysteine in the presence of 4 μmCu2+ (▾) for the indicated time periods. Cell growth was assessed by counting aliquots of the cell suspensions. Values represent the means ± S.E. of three separate experiments. *,p < 0.01 in growth rates of control cellsversus cells exposed to 5 mm homocysteine or 1 mm homocysteine plus 4 μmCu2+.

       Identification of Cytochrome c Oxidase/ATPase 6,8 as a Homocysteine-inducible Gene

      To identify changes in gene expression induced by homocysteine, we compared mRNA differential display patterns from DAMI cells exposed to 1 mmhomocysteine for 18 h with those from cells cultured in the absence of homocysteine. After screening through 48 primer combinations, a total of 10 differentially expressed cDNA fragments were identified on sequencing gels. Among these, a cDNA fragment amplified with T12MG and AP-1 primers, designated dhc-1 (Fig. 2 A), was shown to be reproducibly up-regulated in DAMI cells exposed to homocysteine. Initial Northern blot hybridization experiments using RNA from control or homocysteine-treated DAMI cells indicated that the dhc-1 cDNA fragment hybridized to a homocysteine-inducible transcript of ∼1.8 kb (Fig. 2 B). This 1.8-kb transcript was also shown to be up-regulated in human umbilical vein ECs exposed to homocysteine (data not shown).
      Figure thumbnail gr2
      Figure 2mRNA differential display and Northern blot analysis of the effects of homocysteine on mitochondrial gene expression. A, identification of a homocysteine-inducible gene in DAMI cells by mRNA differential display. Total RNA isolated from DAMI cells treated for 18 h in the presence or absence of 1 mm homocysteine (Hcy) was subjected to mRNA differential display using primers 5′-T11MG-3′ and 5′-AGCCAGGGAA-3′.35S-Labeled PCR products were separated on a 6% denaturing sequencing gel and visualized by autoradiography. Thearrowhead indicates the position of a cDNA fragment (designated dhc-1) that was shown to be reproducibly induced by homocysteine. B, Northern blot analysis of total RNA (10 μg/lane) from DAMI cells treated for 18 h in the presence or absence of 1 mm homocysteine using a radiolabeled dhc-1 cDNA probe, followed by autoradiography (top panel). The position of a 1.8-kb mRNA transcript induced by homocysteine is indicated by the arrowhead. C, Northern blot analysis of total RNA (10 μg/lane) from DAMI cells treated for 18 h in the absence or presence of varying concentrations of homocysteine using a radiolabeled 16–1 cDNA probe against CO3/ATPase 6,8 (top panel). The arrowhead corresponds to the primary 1.8-kb CO3/ATPase 6,8 mRNA transcript, and the asteriskcorresponds to the mature 0.9-kb CO3/ATPase 6,8 mRNA transcript.D, induction of mitochondrial RNA levels by homocysteine. Mitochondrial RNA levels were assessed by Northern blot hybridization using the following mitochondrial cDNA probes: ND5; 16Sand 12S, mitochondrial rRNAs; HSP60, 60-kDa mitochondrial heat shock protein. Control for RNA loading was assessed using a GAPDH cDNA probe (middle panels in Band D) and ethidium bromide staining of the 28S and 18S rRNA bands (bottom panels in B–D).
      Searching of the GenBank™ and EMBL data bases revealed that dhc-1 had >96% sequence similarity to a human cDNA clone designated HHCJ64 (GenBank™ accession number M62278). To identify this cDNA, a human EC library in λgt11 was screened with the dhc-1 cDNA fragment to obtain larger cDNA clones. Five positive phage clones were plaque-purified. The largest of the cDNA inserts (designated 16-1), which was ∼1.8 kb in size, was subcloned, sequenced, and shown to encode cytochrome c oxidase III/ATPase 6,8 (CO3/ATPase 6,8), a mitochondrial electron transport chain transcript encoded by mitochondrial DNA.
      To confirm that expression of CO3/ATPase 6,8 in DAMI cells was induced by homocysteine, total RNA from cells grown in the absence or presence of increasing concentrations of homocysteine was analyzed by Northern blot hybridization, using the 16-1 cDNA as a probe. Of significance, 16-1 was shown to hybridize to two major transcripts of approximately 1.8 and 0.9 kb, respectively (Fig. 2 C), which correspond to the primary and mature transcripts of CO3/ATPase 6,8 (
      • Whitfield C.D.
      • Jefferson L.M.
      ), respectively. Exposure of DAMI cells to increasing concentrations of homocysteine for 18 h caused a dose-dependent increase in the steady-state levels of both transcripts (Fig. 2 C). Even exposure to as little as 0.1 mmhomocysteine for 18 h increased both the primary and mature CO3/ATPase 6,8 mRNA transcripts by 2- and 3-fold, respectively. In addition to homocysteine, other thiol-containing agents, such as dithiothreitol, NAC, and cysteine were shown to induce CO3/ATPase 6,8 mRNA levels (data not shown). However, this effect was not solely dependent on the thiol group because mRNA levels induced by these agents were lower than those observed for homocysteine.
      To determine whether homocysteine increases the expression of other mitochondrial genes, total RNA from control and homocysteine-treated DAMI cells was subjected to Northern blot analysis using the following mitochondrial probes: ND5 (component of the electron transport chain), 12S rRNA, 16S rRNA, and HSP60 (nuclear-encoded mitochondrial chaperone). Fig. 2 D shows that the steady-state levels of these transcripts also are up-regulated by homocysteine. In contrast, there was no change in the steady-state mRNA levels of GAPDH. Although HSP60 was induced, HSP70 mRNA levels remained unchanged (data not shown), suggesting that induction of HSP60 is not due oxidative stress, a finding consistent with previous studies (
      • Outinen P.A.
      • Sood S.K.
      • Liaw P.C.Y.
      • Sarge K.D.
      • Maeda N.
      • Hirsh J.
      • Ribau J.
      • Podor T.J.
      • Weitz J.I.
      • Austin R.C.
      ). Despite an increase in mitochondrial RNA levels upon exposure to homocysteine for 18 h, Western blot analysis using antibodies against cytochrome C oxidase subunits I, II, IV, Vb, or HSP60 indicated no significant increase in mitochondrial protein levels at 18 h (Fig. 3 A).
      Figure thumbnail gr3
      Figure 3Effect of homocysteine ± Cu2+ on mitochondrial protein levels. Total protein lysates (30 μg/lane) from DAMI cells exposed to various concentrations of homocysteine in the absence (A) or presence of Cu2+ (B) were separated on 10% SDS-polyacrylamide gels and immunostained with antibodies to either HSP60 or cytochrome c oxidase (COX) subunits I, II, IV, and Vb.

       Homocysteine and Cu2+ Decrease Mitochondrial RNA and Protein Levels

      To determine whether altered mitochondrial gene expression induced by homocysteine involves reactive oxygen species generated by Cu2+-catalyzed oxidation of homocysteine, Northern blot analysis was performed on total RNA isolated from DAMI cells exposed to 1 mm homocysteine, in the absence or presence of 4 μm Cu2+, for up to 18 h (Fig. 4). As expected, exposure of DAMI cells to 1 mm homocysteine alone caused a time-dependent increase in the mRNA levels of both CO3/ATPase 6,8 and HSP60 (Fig. 4 A). In contrast, the steady-state mRNA levels of CO3/ATPase 6,8 and HSP60 decreased in a time-dependent manner when DAMI cells were exposed to both 1 mm homocysteine and Cu2+ (Fig. 4 B). The absence of smearing of the bands suggests that the decrease in the mRNA levels is likely due to a reduced transcription rate and not degradation. By 18 h, mRNA levels of CO3/ATPase 6,8 and HSP60 were <10% of control levels. However, this effect was completely blocked by the addition of catalase (Fig. 4 C). Additional mitochondrial transcripts, including ND5, 12S, and 16S rRNAs, also decreased to a similar extent when DAMI cells were exposed to homocysteine and Cu2+; however, GAPDH levels were unchanged (data not shown). Exposure of DAMI cells to either Cu2+ or catalase alone had no effect on mitochondrial gene expression. Mitochondrial protein levels were also decreased by 18 h in the presence of at least 1 mmhomocysteine and Cu2+ (Fig. 3 B); however, the decrease was not as dramatic as that observed with RNA levels.
      Figure thumbnail gr4
      Figure 4Homocysteine and Cu2+ decrease mitochondrial mRNA levels in DAMI cells. Total RNA (10 μg/lane) was analyzed by Northern blot hybridization using cDNA probes against either CO3/ATPase 6,8 or HSP60. A, time-dependent increase in the steady-state mRNA levels of CO3/ATPase 6,8 and HSP60 in DAMI cells exposed to homocysteine.B, time-dependent decrease in the steady-state mRNA levels of CO3/ATPase 6,8 and HSP60 in DAMI cells exposed to homocysteine and Cu2+. C, Ability of catalase to prevent a decrease in the steady-state mRNA levels of CO3/ATPase 6,8 in DAMI cells exposed to homocysteine and Cu2+ for 18 h. Control for RNA loading and integrity was assessed by ethidium bromide staining of the 28S and 18S rRNA bands (lower panels).
      Having shown that homocysteine and Cu2+ decrease mitochondrial mRNA levels, we next determined the minimal concentrations of homocysteine and Cu2+ necessary to cause this effect. In the presence of 4 μm Cu2+, concentrations of homocysteine less than 1 mm did not significantly decrease CO3/ATPase 6,8 mRNA levels (Fig. 5 A). Furthermore, concentrations of at least 1 μm Cu2+ together with 1 mm homocysteine are needed to decrease mRNA levels to <10% of control levels (Fig. 5 A).
      Figure thumbnail gr5
      Figure 5Effect of various conditions on the mRNA levels of CO3/ATPase 6,8. Northern blot analysis of total RNA (10 μg/lane) from DAMI cells probed with a radiolabeled cDNA against CO3/ATPase 6,8 (upper panels). A, exposure of cells to various concentrations of homocysteine and Cu2+for 18 h. B, exposure of cells to 1 mmconcentrations of either methionine, cysteine, homoserine, SAM, SAH, or cystathionine, in the absence or presence of 4 μmCu2+, for 18 h. Control for RNA loading and integrity was assessed by ethidium bromide staining of the 28S and 18S rRNA bands (lower panels).
      In addition to homocysteine, other structurally related molecules were examined for their ability to decrease CO3/ATPase 6,8 mRNA levels in the presence of Cu2+ (Fig. 5 B). Cysteine, which like homocysteine contains a free thiol group, also causes a decrease in CO3/ATPase 6,8 mRNA levels, albeit to a lesser extent than equimolar concentrations of homocysteine. In contrast, methionine, SAH, SAM, cystathionine, or homoserine had little or no effect on mRNA levels (Fig. 5 B) or DAMI cell viability, even in the presence of Cu2+ (Table II).
      Table IIEffect of homocysteine and other related compounds, in the absence or presence of 4 μm Cu2+, on DAMI cell viability
      TreatmentLive cells
      %
      Control82 ± 0.3
      Control + 4 μm Cu2+76 ± 1.9
      1 mmhomocysteine77 ± 1.6
      1 mm homocysteine + 4 μm Cu2+74 ± 0.1
      1 mmcysteine78 ± 2.9
      1 mm cysteine + 4 μm Cu2+78 ± 0.8
      1 mmmethionine74 ± 1.5
      1 mm methionine + 4 μm Cu2+83 ± 0.2
      1 mmhomoserine79 ± 1.4
      1 mm homoserine + 4 μm Cu2+79 ± 3.6
      1 mmSAM82 ± 1.2
      1 mm SAM + 4 μmCu2+75 ± 1.1
      1 mm SAH69 ± 1.3
      1 mm SAH + 4 μm Cu2+71 ± 2.0
      1 mm cystathionine81 ± 0.9
      1 mm cystathionine + 4 μm Cu2+72 ± 3.4
      DAMI cells (1 × 106 cells/ml) were treated as described in Table I, and cell viability was assessed by counting cells on a hemocytometer after suspension in PBS containing 0.5% trypan blue stain. The data represent the means ± S.E. from three independent experiments.

       Homocysteine and H2O2 Act Synergistically to Decrease Mitochondrial RNA

      To determine whether homocysteine and H2O2 act synergistically to decrease steady-state mRNA levels of CO3/ATPase 6,8, DAMI cells were exposed to homocysteine in the presence or absence of exogenous H2O2. Cells exposed to 0.1 to 1.0 mm H2O2 for 18 h showed no change in CO3/ATPase 6,8 mRNA levels (Fig. 6 A). In contrast, when cells were exposed to 1 mm homocysteine together with H2O2 in concentrations ranging from 0.1 to 1.0 mm, there was a concentration-dependent reduction in steady-state mRNA levels of CO3/ATPase 6,8 (Fig. 6 B), an effect prevented by the addition of catalase (Fig. 6 C).
      Figure thumbnail gr6
      Figure 6Homocysteine, in the presence of H2O2, decreases the mRNA levels of CO3/ATPase 6,8 in DAMI cells. Total RNA isolated from DAMI cells treated for 18 h either in the presence of increasing concentrations of H2O2 (A), in the presence of homocysteine and increasing concentrations of H2O2 (B), or in the presence of homocysteine, catalase, and increasing concentrations of H2O2 (C) was analyzed by Northern blot hybridization using a CO3/ATPase 6,8 cDNA probe. Lower panels in A, B, and C correspond to controls for RNA loading and integrity as assessed by ethidium bromide staining of the 28S and 18S ribosomal RNA bands.

       Effect of Homocysteine ± Cu2+ on Mitochondrial Respiration Rates

      Mitochondrial respiration rates were measured to determine whether homocysteine, in the absence or presence of Cu2+, altered mitochondrial function (Fig. 7). Although exposure to 1 mmhomocysteine for 18 or 36 h had no effect on mitochondrial respiration rates, cells exposed to 5.0 mm homocysteine for 36 h showed a significant decrease (p < 0.01), when compared with control cells or cells exposed to 1 mmhomocysteine (Fig. 7 B). When cells were exposed to 1 mm homocysteine together with Cu2+, respiration rates were virtually undetectable at 18 h. This effect likely involves depolarization of the mitochondria because the decrease in respiration rates did not correlate with a significant decrease in mitochondrial protein levels (Fig. 3 B).
      Figure thumbnail gr7
      Figure 7Effect of homocysteine ± Cu2+ on mitochondrial respiration rates. Mitochondrial respiration rates were determined for control and DAMI cells treated with 1 or 5 mm homocysteine continuously for 18 (A) or 36 h (B) as described under “Experimental Procedures.” Treatment of DAMI cells with 1 mm homocysteine had no significant effect on mitochondrial respiration rates at 18 or 36 h, whereas treatment with 5 mm homocysteine for 36 h reduced respiration rates significantly (p < 0.01) compared with both control cells and cells treated with 1 mm homocysteine. Mitochondrial respiration rates in DAMI cells exposed to 1 mm homocysteine and 4 μm Cu2+were undetectable by 18 h. Values represent the means ± S.E. from three separate experiments.

       Ultrastructural Changes in Mitochondria from DAMI Cells Exposed to Homocysteine ± Cu2+

      Based on our observation that homocysteine influences mitochondrial gene expression and function, we examined mitochondrial ultrastructure in DAMI cells exposed to 1 mm homocysteine for 18 h, in the presence or absence of Cu2+ (Fig. 8). Compared with controls, cells exposed to homocysteine contained a subpopulation of enlarged mitochondria with fractured cristae (Fig. 8, compare A and B). Mitochondria also tended to be clustered toward the cell center. In addition, large intracytoplasmic vesicles were observed in DAMI cells exposed to homocysteine; structures not found in control cells. Cells exposed to both homocysteine and Cu2+ contained grossly enlarged mitochondria with the general organization of the inner membrane cristae virtually absent (Fig. 8 C). The addition of catalase prevented these gross morphological changes to the mitochondria (Fig. 8 D); however, the majority of mitochondria remained clustered toward the cell center. Despite these changes in mitochondrial ultrastructure, mitochondrial mass, as measured by cardiolipin staining with nonyl acridine orange, was unaltered in DAMI cells exposed to 1 or 5 mm homocysteine in the absence or presence Cu2+, for up to 18 h (data not shown).
      Figure thumbnail gr8
      Figure 8Ultrastructural analysis of DAMI cells demonstrating homocysteine-induced changes in mitochondria.Transmission electron microscopy of control DAMI cells (A), DAMI cells exposed to 1 mm homocysteine for 18 h (B), DAMI cells exposed to 1 mm homocysteine and 4 μm Cu2+ for 18 h (C), or DAMI cells exposed to 1 mm homocysteine, 4 μmCu2+, and 2500 units/ml catalase for 18 h (D). m, mitochondria; v, intracytoplasmic vesicles; er, endoplasmic reticulum. Magnification, × 5000.

       Homocysteine ± Cu2+ Increase Intracellular Levels of H2O2 in DAMI Cells

      To determine whether homocysteine, in the absence or presence of Cu2+, increases intracellular levels of H2O2, we measured DCF fluorescence using flow cytometry (
      • Cathcart R.
      • Schwiers E.
      • Ames B.N.
      ). As shown in Fig. 9, incubation of DAMI cells with 1 or 5 mm homocysteine did not significantly increase DCF fluorescence above that found in control or Cu2+-treated cells. In contrast, DCF fluorescence was increased 2–3-fold (p < 0.01) in cells exposed to homocysteine and Cu2+. As a positive control, DCF fluorescence increased approximately 5-fold when DAMI cells were exposed to 1 mmH2O2.
      Figure thumbnail gr9
      Figure 9Homocysteine and Cu2+ increase intracellular levels of H2O2. Following a 60-min pulse with 100 μm DCF-diacetate, DAMI cells were treated for 1 h in the absence (control) or presence of either 4 μm Cu2+, 1 or 5 mmhomocysteine (Hcy), 1 or 5 mm homocysteine and 4 μm Cu2+, or 1 mmH2O2. DCF fluorescence was determined for each sample by flow cytometry as described under “Experimental Procedures.” Results are represented as percentage of control and are the means ± S.E. of four separate cell experiments. *,p < 0.01 versus control cells.

       Precursors of Cellular Glutathione, but Not Free Radical Scavengers, Block the Decrease in Mitochondrial RNA Levels

      We determined whether precursors of glutathione or free radical scavengers blocked the adverse effects of homocysteine and Cu2+ on mitochondria by examining CO3/ATPase 6,8 mRNA levels. High concentrations of homocysteine can potentially enhance cellular glutathione levels and scavenge H2O2 (
      • Starkebaum G.
      • Harlan J.M.
      ). Unlike 1 or 5 mm homocysteine in the presence of Cu2+, 10 mm homocysteine together with Cu2+ blocked the decrease in CO3/ATPase 6,8 mRNA levels (Fig. 10 A). Similarly, the addition of 5 mm NAC, a general antioxidant and precursor of glutathione (
      • Toledano M.M.
      • Leonard W.J.
      ,
      • Abate C.
      • Patel L.
      • Rauscher F.J.
      • Curran T.
      ), to cells exposed to 5 mmhomocysteine and Cu2+, prevented the decrease in mRNA levels. To better define this protective effect, intracellular levels of GSH were measured from DAMI cells exposed to either homocysteine or NAC. As shown in Table III, DAMI cells exposed to 1 or 10 mm homocysteine showed increases in intracellular GSH of 50 and 113%, respectively, compared with control cells, a result consistent with previous studies using bovine aortic endothelial cells (
      • Upchurch Jr., G.R.
      • Welch G.N.
      • Fabian A.J.
      • Freedman J.E.
      • Johnson J.L.
      • Keaney Jr., J.F.
      • Loscalzo J.
      ). Cells exposed to NAC also showed similar increases in GSH levels. These findings suggest that the protective effect of high levels of homocysteine or NAC result, in part, from an increase in intracellular GSH.
      Figure thumbnail gr10
      Figure 10Precursors of intracellular glutathione (A), but not free radical scavengers (B), block the decrease in CO3/ATPase 6,8 mRNA levels in DAMI cells treated with homocysteine and Cu2+. Total RNA (10 μg/lane) isolated from DAMI cells cultured for 18 h was analyzed by Northern hybridization using a radiolabeled CO3/ATPase 6,8 cDNA probe (upper panel). Control for RNA loading and integrity was assessed by ethidium bromide staining of the 28S and 18S rRNA bands (lower panel).
      Table IIIIntracellular GSH levels in DAMI cells treated for 4 h with either homocysteine or NAC
      TreatmentGSHGSH
      nmol/106 cells% of control
      Control0.472 ± 0.011
      1 mm homocysteine0.710 ± 0.02350
      10 mm homocysteine1.005 ± 0.027
      p < 0.01 versus control and 1 mm homocysteine.
      113
      1 mm NAC0.676 ± 0.01643
      10 mm NAC0.928 ± 0.008
      p < 0.01 versus control and 1 mm NAC.
      97
      Intracellular levels of GSH were measured from whole cell lysates and are presented as the means ± S.E. from three independent experiments.
      3-a p < 0.01 versus control and 1 mm homocysteine.
      3-b p < 0.01 versus control and 1 mm NAC.
      Unlike the protective effects of high concentrations of homocysteine or NAC, free radical scavengers such as mannitol, dimethyl sulfoxide, and superoxide dismutase failed to block the decrease in the mRNA levels of CO3/ATPase 6,8 caused by homocysteine and Cu2+ (Fig. 10 B). In addition, superoxide dismutase in the presence of homocysteine alone caused a reduction in mRNA levels. Taken together, these findings suggest that H2O2, but not superoxide anion or hydroxyl radicals, acts preferentially with homocysteine to enhance mitochondrial damage.

       Decrease in Mitochondrial RNA Levels by Homocysteine and Cu2+ Is Blocked by Preexposure of DAMI Cells to Heat Shock

      Previous studies have demonstrated that heat shock pretreatment of U937 cells prevents H2O2-induced oxidative injury to mitochondria (
      • Polla B.S.
      • Kantengwa S.
      • Francois D.
      • Salvioli S.
      • Fancheschi C.
      • Marsac C.
      • Cossarizza A.
      ). To examine whether heat shock pretreatment also protects mitochondria from the effects of homocysteine and Cu2+, DAMI cells preexposed to heat shock (followed by a 6- or 18-h recovery period), were treated with homocysteine and Cu2+. As shown in Fig. 11, heat shock pretreatment prevented the decrease in the mRNA levels of both CO3/ATPase 6,8 and HSP60 (Fig. 11, compare lanes 1 and 2 withlanes 4–7). Preexposure to heat shock alone did not alter mitochondrial RNA levels (compare lanes 1 and 3).
      Figure thumbnail gr11
      Figure 11Preexposure to heat shock blocks the decrease in CO3/ATPase 6,8 and HSP60 mRNA levels in DAMI cells treated with homocysteine and Cu2+. DAMI cells were preexposed for 2 h at 42 °C and allowed to recover for 6 (lanes 4 and 5) or 18 (lanes 6 and 7) h at 37 °C. Following recovery, cells were exposed to homocysteine and Cu2+ for 18 h, and total RNA was isolated and analyzed by Northern hybridization using radiolabeled cDNA probes to either CO3/ATPase 6,8 (A) or HSP60 (B) (upper panel). Control for RNA loading and integrity was assessed by ethidium bromide staining of the 28S and 18S rRNA bands (lower panel).

      DISCUSSION

      Previous studies have shown mitochondrial abnormalities in ECs from the aortas of hypertensive rats with diet-induced HH (
      • Matthias D.
      • Becker C.-H.
      • Riezler R.
      • Kindling P.H.
      ) and in hepatocytes of patients with HH (
      • McCully K.S.
      ,
      • McCully K.S.
      ). Although these studies suggest that homocysteine adversely affects mitochondria, the mechanism of action is unknown. In this study, we demonstrate that homocysteine alters mitochondrial gene expression, function, and structure and provide evidence that homocysteine and H2O2 act synergistically to enhance mitochondrial damage. Furthermore, our findings suggest that intracellular glutathione levels and heat shock proteins play an important role in protecting mitochondria from the damaging effects of homocysteine and H2O2.
      Although 1 mm homocysteine had no effect on cell growth or mitochondrial respiration rates, it induced changes in mitochondrial ultrastructure and increased mitochondrial RNA levels. Similar increases in mitochondrial RNA levels have also been observed in the livers of cystathionine β-synthase deficient mice having HH.
      R. C. Austin and S. K. Sood, unpublished observations.
      This effect likely involves, in part, the thiol group of homocysteine because other thiol-containing agents show similar but less dramatic effects on mitochondrial mRNA levels. These findings suggest that homocysteine causes minor mitochondrial damage and/or turnover to which cells respond by increasing mitochondrial gene expression, thereby maintaining growth and viability. In support of this concept, the homocysteine-induced expression of NAD-dependent methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase in human vascular ECs (
      • Kokame K.
      • Kato H.
      • Miyata T.
      ) is associated with increased mitochondrial biogenesis (
      • Yang X.
      • MacKenzie R.E.
      ).
      Increased mitochondrial RNA levels may also reflect a cellular response involving either the metabolism of homocysteine or its removal from the cell, both of which require cellular ATP. It is well established that the mitochondrial folate metabolism pathway is an important component of the homocysteine remethylation pathway (
      • Mayer E.L.
      • Jacobsen D.W.
      • Robinson K.
      ,
      • Ueland P.M.
      • Refsum H.
      • Brattstrom L.
      ,
      • Welch G.N.
      • Loscalzo J.
      ,
      • McCully K.S.
      ,
      • Girgis S.
      • Suh J.R.
      • Jolivet J.
      • Stover P.J.
      ). Thus, an increase in intracellular levels of homocysteine, either endogenously (formed in the methionine biosynthetic pathway) or exogenously (added to the culture medium), would be expected to activate genes encoding enzymes within this pathway. The finding that homocysteine induces the expression of NAD-dependent methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, a bifunctional mitochondrial enzyme involved in homocysteine remethylation (
      • Kokame K.
      • Kato H.
      • Miyata T.
      ), supports this concept. Because the mitochondrial folate and homocysteine remethylation pathways require cellular ATP, increased intracellular levels of homocysteine would be expected to deplete cellular ATP levels. This is consistent with recent findings indicating that overexpression of SAM in Chinese hamster ovary cells causes depletion of ATP (
      • Sanchez-Gongora E.
      • Pastorino J.G.
      • Alvarez L.
      • Pajares M.A.
      • Garcia C.
      • Vina J.R.
      • Mato J.M.
      • Farber J.L.
      ). Depletion of cellular ATP could also result from the conversion of excess intracellular homocysteine to homocysteine thiolactone, an ATP-dependent reaction catalyzed by methionyl-tRNA synthase (
      • Jakubowski H.
      ). Thus, as homocysteine accumulates within the cell, a futile cycle of synthesis and hydrolysis of homocysteine thiolactone occurs, which could potentially deplete intracellular ATP levels. To compensate for this decrease, cells would need to generate more ATP, presumably by increasing both mitochondrial activity and gene expression. This is consistent with our observation that homocysteine induces the expression of CO3/ATPase 6,8 and ND5, both of which are components of the respiratory transport chain.
      Earlier in vitro studies have demonstrated that Cu2+ or ceruloplasmin catalyzes the auto-oxidation of homocysteine, thereby generating H2O2, increasing intracellular levels of homocysteine, and leading to cell injury (
      • Starkebaum G.
      • Harlan J.M.
      ,
      • de Groot P.G.
      • Willems C.
      • Boers G.H.J.
      • Gonsalves M.D.
      • VanAken W.G.
      • Mourik J.A.
      ,
      • Hultberg B.
      • Andersson A.
      • Isaksson A.
      ). Because catalase prevents homocysteine-induced cell injury, it has been suggested that cell injury is largely due to H2O2 and not homocysteine. Our findings also confirm the importance of H2O2; however, we support a mechanism whereby homocysteine and H2O2 act synergistically to cause mitochondrial damage and to decrease cell growth and viability. Thus, we have shown that homocysteine in the presence of Cu2+ causes severe changes in mitochondrial ultrastructure, decreases mitochondrial RNA levels (marker of mitochondrial damage) and inhibits both cell growth and mitochondrial respiration rates. Furthermore, intracellular levels of H2O2 are significantly increased in the presence of homocysteine and Cu2+, and homocysteine together with H2O2, but not H2O2 alone, decreases mitochondrial RNA levels. The observation that catalase, but not free radical scavengers, blocks the decrease in mitochondrial RNA levels and prevents changes in mitochondrial ultrastructure indicates that this effect is specific for H2O2 and not superoxide anions or hydroxyl radicals. These findings are also consistent with previous studies demonstrating that extracellularly generated free radicals are unlikely to contribute to homocysteine-induced cytotoxicity (
      • Blundell G.
      • Jones B.G.
      • Rose F.A.
      • Tudball N.
      ). Whether these increased intracellular levels of H2O2 directly cause mitochondrial damage or lead to an increase in intracellular free radicals, which have been shown to play a role in homocysteine-induced cytotoxicity (
      • Blundell G.
      • Jones B.G.
      • Rose F.A.
      • Tudball N.
      ), is not known. However, the observation that H2O2 fails to increase mitochondrial damage and does not inactivate cytochrome c oxidase (
      • Higuchi Y.
      • Linn S.
      ) suggests the involvement of intracellular free radicals generated from H2O2.
      How homocysteine and H2O2 act together to enhance mitochondrial damage is not entirely clear; however, several possibilities exist. The fact that homocysteine does not elicit an oxidative stress response (
      • Outinen P.A.
      • Sood S.K.
      • Liaw P.C.Y.
      • Sarge K.D.
      • Maeda N.
      • Hirsh J.
      • Ribau J.
      • Podor T.J.
      • Weitz J.I.
      • Austin R.C.
      ) and decreases the activity of several antioxidant enzymes both in vitro and in vivo(
      • Outinen P.A.
      • Sood S.K.
      • Liaw P.C.Y.
      • Sarge K.D.
      • Maeda N.
      • Hirsh J.
      • Ribau J.
      • Podor T.J.
      • Weitz J.I.
      • Austin R.C.
      ,
      • Upchurch Jr., G.R.
      • Welch G.N.
      • Fabian A.J.
      • Freedman J.E.
      • Johnson J.L.
      • Keaney Jr., J.F.
      • Loscalzo J.
      ,
      • Toborek M.
      • Kopieczna-Grzebieniak E.
      • Drozdz M.
      • Wieczorek M.
      ) suggests that homocysteine could potentially enhance the susceptibility of cells to H2O2. This is particularly relevant because heat shock pretreatment, which induces the expression of HSPs and is known to protect mitochondria from H2O2-induced cell injury (
      • Polla B.S.
      • Kantengwa S.
      • Francois D.
      • Salvioli S.
      • Fancheschi C.
      • Marsac C.
      • Cossarizza A.
      ), prevents the decrease in mitochondrial RNA levels caused by homocysteine and Cu2+. The ability of homocysteine to deplete cellular levels of NAD and ATP could potentially lead to de-energization of mitochondria, resulting in increased susceptibility to oxidative stress. Indeed, overexpression of SAM in Chinese hamster ovary cells leads to NAD and ATP depletion and enhanced H2O2-induced oxidative injury (
      • Sanchez-Gongora E.
      • Pastorino J.G.
      • Alvarez L.
      • Pajares M.A.
      • Garcia C.
      • Vina J.R.
      • Mato J.M.
      • Farber J.L.
      ). Alternatively, homocysteine could potentially affect bcl-2expression or cause cytochrome c release from mitochondria, key components involved in mitochondria-mediated cell death (
      • Wyllie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      ).
      Our findings, as well as those of others (
      • Wall R.T.
      • Harlan J.M.
      • Harker L.A.
      • Striker G.E.
      ,
      • Starkebaum G.
      • Harlan J.M.
      ,
      • de Groot P.G.
      • Willems C.
      • Boers G.H.J.
      • Gonsalves M.D.
      • VanAken W.G.
      • Mourik J.A.
      ,
      • Hultberg B.
      • Andersson A.
      • Isaksson A.
      ,
      • Blundell G.
      • Jones B.G.
      • Rose F.A.
      • Tudball N.
      ), suggest that homocysteine-induced cell toxicity is dependent on H2O2. Although the relationship between homocysteine-induced vascular disease and H2O2remains unknown, recent studies suggest that H2O2 likely plays a role in the progression of vascular disease and that homocysteine may accelerate this process. Thus, the expression and synthesis of extracellular superoxide dismutase are increased in macrophages within atherosclerotic vessels, which could lead to an accumulation of extracellular H2O2 (
      • Fukai T.
      • Galis Z.S.
      • Meng X.P.
      • Parthasarathy S.
      • Harrison D.G.
      ). Furthermore, rabbits fed a high-methionine diet, which leads to HH, have elevated levels of aortic superoxide dismutase, as well as disturbances in lipid peroxidation and antioxidant processes (
      • Toborek M.
      • Kopieczna-Grzebieniak E.
      • Drozdz M.
      • Wieczorek M.
      ). Whether homocysteine directly alters the expression of superoxide dismutase in these macrophages and/or enhances the effects of H2O2 in atherosclerotic vessels remains to be determined.
      In summary, these studies demonstrate that homocysteine alters mitochondrial gene expression, structure, and function. In addition, homocysteine and H2O2 act synergistically to promote mitochondrial damage, possibly by increasing the susceptibility of cells to oxidative stress. The recent observation that overexpression of SAM in Chinese hamster ovary cells enhances H2O2-induced oxidative injury (
      • Sanchez-Gongora E.
      • Pastorino J.G.
      • Alvarez L.
      • Pajares M.A.
      • Garcia C.
      • Vina J.R.
      • Mato J.M.
      • Farber J.L.
      ) supports this concept and suggests an intricate relationship among homocysteine metabolism, oxidative stress, and mitochondrial function. Although our findings provide evidence for homocysteine-induced mitochondrial damage in DAMI cells, it is unclear whether this occurs in all cell types. Based on the variability in homocysteine metabolism (
      • Jakubowski H.
      ), mitochondrial activity (
      • Pollack J.K.
      • Sutton R.
      ,
      • Valcarce C.
      • Navarrete R.M.
      • Encabo P.
      • Loeches E.
      • Satrustegui J.
      • Cuezva J.M.
      ), and antioxidant status (
      • Sies H.
      ) among mammalian cellsin vitro and in vivo, it is likely that differences in susceptibility exist. Further elucidation of the mechanism by which homocysteine and H2O2 causes mitochondrial damage may allow for a better understanding of the cytotoxic effects elicited by this physiologically relevant thiol-containing amino acid.

      ACKNOWLEDGEMENT

      We thank Dr. Jack Hirsh for critical reading of the manuscript.

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