Postlipopolysaccharide Oxidative Damage of Mitochondrial DNA
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Department of Medicine, Duke University Medical Center, Durham, North Carolina}l, http://www.100md.com
ABSTRACT}l, http://www.100md.com
Selected structural and functional alterations of mitochondria induced by bacterial lipopolysaccharide (LPS) were investigated on the basis of the hypothesis that LPS initiates hepatic mitochondrial DNA (mtDNA) damage by oxidative mechanisms. After a single intraperitoneal injection of Escherichia coli LPS, liver mtDNA copy number decreased, as determined by Southern analysis, within 24 hours relative to nuclear 18S rRNA (p < 0.05). LPS induced a novel oxidant-dependent 3.8-kb mtDNA deletion in the region encoding NADH dehydrogenase subunits 1 and 2 and cytochrome c oxidase subunit I, which correlated with mitochondrial glutathione depletion. Expression of mitochondrial mRNA and transcription of mitochondrial RNA were suppressed, whereas mRNA expression increased for selected nuclear-encoded mitochondrial proteins. Resolution of mtDNA damage was mediated by importation of mitochondrial transcription factor A protein, a central regulator of mtDNA copy number, accompanied by binding of mitochondrial protein extract to the mitochondrial transcription factor A DNA-binding site. Hence, mtDNA integrity and transcriptional capacity after LPS administration appeared to be reinstated by mitochondrial biogenesis. These data provide the first link between LPS-mediated hepatic injury and a specific oxidative mtDNA deletion, which inhibits mitochondrial transcription and is restored by activation of mechanisms that lead to biogenesis.
Key Words: mitochondria • mitochondrial DNA deletion • glutathione • reactive oxygen species • mitochondrial transcription factorr7, http://www.100md.com
In sepsis, the liver contributes to both the pathogenesis of the multiple organ dysfunction syndrome and the body's defenses against it (1). Toxic products of gram-negative bacteria, such as lipopolysaccharide (LPS), can enter the portal system, for example, in the presence of increased intestinal permeability, and injure the liver, which releases acute-phase reactant proteins and proinflammatory mediators (2). These mediators, such as tumor necrosis factor-{alpha} , interleukin-1, and interleukin-6, stimulate the production of reactive oxygen species (ROS) and reactive nitrogen species involved in organ failure (1–4). Severe sepsis and shock also challenge oxygen (O2) supply–demand regulation, impair O2 extraction, and lead to lactic acidosis and a fall in mitochondrial oxidation–reduction (redox) state. Virtually nothing, however, is known of the molecular mechanisms of injury involving mitochondria.
Liver mitochondria are an abundant source of ROS, such as superoxide anion (O2·-), generated by incomplete reduction of molecular O2 (5). Normally, 1–2% of the O2 consumed is reduced to O2·-, which is rapidly converted to a less reactive species, H2O2, by manganese superoxide dismutase (MnSOD). H2O2 is metabolized to O2 and H2O, primarily by glutathione peroxidase and catalase. At low concentrations, H2O2 stimulates cell proliferation; however, at high concentrations, oxidative damage occurs (6–8). Mitochondria therefore generate ROS that not only oxidize other cellular constituents, but they also can be damaged by lipid peroxidation and protein and nucleic acid oxidation (6–8).7y|4*, http://www.100md.com
The circular mitochondrial DNA (mtDNA) genome is susceptible to oxidation because it is proximate to mitochondrial redox sites and lacks protective histones (9, 10). These features, a low copy number, and a lag in repair of mtDNA relative to nuclear DNA make mtDNA damage a sensitive index of oxidative stress. However, mtDNA oxidation may also impair mitochondrial transcription and functional capacity (11). Several laboratories have shown that endogenous ROS mediate mtDNA damage and alter mitochondrial gene expression and function (9–14), and failure to meet cellular energy requirements contributes to diseases of defective mtDNA (11, 14, 15).
The mitochondrial genome encodes 22 tRNAs, 2 rRNAs, and 13 polypeptides of the electron transport complexes. Thus, most of the approximately 200 mitochondrial proteins are encoded in the nucleus. Nuclear genes of respiration respond rapidly to cell activation by the induction of specific transcription factors (16). Nuclear mitochondrial transcription factor (Tfam) A regulates mammalian mtDNA replication and transcription (17, 18). Thus, mitochondrial biogenesis appears to be coordinated by interactions between the two genomes through expression of regulatory proteins such as Tfam. This background led us to the hypothesis that hepatic injury by LPS-associated ROS production causes specific oxidative mtDNA damage and impairs mitochondrial transcription. If true, this mechanism could stimulate Tfam importation and activation of mitochondrial biogenesis.m85[jg&, http://www.100md.com
METHODSm85[jg&, http://www.100md.com
Animal Protocolm85[jg&, http://www.100md.com
The protocol was approved by the Duke University (Durham, NC) Animal Care and Use Committee. Adult male Sprague-Dawley rats (300–400 g) were injected with a sublethal intraperitoneal dose of crude LPS (Escherichia coli 055 B5; Difco, Detroit, MI) (1,000 µg/kg) dissolved in 0.5 ml of sterile, pyrogen-free 0.9% sodium chloride (saline) and the livers were harvested after 1, 2, 5, or 10 days. Control animals were injected with 0.5 ml of sterile saline. The LPS dose was chosen after preliminary studies showed that the histologic changes in the liver returned to normal over this period. The animals were anesthetized with halothane, the abdomens were opened, and the aortas were transected. The livers were excised and placed in cold isolation buffer. Approximately 4 g of tissue was used to isolate mitochondria and 2 g was snap frozen in liquid N2 and stored at -80°C.
Organelle Isolation#:'d@, 百拇医药
Highly purified mitochondria were obtained from liver homogenate by discontinuous Percoll gradient centrifugation, using a method that produces well-coupled organelles in control and LPS-exposed animals (3, 19, 20). The mitochondria-rich band at the interface between Percoll layers was harvested, taking care to collect intact organelles of the same density from all rats. The mitochondria were washed and the pellet resuspended and placed on ice. In some experiments, crude mitochondria were prepared by centrifugation of liver homogenates through 0.25% sucrose and used for respiration studies. Respiratory capacity and respiratory control ratios were measured at 30°C with Clark electrodes as reported (3). Nuclei were obtained by centrifugation of liver homogenates at 1,000 x g for 20 minutes and centrifugation through 1.75 M sucrose for 1 hour at 40,000 x g. Protein content was determined by the bicinchoninic acid method (Pierce, Rockford, IL).
Microscopy and Mitochondrial Ultrastructure', 百拇医药
Microscopy was performed with sections of livers flushed with phosphate-buffered saline (PBS), pH 7.0, and perfusion fixed with 4% paraformaldehyde. The livers were removed, immersed in fixative for 2 hours, and stored in 2% paraformaldehyde at 4°C until paraffin embedding, sectioning, and staining for light microscopy. For electron microscopy, the livers of three control rats and three at each time point after LPS administration were flushed with PBS and perfusion fixed with fresh 2% paraformaldehyde plus 0.2% glutaraldehyde in 0.1 M PBS. These livers were dissected into small blocks and treated with 2% osmium tetroxide in 0.1 M PBS, washed, dehydrated, and embedded in Epon. Random thin sections (0.05 µm) were cut (Ultracut N; Reichert-Nissei, Ratingen, Germany), mounted on nickel grids, stained with uranyl acetate and lead citrate, and photographed on an electron microscope (model CM12; Philips, Eindhoven, The Netherlands).
Glutathione and Lipid Hydroperoxide Measurementsw., 百拇医药
Fresh mitochondria were sonicated in 5% metaphosphoric acid followed by centrifugation at 1,000 x g for 10 minutes at 4°C. The supernatant was snap frozen and stored at -80°C until assay. The total glutathione (GSH) and glutathione disulfide (GSSG) content of isolated mitochondria and liver homogenate was determined by enzymatic recycling (21). GSSG was determined after derivatization of GSH with 2-vinylpyridine. Lipid peroxidation was assessed as malondialdehyde content (Calbiochem, La Jolla, CA) and expressed as nanomoles per milligram of protein.w., 百拇医药
DNA Extractionw., 百拇医药
Mitochondria were suspended in 3 ml of 10 mM TRIS-HCl–1 mM EDTA (TE) and incubated with 330 µl of 10% sodium dodecyl sulfate (SDS) and 400 µl of proteinase K (10 mg/ml) at 50°C for 3 hours. The digest was extracted three times with 3 ml of phenol saturated with TE (1 mM EDTA, 10 mM TRIS-HCl, pH 7.4) by shaking gently for 30 minutes followed by extraction with 3 ml of phenol–chloroform (1:1, vol/vol) (12). DNA was precipitated from the aqueous phase with 3 M sodium acetate in 99.5% ethanol followed by incubation for 30 minutes at -80°C. Precipitate was collected by centrifugation, washed with 70% ethanol, dried, and resuspended in TE. Purified mtDNA was stored at -20°C.
Nuclear Oligonucleosomal DNA Cleavagemn, http://www.100md.com
Fresh liver was homogenized in 10 mM TRIS-HCl lysis buffer (pH 8.0) on ice. Polyethylene glycol (2.5%) and NaCl (to 1 M) were added and samples were cold-centrifuged at 12,000 x g for 15 minutes. Supernatants were incubated with DNase-free ribonuclease (400 µg/ml; Sigma, St. Louis, MO) at 37°C for 1 hour and treated with proteinase K (400 µg/ml; Sigma) for 1 hour. They were precipitated overnight in isopropanol at -20°C and centrifuged, and the pellets were dissolved in TRIS–EDTA (pH 8.0). DNA was electrophoresed on 1.5% agarose containing GelStar (BioWhittaker Molecular Applications [formerly FMC Bioproducts], Rockland, ME) and viewed under ultraviolet light for ladder formation.mn, http://www.100md.com
Mitochondrial DNA Copy Number by Southern Analysismn, http://www.100md.com
Cellular DNA was extracted from liver (in the presence of RNase and proteinase K) and digested with BamHI. mtDNA was prepared from mitochondria suspended in 10 mM TRIS-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 2% SDS and incubated at 37°C for 10 minutes in the presence of proteinase K (50 g/ml). Nucleic acids were extracted, resuspended in 10 mM TRIS-HCl–1 mM EDTA, pH 8, and incubated with RNase A (50 g/ml) at 37°C for 15 minutes. mtDNA was precipitated with ethanol, resuspended in H2O, and cut with endonuclease PvuII. DNA fragments were resolved on 0.8% agarose gels and transferred to nylon membranes (GeneScreen; Du Pont-NEN, Boston, MA) and then incubated with [32P]dCTP-labeled linearized hepatic mtDNA. Probes for the 16.3-kb mtDNA molecule and for nuclear 18S rRNA were used to control for DNA loading. DNA probes were labeled by nick translation at 15°C for 1 hour, using 50 Ci of [32P]dCTP (3,000 Ci/mmol). Hybridization was carried out at 42°C for 18 hours, after which membranes were washed according to standard procedures. Hybridization signals were analyzed within the linear range of the film by laser scanning densitometry.
Semiquantitative Polymerase Chain Reaction19, 百拇医药
mtDNA was analyzed for a deletion between direct repeats (direct repeat 16 corresponding to bp 1095–4905 of rat mtDNA) by polymerase chain reaction (PCR) with the following primers:19, 百拇医药
The number of PCR cycles was optimized during the exponential phase of the PCR by titration of visible products on GelStar-stained agarose gels.19, 百拇医药
Electrophoretic Mobility Shift Assay19, 百拇医药
An electrophoretic mobility shift assay (EMSA) was developed for Tfam, using 32P-labeled oligonucleotide (5'-TTTTAACTTAAATCTTAGCATTGGTA-3') representing the Tfam-binding site (underlined) on the rat light strand promoter (LSP). Mitochondria were suspended in poly(dI-dC) (20 µg/ml), 50 µM pyrophosphate, and 0.5% Triton X-100 in a buffer of 0.1 M MgCl2, 50 mM dithiothreitol, 1 mM spermidine, 1 mM EDTA, and 20 mM TRIS (pH 7.6). The suspension was sonicated for 10 seconds at 4°C, brought to room temperature, and incubated with labeled oligonucleotide. Specific and nonspecific competition assays were performed by preincubating mitochondrial extracts with a 200-fold excess of cold nucleotide for the Tfam LSP-binding site. After the binding reaction, the mixture was separated on a nondenaturing 6% polyacrylamide gel, fixed with acetate–methanol–water (10:30:60, vol/vol/vol), dried, and quantified by image analysis software (Bio-Rad, Hercules, CA).
RNA Isolation and Detection of Nuclear and Mitochondrial mRNA Expressionimpf(1?, 百拇医药
Cytoplasmic RNA was extracted with a TRIzol total RNA isolation kit (GIBCO, Gaithersburg, MD) (22). Total RNA (1 µg) from each sample was reverse transcribed with oligo(dT) or with gene-specific primers for rat 12S rRNA, NADH dehydrogenase subunits 1 and 2 (ND1 and ND2, respectively), cytochrome c oxidase subunits I and IV (COX I and COX IV, respectively), MnSOD, augmenter of liver regeneration (ALR), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA . GAPDH was used to control for nuclear-encoded genes whereas mtRNA gene expression was normalized to 18S rRNA. Quantification of the amplified product was performed in triplicate by gel densitometry.impf(1?, 百拇医药
fig.ommittedimpf(1?, 百拇医药
TABLE 1. Primer sequences used to amplify mitochondrial and nuclear mRNAimpf(1?, 百拇医药
In Organello RNA Synthesisimpf(1?, 百拇医药
RNA synthesis in organello was assessed in mitochondria with and without 1-hour exposures to 0 to 1.0 mM H2O2 at 37°C and compared with identical transcription analysis of mitochondria isolated 24 hours after LPS injection. In organello assays were performed by adding 20 µCi of [{alpha} -32P]UTP (400 to 600 Ci/mmol) to incubation buffer at 37°C for 60–120 minutes in a rotary shaker. Pulse–chase experiments were performed in mitochondria prelabeled with [{alpha} -32P]UTP for 2 hours; mitochondria were centrifuged at 13,000 x g and the supernatant, containing nonincorporated [{alpha} -32P]UTP, was decanted. Mitochondria were resuspended in incubation medium with a 200-fold excess of unlabeled UTP and incubated for various periods of time before harvesting. mtRNA was extracted with a kit (GIBCO-BRL Life Technologies, Gaithersburg, MD) and analyzed by agarose gel electrophoresis. Gels were exposed for autoradiography at -70°C and RNA was quantified with a PhosphorImager screen and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Statistical Analysis%d, http://www.100md.com
Data were expressed as means ± SEM. Statistics were performed by analysis of variance followed by Tukey's post hoc comparison. A probability of p < 0.05 was considered significant. Values of n refer to data from separate experiments. Linear regressions were performed with StatView (version 5.0.1; SAS Institute, Cary, NC).%d, http://www.100md.com
RESULTS%d, http://www.100md.com
Histology and Mitochondrial Structure%d, http://www.100md.com
Representative photomicrographs of random liver sections before and after LPS injection are shown in . By light microscopy, LPS produced small necrotic foci , which tended to involve midzonal cells. Necrotic foci involved several dozen cells and accounted for 5–10% of the sectional area. Small clusters of inflammatory cells were present around these foci and around the central veins, primarily on Day 1. By Day 2, enlarged hepatocytes and nuclei and an increase in mitotic figures were observed . By Day 5, necrotic hepatocytes were no longer present, but scattered mitotic figures and mononuclear cells were apparent . By Day 10 the livers appeared normal (not shown).
fig.ommittedaqc'r;, 百拇医药
Figure 1. Representative histology and ultrastructure of liver of LPS-treated rats. (A) Effect of LPS treatment on liver histology. (a) Day 0: Control liver section. No significant structural abnormalities noted. (b) Day 1: Liver section shows a necrotic focus (arrow) with rim of scattered inflammatory cells. (c) Day 2: Note the increase in mitotic figures (arrowheads). (d) Day 5: Liver section shows the presence of a few infiltrating mononuclear cells and scattered mitotic figures. (A) represents one of four independent experiments. (H&E stain; original magnification, x300.) (B) Representative electron photomicrographs of hepatic mitochondrial ultrastructure. (a) Day 0: Control rat liver. (b) Day 1: Liver 24 hours after LPS treatment shows increased number of mitochondrial profiles associated with condensation and elongated forms (short arrows). Membrane whorls were seen on the surface of many mitochondria (long arrow). Some hepatocytes showed dilated endoplasmic reticulum (arrowhead). (c) Day 2: Liver shows more normal mitochondrial morphology. (d) Day 5: Rat liver 5 days after LPS injection shows normal mitochondrial appearance. m = Mitochondria; n = nucleus. (Original magnification, x20,000.)
On electron microscopy, orderly changes in mitochondrial ultrastructure were identified after LPS injection . In control liver, the mitochondria were smooth with distinct cristae and complete membranes. One day after LPS injection, the mitochondrial profile density in the hepatocytes increased in association with many irregular and elongated forms . Internal structure was hypercondensed and obscured by electron-dense matrix. A few mitochondria were associated with rough endoplasmic reticulum (RER), and alterations of the outer membrane, including surface whorls, were common. Distorted cristae in some organelles indicated degeneration. These changes were typical of mitochondrial condensation and/or proliferation (23), and the dumbbell-shaped organelles suggested biogenesis. Two days after LPS, cisternae of RER were wrapped around many mitochondria . The matrix remained condensed. Five days after LPS, association of mitochondria with RER was no longer abundant . The matrix still appeared condensed, but mitochondrial volume density appeared normal. By 10 days, no consistent abnormalities were seen (not shown).
Oxygen Consumption and Oxidative Stress in Mitochondriacoh#z, http://www.100md.com
State 3 and State 4 respiration rates were measured in quadruplicate in crude and highly purified samples of mitochondria of control and LPS-exposed rats on Day 1. In crude mitochondria of control and LPS-exposed rats, respiratory control ratios (RCRs) ranged from 3 to 5 with glutamate and malate as substrates and from 4 to 7 with succinate as substrate. Average RCRs were not significantly different between crude liver mitochondria of control and LPS-treated rats, but the maximum respiratory capacity (State 3) was reduced by 22 ± 3% in the organelles of LPS-exposed rats after 24 hours (p < 0.05). In highly purified organelles, RCRs were significantly higher relative to the crude preparation only when succinate was used as substrate (5.5 versus 8.1, p < 0.05), but neither RCR nor State 3 was different in rats treated with LPS compared with controls. The remaining studies were performed only in highly purified mitochondria.
To assess oxidative stress after LPS injection, the content of glutathione (GSH), the main low molecular weight antioxidant in mitochondria, was measured. Baseline hepatic GSH levels did not change significantly in the LPS-treated rats, whereas levels of GSH in the mitochondrial compartment decreased significantly 1 day after LPS treatment . GSSG markedly and significantly accumulated in liver homogenate of LPS-treated rats by 1 day and the GSSG:GSH ratio increased significantly before decreasing to normal . Typical of mitochondrial GSSG export after GSH oxidation, the GSSG level inside the mitochondria did not change. The GSSG:GSH ratio in mitochondria increased significantly after LPS administration and then decreased, returning to normal by 5 days . The changes in GSH indicated a substantial oxidative stress in response to LPS.)^.ev{&, 百拇医药
fig.ommitted)^.ev{&, 百拇医药
Figure 2. Oxidative stress in liver of LPS-treated rats. (A) Total GSH and GSSG in liver after LPS treatment. (a) Total GSH; (b) oxidized glutathione (GSSG); (c) GSSG/GSH ratio as a percentage of total GSH; (d) levels of mitochondrial GSH; (e) oxidized mitochondrial glutathione (GSSG); (f) GSSG/GSH ratio as a percentage of total mitochondrial GSH. (B) Malondialdehyde concentration in mitochondria and liver homogenate. (a) Malondialdehyde level in the mitochondrial compartment; (b) Malondialdehyde level in liver total homogenate. Values are expressed as means ± SEM for n = 4 animals per group. *p < 0.05.
The extent of lipid peroxidation, measured as the major aldehyde end product malondialdehyde, indicated cumulative oxidative damage in membrane lipids (24). Malondialdehyde was elevated in both liver homogenate and mitochondria on Day 1 after LPS and then recovered over 2 to 10 days . Thus, LPS induced significant but reversible oxidative stress by depleting GSH and enhancing lipid peroxidation.|\, 百拇医药
DNA Fragmentation|\, 百拇医药
As expected, necrosis was the major cause of hepatocyte death after LPS treatment. shows primarily high molecular weight genomic DNA, detected as smearing of the band without laddering 1 day after LPS administration. DNA fragments larger than 10,000 bp often originate from cells dying via necrosis. Cleavage of oligonucleosomal chromatin in multiples of 180-bp fragments, a hallmark of apoptosis, was absent by ladder detection assay (25, 26).|\, 百拇医药
fig.ommitted|\, 百拇医药
Figure 3. Agarose gel electrophoresis of oligonucleosomal DNA from livers of rats treated with LPS. Illustrated is genomic DNA from whole livers at different time points after LPS injection. The GelStar-stained gel was inverted to improve resolution and to show the integrity of genomic DNA. Each well was loaded with DNA (15 µg/lane). The decrease in the large band of genomic DNA and concomitant classic appearance of smearing of band at 10 kb is typical of necrotic cells. Lane M: Migration of a positive control 1-kb DNA ladder. Data are representative of four independent studies.
Assessment of mtDNA Copy Number and mtDNA Damage6.v^&%), http://www.100md.com
mtDNA copy number was assessed by Southern analysis, and the data were expressed as the ratio of mtDNA to nuclear DNA (18S rRNA) in quadruplicate experiments. The copy number in tightly coupled mitochondria decreased by an average of 28% (p < 0.05) on Day 1 after LPS treatment compared with control rats, followed by recovery (see ) . The location of mtDNA damage was assessed relative to nuclear DNA, using primers encompassing two GC-rich repetitive sequences in the rat mitochondrial genome (see ) . These direct repeats undergo oxidative modification, eventually resulting in a bulky deletion detectable by PCR (12). After LPS administration, the quantity of deleted mtDNA detected by PCR correlated by linear regression analysis with the GSSG:GSH ratio in mitochondria (r2 = 0.88) and the malondialdehyde content of mitochondria (r2 = 0.94). The amount of deletion also correlated with the accumulation of GSSG in whole liver homogenate (r2 = 0.95).
fig.ommitted3ji!, 百拇医药
Figure 4. Southern blot analysis of hepatic mtDNA depletion after LPS injection. Top bands show autoradiography signals from the mtDNA fragment, and bottom bands show signals from the nuclear DNA fragment containing the 18S rRNA gene. The line below the bands shows the mtDNA:18S intensity ratio as a percentage. Data are representative of four independent studies. Values are expressed as means ± SEM for n = 4 animals per group.3ji!, 百拇医药
fig.ommitted3ji!, 百拇医药
Figure 5. Map of the rat mitochondrial genome. The 16.3-kb rat mitochondrial genome is illustrated with 13 mRNA-coding regions, 2 rRNA (12S and 16S)-coding regions, and 22 tRNA-coding regions. mRNA genes are shown as the areas labeled with the codes of the corresponding electron transport chain Complexes I, III, IV, and V. mtDNA deletion primers mtf1 and mtr2 used in the deletion analysis are represented as arrows. PH and PL = promoters of heavy (H) and light (L) strand transcription, respectively.3ji!, 百拇医药
shows the results of PCR amplification carried out for 35 cycles at 94°C for 40 seconds, 50°C for 30 seconds, and 72°C for 2 minutes. The PCR product of the intact wild-type mtDNA is 4669 bp, whereas the size of the deletion between the direct repeats is 3810 bp, producing a predicted PCR deletion product of 859 bp. This deletion was essentially undetectable in control mitochondria . In contrast, the deletion transiently accumulates in liver mitochondria after LPS injection and was most abundant on Day 1, when its band density was always at least an order of magnitude greater than the control background. The density of the PCR deletion product was approximately 20% of total mtDNA, but became progressively less in mitochondria isolated 2 to 10 days after LPS treatment.
fig.ommitted2lc?, 百拇医药
Figure 6. Identification and verification of mtDNA deletion. (A) GelStar-stained 1.5% agarose gel demonstrating semiquantitative PCR detection of deletion in liver mtDNA. (B) Densitometry analysis of (A); results are presented as deletion (expressed as a percentage) by normalizing the optical density of deleted mtDNA to that of wild-type mtDNA and multiplying by 100. The deletion was undetectable in mitochondria isolated from control rats. In contrast, the deletion was readily detectable in hepatic mitochondria 1 and 2 days after LPS injection. Data represent four independent studies. Densitometry values are expressed as means ± SEM for n = 4 animals per group. Symbols indicate p < 0.05 relative to control.2lc?, 百拇医药
Positive identification of deletion bands was confirmed by sequencing after purifying the PCR products by passage through QIAquick PCR purification spin columns (Qiagen, Chatsworth, CA). The purified PCR fragment was sequenced in both directions, using the same primers as those used for PCR, an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA), and an ABI377 DNA sequencer.
Mitochondrial Transcription Factor A Activityw@?/u)f, http://www.100md.com
Tfam is a nuclear-encoded protein imported into mitochondria and required for transcription of mtDNA. The observed decline in copy number suggested the possibility that Tfam might respond to LPS injury. To assess this, EMSA was performed in mitochondrial extract, using an oligonucleotide for the Tfam-binding site in the LSP. In , prominent protein binding to the Tfam-binding site is shown on Day 1 after LPS injection. The binding complex was not detectable after addition of unlabeled LSP oligonucleotide. The formation of binding complex at the Tfam-binding site on the LSP had decreased by 2 days after LPS and disappeared after 5 to 10 days. The increase in complex formation at the mtDNA promoter on Day 1 was associated with recovery of the mitochondrial transcripts, for example, COX I (p < 0.05), on Day 2. These increases in Tfam binding to the mtDNA promoter are consistent with initiation of mitochondrial biogenesis preceding restoration of mitochondrial transcription.
fig.ommitteda*5^9u], 百拇医药
Figure 7. Effects of LPS on mitochondrial transcription factor A (Tfam)–DNA binding. The EMSA was performed with isolated hepatic mitochondria (20 µg) and an oligonucleotide for the Tfam-binding site on the light strand promoter (LSP). The EMSA indicates LPS induction of a Tfam-specific shift of the LSP sequence from the mitochondrial genome 1 day after treatment. Bands detected (arrows) illustrate specific binding to the mtTFA site. Addition of unlabeled LSP competitor oligonucleotide (Comp Oligo+) greatly reduces detectable DNA binding. A lane containing free probe incubated in the absence of mitochondrial extract is shown (Oligo). Data are representative of four independent studies.a*5^9u], 百拇医药
Transcription of Mitochondrial mRNA and Nuclear mRNAa*5^9u], 百拇医药
To determine the extent to which LPS altered mitochondrial transcription, steady state mitochondrial mRNA levels were measured as a function of time . Gene-specific PCR primers were designed to amplify fragments of 12S rRNA; COX I, a mitochondrial-encoded subunit of Complex IV; and two mitochondrial-encoded subunits of Complex I, ND1 and ND2. RT-PCR was used to amplify the mtRNAs in relation to nuclear transcripts represented by 18S rRNA expression as a normalizing factor. The data showed an approximately 30% decrease in the levels of ND1, ND2, COX I, and 12S rRNA transcripts relative to 18S rRNA in the first day after LPS; however, transcript levels in the mitochondria recovered on Day 2 after LPS exposure. COX I showed a twofold increase in steady state mRNA 2 days after LPS injection. Because these mitochondrial mRNAs are transcribed as single polycistronic molecules from the same transcription unit of the heavy chain of mtDNA (27), changes observed in the steady state content of COX I and ND subunit mRNA suggest specific regulation of transcript stability during injury. In addition, transcript recovery tracked the activation of mitochondrial biogenesis by electron microscopy and Tfam binding 1–2 days immediately after LPS treatment.
fig.ommitted}#4)}, http://www.100md.com
Figure 8. (A) Steady state hepatic mtDNA transcription products in LPS-treated rats. Top: GelStar-stained 2% agarose gels demonstrating RT-PCR products from livers of rats. Total cellular RNA was prepared from livers of control rats (Day 0) and after LPS treatment (Days 1–10). Total RNA was reverse transcribed with gene-specific oligonucleotide primers for rat mitochondrial genes ND1, ND2, COX I, and 12S rRNA. Mitochondrial gene expression was then measured by relative RT-PCR. The nuclear mRNA for 18S rRNA was used as a control for the RNA loading levels and the efficiency of the RT-PCR. Bottom: Levels of mitochondrial gene expression after normalization to 18S rRNA levels are shown. Graph represents four independent samples expressed as means ± SEM (n = 4, *p < 0.05). (B) Steady state hepatic mRNA transcription products in LPS-treated rats. Top: GelStar-stained 2% agarose gels demonstrating RT-PCR products from livers of rat. Total cellular RNA was prepared from livers of control rats (Day 0) and after LPS treatment (Days 1–10). Nuclear gene expression for COX IV, MnSOD, and ALR was then measured by relative RT-PCR. GAPDH is constitutively expressed and used as a control for RNA loading and the efficiency of the RT-PCR. Bottom: Levels of nuclear gene expression after normalization to GAPDH levels. Graph represents four independent samples expressed as means ± SEM (n = 4, *p < 0.05).
mRNAs for the nuclear-encoded subunit of complex IV of the respiratory chain, cytochrome c oxidase subunit IV (COX IV), the nuclear-encoded mitochondrial antioxidant enzyme MnSOD, and the nuclear-encoded augmenter of liver regeneration (ALR) were analyzed by RT-PCR . For comparison, the levels of COX IV, MnSOD, and ALR mRNA were normalized for RNA loading, using GAPDH. The mRNA for MnSOD expression was maximally induced (2.5-fold) 1 day after LPS treatment, indicating transcriptional activation. The mRNA levels for ALR were increased 40% by 1 day after LPS injection, whereas COX IV mRNA fell slightly before recovering. LPS effects were mRNA specific compared with nuclear-encoded GAPDH mRNA, which remained stable throughout the analysis. These data show compartment- and gene-specific differences in the effects of LPS. LPS did not stimulate Cu,ZnSOD mRNA (not shown), indicating selective induction of the intramitochondrial SOD isoform.x\, 百拇医药
In Organello RNA Synthesis
Mitochondria were exposed to H2O2 ex vivo to determine whether the 3.8-kb deletion could be produced by a single oxidant associated with GSH depletion. After defining conditions to minimize nonspecific and endogenous oxidant-mediated degradation of mtDNA in control organelles, a 1-hour exposure to H2O2 at 37°C was selected to examine the deletion effect ex vivo. In the absence of H2O2, the 3.8-kb fragment of mtDNA in control mitochondria was usually below the PCR detection limit; however, as in LPS-treated rats, the 3.8-kb deletion was detected after exposure of control mitochondria to 500 to 1,000 µM H2O2. A 1-hour exposure to 1,000 µM H2O2 in control mitochondria produced one-fourth the amount of 3.8-kb mtDNA present in mitochondria isolated from LPS-treated animals after 24 hours . The ability of H2O2 to produce the deletion was attenuated by preexposure to GSH methyl ester.
fig.ommitted.gqe/(, 百拇医药
Figure 9. H2O2-induced mtDNA damage and in organello RNA synthesis. (A) GelStar-stained 1.5% agarose gel demonstrating semiquantitative PCR detection of deletion in liver mtDNA. Densitometry analysis shown as percent deletion by normalizing the optical density of deleted mtDNA to that of wild-type mtDNA and multiplying by 100. The deletion was undetectable in control liver mitochondria but was readily detectable after incubation with 1 mM H2O2. Preincubation of the mitochondria with 500 µM GSH methyl ester (GSHme) prevented most of the deletion. (B) Electrophoresis pattern of nascent RNA in organello under different conditions (lanes 1 and 2, mitochondria from control rats; lanes 3 and 4, mitochondria from 24-hour LPS-treated rats; lanes 5 and 6, control mitochondria treated in vitro with 1 mM H2O2; lanes 7 and 8, control mitochondria incubated in vitro with GSHme followed by 1 mM H2O2). The line at the bottom indicates the overall RNA synthesis rate for mitochondria under different conditions, referenced to the content of labeled mtRNA by normalizing the control value to 100%. Values represent means for n = 4 animals. Asterisks indicate p < 0.05 relative to control.
To ascertain whether a peroxidative mechanism could account for loss of steady state mitochondrial mRNA after LPS treatment in vivo, transcription assays were conducted in organello under different conditions. indicates that mitochondrial transcription was active in the isolated organelles of normal liver. Mitochondria of LPS-exposed rats and control mitochondria exposed ex vivo to H2O2, however, showed 32 and 18% reductions, respectively, in labeled UTP incorporation into RNA relative to normal controls (p < 0.05). The proximity of the deletion to the D-loop initiation site probably accounts for the broad suppression of mitochondrial mRNA transcripts. The transcriptional impairment produced by H2O2 was abrogated by preexposure to GSHme, indicating the importance of the intramitochondrial reduced thiol pool for template and transcriptional integrity.gnw\, 百拇医药
DISCUSSIONgnw\, 百拇医药
Oxidative stress contributes to organ failure in sepsis, but the details of the molecular pathogenesis are unknown (1–3, 15, 28, 29). Because mitochondria are the principal O2-consuming and ROS-generating organelles of the cell, we postulated that cell activation by LPS, which stimulates cytokine and ROS production, would damage mitochondria by oxidation of mtDNA. In testing this hypothesis in the liver, intraperitoneal LPS injection was found to cause a significant decrease in mtDNA copy number. LPS depleted GSH and increased mitochondrial lipid peroxidation in conjunction with increased MnSOD gene expression. This oxidative stress was associated with a 3.8-kb deletion of a region bracketed by GC-rich multiple direct repeats involving coding regions for Complexes I and IV. Accumulation of this deletion was associated with a decline in mitochondrial mRNA transcript levels in vivo.
These observations were made in control and post-LPS mitochondria of the same centrifugation density and equivalent RCR and State 3 in order to avoid gross damage and to exclude large changes in adenine nucleotide pool as a cause of transcriptional impairment. If crude mitochondria of a greater density distribution are included in the analysis then some loss of maximum respiratory capacity is apparent 24 hours after LPS. Thus, the mtDNA genomic damage precedes the loss of bioenergetic function in vitro.svl\o, 百拇医药
The importance of oxidative stress is underscored by the increases in hepatic and mitochondrial lipid peroxidation, and the increase in mitochondrial GSSG:GSH ratio, after LPS treatment, both important cytotoxic effects of oxidants (24). Oxidation of the mitochondrial GSH pool has also been correlated with age-related mtDNA degradation (12, 30). Similarly, the correlation between GSH depletion and the relative ease with which H2O2 drives the 3.8-kb mtDNA deletion implies that loss of mtDNA integrity by LPS-induced ROS generation is more sensitive to degradation than the capacity for oxidative phosphorylation assessed by standard ex vivo methods. When the 3.8-kb mtDNA deletion was detectable in the mitochondria of LPS-exposed animals, there was a significant decline in mtDNA copy number and a loss of steady state mtRNA synthesis.
The molecular pathogenesis of the mtDNA deletion has not been worked out completely, but the mechanism appears to involve elevated oxidant production by mitochondria, which damages regions of high GC content, leading to misannealing and heavy strand loops that are susceptible to strand breakage. After degradation of the excluded loop and ligation of the free ends of the heavy strand, replication can be completed, leading to a normal (16.3 kb) and a deleted (12.5 kb) mtDNA molecule. Molecules of both sizes contain the primer sites, which allows amplification of both the intact molecule and the smaller, deleted molecule.n-zp%, 百拇医药
The mtDNA deletion involved 3810 nucleotides of the rat genome, encompassing coding regions for cytochrome c oxidase subunit I (COX I) and NADH dehydrogenase (ND1 and ND2). This deletion is accompanied by the accumulation of substantial 8-hydroxyguanine (12) initiated by oxidation of DNA bases by H2O2-catalyzed hydroxylation and perhaps other strong oxidants (31–33). Production of other oxidants, such as NO and its products, which are known to be stimulated by LPS, may also be involved in mtDNA damage or inhibition of its repair; this possibility was not investigated in the present study. Moreover, Complex I deficiency increases mitochondrial O2·- production and induces MnSOD expression (34). Mitochondria are iron rich, and reduced iron catalyzes H2O2-driven hydroxylation of DNA bases. Depletion of mtDNA may result from ROS-induced mutations at the origins of replication or from mutations outside the origins that interfere with replication (35). Equivalent damage to nuclear DNA, however, as in this study, may not be apparent.
After LPS treatment, the liver produced many small, dense, dumbbell-shaped mitochondria consistent with organelle biogenesis. At the time these changes were apparent, the mtDNA copy number had decreased by 28%. This decline in mtDNA copy number is not consistent with arrested biogenesis because such an interpretation conflicts with the morphology and the increased Tfam-binding activity, a hallmark of biogenesis (36). Biogenesis thus would begin before recovery of mtDNA copy number, which would be reestablished as new organelles are produced and older ones are degraded.ii/[, http://www.100md.com
During biogenesis, mtDNA replication and transcription in many cells are regulated by transcription factor A (Tfam; previously mtTFA, mtTF1, TCF-6, or TCF6L2) (37). Heterozygous Tfam knockout mice exhibit low levels of mitochondria-encoded mRNA, reduced mtDNA copy number, and respiratory chain deficiencies whereas homozygotes die in early gestation with enlarged mitochondria and highly disorganized cristae (38, 39). LPS in this study led to increased mitochondrial Tfam importation as reflected in Tfam DNA-binding activity by EMSA. Tfam exhibits a concentration-dependent effect on initiation of transcription in vitro (39), and thus its induction after LPS corresponds to a crucial step in restoring transcriptional competence. Replication of the mitochondrial genome is required to synthesize new protein to support biogenesis (40). Although a direct correlation has not been made between Tfam and mitochondrial genome replication, the centrality of Tfam is implicit because RNA synthesis produces an RNA–DNA hybrid at the origin sequence (41). Thus, Tfam coordinates both replication and transcription for organelle biogenesis and ensures an adequate capacity for oxidative phosphorylation.
Mitochondrial transcripts decreased significantly after LPS exposure, and the mRNA for some nuclear-encoded mitochondrial proteins, such as MnSOD and ALR, increased significantly whereas others, such as COX IV, did not. The decrease in mitochondrial transcripts was proportional to the deletion of template and fall in mtDNA copy number. Thus changes in transcription or translation efficiency, although not precluded, are not required to explain the mitochondrial mRNA dynamics. The sentinel importance of the recovery of mRNA for COX I, ND1, and ND2 subunits encoded by mtDNA is highlighted by the lack of simultaneous change in the expression of nuclear 18S rRNA and GAPDH. Although not shown here, detectable decreases do occur in Complex I and IV protein levels 24 hours after LPS treatment (our unpublished observations).(, http://www.100md.com
Depletion of the mtDNA template may not cause immediate respiratory chain dysfunction because of the persistence of functional constitutive proteins. However, the continued loss of mtDNA must ultimately lead to a deficiency of functional respiratory chain proteins and impaired respiration. Thus, the issue remains moot as to what functional effects to attribute to mtDNA changes that precede a decline in respiratory capacity or to simply interpret them as an index of damage (42). In some cells, ongoing injury, for example, ROS generation, may precipitate a catastrophic loss of function, characterized by energy failure or pore transition, and cell lethality, which would not be ascertained readily by ex vivo study.
The cell may avert death if the mtDNA damage can be repaired promptly. Mitochondria perform base excision repair, but do not repair bulky lesions by nucleotide excision repair (43). A 3.8-kb deletion is too large for base excision repair, but if base excision repair is impaired, for example, by ROS or reactive nitrogen species, it may predispose to the deletion. Once the deletion is present, however, degradation of damaged mtDNA with increased mitochondrial turnover is a more attractive explanation for resolution of the deletion.xun(^7, 百拇医药
Hepatocyte proliferation and mitochondrial biogenesis are stimulated by upregulation of ALR, a potent hepatopoietin, and liver growth factor (44). ALR is located in the intermembrane space of the mitochondria and represents an early respondent in the genesis of cytosolic Fe/S proteins (45). Because nuclear MnSOD transcription is predictably increased in response to LPS, oxidative events, not simply inhibition of energy-coupled processes, are implicated in nuclear gene induction. Oxidant activation of protein kinase pathways may also lead to induction via DNA–protein interactions within the intronic enhancers of MnSOD and ALR. Hepatic expression of tumor necrosis factor-{alpha} in gram-negative sepsis poses a danger by increasing ROS production by mitochondria, but also regulates putative survival factors, such as NF-{kappa}
B, and mitochondrial proteins that facilitate proliferation (46, 47).3, http://www.100md.com
In summary, we have elucidated a new mitochondrial mechanism involved in LPS-mediated hepatic injury and investigated some of the key events regulating mitochondrial gene expression afterward. The data indicate that LPS promotes mtDNA oxidation, wherein H2O2 singly or collectively with related oxidants leads to GSH depletion and accumulation of a novel deletion. Energetically functional mitochondria show impaired mtDNA transcription and expression of the encoded respiratory chain components relative to nuclear-encoded proteins. Mitochondrial genomic integrity is restored by activation and importation of Tfam, providing evidence of biogenesis and coordination of nuclear and mitochondrial gene expression.3, http://www.100md.com
These new findings suggest a possible link between mtDNA transcription and organ dysfunction in gram-negative sepsis. Although the relevance to clinical sepsis of using large amounts of LPS to produce mtDNA damage can be questioned, the time course of the effect suggests the influence of LPS involves host inflammatory responses. It seems reasonable to speculate that hepatic mtDNA damage, whatever the sequence of events triggered by LPS, plays an important role not only in the development of energetic and oxidative stress but in adaptation to those stresses by stimulating mitochondrial biogenesis and respiratory protein expression via Tfam activation.
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