Increased Oxidative Damage Is Correlated to Altered Mitochondrial Function in Heterozygous Manganese Superoxide Dismutase Knockout Mice

• DOI: 10.1074/jbc.273.43.28510
• Publication Date: 2002-07-26T15:02:22Z
• Authors: Melissa D. Williams; Holly Van Remmen; Craig C. Conrad; Ting Ting Huang; Charles J. Epstein; Arlan Richardson
• Keywords: SOD2; Dismutase

This study characterizes mitochondria isolated from livers of Sod2 −/+ and +/+ mice. A 50% decrease in manganese superoxide dismutase (MnSOD) activity was observed mice compared withSod2 mice, with no change the activities either glutathione peroxidase or copper/zinc dismutase. However, level total 30% less liver The reduction MnSOD correlated to an increase oxidative damage mitochondria: decreased Fe-S proteins (aconitase NADH oxidoreductase), increased carbonyl groups proteins, levels 8-hydroxydeoxyguanosine mitochondrial DNA. In contrast, there were significant changes cytosolic nuclear altered function. respiratory control ratio for substrates metabolized by complexes I, II, III. addition, showed rate induction permeability transition. Therefore, this provides direct evidence correlating reduced vivo alterations DNA coenzyme Q1 8- hydroxydeoxyguanosine. Under normal physiological conditions, metabolism oxygen aerobic organisms generates a wide variety potentially deleterious reactive species. These species initiate large number reactions cellular systems that lead oxidation macromolecules, e.g. DNA, lipids (1Pacifici R.E. Davies K.J. Gerontology. 1991; 37: 166-180Crossref PubMed Scopus (371) Google Scholar). Over past decade, numerous investigators have argued may contribute chronic diseases (including emphysema, cardiovascular disease, cancer, neurodegenerative such as Alzheimer Parkinson amyotrophic lateral sclerosis) well aging (2Gaulton G.M. Markmann J.F. Immunol. Res. 1988; 7: 113-135Crossref (8) Although it is attractive speculate accumulation be involved decline function disease pathogenesis, concept. Mitochondria are extremely susceptible because 2–4% consumed converted anions electron transport chain (3Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref (4933) Scholar) limited protection stress (4Wallace D.C. Science. 1992; 256: 628-632Crossref (1151) would predicted one components especially vulnerable damage. 20 years, studied effect on subjecting vitro. Many functional properties substantially impaired after exposure vitro include inhibition chain, complex I ATP synthetase (5Hardy L. Clark J.B. Darley-Usmar V.M. Smith D.R. Stone D. Biochem. J. 274: 133-137Crossref (114) Scholar), adenine nucleotide content, translocase (6Duan Karmazyn M. Eur. Pharmacol. 210: 149-157Crossref (55) lipid peroxidation (7Zhang Y. Marcillat O. Giulivi C. Ernster K.J.A. Biol. Chem. 1990; 265: 16330-16336Abstract Full Text PDF swelling (8Takeyama N. Matsuo Tanaka T. 1993; 294: 719-725Crossref (142) not clear studies accurate indication what occurs vivo. other words, does under conditionsin dysfunction? Transgenic mutant animals antioxidant defense system provide model can used test functionin For example, two MnSOD1 knockout mouse models been produced disruption different sections theSod2 gene (9Li Huang T.-T. Carlson E.J. Melov S. Ursell P.C. Olson J.L. Noble L.J. Yoshimura M.P. Berger Chan P.H. Wallace Epstein C.J. Nat. Genet. 1995; 11: 376-381Crossref (1479) Scholar, 10Hillier Becker Chiapelli Chissoe Dietrich DuBuque Favello Gish W. Hawkins Hultman Kucaba Lacy Le Mardis E. Moore Morris Parsons Rifkin Rohlfing Schellenberg K. Tan F. Thierry-Meg Trevaskis Underwood Wohldman P. Waterston R. Wilson Marra Lennon G. Bonaldo M.F. Prange Soares M.B. Genome 1996; 6: 807-828Crossref (393) both models, homozygous (Sod2 −/−) mutants exhibited detectable any tissues studied. TheSod2 −/− phenotype lethal models. (Sod2<tm1>Cje) Li et al. smaller paler hypotonic hypothermic state wild type heterozygous −/+). addition their overall gross appearance, null appeared fatigue more rapidly exertion; however, behavior otherwise normal. After 4–5 days, 37% died, nearly all dead day 10. At death, had enlarged hearts dilated left ventricular cavity wall thickness, which suggested cardiomyopathy. Furthermore, size marked steatosis, deposits skeletal muscle. these extreme myocardium, did show motor disturbances, central nervous injury, ultrastructural (mtDNA) rearrangements. (Sod2<tm1>BCM) Lebovitz (11Lebovitz R.M. Zhang Vogel Cartwright Dionne Lu Matzuk M.M. Proc. Natl. Acad. Sci. U. 93: 9782-9787Crossref (859) could distinguished littermate controls at birth. diminished growth became apparent between 2 7 continued until usually occurred 18 days abnormalities observed, they exhibit reductions adipose tissue Histological analysis revealed hypocellular bone marrow anemia atypical patterns glycogen deposition hepatocytes. brain spinal cord microscopy demonstrated degenerative injury neurons, particularly basal ganglia brainstem, characterized extensive damage, loss polysomes clearing cytoplasm. progressive disturbances weakness, rapid fatigue, circling behavior. Only 10% died Electron micrographs myocardium swollen damaged mitochondria, developed phenotypic differences types shown attributable strain background genes expressed. al.(12Huang Gillespie A.M. Age. 1998; 21: 39-40Crossref (13) 2T. Epstein, unpublished data. phenotypes mutations made resemble another changing genetic backgrounds characterized, relatively little information animals. appear normal, even though former found develop larger cerebral infarcts when subjected middle artery ischemia reperfusion (13Chan Murakami Mikawa Chen S.F. Reola Krieglstein Pharmacology Cerebral Ischemia. Wissenschaftliche Verlagsgesellschaft, Stuttgart, Germany1996: 573-579Google our present study, we activity, protein but cytoplasmic/nuclear compartments cell. function, including respiration designatedSod2<tm1>Cje, originally CD1 Scholar); described backcrossed C57Bl/6 13 generations (B6-Sod2<tm1>Cje). genotype determined polymerase reaction obtained 1-cm portion tail Female fed ad libitum maintained barrier conditions 12-h dark/light cycle. 2–4 months age, sacrificed cervical dislocation, immediately excised placed ice. All procedures followed guidelines approved Institutional Animal Care Use Committee University Texas Health Science Center, San Antonio, TX. according method Johnson Lardy (14Johnson Methods Enzymol. 1967; 10: 94-96Crossref (1220) pellet resuspended buffer containing 250 mm mannitol, 75 sucrose, 500 μm EGTA, 3 HEPES (pH 7.4), suspension measure respiration, membrane potential, enzyme assays Western blots, 50 mmTris-HCl 1 EDTA, 0.1% (v/v) Triton X-100. preparations aconitase sodium citrate added stabilize [Fe-S] clusters (15Hausladen Fridovich I. 269: 37-41Crossref Protein concentrations using Bio-Rad Bradford assay kit. Superoxide measured polyacrylamide gels Beauchamp (16Beauchamp Anal. 1971; 44: 276-287Crossref (10348) staining, gel photographed, photograph scanned quantitated ImageQuant software (Sunnyvale, CA). 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(Tokyo). 113: 738-741Crossref (4) hydroxylamate/min. 5-thio-2-nitrobenzoic acid Anderson (23Anderson M.E. Greenwald Handbook Oxygen Radical Research. CRC Press, Inc., Boca Raton, FL1985: 317-323Google incubated 25 succinate maintain 10 °C before perchloric deproteinize samples. consumption Gilson oxygraph equipped electrode Estabrook (24Estabrook R.W. 1974; 41-47Crossref (1917) Reactions conducted 30 2-ml chamber mg (250 mmsucrose, KH2PO4, Tris-HCl, pH 7.4) following three substrates: glutamate (1.7 mm) malate mm), (2.5 rotenone (5 μg/ml), duroquinol (500 μm). State rates ADP (200 final concentration), 4 presence adequate substrate without ADP. (RCR) calculated rates. There discernible yield betweenSod2 −/+mice: 6.2 ± 0.8 protein/liver 6.3 −/+mice (data expressed mean S.E. six animals). fromSod2 major enzymes, GPX, localized matrix. Fig. shows approximately GPX remained unchanged (Fig. 1) and, therefore, compensate We also CuZnSOD catalase preparations. has reported only cytosol, intermembrane space (25Peeters-Joris Vandevoorde Baudhuin 1975; 150: 31Crossref (131) same 1). data agreement previous report difference whole homogenates extracts unexpected specifically perixosomes (26Halliwell Gutteridge J.M.C Free Radicals Biology Medicine. Clarendon Oxford1995: 86-187Google act directly GPX. 10–15% inside matrix (27Fernandez-Checa J.C. Kaplowitz Garcia-Ruiz Colell Miranda Mari Ardite Morales Am. 1997; 273: G7-G17Crossref possible extracts. significance pool appears important physiologically protecting cells stress. (28Garcia-Ruiz Fernandez-Checa Mol. 48: 825-834PubMed recently (with intact) rendered endogenous induced antimycin situation decreased. Because key component system, if sensitive tissue/cellular (29Gardner P.R. Nguyen D.H. White C.W. 1994; 91: 12248-12252Crossref (376) iron-sulfur inactivated oxidizes [4Fe-4S] cluster leads iron ion (30Gardner Raineri L.B. 270: 13399-13405Abstract (436) reducing agent blot similar; due protein. reactivation dithiothreitol resulted equal +/+mice. result inactivation anions. anion higher than Previously, +/+mice; failed detect rather activity. (31Koen Goodman Biochim. Biophys. Acta. 191: 698-701Crossref (29) oxidoreductase (complex I) 32Moreadith Batshaw M.L. Ohnishi Kerr Knox Jackson Hruban Reyafarje Lehninger Clin. Invest. 1984; 74: 685-697Crossref (117) employ terminal acceptors: Q (Table I). Coenzyme Q, accepts electrons I; inactivation. significantly lower (30%) To determine clusters, substrate. Ferricyanide artificial acceptor flavin mononucleotide by-passes (33Singer, (1974) Anal.123–175Google Using acceptor, +/+mice clusters.Table IThe enzymes Sod2+/+ Sod2−/+ miceSod2+/+Sod2−/+Mitochondrial oxidoreductase1-aMilliunits/mg protein.NADH:coenzyme Q309.6 25.7216.1 9.8*NADH:ferricyanide626.9 118.7626.9 121.7 Fumarase1-aMilliunits/mg protein.161.5 13.8184.2 20.2Cytosolic Aconitase1-aMilliunits/mg protein.38.3 2.338.8 2.0 synthetase1-bUnits/mg protein.52.5 5.454.2 7.1All values represent nine *,p < 0.01 paired Student's t test.1-a Milliunits/mg protein.1-b Units/mg Open table new tab test. iron-dependent (34Woods S.A. Schwartzbach S.D. Guest J.R. 954: 14-26Crossref (166) insensitive (35Patel Day B.J. Crapo J.D. McNamara J.O. Neuron. 16: 345-355Abstract (394) Table glutamine encoded genes; structure conserved, (36Liochev S.I. 25: 369-384Crossref (85) (37Kates Cold Spring Harbor Symp. Quant. 1970; 35: 743-752Crossref 38Hill B.T. Whelan R.D.H. 1978; 24: 326-336Crossref (7) single histidine residue oxidized producing group (39Levine 11828-11833Abstract −/+mice. further characterize −/+mice, specific antibody detects 2,4-dinitrophyenyl adducts (40Keller R.J. Halmes N.C. Hinson J.A. Pumford N.R. Toxicol. 430-433Crossref (109) Carbonyl (aldehydes ketones) formed amino side chains metal catalyzed standard markers (41Stadtman E.R. 257: 1220-1224Crossref (2417) Coomassie Blue stain contrast contain similar ensure detected groups, derivatization included 2,4-dinitrophenyl hydrazine binding (8-OHdG) mtDNA 8-OHdG RCR measurements efficiency movement along coupling phosphorylation through II substrates. greatest (29 33%) glutamate/malate duroquinol, III, respectively. surprising reasons: (a) (42Beyer Cell 70: 390-403Crossref (218) (b) sites (43Cadenas Stoppani A.O.M. Arch. 180: 248-257Crossref (702) (c) cardiolipin, essential biological complexes, (44Veitch Hombroeckx Caucheteux Pouleur Hue 281: 709-715Crossref (145) consistent 30%.Table IIOxygen utilization miceSubstrateState 32-anmol O2/min/mg protein.State 42-anmol protein.RCRGlutamate/malate +/+30.7 3.06.5 0.85.1 −/+22.7 3.96.8 0.93.4 0.5**Succinate +/+39.2 5.714.8 2.02.6 0.1 −/+29.9 3.914.2 1.92.2 0.1**Duroquinol +/+57.0 10.218.7 3.03.1 0.3 −/+32.2 8.3*16.5 5.52.2 0.2*Each value represents experiments, pooled per experiment. *, P 0.05 Student'st **, test.2-a Each respiration. (44%) statistical mice; approached 0.06. essentially identical suggests integrity inner (45Hansford R.G. 726: 41-80Crossref (177) observation substantiated potential safranine O Akerman Wikstrom (46Akerman K.E.O. M.K.F. Lett. 68: 191-197Crossref (682) potentials −173.74 15.61 mV −167.30 23.93 mV, respectively experiments pooling each experiment). respect transition proteinaceous pore, whose opening calcium stress, t-butylhydroperoxide (47Zoratti Szabo 1241: 139-176Crossref (2211) pore sudden small ions molecules (calcium glutathione) complete collapse undergo based upon relative absorbance. transition, cyclosporin A, inhibitor reactions. As illustrated 5, inhibited absorbance, indicating absorbance pore. (calculated 1/2 seconds) statistically significant. Addition botht-butylhydroperoxide (38%) t-butylhydroperoxide. serious integrity. Once longer proton-motive force, unable generate ATP. summary, detailed important. presented several lines showing experience greater stress/damage. reduced, increased, increased. Interestingly, stress/damage nuclei Oxidation (glutamine groups) Thus, impact mutation playing critical role first line against during mitochondria. observations compromised. ratios efficient strong Our over years exposed express gratitude Drs. Claire Department Biochemistry Medical College Wisconsin Paul Srere VA Center Dallas gifts Michael Luther, South Veterans System assistance analysis. thank Dr. Richard Hansford National Institute Aging expert advice Marian Sabia Hong Yang technical assistance. Elaine original breeding