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Folate deprivation promotes mitochondrial oxidative decay: DNA large deletions, cytochrome c oxidase dysfunction, membrane depolarization and superoxide overproduction in rat liver

  • Chun-Min Chang (a1), Chu-Ching Yu (a1), Hsin-Te Lu (a1), Yi-Fang Chou (a1) and Rwei-Fen S. Huang (a1)...


Little is known about the biological effect of folate in the protection against mitochondrial (mt) oxidative decay. The objective of the present study was to examine the consequence of folate deprivation on mt oxidative degeneration, and the mechanistic link underlying the relationship. Male Wistar rats were fed with an amino acid-defined diet containing either 8 (control) or 0 (folate-deficient, FD) mg folic acid/kg diet. After a 4-week FD feeding period, significant elevation in oxidative stress was observed inside the liver mitochondria with a 77 % decrease in mt folate level (P < 0·001), a 28 % reduction in glutathione peroxidase activity (P = 0·0333), a 1·2-fold increase of mt protein carbonyls (P = 0·0278) and an accumulated 4834 bp large-scale deletion in mtDNA. The elicited oxidative injuries in FD liver mitochondria were associated with 30 % reduction of cytochrome c oxidase (CcOX) activity (P = 0·0264). The defective CcOX activity in FD hepatocytes coincided with mt membrane potential dissipation and intracellular superoxide elevation. Exposure of FD hepatocytes to pro-oxidant challenge (32 μm-copper sulphate for 48 h) led to a further loss in CcOX activity and mt membrane potential with a simultaneous increase in superoxide production. Preincubation of pro-oxidant-treated FD hepatocytes with supplemental folic acid (10–1000 μm) reversed the mt oxidative defects described earlier and diminished superoxide overproduction. Increased supplemented levels of folic acid strongly correlated with decreased lipid peroxidation (γ − 0·824, P = 0·0001) and protein oxidative injuries (γ − 0·865, P = 0·0001) in pro-oxidant-challenged FD liver mitochondria. Taken together, the results demonstrated that folate deprivation induces oxidative stress in liver mitochondria, which is associated with CcOX dysfunction, membrane depolarization and superoxide overproduction. The antioxidant activity of supplemental folic acid may partially, if not fully, contribute to the amelioration of pro-oxidant-elicited mt oxidative decay.

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      Folate deprivation promotes mitochondrial oxidative decay: DNA large deletions, cytochrome c oxidase dysfunction, membrane depolarization and superoxide overproduction in rat liver
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Corresponding author

*Corresponding author: Dr Rwei-Fen S. Huang, fax +886 2 29021215, email


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Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.
Branda, RF, Brooks, EM, Chen, Z, Naud, SJ & Nicklas, JA (2002) Dietary modulation of mitochondrial DNA deletions and copy number after chemotherapy in rats. Mutat Res 501, 2936.
Brookes, PS & Baggott, JE (2002) Oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate by complex IV of rat mitochondria. Biochemistry 41, 56335636.
Cadenas, E & Davies, KJA (2000) Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 29, 222230.
Chen, YH, Huang, RFS, Wei, JS & Liu, TZ (2001) Folate deficiency-mediated down regulation of intracellular glutathione and antioxidant enzymes increases susceptibility of human hepatoma HepG2 cells to various oxidant stress-induced cytotoxicity. J Biomed Lab Sci 13, 5257.
Chern, CL, Huang, RFS, Chen, YH, Cheng, JT & Liu, TZ (2001) Folate deficiency-induced oxidative stress and apoptosis are mediated via homocysteine-dependent overproduction of hydrogen peroxide and enhances activation of NF-κB in human HepG2 cells. Biomed Pharmacother 55, 434442.
Coppola, A, D'Angelo, A, Fermo, I, et al. (2005) Reduced in vivo oxidative stress following 5-methyltetrahydrofolate supplementation in patients with early-onset thrombosis and 677TT methylenetetrahydrofolate. Br J Haematol 131, 100108.
Cortopassi, GA, Shibata, DD, Soong, NW & Amheim, N (1992) A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues. Proc Natl Acad Sci U S A 89, 73707374.
Cortopassi, G & Wang, E (1995) Modelling the effects of age-related mtDNA mutation accumulation; complex I deficiency, superoxide and cell death. Biochim Biophys Acta 1271, 171176.
Crott, JW, Choi, SW, Branda, RF & Mason, JB (2005) Accumulation of mitochondrial DNA deletions is age, tissue and folate-dependent in rats. Mutat Res 570, 6370.
D'Aurelio, M, Pallotti, F, Barrientos, A, et al. (2001) In vivo regulation of oxidative phosphorylation in cells harboring a stop-codon mutation in mitochondrial DNA-encoded cytochrome c oxidase subunit I. J Biol Chem 276, 4692546932.
Doshi, SN, McDowell, IFW, Moat, SJ, et al. (2001) Folate improves endothelial function in coronary artery disease. An effect mediated by reduction of intracellular superoxide? Arterioscler Thromb Vasc Biol 21, 11961202.
Duranteau, J, Chandel, NS, Kulioz, A, Shao, Z & Schumacher, PT (1998) Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J Biol Chem 273, 1161911624.
Duthie, SJ, Narayanan, S, Brand, GM, Pirie, L & Grant, G (2002) Impact of folate deficiency on DNA stability. J Nutr 132, 2444S2449S.
Edris, W, Burgett, B, Stine, OC & Filburn, CR (1994) Detection and quantitation by competitive PCR of an age-associated increase in a 4·8 kb deletion in rat mitochondrial DNA. Mutat Res 316, 6978.
Evans, WH (1992) Isolation and characterization of membranes and cell organelles. In Preparative Centrifugation: A Practical Approach, pp. 233270 [Rickwood, D, editor]. New York: Oxford University Press.
Fraga, CG, Leibovitz, BE & Tappel, AL (1988) Lipid peroxidation measured as thiobarbituric-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. Free Radic Biol Med 4, 155161.
Hagen, TM, Yowe, DL, Bartholomew, JC, Wehr, CM, Do, KL, Park, JY & Ames, BN (1997) Mitochondrial decay in hepatocytes from old rats: membrane potential declines, heterogeneity and oxidants increase. Proc Natl Acad Sci U S A 94, 30643069.
Hissin, PJ & Hilf, R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74, 214226.
Ho, PI, Ashline, D, Dhitavat, S, et al. (2003) Folate deprivation induces neurodegeneration; roles of oxidative stress and increased homocysteine. Neurobiol Dis 14, 3242.
Hoffbuhr, KC, Davidson, E, Filiano, BA, et al. (2000) A pathogenic 15-base pair deletion in mitochondrial DNA-encoded cytochrome c oxidase subunit III results in the absence of functional cytochrome c oxidase. J Biol Chem 275, 1399414003.
Horne, DW & Patterson, D (1988) Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clin Chem 34, 23572359.
Huang, RFS, Hsu, YC, Lin, HL & Yang, FL (2001) Folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers. J Nutr 131, 3338.
Huang, RFS, Huang, SM, Lin, BS, Hung, CY & Lu, HT (2002) N-Acetylcysteine, vitamin C and vitamin E diminish homocysteine thiolactone-induced apoptosis in human promyeloid HL-60 cells. J Nutr 132, 21512155.
Huang, RFS, Yaong, HC, Chen, SC & Lu, YF (2004) In vitro folate supplementation alleviates oxidative stress, mitochondria-associated death signaling and apoptosis induced by 7-ketocholesterol. Br J Nutr 92, 887894.
Johnson, D & Lardy, H (1967) Isolation of liver or kidney mitochondria. Methods Enzymol 10, 9496.
Joshi, R, Adhikari, S, Patro, BS, Chattopadhyay, S & Mukherjee, T (2001) Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med 30, 13901399.
Kim, JM, Lee, H & Chang, N (2002) Hyperhomocysteinemia due to short-term folate deprivation is related to electron microscopic changes in the rat brain. J Nutr 132, 34183421.
Ksenzenko, M, Konstantinov, AA, Khomutov, GB, Tikhonov, AN & Ruuge, EK (1983) Effect of electron transfer inhibitors on superoxide generation in the cytochrome bc1 site of the mitochondrial respiratory chain. FEBS Lett 155, 1924.
Lee, HC, Pang, CY, Hsu, HS & Wei, YH (1994) Differential accumulations of 4977 bp deletion in mitochondrial DNA of various tissues in human ageing. Biochim Biophys Acta 1226, 3743.
Lii, CK & Hendrich, S (1993) Selenium deficiency suppresses the S-glutathiolation of carbonic anhydrase III in rat hepatocytes under oxidative stress. J Nutr 123, 14801486.
Lezza, AMS, Mecocci, P, Cormio, A, Beal, FM, Cherubini, A, Cantatore, P, Senin, U & Gadaleta, MN (1999) Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimer's disease patients. FASEB J 13, 10831088.
Lu, CY, Lee, HC, Fahn, HJ & Wei, YH (1999) Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin. Mutat Res 423, 1121.
Marklund, S & Marklund, G (1974) Involvement of the superoxide anion radical in the autoxidation of paragallol and a convenient assay for superoxide dismutase. Eur J Biochem 47, 469474.
Mayer, O Jr, Simon, J, Rosolova, H, et al. (2002) The effects of folate supplementation on some coagulation parameters and oxidative status surrogates. Eur J Clin Pharmacol 58, 15.
Nakano, E, Higgins, JA & Powers, HJ (2001) Folate protects against oxidative modifications of human LDL. Br J Nutr 86, 637639.
Palacios-Callender, M, Quintero, M, Hollis, VS, Springett, RJ & Moncada, S (2004) Endogenous NO regulates superoxide production at low oxygen concentrations by modifying the redox state of cytochrome c oxidase. Proc Natl Acad Sci U S A 101, 76307635.
Paradies, G, Petrosillo, G, Pistolese, M & Ruggiero, FM (2000) The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles. FEBS Lett 466, 323326.
Ravagnan, L, Roumier, T & Kroemer, G (2002) Mitochondria, the killer organelles and their weapons. J Cell Physiol 192, 131137.
Rezk, BM, Haenen, GRMM, van der Vijgh, WJF & Bast, A (2003) Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore. FEBS Lett 555, 601605.
Reznick, AZ & Packer, L (1994) Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 233, 357363.
Schon, EA, Bonilla, E & Dimauro, S (1997) Mitochondrial DNA mutations and pathogenesis. J Bioenerg Biomembr 29, 131149.
Seligman, AM, Karnovsky, MJ, Wasserkrug, HL & Hanker, JS (1968) Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J Cell Biol 38, 114.
Shane, B (1995) Folate chemistry and metabolism. In Folate in Health and Disease, pp. 122 [Bayley, LB, editor]. New York: Marcel Dekker.
Stroes, ES, van Faassen, EE, Yo, M, et al. (2000) Folic acid reverts dysfunction of endothelial nitric oxide synthase. Circ Res 86, 11291134.
Sue, CM, Karadimas, C, Checcarelli, N, et al. (2000) Differential features of patients with mutations in two COX assembly genes, SURF-1 and SCO2. Ann Neurol 47, 589595.
Tappel, AL (1977) Glutathione peroxidase and hydroperoxides. Methods Enzymol 52, 506513.
Trounce, IA, Kim, YL, Jun, AS & Wallace, DC (1996) Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol 264, 484509.
Turrens, JF, Alexandre, A & Lehninger, AL (1985) Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys 237, 408414.
Walzem, RL & Clifford, AJ (1988) Folate deficiency in rats fed diets containing free amino acid or intact proteins. J Nutr 118, 10891096.
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British Journal of Nutrition
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