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• W2003044361 abstract "We determined the activities of NADH dehydrogenase (ND), succinate dehydrogenase, and cytochromec oxidase (COX) in 29 skin fibroblast lines established from donors ranging in age from 12 gestational weeks to 94 years. The results of this study demonstrate that all three of the enzyme activities examined are greater in adult-derived fibroblasts than in the fetal cell lines. The ratio of enzyme activities that control electron entry into and exit from the electron transport chain varied directly with lucigenin-detected chemiluminescence (an indicator of⋅O2− generation) and inversely with H2O2 generation. These results indicate a clear difference in the predominant oxidant species generated during fetal and adult stages of life. We also examined the mRNA abundances of different components of the electron transport chain complexes. We observed higher abundances of mitochondrial encoded mRNAs (COX 1 and ND 4) in cell lines established from adults than in fetal cells. No differences in the mRNA abundances of the nuclear encoded sequences (COX 4 and ND 51) were observed in fetal and postnatal-derived lines. Succinate dehydrogenase mRNA abundance was greater in cell lines established from postnatal donors than in fetal cell lines. No significant differences between cell lines established from young and old adults were detected in any of the parameters examined. We determined the activities of NADH dehydrogenase (ND), succinate dehydrogenase, and cytochromec oxidase (COX) in 29 skin fibroblast lines established from donors ranging in age from 12 gestational weeks to 94 years. The results of this study demonstrate that all three of the enzyme activities examined are greater in adult-derived fibroblasts than in the fetal cell lines. The ratio of enzyme activities that control electron entry into and exit from the electron transport chain varied directly with lucigenin-detected chemiluminescence (an indicator of⋅O2− generation) and inversely with H2O2 generation. These results indicate a clear difference in the predominant oxidant species generated during fetal and adult stages of life. We also examined the mRNA abundances of different components of the electron transport chain complexes. We observed higher abundances of mitochondrial encoded mRNAs (COX 1 and ND 4) in cell lines established from adults than in fetal cells. No differences in the mRNA abundances of the nuclear encoded sequences (COX 4 and ND 51) were observed in fetal and postnatal-derived lines. Succinate dehydrogenase mRNA abundance was greater in cell lines established from postnatal donors than in fetal cell lines. No significant differences between cell lines established from young and old adults were detected in any of the parameters examined. Mitochondria are the primary site of aerobic energy production and are thus the major site of generation of reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; ⋅O2−, superoxide; ETC, electron transport chain; COX, cytochrome c oxidase; COX 1, mitochondrial encoded subunit of cytochrome c oxidase; COX 4, nuclear encoded subunit of cytochrome c oxidase; ND, NADH dehydrogenase; ND 4, mitochondrial encoded subunit of NADH dehydrogenase; ND 51, nuclear encoded subunit of NADH dehydrogenase; SD, succinate dehydrogenase; PBS, phosphate-buffered saline; CCI, calculated chemiluminescent intensity; DCF, 2′,7′-dichlorofluorescein; DCFH, 2′,7′-dichlorofluorescin; DCFH-DA, 2′,7′-dichlorofluorescin diacetate (DCFH-DA); SOD superoxide dismutase.1The abbreviations used are: ROS, reactive oxygen species; ⋅O2−, superoxide; ETC, electron transport chain; COX, cytochrome c oxidase; COX 1, mitochondrial encoded subunit of cytochrome c oxidase; COX 4, nuclear encoded subunit of cytochrome c oxidase; ND, NADH dehydrogenase; ND 4, mitochondrial encoded subunit of NADH dehydrogenase; ND 51, nuclear encoded subunit of NADH dehydrogenase; SD, succinate dehydrogenase; PBS, phosphate-buffered saline; CCI, calculated chemiluminescent intensity; DCF, 2′,7′-dichlorofluorescein; DCFH, 2′,7′-dichlorofluorescin; DCFH-DA, 2′,7′-dichlorofluorescin diacetate (DCFH-DA); SOD superoxide dismutase. such as oxygen-centered free radicals and peroxides, which are by-products of metabolism (1Foreman H.J. Boveris A. Pryor W.A. Free Radicals in Biology. Academic Press, New York1982: 65-90Crossref Google Scholar, 2Foreman H.J. Fischer A.B. Gilbert D.L. Oxygen and Living Processes. Springer-Verlag, New York1981: 65-90Crossref Google Scholar, 3Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4819) Google Scholar, 4Halliwell B. Sohal R.S. Age Pigments. Elsevier/North Holland, Amsterdam1981: 1-62Google Scholar). Estimates of the rate of ROS generation can differ greatly; however, it has been demonstrated repeatedly that the relative rate of oxidant generation increases with age and that this increase correlates strongly with age-associated changes in the cellular redox state (5Noy N. Schwartz H. Gafni A. Mech. Ageing Dev. 1985; 29: 63-69Crossref PubMed Scopus (41) Google Scholar, 6Sohal R.S. Farmer K.J. Allen R.G. Cohen N.R. Mech. Ageing Dev. 1983; 24: 185-195Crossref Scopus (57) Google Scholar, 7Sohal R.S. Svensson I. Brunk U.T. Mech. Ageing Dev. 1990; 53: 209-215Crossref PubMed Scopus (151) Google Scholar, 8Sohal R.S. Svensson I. Sohal B.H. Brunk U.T. Mech. Ageing Dev. 1989; 49: 129-135Crossref PubMed Scopus (138) Google Scholar). For example, the rates of superoxide (⋅O2−) (8Sohal R.S. Svensson I. Sohal B.H. Brunk U.T. Mech. Ageing Dev. 1989; 49: 129-135Crossref PubMed Scopus (138) Google Scholar, 9Sohal R.S. Arnold L.A. Sohal B.H. Free Radical Biol. Med. 1990; 9: 495-500Crossref PubMed Scopus (157) Google Scholar, 10Farmer K.J. Sohal R.S. Free Radical Biol. Med. 1989; 7: 23-29Crossref PubMed Scopus (78) Google Scholar, 11Nohl H. Hegner D. Eur. J. Biochem. 1978; 82: 563-567Crossref PubMed Scopus (468) Google Scholar, 12Nohl H. Johnson J.E. Walford R. Harman D. Miquel J. Free Radicals, Aging, and Degenerative Disease. Alan R. Liss, New York1986: 79-97Google Scholar, 13Hegner D. Mech. Ageing Dev. 1980; 14: 101-118Crossref PubMed Scopus (142) Google Scholar, 14Ku H.-H. Brunk U.T. Sohal R.S. Free Radical Biol. Med. 1993; 15: 621-627Crossref PubMed Scopus (415) Google Scholar) and H2O2 generation (7Sohal R.S. Svensson I. Brunk U.T. Mech. Ageing Dev. 1990; 53: 209-215Crossref PubMed Scopus (151) Google Scholar, 10Farmer K.J. Sohal R.S. Free Radical Biol. Med. 1989; 7: 23-29Crossref PubMed Scopus (78) Google Scholar, 14Ku H.-H. Brunk U.T. Sohal R.S. Free Radical Biol. Med. 1993; 15: 621-627Crossref PubMed Scopus (415) Google Scholar, 15Allen R.G. Farmer K.J. Sohal R.S. Biochem. J. 1983; 216: 503-506Crossref PubMed Scopus (77) Google Scholar, 16Sohal R.S. Free Radical Biol. Med. 1993; 14: 583-588Crossref PubMed Scopus (126) Google Scholar, 17Sohal R.S. Sohal B.H. Mech. Ageing Dev. 1991; 57: 187-202Crossref PubMed Scopus (251) Google Scholar) increase in the cells of aging organisms, whereas the glutathione concentration declines progressively with advancing age (5Noy N. Schwartz H. Gafni A. Mech. Ageing Dev. 1985; 29: 63-69Crossref PubMed Scopus (41) Google Scholar, 6Sohal R.S. Farmer K.J. Allen R.G. Cohen N.R. Mech. Ageing Dev. 1983; 24: 185-195Crossref Scopus (57) Google Scholar, 18Lang C.A. Naryshkin S. Schneider D.L. Mills B.J. Linderman R.D. J. Lab. Clin. Med. 1992; 120: 720-725PubMed Google Scholar, 19Rikans L.E. Moore D.R. Biochim. Biophys. Acta. 1988; 966: 269-275Crossref PubMed Scopus (85) Google Scholar). Differences in ROS metabolism also exist between cells obtained from early developmental stages and from postnatal tissues (for review, see Refs.20Allen R.G. Balin A.K. Free Radical Biol. Med. 1989; 6: 631-661Crossref PubMed Scopus (264) Google Scholar and 21Allen R.G. Proc. Soc. Exp. Biol. Med. 1991; 196: 117-129Crossref PubMed Scopus (159) Google Scholar). The activities of enzymic antioxidant defenses tend to be lower in fetal cells than in cells derived from postnatal tissues (22Allen R.G. Balin A.K. J. Clin. Invest. 1988; 82: 731-734Crossref PubMed Scopus (24) Google Scholar, 23Allen R.G. Keogh B.P. Gerhard G.S. Pignolo R. Horton J. Cristofalo V.J. J. Cell. Physiol. 1995; 165: 576-587Crossref PubMed Scopus (30) Google Scholar, 24Keogh B.P. Allen R.G. Pignolo R. Horton J. Tresini M. Cristofalo V.J. J. Cell. Physiol. 1996; 167: 512-522Crossref PubMed Scopus (40) Google Scholar); however, the GSH concentration is frequently greater in fetal cells than in cells from adults (20Allen R.G. Balin A.K. Free Radical Biol. Med. 1989; 6: 631-661Crossref PubMed Scopus (264) Google Scholar, 24Keogh B.P. Allen R.G. Pignolo R. Horton J. Tresini M. Cristofalo V.J. J. Cell. Physiol. 1996; 167: 512-522Crossref PubMed Scopus (40) Google Scholar). Whether these observations reflect differences in the steady-state level of ROS in fetal and postnatal cells is not presently known (20Allen R.G. Balin A.K. Free Radical Biol. Med. 1989; 6: 631-661Crossref PubMed Scopus (264) Google Scholar, 21Allen R.G. Proc. Soc. Exp. Biol. Med. 1991; 196: 117-129Crossref PubMed Scopus (159) Google Scholar). The rate of mitochondrial ROS generation in cells is largely dependent on the amounts of autoxidizable respiratory carriers and the redox state of electron carriers (1Foreman H.J. Boveris A. Pryor W.A. Free Radicals in Biology. Academic Press, New York1982: 65-90Crossref Google Scholar, 3Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4819) Google Scholar, 25Boveris A. Cadenas E. Oberley L.W. Superoxide Dismutase. CRC Press, Boca Raton, FL1982: 16-30Google Scholar, 26Turrens J.F. McCord J.M. Paulet A.C. Douste-Blazy L. Paoletti R. Free Radicals, Lipoproteins, and Membrane Lipids. Plenum Press, New York1990: 203-212Crossref Google Scholar). All electron carriers located prior to one of low abundance will exhibit a greater propensity to be chemically reduced because of the slowing of electron flux (electron stacking; 1, 16, 25, 27). Thus, either age or development-associated changes in the relative abundance of components of the electron transport chain (ETC) may exert significant effects on ROS generation. A second factor that can alter the rate of electron flow and the rate of cellular oxidant generation is the activity of the enzyme complexes that regulate electron entry and exit from the ETC. For example, an age-dependent decrease in cytochromec oxidase (COX) activity has been implicated as one possible cause of the rise in ROS generation observed in aging insects (16Sohal R.S. Free Radical Biol. Med. 1993; 14: 583-588Crossref PubMed Scopus (126) Google Scholar) and in aging mammalian brains (28Benzi G. Pastoris O. Marzatico R.F. Villa R.F. Curti D. Neurobiol. Aging. 1992; 13: 361-368Crossref PubMed Scopus (111) Google Scholar). Changes in the relative proportions of electron carriers are not well studied in human cells. It has been reported that the activity of COX increases in cell cultures as they approach the mid phases of their proliferative lifespan (29Sun A.S. Aggarwal B.B. Packer L. Arch. Biochem. Biophys. 1975; 170: 1-11Crossref PubMed Scopus (57) Google Scholar). Furthermore, studies reported from this (30Doggett D.L. Rotenberg M.O. Pignolo R.J. Phillips P.D. Cristofalo V.J. Mech. Ageing Dev. 1992; 65: 239-255Crossref PubMed Scopus (40) Google Scholar) and other laboratories (31Kodama S. Yamada H. Annab L. Barrett J.C. Exp. Cell Res. 1995; 219: 82-86Crossref PubMed Scopus (16) Google Scholar) reveal that the mRNA abundance of a mitochondrial encoded subunit of NADH dehydrogenase (ND) increases in cell cultures that have reached advanced proliferative age (cellular senescence). Whether similar changes occur during aging in vivo is presently unknown. It is, however, noteworthy that various pathologies arising from deficiency of complex I (NADH dehydrogenase) have been reported to stimulate⋅O2− generation in human skin fibroblasts (32Pitkänen S. Robinson B.H. J. Clin. Invest. 1996; 98: 345-351Crossref PubMed Scopus (328) Google Scholar). In this study we investigate the relationship of the activities of mitochondrial enzymes that regulate the rate of electron flow in the ETC and the rate of cellular oxidant generation in 29 human skin fibroblast lines established from donors of different ages. We also determined the mRNA abundances of representative sets of nuclear and mitochondrial encoded subunits of ND and COX in these lines. Additionally, we determined the mRNA abundance of succinate dehydrogenase (SD), which is encoded entirely by the nuclear genome (33Bourgeron T. Rustin P. Chretien D. Birch-Machin M. Bourgeois M. Viegas-Péquignot E. Munnich A. Rötig A. Nat. Genet. 1995; 11: 144-149Crossref PubMed Scopus (615) Google Scholar, 34Au H.C. Ream-Robinson D. Bellew L.A. Broomfield P.L.E. Saghbini M. Scheffler I.E. Gene ( Amst. ). 1995; 159: 249-253Crossref PubMed Scopus (49) Google Scholar) for a comparison with the nuclear encoded subunits of the mosaic enzymes examined. We have determined previously that the skin fibroblast model, used in this study, maintains the development and age-dependent differences in antioxidant defenses known to exist in numerous organisms and tissues in vivo(22Allen R.G. Balin A.K. J. Clin. Invest. 1988; 82: 731-734Crossref PubMed Scopus (24) Google Scholar, 23Allen R.G. Keogh B.P. Gerhard G.S. Pignolo R. Horton J. Cristofalo V.J. J. Cell. Physiol. 1995; 165: 576-587Crossref PubMed Scopus (30) Google Scholar, 24Keogh B.P. Allen R.G. Pignolo R. Horton J. Tresini M. Cristofalo V.J. J. Cell. Physiol. 1996; 167: 512-522Crossref PubMed Scopus (40) Google Scholar). Unless otherwise stated chemicals used in this study were obtained from Sigma Chemical Co. and were of the highest purity. The probes for human COX subunit 1 (HHCJ-10), subunit 4 (pCOX4.111), the 51-kDa subunit of NADH dehydrogenase (IB38), and succinate dehydrogenase (HHHCPJ42) were all obtained from ATCC. The probe for ND subunit 4 (ND 4) was isolated previously in the Center for Gerontological Research (30Doggett D.L. Rotenberg M.O. Pignolo R.J. Phillips P.D. Cristofalo V.J. Mech. Ageing Dev. 1992; 65: 239-255Crossref PubMed Scopus (40) Google Scholar). Human skin fibroblast cultures established from skin samples obtained from fetal (12–20 weeks gestational age), young (17–33 years), and old donors (78–92 years) were obtained from the National Institute on Aging cell repository at the Coriell Institute for Medical Research, Camden, NJ. Most of the cell lines were obtained when they had completed fewer than 10 population doublings. The lines were minimally expanded and harvested at the earliest possible population doubling level. The population doubling level of cultures at harvest has been published elsewhere (23Allen R.G. Keogh B.P. Gerhard G.S. Pignolo R. Horton J. Cristofalo V.J. J. Cell. Physiol. 1995; 165: 576-587Crossref PubMed Scopus (30) Google Scholar). When originally obtained none of the lines was known to exhibit genetic defects; however, several of the lines have since been found to exhibit karyotypic anomalies. Chromosome breakage was present in 18% of the cells in AG04449; 6% of the cells in AG08817 exhibited random chromosome loss; 2% of cells in AG011016 exhibited random chromosome gain, and 2% were tetraploid; 8% of the cells in AG08433 exhibited random chromosome loss, 4% had a 47,XY + 7 karyotype, and AG09602 exhibited a 26%/74% mosaic karyotype of 45,X/46,XX. These lines retain a normal appearance and exhibit limited replicative lifespan. All other cell lines used in this study had a normal genotype, i.e. 46,XX or 46,XY. Cells were grown in minimum essential medium without antibiotics and supplemented with 2 mml-glutamine and 10% (v/v) fetal bovine serum according to procedures described previously (35Cristofalo V.J. Charpentier R. J. Tissue Culture Methods. 1980; 6: 117-121Crossref Scopus (116) Google Scholar). Cultures were gassed with a mixture of 5% CO2 with the balance air and were grown at 37 °C. When the cultures had reached stationary phase, they were washed twice, refed with serum-free MCDB 104 (Life Technologies, Inc.), and then incubated at 37 °C for an additional 72 h. This treatment was used to ensure that the cells were in a state of growth arrest. Cultures used for detection of chemiluminescence and H2O2 were maintained in minimum essential medium with 10% fetal bovine serum until the time of assay. All materials used in preparation of Northern blots were made RNase-free either by baking for 6 h at 170 °C or by pretreatment with 0.2% diethyl pyrocarbonate for 24 h followed by autoclaving. RNA samples were denatured by mixing 2 volumes (v/v) of a denaturing mixture and incubating at 50 °C for 15 min. The denaturing mixture was made by mixing 32.4 μl of glyoxal (Fluka, highest grade available), 3 μl of 1 m sodium phosphate, pH 6.5, 3 μl of 10% SDS, 10 μl of H2O, and 150 μl of dimethyl sulfoxide. The samples were resolved on 1.5% agarose gels. After electrophoresis, gels were treated with 50 mm NaOH for 30 min and then neutralized with 100 mm Tris, pH 7.5. Materials used beyond this point were not treated with diethyl pyrocarbonate; instead high quality water was used. Gels were transferred to Nytran Plus nylon membrane (prewetted in H2O and soaked in TAE (40 mm Tris acetate, 1 mm EDTA), pH 7.8) using a model TE-42 electrophoretic transfer apparatus (Hoefer Scientific Instruments) in 1 × TAE, 1.5 h at 25 volts. After transfer, the membrane was rinsed in 1 × TAE and allowed to air dry. It was then exposed to a UV transilluminator (FotoPrep I; Fotodyne Inc.) at the high setting for 8 min. The membrane was baked for 1 h at 80 °C in vacuo. Blots were prehybridized for at least 18 h at 45 °C in 50% formamide, 4 × SSPE (150 mm sodium chloride, 10 mm sodium phosphate, 1 mm EDTA), 0.5% BLOTTO, 1% SDS, 300 μg/ml sheared denatured herring sperm DNA, and then hybridized in 50% formamide, 3 × SSPE, 0.5% BLOTTO, 1% SDS, 300 μg/ml sheared denatured herring sperm DNA, and 10% dextran sulfate with 1–4 × 106 cpm/ml 32P random primer-labeled (Boehringer Mannheim) probe at 45 °C overnight. Blots were washed in three changes of wash buffer (0.1 × SSC, 0.5% SDS) for 15 min each wash. The blots were then exposed to Kodak XAR film using DuPont Lightning Plus intensifying screens. Postnuclear fractions were prepared by a modification of Sun et al. (29Sun A.S. Aggarwal B.B. Packer L. Arch. Biochem. Biophys. 1975; 170: 1-11Crossref PubMed Scopus (57) Google Scholar). Cultures were washed twice with PBS, once with 1 × isolation buffer (70 mmsucrose, 220 mm mannitol, 1 mm Tris, pH 7.4). The isolation buffer used for SD activity also contained 7 mm succinate. Cells were incubated at 4 °C for 5 min in a hypotonic buffer (5 mm Tris, pH 7.4) and then homogenized with seven strokes of a Dounce homogenizer. After homogenization, an equal volume of 2 × isolation buffer was added, and the homogenate was centrifuged at 600 × g for 2 min. The supernatant was collected, and the pellet was resuspended in 0.5 ml of 1 × isolation buffer. The resuspended pellet was centrifuged at 600 × g for 2 min. The supernatants from both fractionations were pooled, sonicated, and debris removed by centrifugation at 3,000 × g. COX activity was determined by a modification of the method of Sun et al.(29Sun A.S. Aggarwal B.B. Packer L. Arch. Biochem. Biophys. 1975; 170: 1-11Crossref PubMed Scopus (57) Google Scholar). A 50 μm cytochrome c solution (in 100 mm Tris, pH 7) was reduced with enough 0.1 mdithionite to yield anA 550/A 565 ratio that was between 6 and 9. The rate of change in A 550 of 100 μl of homogenate and 900 μl of reduced cytochrome cat 37 °C was monitored as a measure of COX activity. Specific activity was calculated as nmol/min/mg of protein using an extinction coefficient of ε = 19.2 mm−1cm−1. Although several groups have reported improved results when detergents were included in the homogenate preparation (29Sun A.S. Aggarwal B.B. Packer L. Arch. Biochem. Biophys. 1975; 170: 1-11Crossref PubMed Scopus (57) Google Scholar, 36Rafael J. Bergmeyer H.U. Methods in Enzymatic Analysis. Academic Press, New York1983: 266-273Google Scholar, 37Madden E.A. Storrie B. Anal. Biochem. 1987; 163: 350-357Crossref PubMed Scopus (36) Google Scholar), we observed no benefit from the addition of detergents to our reaction mixture and therefore included none. ND activity was determined by a slight modification of the method of Galante and Hatefi (38Galante Y.M. Hatefi Y. Methods Enzymol. 1978; 53: 15-21Crossref PubMed Scopus (102) Google Scholar). Postnuclear homogenates were mixed with a solution containing 55 mm Tris, pH 8.0, 130 μm2,6-dichloroindophenol, and 750 μm NADH, and the rate of change at A 600 was monitored at 37 °C. ND activity was expressed as nmol/min/mg of protein based on the extinction coefficient ε = 21 mm−1cm−1 for 2,6-dichloroindophenol. SD activity was determined by a modification of the method described by Hatefi and Stiggall (39Hatefi Y. Stiggall D.L. Methods Enzymol. 1978; 53: 21-27Crossref PubMed Scopus (165) Google Scholar). Solution I contained 55 mm potassium phosphate buffer, pH 7.4, 0.1 mm EDTA, 0.1% bovine serum albumin, and 40 mm sodium succinate (from a stock that was brought to a pH of 7.3–7.4 with 2 m KOH), and 298 μm2,6-dichloroindophenol. Solution II contained 55 mmpotassium phosphate buffer, pH 7.4, 0.1 mm EDTA, 0.1% bovine serum albumin, and 3.25 mm phenazine methosulfate. 200 μl of homogenate was mixed with 0.4 ml of solution I, and 0.4 ml of solution II was then added to start the reaction. After allowing the assay mixture and homogenate to react for 10 min at 37 °C, the change in A 600 was monitored at 37 °C. Activity was calculated as nmol/min/mg of protein using extinction coefficient ε = 21 mm−1 cm−1for 2,6-dichloroindophenol. For this study, lucigenin (bis-N-methylacridinium nitrate) was used as an indicator of ⋅O2−. Cells exposed to lucigenin tend to sequester it in their mitochondria, which should increase specificity. Furthermore, changes in chemiluminescence stimulated by various metabolic inhibitors are consistent with the cytochromec method of⋅O2− determination (40Rembish S.J. Trush M.A. Free Radical Biol. Med. 1994; 17: 117-126Crossref PubMed Scopus (88) Google Scholar). It must be noted that lucigenin is capable of redox cycling reactions that produce ⋅O2− (41Faulkner K. Fridovich I. Free Radical Biol. Med. 1993; 15: 447-451Crossref PubMed Scopus (324) Google Scholar, 42Vasquez-Vivar J. Hogg N. Fritchard K.A. Martasek P. Kalyanaraman B. FEBS Lett. 1997; 403: 127-130Crossref PubMed Scopus (190) Google Scholar, 43Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (202) Google Scholar), which can potentially skew the results of quantitative assays of⋅O2−. Hence, although the assay has limited usefulness for quantitative analysis of⋅O2− concentration, it does provide a qualitative estimate of mitochondrial⋅O2− generation which is rapid and far more sensitive than other methods available. Lucigenin-dependent chemiluminescence was determined in intact cells by a modification of the⋅O2− assay suggested by Aitkenet al. (44Aitken R.J. Buckingham D.W. West K.M. J. Cell. Physiol. 1992; 151: 466-477Crossref PubMed Scopus (212) Google Scholar). Confluent monolayers were released from plastic tissue culture vessels with 0.25% trypsin. The trypsin was neutralized using soybean trypsin inhibitor (1 mg/ml). Cells were pelleted by centrifugation at 500 × g for 7 min, washed once with reaction buffer (PBS, 1 g of glucose/liter, and 2 mm MgCl2). After repelleting the cells were resuspended in fresh reaction buffer. Typically 1.5 × 106 cells were suspended in 0.5 ml of buffer. 50 μl of suspended cells was mixed with 50 μl of 2 mm lucigenin (dissolved in reaction buffer). The reaction was placed in the dark for 10 min and then read using a Turner model 20e luminometer. The instrument was used to determine the integration of light emitted during a 15-s period. The luminometer was calibrated using a uranyl acetate light source (Turner Instruments). After the measurements were collected, CCI was determined as (1/uranyl acetate standard × chemiluminescent intensity)/mg of protein. The rate of H2O2 generation was determined using 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Because it is nonpolar, cell membranes are permeable to DCFH-DA (45Bass D.A. Parce J.W. DeChatelet L.R. Szejda P. Seeds M.C. Thomas M. J. Immunol. 1983; 130: 1910-1917PubMed Google Scholar); once in cells the compound is deacetylated to 2′,7′-dichlorofluorescin (DCFH). DCFH is polar and is thus trapped in cells. DCFH is oxidized by H2O2 to the highly fluorescent 2′,7′-dichlorofluorescein (DCF; 45). The oxidation of DCFH to DCF has been used widely to determine H2O2 generation by flow cytometry and direct visualization techniques (46Rothe G. Valet G. Methods Enzymol. 1994; 233: 539-548Crossref PubMed Scopus (103) Google Scholar, 47Carter W.O. Narayanan P.K. Robinson J.P. J. Leukocyte Biol. 1994; 55: 253-258Crossref PubMed Google Scholar, 48Rothe G. Valet G. J. Leukocyte Biol. 1990; 47: 440-448Crossref PubMed Scopus (781) Google Scholar). In this study, we used DCFH oxidation as a measure of H2O2 generation by entire cultures. Confluent T25 flasks were washed twice with PBS and then incubated 45 min at 37 °C with 3 ml of PBS containing 1 g glucose/liter, 2 mm MgCl2, and 5 μm DCFH-DA. The cultures were then washed twice with cold PBS and incubated for 1 min in cold hypotonic solution (10% PBS). The flasks were then scraped with a rubber policeman to remove any remaining material. The suspension was sonicated for 20 s using a Branson sonicator with a stepped microtip and a setting of 4. 0.5 ml of the homogenate was diluted by adding 1.5 PBS and then read using a Hitachi F2000 fluorometer (excitation = 499, emission = 524). The amount of H2O2 in the samples was calculated using known concentrations of DCF (ACS grade; Sigma). COX is the terminal protein of electron transport and is the sole determinant of the rate of electron exit from the ETC when mitochondria are coupled. The control of COX by cells requires coordinate regulation of both nuclear and mitochondrial encoded subunits (49Hatefi Y. Annu. Rev. Biochem. 1985; 54: 1015-1069Crossref PubMed Google Scholar, 50Attardi G. Chomyn A. King M.P. Kruse B. Polosa P.L. Murdter N.N. Biochem. Soc. Trans. 1990; 18: 509-513Crossref PubMed Scopus (51) Google Scholar). Fig. 1 shows that the mRNA abundance of COX 4 was relatively uniform, whereas the abundance of COX 1 was significantly greater in cultures established from either young or older adults than in cultures derived from fetal skin (Table I). COX activity was also at a significantly lower level in fetal fibroblasts than in postnatal cultures.Table IAnalysis of cytochrome c oxidase, NADH dehydrogenase, and succinate dehydrogenase in fibroblasts from people of different agesEnzyme and groupMeanTotal ANOVA1-aAll effects.Groups comparedLSD1-bPost hoc analysis; LSD, least significant difference. p valueCytochrome c oxidase ActivityFetal7.2Fetal/young0.0002Young28.50.0001Fetal/old0.00007Old30.1Young/old0.7 mRNA abundanceCOX 1Fetal0.3Fetal/young0.007Young0.70.007Fetal/old0.0006Old0.7Young/old0.3COX 4Fetal0.5Young0.40.2Old0.5NADH dehydrogenase ActivityFetal54.8Fetal/young0.03Young90.80.03Fetal/old0.01Old97.5Young/old0.7 mRNA abundanceND 4Fetal0.3Fetal/young0.00001Young0.70.000001Fetal/oldp < 10−7Old0.8Young/old0.2ND 51Fetal0.7Young0.60.4Old0.7Succinate dehydrogenase ActivityFetal6.1Fetal/young0.0006Young10.60.0007Fetal/old0.0005Old10.6Young/old0.9 mRNA abundanceSDFetal0.2Fetal/young0.04Young0.50.004Fetal/old0.001Old0.7Young/old0.1Optical densities were normalized to the greatest before analysis.1-a All effects.1-b Post hoc analysis; LSD, least significant difference. Open table in a new tab Optical densities were normalized to the greatest before analysis. ND is the primary component of complex I and the initial step in the ETC. ND is composed of multiple peptides that are encoded by both the nuclear and mitochondrial genomes (49Hatefi Y. Annu. Rev. Biochem. 1985; 54: 1015-1069Crossref PubMed Google Scholar, 50Attardi G. Chomyn A. King M.P. Kruse B. Polosa P.L. Murdter N.N. Biochem. Soc. Trans. 1990; 18: 509-513Crossref PubMed Scopus (51) Google Scholar, 51Ragan C.I. Biochem. Soc. Trans. 1990; 18: 515-516Crossref PubMed Scopus (19) Google Scholar). As in the case of COX, the mitochondrial encoded subunit (ND 4) was significantly greater in adults than in fetal cultures, whereas the nuclear encoded subunit (ND 51) was not (Fig.2 and Table I). ND activity was significantly greater in adult fibroblasts than in fetal cultures (Fig.2 and Table I). SD is about 50% of the protein component of complex II and provides a second point of entry for electrons into the ETC (52Hatefi Y. Methods Enzymol. 1978; 53: 27-35Crossref PubMed Scopus (45) Google Scholar). Although it is bound to the inner mitochondrial membrane, it is encoded entirely in the nucleus (34Au H.C. Ream-Robinson D. Bellew L.A. Broomfield P.L.E. Saghbini M. Scheffler I.E. Gene ( Amst. ). 1995; 159: 249-253Crossref PubMed Scopus (49) Google Scholar). An examination of fibroblasts established from donors of different ages revealed that fetal lines exhibited a significantly lower mRNA abundance than adults (Fig. 3 and TableI). Furthermore, SD activity was significantly greater in lines established from postnatal donors than in fetal lines (Table I). The ratio of ND and SD to COX was determined as a measure of the magnitude and efficiency of electron flux possible in the three age groups examined. As seen in Fig.4, all but one of the fetal cell lines exhibited a relatively high electron entry to exit potential. On average, the" @default.
• W2003044361 created "2016-06-24" @default.
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• W2003044361 title "Development and Age-associated Differences in Electron Transport Potential and Consequences for Oxidant Generation" @default.
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