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• W2017692041 abstract "With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-α. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-α. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models. With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-α. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-α. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models. infrared aminomethylcoumarin Dulbecco's modified Eagle's medium carbonyl cyanide p-trifluoromethoxyphenylhydrazone lactate dehydrogenase malondialdehyde 10-(6′-ubiquinolyl)decyltriphenylphosphonium 10-(6′-ubiquinonyl)decyltriphenylphosphonium mixture of mitoquinol and mitoquinone ubiquinone-1 ubiquinone-2 thiobarbituric acid-reactive species methyltriphenylphosphonium cation phosphate-buffered saline 4-morpholinepropanesulfonic acid The mitochondrial respiratory chain is a major source of superoxide and, therefore, mitochondria accumulate oxidative damage more rapidly than the rest of the cell, contributing to mitochondrial dysfunction and cell death in degenerative diseases and in aging (1Wallace D.C. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2595) Google Scholar, 2Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5382) Google Scholar, 3Ames B.N. Shigenaga M.K. Hagen T.M. Biochim. Biophys. Acta. 1995; 1271: 165-170Crossref PubMed Scopus (338) Google Scholar, 4Beckman K.B. Ames B.N. Physiol. Rev. 1998; 78: 547-581Crossref PubMed Scopus (3103) Google Scholar, 5Michikawa Y. Mazzucchelli F. Bresolin N. Scarlato G. Attardi G. Science. 1999; 286: 774-779Crossref PubMed Scopus (620) Google Scholar). Mitochondria are also central to activating apoptosis and oxidative damage can lead to cell death, however, the significance of mitochondrial oxidative damage for cell death is unclear (6Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2234) Google Scholar, 7Kroemer G. Dallaporta B. Resche-Rignon M. Ann. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1755) Google Scholar, 8Hampton M.B. Orenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (585) Google Scholar). One approach to this problem is to selectively target antioxidants to mitochondria (9Murphy M.P. Trends Biotechnol. 1997; 15: 326-330Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 10Murphy M.P. Smith R.A.J. Adv. Drug Delivery Rev. 2000; 41: 235-250Crossref PubMed Scopus (380) Google Scholar, 11Matthews R.T. Yang L. Browne S. Bail M. Beal M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8892-8897Crossref PubMed Scopus (506) Google Scholar). This should allow the relative importance of mitochondrial and cytoplasmic oxidative stress for cell death to be distinguished, and also enable the contribution of mitochondrial damage to aging, diabetes, and cancer to be investigated in cell and animal models. Derivatives of ubiquinol are promising antioxidants to target to mitochondria (11Matthews R.T. Yang L. Browne S. Bail M. Beal M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8892-8897Crossref PubMed Scopus (506) Google Scholar, 12Lass A. Forster M.J. Sohal R.S. Free Radic. Biol. Med. 1999; 26: 1357-1382Crossref PubMed Scopus (129) Google Scholar). In mammals ubiquinone comprises a 2,3-dimethoxy-5-methylbenzoquinone core with a hydrophobic 45- to 50-carbon chain at the 6 position (13Crane F.L. Barr R. Methods Enzymol. 1971; 18C: 137-165Crossref Scopus (114) Google Scholar, 14Crane F.L. Annu. Rev. Biochem. 1977; 46: 439-469Crossref PubMed Scopus (113) Google Scholar). Mitochondrial ubiquinone is a respiratory chain component buried within the lipid core of the inner membrane where it accepts two electrons from complexes I or II becoming reduced to ubiquinol, which then donates electrons to complex III (14Crane F.L. Annu. Rev. Biochem. 1977; 46: 439-469Crossref PubMed Scopus (113) Google Scholar). The ubiquinone pool in vivo is largely reduced and ubiquinol is an effective antioxidant, as well as being a mobile electron carrier (15Lass A. Sohal R.S. Arch. Biochem. Biophys. 1998; 352: 229-236Crossref PubMed Scopus (148) Google Scholar, 16Kagan V.E. Serbinova E.A. Stoyanovsky D.A. Khwaja S. Packer L. Methods Enzymol. 1994; 234: 343-354Crossref PubMed Scopus (43) Google Scholar, 17Maguire J.J. Wilson D.S. Packer L. J. Biol. Chem. 1989; 264: 21462-21465Abstract Full Text PDF PubMed Google Scholar, 18Ernster L. Forsmark P. Nordenbrand K. Biofactors. 1992; 3: 241-248PubMed Google Scholar). Ubiquinol acts as an antioxidant by donating a hydrogen atom from one of its hydroxyl groups to a lipid peroxyl radical, thereby decreasing lipid peroxidation within the mitochondrial inner membrane (18Ernster L. Forsmark P. Nordenbrand K. Biofactors. 1992; 3: 241-248PubMed Google Scholar, 19Takada M. Ikenoya S. Yuzuriha T. Katayama K. Methods Enzymol. 1984; 105: 147-155Crossref PubMed Scopus (94) Google Scholar, 20Ingold K.U. Bowry V.W. Stocker R. Walling C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 45-49Crossref PubMed Scopus (315) Google Scholar). The ubisemiquinone radicals thus formed disproportionate to ubiquinone and ubiquinol (21Land E.J. Swallow A.J. J. Biol. Chem. 1970; 245: 1890-1894Abstract Full Text PDF PubMed Google Scholar), or react with oxygen to form superoxide and ubiquinone thereby transferring the radical to the aqueous phase for detoxification by superoxide dismutase and peroxidases (17Maguire J.J. Wilson D.S. Packer L. J. Biol. Chem. 1989; 264: 21462-21465Abstract Full Text PDF PubMed Google Scholar, 20Ingold K.U. Bowry V.W. Stocker R. Walling C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 45-49Crossref PubMed Scopus (315) Google Scholar). The respiratory chain then recycles ubiquinone back to ubiquinol to restore its antioxidant function. Vitamin E is another important antioxidant within the mitochondrial inner membrane, and the tocopheroxyl radical thus formed is regenerated to active vitamin E by reaction with ubiquinol or ubisemiquinone (15Lass A. Sohal R.S. Arch. Biochem. Biophys. 1998; 352: 229-236Crossref PubMed Scopus (148) Google Scholar, 17Maguire J.J. Wilson D.S. Packer L. J. Biol. Chem. 1989; 264: 21462-21465Abstract Full Text PDF PubMed Google Scholar, 20Ingold K.U. Bowry V.W. Stocker R. Walling C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 45-49Crossref PubMed Scopus (315) Google Scholar, 22Stoyanovsky D.A. Osipov A.N. Quinn P.J. Kagan V.E. Arch. Biochem. Biophys. 1995; 323: 343-351Crossref PubMed Scopus (146) Google Scholar,23Mukai K. Kikuchi S. Urano S. Biochim. Biophys. Acta. 1990; 1035: 77-82Crossref PubMed Scopus (109) Google Scholar). Therefore, in vivo ubiquinol probably acts as an antioxidant by direct reaction with peroxyl radicals and by regenerating vitamin E (16Kagan V.E. Serbinova E.A. Stoyanovsky D.A. Khwaja S. Packer L. Methods Enzymol. 1994; 234: 343-354Crossref PubMed Scopus (43) Google Scholar, 17Maguire J.J. Wilson D.S. Packer L. J. Biol. Chem. 1989; 264: 21462-21465Abstract Full Text PDF PubMed Google Scholar, 20Ingold K.U. Bowry V.W. Stocker R. Walling C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 45-49Crossref PubMed Scopus (315) Google Scholar). The low solubility of ubiquinone in water makes it difficult to usein vitro, and animals must be fed ubiquinone-enriched diets for several weeks to increase levels in subsequently isolated mitochondria (11Matthews R.T. Yang L. Browne S. Bail M. Beal M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8892-8897Crossref PubMed Scopus (506) Google Scholar, 14Crane F.L. Annu. Rev. Biochem. 1977; 46: 439-469Crossref PubMed Scopus (113) Google Scholar). Therefore, to manipulate mitochondrial ubiquinone content in vitro we synthesized a ubiquinone analog selectively targeted to mitochondria by addition of a lipophilic triphenylphosphonium cation. Such lipophilic cations easily permeate lipid bilayers and accumulate in mitochondria within cells, driven by the large mitochondrial membrane potential (9Murphy M.P. Trends Biotechnol. 1997; 15: 326-330Abstract Full Text PDF PubMed Scopus (289) Google Scholar,10Murphy M.P. Smith R.A.J. Adv. Drug Delivery Rev. 2000; 41: 235-250Crossref PubMed Scopus (380) Google Scholar, 24Liberman E.A. Topali V.P. Tsofina L.M. Jasaitis A.A. Skulachev V.P. Nature. 1969; 222: 1076-1078Crossref PubMed Scopus (379) Google Scholar). Here we report on the antioxidant and antiapoptotic properties of this mitochondrially targeted ubiquinone derivative. To synthesize 11-bromoundecanoic peroxide (1) 11-bromoundecanoic acid (4.00 g, 15.1 mmol) and SOCl2 (1.6 ml, 21.5 mmol) were heated at 90 °C for 15 min (25Yu C.A. Yu L. Biochemistry. 1982; 21: 4096-4101Crossref PubMed Scopus (70) Google Scholar). Excess SOCl2 was removed by distillation under reduced pressure (15 mm Hg, 90 °C) and the residue (IR,1 1799 cm-1) was dissolved in diethyl ether (20 ml) and cooled to 0 °C. Hydrogen peroxide (30%, 1.8 ml) was added, followed by dropwise addition of pyridine (1.4 ml) over 45 min, then diethyl ether (10 ml) was added and after 1 h at room temperature the product was diluted with diethyl ether (150 ml), washed with H2O (2 × 70 ml), 1.2m HCl (2 × 70 ml), H2O (70 ml), 0.5m NaHCO3 (2 × 70 ml), and H2O (70 ml). After drying over MgSO4 the solvent was removed at room temperature under reduced pressure, giving crude 1 as a white solid (3.51 g), which was used without delay. IR (Nujol mull) 1810, 1782 cm-1. 6-(10-Bromodecyl)ubiquinone (2) was synthesized by stirring crude 1 (3.51 g, 12.5 mmol), 2,3-dimethoxy-5-methyl-1,4-benzoquinone (1.31 g, 7.19 mmol, Aldrich), and acetic acid (60 ml) for 20 h at 100 °C. After cooling to room temperature, the reaction was diluted with diethyl ether (600 ml), washed with H2O (2 × 400 ml), 1 m HCl (2 × 450 ml), 0.5 m NaHCO3 (2 × 450 ml), and H2O (2 × 400 ml), and dried over MgSO4. Removal of the solvent under reduced pressure gave a reddish solid (4.31 g). Column chromatography on silica gel, eluting with CH2Cl2, gave 2 as a red oil (809 mg, 28%) and unreacted 2,3-dimethoxy-5-methyl-1,4-benzoquinone (300 mg, 1.6 mmol, 13%). 2: TLC: R F(CH2Cl2, diethyl ether 20:1) 0.46; IR (film) 2928, 2854, 1650, 1611, 1456, 1288 cm-1; λmax(ethanol): 278 nm; 1H NMR (299.9 MHz) 3.99 (s, 6H, 2 x-OCH 3), 3.41 (t, J = 6.8 Hz, 2H, -CH 2-Br), 2.45 (t, J = 7.7 Hz, 2H, ubquinone-CH 2-), 2.02, (s, 3H, -CH 3). 1.89 (quin, J = 7.4 Hz, 2H, -CH 2 -CH2-Br), 1.42–1.28 (m, 14H, -(CH 2)7-) ppm;13C NMR (125.7 MHz) 184.7 (C = O), 184.2 (C = O), 144.3 (2C, ring), 143.1 (ring), 138.7 (ring), 61.2 (2× -OCH3), 34.0 (-CH2-), 32.8 (-CH2-), 29.8 (-CH2-), 29.4 (2× -CH2-), 29.3 (-CH2-), 28.7 (2× -CH2-), 28.2 (-CH2-), 26.4 (-CH2-), 11.9 (-CH3) ppm. Anal. calcd. for C19H29O4Br: C, 56.86; H, 7.28; found: C, 56.49, H, 7.34%; mass spectrum: calcd. for C19H29O4Br 400/402; found 400/402. To form the quinol, 6-(10-bromodecyl)-ubiquinol (3), NaBH4 (295 mg, 7.80 mmol) was added to 2 (649 mg, 1.62 mmol) in methanol (6 ml) and stirred under argon for 10 min (Scheme FS1). Excess NaBH4 was quenched with 5% HCl (2 ml), diluted with diethyl ether (40 ml), washed with 1.2 m HCl (40 ml), saturated NaCl (2 × 40 ml), and dried over MgSO4. Removal of the solvent under reduced pressure gave 3 as a yellow oily solid (541 mg, 83%). 1H NMR (299.9 MHz) 5.31 (s, 1H, -OH), 5.26 (s, 1H, -OH), 3.89 (s, 6H, 2× -OCH 3), 3.41 (t, J = 6.8 Hz, 2H, -CH 2-Br), 2.59 (t, J = 7.7 Hz, 2H ubquinol-CH 2-), 2.15 (s,3H, CH 3), 1.85 (quin,J = 7.4 Hz, 2H, -CH 2-CH2-Br), 1.44–1.21 (m, 14H, -(CH 2)7-) ppm. To synthesize 10-(6′-ubiquinolyl)decyltriphenylphosphonium bromide (4), triphenylphosphine (387 mg, 1.48 mmol), 3(541 mg, 1.34 mmol), and ethanol (95%, 2.5 ml) were sealed under argon in a 15-ml Kimax tube and stirred in the dark for 88 h at 85 °C. Removal of the solvent under reduced pressure gave an oily orange residue, which was dissolved in CH2Cl2(2 ml). Addition of diethyl ether (20 ml) gave a suspension, and after 5 min the supernatant was decanted. The residue was dissolved in CH2Cl2 (2 ml) followed by addition of diethyl ether (20 ml), and the supernatant was decanted. The CH2Cl2/diethyl ether extraction was repeated twice more, and residual solvent was removed under reduced pressure, giving crude 4 as a cream solid (507 mg). 1H NMR (299.9 MHz) 7.9–7.6 (m, 15H, -P+ Ph 3), 3.89 (s, 6H, 2× -OCH 3), 3.91–3.77 (m, 2H, -CH 2-P+Ph3), 2.57 (t,J = 7.8 Hz, 2H ubquinol-CH 2-), 2.14 (s, 3H, CH 3), 1.6–1.2 (m, 16H, -(CH 2)8-) ppm. 31P NMR (121.4 MHz) 25.1 ppm. Crude 4 (200 mg) was oxidized to 10-(6′-ubiquinonyl)decyltriphenyl-phosphonium bromide (5) by stirring in CDCl3 at room temperature under an oxygen atmosphere. The solvent was removed under reduced pressure, the residue was dissolved in CH2Cl2 (5 ml), diethyl ether (15 ml) was added, and the resultant suspension was stirred for 5 min. The supernatant was decanted, and the CH2Cl2/diethyl ether precipitation was repeated twice more. Residual solvent was removed under reduced pressure, giving crude 5 as a brown sticky solid (173 mg). IR (film) 3357, 2927, 2857, 1650, 1609, 1438, 1266, 1113 cm-1.1H NMR (299.9 MHz) 7.9–7.6 (m, 15H-P+ Ph 3), 3.98 (s, 6H, 2× -OCH 3), 3.93–3.8 (m, 2H, -CH 2-P+Ph3), 2.42 (t, J = 7.4 Hz, 2H, ubiquinone-CH 2-), 2.00 (s, 3H, CH 3), 1.6–1.2 (m, 16H, (CH 2)8-) ppm; 13C NMR (75.4 MHz) 184.8 (C=O), 184.2 (C=O) 144.3 (2C, ring), 143.1 (ring), 138.8 (ring). 135.0 (d,J = 2.4 Hz, -P+ Ph 3para), 133.8 (d, J = 85.0 Hz, -P+ Ph 3 ortho/meta), 130.5 (d,J = 13.3 Hz, P+ Ph 3ortho/meta), 118.6 (d, J = 85.0 Hz, P+ Ph 3 ipso); 30.4 (d,J = 15.8 Hz, -CH2-CH2-CH2-P+Ph3), 29.8 (-CH2-), 29.3 (−CH2-), 29.2 (2× -CH2-), 29.1 (-CH2-), 28.7 (-CH2-), 26.4 (-CH2-), 22.9 (d, J = 48.5 Hz, -CH2-P+Ph3), 22.7 (d,J = 4.9 Hz, -CH2-CH2-P+Ph3), 11.9 (-CH3) ppm. 31P NMR (121.4 MHz) 25.1 ppm. Anal. calcd. for C37H44O4PBr: C, 66.97; H 6.68; found: C, 66.69; H, 6.99; mass spectrum: calcd. for C37H44O4P 583.2977; found 583.2972. To synthesize 3H-enriched 10-(6′-ubiquinolyl)decyltriphenylphosphonium bromide, triphenylphosphine (4.09 mg; 15.6 μmol), 3 (6.3 mg; 15.6 μmol), and 250 μl of ethanol containing [3H]triphenylphosphine (74 μCi, Moravek Biochemicals, Brea, CA, 1 Ci/mmol) were sealed under argon in a Kimax tube and stirred in the dark for 55 h at 80 °C. After cooling the product was precipitated by addition of diethyl ether, and the orange solid was dissolved in a few drops of CH2Cl2and precipitated with diethyl ether. This was repeated four times to remove unreacted triphenylphosphine and 3. Two separate syntheses of 3H-enriched 10-(6′-ubiquinonyl)decyltriphenylphosphonium bromide were carried out giving products of 2.6 and 2.46 mCi/mmol, respectively, which gave the same results in experiments with isolated mitochondria, and their UV absorption spectra were as expected for a mixture of 4 and5. TLC followed by scintillation counting of sectioned plates and comparison with the R F values of the unlabeled compounds confirmed radiopurity. Stock solutions containing a mixture of 4 and 5 in ethanol were stored at -80 °C, and their concentrations were confirmed by 31P NMR. Fully oxidized solutions were generated by incubation in basic 95% ethanol on ice (13Crane F.L. Barr R. Methods Enzymol. 1971; 18C: 137-165Crossref Scopus (114) Google Scholar) or with beef heart mitochondrial membranes at room temperature. Both procedures gave an extinction coefficient of 10,400 m-1 cm-1 at 275 nm for the quinone, with shoulders at 263 and 268 nm corresponding to the triphenylphosphonium moiety (26Smith R.A.J. Porteous C.M. Coulter C.V. Murphy M.P. Eur. J. Biochem. 1999; 263: 709-716Crossref PubMed Scopus (409) Google Scholar, 27Burns R.J. Smith R.A.J. Murphy M.P. Arch. Biochem. Biophys. 1995; 322: 60-68Crossref PubMed Scopus (86) Google Scholar) and a broad shoulder at 290 nm due to the quinone (13Crane F.L. Barr R. Methods Enzymol. 1971; 18C: 137-165Crossref Scopus (114) Google Scholar) (Fig. 1 A). Reduction with NaBH4 gave the quinol, which had local maxima at 290 nm (ε = 1800 m-1 cm-1) and at 268 nm (ε = 3000 m-1 cm-1) (27Burns R.J. Smith R.A.J. Murphy M.P. Arch. Biochem. Biophys. 1995; 322: 60-68Crossref PubMed Scopus (86) Google Scholar). The Δεox-red at 275 nm in 50 mmsodium phosphate, pH 7.2, was 7000 m-1cm-1. The quinone extinction coefficient (10,400m-1 cm-1 at 275 nm) was slightly lower than that reported for other quinones (12,250m-1 cm-1) in aqueous buffer (28Cabrini L. Landi L. Pasquali P. Lenaz G. Arch. Biochem. Biophys. 1981; 208: 11-19Crossref PubMed Scopus (23) Google Scholar). This difference was not due to an intermolecular interaction between the phosphonium and the quinone, because the absorbances of2 and the simple phosphonium methyltriphenylphosphonium (TPMP) were additive when 50 μm of each were mixed together in either ethanol or aqueous buffer. To prepare mitoquinol an ethanolic solution was diluted in ∼0.5–1 ml of water and a few grains of NaBH4 were added. After incubation on ice in the dark for 5 min, excess NaBH4 was quenched with 5% HBr (0.2 ml) and the quinol was extracted into CH2Cl2(3 × 0.5 ml). The extract was then washed with water and 2m NaCl, then the CH2Cl2 was removed under a stream of nitrogen. The pale yellow solid residue was dissolved in acidified 96% ethanol. The yield was typically 70–80% and, as the quinol slowly oxidized in air, it was freshly prepared and stored on ice under argon in the dark. To determine partition coefficients, compounds were added to 2 ml each of 1-octanol-saturated PBS and PBS-saturated 1-octanol then shaken at 37 °C for 30 min in the dark. After separation by centrifugation, the amounts in each phase were determined by absorption relative to standard curves in 1-octanol-saturated PBS or PBS-saturated 1-octanol. Rat liver mitochondria were prepared by homogenization followed by differential centrifugation (29Chappell J.B. Hansford R.G. Birnie G.D. Subcellular Components: Preparation and Fractionation. Butterworths, London1972: 77-91Google Scholar). Beef heart mitochondria were isolated by standard procedures, and membrane fragments were prepared by sonication followed by centrifugation (30Smith A.L. Methods Enzymol. 1967; 10: 81-86Crossref Scopus (470) Google Scholar). Protein concentrations were determined by the biuret assay using bovine serum albumin as a standard (31Gornall A.G. Bardawill C.J. David M.M. J. Biol. Chem. 1949; 177: 751-766Abstract Full Text PDF PubMed Google Scholar). Endogenous ubiquinone was removed from lyophilized beef heart mitochondria by pentane extraction, and complete extraction was confirmed by the inability of these mitochondria to oxidize NADH in the absence of added Q1 (13Crane F.L. Barr R. Methods Enzymol. 1971; 18C: 137-165Crossref Scopus (114) Google Scholar). For respiration measurements, rat liver mitochondria (2 mg of protein/ml) were suspended in KCl medium (120 mm KCl, 10 mm Hepes, 1 mm EGTA, pH 7.2) at 25 °C supplemented with respiratory substrates and 1 mm phosphate in a 3-ml oxygen electrode (Rank Brothers, Bottisham, Cambridge, UK). After measuring the rate of coupled respiration 200 μmADP was added, the rate of phosphorylating respiration was measured and then FCCP (300 nm) was added and the rate of uncoupled respiration determined. To measure membrane potential, rat liver mitochondria (2 mg of protein/ml) were incubated for 3 min in 0.5 ml of KCl medium supplemented with nigericin (1 μg/ml), 5 mmeach of glutamate and malate, 1 μm TPMP, and 100 nCi/ml [3H]TPMP. After incubation the mitochondria were pelleted by centrifugation, the radioactivity in the pellet and supernatant were measured by scintillation counting, and the membrane potential was calculated using the Nernst equation, assuming a mitochondrial volume of 0.5 μl/mg of protein and that 60% of the intramitochondrial TPMP was membrane-bound (32Scott I.D. Nicholls D.G. Biochem. J. 1980; 186: 21-33Crossref PubMed Scopus (334) Google Scholar, 33Brown G.C. Brand M.D. Biochem. J. 1985; 225: 399-405Crossref PubMed Scopus (84) Google Scholar). The uptake of [3H]mitoQ by rat liver mitochondria was measured under the same conditions. Scanning spectra and kinetic measurements were made with an Aminco DW-2000 dual beam spectrophotometer using matched 1-ml quartz cuvettes at 20 °C. Beef heart mitochondrial membranes and freeze-thawed yeast mitochondria were incubated in 50 mm potassium phosphate, pH 7.2. Rat liver mitochondria were incubated in KCl medium. To measure thiobarbituric acid reactive species (TBARS), rat liver mitochondria (2 mg of protein/ml) were incubated at 37 °C with shaking for 45 min in 100 mm KCl, 10 mm Tris-HCl, pH 7.6. Then 0.8-ml aliquots were mixed with 400 μl of 0.5% thiobarbituric acid in 35% HClO4, heated at 100 °C for 15 min, diluted with 3 ml of water, and extracted into 3 ml of n-butanol. TBARS were determined fluorometrically (λexcite = 515 nm; λemission = 553 nm) and expressed as nanomoles of MDA by comparison with standard solutions of 1,1,3,3-tetraethoxypropane processed as above. Prior to analyzing samples, their mitoQ contents were brought to the same concentration to eliminate differences in MDA formation during heating and processing. To measure the membrane potential after exposure to oxidative damage, mitochondria were pelleted by centrifugation and resuspended in KCl medium, and their membrane potentials were determined as described above. To measure cis-parinaric acid oxidation, mitochondria (2 mg of protein/ml) were suspended in a 3-ml fluorimeter cuvette in 100 mm KCl and 10 mm Tris-HCl, pH 7.6, at 37 °C. The oxidation of cis-parinaric acid (Molecular Probes) was monitored fluorometrically (λexcite = 324 nm; λemission = 413 nm). A control experiment showed that titration of cis-parinaric acid into a mitochondrial suspension initially increased the fluorescence as it partitioned into membranes, but the increase became nonlinear after about 3 μm and declined above 10 μm due to self quenching (34Tribble D.L. van den Berg J.J.M. Motchnik P.A. Ames B.N. Lewis D.M. Chait A. Krauss R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1183-1187Crossref PubMed Scopus (148) Google Scholar). Therefore, 3 μm cis-parinaric acid was used for all experiments. Peroxynitrite was synthesized from acidified H2O2 and NaNO2 in a simple flow reactor as described previously (35Packer M.A. Murphy M.P. FEBS Lett. 1994; 345: 237-240Crossref PubMed Scopus (144) Google Scholar), concentrated by freeze fractionation and stock solutions in 1.5 m NaOH quantitated [ε302 = 1.67 mm-1.cm-1 (36Hughes M.N. Nicklin H.G. J. Chem. Soc. 1968; A: 450-452Crossref Google Scholar)]. The Saccharomyces cerevisiae strains used were: CY4-ΔCOQ3 (MATaura3-52 leu2-3 leu2-112 trp1-1 ade2-1 his3-11 can1-100 coq3::HIS3), kindly supplied by Prof. Ian W. Dawes, University of New South Wales, Australia (37Grant C.M. MacIver F.H. Dawes I.W. FEBS Lett. 1997; 410: 219-222Crossref PubMed Scopus (125) Google Scholar) and CEN.PK2–1C-ΔCOQ3 (CEN.PK2–1Ccoq3::LEU2) kindly supplied by Prof. Cathy Clarke, UCLA. Both ΔCOQ3 yeast strains were auxotrophic for ubiquinone when grown on nonfermentable medium. Yeasts were grown in Erlenmeyer flasks at 28 °C under air with shaking at 200 rpm. For growth analysis, cultures in YPD (1% bacto yeast extract, 2% bactopeptone, 2% dextrose) were diluted into YPEG (1% bacto yeast extract, 2% bactopeptone, 3% ethanol, 3% glycerol) to anA 600 of 0.1 and then grown in the dark while theA 600 was monitored. For studies on yeast mitochondria, mitochondria were prepared from lactate-grown yeast of the CY4-ΔCOQ3 and CY4 wild type strains (38Glick B.S. Pon L. Methods Enzymol. 1995; 260: 213-233Crossref PubMed Scopus (286) Google Scholar). Briefly, lactate-grown yeast were isolated by centrifugation, the cell wall was removed by digestion with Zymolyase, spheroplasts were homogenized, and mitochondria were isolated by differential centrifugation. Mitochondria were stored at -80 °C in 0.6 m sorbitol, 20 mm HEPES, pH 7.4, supplemented with 10 mg/ml fatty acid-free bovine serum albumin. For spectrophotometric assays, yeast mitochondria were washed in 0.6 m sorbitol, 20 mm HEPES and freeze-thawed in 50 mm potassium phosphate, pH 7.2. Human osteosarcoma 143B cells were cultured at 37 °C under humidified 95% air/5% CO2 in DMEM supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml), and 10% fetal calf serum. For toxicity studies, cells were grown to confluence in 24-well tissue culture dishes and incubated for 24 h with DMEM/serum containing the compound. The supernatants were then harvested, and the amount of LDH released was assayed and compared with that present in untreated wells lysed with 0.1% Triton. For uptake studies cells were suspended in 0.5 ml of DMEM supplemented with 10 mm HEPES and 5 μm[3H]mitoQ. After incubation, cells were pelleted by centrifugation, and the radioactivity in the pellet was quantitated by scintillation counting. For digitonin fractionation, cells were incubated as above, and then 500 μl of the cell suspension was mixed rapidly with 1.2 ml of ice-cold 250 mm sucrose, 20 mm MOPS pH 6.7, 3 mm EDTA, and 1 mg of digitonin, then 1 ml was layered onto 350 μl of oil (66% silicone oil/34% dioctyl pthalate) over 100 μl of 500 mm sucrose, 0.1% Triton and separated into mitochondrial and cytoplasmic fractions by centrifugation. The two fractions were assayed for citrate synthase and LDH activity or for content of radioactivity by scintillation counting (39Burns R.J. Murphy M.P. Arch. Biochem. Biophys. 1997; 339: 33-39Crossref PubMed Scopus (42) Google Scholar). The Jurkat human T lymphocyte line was grown at 37 °C under humidified 95% air/5% CO2 in RPMI 1640 supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml), and 10% fetal calf serum. Apoptosis was induced by addition of hydrogen peroxide (8Hampton M.B. Orenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (585) Google Scholar). Caspase activation in lysed cell pellets was measured fluorometrically by the cleavage of the peptide DEVD labeled with AMC (DEVD-AMC) and calibrated using an AMC standard curve (40Scarlett J.L. Sheard P.W. Hughes G. Ledgerwood E.C. Ku H.-H. Murphy M.P. FEBS Lett. 2000; 475: 267-272Crossref PubMed Scopus (207) Google Scholar). The proportion of cells undergoing apoptosis was quantitated by annexin V-fluorescein isothiocyanate staining followed by detection of annexin-positive cells using a Becton Dickinson FACScan flow cytometer. Column chromatography was on Silica Gel type 60, 200–400 mesh, 40–63 μm (Me" @default.
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• W2017692041 title "Selective Targeting of a Redox-active Ubiquinone to Mitochondria within Cells" @default.
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