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N-Acetylcysteine elicited increase in cytochrome c oxidase activity in mice synaptic mitochondria
Brain Research 842 Ž1999. 249–251 www.elsevier.comrlocaterbres
Short communication
N-Acetylcysteine elicited increase in cytochrome c oxidase activity in mice synaptic mitochondria Marcos Martınez ´ Banaclocha a
a, )
, Natividad Martınez ´
b
Department of Pathology, Hospital UniÕersitario La Paz, Madrid, Spain b Department of Oncology, Hospital General, Valencia, Spain Accepted 29 June 1999
Abstract It has been suggested that thiolic groups are essential for cytochrome c oxidase ŽCOX. activity and other respiratory mitochondrial enzymes. Recent experiments showed that the thiolic antioxidant N-acetylcysteine ŽNAC. can protect against age-related impairment in COX activity in mice hepatic mitochondria. The present paper shows that NAC enhances COX activity in vitro in synaptic mitochondria isolated from young and old mice. The optimum NAC concentration for maximum COX activity was 5 mM in young and 10 mM in old synaptic preparations. Our data suggest that mitochondrial thiolic groups, which are essentials to oxidative phosphorylation, are impaired by aging. q 1999 Elsevier Science B.V. All rights reserved. Keywords: N-Acetylcysteine; Aging; Thiol; Cytochrome c oxidase; Mitochondria; Complex IV
Postmitotic cells, such as neurons, are dependent on mitochondrial oxidative phosphorylation which produces ATP in the aerobic metabolism. Mono- and dithiols appear to be involved in oxidative phosphorylation and in the maintenance of membrane integrity in mitochondria w12,23,27x. Among the mitochondrial components which contain thiol groups essential for oxidative phosphorylation is cytochrome c oxidase ŽCOX. w6x. It is the terminal enzyme complex ŽComplex IV. in the mitochondrial electron transport chain and the major molecular oxygen reduction site in cells. There is increasing evidence of ageassociated impairment in the activity of COX and other electron transport chain enzymes w3,5,9,11,16x. Moreover, deficient COX activity has been implicated in age-related neurodegenerative diseases such as Alzheimer’s disease w2x. Of the different chemical compounds shown to increase COX in vitro and in vivo activity, thiolic substances seem to have special importance w25x. N-Acetylcysteine ŽNAC. is a thiolic antioxidant w1,10x that has proven to be an invaluable agent in the treatment of chronic bronchitis and paracetamol poisoning. Its spectrum of use also includes the treatment of hemorrhagic cystitis caused by oxazaphos-
)
Corresponding author. Cr Isla de Java, 70, 4-B, E-28034 Madrid, Spain. Fax: q34-1-7292280
phorines, and cardioprotection against doxorubicin, of renewed interest to clinical toxicologists. New experimental and clinical data have shown its ability to exert beneficial effects in human immunodeficiency virus infection, heart disease and cancer, and at present, it is one of the most promising antioxidant substances w18x. Despite the above, practically nothing is known about the action of NAC at the level of the subcellular organelles. In an earlier communication, we reported the in vivo effect of NAC on mitochondrial complex activities in mice liver mitochondria. The dietary administration of NAC Ž3% wrw. during 6 months to mice, resulted in a protection against age-related decreases in hepatic mitochondrial respiratory complex activities w20x. We have also recently shown that chronic NAC administration can protect against the agedependent increase of oxidised proteins in mice synaptic mitochondria w17x. In view of the above, and because synaptic mitochondria represent a homogeneous population of mitochondria derived from postmitotic cells, we investigated the possible action of NAC on COX activity in mice synaptic mitochondria. In the present paper, we report that NAC enhances the in vitro COX activity in synaptic mitochondria isolated from young and old mice. Female OF-1 mice, distributed in two groups of 24 " 1 Ž n s 6. and 72 " 2 week Ž n s 6. old animals, were maintained under controlled environmental conditions of tem-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 8 1 9 - 3
M. Martınez Brain Research 842 (1999) 249–251 ´ Banaclocha, N. Martınezr ´
250
perature Ž24 " 18C., light Ž12 h light:12 h dark. and fed ‘‘ad libitum’’ standard maintenance pellets. The animals were sacrificed by cervical dislocation and their brains were quickly dissected out in ice-cold isolation medium Žin mM: sucrose, 320; K-EDTA, 1; Tris–HCl, 10, pH 7.4.. Whole brain homogenates were obtained using a Teflonglass homogeniser ŽBraun. by six up and down passes of the pestle Žclearance 0.1 mm. at 800 rpm, and synaptosomes were isolated according to Lai and Clark w14x. Synaptosomal fractions obtained by discontinuous Ficoll gradient centrifugation were resuspended in isolation medium with digitonin Ž0.15 mgrmg of synaptosomal protein., and synaptic brain mitochondria were prepared according to the method of Nicholls w21x with minor modifications w16x. Freshly isolated synaptic mitochondria were removed and stored at y808C for further analysis. Protein content was evaluated w15x and COX activity measured w26x with growing concentrations of NAC in the enzymatic assay of mitochondrial preparation samples. Enzymatic activities were recorded ŽBeckman DU 7500 Spectrophotometer Recorder. and calculated using the straight portion of the reaction curves. The results were expressed as specific activities on a protein basis Žnmol miny1 mgy1 of mitochondrial protein. and then statistically analyzed by Student’s t-test. This study was performed in accordance with the guidelines of the CEE for the care and use of laboratory animals and was approved by the local animal care committee. All reagents were purchased from Sigma ŽSt. Louis, MO, USA.. Table 1 gives the values for COX specific activities assayed in the synaptic mitochondrial fraction of young and old mice brains without and with growing concentrations of NAC in the assay. The addition of NAC to the
Table 1 COX specific activities in synaptic mitochondria from young and old mice. Effect of in vitro addition of NAC COX activities Žnmol miny1 mgy1 of mitochondrial protein. are expressed as mean"S.D. COX activity
Control 0.01 mM 0.1 mM 1 mM 5 mM 10 mM 20 mM 50 mM 100 mM a
Young mice Ž ns6.
Old mice Ž ns6.
446"63 448"67 473"71 538"70 a 688"76 b 560"72 a 402"56 220"66 b 44"9 c
309"65 301"60 330"62 342"73 425"73a 450"79 b 382"64a 170"65 b 28"6 c
p- 0.05 for statistically significant differences between enzymatic activities obtained in the presence of NAC and control values. b p- 0.01 for statistically significant differences between enzymatic activities obtained in the presence of NAC and control values. c p- 0.001 for statistically significant differences between enzymatic activities obtained in the presence of NAC and control values.
reaction mixture enhanced COX activity in synaptic mitochondria isolated from young and old mice. The optimum NAC concentrations to produce the maximum COX activity in synaptic mitochondria were 5 mM in young Ž31% activity increase. and 10 mM in old Ž45% activity increase. mice. The percentage range of concentrations which significantly increase COX activity in vitro ranged from 1 mM to 10 mM in young mice, and from 5 mM to 20 mM in old mice. Our results also show that high concentrations of NAC Ž) 20 mM. decrease COX activity in synaptic mitochondria in both young and old mice. The present studies reveal that the thiolic antioxidant NAC can increase COX activity in synaptic mitochondrial fractions isolated from young and old mice, eliciting a stronger increase in old Ž45%. than in young Ž31%. mice. These results suggest that there is an age-related impairment in the turnover of mitochondrial thiolic groups that may affect the activity of key metabolic enzymes. Mitochondrial COX is of transcendental importance since this enzyme plays a central role in the energetic metabolism. Since previously published data support the concept that thiolic groups are lost during the aging process, we feel that thiolic antioxidants such as NAC may help prevent the age-related decrease in mitochondrial respiratory activity. There are several potential mechanisms by which NAC may increase COX activity in vitro. First, the NAC thiolic group may play a role in catalysis since the catalytic core of the enzyme contains the prosthetic groups and metals, in which seem to have critical importance the existence of sulfur-containing residues w6x. Second, thiolic groups may participate in cofactor and substrate binding by the formation of covalent addition products or by the formation of charge transfer complexes. Third, thiolic groups may help maintain the tertiary structure acting as donors for weak hydrogen bonds. These, and other potential mechanisms of action, may change the affinity for the substrate, impair the internal electron transfer rate or change the affinity for oxygen in the enzyme. It may be noted that the optimum concentrations of NAC Ž5–10 mM. found in the present experiments are similar to those previously reported in in vitro w1,7,8,19,22x and in vivo w4,10x studies. Lower concentrations seem to have no effect, whereas higher concentrations Ž) 20 mM. can be inhibitory. The physiological cellular concentrations of glutathione have been reported to be in the range of 0.5–7 mM w13x. Thiol homeostasis is crucial for cell survival and concentrations as high as 25 mM are deleterious to the cells. In agreement with our present results with NAC, similar concentration-dependent response curves for glutathione have also been reported in other systems w24x. The deleterious effect of higher thiol levels is probably due to their interference with the delicate redox balance of cysteine residues and the reduction of disulfide bridges which impair structural proteins and enzymatic catalysis. Our present data show 50% inhibition of COX activity at 50 mM of NAC, and 90% inhibition at 100 mM of the
M. Martınez Brain Research 842 (1999) 249–251 ´ Banaclocha, N. Martınezr ´
antioxidant. The relevance of the present data needs to be confirmed in vivo. Our results highlight the primary importance of thiolic groups for the integrity of mitochondrial energy-linked processes.
w13x w14x w15x
References w16x w1x O.I. Aruoma, B. Halliwell, B.M. Hoey, J. Butler, The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid, Free Rad. Biol. Med. 6 Ž1989. 593–597. w2x M.F. Beal, Mitochondria, free radicals, and neurodegeneration, Curr. Opin. Neurobiol. 6 Ž1996. 661–666. w3x G. Benzi, O. Pastoris, F. Marzatico, R.F. Villa, F. Dagani, D. Curti, The mitochondrial electron transfer alteration as a factor involved in the brain aging, Neurobiol. Aging 13 Ž1992. 361–368. w4x S. Boesgaard, J. Aldershvile, H.E. Poulsen, S. Christensen, H. Digepetersen, J. Giese, N-acetylcysteine inhibits angiotensin converting enzyme in vivo, J. Pharmacol. Exp. Ther. 265 Ž1993. 1239–1244. w5x A.C. Bowling, E.M. Mutisya, L.C. Walker, D.L. Price, L.C. Cork, M.F. Beal, Age-dependent impairment of mitochondrial function in primate brain, J. Neurochem. 60 Ž1993. 1964–1967. w6x R.A. Capaldi, Structure and function of cytochrome c oxidase, Annu. Rev. Biochem. 59 Ž1990. 569–596. w7x C.A. Colton, F. Pagan, J. Snell, J.S. Colton, A. Cummins, D.L. Gilbert, Protection from oxidation enhances the survival of cultured mesencephalic neurons, 132 Ž1995. 54–61. w8x A. Cossarizza, C. Franceschi, D. Monti, S. Salvioli, E. Bellesia, R. Rivabene, L. Biondo, G. Rainaldi, A. Tinari, W. Malorni, Protective effect of N-acetylcysteine in tumour necrosis factor-a-induced apoptosis in U937 cells: the role of mitochondria, Exp. Cell. Res. 220 Ž1995. 232–240. w9x D. Curti, M.C. Giangare, M.E. Redolfi, I. Fugaccia, G. Benzi, Age-related modifications of cytochrome c oxidase activity in discrete brain regions, Mech. Ageing Dev. 55 Ž1990. 171–180. w10x S. De Flora, A. Izzotti, F. D’Agostini, C.F. Cesarone, Antioxidant activity and other mechanisms of thiols involved in chemoprevention of mutation and cancer, Am. J. Med. 91 Ž1991. 122–130. w11x H.J. Harmon, S. Nank, R.A. Floyd, Age-dependent changes in rat brain mitochondria of synaptic and non-synaptic origins, Mech. Ageing Dev. 38 Ž1987. 167–177. w12x N. Haugaard, N.H. Lee, R. Kostrzewa, R.S. Horn, E.S. Haugaard, The role of sulfhydryl groups in oxidative phosphorylation and ion
w17x
w18x
w19x
w20x
w21x
w22x
w23x
w24x
w25x w26x w27x
251
transport by rat liver mitochondria, Biochem. Biophys. Acta 172 Ž1969. 198–204. P.D. Jones, Glutathione distribution in natural products: absorption and tissue distribution, Methods Enzymol. 252 Ž1995. 3–25. J.C.K. Lai, J.B. Clark, Preparation and properties of mitochondria derived from synaptosomes, Biochem. J. 154 Ž1976. 421–432. O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. M. Martınez, M.L. Ferrandiz, E. De Juan, J. Miquel, Age related ´ ´ changes in glutathione and lipid peroxide content in mouse synaptic mitochondria: relationship to cytochrome c oxidase decline, Neurosci. Lett. 170 Ž1994. 121–124. M. Martınez, A.I. Hernandez, N. Martınez, M.L. Ferrandiz, N´ ´ ´ ´ acetylcysteine protects against age-related increase in oxidized proteins in mouse synaptic mitochondria, Brain Res. 762 Ž1997. 256– 258. M. Martınez, N. Martınez, A.I. Hernandez, M.L. Ferrandiz, Hypoth´ ´ ´ ´ esis: can N-acetylcysteine be beneficial in Parkinson’s disease?, Life Sci. 64 Ž1999. 1253–1257. M. Mayer, M. Noble, N-acetyl-L-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 7496–7500. J. Miquel, M.L. Ferrandiz, E. De Juan, I. Sevila, M. Martınez, ´ ´ N-acetylcysteine protects against age-related decline of oxidative phosphorylation in liver mitochondria, Eur. J. Pharmacol. 292 Ž1995. 333–335. D.G. Nicholls, Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart, Biochem. J. 170 Ž1978. 511–522. D. Offen, I. Ziv, H. Sternin, E. Melamed, A. Hochman, Prevention of dopamine-induced cell death by thiol antioxidants: possible implications for treatment of Parkinson’s disease, Exp. Neurol. 141 Ž1996. 32–39. D. Siliprandi, N. Siliprandi, G. Scutari, F. Zoccarato, Restoration of some energy linked processes lost during the ageing of rat liver mitochondria, Biochem. Biophys. Res. Commun. 55 Ž1973. 563–567. M. Vincent, M. Lafleur, J.J. Hoorweg, H. Joenje, E.J. Westmijze, J. Retel, The ambivalent role of glutathione in the protection of DNA against singlet oxygen, Free Rad. Res. 21 Ž1994. 9–17. W.W. Wainio, V. Nikodem, Sulfhydryl groups of cytochrome oxidase, J. Bioenerg. 4 Ž1973. 579–590. D.C. Wharton, A. Tzagoloff, Cytochrome oxidase from beef heart mitochondria, Methods Enzymol. 10 Ž1967. 245–250. T. Yagi, Y. Hatefi, Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers, Biochemistry 23 Ž1984. 2449–2455.
Short communication
N-Acetylcysteine elicited increase in cytochrome c oxidase activity in mice synaptic mitochondria Marcos Martınez ´ Banaclocha a
a, )
, Natividad Martınez ´
b
Department of Pathology, Hospital UniÕersitario La Paz, Madrid, Spain b Department of Oncology, Hospital General, Valencia, Spain Accepted 29 June 1999
Abstract It has been suggested that thiolic groups are essential for cytochrome c oxidase ŽCOX. activity and other respiratory mitochondrial enzymes. Recent experiments showed that the thiolic antioxidant N-acetylcysteine ŽNAC. can protect against age-related impairment in COX activity in mice hepatic mitochondria. The present paper shows that NAC enhances COX activity in vitro in synaptic mitochondria isolated from young and old mice. The optimum NAC concentration for maximum COX activity was 5 mM in young and 10 mM in old synaptic preparations. Our data suggest that mitochondrial thiolic groups, which are essentials to oxidative phosphorylation, are impaired by aging. q 1999 Elsevier Science B.V. All rights reserved. Keywords: N-Acetylcysteine; Aging; Thiol; Cytochrome c oxidase; Mitochondria; Complex IV
Postmitotic cells, such as neurons, are dependent on mitochondrial oxidative phosphorylation which produces ATP in the aerobic metabolism. Mono- and dithiols appear to be involved in oxidative phosphorylation and in the maintenance of membrane integrity in mitochondria w12,23,27x. Among the mitochondrial components which contain thiol groups essential for oxidative phosphorylation is cytochrome c oxidase ŽCOX. w6x. It is the terminal enzyme complex ŽComplex IV. in the mitochondrial electron transport chain and the major molecular oxygen reduction site in cells. There is increasing evidence of ageassociated impairment in the activity of COX and other electron transport chain enzymes w3,5,9,11,16x. Moreover, deficient COX activity has been implicated in age-related neurodegenerative diseases such as Alzheimer’s disease w2x. Of the different chemical compounds shown to increase COX in vitro and in vivo activity, thiolic substances seem to have special importance w25x. N-Acetylcysteine ŽNAC. is a thiolic antioxidant w1,10x that has proven to be an invaluable agent in the treatment of chronic bronchitis and paracetamol poisoning. Its spectrum of use also includes the treatment of hemorrhagic cystitis caused by oxazaphos-
)
Corresponding author. Cr Isla de Java, 70, 4-B, E-28034 Madrid, Spain. Fax: q34-1-7292280
phorines, and cardioprotection against doxorubicin, of renewed interest to clinical toxicologists. New experimental and clinical data have shown its ability to exert beneficial effects in human immunodeficiency virus infection, heart disease and cancer, and at present, it is one of the most promising antioxidant substances w18x. Despite the above, practically nothing is known about the action of NAC at the level of the subcellular organelles. In an earlier communication, we reported the in vivo effect of NAC on mitochondrial complex activities in mice liver mitochondria. The dietary administration of NAC Ž3% wrw. during 6 months to mice, resulted in a protection against age-related decreases in hepatic mitochondrial respiratory complex activities w20x. We have also recently shown that chronic NAC administration can protect against the agedependent increase of oxidised proteins in mice synaptic mitochondria w17x. In view of the above, and because synaptic mitochondria represent a homogeneous population of mitochondria derived from postmitotic cells, we investigated the possible action of NAC on COX activity in mice synaptic mitochondria. In the present paper, we report that NAC enhances the in vitro COX activity in synaptic mitochondria isolated from young and old mice. Female OF-1 mice, distributed in two groups of 24 " 1 Ž n s 6. and 72 " 2 week Ž n s 6. old animals, were maintained under controlled environmental conditions of tem-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 8 1 9 - 3
M. Martınez Brain Research 842 (1999) 249–251 ´ Banaclocha, N. Martınezr ´
250
perature Ž24 " 18C., light Ž12 h light:12 h dark. and fed ‘‘ad libitum’’ standard maintenance pellets. The animals were sacrificed by cervical dislocation and their brains were quickly dissected out in ice-cold isolation medium Žin mM: sucrose, 320; K-EDTA, 1; Tris–HCl, 10, pH 7.4.. Whole brain homogenates were obtained using a Teflonglass homogeniser ŽBraun. by six up and down passes of the pestle Žclearance 0.1 mm. at 800 rpm, and synaptosomes were isolated according to Lai and Clark w14x. Synaptosomal fractions obtained by discontinuous Ficoll gradient centrifugation were resuspended in isolation medium with digitonin Ž0.15 mgrmg of synaptosomal protein., and synaptic brain mitochondria were prepared according to the method of Nicholls w21x with minor modifications w16x. Freshly isolated synaptic mitochondria were removed and stored at y808C for further analysis. Protein content was evaluated w15x and COX activity measured w26x with growing concentrations of NAC in the enzymatic assay of mitochondrial preparation samples. Enzymatic activities were recorded ŽBeckman DU 7500 Spectrophotometer Recorder. and calculated using the straight portion of the reaction curves. The results were expressed as specific activities on a protein basis Žnmol miny1 mgy1 of mitochondrial protein. and then statistically analyzed by Student’s t-test. This study was performed in accordance with the guidelines of the CEE for the care and use of laboratory animals and was approved by the local animal care committee. All reagents were purchased from Sigma ŽSt. Louis, MO, USA.. Table 1 gives the values for COX specific activities assayed in the synaptic mitochondrial fraction of young and old mice brains without and with growing concentrations of NAC in the assay. The addition of NAC to the
Table 1 COX specific activities in synaptic mitochondria from young and old mice. Effect of in vitro addition of NAC COX activities Žnmol miny1 mgy1 of mitochondrial protein. are expressed as mean"S.D. COX activity
Control 0.01 mM 0.1 mM 1 mM 5 mM 10 mM 20 mM 50 mM 100 mM a
Young mice Ž ns6.
Old mice Ž ns6.
446"63 448"67 473"71 538"70 a 688"76 b 560"72 a 402"56 220"66 b 44"9 c
309"65 301"60 330"62 342"73 425"73a 450"79 b 382"64a 170"65 b 28"6 c
p- 0.05 for statistically significant differences between enzymatic activities obtained in the presence of NAC and control values. b p- 0.01 for statistically significant differences between enzymatic activities obtained in the presence of NAC and control values. c p- 0.001 for statistically significant differences between enzymatic activities obtained in the presence of NAC and control values.
reaction mixture enhanced COX activity in synaptic mitochondria isolated from young and old mice. The optimum NAC concentrations to produce the maximum COX activity in synaptic mitochondria were 5 mM in young Ž31% activity increase. and 10 mM in old Ž45% activity increase. mice. The percentage range of concentrations which significantly increase COX activity in vitro ranged from 1 mM to 10 mM in young mice, and from 5 mM to 20 mM in old mice. Our results also show that high concentrations of NAC Ž) 20 mM. decrease COX activity in synaptic mitochondria in both young and old mice. The present studies reveal that the thiolic antioxidant NAC can increase COX activity in synaptic mitochondrial fractions isolated from young and old mice, eliciting a stronger increase in old Ž45%. than in young Ž31%. mice. These results suggest that there is an age-related impairment in the turnover of mitochondrial thiolic groups that may affect the activity of key metabolic enzymes. Mitochondrial COX is of transcendental importance since this enzyme plays a central role in the energetic metabolism. Since previously published data support the concept that thiolic groups are lost during the aging process, we feel that thiolic antioxidants such as NAC may help prevent the age-related decrease in mitochondrial respiratory activity. There are several potential mechanisms by which NAC may increase COX activity in vitro. First, the NAC thiolic group may play a role in catalysis since the catalytic core of the enzyme contains the prosthetic groups and metals, in which seem to have critical importance the existence of sulfur-containing residues w6x. Second, thiolic groups may participate in cofactor and substrate binding by the formation of covalent addition products or by the formation of charge transfer complexes. Third, thiolic groups may help maintain the tertiary structure acting as donors for weak hydrogen bonds. These, and other potential mechanisms of action, may change the affinity for the substrate, impair the internal electron transfer rate or change the affinity for oxygen in the enzyme. It may be noted that the optimum concentrations of NAC Ž5–10 mM. found in the present experiments are similar to those previously reported in in vitro w1,7,8,19,22x and in vivo w4,10x studies. Lower concentrations seem to have no effect, whereas higher concentrations Ž) 20 mM. can be inhibitory. The physiological cellular concentrations of glutathione have been reported to be in the range of 0.5–7 mM w13x. Thiol homeostasis is crucial for cell survival and concentrations as high as 25 mM are deleterious to the cells. In agreement with our present results with NAC, similar concentration-dependent response curves for glutathione have also been reported in other systems w24x. The deleterious effect of higher thiol levels is probably due to their interference with the delicate redox balance of cysteine residues and the reduction of disulfide bridges which impair structural proteins and enzymatic catalysis. Our present data show 50% inhibition of COX activity at 50 mM of NAC, and 90% inhibition at 100 mM of the
M. Martınez Brain Research 842 (1999) 249–251 ´ Banaclocha, N. Martınezr ´
antioxidant. The relevance of the present data needs to be confirmed in vivo. Our results highlight the primary importance of thiolic groups for the integrity of mitochondrial energy-linked processes.
w13x w14x w15x
References w16x w1x O.I. Aruoma, B. Halliwell, B.M. Hoey, J. Butler, The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid, Free Rad. Biol. Med. 6 Ž1989. 593–597. w2x M.F. Beal, Mitochondria, free radicals, and neurodegeneration, Curr. Opin. Neurobiol. 6 Ž1996. 661–666. w3x G. Benzi, O. Pastoris, F. Marzatico, R.F. Villa, F. Dagani, D. Curti, The mitochondrial electron transfer alteration as a factor involved in the brain aging, Neurobiol. Aging 13 Ž1992. 361–368. w4x S. Boesgaard, J. Aldershvile, H.E. Poulsen, S. Christensen, H. Digepetersen, J. Giese, N-acetylcysteine inhibits angiotensin converting enzyme in vivo, J. Pharmacol. Exp. Ther. 265 Ž1993. 1239–1244. w5x A.C. Bowling, E.M. Mutisya, L.C. Walker, D.L. Price, L.C. Cork, M.F. Beal, Age-dependent impairment of mitochondrial function in primate brain, J. Neurochem. 60 Ž1993. 1964–1967. w6x R.A. Capaldi, Structure and function of cytochrome c oxidase, Annu. Rev. Biochem. 59 Ž1990. 569–596. w7x C.A. Colton, F. Pagan, J. Snell, J.S. Colton, A. Cummins, D.L. Gilbert, Protection from oxidation enhances the survival of cultured mesencephalic neurons, 132 Ž1995. 54–61. w8x A. Cossarizza, C. Franceschi, D. Monti, S. Salvioli, E. Bellesia, R. Rivabene, L. Biondo, G. Rainaldi, A. Tinari, W. Malorni, Protective effect of N-acetylcysteine in tumour necrosis factor-a-induced apoptosis in U937 cells: the role of mitochondria, Exp. Cell. Res. 220 Ž1995. 232–240. w9x D. Curti, M.C. Giangare, M.E. Redolfi, I. Fugaccia, G. Benzi, Age-related modifications of cytochrome c oxidase activity in discrete brain regions, Mech. Ageing Dev. 55 Ž1990. 171–180. w10x S. De Flora, A. Izzotti, F. D’Agostini, C.F. Cesarone, Antioxidant activity and other mechanisms of thiols involved in chemoprevention of mutation and cancer, Am. J. Med. 91 Ž1991. 122–130. w11x H.J. Harmon, S. Nank, R.A. Floyd, Age-dependent changes in rat brain mitochondria of synaptic and non-synaptic origins, Mech. Ageing Dev. 38 Ž1987. 167–177. w12x N. Haugaard, N.H. Lee, R. Kostrzewa, R.S. Horn, E.S. Haugaard, The role of sulfhydryl groups in oxidative phosphorylation and ion
w17x
w18x
w19x
w20x
w21x
w22x
w23x
w24x
w25x w26x w27x
251
transport by rat liver mitochondria, Biochem. Biophys. Acta 172 Ž1969. 198–204. P.D. Jones, Glutathione distribution in natural products: absorption and tissue distribution, Methods Enzymol. 252 Ž1995. 3–25. J.C.K. Lai, J.B. Clark, Preparation and properties of mitochondria derived from synaptosomes, Biochem. J. 154 Ž1976. 421–432. O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. M. Martınez, M.L. Ferrandiz, E. De Juan, J. Miquel, Age related ´ ´ changes in glutathione and lipid peroxide content in mouse synaptic mitochondria: relationship to cytochrome c oxidase decline, Neurosci. Lett. 170 Ž1994. 121–124. M. Martınez, A.I. Hernandez, N. Martınez, M.L. Ferrandiz, N´ ´ ´ ´ acetylcysteine protects against age-related increase in oxidized proteins in mouse synaptic mitochondria, Brain Res. 762 Ž1997. 256– 258. M. Martınez, N. Martınez, A.I. Hernandez, M.L. Ferrandiz, Hypoth´ ´ ´ ´ esis: can N-acetylcysteine be beneficial in Parkinson’s disease?, Life Sci. 64 Ž1999. 1253–1257. M. Mayer, M. Noble, N-acetyl-L-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 7496–7500. J. Miquel, M.L. Ferrandiz, E. De Juan, I. Sevila, M. Martınez, ´ ´ N-acetylcysteine protects against age-related decline of oxidative phosphorylation in liver mitochondria, Eur. J. Pharmacol. 292 Ž1995. 333–335. D.G. Nicholls, Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart, Biochem. J. 170 Ž1978. 511–522. D. Offen, I. Ziv, H. Sternin, E. Melamed, A. Hochman, Prevention of dopamine-induced cell death by thiol antioxidants: possible implications for treatment of Parkinson’s disease, Exp. Neurol. 141 Ž1996. 32–39. D. Siliprandi, N. Siliprandi, G. Scutari, F. Zoccarato, Restoration of some energy linked processes lost during the ageing of rat liver mitochondria, Biochem. Biophys. Res. Commun. 55 Ž1973. 563–567. M. Vincent, M. Lafleur, J.J. Hoorweg, H. Joenje, E.J. Westmijze, J. Retel, The ambivalent role of glutathione in the protection of DNA against singlet oxygen, Free Rad. Res. 21 Ž1994. 9–17. W.W. Wainio, V. Nikodem, Sulfhydryl groups of cytochrome oxidase, J. Bioenerg. 4 Ž1973. 579–590. D.C. Wharton, A. Tzagoloff, Cytochrome oxidase from beef heart mitochondria, Methods Enzymol. 10 Ž1967. 245–250. T. Yagi, Y. Hatefi, Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers, Biochemistry 23 Ž1984. 2449–2455.