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Age-dependent increase of mitochondrial DNA deletions together with lipid peroxides and superoxide dismutase in human liver mitochondria
Free RadicalBiology& Medicine,Vol. 16, No. 2, pp. 207-214, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0891-5849/94$6.00 + .00
Pergamon
0891-5849(93)E0036-5
Original Contribution AGE-DEPENDENT INCREASE OF MITOCHONDRIAL DNA DELETIONS TOGETHER WITH LIPID PEROXIDES AND SUPEROXIDE DISMUTASE IN HUMAN LIVER MITOCHONDRIA
TzU-CHEN YEN, *¢ KWANG-LIANG KING, t HSIN-CHEN LEE, $ SHIN-HWA YEH,* and YAU-HUEI WEI ~t *Department and Division of Nuclear Medicine; *Division of General Surgery; Veterans General Hospital--Taipei, and *Department of Biochemistry, National Yang-Ming Medical College, Taipei 112, Taiwan
(Received 2 April 1993; Revised 1 July 1993;Accepted 13 July 1993) Abstract--We previously reported an age-dependent deterioration of mitochondrial respiration as well as two age-associated mitochondrial DNA (mtDNA) deletions in the human liver. In this study, we further determined the relative quantities of the deleted mtDNAs in liver biopsies from 64 subjects of different ages. The results showed that both mtDNA deletions increase in frequency and quantity with age. Moreover, we measured hepatic lipid peroxides (malondialdehyde; MDA) of isolated mitochondria and manganese superoxide dismutase (Mn-SOD) activity of submitochondrial particles. We found a significant age-dependent increase in both MDA and Mn-SOD levels in liver mitochondria. These results confirm the previous contention that enhanced generation of lipid peroxides in the mitochondria during the aging process may damage mtDNA, and mtDNA deletions may be one of the important factors contributingto aging in humans. Keywords--Aging, Mitochondrial DNA, Lipid peroxides, Manganese superoxide dismutase, Free radicals
tem. 16-20Mitochondria are also subject to high oxidative stress and are active in continuous generation of semiquinone radicals and reactive oxygen species. 9'14'21 These characteristics render mammalian mtDNA vulnerable to attack by mutagens, as well as reactive oxygen species and various free radicals. ~°-15 Effects of free radicals on mitochondrial structure and functions were extensively studied in the 1960s with inconclusive results. Some researchers failed to find any significant effect of irradiation on the oxidative phosphorylation of mitochondria, regardless of the dose involved. 22-26 However, several reports demonstrated that mitochondrial respiration and oxidative phosphorylation of tissue cells decreased when the radiation dose exceeded 8 Gy. 27-33 Besides these ionized irradiation-generated free radicals, there are many other biological sources of free radicals inside the tissue cells. 14'2~ It is now generally accepted that mtDNA is continuously exposed to reactive oxygen species and free radicals in mitochondrial matrix under normal physiological conditions, and may thus be more susceptible to damage than nuclear DNA.14 In previous studies, we observed that liver mitochondrial respiratory functions decline with age, and also
INTRODUCTION
Aging has been defined as the progressive accumulation of changes associated with or responsible for the ever-increasing susceptibility of the human to diseases and death that accompany advancing age. ~'2 Most modern theories of aging have centered around the notion that age-related deterioration of physiological functions is primarily due to functional decline caused by modifications of cellular constituents. Endogenous damage to mitochondrial DNA (mtDNA) by free radicals is believed to be a major contributory factor to aging. 3-15 Mitochondria are unique among intraceUular organelles in mammalian cells. They contain their own DNA, which can be transcribed and translated to form proteins that are involved in mitochondrial respiration and oxidative phosphorylation. 15'16 It is known that mtDNA is not associated with histones or other DNA-binding proteins, and is replicated rapidly by DNA polymerase gamma without proofreading or an efficient DNA repair sysAddress correspondence to: Yau-Huei Wei, Department of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan. 207
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found that there are at least two types of agingassociated mtDNA deletions (5 kb and 6 kb) in the liver. 3-5 We believe that the enhanced generation of free radicals during the aging process may damage mtDNA, and that mtDNA deletions may be one of the many factors leading to aging in humans. In this study, we further quantitated the relative proportions of these deleted mtDNAs in human liver tissues. Furthermore, we analyzed the hepatic lipid peroxides (malondialdehyde; MDA) of mitochondria and manganese superoxide dismutase (Mn-SOD) activity of submitochondrial particles (SMP) from 64 liver biopsies obtained from subjects of various ages. The association of free radicals, scavenging enzyme activities, deletions in mtDNA, and functional deteriorations of liver and other organs or tissues during the aging process of the human is discussed. MATERIALS AND METHODS
Patients Sixty-four human liver biopsies were collected in the Veterans General Hospital--Taipei (age range: 27-78 years; mean: 54 years; 47 male and 17 female) between June 1991 and December 1992. The liver biopsies (0.5-1.0 g) were obtained with informed consent of patients with nonliver diseases during exploratory laparotomy. The patients had been screened to exclude liver diseases by clinical, histological, and biochemical criteria (SMA-12 analysis).
Preparation of liver mitochondria Human liver mitochondria were prepared by the method of Vercesi et al., 34 with some modifications. The liver samples were quickly excised and thoroughly washed with ice cold 0.25 M sucrose. Connective tissues and fats were trimmed offthe liver, which was then minced into small pieces. The tissues were then gently homogenized with a loose-fit Potter-E1vehjem homogenizer into a buffer containing 0.25 M sucrose, 0.5 mM EGTA, and 3 mM Hepes, pH 7.2 (SEH buffer) at the ratio of 10 ml per g wet weight of the liver. The homogenate was centrifuged at 800 X g for 10 min. The supernatant was further centrifuged at 9500 × g for 10 min. The pellet was collected and washed twice with the SEH buffer by repeating the above centrifugations. The final mitochondrial pellet was suspended in the SEH buffer. The entire procedure was carried out at 4°C. Usually 10-15 mg of mitochondria were obtained from 1 g of human liver.
Preparation of SMP SMP were prepared by sonicating human liver mitochondria with a polytron (Kinematic, Switzerland) in 50 mM Na-K-phosphate buffer (pH 7.4) over ice water. The sonication was carried out at the power output scale of 4 for 10 min, and separated by 1-2 min intervals to reduce sample heating. The suspension was then centrifuged at 136000 × g for 30 min in a Beckman (Palo Alto, CA) L80M ultracentrifuge. The firmly packed pellet was suspended in 50 mM Na-K-phosphate buffer (pH 7.4) by gentle homogenization. The yield was usually 40-50% of the protein of the starting mitochondrial suspension.
Analysis of deletions in mitochondrial DNA Human liver mitochondrial DNA was prepared from isolated mitochondria by alkaline-SDS lysis and phenol/chloroform extraction as described elsewhere: '5 Deletions of mtDNA were analyzed by polymerase chain reaction (PCR) techniques and agarose gel electrophoresis as previously described: ,s The nucleotide sequences of the primers L 7901-L 7920 and H 13631-H 13650 (for the detection of 4977 bp deletion), and L 7293-L 7316 and H 13905-H 13928 (for the detection of 6063 bp deletion) are the same as reported previously. 4'5 The quantity of deleted mtDNA was determined using a Micro Computer Imaging Device (MCID) System (Imaging Research Inc., Brock University, Canada) with M1 features. The densitometric readings of specific bands were made according to the following steps. First, the optic density of each of the DNA standards with known amounts in the gel were input into the computer. Second, the background area of the negative film of the target DNA bands and the standards were read by the densitometer to construct a correlation curve between the optic density and the amount of the target DNA band. Third, all the above data were entered into a computer (IBM PC AT equipped with an 80287 CPU) to get the ratio between the amount of target DNA band and the standards for each sample.
Assay of manganese superoxide dismutase activity Mn-SOD activity was measured by the method of Crapo et al. 35 The assay system in 1 ml contained 0.5 mg/ml adrenaline, 0.25 M sucrose, and 50 mM Hepes/NaOH, pH 7.5. After preincubation at 25°C for 5 min, the reaction under the conditions as indicated was initiated by the addition of 0.1 mM NADH and the adrenochrome product was determined at
Mitochondrial DNA deletions and MnSOD in aging
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Fig. 1. PCR analysisof 4977 bp deletion in human liver mitochondrialDNA. The mtDNAs from the liver tissues of a stillbirth (lane 1) and the liverbiopsies of the subjectsof agesof 35 (lane 3), 42 (lane 4), 52 (lane 5), 64 (lane 6), and 78 (lane 7) years,were both amplifiedby PCR for 30 cyclesand analyzedby 0.8% agarosegel electrophoresisas previouslydescribed.4 Lane 2 was the PCR product of the blood cell mtDNA of a 79 year old subject. M representsthe size marker of BRL 1 kb DNA ladder (Life Technologies,Inc., Gaithersburg, MD).
485-575 n m in a double-beam ultraviolet (UV) visible spectrophotometer.
Measurement of lipid peroxides Lipid peroxides were measured as MDA by the method of Ohkawa et al. 36 The breakdown product of 1,1,3,3-tetraethoxypropane was used as standard and the measured a m o u n t of lipid peroxides in the mitochondria was expressed as pmol M D A / m g protein. The protein concentration of mitochondrial suspension or SMP was determined by the method of Lowry et al. 37
Data analysis All the data were fitted into a correlation line by simple linear regression analysis.
could obtain a 5.75 kb PCR product o f the wild-type m t D N A in the liver tissues of all the study subjects (lanes 1-7) and a shorter PCR product of 773 bp in size from the 4977 bp-deleted m t D N A in the liver tissues of older humans (lanes 3-7). In addition, we also detected the previously reported 6063 bp deletion in the liver mitochondria o f aged individuals (data not shown). By using a computerized scanning imaging system, we determined the relative proportion o f the 4977-bp and 6063-bp deleted mtDNAs in the liver mitochondria from h u m a n subjects of different ages. The results showed that both deletions of m t D N A in liver mitochondria increased concomitantly with age (Figs. 2 and 3). The a m o u n t of lipid peroxides and Mn-SOD activity in the liver mitochondria from subjects of different ages are shown in Figures 4 and 5. The results clearly demonstrate that both MDA and Mn-SOD levels in the liver mitochondria increase with the age of the human subject.
RESULTS We confirmed, by use of 30 cycles o f PCR, that a 4977 bp deletion occurs in the liver m t D N A from subjects above 35 years old, and is not seen in the liver m t D N A of younger individuals (Fig. 1). Using the primers L 7901-L 7920 and H 13631-H 13650, we
DISCUSSION A comprehensive hypothesis concerning the contribution of m t D N A mutations to the h u m a n aging process TM has been further tested in this study. The central concept is that random mutations in the popu-
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Cortopassi et al. 38 detected a specific 5 kb deletion o f m t D N A in various adult tissues after 30 cycles o f P C R , b u t this deletion was n o t detected in infant tissues until after 60 cycles o f P C R amplification. U p to now, m o r e t h a n ten different kinds o f m t D N A deletions have been identified in various tissues o f aged
lation o f m t D N A molecules for each cell o c c u r t h r o u g h o u t a h u m a n ' s lifespan, a n d that it is a m a j o r contributor to age-dependent decline in cellular bioenergetic functions. This can initiate a n d / o r p r o m o t e various degenerative diseases that are usually associated with aging.
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individuals without any of the known mitochondrial diseases, and the proportion of some of these deleted mtDNAs was found to increase with age. 4's'13'38-45 Thus, these age-dependent deletions in mtDNA presumably occur naturally in normal human tissues. Figure l elaborates one of such deletions in the liver mtDNA. Because mtDNA is replicated by DNA poly-
merase gamma that is lacking proofreading and efficient DNA repair mechanisms, the sensitivity of mtDNA to damage by mutagens predisposes mtDNA to mutation and/or deletion upon exposure of cells to genotoxins or oxidative stress, 10'46'47 The accumulation of these damages in the mitochondrial genome during a human's lifespan will in turn cause muta-
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212
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tions of certain genes or loss of mitochondrial gene products, which are manifested as a decline in mitochondrial respiratory functions. 3'1~ The decreases in the bioenergetic capability and capacity of the cell will lead to a concomitant decrease in ATP-dependent synthesis of various important biomacromolecules that are essential for specific physiological functions of the cell. These deteriorating processes may contribute to the aging of the human and other a n i m a l s . 3'48,49 Under normal physiological conditions, the reactive oxygen species or free radicals produced as side products of oxidative metabolism can be efficiently disposed of by various antioxidants such as glutathione and scavenging enzyme systems, including SOD and catalase, to prevent lipid peroxidation and other detrimental effects to the tissue cells. However, when lipid peroxides are generated beyond normal levels due to accelerated degradation of membranes and other components of the cell in various tissues of aged individuals, the amounts and activities of detoxification enzymes such as Mn-SOD and catalase in the mitochondria are increased in many possible ways (enhanced gene expression or substrate/modulator induced activation), to meet the cellular needs to clear up the detrimental free radicals to avoid further tissue damage. This notion is supported by the data illustrated in Figures 4 and 5, where we clearly show that both lipid peroxides and Mn-SOD activity are increased with age in liver mitochondria. Although several groups have identified specific mtDNA deletions in various tissues of older humans, the reported proportion of these deleted mtDNAs in the mitochondria has differed greatly among different laboratories. 45,5°-5z Cortopassi et al. 38'5° estimated the deletion by PCR, and found that the 4977 bp-deleted mtDNA was about 0. 1% in the heart muscle of subjects beyond middle age. However, Corral-Debrinski et al. 51 used a dilution-PCR method to show that the amount of the 4977-bp deleted mtDNA in the heart mitochondria of human subjects of 63 and 78 years old was 0.0025% and 0.0035%, respectively. Very recently, Simonetti and coworkers 52 developed a quantitative PCR method and determined the proportion of 4977-bp deleted mtDNA to be about 0.1% in the muscle of old humans. In this study, we undertook a different approach to measure the proportion of 4977-bp deleted mtDNA relative to the wild-type mtDNA, and the results showed that both 4977-bp and 6063-bp deletions increased concomitantly with age (Figs. 4 and 5). While this determination is based on relative optical density of the DNA bands in the gel, we could not obtain the absolute quantity of deleted mtDNA in the liver tissues. It is worth mentioning that the relative optical intensity of the 773 bp to
that of 5.75 Kb PCR product (see Fig. 1 and Fig. 2) was consistently higher than that estimated for the 6063 bp-deleted mtDNA (Fig. 3). This finding indicates that the 4977 bp-deleted mtDNA is more abuntant than 6063 bp-deleted mtDNA in the mitochondria of human liver during the aging process. Replacement therapy and pharmacological support using appropriate redox-active compounds may be useful for the alleviation of aging-associated increase of oxidative stress. Although there is no available evidence to show that a defect in antioxidant enzymes will lead to accelerated aging, it has been demonstrated that there is a significant decrease of lipid peroxides in the mitochondria in animals administered with antioxidants. 49'53-55However, further work is necessary to elucidate the mechanism of antioxidant effects on the human aging process before a rational strategy can be designed for clinical use. Although aging-associated alterations of mtDNA have been observed in humans and other mammals, 4'5'13'39-45'56 n o age-related change of the level of free radicals in living human organs during aging has ever been reported. In this study, we confirmed the two previously reported mtDNA deletions and found that the lipid peroxide content and Mn-SOD activity of the liver mitochondria increased with the age of the human subject. Although Semsei et al. 57 observed an age-dependent decrease in the activity and mRNA level of Mn-SOD in rat brain tissues, a significant ageassociated increase of MDA and Mn-SOD activity was observed in the mitochondria of rat brain, 58-61rat liver and h e a r t , 62 and human retinal pigment epithelium. 63 It is possible that oxygen free radicals cause damage to the genes of free radical scavenging enzymes and reduce their activity during aging. However, our studies clearly demonstrated that free radical scavenger Mn-SOD is increased to cope with the increased oxygen free radicals in tissue cells in senescence. The increased Mn-SOD activity may be one of the self-protection mechanisms to alleviate damage to biomolecules by free radicals in normal aging liver. In conclusion, these results, together with our previous findings, support the hypothesis that the everincreasing mutations and deletions in mtDNA affecting mitochondrially synthesized polypeptides involved in mitochondrial respiration and oxidative phosphorylation in advancing age, may be due to the accumulation of free radicals during the aging process of the human. - - This work was supported by grants No. NSC 81-0412-B075-533 and No. NSC 82-0412-B010-024 from the National Science Council, Republic of China. It was also partially supported by the Institute of Biomedical Sciences, Academia Sinica, and a grant (DOH 82-HR-L01) from the Department of Health, Acknowledgements
Mitochondrial DNA deletions and MnSOD in aging Executive Yuan, Republic of China. One of the authors, Yau-Huei Wei, would like to express his gratitude to the National Science Council for the Outstanding Research Award received in the course of this study.
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try and health benefits. Annals New York Acad. Sci. 570:1-6; 1989. Piko, L.; Hougham, A. J.; Bulpitt, K. J. Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: Evidence of an increased frequency of deletions/additions with ageing. Mech. Ageing Dev. 43:279-293; 1988. Semsei, I.; Rao, G.; Richardson, A. Expression of superoxide dismutase and catalase in rat brain as a function of age. Mech. Ageing Dev. 58:13-19; 1991. Santiago, L. A.; Osato, J. A.; Liu, J.; Moil, A. Age-related increases in superoxide dismutase activity and thiobarbituric acid-reactive substances: Effect of bio-catalyzer in aged rat brain. Neurochem. Res. 18:711-718; 1993. Hiramatsu, M.; Kohno, M.; Edamatsu, R.; Mitsuta, K.; Moil, A. Increased superoxide dismutase activity in aged human cerebrospinal fluid and rat brain determined by electron spin resonance spectrometry using the spin trap method. Z Neurochem. 58:1160-1164; 1992. Nistico, G.; Ciriolo, M. R.; Fiskin, K.; Iannone, M.; de Martino, A.; Rotilio, G. NGF restores decrease activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of ages rat. FreeRadic. Biol. Med. 12:177-181; 1992. VeneUa, A.; Geremia, E.; D'Urso, G.; et al. Superoxide dismutase activities in aging rat brain. Gerontology 228:108-113; 1982. Ji, L. L.; Dillon, D.; Wu, E. Myocardial aging: Antioxidant enzyme systems and related biochemical properties. Am. J. Physiol. 261:R386-R392; 1991. Oliver, P. D.; Newsome, D. A. Mitochondrial superoxide dismutase in mature and developing human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 33:1909-1918; 1992.
Pergamon
0891-5849(93)E0036-5
Original Contribution AGE-DEPENDENT INCREASE OF MITOCHONDRIAL DNA DELETIONS TOGETHER WITH LIPID PEROXIDES AND SUPEROXIDE DISMUTASE IN HUMAN LIVER MITOCHONDRIA
TzU-CHEN YEN, *¢ KWANG-LIANG KING, t HSIN-CHEN LEE, $ SHIN-HWA YEH,* and YAU-HUEI WEI ~t *Department and Division of Nuclear Medicine; *Division of General Surgery; Veterans General Hospital--Taipei, and *Department of Biochemistry, National Yang-Ming Medical College, Taipei 112, Taiwan
(Received 2 April 1993; Revised 1 July 1993;Accepted 13 July 1993) Abstract--We previously reported an age-dependent deterioration of mitochondrial respiration as well as two age-associated mitochondrial DNA (mtDNA) deletions in the human liver. In this study, we further determined the relative quantities of the deleted mtDNAs in liver biopsies from 64 subjects of different ages. The results showed that both mtDNA deletions increase in frequency and quantity with age. Moreover, we measured hepatic lipid peroxides (malondialdehyde; MDA) of isolated mitochondria and manganese superoxide dismutase (Mn-SOD) activity of submitochondrial particles. We found a significant age-dependent increase in both MDA and Mn-SOD levels in liver mitochondria. These results confirm the previous contention that enhanced generation of lipid peroxides in the mitochondria during the aging process may damage mtDNA, and mtDNA deletions may be one of the important factors contributingto aging in humans. Keywords--Aging, Mitochondrial DNA, Lipid peroxides, Manganese superoxide dismutase, Free radicals
tem. 16-20Mitochondria are also subject to high oxidative stress and are active in continuous generation of semiquinone radicals and reactive oxygen species. 9'14'21 These characteristics render mammalian mtDNA vulnerable to attack by mutagens, as well as reactive oxygen species and various free radicals. ~°-15 Effects of free radicals on mitochondrial structure and functions were extensively studied in the 1960s with inconclusive results. Some researchers failed to find any significant effect of irradiation on the oxidative phosphorylation of mitochondria, regardless of the dose involved. 22-26 However, several reports demonstrated that mitochondrial respiration and oxidative phosphorylation of tissue cells decreased when the radiation dose exceeded 8 Gy. 27-33 Besides these ionized irradiation-generated free radicals, there are many other biological sources of free radicals inside the tissue cells. 14'2~ It is now generally accepted that mtDNA is continuously exposed to reactive oxygen species and free radicals in mitochondrial matrix under normal physiological conditions, and may thus be more susceptible to damage than nuclear DNA.14 In previous studies, we observed that liver mitochondrial respiratory functions decline with age, and also
INTRODUCTION
Aging has been defined as the progressive accumulation of changes associated with or responsible for the ever-increasing susceptibility of the human to diseases and death that accompany advancing age. ~'2 Most modern theories of aging have centered around the notion that age-related deterioration of physiological functions is primarily due to functional decline caused by modifications of cellular constituents. Endogenous damage to mitochondrial DNA (mtDNA) by free radicals is believed to be a major contributory factor to aging. 3-15 Mitochondria are unique among intraceUular organelles in mammalian cells. They contain their own DNA, which can be transcribed and translated to form proteins that are involved in mitochondrial respiration and oxidative phosphorylation. 15'16 It is known that mtDNA is not associated with histones or other DNA-binding proteins, and is replicated rapidly by DNA polymerase gamma without proofreading or an efficient DNA repair sysAddress correspondence to: Yau-Huei Wei, Department of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan. 207
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found that there are at least two types of agingassociated mtDNA deletions (5 kb and 6 kb) in the liver. 3-5 We believe that the enhanced generation of free radicals during the aging process may damage mtDNA, and that mtDNA deletions may be one of the many factors leading to aging in humans. In this study, we further quantitated the relative proportions of these deleted mtDNAs in human liver tissues. Furthermore, we analyzed the hepatic lipid peroxides (malondialdehyde; MDA) of mitochondria and manganese superoxide dismutase (Mn-SOD) activity of submitochondrial particles (SMP) from 64 liver biopsies obtained from subjects of various ages. The association of free radicals, scavenging enzyme activities, deletions in mtDNA, and functional deteriorations of liver and other organs or tissues during the aging process of the human is discussed. MATERIALS AND METHODS
Patients Sixty-four human liver biopsies were collected in the Veterans General Hospital--Taipei (age range: 27-78 years; mean: 54 years; 47 male and 17 female) between June 1991 and December 1992. The liver biopsies (0.5-1.0 g) were obtained with informed consent of patients with nonliver diseases during exploratory laparotomy. The patients had been screened to exclude liver diseases by clinical, histological, and biochemical criteria (SMA-12 analysis).
Preparation of liver mitochondria Human liver mitochondria were prepared by the method of Vercesi et al., 34 with some modifications. The liver samples were quickly excised and thoroughly washed with ice cold 0.25 M sucrose. Connective tissues and fats were trimmed offthe liver, which was then minced into small pieces. The tissues were then gently homogenized with a loose-fit Potter-E1vehjem homogenizer into a buffer containing 0.25 M sucrose, 0.5 mM EGTA, and 3 mM Hepes, pH 7.2 (SEH buffer) at the ratio of 10 ml per g wet weight of the liver. The homogenate was centrifuged at 800 X g for 10 min. The supernatant was further centrifuged at 9500 × g for 10 min. The pellet was collected and washed twice with the SEH buffer by repeating the above centrifugations. The final mitochondrial pellet was suspended in the SEH buffer. The entire procedure was carried out at 4°C. Usually 10-15 mg of mitochondria were obtained from 1 g of human liver.
Preparation of SMP SMP were prepared by sonicating human liver mitochondria with a polytron (Kinematic, Switzerland) in 50 mM Na-K-phosphate buffer (pH 7.4) over ice water. The sonication was carried out at the power output scale of 4 for 10 min, and separated by 1-2 min intervals to reduce sample heating. The suspension was then centrifuged at 136000 × g for 30 min in a Beckman (Palo Alto, CA) L80M ultracentrifuge. The firmly packed pellet was suspended in 50 mM Na-K-phosphate buffer (pH 7.4) by gentle homogenization. The yield was usually 40-50% of the protein of the starting mitochondrial suspension.
Analysis of deletions in mitochondrial DNA Human liver mitochondrial DNA was prepared from isolated mitochondria by alkaline-SDS lysis and phenol/chloroform extraction as described elsewhere: '5 Deletions of mtDNA were analyzed by polymerase chain reaction (PCR) techniques and agarose gel electrophoresis as previously described: ,s The nucleotide sequences of the primers L 7901-L 7920 and H 13631-H 13650 (for the detection of 4977 bp deletion), and L 7293-L 7316 and H 13905-H 13928 (for the detection of 6063 bp deletion) are the same as reported previously. 4'5 The quantity of deleted mtDNA was determined using a Micro Computer Imaging Device (MCID) System (Imaging Research Inc., Brock University, Canada) with M1 features. The densitometric readings of specific bands were made according to the following steps. First, the optic density of each of the DNA standards with known amounts in the gel were input into the computer. Second, the background area of the negative film of the target DNA bands and the standards were read by the densitometer to construct a correlation curve between the optic density and the amount of the target DNA band. Third, all the above data were entered into a computer (IBM PC AT equipped with an 80287 CPU) to get the ratio between the amount of target DNA band and the standards for each sample.
Assay of manganese superoxide dismutase activity Mn-SOD activity was measured by the method of Crapo et al. 35 The assay system in 1 ml contained 0.5 mg/ml adrenaline, 0.25 M sucrose, and 50 mM Hepes/NaOH, pH 7.5. After preincubation at 25°C for 5 min, the reaction under the conditions as indicated was initiated by the addition of 0.1 mM NADH and the adrenochrome product was determined at
Mitochondrial DNA deletions and MnSOD in aging
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Fig. 1. PCR analysisof 4977 bp deletion in human liver mitochondrialDNA. The mtDNAs from the liver tissues of a stillbirth (lane 1) and the liverbiopsies of the subjectsof agesof 35 (lane 3), 42 (lane 4), 52 (lane 5), 64 (lane 6), and 78 (lane 7) years,were both amplifiedby PCR for 30 cyclesand analyzedby 0.8% agarosegel electrophoresisas previouslydescribed.4 Lane 2 was the PCR product of the blood cell mtDNA of a 79 year old subject. M representsthe size marker of BRL 1 kb DNA ladder (Life Technologies,Inc., Gaithersburg, MD).
485-575 n m in a double-beam ultraviolet (UV) visible spectrophotometer.
Measurement of lipid peroxides Lipid peroxides were measured as MDA by the method of Ohkawa et al. 36 The breakdown product of 1,1,3,3-tetraethoxypropane was used as standard and the measured a m o u n t of lipid peroxides in the mitochondria was expressed as pmol M D A / m g protein. The protein concentration of mitochondrial suspension or SMP was determined by the method of Lowry et al. 37
Data analysis All the data were fitted into a correlation line by simple linear regression analysis.
could obtain a 5.75 kb PCR product o f the wild-type m t D N A in the liver tissues of all the study subjects (lanes 1-7) and a shorter PCR product of 773 bp in size from the 4977 bp-deleted m t D N A in the liver tissues of older humans (lanes 3-7). In addition, we also detected the previously reported 6063 bp deletion in the liver mitochondria o f aged individuals (data not shown). By using a computerized scanning imaging system, we determined the relative proportion o f the 4977-bp and 6063-bp deleted mtDNAs in the liver mitochondria from h u m a n subjects of different ages. The results showed that both deletions of m t D N A in liver mitochondria increased concomitantly with age (Figs. 2 and 3). The a m o u n t of lipid peroxides and Mn-SOD activity in the liver mitochondria from subjects of different ages are shown in Figures 4 and 5. The results clearly demonstrate that both MDA and Mn-SOD levels in the liver mitochondria increase with the age of the human subject.
RESULTS We confirmed, by use of 30 cycles o f PCR, that a 4977 bp deletion occurs in the liver m t D N A from subjects above 35 years old, and is not seen in the liver m t D N A of younger individuals (Fig. 1). Using the primers L 7901-L 7920 and H 13631-H 13650, we
DISCUSSION A comprehensive hypothesis concerning the contribution of m t D N A mutations to the h u m a n aging process TM has been further tested in this study. The central concept is that random mutations in the popu-
210
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Cortopassi et al. 38 detected a specific 5 kb deletion o f m t D N A in various adult tissues after 30 cycles o f P C R , b u t this deletion was n o t detected in infant tissues until after 60 cycles o f P C R amplification. U p to now, m o r e t h a n ten different kinds o f m t D N A deletions have been identified in various tissues o f aged
lation o f m t D N A molecules for each cell o c c u r t h r o u g h o u t a h u m a n ' s lifespan, a n d that it is a m a j o r contributor to age-dependent decline in cellular bioenergetic functions. This can initiate a n d / o r p r o m o t e various degenerative diseases that are usually associated with aging.
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individuals without any of the known mitochondrial diseases, and the proportion of some of these deleted mtDNAs was found to increase with age. 4's'13'38-45 Thus, these age-dependent deletions in mtDNA presumably occur naturally in normal human tissues. Figure l elaborates one of such deletions in the liver mtDNA. Because mtDNA is replicated by DNA poly-
merase gamma that is lacking proofreading and efficient DNA repair mechanisms, the sensitivity of mtDNA to damage by mutagens predisposes mtDNA to mutation and/or deletion upon exposure of cells to genotoxins or oxidative stress, 10'46'47 The accumulation of these damages in the mitochondrial genome during a human's lifespan will in turn cause muta-
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212
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tions of certain genes or loss of mitochondrial gene products, which are manifested as a decline in mitochondrial respiratory functions. 3'1~ The decreases in the bioenergetic capability and capacity of the cell will lead to a concomitant decrease in ATP-dependent synthesis of various important biomacromolecules that are essential for specific physiological functions of the cell. These deteriorating processes may contribute to the aging of the human and other a n i m a l s . 3'48,49 Under normal physiological conditions, the reactive oxygen species or free radicals produced as side products of oxidative metabolism can be efficiently disposed of by various antioxidants such as glutathione and scavenging enzyme systems, including SOD and catalase, to prevent lipid peroxidation and other detrimental effects to the tissue cells. However, when lipid peroxides are generated beyond normal levels due to accelerated degradation of membranes and other components of the cell in various tissues of aged individuals, the amounts and activities of detoxification enzymes such as Mn-SOD and catalase in the mitochondria are increased in many possible ways (enhanced gene expression or substrate/modulator induced activation), to meet the cellular needs to clear up the detrimental free radicals to avoid further tissue damage. This notion is supported by the data illustrated in Figures 4 and 5, where we clearly show that both lipid peroxides and Mn-SOD activity are increased with age in liver mitochondria. Although several groups have identified specific mtDNA deletions in various tissues of older humans, the reported proportion of these deleted mtDNAs in the mitochondria has differed greatly among different laboratories. 45,5°-5z Cortopassi et al. 38'5° estimated the deletion by PCR, and found that the 4977 bp-deleted mtDNA was about 0. 1% in the heart muscle of subjects beyond middle age. However, Corral-Debrinski et al. 51 used a dilution-PCR method to show that the amount of the 4977-bp deleted mtDNA in the heart mitochondria of human subjects of 63 and 78 years old was 0.0025% and 0.0035%, respectively. Very recently, Simonetti and coworkers 52 developed a quantitative PCR method and determined the proportion of 4977-bp deleted mtDNA to be about 0.1% in the muscle of old humans. In this study, we undertook a different approach to measure the proportion of 4977-bp deleted mtDNA relative to the wild-type mtDNA, and the results showed that both 4977-bp and 6063-bp deletions increased concomitantly with age (Figs. 4 and 5). While this determination is based on relative optical density of the DNA bands in the gel, we could not obtain the absolute quantity of deleted mtDNA in the liver tissues. It is worth mentioning that the relative optical intensity of the 773 bp to
that of 5.75 Kb PCR product (see Fig. 1 and Fig. 2) was consistently higher than that estimated for the 6063 bp-deleted mtDNA (Fig. 3). This finding indicates that the 4977 bp-deleted mtDNA is more abuntant than 6063 bp-deleted mtDNA in the mitochondria of human liver during the aging process. Replacement therapy and pharmacological support using appropriate redox-active compounds may be useful for the alleviation of aging-associated increase of oxidative stress. Although there is no available evidence to show that a defect in antioxidant enzymes will lead to accelerated aging, it has been demonstrated that there is a significant decrease of lipid peroxides in the mitochondria in animals administered with antioxidants. 49'53-55However, further work is necessary to elucidate the mechanism of antioxidant effects on the human aging process before a rational strategy can be designed for clinical use. Although aging-associated alterations of mtDNA have been observed in humans and other mammals, 4'5'13'39-45'56 n o age-related change of the level of free radicals in living human organs during aging has ever been reported. In this study, we confirmed the two previously reported mtDNA deletions and found that the lipid peroxide content and Mn-SOD activity of the liver mitochondria increased with the age of the human subject. Although Semsei et al. 57 observed an age-dependent decrease in the activity and mRNA level of Mn-SOD in rat brain tissues, a significant ageassociated increase of MDA and Mn-SOD activity was observed in the mitochondria of rat brain, 58-61rat liver and h e a r t , 62 and human retinal pigment epithelium. 63 It is possible that oxygen free radicals cause damage to the genes of free radical scavenging enzymes and reduce their activity during aging. However, our studies clearly demonstrated that free radical scavenger Mn-SOD is increased to cope with the increased oxygen free radicals in tissue cells in senescence. The increased Mn-SOD activity may be one of the self-protection mechanisms to alleviate damage to biomolecules by free radicals in normal aging liver. In conclusion, these results, together with our previous findings, support the hypothesis that the everincreasing mutations and deletions in mtDNA affecting mitochondrially synthesized polypeptides involved in mitochondrial respiration and oxidative phosphorylation in advancing age, may be due to the accumulation of free radicals during the aging process of the human. - - This work was supported by grants No. NSC 81-0412-B075-533 and No. NSC 82-0412-B010-024 from the National Science Council, Republic of China. It was also partially supported by the Institute of Biomedical Sciences, Academia Sinica, and a grant (DOH 82-HR-L01) from the Department of Health, Acknowledgements
Mitochondrial DNA deletions and MnSOD in aging Executive Yuan, Republic of China. One of the authors, Yau-Huei Wei, would like to express his gratitude to the National Science Council for the Outstanding Research Award received in the course of this study.
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