M-H, Wei V-H. Age-related 4,977 bp deletion in human lung mitochondrial DNA. AM J RESPIR CRIT CARE MED 1996;154:1141-5. Aging has long been defined ...
Brief Communications A~e-related 4,977
bp Deletion in Human Lung Mitochondrial DNA
HUEI-JYH FAHN, LIANG-SHUN WANG, RONG-HONG HSIEH, SHI-CHUAN CHANG, SHU-HUEI KAO, MIN-HSIUNG HUANG, and YAU-HUEI WEI Institute of Clinical Medicine, School of Medicine, Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University and Division of Thoracic Surgery, Department of Surgery, and Department of Chest Disease, Veterans General Hospital-Taipei, Taiwan, Republic of China
The accumulation of mitochondrial DNA (mtDNA) mutations has been suggested to be an important contributor to human aging and degenerative diseases. The lung is exposed to ambient air and makes direct contact with the external environment. Numerous potentially noxious agents may damage lung tissues directly or indirectly through free-radical-mediated reactions. In previous studies, we demonstrated an age-dependent increase of mtDNA mutations in various human tissues. We hypothesize that the accumulation of the 4,977 bp (base pairs) deleted mtDNA in human lung tissues is also agedependent. Using the polymerase chain reaction technique, we determined the incidence of the 4,977 bp-deleted mtDNA in 127 human lung specimens from 34-wk gestation to 79 yr of age. The results showed that 77 lung biopsies (60.6%) contained the 4,977 bp-deleted mtDNA, which started to appear in lung tissues after the fourth decade of life. The incidence apparently increased from 14.3% (one of seven) of the subjects in the 30- to 39-yr age group to 77.8% (two of 27) of the subjects in the 70- to 79-yr age group (p < 0.0001). The mean (± SEM) proportion ofthe 4,977 bp-deleted mtDNA in lung tissues significantly increased from 0.007 ± 0.007% of the subjects in the 30- to 39-yr age group to 0.833 ± 0.330% of those in the 70- to 79-yr age group (p < 0.005). Other factors such as sex, pulmonary function indices, and smoking status did not have statistically significant impact on the amount of the deleted mtDNA. These findings suggest that the accumulation of the 4,977 bp-deleted mtDNA is associated with aging human lung. Fahn H-J, Wang L-S, Hsieh R-H, Chang SoC, Kao SoH, Huang M-H, Wei V-H. Age-related 4,977 bp deletion in human lung mitochondrial DNA. AM
Aging has long been defined as the progressive accumulation of various changes in tissues that are either associated with or responsible for susceptibility of humans to disease and ultimate death that accompanies advancing age (1). However, the molecular mechanism underlying the aging process remains to be elucidated. About four decades ago, Harman (2) proposed the so-called free radical theory ofaging, which campaigned the concept that the key target of the ever-increasing reactive oxygen species (ROS) and other types of free radicals along with a human's life-span is the DNA rather than biomembranes. He further suggested that mitochondrial DNA (mtDNA) is a possible target of free radical attack in tissue cells during the aging process (3).
(Received in original form October 17, 1995 and in revised form March 4, 1996)
Supported by Research Grants No. NSC84-2331-B-Oll-080 and NSC85-2331B-Ol0-102 from the National Science Council and in part by Grant DOH 84HR-219 from the Department of Health, Executive Yuan, Republic of China. Correspondence and requests for reprints should be addressed to Professor Yau-Huei Wei, Ph.D., Department of Biochemistry, School of life Science, National Yang-Ming University, Taipei 112, Taiwan, Republic of China. Am) Respir Crit Care Med Vol 154. pp 1141-1145, 1996
J RESPIR
CRIT CARE MED 1996;154:1141-5.
Human mtNDA is a 16,569-bp(base pairs) naked circular doublestranded extrachromosomal genetic element (4) that is continually exposed to high levelsof ROS and free radicals in the matrix of mitochondria (5). In contrast to nuclear DNA, mtDNA is intronless and replicates much faster than nuclear DNA without proofreading and efficient DNA repair systems (6). There are about 800 mtDNA molecules in a human lung fibroblast (7). These molecules are located near the inner membrane of mitochondria and thus are vulnerable to oxidative damage by the ROS generated from the respiratory chain in the inner membrane of the organelle or extrinsic sources. In recent years, an increasing number of reports have shown that mtDNA mutations are associated with human aging and mitochondrial diseases (8, 9). More than a dozen mtDNA deletions have been identified in various tissues of elderly human subjects. Most of these deletions were detected in a particular tissue, but others were detected in more than one tissue (8). Among the reported mtDNA deletions, the 4,977 bp deletion is the most common one that occurs in almost all the tissues of elderly humans (10-12). Moreover, multiple deletions of mtDNA were found to exist in the same tissues of some elderly subjects (13, 14). These findings suggest that mtDNA indeed undergoes large-scale deletions in various human tissues during the aging process.
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 154
1142
The lung is different from the other internal organs in that it is directly exposed to the environment. A variety of factors may adversely affect lung function such as environmental or industrial exposures, the disturbed microenvironment associated with smoking, altered immune mechanisms, weakened defense mechanisms, or the injurious effects of previous diseases that may have occurred in childhood. The changes that occur in the lung with age include: increase in alveolar duct air, decrease in either complexity of the alveolar surface or surface-to-volume ratio (15), loss of alveolar wall tissue and the elastic recoil properties (16), increase in closing volume (17), progressive reduction of ve, FEV I , and FEV,/FVe ratio (18, 19), maximal expiratory flows, decrease in diffusing capacity of the lung and arterial oxygen tension, and changes in the response to stimuli (20). To the best of our knowledge, there is no published information about mtDNA mutations in lung tissues of elderly humans. Wehypothesize that the accumulation of mtDNA mutations in the lung is also associated with the aging process. To test this hypothesis, we investigated the incidence and the amount of the 4,977 bpdeleted mtDNA in lung tissues from subjects of different ages. We collected lung tissue from one stillborn baby and from 126 patient s who had undergone resection for various pulmonary disorders such as spontaneous pneumothorax, metastatic lung tumors, and bronchogenic carcinoma at the Division of Thoracic Surgery, VeteransGeneral Hospital-Taipei. Pat ients with infectious disea ses such as pneumonia, fungal infection, and pulmonary tuberculosi s were excluded from this study. None had a known history of industrial or occupational exposure to asbesto s or organic solvent. A sma ll piece of the lung tissue was collected from the grossl y healthy region of the resected specimen and frozen in liquid nitrogen until analysis. Pat ient data, including sex, age, and history of major systemic disease and surgery were collected prospectively. Smoking was classified into three categories: (1) current smokers: subjects who had smoked more than one cigarette daily for more than I yr; (2) ex-smokers: subjects who had previously been reported as smo kers but who had not smoked for more than I yr; and (3) nonsmokers: subjects who had never smoked. The smoking index is expressed as pack-year (cigarettes smoked per day times years of smokingI20). Spirometry was performed in the standa rd (seated) position in 123 pat ients a minimum of three times using a CPI 5000 IV spirometer (Gould, Houston, TX) before exploratory thoracotomy. The best values of FEV I and FVC were selected for analysis according to the criteria set by the American Thoracic Society (21). The results of pulmonary function testing were reviewed by a senior chest physician (Dr. S. C. Ch ang) . Because only a small amount of lung tissue was available (50 to 100 mg) from each patient, total DNA was isolated by proteinase K/SDS lysis followed by phenol/chloroform extraction as previously described
TABLE 1 OLIGONUCLEOTIDE PRIMERS USED FOR POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION OF HUMAN LUNG MITOCHONDRIAL DNA WITH THE 4,977 bp DELETION
5' - 3'
Length (bp)
Predicted Length of peR Product Amplified from the 4,977 bp-deleted mtDNA (bp)
3,108-3,717 8,251-14,020 8,251 -13,845 8,282-13,845 8,251-13,650
610 5,770 5,595 5,564 5,400
610 793 618 587 423
Amplified Position Prim er Pair Ll -H1*
I2-H2 I2 -H3 L3·H3 I2-H4
• The prim er set 11, H1 was used for the determination of total mitochondrial DNA. 11 (3,108-3,127) = 5'-TICAAATICCTCCCTGTACG·3' L2 (8,251-8,270) = 5'-GCCCGTATTTACCCTATAGC·3' L3 (8,282-8,305) = 5'-CCCCTCTAGAGCCCACTGTAAAGC-3' H1 (3,717 - 3,701) = 5'-GGCTACTGCTCGCAGTG-3' H2 (14,020-14,001) = 5'-ATAGCTTTTCTAGTCAGGTI-3' H3 (13,845-13,826) = 5'-GTCTAGGGCTGTIAGAAGTC-3' H4 (13,650-13,631) = 5'-GGGGAAGCGAGGTIGACCTG-3'
1996
(22). After ethanol precipitation, th e DNA pellet was dissolved in 100 III sterilized doubly distilled water and frozen at - 30° C until use. Oligonucleotide primers were synthesized by Bio-Synthesis, Inc. (Lewisville, TX). The sequences of the oligonucleotide pr imers and the sizes of the polymerase chain reaction (PCR) products amplified from normal mtDNA and the 4,977 bp-deleted mtDNA with the indicated primer pairs are shown in Table I. PCR was carried out for 31 cycles in a 100-111 reaction mixture containing 200 ng template DNA, 200 11M of each dNTP, 40 pmol of each primer, 1.0 U of Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT) , 50 mM KCI, 1.5 mM MgCl" 10 mM TRIS-HCI at 25° C (pH, 8.3), 0.1% Triton X-IOO, and 0.01070 (wt/vol) gelatin. The first cycle consisted of 5-min denaturation at 94° C, 5-min annealing at 55° C, and 3-min primer extension at 72° C. The the rmal profile of the following 30 cycles was denaturation at 94° C for 40 s, annealing at 56° C for 40 s, and extension at 72° C for 40 s. The amplified PCR products were separated on a 1.5% agarose gel at 150 volts for I h, and DNA bands were visualized under UV light transillumination after staining with ethidium bromide (0.1 mg/ml). A primer-shift PCR method (23) was used to ascertain the authenticity of the 4,977 bp mtDNA deletion. By using the different primer sets listed in Table I, the length of the amplified DNA changed in parallel to the shift of the distance between each primer pair. Because no DNA fragment could be amplified from the undeleted mtDNA under the PCR thermal profile (short extension time), as described above , the only DNA fragment was that amplified from the 4,977 bp-deleted mtDNA. The sizes of the mtDNA fragments amplified by different primer pair s were 793 bp (Li-H2), 618 bp (L2-H3), 587 bp (L3-H3), and 423 bp (L2-H4), respectively(Figure I). Th e proportion of the 4,977 bp-deleted mtDNA in each lung sample was determined by a semiquantitative PCR meth od as described previou sly (12). The frequencies of occurrence of the 4,977 bp-deleted mtDNA in the lung tissues of the subjects in different age cohorts were compared using the chi-square test and Fishe r's exact test for occurrence less than 5. The differences in continuous varia bles were compared by using Student 's independent t test between two groups or one-way analysis of variance (ANOVA) in more than two groups. The differences in the amount of the 4,977 bp-deleted mtDNA relative to the wild-type mtDNA between age groups were compared by using the nonparametric KruskalWallis H method. Multivariate regression analysis using the stepwise
M
1
2
3
4
-I --.-... . ..
...--
793 bp
...-...--
6 18bll 5117 bll
...--
·U J bJl
Figure 1. Electrophoretogram of PCRproducts amplified from mtDNA with the specific 4,977 bp deletion from human lung. Lanes 1 to 4 indicate the 793, 618, 587, and 423 bp PCR products amplified by primer pairs L2-H2, L2-H3, L3-H3, and L2-H4, respectively, from the specific 4,977 bp-deleted mtDNA in the lung tissues of a 69-yr-old subject. Lane M represents the 100 bp ladder size marker.
1143
Brief Communication TABLE 2 THE FREQUENCY OF OCCURRENCE OF THE 4,977 bp-DELETED mtDNA IN HUMAN LUNGS OF DIFFERENT AGE COHORTS Age Cohort (yr)
Subjects without 4,977 bp-deleted mtDNA
0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 Total
Subjects with 4,977 bp-deleted mtDNA
1 7 7 6 4
o o o
6 13
Total Number of Subjects
Frequency of Occurrence (%)
6
8 40 21
7 7 7 11 14 53 27
0.0 0.0 0.0 14.3 63.6 57.1 75.5 77.8
50
77
127
60.6
1
1 7
method was adopted to decide the variable with statistical significance. A p value less than 0.05 was considered statistically significant. There were 89 male and 38 female subjects recruited in this study. Their ages rangedfrom 34-wk gestation to 79 yr of age, with a mean (± SD) of 57 ± 18yr. The mtDNA samples obtained from the 127subjects were examined using agarose gel electrophoresis of the PCR products amplified with four specific pairs of primers (Table I). The "primershift PCR" experiment clearly demonstrated that there was a specific 4,977 bp deletion in the mtDNA of a 69-yr-old human lung (Figure 1). Seventy-seven of the 127samples (60.6%) showed the 4,977 bp deletion of mtDNA from subjects with a mean age of 64 ± 10yr. The frequency of occurrences of the 4,977 bp-deleted mtDNA in the subjects in different age cohorts are presented in Table 2. The incidence increased significantly, from 14.3070 (one of seven) in the fourth decade to 77.8% (21 of 27) in the eighth decade (p < 0.0001, chi-square test). No 4,977 bpdeleted mtDNA was found in human lungs from subjects younger than 30 yr of age. We further analyzed the impact of possible confounding factors, including sex, disease category, smoking habit, smoking index, and pulmonary function indices in the 94 subjects older than the age of 50. No single factor was found to have statistically significant effect on either the frequency of occurrence or the proportion of the 4,977 bp-deleted mtDNA, as shown in Tables 3 and 4. The determination of the relative amount of the 4,977 bp-deleted mtDNA in one of the 77 subjects with 4,977 bp mtDNA deletion is shown in Figure 2. The proportion of the 4,977 bp-deleted mtDNA in human lung tissues was found to increase with age (Figure 3). The mean
(± SEM) proportions of the 4,977 bp-deleted mtDNA in lung tissues of the subjects in the 30 to 39, 49 to 49, 50 to 59, 60 to 69 and 70 to 79 age groups are 0.007 ± 0.007070, 0.006 ± 0.002070, 0.066 ± 0.030070, 0.544 ± 0.130070, and 0.833 ± 0.330070, respectively (p < 0.005, KruskalWallis H test). Multivariate regression using the stepwise method was applied to analyze the relationship between the proportion of the 4,977 bp-deleted mtDNA and age as well as all possible confounding factors, including sex, disease category, smoking habit, smoking index, and pulmonary function indices. Age was found to be the only significant independent factor related to the proportion of the 4,977 bp-deleted mtDNA in human lungs.
* * ** Oxidative damage to the mitochondrial genome has been implicated as a major contributory factor of age-related functional deficits, particularly in nondividing postmitotic cells (5). The high mutation rate, the small size and rich information content of human mtDNA, and a lack of efficient mechanisms for the repair of damaged mtDNA all contribute to the age-dependent accumulation of mutant mtDNA within tissue cells (8, 9). Damages to cells with limited life-spans such as erythrocytes, leukocytes, or epithelial cells can be rapidly replaced by newly regenerated cells. However, for cells that cannot be regenerated such as adult neurons, cardiomyocytes or cells with limited replicativelife-spans
TABLE 3 THE INCIDENCE OF THE 4,977 bp-DELETED mtDNA IN THE LUNG TISSUE OF 94 SUBJECTS OLDER THAN SO YR OF AGE
----------------
Subjects without 4,977 bp-deleted mtDNA Pertinent Factors Sex Male Female Disease category Primary lung cancer Metastatic tumors Smoking habit Nonsmoker Ex-smoker Current smoker Mean age (± SD) Smoking index (± SD) Pulmonary function indices FVC, % pred FEV], % pred FEV1/FVC, % * Chi-square test t Student's independent t test.
(n
= 25)
Subjects with 4,977 bp-deleted mtDNA (n
= 69)
P Value 0.28*
16 9
52 17
23 2
66 3
8 5 12
20 12 37
64.9 ± 6.6 30.3 ± 27.4
66.4 ± 6.4 33.2 ± 32.1
0.32t 0.68t
89.8 ± 17.7 84.8 ± 19.3 71.2 ± 10.2
91.7 ± 15.5 84.5 ± 18.7 68.1 ± 12.0
0.63t 0.9St O.27t
0.49*
0.89*
1144
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
1.2
TABLE 4 THE PROPORTION OF THE 4,977 bp-DELETED mtDNA IN THE LUNG TISSUES OF 94 SUBJECTS OLDER THAN 50 YR OF AGE
Pertinent Factors Sex Male Female Disease category Primary lung cancer Metastatic lung cancer Smoking habit Nonsmoker Ex-smoker Current smoker Pulmonary function indices FVC, % pred F\lC > 80% FVC < 80% FEV!, % pred FEVI > 80% FEVI < 80% FEV1/FVC ratio FEV1/FVC ratio> 70% FEV1/FVC ratio < 70%
Subjects
Percentage of 4,977 bp-deleted mtDNA
(n)
(mean±SEM)
68 26 89 5
VOL 154 1996
~ ~
< Z C
p Value'
0.53 ± 0.26 0.72 ± 0.39 0.51 ±0.12
74 18
0.56 ± 0.13 0.62 ±0.35
0.50
'tS
0.27
l"I"-
0.17 0.44 ± 0.12 0.80 ± 0.28
52 40
0.63 ± 0.17 0.48 ± 0.18
~
0.8
Cii
0.85
60 32
E
'tS
0.58 ±0.13 0.21±0.15
28 17 49
....
0.24 0.64 ± 0.15 0.33±0.17
1
0.55
, The p values were determined by Student's independent t test between two groups and by ANOVA for more than three groups.
such as human fibroblasts, the oxidative damages have to be repaired by that very cell (24). Nevertheless, the damage to mtDNA is likely to be accumulated in the injured cells, which results in the functional decline of respiration and oxidative phosphoryM I 2 3 .J 5 6 7 8 9 10 II 1213 I.J 15
A
B
Figure 2. Semiquantitative PCR analysis of the 4,977 bp-deleted mtDNA in human lung. Panel A. PCR amplification of total mtDNA by 2-fold dilutions (lanes 1 to 15 indicate dilutions of 2 5_ to 219_fold, respectively) of total DNA from human lung tissues of a 69-yr-old subject using primers L1 and H1 under conditions described in text. Panel B. peR amplification of the 4,977 bp-deleted mtDNA by 2-fold dilutions (lanes 1 to 15 indicate dilutions of 2 1_ to 2 15_fold, respectively) of total DNA using primers L2 and H3 under identical conditions. Lane M, 100 bp ladder size marker.
,g- 0.6 en
..; '0 c o
0.4
&.
0.2
:e
e a.
O+-----,~-~--..--.......'-.---~
0-9
10-19 20-29 30-39 40-49 50-59 60-69 70-79
Age cohort Figure 3. Age-dependent increase in the proportion of the 4,977 bpdeleted mtDNA in human lung. The black bar in each age cohort represents the average proportion (mean ± SEM) of the 4,977 bpdeleted mtDNA in the lung tissues from subjects in the cohort. The proportions of the 4,977 bp-deleted mtDNA were all determined under identical condition, as described in Figure 2.
lation. Age-dependent accumulation of the 4,977 bp-deleted mtDNA has been demonstrated in brain (10),liver (ll), testis (12), muscle (12), heart (13),skin (14), and ovary (25) tissues of elderly subjects. Increased proportion of the 4,977 bp-deleted mtDNA has also been found in disorders associated with hypoxemia such as ischemic heart conditions (26) or male infertility (27). The 4,977 bp deletion of mtDNA causes the truncation or removal of major structural genes of cytochrome oxidase (CO III), Fo-F,-ATPase (ATPase 6 and 6), and NADH-CoQ oxidoreductase (ND3, ND4, ND4L, and ND5) (8). Thus, the deleted or truncated genes in the 4,977 bp-deleted mtDNA will result in impaired gene expression by producing fused transcript of ATPase 8 and ND5 genes and lower amounts of the transcripts of the deleted genes. Accumulation of the 4,977 bp deletion and other oxidative damages to mtDNA have been associated with the decline of mitochondrial respiratory functions (5, 8), diminished fertility and motility of human sperm (27), and increase in both lipid peroxides and manganese superoxide dismutase levels in human liver (28). Both endogenous ROS and free radicals generated by the electron leak of the respiratory chain and exogenous oxidative stresses may elicit the generation and accumulation of mutant mtDNAs. The lung is unique compared with the other internal organs in that it is directly in contact with ambient air and thus is exposed to a wide variety of environmental noxious agents. Moreover, it is also a metabolically active organ dealing with various chemicals passing through the pulmonary circulation. Thus, it is conceivable that mtDNA in the lung tissues is quite vulnerable to oxidative damages. Indeed, we clearly demonstrated in this study that the frequency of occurrence and proportion of the 4,977 bp-deleted mtDNA in human lung increase with age. The incidence of the 4,977 bp mtDNA deletion in human lung was no less than other energy-demanding tissues such as those of the brain (10), muscle
Brief Communication
(12), and heart (26).The relative proportion of the 4,977 bp-deleted mtDNA in the lung was about 10times higher than those of the tissues examined in our previous studies such as liver, muscle and testis (12), skin (14), and sperm (27). Our findings that aged human lung tissues contain relativelyhigh proportions of mutated mtDNA without causing clinically identifiable symptoms and significant functional deterioration may be explained by the fact that little energy is required for gas exchange, and that complementation between wild-type and mutant mtDNA in each cell may remedy the possible defects in energy metabolism of the lung. However, relatively high oxygen tension in the alveoli, the potential noxious agents from the environment, and the defense systems (e.g., alveolar macrophages) of the lung may increase the generation of free radical damages to mtDNA and other biomolecules. These may partly explain the reason why the proportion of the 4,977 bp-deleted mtDNA in the lung is higher than those of the other energy-demanding tissues. Although one may perceive that some exogenous factors will cause enhanced oxidative damages to the lung mtDNA, we could find only that age is the sole independent factor related to the incidence and proportion of the 4,977 bp-deleted mtDNA in human lung. It remains to be determined whether other injurious factors leading to increased oxidative stress such as cigarette smoking, inflammation, infection, irradiation, or environmental carcinogens will elicit other types of mtDNA mutations in the lung tissues. Taking these results together, we conclude that the 4,977 bp-deleted mtDNA is generated and accumulated in the human lung during the aging process. References I. Harman, D. 1991. The aging process: major risk factor for disease and death. Proc. Natl. A cad. Sci. U.S.A. 88:5360-5363. 2. Harman, D. 1956. Ageing: theory based on free radical and radiation chemistry. J. Gerontol. 11:298-300. 3. Harman, D. 1972.The biological clock: the mitochondria? J. Am. Geriatr. Soc. 20:145-147. 4. Anderson, S., A. T. Bankier, B. G. Barrell, M. H. L. de Bruijn, A. R. Coulson, J. Drouin, l. C. Eperon, D. P. Nierlich, B. A. Roe, F. Sanger, P. H. Schreier, A. J. H. Smith, R. Staden, and I. G. Young. 1981. Sequence and organization of the human mitochondrial genome. Nature 290:457-465. 5. Shigenaga, M. K., T. M. Hagen, and B. N. Ames. 1994. Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. U.S.A. 91: 10771-10778. 6. Clayton, D. A., J. N. Doda, and E. C. Friedberg. 1974. The absence of a pyrimidine dimer repair mechanism in mammalian mitochondria. Proc. Nat!. A cad. Sci. U.S.A. 71:2777-2781. 7. Robin, E.D., and R. Wong. 1988. Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J. Cell. Physiol. 136:507-513. 8. Wei, Y. H. 1992. Mitochondrial DNA alterations as ageing-associated molecular events. Mutat. Res. 275:145-155. 9. Wallace, D. C. 1992. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science 256:628-632. 10. Ikebe, S., M. Tanaka, K. Ohno, W. Sato, K. Hattori, T. Knodo, Y. Mizuno, and T. Ozawa. 1990. Increase of deleted mitochondrial DNA
1145 in the striatum in Parkinson's disease and senescence. Biochem. Biophys. Res. Commun. 170:1044-1048. II. Yen, T. C., J. H. Su, K. L. King, and Y. H. Wei. 1991.Ageing-associated 5 kb deletion in human liver mitochondrial DNA. Biochem. Biophys. Res. Commun. 178:124-131. 12. Lee, H. C., C. Y. Pang, H. S. Hsu, and Y. H. Wei. 1994. Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing. Biochim. Biophys. Acta 1226:37-43. 13. Katsumata, K., M. Hayakawa, M. Tanaka, S. Sugiyama, and T. Ozawa. 1994. Fragmentation of human heart mitochondrial DNA associated with premature aging. Biochem. Biophys. Res. Commun. 202:102-110. 14. Yang, J. H., H. C. Lee, and Y. H. Wei. 1995. Photoageing-associated mitochondrial DNA length mutations in human skin. Arch. Dermatol. Res. 287:641-648. 15. Thurlbeck, W. M. 1991. Morphology of the aging lung. In R. G. Crystal and J. B. West, Editors-in-Chief; P. J. Barnes, N. S. Cherniack, and E. R. Weibel, Associate Editors. The Lung: Scientific Foundations. Raven Press, New York. 1743-1748. 16. Pierce, J. A., R. V. Ebert, and L. R. Ark. 1958. The elastic properties of the lungs in the aged. J. Lab. Clin. Med. 51:63-71. 17. Jones, R. L., T. R. Overton, D. M. Hammerlindl, and B. J. Sproule. 1978. Effects of age on regional residual volume. J. Appl. Physiol. 44:195-199. 18. Sherrill, D. L., L. R. J. Knudson, and B. Burrows. 1993. Longitudinal methods for describing the relationship between pulmonary function, respiratory symptoms and smoking in elderly subjects. The Tucson Study. Eur. Respir. J. 6:342-348. 19. Ware, J. H., D. W. Dockery, T. A. Louis, X. Xu, B. G. Ferris, Jr., and F. E. Speizer. 1990. Longitudinal and cross-sectional estimates of pulmonary function decline in never-smoking adults. Am. J. Epidemiol. 132:685-700. 20. Kundson, R. J. 1991. Physiology of the aging lung. In R. G. Crystal, J. B. West, Editors-in-Chief; P. J. Barnes, N. S. Cherniack, and E. R. Weibel, Associate Editors. The Lung: Scientific Foundations. Raven Press, New York. 1749-1759. 21. American Thoracic Society. 1987. Standardization of spirometry: 1987 update. Am. Rev. Respir. Dis. 136:1285-1298. 22. Wallace, D. c., X. Zheng, M. T. Lott, J. M. Shoffner, J. A. Hodge, R. I. Kelly, C. M. Epstein, and L. C. Hopkins. 1988. Familial mitochondrial encephalomyopathy (MERRF): genetic, pathophysiological, and biochemical characterization of a mitochondrial DNA disease. Cell 55:601-610. 23. Tanaka, M., and T. Ozawa. 1992. Analysis of mitochondrial DNA mutations.In A. Longstaff and P. Revest, editors. Methods in Molecular Biology, Vol. 13: Protocols in Molecular Neurobiology. Humana Press, Totowa, NJ. 1-28. 24. Goldstein, S. 1993. The biology of aging: looking to defuse the genetic time bomb. Geriatrics 48:76-82. 25. Kitagawa, T., N. Suganuma, A. Nawa, F. Kikkawa, M. Tanaka, T. Ozawa, and Y. Tomoda. 1993. Rapid accumulation of deleted mitochondrial deoxyribonucleic acid in postmenopausal ovaries. BioI. Reprod. 49:730-736. 26. Corral-Debrinski, M., G. Stepien, J. M. Shoffner, M. T. Lott, K. Kanter, and D. C. Wallace. 1991. Hypoxemia is associated with mitochondrial DNA damage and gene induction. J, A. M. A. 266: 1812-1816. 27. Kao, S. H., H. T. Chao, and Y. H. Wei. 1995. Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm. Bioi. Reprod. 52:729-736. 28. Yen, T. C. K. L. King, H. C. Lee, S. H. Yeh, and Y. H. Wei. 1994. Age-dependent increase of mitochondrial DNA deletions together with lipid peroxides and superoxide dismutase in human liver mitochondria. Free Radic. Bioi. Med. 16:207-214.
