Mitochondrial Dysfunction in the Senescence Accelerated Mouse (SAM)

Free Radical Biology & Medicine, Vol. 24, No. 1, pp. 85–92, 1998 Published by Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 / .00

PII S0891-5849(97)00164-0

Original Contribution MITOCHONDRIAL DYSFUNCTION IN THE SENESCENCE ACCELERATED MOUSE (SAM) HIROKO NAK AHARA,* TOMOKO KANNO,* YOKO INAI,* KOZO UTSUMI,* MIDORI HIRAMATSU, † AKITANE MORI, ‡ and LESTER PACKER ‡ *Institute of Medical Science, Center for Adult Diseases, Kuraashiki, Kurashiki 710, Japan, † Institute for Life Support Technology, Yamagata Technopolis Foundation, Yamagata 990, Japan, and ‡ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA (Received 23 December 1996; Revised 27 March 1997; Re-Revised 6 May 1997; Accepted 7 May 1997)

Abstract—Oxidative damage to DNA, proteins, and lipids in mitochondria caused by free radicals may be one factor in aging. Oxidative phosphorylation was estimated in liver mitochondria from senescence accelerated mice (SAMP8) and a senescence resistant substrain (SAMR1). The respiratory control ratio decreased in liver mitochondria of SAMP8 during aging, and it was estimated that at 18 months of age this respiratory control value suggested that it might be insufficient to provide ATP synthesis necessary for normal cell metabolism. In addition, the ADP/O, an index of efficiency of ATP synthesis, was depressed at 18 months of age. Dinitrophenol-dependent uncoupled respiration in liver mitochondria of SAMP8 mice was markedly decreased with aging, suggesting a dysfunctional energy transfer mechanism in mitochondria of aged SAMP8 mice. Active uptake of calcium in liver mitochondria was markedly dysfunctional in SAMP8 mice with aging, and uncoupling of respiration was induced more easily in aged mitochondria. Milder effects on these functional parameters were observed in SAMR1 mice. A similar dysfunction was also observed in heart mitochondria of SAMP8 mice at 12 months of age. The amount of Bcl-x in liver mitochondria was slightly decreased in SAMP8. We suggest that these changes in mitochondrial function may be related to the shorter life span of the senescence accelerated mouse. Published by Elsevier Science Inc. Keywords—Aging, Senescence accelerated mouse (SAM), Mitochondria, Mitochondrial dysfunction, Free radicals, Hydrogen peroxide, Bcl-x

In particular, oxidative damage to mitochondria may play a key role in aging.5 The univalent reduction of oxygen through the respiratory chain results in the production of reactive oxygen species like the superoxide radical (O2 i 0 ) hydrogen peroxide and the highly reactive hydroxyl radical (OH i 0 ).6,7 Because mitochondrial DNA (mtDNA) and proteins are close to the electron transport chain, the source of reactive oxygen species, they become oxidatively damaged, and this damage accumulates with time. Liver and brain mitochondria display levels of mtDNA oxidative damage that are 10-fold those of nuclear DNA.8,9 8-oxo-2 *-deoxyguanosine levels (a marker of DNA damage) increase threefold in mtDNA over the course of 24 months in rats.10 Similar age-related increases in oxidative damage are found in mitochondrial protein.11 mtDNA deletions have been proposed to be factors in the neurological, cardiovascular, and muscle degeneration seen in aging.5

INTRODUCTION

Aging is influenced by various environmental factors, of which free radicals are thought to be the most important, as proposed by Harman in 1956.1 Recently, much evidence supporting this hypothesis has accumulated, for example, the formation of 8-hydroxyguanine from bases in DNA molecules by hydroxyl radicals, 2 age-related acceleration of peroxidation in the lens body, 3 and decreased superoxide dismutase (SOD) activity in the lens of patients with cataracts.4 H. Nakahara is on leave from Department of Cell Chemistry, Institute of Molecular and Cell Biology, Okayama University Medical School, Okayama 700, Japan. T. Kanno is on leave from Doonan Institute of Medical Science, 41-9, Ishikawa-cho, Hakodate 041, Japan. Address correspondence to: L. Packer, 251 Life Sciences Addition, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200. 85

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The senescence accelerated mouse (SAM) is a pure strain developed as a model animal for aging as described by Takeda.12,13 The half-lifespan of SAM is about one and a half years, i.e., about 25% shorter than that of other common strains of mice. Recently, Edamatsu et al.14 observed that N-tert-butyl-a-phenylnitrone (PBN), a free radical spin-trapping agent, significantly increased the lifespan of SAMP8 mice, suggesting that the shorter lifespan of SAMP8 is related, at least in part, to free radical damage. Accumulated mutations in mitochondrial DNA caused by free radicals in terminally differentiated cells, such as nerve, cardiac muscle, and skeletal muscle, are thought to be important in the mechanism of aging. Decreased activity of the mitochondrial electron transport complex has been demonstrated in Parkinson’s disease and also in aging.15 Moreover, the mitochondrion is known to be the principal superoxide generating organelle in cells. Mitochondrial Mn 2/ -SOD, which maintains a low level of superoxide, should minimize injury by oxygen free radicals in mitochondria. Nevertheless, even slight oxidative stress may induce functional damage to mitochondria and may lead to apoptosis and/or accelerated aging. Recently, studies have shown that mitochondria are affected by a cell death program that is related to active oxygen generation, and that dysfunction of mitochondria may be involved in apoptotic cell death.17 – 20 For this reason we investigated changes in mitochondrial function in the liver and heart of SAMP8 mice during the course of aging. MATERIALS AND METHODS

Chemicals Scopoletin, ADP, horseradish peroxidase, hypoxanthine, xanthine oxidase, ferricytochrome c , and superoxide dismutase (SOD) were purchased from Sigma Co. (St. Louis, MO). Anti-Bcl-xS / L (S-18)-G was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and peroxidase-conjugated rabbit antimouse IgG from EY Lab Inc. (San Mateo, CA). ECL Western blotting detection system was from Amersham, Buckinghamshire (UK) and molecular mass standards were from Daiichi-Kagaku Standards (Tokyo, Japan) and Bio-Rad Standards (CA). All other chemicals were from Nacalai Tesque (Kyoto).

eases, Kyoto University, Kyoto, Japan. They were housed for 6 months, 1- 12 years under standard conditions, 257C with a 12 h light/dark cycle, and allowed free access to water and a standard diet. Male mice (ICR), 3 months of age, were housed under the same conditions for control experiments. Isolation of mitochondria Liver mitochondria of male SAM and ICR mice (18–20 g) were isolated by the method of Hogeboom.21 Briefly, liver mitochondria were isolated in a medium containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), and 0.1 mM EDTA (Medium A). EDTA was omitted in the final wash and the final mitochondrial pellet was resuspended in 0.25 M sucrose–10 mM Tris-HCl (pH 7.4) medium at 10–30 mg protein/ml at 47C. Heart mitochondria were isolated by the modified method of Tyler and Gonze.22 Mitochondrial protein was determined by Bradford’s method using bovine serum albumin as standard.23 Measurement of mitochondrial respiration and oxidative phosphorylation Respiration and oxidative phosphorylation were measured polarographically using a Clark type oxygen electrode fitted to a 2 ml water-jacketed closed chamber maintained at 257C.24 Isolated liver mitochondria (0.5 mg protein/ml) were suspended in a medium consisting of 0.2 M sucrose, 10 mM KCl, 3 mM MgCl2 , 2 mM sodium phosphate, 5 mM Tris-HCl (pH 7.4), and 5 mM succinate at 257C. State 3 respiration was initiated by adding ADP. The respiratory control ratio (State 3 respiration/State 4 respiration, RCR) and the ADP/O ratio after adding 200 mM ADP were determined as described by Estabrook.25 Uncoupled respiration was induced by adding 25 mM DNP. The Ca 2/ induced respiratory control was measured by adding 60 mM Ca 2/ . Isolated heart mitochondria (50 mg protein/ ml) were incubated in a medium containing 0.3 M mannitol, 10 mM KCl, 5 mM Tris-HCl (pH 7.4), 3 mM MgCl2 at 257C. The concentration of succinate, phosphate, and ADP were 5 mM, 2 mM, and 150 mM, respectively. Uncoupled respiration was measured in the presence of 10 mM DNP.

Mice

Measurement of hydrogen peroxide (H2O2 ) generation

SAMP8, senescence accelerated mice, and SAMR1, mice from the senescence resistant substrain of SAM, 12,13 were originally donated by Drs. Toshio Takeda and Masamori Hosokawa, Institute of Chest Dis-

Production of H2O2 was assayed at 257C in a fluorospectrophotometer (Hitachi, Type 650-10LC) as described previously.16 Mitochondria (0.5 mg protein/ ml) were suspended in a medium of 0.23 M mannitol,

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70 mM sucrose, 30 mM Tris-HCl (pH 7.4) containing 0.5 mM horseradish peroxidase and 0.5 mM scopoletin at 257C. Succinate and antimycin A were 5 mM and 10 nM, respectively. The change in fluorescence intensity was measured at 450 nm, with excitation at 360 nm. Measurement of SOD activity in mitochondria SOD (Mn 2/ -SOD) activity in mitochondria was measured by the method of Takesige and Minakami 26 using acetylated cytochrome c . Briefly, cytochrome c reduction was followed by recording absorbance changes at 550–540 nm (A550 – 540 , 27 ) in a 2-ml suspension of mitochondria at 40–80 mg protein/ml at 377C, using a dual beam spectrophotometer (Shimadzu UV 3000) equipped with a water-jacketed cell holder and a magnetic stirrer. The reaction medium was a KrebsRinger-Phosphate solution containing 50 mM hypoxanthine and 20 mM acetylated cytochrome c. Reaction was started by adding xanthine oxidase to a final concentration of 1.25 mU/ml. Detection of Bcl-xS / L Mitochondria were added in the medium sodium dodecyl sulfate (SDS) sample buffer (65.2 M Tris-HCl buffer pH 6.8, 2% SDS, 5% b-mercaptoethanol, 10% glycerol, 0.001% bromphenol blue and 1 mM phenylmethylsulfonyl fluoride (PMSF) and subjected to SDSpolyacrylamide gel electrophoresis (SDS-PAGE) (7.5% gel) by the method of Laemmli.28 Proteins were transferred to an Immobilon-P filter (Nippon Millipore Ltd., Tokyo, Japan) using the Sartorious semidry blotting apparatus. After blocking with 5% skim powdered milk, Bcl-xS / L on the filter were detected with Anti-BclxS / L antibodies and peroxidase-conjugated rabbit antimouse IgG.29 Positive bands were visualized by using the ECL Western blotting detection system. Intensity analysis of positive bands was performed on a Macintosh computer using the public domain NIH Image program. Molecular masses were determined using Daiichi-Kagaku standards and Bio-Rad standards.

87 RESULTS

Aging and oxidative phosphorylation Oxidative phosphorylation was measured in liver mitochondria of SAMR1 at 6, 12, and 18 months of age, SAMP8 mice at 6, 12, and 18 months of age, and ICR mice at 3 months of age (control). Mitochondrial respiration (calculated as nmol O2 /min/mg protein) in the presence of succinate and inorganic phosphate (State 4 respiration) was increased by adding ADP (State 3 respiration), and then decreased (State 4 respiration), corresponding to completion of ATP synthesis. Typical traces from these procedures are shown for all strains of mice in Fig. 1. Based on this data, ADP/ O and RCR of liver mitochondria from ICR mice were calculated to be 1.7 and 2.8, respectively. The ADP/O and RCR of SAM mice were each 1.3 and around 2.6, respectively for mitochondrial preparations from SAMP8 and SAMR1 at 6 months old. No significant difference was observed between SAMP8 and SAMR1 at this age. No significant differences between the two groups was observed in State 3 and 4 respiration at 6 months of age. However, State 4 respiration was markedly increased and RCR tended to decline for SAMP8 after 12 months of age. In SAMP8 mice, at 12 months of age, RCR decreased dramatically to about 1.5, and remained at this value at 18 months of age. This decrease was due to an increase in State 4 respiration,

Measurement of lipid peroxidation Lipid peroxidation of mitochondria in vitro was assayed by the thiobarbituric acid reaction as described previously and expressed as the amount of TBA reactive substances (TBARS).30 Statistical analysis Statistically significant differences were determined by Student’s t-test.

Fig. 1. Oxidative phosphorylation of liver mitochondria in SAM mice. Mouse liver mitochondria (0.5 mg/ml) isolated by the method of Hogeboom were incubated in a medium of 0.2 M sucrose, 10 mM KCl, 3 mM MgCl2 , 2 mM sodium phosphate 5mM TRIS-HCl (pH 7.4) and 5mM succinate at 257C. State 3 respiration was induced by adding 200 mM ADP. Oxygen consuption was polarographically recorded using a Clark type oxygen electrode fitted to a 2-ml waterjacketed closed chamber. — ICR (3 months old); ------------, SAMR1 (18 months old); — · — · — ·, SAMP8 (18 months old).

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H. NAK AHARA et al. Table 1. Functional Characteristics of Liver Mitochondria From SAMR1 and SAMP8 Mice at Various Ages SAMR1 Mice

Characteristic ADP/O RCR State 3 respiration State 4 respiration DNP uncoupled O2 uptake RCR by Ca2/

SAMP8 Mice

6 Month

12 Month

18 Month

6 Month

12 Month

18 Month

1.34 { 0.10 2.63 { 0.38

1.39 { 0.15 2.19 { 0.27

1.27 { 0.16 2.56 { 0.39

1.33 { 0.04 2.42 { 0.51

1.00 { 0.12 1.48 { 0.11*

1.11 { 0.04 1.52 { 0.23

41.23 { 5.40

59.85 { 11.97

68.05 { 9.08

42.53 { 7.70

38.63 { 9.56*

37.35 { 2.67†

15.38 { 2.52

27.30 { 4.52

27.02 { 5.87

18.03 { 5.01

26.10 { 6.37

24.69 { 2.47

43.70 { 5.96 3.38 { 0.38

55.65 { 14.59 2.71 { 0.14

64.65 { 72.7 2.93 { 0.28

43.20 { 8.97 3.04 { 0.30

33.04 { 12.34 2.11 { 0.37*

16.33 { 4.95† 1.82 { 0.10

n Å 3, 7, and 6 for 6, 12, 18 months of age SAMR1 mice, respectively, and n Å 3, 6, and 3 for 6, 12, 18 months of age SAMP8 mice, respectively. * p õ .05, † p õ .01: significant difference from SAMR1.

while State 3 respiration remained constant. The ADP/ O ratio also declined at 12 months of age and remained depressed at 18 months of age in SAMP8 mice. In contrast, increased State 3 and 4 respiration were observed in SAMR1 mice at 12 and 18 months of age compared to that seen at 6 months of age, while the ADP/O and RCR showed no significant change at 12 and 18 months (Table 1). The activity of oxidative phosphorylation of heart mitochondria from SAM and ICR mice was also measured. State 3, State 4, and uncoupled respiration of heart mitochondria from SAMP8 mice at 12 months of age were markedly depressed compared to SAMR1 mice of the same age (Table 2). Because both State 3 and State 4 respiration were depressed in SAMP8 mice, the RCR was similar to that for SAMR1 mice. The ADP/O ratio was also similar for SAMP8 and SAMR1 mice.

Aging and uncoupled respiration in liver mitochondria Respiration in mitochondria is uncoupled by agents such as dinitrophenol (DNP) and oxygen consumption increases, reflecting an accelerated electron transport activity. Uncoupled oxygen consumption is parallel

with State 3 respiration, which is the phosphorylating respiration induced by addition of ADP (Fig. 2). DNP-induced uncoupled respiration in liver mitochondria of SAM mice showed values similar to those for State 3 respiration both in SAMP8 and SAMR1 at 6 months of age (Table 1). In SAMR1 mice mitochondria, uncoupled respiration increased at 12 and 18 months of age, again paralleling increases in State 3 respiration seen at those ages. In contrast, uncoupled respiration declined at 12 months of age in SAMP8 mice mitochondria, and dramatically declined at 18 months of age. The uncoupled respiration at 18 months of age was less than half of State 3 respiration at this age (Table 1).

Aging and accelerated respiration induced by calcium Ca 2/ is actively taken up into normal mitochondria through the inner membrane, utilizing energy transfer reactions which induce a transitory accelerated respiration (Fig. 3). Ca 2/ added to mitochondria forms calcium apatite by reaction with inorganic phosphate, leading to membrane injury, which is accompanied by uncoupling of energy transfer, and increased in oxygen consumption.31 The accelerated respiration allows for

Table 2. Functional Characteristics of Heart Mitochondria From ICR and SAM Mice Characteristic ADP/O RCR State 3 respiration State 4 respiration DNP uncoupled O2 uptake

ICR (3 Months of Age) 1.60 { 1.76 { 246.72 { 141.68 { 214.00 {

SAMR1 (12 Months of Age)

0.16 0.29 45.24 27.71 27.71

1.65 { 2.10 { 309.29 { 143.49 { 259.40 {

0.22 0.46 87.64 28.49 85.13

Data are mean { SD from five separate experiments. * p õ .01: significant difference from SAMR1.

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SAMP8 (12 Months of Age) 1.42 { 2.13 { 153.32 { 69.54 { 116.48 {

0.32 0.37 87.36* 36.94* 57.31*

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age (Table 1). Similar responses were observed in mitochondria of both SAMR1 and ICR mice. A single addition of 60 mM CaCl2 to mitochondria increased the respiratory activity after 4–8 min in SAMP8 mice, i.e., an unusually high sensitivity to uncoupling by Ca 2/ was observed in SAMP8 mice (Fig. 3). Changes in H2O2 generation and lipid peroxidation of mitochondria in aged SAMP8

Fig. 2. Uncoupling by DNP of oxidative phosphorylation in liver mitochondria of ICR and SAM mice. Experimental conditions were as described in Fig. 1 except that 25 mM DNP was added instead of ADP. — , ICR, ---------- SAMR1, — · — · — ·, SAMP8.

calculation of an ‘‘RCR’’ similar to that calculated for increased respiration due to the addition of ADP. The RCR induced by Ca 2/ in liver mitochondria of SAMP8 had a high value (3.0) at 6 months of age, but decreased markedly at 12 months old and persisted at a low rate until 18 months of age (Fig. 3), i.e., active Ca 2/ transport was extremely decreased in SAMP8 with age. RCR induced by Ca 2/ in liver mitochondria of SAMR1 was not significantly decreased at 12 and 18 months of

To gain further insight into the mechanism of mitochondrial functional changes in SAMP8 mice, the generation of active oxygen species and lipid peroxidation of mitochondria were examined. One cannot measure the O2 i 0 generated in mitochondria due to the activity of mitochondrial Mn 2/ -SOD. Indeed, because of high SOD activity and the low permeability of intact mitochondrial membrane to O2 i 0 , this species is not observed to be extruded by intact mitochondria. However, H2O2 generation can be measured by following the fluorescence intensity change of scopoletin in the presence of horseradish peroxidase. Using this method, no significant increase in H2O2 generation was observed in mitochondria of SAMP8 mice at 12 months of age in the presence of succinate, compared to SAMR1 mice (data not shown). The sensitivity of this method was 1 mM H2O2 . However, the presence of mitochondria of TBARS in nmol/mg protein showed an increase, though nonsignificant, in the mitochondria of SAMP8 mice at 12 months of age (2.77 { 1.22) compared to that of SAMR1 at 12 months of age (1.69 { 0.31) or of ICR mice at 3 months of age (1.56 { 0.32). Content of SOD in mitochondria Because there was a trend toward increased TBARS in mitochondria of SAMP8, the SOD activity of mitochondria was measured using acetylated cytochrome c. SOD activity was not different between liver mitochondria isolated from SAMP8, SAMR1, and ICR mice (data not shown). This result indicates that the increased lipid peroxidation of mitochondria in SAMP8 liver is not due to changes in SOD activity. Distribution of Bcl-x

Fig. 3. Calcium-induced increase in mitochondrial respiration of ICR and SAM mice. Experimental conditions were as described in Fig. 1 except that 60 mM calcium was added instead of ADP. — ICR (6 months old); ------------, SAMR1 (18 months old); — · — · — ·, SAMP8 (18 months old).

Bcl-x is an oncogene product of Bcl-family protein, 20,29 distributed on the outer membrane of liver mitochondria, which inhibits apoptotic cell death. To investigate the involvement of protective action of Bclx against the mitochondrial dysfunction in SAMP8, expression of the Bcl-xS / L in liver mitochondria of SAMP8 was studied by immunoblotting method using

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anti-Bcl-xS / L antibody. Figure 4 shows the immunoblotting pattern of Bcl-xS / L in the SDS-PAGE of mitochondrial proteins. A decrease in Bcl-xS / L in liver mitochondria of SAMP8 was observed. DISCUSSION

The senescence accelerated mouse (SAM) displays a decreased lifespan compared to normal mice.12,13 Because mitochondrial dysfunction induced by accumulated oxidative damage has been associated with aging, 5 we chose to study mitochondrial function in SAM at various ages and compare it with mitochondrial function in nonage accelerated mice. In addition, we measured indices of oxidative stress in mitochondria and Bcl-xS / L gene products. By 12 months of age, SAMP8 mice mitochondria displayed markedly decreased mitochondrial RCR, as the result of an increase in State 4 respiration. In contrast, the senescence-resistant SAMR1 displayed no decrease in RCR, and age-related increases in both State 4 and State 3 respiration. The increase of State 4 respiration in SAMP8 mice may be due to uncoupling due to membrane damage. Although our finding of higher mitochondrial TBARS in SAMP8 mice at 12 months of age compared to those of SAMR1 mice was not statistically significant, others have reported a significant increase in liver lipid peroxide levels of 2-monthold SAMP1 mice compared with SAMR1 mice.32,33 Furthermore, it has been reported that the content of malondialdehyde was significantly higher in the liver and brain of 11–12-month-old SAMP8 mice compared to SAMR1 mice.32 Moreover, SAMP8 liver homogenates showed less SOD activity than those of SAMR1.32 However, in our study we observed no difference in the SOD content of liver mitochondria from SAMP8, SAMR1, or ICR mice, indicating that any increased lipid peroxidation of mitochondria in SAMP8 liver is not due to changes in SOD activity. Overall, our results suggest that oxidative stress, possibly due to free radicals, might be a cause of the observed mitochondrial dysfunction. Although Sohal has reported age-related increases in O2 i 0 and H2O2 production in mitochondria of insects and rats, 34 we did not observe an age-related increase of H2O2 production in mitochondria from SAMP8 mice. The lack of an observed difference in H2O2 production in mitochondria between the two strains may have been due to a lack of sensitivity of the assay procedure. Small differences in H2O2 production could lead to large differences in oxidative damage over time. The decrease in the ADP/O ratio in the SAMP8 mice at 12 and 18 months can also be explained by their age-related uncoupling. This decrease may ex-

Fig. 4. Distribution of Bcl-x in liver mitochondria of SAMP8 and ICR mice. SDS PAGE of mitochondrial proteins (35 mg/ml) was carried out and transferred to Immobilon-P filter immobile membrane. Bcl-x was detected by anti-Bcl-xS / L antibody and peroxidase-conjugated rabbit anti-mouse IgG. SAMP8 and ICR mice were 12 and 3 months of age, respectively.

plain the functional decline in this strain, because less ATP is made per unit of oxygen consumed. Possibly, this results in less energy for cell maintenance and organismal function. DNP induces uncoupled respiration in normal mitochondria. However, the increased rate of oxygen consumption induced by DNP declined in liver mitochondria from SAMP8 mice at 18 months old to a value less than half that seen in liver mitochondria from SAMP8 mice at 6 months old. This suggests that the coupling mechanism for energy transfer reactions of the electron transport system may be altered in SAMP8 at 18 months of age, which may be due to damage to the components of the electron and energy transfer system. Oxidized protein accumulation has been observed in mitochondria from aged animals.11 The increase in TBARS in mitochondrial membranes of SAMP8 mice suggests that oxidative damage in these mice is increased, possibly altering inner mitochondrial membrane functions. Both the trend toward a decrease in State 3 respiration and DNP-induced uncoupled respiration suggest that compartmentation of mitochondrial inner membrane to the proton electrochemical gradient might be altered. However, an explanation of the mechanism of this finding is not yet known. Similar decreases in mitochondrial respiration were observed in heart mitochondria of SAMP8 mice at 12 months of age, and no significant differences were observed between liver and heart mitochondria of SAMP8 mice. This similarity between liver, a mitotic tissue, and heart, a postmitotic tissue suggests that the accumulated mutations of mtDNA is not likely the cause of increased mitochondrial dysfunction in SAMP8 mice.

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Active transport of calcium in liver mitochondria decreased with age in SAMP8 mice. Uncoupled respiration induced by calcium addition was slightly decreased at 12 months old in SAMP8, but it was almost the same rate as the normal control ICR at 18 months in SAMR1 mice (Fig. 3). However, uncoupled respiration induced by Ca 2/ in SAMP8 mice at 18 months of age was very high. This suggests that the enzymes themselves of the electron transfer system were not damaged in this stage, and rather that activity of the calcium transporter was altered, perhaps because of membrane potential changes. Shigenaga et al., summarizing other experimental results and related studies, conclude that defects in mitochondrial function with age reflect changes in the proton electrochemical gradient across the inner membrane and that this affects many activities dependent upon the membrane potential.5,35 These changes in the proton electrochemical gradient may reflect accumulated oxidative damage to the inner membrane. Indeed, in a recent study of mitochondria isolated from brain and from liver, a potent antioxidant mixture, Ginkgo biloba extract EGb 761, administered to young and old rats, was found to protect against the age-associated decline in mitochondrial membrane potential (J. Sastre, A. Milla´n, J. Garcı´a de la Asuncio´n, R. Pla´, G. Juan, F. Pallardo´, E. O’Connor, J. Martin, M-T. Droy-Lefaix, and J. Vin˜a, personal communication). Recently it has been found that Bcl-2 protected cells from hydrogen peroxide- and menadione-induced oxidative death.20 Bcl-x belong to the Bcl-2 family of proteins and are distributed on the outer membrane of liver mitochondria.29 SAMP8 mice were found to have lower expression of Bcl-x than did ICR mice, suggesting that the decreased expression of Bcl-x may be involved in the dysfunction of mitochondria in SAMP8. In summary, functional damage to oxidative phosphorylation during aging in liver mitochondria of SAMP8 mice, especially in the energy transfer functions for ATP synthesis and active uptake of calcium, was demonstrated in this study. These functional changes might be related to the shorter life span of the senescence accelerated mouse. REFERENCES 1. Harman, D. Aging, a theory based on free radical and radiation chemistry. J. Gerontol. 11:298–300; 1956. 2. Hayakawa, M.; Torii, K.; Sugiyama, S.; Tanaka, M.; Ozawa, T. Age-associated accumulation of 8-hydroxydeoxyguanosine in mitochondrial DNA of human diphragm. Biochem. Biophys. Res. Commun. 178:1023–1029; 1991. 3. Bhuyan, K. C.; Bhuyan, D. K.; Podos, S. M. Lipid peroxidation in cataract of the human. Life Sci. 38:1463–1471; 1986. 4. Scharf, J.; Dorvarat, A. Superoxide dismutase molecules in human cataractous lenses. Opthal. Res. 18:332–337; 1986.

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