Mitochondrial oxidative damage, hydrogen peroxide release, and aging

Free RadicalBiology& Medicine, Vol. 16, No. 5, pp. 621-626, 1994 Copyright© 1994Elsevier ScienceLtd Printed in the USA. All rights reserved 0891-5849/94 $6.00 + .00

Pergamon

Original Contribution MITOCHONDRIAL

OXIDATIVE RELEASE,

DAMAGE, AND

HYDROGEN

PEROXIDE

AGING

RAJINDAR S. SOHAL and ANJU DUBEY Department of Biological Sciences, Southern Methodist University, Dallas, TX, USA (Received 12 July 1993; Revised 27 September 1993; Accepted 1 October 1993) Abstract--The objective of this study was to elucidate the possible nature of the mechanism underlying the widely observed phenomenon that the rate of H202production by mitochondda increases during the aging process, using flight muscle mitochondria of the male housefly as a model system. The protein carbonyl content of mitochondria increased linearly with age of the flies, and was also inversely associated with the life expectancy of flies. Exposure of flies to 100% oxygen caused a progressive increase in the level of mitochondrial carbonyl content. The rate of H202 release by such oxidatively damaged mitochondria was higher than the controls. Similarly, X-irradiation of submitochondrial particles simultaneously resulted in increased rate of 1-1202production and elevated level of carbonyl content. Results of this and previous studies indicate that oxidative damage to mitochondrial membranes may be responsible for the age-related increase in mitochondrial H202 generation.

Keywords---Aging, Free radicals, Mitochondria, Muscle, Oxidative damage

INTRODUCTION

by mitochondria. Adult male housefly was employed as a model organism and protein carbonyl content was used as an indicator of oxidative damage. Stadtman and Oliver 7 have demonstrated that protein carbonyl modifications result primarily from metal catalyzed oxidations. It has also been postulated that mitochondria may act as a biological clock for the aging process. 1~'12Accordingly, it was also determined whether the oxidative damage to mitochondria was associated with the mortality or life expectancy o f the flies rather than their chronological age. Distinction between life expectancy and chronological age was made in two different ways, (1) by experimentally altering the life spans o f flies by varying the level o f physical activity, and (2) by the selection o f relatively short- and long-lived flies on the basis o f the senescence-linked loss of flying ability, which occurs in all flies a few days prior to death. 6'13A4

The possible role of mitochondria in the aging process has been the subject of considerable attention recently. 1'2 It is now well established that the rate o f mitochondrial superoxide anion radical (02"-) and H202 generation increases in the latter part o f life in insects 3 as well as mammais. 4"5 Concomitantly, the concentrations o f the products o f oxidative damage, such as protein carbonyls, 6'7 lipofuscin, s and n-pentane exhalation, 9'1° are also elevated with age. The mechanism underlying the age-related increase in the mitochondrial generation o f 02"- and H202 generation is presently unknown. It has been hypothesized that the reactive oxygen species (ROS), produced by mitochondria, also inflict damage on mitochondrial components leading to the elevation o f ROS production. 2'3 The objective of this study was to test this hypothesis by determining if mitochondria, the main intracellular generators o f ROS, are also themselves the targets o f oxidative damage, and whether such damage, in turn, elevates the rate o f ROS generation

Animals

This research was supported by the grant R01AG7657-05 from the National Institutes of Health-National Institute on Aging, Bethesda, MD. Address correspondence to: R. S. Sohal, Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA.

After emergence from the pupae, adult male houseflies were segregated by sex and kept at 25°C and 50% relative humidity. They were fed on sucrose and water, which assures a longer life span than a diet additionally containing lipids and proteins.

MATERIALS AND METHODS

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R.S. SOHALand A. DUBEY

Variations in life expectancy of flies Two regimes were used to distinguish between life expectancy or physiological age and chronological age. One regime was to experimentally alter the level of physical activity of flies by varying the size of the housing containers, which affects the flying activity. It has been previously reported by US6'13'14 that the average and maximum life spans of the houseflies confined singly within 150 ml glass urine specimen jars, where they are unable to fly due to restriction of space (designated as low activity [LA] flies), is more than 2-fold longer than those kept in 1-cubic-foot cages, 200 flies/ cage, where they are able to fly (designated as high activity [HA] flies). In the other regime, the loss of flying ability, which occurs in all flies as a senescent feature a few days before death, was used to distinguish between life expectancy and chronological age. 6'~3'14 Flies unable to fly, and referred to as "crawlers," were isolated from their flying cohorts, the "fliers," at 10 days of age. The two groups were subsequently housed separately in 1-cubic-foot cages.

Exposure to hyperoxia Flies confined within a 1-cubic-foot cage were placed in a sealed Plexiglas ® container, connected via a gas manometer to a gas cylinder containing 100% oxygen. Oxygen was bubbled through water and then passed into the Plexiglas ® chamber under a low, steady, positive pressure.

Isolation of mitochondria and biochemical assays Procedures used in this study have been described in detail previously and therefore, for the sake of economy, are only referred to here. Mitochondria were isolated from the thoracic flight muscles of male houseflies as described previously) Protein carbonyl content was measured by the method of Levine et al. ~5 using dinitrophenylhydrazine (DPNH) as described elsewhere. 6 The rate of H202 released by mitochondria was measured fluorometrically, according to Hyslop and Sklar, 16 by monitoring the oxidation ofp-hydroxyphenylacetate (PHPA), coupled to enzymatic reduction of H202 by horseradish peroxidase and using a-glycerophosphate as a substrate, as described previously) Submitochondrial particles (smps) were prepared as described previously3 and exposed to X-rays, using Phillips RT5 X-ray generator operating at 300 kV, 10 mA, and dose rate of 200 rads/min with a total dosage of 4 kr.

RESULTS

Effect of age on mitochondrial protein carbonyl content The mortality characteristics of housefly populations have been previously reported from this laboratory a number of times and are therefore only indicated here. 6'~3'14 The average life span of male flies, kept in 1-cubic-foot cages with 200 flies/cage (HA conditions) is about 20 days; however, the dying phase or period of high mortality starts around 15-16 days of age. To avoid cross-sectional sampling errors, age-related changes were limited to 16 days of age. As shown in Figure 1, the protein carbonyl content of isolated mitochondria increased progressively with age of the flies (one-way analysis of variance [ANOVA], p < 0.0001). The magnitude of increase between 5 to 16 days of age was about 55%. It can be calculated that the carbonyl content doubling time is around 24 days. Flies younger than 5 days were avoided because mitochondria undergo considerable maturational changes following emergence. 17 Data presented in Figure 1 are also compatible with interpretation that the age-related pattern of increase in carbonyl content may be sigmoid rather than linear.

Life expectancy and mitochondrial protein carbonyl content The mitochondrial carbonyl content was compared between flies kept under conditions of relatively high levels of physical activity (HA) with those kept under low levels of physical activity (LA), at 14 days of age. The average life span of flies under LA conditions is usually about 45 days, as compared to 20 days under

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Fig. 1. Comparison of protein carbonyl content of mitochondria isolated from the thoracic flightmusclesof adult male housefliesof differentages. Carbonylcontentwas measuredby the DNPHmethod of Levineet al.15Values are average +_SEM of 3-5 determinations.

Mitochondrial damage

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Fig. 2. Comparison of the protein carbonyl content in the mitochondria from 14-day old houseflies kept under conditions of HA and LA levels of physical activity. HA flies were housed within relatively large containers where they could fly, whereas LA flies were confined such that they were unable to undertake a flight. Life spans of flies are more than 2-fold longer under LA than HA conditions. Values are mean ± SEM of 4 determinations.

HA conditions.6'14 Thus, at 14 days of age, the HA flies had reached 0.7 of their average life span, whereas the LA flies had achieved only 0.31 of their average life span. The protein carbonyl content of mitochondria from the 14-day HA flies was about 52% higher (p = 0.0002) than in LA flies of the same age (Fig. 2). Mitochondrial protein carbonyl level was also compared between crawlers, which have an average life span of about 17 days, and fliers, which have an average life span of about 25 days, at 12 days of age. Thus, at this age of sampling, the crawlers had reached 0.70 and fliers 0.48 of their average life span. As shown in Figure 3, the carbonyl level of mitochondrial proteins was significantly higher in the short-lived crawlers than the longer-lived fliers of the same age (p = 0.04).

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Fig. 3, Comparison of protein carbonyl content in the mitoehondria of 12-day-old crawlers and fliers. Crawlers are the flies that have lost their flight ability, a characteristic of senescence, whereas fliers are their cohorts that still retained flying ability. Fliers have about 23-35% longer life expectancy than crawlers. Values are mean --+ SEM of 3 - 4 determinations.

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Fig. 4. Effect of hyperoxia on protein carbonyl content of mitochondria in the houseflies. Five-day-old flies, kept under HA conditions, were exposed to 100% oxygen and a sample of flies was removed each day for determination of protein carbonyls. The amount of carbonyls increased progressively in response to hyperoxia, and differences between the controls and the exposed group widened with the length of exposure. Values are mean ___SEM of 4 - 5 determinations.

Effect of hyperoxic exposure on mitochondrial protein carbonyl content and mitochondrial H202 release Five-day-old flies were placed either under 100% oxygen or in air. We have previously reported that such hyperoxic exposure induces rapid mortality in flies, usually on the fourth or fifth day of treatment.6 Protein carbonyl content of mitochondria, isolated from the flies after 1, 3, and 5 days of hyperoxic exposure, is presented in Figure 4. The protein carbonyl content was higher in the hyperoxia-exposed flies than the controls (p < 0.0001), and the percent difference between the exposed and the controls increased with the length of exposure. For example, the absolute differences between the experimental and the control through days 1, 3, and 5 of exposure were respectively 0.43, 0.57, and 0.85 nmoles/mg mitochondrial protein, indicating that hyperoxia accelerated the rate of accumulation of protein oxidative damage. To ascertain whether oxidative damage affects ROS production, the effects of hyperoxia on the rate of H202 release by mitochondria, isolated from flies exposed to hyperoxia, for 1, 3, and 5 days, was compared with the controls kept in air. H202 release in both the experimental and the control group was measured in the air. As shown in Figure 5, the rate of H202 release was greater in mitochondria obtained from hyperoxic flies than in controls, and the magnitude of the difference between the two groups increased progressively with the length of hyperoxic exposure. After 5 days of hyp-

624

R.S. SOHALand A. DUBEY

1.0

crease in the rate of H202 generation, smps, obtained from 10-day old flies, were exposed to 4 kr of Xirradiation. As shown in Figure 6, irradiated smps exhibited both an enhanced concentration of protein carbonyls (p -- 0.0006) as well as an increased rate of H202 generation (p = 0.0001) as compared to the unirradiated controls.

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Fig. 5. Comparison of the rate of H202 release by mitochondria isolated from the houseflies exposed to 100% oxygen and the controls. Flies were removed from hyperoxia after 5 days of exposure and placed in air (indicated by arrowhead). Controls were kept in air throughout. Mitochondrial H202 release was measured in air. The reaction mixture consisted of KCl-phosphate buffer, 3 mM MgC12, 0.1 mM EGTA, 166 #g PHPA'ml-~, 83 units horseradish peroxidase, about 20-30 #g mitochondrial protein, and 7 mM a-glycerophosphate. Values are mean +_ SEM of 4-6 determinations. eroxic exposure, flies were transferred to air. The differences in mitochondrial H202 generation between the hyperoxia-exposed and controls persisted when examined at 2 and 4 days, in the postexposure period, albeit the magnitude of the difference between the two groups declined.

Effect of X-irradiation on carbonyl content and H202 generation by stops To determine whether oxidative damage to the inner mitochondrial membrane alone could induce an in-

Results of this study indicate that the oxidative damage to mitochondria, as indicated by the protein carbonyl content, increases during aging and is correlated with the life expectancy o f the flies. Such oxidative damage appears to be linked in a positive feedback fashion to further increases in the rate of ROS production by the mitochondria. We have previously reported that the rate o f mitochondrial H202 release not only increases with age, 3 but is also correlated with life expectancy of the flies. 14 For example, rates of H202 release by mitochondria from the H A flies and from crawlers were found to be, respectively, higher than those from the L A flies and fliers o f comparable ages. Results o f this study indicate that the level of mitochondrial oxidative damage, as reflected by protein carbonyl content, parallels the rate o f mitochondrial H202 release, and is also inversely related to life expectancy of the flies albeit that the comparison between H A and L A flies was made at one age only. This inference is further corroborated by our previous finding that the concentration of thiobarbituric acid-reactive material in the flight muscle mitochondria increases with age and corresponds 1000

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Fig. 6. Effect of X-irradiation on the rate of H202 generation and the concentration of protein carbonyls in smps. Mitochondria from 5-day-old flies were used to obtain stops, which were suspended in a buffer consisting of 154 mM KC1/5 mM potassium phosphate/3 mM MgCl2 and 0.1 mM EDTA, and exposed to 4 kr of X-rays. Rate of H202 generation by smps was measured as described in Figure 5, with the exception that 167 units of SOD/ml was added to the reaction mixture to catalyze the conversion of 02 °- tO H202. Results for H202 and carbonyl measurements are mean -4- SEM of 4-6 and 4 determinations, respectively.

Mitoehondrial damage

to the life expectancy of the flies, using similar regimes to distinguish between chronological and physiological ages. ~8 The rate of mitochondrial H202 release has been reported by Turrens et al. 19'2° to be directly related to the ambient oxygen concentration. Consequently, it can be assumed that mitochondriai H202 generation during the period flies were exposed to 100% oxygen was relatively higher than the controls. This assumption is in accordance with the finding that the protein carbonyl content of mitochondria was higher in the hyperoxia-exposed flies than the controls. The view that mitochondrial oxidative damage may be responsible for the increase in H202 production, in a positive feedback fashion, is supported by the observation that the differences between the two groups, in both the rate of mitochondriai H202 release as well as mitochondrial carbonyl content, widened progressively and concurrently with the length of hyperoxic exposure. The nature of the mechanism linking the mitochondrial oxidative damage with elevated production of H202 by mitochondria has been obscure; however, two different hypotheses have been proposed. One hypothesis, advanced by us, 2"3"14implicates the involvement of oxidative damage to the inner mitochondrial membrane and intermolecular cross-linking. This hypothesis is based on the evidence that brief experimental exposures of isolated mitochondria to ROS-generating model systems such as Fe-ADP/ascorbate, t-butyl hydroperoxide, xanthine-xanthine oxidase, and AAPH were found to cause a notable increase in H202 release in mitochondria of the housefly.TM The present finding that X-irradiation of smps, which are mainly composed by the inner mitochondrial membranes, causes an enhancement of both H202 generation and carbonyl content, provides direct evidence that oxidative membrane damage can cause an increase in mitochondrial H202 production. The other hypothesis regards mitochondrial DNA damage to play a central role. 21 It is envisioned that components of the electron transport chain, whose synthesis is dependent on the mitochondrial genome, would be relatively more vulnerable to alterations than those dependent on nuclear genome because mitochondrial genome is more susceptible to oxidative damage due to its proximity to the sites of ROS generation. Mitochondrial DNA damage could cause an imbalance between the components of the mitochondrial respiratory chain encoded by the nuclear and the mitochondrial genomes, creating conditions that enhance the possibility of autoxidation of certain electron carders and the resultant formation of 02"-. An age-associated decline in cytochrome oxidase activity has been reported in mammals as well as the housefly,22'23 which

625

can be regarded as supportive of this hypothesis. However, a specific linkage between mitochondrial DNA damage and enhanced ROS generation by mitochondria has not as yet been established. Nonetheless, on the basis of present results it remains possible that both mechanisms, i.e., either direct oxidative membrane damage or mitochondrial DNA-mediated alterations in the respiratory chain, may be responsible for the ageassociated enhancement of mitochondrial H202 release. An important implication of the present study concerns the consequences of age-related oxidative damage on mitochondrial function, particularly the ability to synthesize ATP. Experimental oxidative damage has been shown to deleteriously affect respiratory efficiency of mitochondria.24 However, there is presently no consensus about whether mitochondrial loss of function is either a significant or a uniform feature of aging. 25 In conclusion, results of this study lead to the hypothesis that mitochondrial proteins undergo age-associated oxidative damage, most probably inflicted by the self-generated ROS, and such damage, in turn, can cause an elevation in the mitochondrial ROS generation. Such a positive feedback mechanism may underlie the widely observed age-associated increase in H202 release by mitochondria. It should however be pointed out that protein oxidative damage during aging is not strictly restricted to mitochondria, although, being primary producers of ROS, they may be particularly vulnerable to such damage. For example, protein carbonyl content in whole body homogenates of the houseflies was also found to be correlated with life expectancy of flies, as well as the ambient oxygen concentration.6 Acknowledgements - - We are indebted to Mr. Richard C. Kebart

and Dr. G. Mues, Mary C. Crowley Medical Research Program, Baylor Research Institute, Dallas, TX, for the use of the X-ray source.

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R.S. SOHALand A. DUBEY tive damage is associated with life expectancy. Proc. Natl. Acad. Sci. USA 90:7255-7259; 1993. Stadtman, E. R.; Oliver, C. N. Metal-catalyzed oxidation of proteins. J. Biol. Chem. 266:2005-2008; 1991. Sohal, R. S.; Brunk, U. T. Lipofuscin as an indicator of oxidative stress and aging. In: Porta, E. A., ed. Lipofuscin and ceroid pigments. New York: Plenum Press; 1989:17-26. Sagai, M.; Ishinose, T. Age-related changes in lipid peroxidation as measured by ethane, ethylene, butane and pentane in respired gases of rats. Life Sci. 27:731-738; 1980. Sohal, R. S.; Muller, A.; Koletzko, B.; Sies, H. Effect of age and ambient temperature on n-pentane production in adult housefly, Musca domestica. Mech. Ageing Dev. 29:317-326; 1985. Harman, D. The biological clock: The mitochondria? JAGS 20:145-147; 1972. Miquel, J.; Fleming, J. Atwo-step hypothesis of the mechanisms of in vitro cell aging: Cell differentiation followed by intrinsic mitochondrial mutagenesis. Exp. Geront. 19:31-36; 1984. Ragland, S. S.; Sohal, R. S. Mating behavior, physical activity and aging in the housefly, Musca domestica. Exp. Geront. 8:135-145; 1973. Sohal, R. S. Hydrogen peroxide production by mitochondria may be a biomarker of aging. Mech. Ageing Dev. 60:189-198; 1991. Levine, R. L.; Garland, D.; Oliver, C. N.; Amici, A.; Climent, I.; Lenz, A. G.; Ahn, B. W.; Shaltiel, S.; Stadtman, E. Determination of carbonyl content in oxidatively modified proteins. Meth. EnzymoL 186:464-478; 1990. Hyslop, P. A.; Sklar, L. A. A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: Its use in the simultaneous fluorimetric assay of cellular activation processes. Anal Biochem. 141:280-286; 1984. Sohal, R. S. Aging in insects. In: Kerkut, G. A.; Gilbert, L. I., eds. Comprehensive insect physiology, biochemistry and pharmacology. Vol. 10.; Oxford: Pergamon; 1985:595-631.

18. Farmer, K. J.; Sohal, R. S. Relationship between superoxide anion radical generation and aging in the housefly, Musca domestica. Free Radic. Biol. Med. 7:23-29; 1989. 19. Turrens, J. F.; Freeman, B. A.; Crapo, J. D. Hyperoxia increases HzOz release by lung mitochondria and microsomes. Arch. Biochem. Biophys. 217:411-421; 1982. 20. Turrens, J. F.; McCord, J. M. Mitochondrial generation of reactive oxygen species. In: Panlet, A. C.; Douste-Blazy, L.; Paoletti, R., eds. Free radicals, lipoproteins, and membrane lipids. New York: Plenum Press; 1990:203-212. 21. Bandy, B.; Davison, A. J. Mitochondrial mutations may increase oxidative stress: Implications for carcinogenesis and aging? Free Radic. Biol. Med. 8:523-539; 1990. 22. Benzi, G.; Pastoris, O.; Marzatico, R. F.; Villa, R. F.; Curti, D. The mitochondrial electron transfer alteration as a factor involved in the brain aging. Neurobiol. Aging 13:361-368; 1992. 23. Sohal, R. S. Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria. Free Radic. Biol. Med. 14:583-588; 1993. 24. Hillered, L.; Ernster, L. Respiratory activity of isolated rat brain mitochondria following in vitro exposure to oxygen radicals. J. Cereb. Blood Flow Metabol. 3:207-214; 1983. 25. Hansford, R. D. Bioenergetics in aging. Biochim. Biophys. Acta 726:41-80; 1983. ABBREVIATIONS

AAPH-- 2,2-azobis(2-aminopropane)dihydrochloride DNPH-- 2,4-dinitrophenylhydrazine HA--high levels of activity LA--low levels of activity PHPA--p-hydroxyphenylacetate R O S - - r e a c t i v e o x y g e n species SOD--superoxide dismutase