Mechanisms of Ageing and Development, 57 (1991) 187--202 Elseiver Scientific Publishers Ireland Ltd.

HYDROGEN PEROXIDE DURING AGING

RELEASE BY MITOCHONDRIA

187

INCREASES

R.S. SOHAL and BARBARA H. SOHAL Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275(U.S.A.)

(Received August 6th, 1990) SUMMARY

The effect of aging on the release of H202 by mitochondria was studied in the housefly in order to elucidate the causes of previously observed age-related increase in the level of oxidative stress. Intact flight muscle mitochondria of the housefly, supplemented with a-glycerophosphate, produce 1--2 nmol H202/min per mg protein, even in the absence of respiratory inhibitors. The rate of H202 secretion progressively increases approximately 2-fold during aging of the fly. Neither uncoupling of oxidative phosphorylation nor mechanical damage to mitochondria during the isolation procedure appear to be responsible for the age-related increase in H202 production. Activities of NADH-ferricyanide reductase, succinate-ubiquinone reductase, and NADH-, succinate- and a-glycerophosphate-cytochrome c reductases, were approximately 2-fold higher in mitochondria from the old than those from the young flies. However, the concentration of enzymatically reducible ubiquinone remained unchanged with age. Infliction of damage by exposure of mitochondria to free radical-generating systems in vitro caused an increase in the rate of H202 generation. Glutaraldehyde, an intermolecular crosslinking agent, induced an increase in the rate of H202 generation by mitochondria. Results of this study demonstrate that aging in the housefly is associated with an increase in the rate of H202 generation by mitochondria probably due, at least in part, to self-inflicted damage by mitochondria. Intermolecular cross-linking in the inner mitochondrial membrane can contribute towards the increased H202 generation.

Key words: Oxygen free radicals; Aging; Oxidative stress; Mitochondrial damage Correspondence to: Prof. R.S. Sohal, Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, U.S.A. Abbreviations: AAPH, 2,2-azobis (2-aminopropane) dihydrochloride; buffer A, 154 mM KCI/I.0 mM EDTA; buffer B, 154 mM KCI/5 mM potassium phosphate/3 mM MgCi2/0.1 mM EDTA; CCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; GSH, reduced glutathione; GSSG, oxidized glutathione; O2-, superoxide anion radical; PHPA, p-hydroxyphenylacetate; SOD, superoxide dismutase; TBA, thiobarbituric acid.

0047-6374/91/$03.50 Printed and Published in Ireland

© 1991 Elsevier Scientific Publishers Ireland Ltd.

188 INTRODUCTION A current hypothesis of aging postulates that deleterious reactions initiated by partially reduced oxygen species are an underlying factor in the causation of the aging process in multicellular organisms [1,2]. The initial molecular species in the one electron reduction of dioxygen is the superoxide anion radical (02-), which is converted into H202 either by spontaneous disproportionation or enzymically by superoxide dismutase activity [3]. H202 is thus the first stable product of the univalent pathway of oxygen reduction. The main site of 02- and H202 production in eukaryotic cells is the electron transport chain in mitochondria [4,5]. Previous studies in this laboratory have suggested that the level of oxidative stress, i.e., the balance between pro-oxidants and antioxidants, shifts towards a relatively more pro-oxidizing status during aging [2]. Steady state concentrations of reaction products of partially reduced oxygen species such as inorganic peroxides, oxidized glutathione (GSSG), thiobarbituric acid (TBA)-reactive material and lipidsoluble Schiff's base-like fluorescent material (presumably consisting of lipofuscinderived fluorophores) were found to increase during aging in the adult housefly [6,7]. The ratios of redox-sensitive indicators such as G S H / G S S G , N A D H / N A D ÷ and N A D P H / N A D P ÷ in the whole body homogenates of flies decreased or became more pro-oxidizing with age [8]. Furthermore, the rate of n-pentane exhalation (a product of lipid peroxidation) by flies significantly increased with age suggesting that the net rate of lipid peroxidation in vivo accelerates during aging [9]. The main objective of the present study was to understand the underlying causes of the age-related increase in the level of oxidative stress in the housefly. The hypothesis that a corresponding age-related increase in the production of oxidants, specifically H202, by mitochondria was one of the factors responsible for the increase in the level of oxidative stress was tested in flight muscle mitochondria of the adult houseflies. Flight muscles of the housefly provide an excellent model for studies on the aging process; they exhibit a clear-cut and universal loss of function during aging [10,11]. Flight performance, as measured by duration, distance flown and speed of flying, decreases during aging to the extent that the very ability to fly is completely lost during the terminal phase of life [12]. The present study examines the effect of age on the generation of H202 by mitochondria and also explores the possible causes of the observed increase in the rate of H202 production.

MATERIALSAND METHODS

Materials Fly larval medium was obtained from Ralston Purina Company, Richmond, IND. Superoxide dismutase, catalase, horseradish peroxidase, cytochrome c (Type VI), antimycin A, rotenone, N A D H , dichloroindolphosphate and H202 were pur-

189

chased from Sigma Chemical Co., St. Louis, MO. AAPH was obtained from Polysciences, Warrington, PA. Glutaraldehyde was purchased from Fullam Inc., Latham, NY. All the chemicals were of analytical grade.

Animals Studies were conducted on adult male houseflies, Musca domestica, belonging to a stock originally obtained from the Department of Zoology, University of Cambridge (U.K.). Eggs were obtained from 7- to 10-day-old flies and placed in moist CSMA (Chemical Specialties Manufacturers Association) fly larval medium. After emergence from pupae, adult flies were segregated by sex and housed in one-cubicfoot cages, with 200 male flies per cage, at 25°C, 45°70 relative humidity and 24 h light. Flies were fed on sucrose and water. Male houseflies have been shown to live longer on such a diet than on a more complex protein- containing diet [13]. Average life span of the male flies was about 20 days. All biochemical measurements were made at 30°C.

Isolation of mitochondria Mitochondria were isolated from the thoracic flight muscles of flies of different ages by a modification of the procedure described by Wood and Nordin [141. Thoraces were severed from the head and the abdomen of flies that had been immobilized by chilling atop a glass plate resting on a bed of ice. After a desired number of thoraces were removed (usually 50---200), they were placed in a chilled mortar con taining buffer A (0.1 ml/thorax) composed of 154 mM KCI, 1.0 mM EDTA (pH 7.0) and gently pounded, avoiding grinding action. The resulting mash was filtered by suction through eight layers of cheesecloth. The filtrate was centrifuged at 125 x g for 3 min and the pellet was discarded. The supernatant was centrifuged at 3000 x g for 8 min. The pellet was resuspended in the specified buffer. Light microscopy, using a phase contrast microscope and electron microscopical examination indicated that this pellet consisted of mitochondria without any/or rare nuclei or myofibrils. Each fly yielded approximately 60 t~g of mitochondrial protein.

Preparation of submitochondrial particles Mitochondria were suspended in 30 mM potassium phosphate buffer (pH 7.0) and sonicated at 10 W, using a Branson Sonifier (Model W185), four times for 30 s each at 1-min intervals in ice-cold water. The sonicated mitochondria were centrifuged at 8250 x g to sediment unfragmented organelles and the supernatant was centrifuged at 80 000 × g for 40 min to sediment submitochondrial particles, which were resuspended, unless specified otherwise, in buffer B consisting of 154 mM KC1, 5 mM potassium phosphate, 3 mM MgCI2 and 0.1 mM EDTA (pH 7.4).

Measurement of H20 ~production by mitochondria The rate of H202 generation was measured in intact mitochondria fluorometri-

190 cally by the method of Hyslop and Sklar [15] which is based on the oxidation of p-hydroxyphenylacetate ( P H P A ) during the enzymatic reduction of H202 by horseradish peroxidase. The reaction mixture consisted of buffer B (pH 7.4), mitochondria (20/zg protein), 166/~g/ml P H P A , 83 units of horseradish peroxidase/ml, and 7 mM ot-glycerophosphate. Intact insect mitochondria are quite impermeable to succinate, however, sonicated mitochondria can readily utilize this substrate [ 16]. The rate of H202 generation was monitored by following the increase in fluorescence at an excitation maximum of 317 nm and an emission maximum of 400 nm using Perkin Elmer LS-5 spectrofluorometer. Known concentrations of H202 were used to establish the standard concentration curve.

Activity of mitochondrial respiratory complexes Activity of NADH-ferricyanide reductase was measured by the adaptation of the method described by Galante and Hatefi [17]. The reaction mixture consisted of 40 mM Tris--HC1 (pH 7.5), 1.6 mM K3Fe(CN) 6, 0.15 mM N A D H and sonicated mitochondria (20--160/~g protein/ml). The rate o f the reaction was monitored by the decrease in absorbance due to reduction o f ferricyanide at 410 nm using a Beckman DU 70 spectrophotometer. The activity was calculated using an extinction coefficient o f 1 . 0 / m M per cm. NADH-cytochrome c reductase activity was measured by a modification of the method of Hatefi and Stiggal [18]. The reaction mixture consisted of 156 mM phosphate buffer (pH 7.2), 0.4 mM KCN, 0.08 mM cytochrome e ÷, 0.4 mM N A D H and sonicated mitochondria (25--140/ag protein/ml). The rate o f the reaction was monitored by following an increase in absorbance at 550 nm (E550 = 29.5 mM-~ cm-l). Similar procedures were followed for the measurement of succinate - - and a-glycerophosphate-cytochrome c reductase except that N A D H was replaced with 7 mM succinate and a-glycerophosphate, respectively. Activity of succinate-ubiquinone reductase was measured by following the reduction of 2, 6-dichloroindolphosphate at 600 nm [19]. The reaction mixture consisted of 50 mM phosphate buffer (pH 7.4), 0.1 mM EDTA, 0.05 mM DCIP, 2 mM 2-thenoyltrifluoroacetone, 7 mM succinate and sonicated mitochondria (2--8/~g protein/ml). The specific activity o f the enzyme was calculated using an extinction coefficient of 21 mM -~ cm-'. Enzymatically-reducible ubiquinone was measured directly at E275--E300 (AE = 12 mM -I cm-~). The reaction mixture consisted of submitochondrial particles (60-160/~g protein), 0.2/zM antimycin A, 7 mM succinate and 230 mM-mannitol/70 mM-sucrose/30 mM Tris buffer (pH 7.4) [20]. RESULTS

Quantitation of H202 and verification of the method The assay for H202 determination is based on the increase in the fluorescence of the non-fluorescent substrate p-hydroxyphenylacetate (PHPA), which is enzymatically oxidized to a stable fluorescent form (PHPA) 2 (Eq. 1) [15].

191

(1)

H202 + 2 ( P H P A ) ~ 2 H 2 0 + (PHPA)2

The relationship between (PHPA) 2 fluorescence and known concentrations of H202 was linear from 0 to 166nM. Figure 1 demonstrates that the addition of a-glycerophosphate to the mitochondrial preparation stimulated a steady increase in the level of (PHPA) 2 fluorescence. The rate of increase in the fluorescence intensity was depressed sequentially by each addition of 83 units of catalase/ml. It was therefore concluded that the employed method provided a sensitive and reliable measurement of the rate of H202 release.

Mitochondrial production of H202 It has been previously shown that there are two main sites in the mammalian mitochondrial respiratory chain for 02- production by autoxidation, namely the NADH dehydrogenase and ubiquinone-cytochrome b region [21,22]. Among the NADH-linked substrates proline plus pyruvate have been demonstrated to promote the highest rate of in vitro mitochondrial respiration in the dipteran insects [23]. Intact fly mitochondria are relatively impermeable to succinate but are highly efficient in the utilization of a-glycerophosphate, which also delivers electrons at the flavin site [24]. In order to identify the main site of oxidant production in the housefly mitochondria, rates of H202 generation were compared following proline plus pyruvate and a-glycerophosphate supplementation (Table I). Proline plus pyruvate were found to support only a relatively low rate of H202 generation, however, the addition of rotenone (an inhibitor of NADH dehydrogenase activity) increased the

O~

¢q

c

o

t'Xl :I:

ta

O

t-

O

~

.--

t--

0~ _

0

t~

--

D "~

oO

/

0 v

I

I

I

I

I

I

0

2

4

6

8

10

Time (rain) Fig. 1. Verification of H202 secretion by mitochondria. Recording of fluorescence exhibited by a mitochondrial preparation initially consisting of buffer B (pH 7.4), 166/~g/mlPHPA, 83 units of horseradish peroxidase/ml and mitochondria (20 ~g protein). When indicated, 7 mM a-glycerophosphate and 83 units of catalasv/ml were added.

192 TABLE I COMPARISON OF THE RATES OF H202 RELEASE BY FLIGHT MUSCLE MITOCHONDRIA ISOLATED FROM 9-DAY-OLD FLIES IN RESPONSE TO DIFFERENT SUBSTRATES AND RESPIRATORY INHIBITORS

Substrate/inhibitor

Rate of 1-1202 release (nmol/min per mg protein)

1. 2.

0.07 _+ 0.0009 0.13 _+ 0.016

3. 4. 5. 6.

5 mM Proline + 1 mM pyruvate 5 mM Proline + 1 mM pyruvate + 1.5 tam rotenone 7 mM a-glycerophosphate 7 mM tr-glycerophosphate + 1.5/~M rotenone 7 mM a-glycerophosphate + 2 taM antimycin A 7 mM a-glycerophosphate + 1.5 tam rotenone + 2taM antimycin A

1.14 _+ 0.06 1.45 _+ 0.18 2.27 _+ 0.12 2.57 _ 0.03

The reaction mixture consisted of buffer B (pH 7.4), 166 tag/ml PHPA, 83 units horseradish peroxidase/ ml, mitochondreia (25 tag protein) and indicated amounts of substrates. Values represent mean ± S.D. of 3--5 measurements.

rate of H202generation by 85070. Relative to proline plus pyruvate, the rate of H202 generation by mitochondria was about 16-fold greater following supplementation with a-glycerophosphate. Addition of rotenone to a-glycerophosphate-supplemented mitochondria, to prevent the reverse electron flow, had only a small effect on the rate of H 2 0 2 production; however addition of antimycin A, which inhibits electron flow in the ubiquinone-cytochrome b region almost doubled the rate of H20 2 release. It therefore seems that the main site of H 2 0 2 generation in the housefly mitochondria lies between the rotenone-sensitive and antimycin A-sensitive region of the electron transport chain. Neither pyruvate, proline, or- glycerophosphate, rotenone nor antimycin A had any direct effect on (PHPA) 2 fluorescence suggesting that these compounds are not involved in the oxidation of PHPA.

Effect of age on the mitochondrial H202 release Because housefly mitochondria release relatively copious amounts of H202, when supplemented with ot-glycerophosphate, even in the absence of respiratory inhibitors and because the rates of H 2 0 2 generation in the absence of respiratory inhibitors would be a better approximation of the physiological condition, it was decided to study the effect of aging o n H 2 0 2 generation using a-glycerophosphate alone without antimycin A. The rate of H 2 0 2 production was measured in mitochondria obtained from flies of different ages ranging from 4 to 16 days of age. The lower age limit was selected becatise flight muscle mitochondria exhibit biochemical maturation changes during the period immediately following emergence of the adult

193

[25,26]. Regarding the upper age limit, it has been previously emphasized by us that cross-sectional sampling of aging populations should be restricted to a period prior to the onset of dying phase [27]. In any aging population the survivors progressively represent subsets of the population undergoing relatively slower rates of aging. Sixteen--eighteen days of age approximated the beginning of the dying phase in the housefly populations. The rate of mitochondrial H202 production was found to increase progressively with age of the flies (r = 0.94) with a > 2-fold difference between the young and the old flies (Fig. 2).

Effect of mitochondrial uncoupling on H202production To investigate if age-related uncoupling of oxidative phosphorylation or mitochondrial damage during isolation might be responsible for the observed increase in H202 production, two lines of investigation were pursued. First, the effects of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (CCP), a potent uncoupler of oxidative phosphorylation on H202 production were determined. Exposure of mitochondria to 0.16/~M, 8.3/~M and 50/~M CCP was found to reduce H202 production by 12070, 30070 and 58070, respectively. Secondly, as described below, rates of H202 production were determined in submitochondrial particles, prepared by sonication of the intact isolated organelles.

H2Ozproduction by submitochondrialparticles Age-related alterations in mitochondrial permeability to substrates, as well as intramitochondrial SOD activity may potentially alter the rate of H20 2 secretion by

i

C

4

12

16

Age (days) Fig. 2. Rates of H202 generation by mitochondria from flies of different ages. The reaction mixtures consisted of buffer B (pH 7.4), 166/~g/ml PHPA, 83 units/ml horseradish peroxidase, 7 mM a-glycerophosphate and 20 /ag of mitochondrial protein. Each value represents the mean _+ S.E.M. of 3--5 measurements.

194 m i t o c h o n d r i a . T o a v o i d such a c o m p l i c a t i o n the rates o f H 2 0 2 g e n e r a t e d by submit o c h o n d r i a l particles were c o m p a r e d b e t w e e n y o u n g a n d old flies. T h e rate o f a-glyc e r o p h o s p h a t e - s u p p o r t e d HzO 2 g e n e r a t i o n b y s u b m i t o c h o n d r i a l particles was f o u n d t o be 70°7o greater in 18-day o l d flies t h a n in 5 - d a y - o l d flies.

Effect o f age on mitochondrial respiratory components A g e - r e l a t e d increase in H 2 0 2 g e n e r a t i o n b y s u b m i t o c h o n d r i a l particles is indicative o f a l t e r a t i o n s in the e l e c t r o n t r a n s p o r t chain. A n a g e - r e l a t e d increase in the rate o f State 4 r e s p i r a t i o n in flight muscle m i t o c h o n d r i a o f the h o u s e f l y , p r e v i o u s l y r e p o r t e d b y us, also p o i n t s t o w a r d s a l t e r a t i o n s in the r e s p i r a t o r y chain d u r i n g aging [28]. T o investigate this possibility, activities o f N A D H - f e r r i c y a n i d e reductase, succ i n a t e - u b i q u i n o n e r e d u c t a s e a n d N A D H - c y t o c h r o m e c r e d u c t a s e a n d succinate- a n d a - g l y c e r o p h o s p h a t e - c y t o c h r o m e c r e d u c t a s e s were c o m p a r e d in m i t o c h o n d r i a b e t w e e n 5 - d a y a n d 18-day-old flies. E n z y m a t i c activities o f all o f these complexes were n o t a b l y higher in the o l d e r t h a n in the y o u n g e r flies (Table II). Since the changes in the relative c o n t e n t o f r e s p i r a t o r y c o m p o n e n t s can also affect the rate o f electron flow as well as the r e d o x state o f the carriers [ 2 0 ] , which in t u r n could influence the rate o f 0 2 a n d H 2 0 2 p r o d u c t i o n , it was d e e m e d desirable to c o m p a r e the a m o u n t o f e n z y m a t i c a l l y r e d u c i b l e - u b i q u i n o n e in the y o u n g a n d old

TABLE II AGE-RELATED DIFFERENCES IN MITOCHONDRIAL RESPIRATORY COMPONENTS IN THE HOUSEFLY

Component

Age of flies 5-day

a. b. c. d.

e. f.

NADH-ferricyanide 0.27 ± 0.03 reductase ~mol/min per mg protein) NADH-cytochrome c reductase (nmol/min per mg protein) 63.00 ± 3 + 1.5/aM rotenone 11.00 ± 0.8 Succinate-ubiquinone reductase 89.00 _ 17 (9) (~mol/min per mg protein) Succinate-cytochrome c reductase (nmol/min per mg protein) 38.00 ± 3 + 2/aM antimycin A undetectable a-glycerophosphate-cytochromec reductase (nmol/min per mg protein) 30.00 ± 2 + 2/aM antimycin A 2.40 Reducibleubiquinone 23.10 ± 5.9 (nmol/mg protein)

18-day (6)

0.69 ± 0.07

(4)

(3) (4) (9)

110.00 ± 11 8.70 ± 2,4 177.00 ± 8

(4) (4) (4)

(3) (3)

66.00 ± 4 2.80 ± 0.9

(4) (3)

(4) (1) (9)

67.00 ± 2 2.70 23.70 ± 1.6

(4) (2) (19)

Measurements were made in sonicated mitochondria. Values represent mean ± S.D. Values in parenthesis indicate the number o f determinations.

195 flies. As also shown in Table II, the amount of NADH-reducible ubiquinone is quite similar in young and old flies.

Effect of experimental oxidative stress on mitochondrial H202 generation Previous studies in this laboratory have indicated that the flight muscle mitochondria in the housefly exhibit an age-dependent increase in oxidative damage as indicated by the concentration of TBA-reactive material [28]. H202,generated in mitochondria, would be expected to also expose mitochondria itself to potential oxidative damage. To investigate the possibility that oxidative damage could itself be responsible for the increase in H202production, the effects of experimental exposure to oxidative stress on the rate of mitochondrial H202production were examined Mitochondria were exposed to the following free radical-generating systems: (i) aqueous decomposition of the azo compound 2,2-azobis (2-aminopropane) dihydrochloride (AAPH) [29], (ii) tert-butyl hydroperoxide, and Off) ADP-Fe/ascorbate. Isolated mitochondria were incubated in these free radical generating systems for specified periods and washed twice prior to the determination of the rate of H202 generation. The rate of H20 2 production by mitochondria, exposed to AAPH for 10 min, was found to increase linearly with concentration of AAPH (Fig. 3a). Treatment with 75 mM AAPH caused a 55070 increase in H202generation. The rate of H202release by mitochondria incubated with 0.5 mM tert- butyl hydroperoxide for different durations increased about 20°70 after 2.5 min with no further increase observed up to 20 rain of exposure (Fig. 3b). Incubation of mitochondria in ADP-Fe/ascorbate for different lengths of time resulted in an increase in the rate of H202production by about 155070 after 10 min exposure and remaining at the same level after 20 min exposure (Fig. 3b). These results demonstrate that exposure of mitochondria to oxidative stress can induce an increase in H202generation by these organelles.

Effect of glutaraldehyde on H202 production To further investigate the possible mechanism underlying the increase in mitochondrial H202 production during aging and in response to experimental oxidative stress, it was hypothesized that the structural organization of the mitochondrial respiratory components may be altered due to molecular cross-linking reactions, as reported in the red blood cell membranes exposed to oxidants [29,30]. To test this hypothesis, intact mitochondriafrom relatively young and old flies were exposed to various concentrations of glutaraldehyde. As expected, unexposed control mitochondria from the older flies generated H202 at a higher rate than those from the younger flies (Fig. 4). However, exposure to glutaraldehyde caused a greater increase in the mitochondria from the young than those from the old flies. The maximal rate of H20 z production in response to glutaraldehyde was comparable in the two ages.

2.5 c

2

2.0

E

1.5

e-

tN o z 0

E

1.0 0.5

c

0.0 1

10

75

30

AAPH (mM)

200

b

t-, O

~8

150

1~ 100 Z .c_ ~

50

0 0

i

i

i

I

5

10

15

20

Duration

of Exposure

25

(min)

Fig. 3. H202 secretion by mitochondria pretreated with different free radical-generating systems prior to the measurement of the rates of H20~release. (a) Mitochondria (1 mg protein/ml) were exposed to different concentrations of AAPH for 20 min in buffer B, centrifuged and washed twice in buffer B. Rates of H202 secretion were measured under conditions similar to those described in Fig. 2. (b) Mitochondria (1 mg protein/ml) were exposed to 0.5/aM-butyl hydroperoxide in buffer B ( ~ - - , ) , or in buffer B (minus EDTA) containing 2 mM ADP, 16/aM FeC13and 0.5 mM ascorbate (Fl--lzl) for varying periods, centrifuged and washed twice in buffer prior to the measurement of H20~production.

A d d i t i o n of g l u t a r a l d e h y d e to the reaction mixture, c o n t a i n i n g all the ingredients except m i t o c h o n d r i a , did n o t alter the intensity o f fluorescence, i n d i c a t i n g that glut a r a l d e h y d e does n o t oxidize P H P A . Similarly, a d d i t i o n o f t h e r m a l l y - d e n a t u r e d m i t o c h o n d r i a to the g l u t a r a l d e h y d e a n d P H P A - c o n t a i n i n g reaction m i x t u r e did n o t alter the level o f fluorescence, thus ruling o u t the possibility that g l u t a r a l d e h y d e m a y be directly involved in P H P A o x i d a t i o n .

19"/

e-

3.0

.= o

o~

g

.E

2.0

0(N 1.0 m

E e-

•

0.0 0.0

3-days 13-days

I

l

I

I

0.1

02.

0.3

0.4

0.5

Glutaraldehyde (mM) Fig. 4. Effect of glutaraldehyde on H202 secretion by mitochondria from relatively young (3-day-old) and old (13-day-old) flies. The basic assay conditions were similar to those indicated in Fig. 2. Glutaradehyde was directly added to the cuvette after the initial rates of H202 secretion had been established. Valuesxepresent means ± S.D. of 4 determinations.

Effect of glutaraldehyde on 1-1202production by submitochondrial particles To determine if the observed increase in H202 production by mitochondria in response to glutaraldehyde exposure was due to intrinsic alteration in the inner mitochondrial membrane and not the inactivation of matrix proteins, the effect of glu8

150

Q.

04 100 0 04

=6 50 E

8

z~

17-days

5-days AGE

Fig. 5. Effect of glutaraldehyde on H202 production by submitochondrial particles from young and old flies. The reaction mixture consisted of submitochondrial particles (23~g protein), buffer B (pH 7.4), 166 /ag/ml PHPA, 83 units of horseradish peroxidase/ml and 7 mM a-glycerophosphate. Glutaraldehyde (0.04 raM) was added directly to the cuvette after which the rate of H202 release was monitored for about 3 min. The values represent average ± S.D. of 4--7 determinations, ( I , control; m, glutaraldehydeexposed).

198 taraldehyde on H202 production by submitochondrial particles, which primarily consist of inner membrane vesicles lacking matrix, was investigated. Exposure of submitochondrial particles to 0.04 mM glutaraldehyde was found to increase the rate of H202 production by 30.4°70 in 5-day-old flies and only 12.5070 in 17-day-old flies (Fig. 5). This suggests that putative molecular cross-linking may be a factor in increased H20 2 production. Furthermore, submitochondrial particles from younger flies, which presumably have undergone relatively lesser age-related cross-linking, are more sensitive to glutaraldehyde than those from older flies. DISCUSSION The main site of 0 2- and H202 production in the flight muscle mitochondria seems to lie between the rotenone-and antimycin A-sensitive region of the mitochondrial respiratory chain. Using a variety of inhibitors and substrates, Turrens et al. [22] have identified ubisemiquinone to be the major site of 02-, and consequently of H2OE-generation in mammalian mitochondria. Insect mitochondria thus exhibit an apparent similarity to mammalian mitochondria in the site o f oxidant production. However, the rate of H202generation by insect mitochondria is several times greater than in mammalian mitochondria [31]. The observed age-related increase in mitochondrial H202production is not due to uncoupling of oxidative phosphorylation or mechanical damage to mitochondria or a variation in the amount of enzymatically-reducible ubiquinone. We have previously reported that the amount of total ubiquinone and of cytochrome c in the flight muscle mitochondria does not vary with age [28]. Such data suggest that the age-related increase in H202production may not be due to changes in the molar ratios of these mitochondrial respiratory components. On the basis of the results of this investigation, the underlying cause of the agedependent increase in H202production would appear to be related to structural alterations in the organization of the respiratory chain. The activities of mitochondrial NADH-ferricyanide reductase, succinate-ubiquinone reductase, succinatecytochrome c reductase, a-glycerophosphate-cytochrome c reductase and NADHcytochrome c reductase were all found to increase with age of the flies. The rate of state 4 respiration of flight muscle mitochondria in the fly was previously reported by us to increase during aging [28]. Together, such alterations suggest that the rate of electron flow in the mitochondrial respiratory chain of the fly may be speeded up during aging. Further evidence fostering the view that the observed age-related increase in the rate of H202production may be due to structural alterations in the inner mitochondrial membrane is provided by the fact that increased H202production is not only exhibited by intact mitochondria but also by the submitochondrial particles, which are primarily composed of inner mitochondrial membrane. Furthermore, the rate of 0 2- generation, a stoichiometric precursor of mitochondrial H202 [32], by submitochondrial particles, also increases during aging of the fly, as

199

reported previously from this laboratory [28], which strengthens the inference that the structural organization of Complex III within the inner mitochondrial membrane in the flight muscles of the fly is altered in a manner that enhances the autoxidation of a component of the respiratory chain, widely believed to be the ubisemiquinone. On the basis of the results of this study it can be hypothesized that age-related structural alterations in the inner mitochondrial membrane may be due to oxidative damage. Two different lines of evidence support this hypothesis. Firstly, as demonstrated in this study, experimental exposure of mitochondria in vitro to free radicalgenerating systems induces an increase in the rate of H202generation. Secondly, the flight muscle mitochondria exhibit age-related oxidative damage during the normal course of aging [28]; furthermore, the overall level of oxidative stress increases during aging of the housefly. For example, the concentration of lipid peroxidation products, as indicated by the concentration of TBA-reactive materials, increases during aging in the flight muscle mitochondria [28]. The rate of n-pentane exhalation by the flies, which is a sensitive indicator of lipid peroxidation in vivo [33], also increases during aging [9]. Furthermore, the relative concentration of esterified polyunsaturated fatty acids, associated with the cellular membranes, decreases during aging in houseflies [34]. Other indications of an increase in the level of oxidative stress during aging in the housefly include an elevation in the concentration of inorganic peroxides, oxidized glutathione and Schiff's base-like fluorescent products in the aging flies [34,35]. It is not unreasonable to expect that the 02- and H202 generated in mitochondria may also cause some damage to the mitochondrial membranes, which, in turn, would further increase the rate of 02- and H202 production. Such a mechanism could be responsible for the age-related increase in the rate of H202 production. 02and H202-generating systems have been shown in vitro to induce peroxidation of polyunsaturated fatty acids [36], damage the secondary and tertiary structure of proteins [37], and cross-linking of proteins and of lipids and proteins [30,31,38] in cellular membranes. Indeed, it has been shown by Tappel [38] that malonaldehyde, a product of lipid peroxidation, can form conjugated imine linkages between amino groups to form products of higher molecular weight. The hypothesis that cross-linking reactions in the inner mitochondrial membrane may be responsible for the agerelated increase in the rate of mitochondrial H202 production is supported by two different lines of evidence. Firstly, in the present study, the exposure of submitochondrial particles to glutaraldehyde, a cross-linking agent, was found to increase the rate of H202 production. This would be expected if cross-linking reactions were in fact involved in vivo in the increase of oxidant production by mitochondria. Secondly, as also pointed out above, in vitro studies have clearly shown that 02 - and H202-generating systems can cause intermolecular cross-linking of proteins and lipids in cellular membranes [29,30]. Such intermolecular cross-linking in the components of the electron transport chain can result in the speeding up of the electron

200 movement which may explain the observed age-related increases in the activity of respiratory complexes, State 4 respiration and 0 2- generation [28]. Age-related increase in mitochondrial H20 2 production m a y also occur in m a m mals. Nohl and Hegner [39] have reported a 34% higher rate of H202production by heart mitochondria from 23-month-old rats as compared to those from 3-month-old rats. However, in contrast to the present study, these authors measured H20 2 production only in the presence of the respiratory inhibitor antimycin A. Whether the reported increase also occurs in the absence of antimycin A or under physiological conditions is unknown. Notwithstanding, the level of in vivo oxidative stress does seem to increase during aging in m a m m a l s as indicated by an age-related increase in the exhalation of alkanes in rats [40]. Finally, the results of this study emphasize the potential role of mitochondria in the aging process. The cause of the age-related increase in the level of oxidative stress in the housefly can now, at least, be partially assigned to the progressive increase in the rate of H202production by mitochondria. If oxygen free radicals play a causal factor in the aging process, as is widely postulated, then mitochondria are not only the initial generators of oxy-radicals and hydroperoxides but also their target of damage, which, in turn, may further exacerbate the generation of oxy-radicals. Further evidence supporting the possible role of mitochondria in the aging process is provided by the comparative studies on the rates of mitochondrial O2- and H20 2 generation in different m a m m a l i a n species. The rates of both 0 2- and H202, produced by liver mitochondria, were found to be inversely correlated with the maxi m u m life potential of the species (r = - 0.92) [31,41]. The propensity of mitochondria, from the same cell type, to generate 0 2- and H202 at differential rates in different species suggests the existence of, as yet, unknown differences in the organization o f the inner mitochondrial m e m b r a n e a m o n g different species. ACKNOWLEDGMENTS This research was supported by the National Institutes of Health-National Institute on Aging grant RO1 AG7657. REFERENCES 1 D. Harman, Aging: A theory based on free radical and radiation chemistry, aT.Gerontol., 11 (1956) 298--300. 2 R.S. Sohal and R.G. Allen, Relationship between oxygenmetabolism, aging and development.Adv. Free Rad. Biol. Med., 2 (1986) 117--160. 3 I. Fridovich, The biology of oxygenradicals. Science, 201 (1978) 875--880. 4 G. Loschen, L. Flohe and B. Chance, Respiratory chain linked H202 production in pigeon heart mitochondria. FEBSLett,., 18 (1971) 261--264. 5 B. Chance, H. Sies and A. Boveris, Hydroperoxide metabolism in mammalian organs. Physiol. Rev., 59 (1979) 527--603.

201 6 7 8 9 10 11 12 13 14 15

16 17 18 19 20 21 22

23 24 25 26 27 28 29

30 31

R.S. Sohal, H. Donato and E.R. Biehl, Effect of age and metabolic rate on lipid peroxidation in the housefly, Musca domestica. Mech. Ageing Dev., 16 (1981) 159--167. R.S. Sohal and B. Donato, Effects of experimentally altered life spans on the accumulation of fluorescent age pigments in the housefly, Musca domestica. Exp. Gerontol., 13 (1978) 335--341. R.S. Sohal, P.L. Toy and K.J. Farmer, Age-related changes in the redox status of the housefly, Musca domestica. Arch. Geront. Geriatr., 6 (1987) 95--100. R.S. Sohal, A. Mfiller, B. Kolc~zko and H. Sies, Effect of age and ambient temperature on n-pentane production in adult housefly, Musca domestica. Mech. Ageing Dev., 29 (1985) 317--326. M. Rockstein and P.L. Bhatnagar, Duration and frequency of wing beat in the aging housefly, Musca domestica. L. Biol. Bull., 131 (1966) 479--486. S.S. Ragland and R.S.Sohal, Mating behavior, physical activity and aging in the housefly, Musca domestica. Exp. Gerontol., 8 (1973) 135--145. R.S. Sohal and J.H. Runnels, Effect of experimentally prolonged life span on flight performance of houseflies. Exp. Geront., 21 (1986) 509--514. S.S. Ragland, Ph.D. Dissertation, Southern Methodist University, Dallas (1984). F.E. Wood and J.H. Nordin, Temperature effects on mitochondrial respiration of Protophormia terranovae and Musca domestica. Insect Biochem., 10 (1980) 95--99. P.A. Hyslop and L.A. Sklar, A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: Its use in the simultaneous fluorimetric processes. Anal. Biochem., 141 (1984) 280--286. S.G. Van den Bergh and E.C. Slater, The respiratory activity and permeability of housefly sarcosomes. J. Biochem., 82 (1962) 362--371. Y.M. Galante and Y. Hatefi, Resolution of complex I and isolation of NADH dehydrogenase and an iron-sulfur protein. MethodsEnzymol., 53 (1978) 15--21. Y. Hatefi and S. Stiggall, Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Methods Enzymol., 53 (1978) 21--27. Y. Hatefi and S. Stiggall, Preparation and properties of NADH: cytochrome c oxidoreductase. Methods Enzymol., 53, (1978) 5--10. A.E. Boveris, E. Cadenas and A.O.M. Stoppani, Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem. J., 156 (1976) 435--444. A. Boveris, Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods Enzymol., 105, (1984)429--435. J.F. Turrens, A. Alexandre and A.L. Lehninger, Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch. Biochem. Biophys., 237, (1985) 408-414. B. Sacktor and C.C. Childress, Metabolism of proline in insect flight muscle and its significance in stimulating the oxidation of pyruvate. Arch. Biochem. Biophys., 120 (1967) 583-- 588. R.W. Estabrook and B. Sacktor, a-Giycerophosphate oxidase of flight muscle mitochondria. J. Biol. Chem., 233 (1958) 1014-- 1019. M. Rockstein, Metachemogenesis postemergence chemical maturation in holometabolous insects. Smithsonian Inst. Misc. Collections, 13 7 (1959) 263--286. R.S. Sohal, J.L. McCarthy and V.F. Allison, The formation of "giant" mitochondria in the fibriller flight muscles of the housefly, Musca domestica. J. Ultrastruct. Res., 39 (1972) 484-- 495. H. Donato, M.A. Hoselton and R.S. Sohal, An analysis of the effects of individual variation and selective mortality on population averages in aging populations. Exp. Gerontol. 14 (1979) 133--140. K.J. Farmer and R.S. Sohal, Relationship between superoxide anion radical generation and ageing in the housefly, Musca domestica. Free Rad. Biol. Med., 7 (1989) 23--29. M. Miki, H. Tamai, M. Mino, Y. Yamamoto and E. Niki, Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibitor by a-tocopherol. Arch. Biochem. Biophys., 258 (1987) 373--380. A.W. Girotti, J.P. Thomas and J.E. Jordan, Xanthine oxidase-catalyzed cross-linking of cell membrane proteins. Arch. Biochem. Biophys.,251 (1986)639--653. R.S. Sohal, I. Svensson and U.T. Brunk, Hydrogen peroxide production by liver mitochondria in different species. Mech. Ageing Dev., 53 (1990) 209--215.

202 32

33 34 35

36 37 38 39 40 41

O. Dionisi, T. Gleotti, T. Terranova and A. Azzi, Superoxide radicals and hydrogen peroxide formation in mitochondria from normal and neoplastic tissues. Biochim. Biophys. Acta, 403 (1975) 292--300. C. Riley, G. Cohen and M. Lieberman, Ethane evoluation: a new index of lipid peroxidation. Science, 183(1974) 208--210. R.G. Bridges and R.S. Sohal, Relationship between age-associated fluorescence and linoleic acid in the housefly, Musca domestica. Insect Biochem, 10 (1980) 557--562. R.S. Sohal, K.J. Farmer, R.G. Allen and N.R. Cohen, Effect of age on oxygen consumption, superoxide dismutase, catalase, glutathione, inorganic peroxides and chloroform-soluble antioxidants in the adult male housefly, Musca domestica. Mech, Ageing Dev., 24 (1984) 185--195. E.W. Kellogg and I. Fridovich, Superoxide, hydrogen peroxide and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. Biol. Chem., 250 (1975) 8812--8817. K.J.A. Davies and M.E. Delsignore, Protein damage and degradation by oxygen radicals: 111 Modification of secondary and tertiary structures. J. Biol Chem., 262 (1987) 9908--9913. A.L. Tappel, Measurement of and protection from in vivo lipid peroxidation. In: W.A. Pryor (ed.), Free Radicals in Biology, Vol. 4, Academic Press, New York, 1980, pp. 1--47. H. Nohl and D. Hegner, Do mitochondria produce oxygen radicals in vivo? Eur. J. Biochem., 82 (1978) 563--567. M. Sagai and T. Ishinose, Age-related changes in lipid peroxidation as measured by ethane, ethylene, butane and pentane in respired gases of rats. Life Sci., 27(1980) 731--738. R.S. Sohal, 1. Svensson, B.H. Sohal and U.T. Brunk, Superoxide anion radical production in different species. Mech. Ageing Dev., 49 (1989) 129--135.