Superoxide anion radical production in different animal species

Mechanisms of Ageing and Development, 49 (1989) 129-- 135

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Elsevier Scientific Publishers Ireland Ltd.

S U P E R O X I D E A N I O N R A D I C A L P R O D U C T I O N IN D I F F E R E N T A N I M A L SPECIES

R.S. S O H A L * , I. SVENSSON, B.H. S O H A L and U.T. BRUNK Department of Pathology I1, University of Linkb)~ing, S-581 85 Link6ping (Sweden)

(Received November 9th, 1988) SUMMARY The general objective of this study was to examine the relationship between oxygen free radicals and the aging process. The rate of superoxide anion radical (Oi) generation was measured in liver sub-mitochondrial particles from mouse, rat, rabbit, pig and cow, and in flight muscle sub-mitochondrial particles from the housefly. The rate of O~ generation was determined as superoxide dismutase inhibitable reduction of ferricytochrome c in the presence of antimycin A and KCN. O i generation was inversely related to m a x i m u m species life span potential (MLSP) (r = - 0 . 9 2 ) . A 24-fold difference in the rate of O i production was observed between the cow and the fly while a 6-fold difference existed among the mammals. The results are interpreted to indicate that under identical conditions, mitochondria from organisms with low M L S P have a relatively greater propensity to generate O~. This may be suggestive of innate differences in the molecular organization of the inner mitochondrial m e m b r a n e among different species.

Key words." Oxygen free radicals; Aging; Mitochondria; Superoxide radical; Oxida-

tive stress; Life span. INTRODUCTION It has now been clearly demonstrated that partially reduced oxygen species are produced in aerobic ceils under physiological conditions [1]. According to one estimate, approximately 2°7o of the oxygen consumed by heart mitochondria is diverted to form superoxide anion radicals (Oi), mainly by the autoxidation of ubisemiquinone of the Q cycle in the electron transport chain Ill. It has also been

*Present address." Department of Biological Sciences, Southern Methodist University, Dallas, Texas

75275, U.S.A. 0047-6374/89/$03.50 Printed and Published in Ireland

C> 1989Elsevier Scientific Publishers Ireland Ltd.

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shown that cellular components such as lipids, proteins and nucleic acids undergo oxidant damage under normal conditions [2]. For example, exhalation of alkanes 13t and urinary excretion of thymine glycol [4] are indicative of in vivo oxidant damage to polyunsaturated fatty acids and DNA, respectively. It has been proposed that accrual of unrepaired oxidant damage may constitute one of the underlying causes of the aging process [ 5 ] . . ~ l t h o u g h this hypothesis has attracted considerable attention, the supportive evidence is rather desultory. One of the investigative approaches, that has often been employed to elucidate the possible causal factors in the aging process, involves the comparison ol the suspected factor in different species with varying maximum life span potential (MLSP). The purpose of the present study was to explore the relationship between the rat~ of free radical generation and MLSP in different species. Rates of ()~ production by mitochondrial membranes were compared in five different mammalian species. namely, mouse, rat, rabbit, pig and cow, which vary in their MLSP from approximately 3.5 to 30 years. In addition, for the sake of comparison with ver~ short-lived species, rate of O] generation was also determined in the flight muscle mitochondria of the housefly, which has an MLSP of about 3 months. The results indicate that rate of mitochondrial O~ generation is inversely related to M L S P MATERIAL AND METHODS

Animals Mitochondria were isolated f r o m the liver of mouse, rat, rabbit, pig and cow and the flight muscles of houseflies. The strains of the animals were: mouse, N.M.R.I. (Naval Medical Research Institute, Bethesda); rat, Sprague--Dawley; rabbit, white mixed; pig, Yorkshire; cow, Holstein; houseflies, W . H . O (World Health Organization). All of the m a m m a l s used were young adults. Flies were 10 days old Chemicals" Cytochrome c type VI and antimycin A were obtained from the Sigma Chemicai C o m p a n y (St. Louis, MO, U.S.A.). All the other reagents were of analytical grade. Isolation of mitochondria and preparation of sub-mitochondrial particles from rivet Livers were homogenized in 10 volumes (w/v) buffer consisting of 220 mM mannitol, 70 mM sucrose, 1 mM Tris and 3 mM EDTA, pH 7.4. The homogenate was centrifuged at 271 g for 10 rain, the pellet was discarded and the supernatam recentrifuged at 1086 g for 20 min. The pellet was again discarded and the supernatant recentrifuged at 3015 g for 10 min. The pellet was resuspended in buffer and recentrifuged at 3015 g for 10 min. Subsequently, the pellet was resuspended in 3!) mM potassium phosphate buffer, p H 7.0 and recentrifuged at 3015 g for 10 rain The pellet was resuspended in 6 ml phosphate buffer and sonicated four times for

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30 s each at l-rain intervals, in ice. The sonicated mitochondria were centrifuged at 7719 g for 10 min to sediment any unfragmented mitochondria. The supernatant was centrifuged at 80 000 g for 40 min to sediment sub-mitochondrial particles, which were resuspended in 30 mM potassium phosphate buffer, pH 7.0.

Isolation of mitochondria and preparation of sub-mitochondrial particles from flight muscles of the housefly Mitochondria were isolated from thoraces according to the procedure described previously [71. Thoraces were removed using razor blades and gently crushed in a chilled mortar and pestle in 10 volumes (w/v) of mannitol buffer. The homogenate was filtered by suction through five layers of sucrose-soaked gauze. As reported previously, examination of filterate by phase contrast microscopy indicated the presence of relatively pure mitochondria [7]. Sub-mitochondrial particles were prepared in a manner similar to that in the liver.

Measurement of 0~ generation The rate of O 5 production is usually measured in the sub-mitochondrial particles because O 5 is unable to pass through the inner mitochondrial membrane and superoxide dismutase (SOD) present in the mitochondrial matrix converts O 5 to H202 [8,9]. There are at present no known inhibitors of mitochondrial SOD activity. Sonication of mitochondria into sub-mitochondrial particles with reversed membrane surfaces tends to circumvent these problems [8,9]. The rate of O 5 production by sub-mitochondrial particles was measured as SOD inhibitable reduction of ferricytochrome c. Both the test and the reference cuvette contained 0 . 2 - - 1 . 0 mg sub-mitochondrial protein, 0.1 M potassium phosphate buffer, p H 7.4, 75/aM cytochrome c, 0.6/aM antimycin A, 1.3 mM KCN and 7 m M succinate; 200 units o f S O D / m l were added to the reference cuvette. The reduction of ferricytochrome c was spectrophotometrically monitored at 550 nm. In a reaction mixture, that contains sub-mitochondrial particles, cytochrome c is reduced by O 3 as well as by cytochrome c reductase, present in the mitochondrial membrane [8]. In the present study the reference and the test cuvette contained identical ingredients except that the former included SOD. Thus the measured rate of cytochrome c reduction is specifically due to its reaction with 05.

Superoxide dismutase activity SOD activity was measured by the method of Crapo et al. [10] using xanthinexanthine oxidase, as reported previously [11]. RESULTS

The maximal rates of O 5 generation by sub-mitochondrial particles from the liver of mouse, rat, rabbit, pig and cow and f r o m the flight muscles of the housefly are

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TABLE 1 RATE OF SUPEROXIDE ANION R A D I C A l _ (O~) P R O D U C T I O N AND SUPEROXII)t D I S M U T A S E ( S O D ) A C T I V I T Y IN S U B - M I T O C H O N D R I A L PARTICLES FROM DIFFEREN1 SPECIES OF VARIOUS MAXIMUM LIFE SPAN POTENTIAL (MLSP) Animal

O~ p r o d u c t i o n ~ ( n m o l O~ / m i n / r n g prot.)

M L SI ~' (vears)

S O D at "ti vit v ( u n i t s mg p r o t J

Housefly Mouse Rat Rabbit Pig Cow

3.30 0.66 0.77 0.46 0.42 0.13

0.25 3.5 4.5 18 27 30

55,5 50. I 55 ~ 244 29 1 22.2

± ± ± ± ± ±

0.36 0.05 0.06 0.05 0.06 0.01

± 1~ ._~ 1 ~ ± 3.t! *__ 0 " :+_ I " ± ~ i

aValues a r e a n a v e r a g e o f 4 - - 6 d e t e r m i n a t i o n s ± S D . b M L S P o f m o u s e , r a t , pig a n d c o w were o b t a i n e d f r o m Ref. 14, o f r a b b i t f r o m Ref f r o m Ref. 12. ' V a l u e s a r e a n a v e r a g e o f 2 or 3 d e t e r m i n a t i o n s ± r a n g e or S.I)., re~,pectivel;,

t3, a n d ol houscll3

presented in Table I. The rate of O.~ production was found to be highest in the h o u sefly and lowest in the cow, being 24-fold higher in the housefly than in the cox,. Among the m a m m a l s , the rate of O~ generation was highest in the rat, being about 6 times greater than in the cow. Data on MLSP, shown in Table I, were culled from the literature [12--14] ~I should be added that M L S P of a species is an approximate rather than a precise determination. Furthermore, MLSP reported by different authors varies. For the purpose of this study the longest reported MLSPs were selected. A comparison of O~ production in relation to M L S P indicated that, in general, the rate of O: generation decreases as MLSP increases (correlation coefficient . . . . . 0.92),

Residual SOD activity in sub-mitochondrial particles As also noted above, the presence of any SOD activity in sub-mitochondrial particles tends to depress the actual rate of O~ production [8,9]. Unequal levels or SOD activity in sub-mitochondriai particles from different species can therefore, potentially, confound the interpretation of the results. We compared the total SOl) activity in the various samples of sub-mitochondrial particles used for the measurement of the rate of O~ generation. As shown in Table 1, sub-mitochondrial particles exhibited residual SOD activity but in a fashion that strengthens rather than confounds the observed disparity in the rates of O~ generation. Relatively higher levels of SOD activity were observed in species exhibiting relatively greater rates of O~ generation. Since SOD tends to decrease the rate of cytochrome c reduction by O 3 the observed rates are an underestimate of the absolute disparity among species.

133 DISCUSSION The results of this study indicate that under identical in vitro conditions the maximal rates of O~ production by sub-mitochondrial particles exhibit a 6-fold difference among mammals and a 24-fold difference if the housefly is included in the comparison. This points to certain, presently unknown, differences in the respiratory chain components in different species which modulate the autoxidation rates of ubisemiquinone. Potentially, differences in the rates of succinate utilization or structural organization of succinate dehydrogenase-ubiquinol segment of the respiratory chain or factors affecting the ratio of reduced to oxidized form of ubiquinone can effect the O~ production in ubiquinone-cytochrome c region [15]. It has been previously postulated by us [16] and others [5] that oxidative stress may play a causal role in the aging process. The level of oxidative stress is, by definition, dependent on the ratio between pro-oxidants and antioxidants [2]. If the observed in vitro differences in O~ generation are reflective of corresponding in vivo differences, the organisms with relatively high rates of O~ generation would produce correspondingly higher amounts of H202, which is the progenator of the highly reactive hydroxyl free radical (HO'), believed to be the damaging agent in oxygen toxicity [16]. It is possible that the relatively elevated level of oxidant damage observed in species with low MLSP [17] could be partially due to an enhanced rate of free radical generation. The ratio of pro-oxidants to antioxidants has been suggested to be a longevity determinant in mammals. Tolmasoff et al. [6] compared SOD activity in tissues of two rodent and 12 primate species with MLSP varying from about 4 to 100 years. No correlation was found between SOD activity and MLSP, however, the ratio of SOD activity to basal metabolic rate was found to increase with MLSP. The interpretation of these results was that animals with more efficient protection against O~ tend to have longer MLSP. The inverse relationship between metabolic rate and life span has been recognized since 1908 when Rubner [19] reported that the total amount of energy consumed by six different domesticated mammalian species which exhibited 6-fold differences in life span was constant around 200 kcal/life span, that is, species with longer MLSP expended their metabolic potential at a lower rate. In terms of the free radical hypothesis of aging, this relationship has been usually interpreted to imply that animals with relatively higher metabolic rates produce correspondingly high levels of O~ and other reactive oxygen species. It is widely regarded, also implicitly by Tolmasoff et al., but without evidence, that a fixed proportion of oxygen consumed by cells is diverted to O i generation. Paradoxically, the rate of O~ generation in mitochondria, which are the main sources of O~ production, is higher during state 4 when respiratory components are in a relatively more reduced state than in state 3, where ADP-stimulated phosphorylation is taking place [8,9]. Thus "resting" mitochondria produce relatively more O 3 than mitochondria engaged in ATP synthesis. Results of this study indicate that Oi

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production at the ubiquinone-cytochrome b region is independent of metabolic rate of the organism. For example, O~ production in rabbit is comparable to that in the pig, but the metabolic rate o f a rabbit is significantly lesser than that of a pig. ~X similar situation exists in the rat and mouse which have a somewhat comparable rate of O~ generation but significantly different metabolic rates. It can be speculated that characteristics of the mitochondrial respiratory chain may modulate the rate of Oi generation in different species. In conclusion, results of this study indicate that rate of O~ generation at the ubi quinone-cytochrome b site is inversely correlated with MLSP and is not strictl~ dependent on the specific metabolic rate of the animals. ACKNOWLEDGEMENTS

This research was supported by grants from the Swedish Medical Research Council (No. 4481) and the National Institutes of Health-National Institute c~t~ Aging (R01 AG7657). REFERENCES

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