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Effect of physical activity on superoxide dismutase, catalase, inorganic peroxides and glutathione in the adult male housefly, Musca domestica
Mechanisms of Ageing and Development, 26 (1984) 75 -81
75
Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF PHYSICAL ACTIVITY ON SUPEROXIDE DISMUTASE, CATALASE, INORGANIC PEROXIDES AND GLUTATHIONE IN THE ADULT MALE HOUSEFLY, MUSCA DOMESTICA
R.S. SOHAL*, R.G. ALLEN, K.J. FARMER and J. PROCTER Department of Biology, Southern Methodist University, Dallas, Texas 75275 (U.S.A.)
(Received September 20~h, 1983)
SUMMARY Average and maximum life spans of the adult male housefly are considerably prolonged by the elimination of flying activity. The objective of this study was to elucidate further the biochemical basis of this phenomenon. Activities of the enzymes superoxide dismutase and catalase, and the concentrations of inorganic peroxides and glutathione were compared between houseflies kept under conditions of relatively high and low levels of physical activity at different ages. The activities of superoxide dismutase and catalase were not appreciably affected by the level of physical activity whereas the concentrations of inorganic peroxides and glutathione were higher in flies undergoing relatively high level of physical activity. These results are interpreted to support the concept that by-products of oxygen metabolism may play a causal role in the aging process.
Key words: Aging; Superoxide dismutase; Catalase; Glutathione; Physical activity;
Inorganic peroxides
INTRODUCTION There is a considerable body of evidence to suggest that metabolic rate (rate of oxygen consumption) may be a factor in governing the rate of aging in animals. Although there is a correlational tendency towards an inverse relationship between basal metabolic rate and species-specific longevity in homeothermic mammals [1,2], such a relationship is most demonstrable in poikilotherms [3-5] and hibernating mammals [6] whose metabolic rates are readily affected by ambient temperature. Previous studies in this laboratory
*To whom correspondence should be directed.
0047-6374/84/$03.00 Printed and Published in Ireland
© 1984 ElsevierScientific Publishers Ireland Ltd.
76
(see refs. 3 and 4 for review), dealing with the relationship between oxygen consumption and aging, have employed physical activity rather than ambient temperature to vary metabolic rates because many cellular processes behave differently under different thermal conditions (see refs. 3 and 7 for review). Results of these studies have indicated that reduction in the level of physical activity prolongs the average and the maximum life span of the adult houseflies [3,4,8] and also retards the rate of age-associated changes such as the accumulation of lipofuscin granules [9,10] and lipid-soluble fluorescent material [4,11 ]. The mechanism by which oxygen utilization affects life span is presently obscure, although it is widely recognized that a small proportion of oxygen consumed by cells is reduced univalently generating, first, superoxide radical (O~-) which dismutates into H202 [12,13]. The dismutation of O~- is catalyzed by the enzyme superoxide dismutase, while H202 is primarily eliminated by the activities of catalase and peroxidases [ 12,13]. In insects, however, the enzyme glutathione peroxidase, which removes peroxides by catalyzing their reaction with glutathione, is notably absent [14,15]. The cytotoxic effects of oxygen are widely believed to be due to iron-catalyzed interaction between O~- and H202 which results in the production of highly reactive hydroxyl radicals (OH ") [12,13,16]. Deleterious effects of O~- fluxes have been amply demonstrated under in vitro conditions [ 12,13,17 ]. In addition to the enzymes superoxide dismutase, catalase and peroxidases, whose combined functions tend to minimize the probability of OH • generation, the cells also possess non-enzymatic defenses against free radicals. Glutathione, ~3-carotene, a-tocopherol and ascorbic acid are the best known endogenous antioxidants [18]. Previous studies in this laboratory on the housefly have indicated that, under conditions of oxidative stress, glutathione may play an important adaptive function [19,20]. The present study was undertaken in an effort to gain further understanding.of the biochemical basis of the inverse relationship between physical activity and life span observed in the adult housefly. Thus, activities of superoxide dismutase and catalase and the levels of inorganic peroxides and glutathione were compared between houseflies kept under conditions of relatively high and low levels of physical activity. MATERIALS AND METHODS
Rearing of houseflies Eggs, obtained from 8- to 10-day-old flies (Cambridge University strain), were placed in moist CSMA (Chemical Specialties Manufacturer's Association) fly larval medium obtained from Ralston Purina Co., Richmond, IN. After emergence of the pupa, adult flies were fed on sucrose and water. Throughout the life cycle, ambient temperature was maintained at 25°C and relative humidity at about 45%. Only male flies were used for experimental studies. Variation of physical activity Levels of physical activity of the flies were varied by affecting the flying activity of
77 the flies. Environmental conditions governing the flying activity of housefly populations have been defined previously in this laboratory [4,8]. For achieving a relatively high level of physical activity, 200 male flies were housed in 1-cubic foot (0.03 m) cages where flying is possible. In the second group, flying activity was prevented by confining each fly, individually, in urine specimen glass bottles (about 250 ml) partitioned with a cardboard maze in such a manner that flies could walk but were unable to fly due to limitation of space. Radar-Doppler monitoring of flies confined in such a manner has previously been shown to prevent flying [8,10]. It may be added that solitary confinement of the male flies was necessary because they make persistent homosexual copulatory attempts on each other which enhances their physical activity [8]. Experimental procedures Procedures for the measurement of the various parameters examined in this study were recently reported in detail and therefore will be only briefly described here. The rate of oxygen consumption was measured by placing cages or bottles, containing the flies, within a plexiglas respiration chamber and determining the oxygen concentration during 3-h periods. Oxygen concentration was detected by a Beckman OM14 oxygen analyzer. Activity of superoxide dismutase was measured by the indirect method suggested by Crapo et al. [21]. Catalase activity was determined by the method recommended by Luck [22]. Concentration of inorganic peroxides was measured by the method of Bernt and Bergmeyer [23]. The levels of total glutathione, i.e. reduced (GSH) and oxidized (GSSG) forms, were measured by a modification [24] of the method described by Tietze
[251. RESULTS Life span and metabolic rate The average life span of the houseflies was 19.5 + 6.7 days under conditions of relatively high physical activity and 33.3 -+ 14.7 days under low activity conditions, whereas
TABLE I ACTIVITIES OF SUPEROXIDE DISMUTASE AND CATALASE IN ADULT MALE HOUSEFLIES MAINTAINED UNDER CONDITIONS OF RELATIVELY HIGH (HA) AND LOW (LA) PHYSICAL ACTIVITY Age (days}
7 9 14
a-+S.D. *P < 0.05.
Supero xide dismutase (units/mg protein}
Catalase {units/mg protein)
HA
LA
HA
LA
97 +-4 a 145 -+6 103 -+5
106 -+3 146 -+5 114 -+8
29.8 -+0.5 32.4 -+0.5 27.3 _+0.4
26.7 -+0.5* 30.0 -+0.4* 26.8 -+0.5
78 TABLE II CONCENTRATIONS OF INORGANIC PEROXIDES IN ADULT MALEHOUSEFLIESKEPT UNDER CONDITIONS OF RELATIVELY HIGH (HA)AND LOW (LA) LEVELS OF PHYSICALACTIVITY Age (days)
6 12 17
Inorganic peroxides (tagH202 per g wet weighU HA
LA
28.3 -+ 1.6a 32.4 -+ 1.9 39.4 +-3.2
18.3 _+0.8** 27.1 +_1.8" 33.7 +-1.2"*
a+_S.D. *P < 0.05. **P < 0.01. the maximum life spans in the two groups were 28 and 65 days, respectively. The average rate of oxygen consumption, measured on 7, 8 and 9 days of age, was 13.9 gl/h/mg wet weight in.high activity flies and 8.8 #l/h/rag in low activity flies. Superoxide dismutase Activity of superoxide dismutase, presented in Table I, was measured in 7- 9- and 14-day-old flies. No significant differences were found between the groups maintained under the regimes of relatively high and low physical activity. Catalase As shown in Table I, catalase activity measured on 7, 9 and 14 days of age, was slightly higher in the flies kept under conditions of high physical activity as compared to those kept under low activity conditions. Inorganic peroxides The concentration of inorganic peroxides was measured in the whole body homogenates of 6-, 12- and 17-day-old flies. Inorganic peroxide levels increased with age in both groups of flies; however, at all ages, flies kept under conditions of relatively higher physical activity contained a greater concentration of inorganic peroxides than those kept under the low activity regime (Table II). Glutathione The concentration of total glutathione (GSH + GSSG) and GSSG was measured in 7-, 10- and 14-day-old flies. GSH levels were calculated by subtracting GSSG values from the total amount. At all ages, concentrations of total glutathione and GSSG were greater in the flies kept under conditions of relatively high activity than in the low activity flies (Table III). GSH levels were similar in the two groups at 7 days of age but were higher in the high activity flies at 10 and 14 days of age.
79 TABLE III LEVELS OF TOTAL GLUTATHIONE, OXIDIZED GLUTATHIONE (GSSG) AND REDUCED GLUTATHIONE (GSH) IN ADULT HOUSEFLIES MAINTAINEDUNDER REGIMESOF RELATIVELY HIGH (HA) AND LOW (LA) LEVELS OF PHYSICALACTIVITY Age (days)
7 10 14
Glutathione {mg/l O g wet weight) Total (GSH + GSSG)
GSSG
GSH
HA
LA
HA
LA
HA
LA
3.4±0.1 a 3.9±0.1 3.3±0.05
3.0±0.1 2.8±0.06* 2.2±0.09*
1.0±0.05 1.2±0.06 1.2±0.02
0.7~0.03" 0.7±0.04* 0.9±0.09*
2.4 2.7 2.1
2.2 2.l 1.3
a±S.D. *P < 0.01.
DISCUSSION Results of this study indicate that elevation in the level of physical activity and the resultant rate of oxygen consumption causes an increase in concentrations of inorganic peroxides and glutathione whereas the activity of superoxide dismutase is apparently unaffected and catalase activity is only slightly increased. Due to the apparent absence of glutathione peroxidase in insects, the main enzymatic defenses against the initial products of univalent reduction of oxygen, i.e. 0~- and H202, are presumably provided by superoxide dismutase and catalase. The activities of these enzymes, however, were not appreciably altered by an increase in the level of physical activity of the flies. On the other hand, the concentration of inorganic peroxides in the tissues of the houseflies was elevated under conditions of relatively high level of physical activity. The apparent imbalance between the rates of production and elimination of inorganic peroxides (presumably H202) may be a causal factor in shortening of the life span of houseflies kept under conditions of relatively high physical activity. Higher concentration of H202 would, tend to increase the risk of OH • generation. In this regard the studies by Davies et al. [26,27] are of interest. They tbund that physical exertion in the rat caused a 2- to 3-fold increase in the concentration of free radicals in muscles and liver. Physical exercise also caused an increase in the levels of lipid peroxidation products, loss of sarcoplasmic reticulum and decrease in mitochondrial respiratory control values. Elevation in GSH and total glutathione levels was the primary adaptive response observed in the houseflies to higher levels of physical activity. A similar increase in glutathione concentration was previously observed in the houseflies in response to oxidative stress induced by inhibition of superoxide dismutase [19] and catalase [20]. Although GSH has been shown to quench O~- and OH • [ 18], it is at present unknown if
80 it can also react with H202 directly in the absence o f glutathione peroxidase, albeit two different m e c h a n i s m s for such a reaction have b e e n p r o p o s e d [28]. Overall, the results o f this study are consistent with the c o n c e p t that by-products o f o x y g e n m e t a b o l i s m m a y play a causal role in the aging process. ACKNOWLEDGEMENTS The research was s u p p o r t e d by grants f r o m the National Institutes o f Health, National Institute o n Aging (R01 A G 0 0 1 7 1 ) and the G l e n n F o u n d a t i o n for Medical Research. REFERENCES 1 G.A. Sacher, Life table modification and life prolongation. In C.E. Finch and L. Hayflick (eds.), The Biology of Aging, Van Nostrand Reinhold, New York, 1977, pp. 582-638. 2 J.M. Tolmasoff, T. Ono and R.G. Cutler, Superoxide dismutase: correlation with life-span and specific metabolic rate in primate species. Proc. Natl. Acad. ScL USA, 27 (1980) 2777-2781. 3 R.S. Sohal, Metabolic rate and life span. Interdiscip. Top. Gerontol., 9 (1976) 25-40: 4 R.S. Sohal, Metabolic rate, aging and lipofuscin accumulation. In R.S. Sohal (ed.), Age Pigments, Elsevier/North Holland, Amsterdam, 1981, pp. 303- 316. 5 J. Miquel, P.R. Lundgren, K.G. Bensch and H. Atlan, Effect of temperature on the life span, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Dev., 5 (1976) 347-370. 6 C.P. Lyman, R.C. Green and E.D. Papafrangos, Hibernation and longevity in the Turkish hamster Mesocricetus brandti. Science, 201 (1978) 875-880. 7 C.L. Prosser, Temperature. In C.L. Prosser (ed.), Comparative Animal Physiology, 3rd Edn., Sanders, Philadelphia, 1973, pp. 362-428. 8 R.S. Sohal and P.B. Buchan, Relationship between physical activity and life span in the adult ho usefly , Musca domestica. Exp. Gerontol., 15 (1981) 243-249. 9 R.S. Sohal and H. Donato, Effect of experimental prolongation of life span on lipofuscin content and lysosomal enzyme activity in the brain of the housefly, Musca domestica. £ Gerontol., 34 (1979) 489-496. 10 R.S. Sohal, Relationship between metabolic rate, lipofuscin accumulation and lysosomal enzyme activity during aging in the adult housefly, Musca domestica. Exp. Gerontol., 16 (1981) 347-355. 11 R.S. Sohal and H. Donato, Effects of experimentally altered life spans on the accumulation of fluorescent age pigment in the housefly, Musca domestica. Exp. Gerontol., 13 (1978) 335-341. 12 I. Fridovich, Superoxide radical and superoxide dismutases. In D.L. Gilbert (ed.), Oxygen and Living Processes: An Interdisciplinary Approach, Springer-Verlag, New York, 1981, pp. 250-272. 13 B. HalliweU, Oxygen toxicity, free radicals and aging. In R.S. Sohal (ed.), Age Pigments, Elsevier/ North Holland, Amsterdam, 1981, pp. 1-62. 14 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., in press. 15 J. Smith and A. Shrift, Phylogenetic distribution of glutathione peroxidase. Comp. Biochem. Physiol., 63B (1979) 39-44. 16 B. Halliwell, The effects of the superoxide radical and its products. Bull. Eur. Physiopathol. Resp. Suppl., 1 7 (1981) 21-28. 17 K. Brawn and I. Fridovich, Superoxide radical and superoxide dismutases: threat and defence. Acta Physiol. Scand. Suppl., 492 (1980) 9-18. 18 H.J. Forman and A.B. Fischer, Antioxidant defences. In D.L. Gilbert (ed.), Oxygen and Living Processes, Springer-Verlag, New York, 1981, pp. 235-249. 19 R.S. Sohal, K.J. Farmer, R.G. Allen and S.S. Ragland, Effects of diethyldithiocarbamate on life span, metabolic rate, superoxide dismutase, catalase, inorganic peroxides and glutathione in the adult male housefly, Musca domestica. Mech. Ageing Dev., in press.
81 20 R.G. Alien, K.J. Farmer and R,S. Sohal, Effect of catalase inactivation on levels of inorganic peroxides, superoxide dismutase, glutathione, oxygen consumption and life span in adult houseflies, lh'ochem. J., 216 (1983) 5 0 3 - 5 0 6 . 21 J.D. Crapo, J.M. McCord and I. Fridovich, Preparation and assay of superoxide dismutases. Methods Enzymol,, 53 (1978) 382-393. 22 H. Luck, Catalase. In H. Bergmeyer (ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1965, pp. 885-894. 23 E. Bernt and H.U. Bergmeyer, Inorganic peroxides. In H.U. Bergmeyer (ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1976, pp. 2246-2248. 24 O.W. Griffith, Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem., 106 (1980) 207-212. 25 F. Tietze, Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Anal. Biochem., 27 (1969) 5 0 2 - 5 2 2 . 26 K.J,A. Davies, A.J. Quintanilha, G.A. Brookes and L. Packer, Free radicals and tissue damage during exercise. Biochem, Biophys. Res. Commun.. 107 (1982) 1198-1205. 27 A.T. Quintanilha, L. Packer, J.M.C. Davies, T.L. Racanelli and K.J.A. Davies, Membrane effects of vitamin E deficiency: bioenergetic and surface charge density studies on skeletal muscle and liver mitochondria. Ann. N. Y. Acad. ScL, 393 (1982) 3 2 - 3 7 . 28 J.W. Finley and R.E. Lundin, Lipid hydroperoxide induced oxidation of cysteine in peptides. In M.G. Simic and M. Karel (eds.), Autoxidation in Food and Biological Systems, Plenum Press, London, 1980, pp. 223-235.
75
Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF PHYSICAL ACTIVITY ON SUPEROXIDE DISMUTASE, CATALASE, INORGANIC PEROXIDES AND GLUTATHIONE IN THE ADULT MALE HOUSEFLY, MUSCA DOMESTICA
R.S. SOHAL*, R.G. ALLEN, K.J. FARMER and J. PROCTER Department of Biology, Southern Methodist University, Dallas, Texas 75275 (U.S.A.)
(Received September 20~h, 1983)
SUMMARY Average and maximum life spans of the adult male housefly are considerably prolonged by the elimination of flying activity. The objective of this study was to elucidate further the biochemical basis of this phenomenon. Activities of the enzymes superoxide dismutase and catalase, and the concentrations of inorganic peroxides and glutathione were compared between houseflies kept under conditions of relatively high and low levels of physical activity at different ages. The activities of superoxide dismutase and catalase were not appreciably affected by the level of physical activity whereas the concentrations of inorganic peroxides and glutathione were higher in flies undergoing relatively high level of physical activity. These results are interpreted to support the concept that by-products of oxygen metabolism may play a causal role in the aging process.
Key words: Aging; Superoxide dismutase; Catalase; Glutathione; Physical activity;
Inorganic peroxides
INTRODUCTION There is a considerable body of evidence to suggest that metabolic rate (rate of oxygen consumption) may be a factor in governing the rate of aging in animals. Although there is a correlational tendency towards an inverse relationship between basal metabolic rate and species-specific longevity in homeothermic mammals [1,2], such a relationship is most demonstrable in poikilotherms [3-5] and hibernating mammals [6] whose metabolic rates are readily affected by ambient temperature. Previous studies in this laboratory
*To whom correspondence should be directed.
0047-6374/84/$03.00 Printed and Published in Ireland
© 1984 ElsevierScientific Publishers Ireland Ltd.
76
(see refs. 3 and 4 for review), dealing with the relationship between oxygen consumption and aging, have employed physical activity rather than ambient temperature to vary metabolic rates because many cellular processes behave differently under different thermal conditions (see refs. 3 and 7 for review). Results of these studies have indicated that reduction in the level of physical activity prolongs the average and the maximum life span of the adult houseflies [3,4,8] and also retards the rate of age-associated changes such as the accumulation of lipofuscin granules [9,10] and lipid-soluble fluorescent material [4,11 ]. The mechanism by which oxygen utilization affects life span is presently obscure, although it is widely recognized that a small proportion of oxygen consumed by cells is reduced univalently generating, first, superoxide radical (O~-) which dismutates into H202 [12,13]. The dismutation of O~- is catalyzed by the enzyme superoxide dismutase, while H202 is primarily eliminated by the activities of catalase and peroxidases [ 12,13]. In insects, however, the enzyme glutathione peroxidase, which removes peroxides by catalyzing their reaction with glutathione, is notably absent [14,15]. The cytotoxic effects of oxygen are widely believed to be due to iron-catalyzed interaction between O~- and H202 which results in the production of highly reactive hydroxyl radicals (OH ") [12,13,16]. Deleterious effects of O~- fluxes have been amply demonstrated under in vitro conditions [ 12,13,17 ]. In addition to the enzymes superoxide dismutase, catalase and peroxidases, whose combined functions tend to minimize the probability of OH • generation, the cells also possess non-enzymatic defenses against free radicals. Glutathione, ~3-carotene, a-tocopherol and ascorbic acid are the best known endogenous antioxidants [18]. Previous studies in this laboratory on the housefly have indicated that, under conditions of oxidative stress, glutathione may play an important adaptive function [19,20]. The present study was undertaken in an effort to gain further understanding.of the biochemical basis of the inverse relationship between physical activity and life span observed in the adult housefly. Thus, activities of superoxide dismutase and catalase and the levels of inorganic peroxides and glutathione were compared between houseflies kept under conditions of relatively high and low levels of physical activity. MATERIALS AND METHODS
Rearing of houseflies Eggs, obtained from 8- to 10-day-old flies (Cambridge University strain), were placed in moist CSMA (Chemical Specialties Manufacturer's Association) fly larval medium obtained from Ralston Purina Co., Richmond, IN. After emergence of the pupa, adult flies were fed on sucrose and water. Throughout the life cycle, ambient temperature was maintained at 25°C and relative humidity at about 45%. Only male flies were used for experimental studies. Variation of physical activity Levels of physical activity of the flies were varied by affecting the flying activity of
77 the flies. Environmental conditions governing the flying activity of housefly populations have been defined previously in this laboratory [4,8]. For achieving a relatively high level of physical activity, 200 male flies were housed in 1-cubic foot (0.03 m) cages where flying is possible. In the second group, flying activity was prevented by confining each fly, individually, in urine specimen glass bottles (about 250 ml) partitioned with a cardboard maze in such a manner that flies could walk but were unable to fly due to limitation of space. Radar-Doppler monitoring of flies confined in such a manner has previously been shown to prevent flying [8,10]. It may be added that solitary confinement of the male flies was necessary because they make persistent homosexual copulatory attempts on each other which enhances their physical activity [8]. Experimental procedures Procedures for the measurement of the various parameters examined in this study were recently reported in detail and therefore will be only briefly described here. The rate of oxygen consumption was measured by placing cages or bottles, containing the flies, within a plexiglas respiration chamber and determining the oxygen concentration during 3-h periods. Oxygen concentration was detected by a Beckman OM14 oxygen analyzer. Activity of superoxide dismutase was measured by the indirect method suggested by Crapo et al. [21]. Catalase activity was determined by the method recommended by Luck [22]. Concentration of inorganic peroxides was measured by the method of Bernt and Bergmeyer [23]. The levels of total glutathione, i.e. reduced (GSH) and oxidized (GSSG) forms, were measured by a modification [24] of the method described by Tietze
[251. RESULTS Life span and metabolic rate The average life span of the houseflies was 19.5 + 6.7 days under conditions of relatively high physical activity and 33.3 -+ 14.7 days under low activity conditions, whereas
TABLE I ACTIVITIES OF SUPEROXIDE DISMUTASE AND CATALASE IN ADULT MALE HOUSEFLIES MAINTAINED UNDER CONDITIONS OF RELATIVELY HIGH (HA) AND LOW (LA) PHYSICAL ACTIVITY Age (days}
7 9 14
a-+S.D. *P < 0.05.
Supero xide dismutase (units/mg protein}
Catalase {units/mg protein)
HA
LA
HA
LA
97 +-4 a 145 -+6 103 -+5
106 -+3 146 -+5 114 -+8
29.8 -+0.5 32.4 -+0.5 27.3 _+0.4
26.7 -+0.5* 30.0 -+0.4* 26.8 -+0.5
78 TABLE II CONCENTRATIONS OF INORGANIC PEROXIDES IN ADULT MALEHOUSEFLIESKEPT UNDER CONDITIONS OF RELATIVELY HIGH (HA)AND LOW (LA) LEVELS OF PHYSICALACTIVITY Age (days)
6 12 17
Inorganic peroxides (tagH202 per g wet weighU HA
LA
28.3 -+ 1.6a 32.4 -+ 1.9 39.4 +-3.2
18.3 _+0.8** 27.1 +_1.8" 33.7 +-1.2"*
a+_S.D. *P < 0.05. **P < 0.01. the maximum life spans in the two groups were 28 and 65 days, respectively. The average rate of oxygen consumption, measured on 7, 8 and 9 days of age, was 13.9 gl/h/mg wet weight in.high activity flies and 8.8 #l/h/rag in low activity flies. Superoxide dismutase Activity of superoxide dismutase, presented in Table I, was measured in 7- 9- and 14-day-old flies. No significant differences were found between the groups maintained under the regimes of relatively high and low physical activity. Catalase As shown in Table I, catalase activity measured on 7, 9 and 14 days of age, was slightly higher in the flies kept under conditions of high physical activity as compared to those kept under low activity conditions. Inorganic peroxides The concentration of inorganic peroxides was measured in the whole body homogenates of 6-, 12- and 17-day-old flies. Inorganic peroxide levels increased with age in both groups of flies; however, at all ages, flies kept under conditions of relatively higher physical activity contained a greater concentration of inorganic peroxides than those kept under the low activity regime (Table II). Glutathione The concentration of total glutathione (GSH + GSSG) and GSSG was measured in 7-, 10- and 14-day-old flies. GSH levels were calculated by subtracting GSSG values from the total amount. At all ages, concentrations of total glutathione and GSSG were greater in the flies kept under conditions of relatively high activity than in the low activity flies (Table III). GSH levels were similar in the two groups at 7 days of age but were higher in the high activity flies at 10 and 14 days of age.
79 TABLE III LEVELS OF TOTAL GLUTATHIONE, OXIDIZED GLUTATHIONE (GSSG) AND REDUCED GLUTATHIONE (GSH) IN ADULT HOUSEFLIES MAINTAINEDUNDER REGIMESOF RELATIVELY HIGH (HA) AND LOW (LA) LEVELS OF PHYSICALACTIVITY Age (days)
7 10 14
Glutathione {mg/l O g wet weight) Total (GSH + GSSG)
GSSG
GSH
HA
LA
HA
LA
HA
LA
3.4±0.1 a 3.9±0.1 3.3±0.05
3.0±0.1 2.8±0.06* 2.2±0.09*
1.0±0.05 1.2±0.06 1.2±0.02
0.7~0.03" 0.7±0.04* 0.9±0.09*
2.4 2.7 2.1
2.2 2.l 1.3
a±S.D. *P < 0.01.
DISCUSSION Results of this study indicate that elevation in the level of physical activity and the resultant rate of oxygen consumption causes an increase in concentrations of inorganic peroxides and glutathione whereas the activity of superoxide dismutase is apparently unaffected and catalase activity is only slightly increased. Due to the apparent absence of glutathione peroxidase in insects, the main enzymatic defenses against the initial products of univalent reduction of oxygen, i.e. 0~- and H202, are presumably provided by superoxide dismutase and catalase. The activities of these enzymes, however, were not appreciably altered by an increase in the level of physical activity of the flies. On the other hand, the concentration of inorganic peroxides in the tissues of the houseflies was elevated under conditions of relatively high level of physical activity. The apparent imbalance between the rates of production and elimination of inorganic peroxides (presumably H202) may be a causal factor in shortening of the life span of houseflies kept under conditions of relatively high physical activity. Higher concentration of H202 would, tend to increase the risk of OH • generation. In this regard the studies by Davies et al. [26,27] are of interest. They tbund that physical exertion in the rat caused a 2- to 3-fold increase in the concentration of free radicals in muscles and liver. Physical exercise also caused an increase in the levels of lipid peroxidation products, loss of sarcoplasmic reticulum and decrease in mitochondrial respiratory control values. Elevation in GSH and total glutathione levels was the primary adaptive response observed in the houseflies to higher levels of physical activity. A similar increase in glutathione concentration was previously observed in the houseflies in response to oxidative stress induced by inhibition of superoxide dismutase [19] and catalase [20]. Although GSH has been shown to quench O~- and OH • [ 18], it is at present unknown if
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