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Effects of exogenous antioxidants on the levels of endogenous antioxidants, lipid-soluble fluorescent material and life span in the housefly, Musca domestica
Mechanisms of Ageing and Development, 31 (1985) 329- 336 Elsevier Scientific Publishers Ireland Ltd.
329
EFFECTS OF EXOGENOUS ANTIOXIDANTS ON THE LEVELS OF ENDOGENOUS ANTIOXIDANTS, LIPID-SOLUBLE FLUORESCENT MATERIAL AND LIFE SPAN IN THE HOUSEFLY, MUSCA DOMESTICA
R.S. SOHAL*, R.G. ALLEN, K.J. FARMER, R.K. NEWTON and P.L. TOY Department of Biology, Southern Methodist University, Dallas, TX 75275 (U.S.A.) (Received March 28th, 1985)
SUMMARY Effects of exogenous antioxidant administration (0.5% and 2% ascorbate,/j-carotene and a-tocopherol in sucrose) on life-span, metabolic rate, activities of superoxide dismutase and catalase, levels of glutathione, inorganic peroxides and chloroform-soluble fluorescent material (lipofuscin) were examined in adult male houseflies. Administration of antioxidants at a level of 0.5% did not affect life-span, whereas, 2% ascorbate and atocopherol decreased average life-span. Metabolic rate of flies was unaffected, except by 2% ascorbate, which caused a decrease. Superoxide dismutase activity Was depressed by 2% ascorbate at all ages, and by ~-carotene and a-tocopherol in older flies. Catalase activity was unaffected except by a-tocopherol at younger ages. Glutathione concentration was decreased by ascorbate and/j-carotene at both concentrations administered. Inorganic peroxides (H202) were increased by 2%/j-carotene and o~-tocopherol. Only high concentrations of ascorbate and/J-carotene decreased the level of soluble fluorescent material. Results suggest that administration of exogenous antioxidants causes a compensatory depression of endogenous defenses.
Key words: Antioxidants; Aging; Insects; Superoxide dismutase; Glutathione; Catalase
INTRODUC~ON The postulate that free radicals, generated during cellular metabolism, are the main cause of molecular damage underlying the aging process was first enunciated by Harman [1]. In a recent review [2], he has cited the life-lengthening effects of antioxidant administration to constitute experimental support of this hypothesis. However, results of numerous studies purporting to test the free radical theory of aging, based on the *To whom all correspondence should be addressed. 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 ElsevierScientific Publishers Ireland Ltd.
330 effects of a variety of natural and synthetic antioxidants, have not clearly supported this claim [ 3 - 5 ] . For example, Harman [6] had originally reported that two antioxidants, 2-mercaptoethylamine hydrochloride and butylated hydroxytoluene, increased the lifespan of mice. In a reinvestigation, Kohn [7] found that when survival of control mice was optimal, the same antioxidants had no effect on life-span. Only when the life-span of control groups was below the optimal level, antioxidants extended the median lifespan. Furthermore, it has been widely observed that antioxidant intake does not prolong the maximum life-span of experimental animals [3-5,7]. Since regimes which extend the average but not the maximum life-span of populations seem to do so by reducing the vulnerability of animals to age-independent causes of death rather than age-dependent death rate [8], it can be inferred that administration of exogenous antioxidants has no significant effect on the rate of aging. The matter of the interpretation of these results - e.g., whether or not they negate the predictions of free radical theory of aging - was recently addressed by Cutler [4,5]. Hy hypothesized that the net level of antioxidants, within a given cell type, remains relatively constant due to compensatory or homeostatic regulatory control of individual antioxidants. It was postulated that perturbations, induced by administration of exogenous antioxidants, would produce a compensatory decrease in a related endogenous antioxidant. The present study was conducted to test Cutler's hypothesis. Effects of the administration of ascorbate, /3-carotene, and a-tocopherol (which are natural antioxidants) on life-span, and levels of endogenous antioxidants, inorganic peroxides, and chloroform-soluble fluorescent material (presumably related to lipofuscin) are reported. Endogenous antioxidants investigated were: superoxide dismutase, catalase, and glutathione. MATERIALS AND METHODS
Chemicals Chemicals were obtained from Sigma, St. Louis, Missouri. Rearing and maintenance of insects Eggs were obtained from 7- to 10-day-old houseflies, belonging to a stock originally obtained from the Department of Zoology, the University of Cambridge (U.K.). Larvae were fed on moist CSMA (Chemical Specialties Manufacturer's Association) fly larval medium, obtained from Ralston Purina Co., Richmond, IN. After emergence from pupae, flies were segregated by sex and males were housed in 1-cubic-foot cages with 200 flies/ cage. Experimental flies were administered 0.5% or 2% ascorbate, 0-carotene, or atocopherol in sucrose. Controls were provided with pure sucrose as food. All groups of flies were maintained at 25°C with a relative humidity of about 40%. Methods for the determination of total glutathione, inorganic peroxide concentration and superoxide dismutase (SOD) activity have been previously described in detail [9] and shall only briefly be outlined here. Inorganic peroxide (H202) concentration was
331 determined by the method o f Bernt and Bergmeyer [10]; glutathione was measured according to the method o f Tietze [ 11 ] ; catalase activity was determined by the method ffescribed by Luck [12] ; total SOD activity was estimated by the direct method of Misra and Fridovich [13]; the activity of the mangano-isozyme was also measured by this method except that the assay mixture contained 1 mM KCN. RESULTS
Life-span Effects o f the intake of 0.5% and 2% ascorbate,//-carotene and a-tocopherol, in sucrose, on the longevity o f the male housefly are shown in Table I. As compared to the controls, there was no change in the life-span o f flies administered 0.5% of these antioxidants. However, at 2% concentration, ascorbate decreased life-span by about half and a-tocopherol by about a fourth, fl-Carotene at this high concentration had no effect on longevity.
Metabolic rate Rate o f oxygen utilization was measured at 48-h intervals between 2 and 12 days o f age. Ascorbate at 2% concentration significantly decreased (P < 0.05) the rate o f oxygen consumption in flies (Table I). As compared to controls, no significant differences in metabolic rate were observed in other groups.
Superoxide dismutase (SOD) Total SOD activity, i.e. cyanide-insensitive plus cyanide-sensitive activity, was measured in 3-, 6-, 9-, and 12-day-old flies. At a 0.5% concentration, none of these antioxidants had an appreciable effect on SOD activity (Table II). However, at a 2% concentra-
TABLE I EFFECT OF EXOGENOUS ANTIOXIDANT ADMINISTRATION ON LIFE-SPAN AND METABOLIC RATE OF ADULT MALE HOUSEFLIES
Group
Average life-span (days)
Metabolic rate (M OJh/mg body wt)
Control (for 0.5%) 0.5% Ascorbate 0.5% &Carotene 0.5% a-Tocopherol Control (for 2%) 2% Ascorbate 2% fl-Carotene 2% c~-Toeopherol
21.6 ± 7.5 18.6 + 5.4 19.7 ± 7.1 19.6 ± 6.7 20.7 ± 5.9 9.9 ± 3.8* 20.1 ± 8.8 15.3 ± 4.2*
7.8 _4-1.1 7.2 _+2.6 9.4 ± 3.7 7.1 ± 1.3 8.0 ± 2.5 5.6 ± 2.7* 7.1 ± 2.1 7.7 ± 2.6
+S.D. *P < 0.05
332 TABLE II EFFECT OF ANTIOXIDANT ADMINISTRATION ON SUPEROXIDE DISMUTASE (SOD) ACTIVITY IN MALE HOUSEFLIES AT DIFFERENT AGES
Group
Control (for 5%) 0.5% Aseorbate 0.5% a-Carotene 0.5% a-Toeopherol Control (for 2%) 2% Aseorbate 2% B-Carotene 2% a-Tocopherol
SOD activity (units/ragprotein) 3-day
6-day
9-day
12-day
18.8 17.7 22.4 18.2 20.5 17.3 18.1 20.0
26.0 27.3 26.0 27.3 26.1 21.6 27.1 20.0
31.0 29.3 28.0 31.7 26.5 19.6 23.6 19.9
27.4 24.9 26.5 28.0 26.3
_+0.8 _+1.4 _+0.4 + 1.5 _+1.1 _+1.1" _+1.4 _+1.1
_+0.6 _+1.3 -+ 0.6 _+0.3 -+ 1.0 _+2.0* + 1.0 -+0.7*
_+0.7 _+0.5 _+0.4 _+0.5 + 0.7 -+0.6* _+0.5* _+0.6*
_+1.1 _+0.5 -+0.4 _+1.6 _+0.7
24.6 -+ 1.0 20.8 _+1.7*
~.D.
*P < 0.05.
tion, SOD activity was decreased. Ascorbate a d m i n i s t r a t i o n significantly (P < 0.05) reduced S O D a c t i v i t y at all ages e x a m i n e d , l$Carotene and a - t o c o p h e r o l decreased SOD activity in older flies.
Catalase Catalase activity was m e a s u r e d in 4-, 8-, and 12-day-old flies (Table III). ot-Tocopherol a d m i n i s t r a t i o n at b o t h 0.5% and 2% i n d u c e d a slight decrease in catalase activity at 4 days o f age, b u t no appreciable changes w e r e observed at o t h e r ages or in o t h e r groups.
TABLE III EFFECT OF ANTIOXIDANT ADMINISTRATION ON CATALASE ACTWITY IN ADULT MALE HOUSEFLIES AT VARIOUS AGES
Group
Catala~ activity (units/ragprotein) 4~y
Control (for 0.5%) 0.5% Ascorbate 0.5% &Carotene 0.5% a-Tocopherol Control (for 2%) 2% Ascorbate 2% a-Carotene 2% a-Toeopherol _+S.D.
12.3 10.7 9.2 7.9 12.6 11.1 12.4 10.7
+ 0.4 + 0.7 _+0.3 _+0.2 _+0.7 _+0.1 _+0.6 -+0.6
8~y
12~y
8.7 8.2 8.9 7.6 8.1 7.6 7.5 7.1
7.3 8.8 7.4 7.9 6.5 6.8 6.7 6.6
_+0.2 _+0.2 + 0.2 + 0.2 _+0.3 _+1.9 _+0.3 -+0.4
+ 0.2 _+0.3 _+0.4 _+0.3 +0.3 _+0.2 _+0.3 -+0.7
333 TABLE IV EFFECT OF ANTIOXIDANT INTAKE ON THE LEVEL OF TOTAL GLUTATHIONE IN HOUSEFLIES OF VARIOUS AGES
Group
Control (for 0.5%) 0.5% Ascorbate 0.5% f-Carotene 0.5% a-Tocopherol Control (for 2%) 2% Ascorbate 2% f-Carotene 2% a-Tocopherol
Glutathione concentration (~g/g body wt) 4-day
8-day
12-day
342 290 295 327 340 270 282 332
358 319 373 418 346 285 334 369
300 277 293 287 374 226 340 345
+5 + 12" _+9* _+ 11 _+4 _+ 1" + 9* -+ 10
_+9 _+2* _+5 _+6 _+2 _+3* -+ 2 _+6
_+6 _+8* _+6 _+8 _+11 -+4* -+ 11 +6
_+S.D. *P < 0.05.
Glutathione G l u t a t h i o n e was m e a s u r e d at 4-, 8-, and 12-days o f age (Table IV). A d m i n i s t r a t i o n o f ascorbate at 0.5% and 2% decreased glutathione level, fl-Carotene also appeared to decrease glutathione levels in y o u n g e r flies at b o t h c o n c e n t r a t i o n s used. et-Tocopherol had n o e f f e c t on g l u t a t h i o n e c o n c e n t r a t i o n .
Inorganic peroxides C o n c e n t r a t i o n o f inorganic peroxides 0-I202) was c o m p a r e d in 12-day-old flies (Table V). A s c o r b a t e a d m i n i s t r a t i o n decreased inorganic p e r o x i d e c o n c e n t r a t i o n in p r o p o r t i o n TABLE V EFFECT OF EXOGENOUS ANTIOXIDANTS ON CONCENTRATION OF INORGANIC PEROXIDES AND CHLOROFORM-SOLUBLE FLUORESCENT MATERIAL (SFM) IN 12-DAY-OLD MALE HOUSEFLIES
Group
Inorganic peroxides (l~g/gbody wt}
SFM (Units/rag body wt)
Control (for 0.5%) 0.5% Ascorbate 0.5% #-Carotene 0.5% a-Toeopherol Control (for 2%) 2% Ascorbate 2% #-Carotene 2% a-Toeopheml
19.8 11.6 17.0 22.3 30.1 9.8 39.8 36.1
0.020 0.024 0.022 0.021 0.023 0.018 0.016 0.025
+ S.D. *P < 0.05.
+ 0.3 _+0.8* + 1.4 + 0.5 _+0.9 + 0.9* + 1.3" _+1.5*
_+0.002 + 0.004 _+0.004 _+0.002 _+0.002 _+0.004* _+0.001" _+0.005
334 to dose./I-Carotene had no effect at 0.5%, but increased the organic peroxide level when administered at a concentration of 2%. On the other hand, a-tocopherol increased inorganic peroxide concentration in proportion to dose.
Soluble fluorescent material (SFM) The concentration of SFM in chloroform extracts of tissues was compared at 12 days of age (Table V). SFM level was unaffected by the 0.5% concentration of antioxidants. A concentration of 2% ascorbate and/3-carotene decreased SFM concentration while atocopherol had no appreciable effect. DISCUSSION In order to evaluate the possible involvement of free radicals in the aging process, it is imperative to recognize both the overlapping and the compensational nature of various endogenous antioxidants. For example, superoxide dismutase and glutathione are both effective scavengers of superoxide; catalase and glutathione can independently eliminate HzO2 [14]. GSH and ascorbate can both donate reducing equivalents to oxidants and can also react with a-tocopherol radical [14,15]. It should also be recognized that various enzymic and non-enzymic antioxidants are compartmentalized in specific intracellular domains, e.g. glutathione and ascorbate are present in the aqueous phase, whereas, t3carotene and a-tocopherol exist in the hydrocarbon core of the membranes [ 15]. Results of this study indicate that the average life-span of flies is not lengthened by antioxidant administration and relatively high intake of ascorbate and a-tocopherol may indeed be toxic. The most pronounced effects on mortality were produced by ascorbate which also depressed SOD activity and glutathione concentration in the body. Inorganic peroxide (H2Oz) concentration was lower in ascorbate-fed flies than in the control, possibly due to an increase in intracellular reducing equivalents. The life-shortening effect of ascorbate may have resulted from an unfavorable alteration in cellular redox state. Since inorganic peroxides and SFM were both lowered in the ascorbate-administered insects, it would seem improbable that the deleterious effects resulted from ascorbatedependent iron-catalyzed free radical reactions [ 16]. Present results deafly indicate the compensatory effects of exogenous antioxidants on endogenous antioxidants, as hypothesized by Cutler [4,5]. Administration ofascorbate, which has an overlapping function with glutathione and SOD was found to depress cellular levels of both. Effects of/I-carotene and a-tocophcrol were less pronounced, but they also tended to decrease SOD activity. In addition, ~carotene decreased ghitathione concentration. Although these lipid-soluble antioxidants did not appear to affect catalase activity, inorganic peroxide levels were elevated by an unknown mechanism in treated flies. Chloroform-soluble fluorescent material (SFM) is widely believed to be arLend-product of free radical-induced oxidation of polyunsaturated fatty acids and other macromolecules [17]. SFM has been shown to progressively accumulate with age in a variety of organisms [ 18,19]. Results of this study indicate that at low concentrations exogenous antioxidants
335 have little effect on SFM; higher doses tend to decrease SFM accumulation without beneficial effect on life-span. Blackett and Hall [20] have also observed this phenomenon in rats. Results o f our previous studies which examined the effects o f experimentally-induced oxidative stress on endogenous antioxidants also support the compensational hypothesis [21]. F o r example, inhibition o f SOD [22] and catalase [9] in the houseflies caused an increase in the level o f glutathione and depression o f metabolic rate. This suggests that organisms adapt to the imposed oxidative stress b y modulation o f endogenous antioxidants and b y decreasing the rate o f oxidative metabolism, which is a major source o f oxygen radicals. In conclusion, results o f this study neither support nor negate the postulates o f the free radical theory o f aging. However, they do indicate, that due to the compensational nature o f antioxidant defense mechanisms, the use o f a high antioxidant regime to test the predictions o f the free radical theory o f aging is invalid and may in fact be misleading. ACKNOWLEDGEMENTS This study was supported b y a grant from the Glenn F o u n d a t i o n for Medical Research. REFERENCES 1 D.H. Harman, Aging: A theory based on free radical and radiation chemistry. J. GerontoL, 11 (1980) 298-300. 2 D.H. Harman, The free radical theory of aging. In W.A. Pryor (ed.), Free Radicals in Biology, Vol. V, Academic Press, New York, 1982, pp. 255-275. 3 A.K. Balin, Testing the free radical theory of aging. In R.C. Adelman and G.C. Roth (eds.), Testing the Theories o/Aging, CRC Press, Boca Raton, Florida, 1982, pp. 137-182. 4 R.G. Cutler, Evolutionary biology of aging and longevity in mammalian species. In J.E. Johnson Jr. (ed.), Aging and Cell Function, Plenum Press, New York, 1984, pp. 1-147. 5 R.G. Cutler, Antioxidants, aging, and longevity. In W.A. Pryor (ed.), b3"eeRadicals in Biology, Vol. VI, Academic Press, New York, 1984, pp. 371-437. 6 D.H. Harman, Free radical theory of aging: Effects of free radical reaction inhibitors on the mortality rate of male LAF, mice. J. Gerontol., 23 (1968) 475-482. 7 R.R. Kohn, Effect of antioxidants on life-span of C57BL mice. J. GerontoL, 26 (1971) 378-380. 8 G.A. Sacher, Life table modifications and life prolongation. In C.E. Finch and L. Hay/lick (eds.), Handbook o/the Biology o/Aging, Van Nostrand, New York, 1977, pp. 582-638. 9 R.G. Allen, 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. Biochem. J., 216 (1983) 503-506. 10 E. Bernt and H.U. Bersmeyer, Inorganic peroxides. In H.U. Bergmeyer (ed.), Methods of Enzymatic Analysis, Vol. IV, Academic Press, New York, 1976. pp. 2246-2248. 11 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) 502-522. 12 H. Luck, Catalase. In H.U. Bergmeyer (ed.), Methods o f Enzymatic Analysis, Academic Press, New York, 1965, pp. 885-894. 13 H.P. Misra and I. Fridovieh, Superoxide dismutase; "positive" spectrophotometric assays. Anal Biochem., 79 (1976) 553-560.
336 14 H.J. Forman and A.B. Fisher, Antioxidant defenses. In D.L. Gilbert (ed.), Oxygen and Living Pro. cesses, Springer-Verlag, New York, 1981, pp. 235-249. 15 B. Chance, H. Sies and A. Boveris, Hydroperoxide metabolism in mammalian organs. Physiol. Rev., 59 (1979) 527-605. 16 B. Halliwell and J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J., 219 (1984) 1-14. 17 A.L. Tappel, Lipid peroxidation and fluorescent moleculax damage to membranes. In B.F. Trump and A.V. Arstila (eds.), Pathology o f Cell Membranes, Vol. I, Academic Press, New York, 1975, pp. 145-170. 18 H. Donate and R.S. Sohal, Lipofuscin. In J. Florini (¢d.), Handbook of Biochemistry in Aging. CRC Press, Cleveland, 1981, pp. 221-227. 19 R.S. Sohal, Metabolic rate, aging and lipofusein accumulation. In R.S. Sohal (ed.), Age Pigments, Elsevier/North Holland, Amsterdam, 1981, pp. 303- 316. 20 A.D. Blackett and D.A. Hall, The effects of vitamin E on mouse fitness and survival. Gerontology, 27(1981) 133-139. 21 R.S. Sohal and R.G. Allen, Relationship between metabolic rate, free radicals, differentiation and aging: a unified theory. In A.D. Woodhead (ed.), The Molecular Basis o f Aging, Plenum Press, New York (in press). 22 R.S. Sohal, K.J. Farmer, R.G. Allen and S.S. Ragiand, Effects of diethyldithiocaxbamate on life span, metabolic rate, superoxide dismutase, catalase, inorganic peroxides and glutathione in the adult male housefly, Musca domestica. Mech. Ageing Dev., 24 (1981) 175-183.
329
EFFECTS OF EXOGENOUS ANTIOXIDANTS ON THE LEVELS OF ENDOGENOUS ANTIOXIDANTS, LIPID-SOLUBLE FLUORESCENT MATERIAL AND LIFE SPAN IN THE HOUSEFLY, MUSCA DOMESTICA
R.S. SOHAL*, R.G. ALLEN, K.J. FARMER, R.K. NEWTON and P.L. TOY Department of Biology, Southern Methodist University, Dallas, TX 75275 (U.S.A.) (Received March 28th, 1985)
SUMMARY Effects of exogenous antioxidant administration (0.5% and 2% ascorbate,/j-carotene and a-tocopherol in sucrose) on life-span, metabolic rate, activities of superoxide dismutase and catalase, levels of glutathione, inorganic peroxides and chloroform-soluble fluorescent material (lipofuscin) were examined in adult male houseflies. Administration of antioxidants at a level of 0.5% did not affect life-span, whereas, 2% ascorbate and atocopherol decreased average life-span. Metabolic rate of flies was unaffected, except by 2% ascorbate, which caused a decrease. Superoxide dismutase activity Was depressed by 2% ascorbate at all ages, and by ~-carotene and a-tocopherol in older flies. Catalase activity was unaffected except by a-tocopherol at younger ages. Glutathione concentration was decreased by ascorbate and/j-carotene at both concentrations administered. Inorganic peroxides (H202) were increased by 2%/j-carotene and o~-tocopherol. Only high concentrations of ascorbate and/J-carotene decreased the level of soluble fluorescent material. Results suggest that administration of exogenous antioxidants causes a compensatory depression of endogenous defenses.
Key words: Antioxidants; Aging; Insects; Superoxide dismutase; Glutathione; Catalase
INTRODUC~ON The postulate that free radicals, generated during cellular metabolism, are the main cause of molecular damage underlying the aging process was first enunciated by Harman [1]. In a recent review [2], he has cited the life-lengthening effects of antioxidant administration to constitute experimental support of this hypothesis. However, results of numerous studies purporting to test the free radical theory of aging, based on the *To whom all correspondence should be addressed. 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 ElsevierScientific Publishers Ireland Ltd.
330 effects of a variety of natural and synthetic antioxidants, have not clearly supported this claim [ 3 - 5 ] . For example, Harman [6] had originally reported that two antioxidants, 2-mercaptoethylamine hydrochloride and butylated hydroxytoluene, increased the lifespan of mice. In a reinvestigation, Kohn [7] found that when survival of control mice was optimal, the same antioxidants had no effect on life-span. Only when the life-span of control groups was below the optimal level, antioxidants extended the median lifespan. Furthermore, it has been widely observed that antioxidant intake does not prolong the maximum life-span of experimental animals [3-5,7]. Since regimes which extend the average but not the maximum life-span of populations seem to do so by reducing the vulnerability of animals to age-independent causes of death rather than age-dependent death rate [8], it can be inferred that administration of exogenous antioxidants has no significant effect on the rate of aging. The matter of the interpretation of these results - e.g., whether or not they negate the predictions of free radical theory of aging - was recently addressed by Cutler [4,5]. Hy hypothesized that the net level of antioxidants, within a given cell type, remains relatively constant due to compensatory or homeostatic regulatory control of individual antioxidants. It was postulated that perturbations, induced by administration of exogenous antioxidants, would produce a compensatory decrease in a related endogenous antioxidant. The present study was conducted to test Cutler's hypothesis. Effects of the administration of ascorbate, /3-carotene, and a-tocopherol (which are natural antioxidants) on life-span, and levels of endogenous antioxidants, inorganic peroxides, and chloroform-soluble fluorescent material (presumably related to lipofuscin) are reported. Endogenous antioxidants investigated were: superoxide dismutase, catalase, and glutathione. MATERIALS AND METHODS
Chemicals Chemicals were obtained from Sigma, St. Louis, Missouri. Rearing and maintenance of insects Eggs were obtained from 7- to 10-day-old houseflies, belonging to a stock originally obtained from the Department of Zoology, the University of Cambridge (U.K.). Larvae were fed on moist CSMA (Chemical Specialties Manufacturer's Association) fly larval medium, obtained from Ralston Purina Co., Richmond, IN. After emergence from pupae, flies were segregated by sex and males were housed in 1-cubic-foot cages with 200 flies/ cage. Experimental flies were administered 0.5% or 2% ascorbate, 0-carotene, or atocopherol in sucrose. Controls were provided with pure sucrose as food. All groups of flies were maintained at 25°C with a relative humidity of about 40%. Methods for the determination of total glutathione, inorganic peroxide concentration and superoxide dismutase (SOD) activity have been previously described in detail [9] and shall only briefly be outlined here. Inorganic peroxide (H202) concentration was
331 determined by the method o f Bernt and Bergmeyer [10]; glutathione was measured according to the method o f Tietze [ 11 ] ; catalase activity was determined by the method ffescribed by Luck [12] ; total SOD activity was estimated by the direct method of Misra and Fridovich [13]; the activity of the mangano-isozyme was also measured by this method except that the assay mixture contained 1 mM KCN. RESULTS
Life-span Effects o f the intake of 0.5% and 2% ascorbate,//-carotene and a-tocopherol, in sucrose, on the longevity o f the male housefly are shown in Table I. As compared to the controls, there was no change in the life-span o f flies administered 0.5% of these antioxidants. However, at 2% concentration, ascorbate decreased life-span by about half and a-tocopherol by about a fourth, fl-Carotene at this high concentration had no effect on longevity.
Metabolic rate Rate o f oxygen utilization was measured at 48-h intervals between 2 and 12 days o f age. Ascorbate at 2% concentration significantly decreased (P < 0.05) the rate o f oxygen consumption in flies (Table I). As compared to controls, no significant differences in metabolic rate were observed in other groups.
Superoxide dismutase (SOD) Total SOD activity, i.e. cyanide-insensitive plus cyanide-sensitive activity, was measured in 3-, 6-, 9-, and 12-day-old flies. At a 0.5% concentration, none of these antioxidants had an appreciable effect on SOD activity (Table II). However, at a 2% concentra-
TABLE I EFFECT OF EXOGENOUS ANTIOXIDANT ADMINISTRATION ON LIFE-SPAN AND METABOLIC RATE OF ADULT MALE HOUSEFLIES
Group
Average life-span (days)
Metabolic rate (M OJh/mg body wt)
Control (for 0.5%) 0.5% Ascorbate 0.5% &Carotene 0.5% a-Tocopherol Control (for 2%) 2% Ascorbate 2% fl-Carotene 2% c~-Toeopherol
21.6 ± 7.5 18.6 + 5.4 19.7 ± 7.1 19.6 ± 6.7 20.7 ± 5.9 9.9 ± 3.8* 20.1 ± 8.8 15.3 ± 4.2*
7.8 _4-1.1 7.2 _+2.6 9.4 ± 3.7 7.1 ± 1.3 8.0 ± 2.5 5.6 ± 2.7* 7.1 ± 2.1 7.7 ± 2.6
+S.D. *P < 0.05
332 TABLE II EFFECT OF ANTIOXIDANT ADMINISTRATION ON SUPEROXIDE DISMUTASE (SOD) ACTIVITY IN MALE HOUSEFLIES AT DIFFERENT AGES
Group
Control (for 5%) 0.5% Aseorbate 0.5% a-Carotene 0.5% a-Toeopherol Control (for 2%) 2% Aseorbate 2% B-Carotene 2% a-Tocopherol
SOD activity (units/ragprotein) 3-day
6-day
9-day
12-day
18.8 17.7 22.4 18.2 20.5 17.3 18.1 20.0
26.0 27.3 26.0 27.3 26.1 21.6 27.1 20.0
31.0 29.3 28.0 31.7 26.5 19.6 23.6 19.9
27.4 24.9 26.5 28.0 26.3
_+0.8 _+1.4 _+0.4 + 1.5 _+1.1 _+1.1" _+1.4 _+1.1
_+0.6 _+1.3 -+ 0.6 _+0.3 -+ 1.0 _+2.0* + 1.0 -+0.7*
_+0.7 _+0.5 _+0.4 _+0.5 + 0.7 -+0.6* _+0.5* _+0.6*
_+1.1 _+0.5 -+0.4 _+1.6 _+0.7
24.6 -+ 1.0 20.8 _+1.7*
~.D.
*P < 0.05.
tion, SOD activity was decreased. Ascorbate a d m i n i s t r a t i o n significantly (P < 0.05) reduced S O D a c t i v i t y at all ages e x a m i n e d , l$Carotene and a - t o c o p h e r o l decreased SOD activity in older flies.
Catalase Catalase activity was m e a s u r e d in 4-, 8-, and 12-day-old flies (Table III). ot-Tocopherol a d m i n i s t r a t i o n at b o t h 0.5% and 2% i n d u c e d a slight decrease in catalase activity at 4 days o f age, b u t no appreciable changes w e r e observed at o t h e r ages or in o t h e r groups.
TABLE III EFFECT OF ANTIOXIDANT ADMINISTRATION ON CATALASE ACTWITY IN ADULT MALE HOUSEFLIES AT VARIOUS AGES
Group
Catala~ activity (units/ragprotein) 4~y
Control (for 0.5%) 0.5% Ascorbate 0.5% &Carotene 0.5% a-Tocopherol Control (for 2%) 2% Ascorbate 2% a-Carotene 2% a-Toeopherol _+S.D.
12.3 10.7 9.2 7.9 12.6 11.1 12.4 10.7
+ 0.4 + 0.7 _+0.3 _+0.2 _+0.7 _+0.1 _+0.6 -+0.6
8~y
12~y
8.7 8.2 8.9 7.6 8.1 7.6 7.5 7.1
7.3 8.8 7.4 7.9 6.5 6.8 6.7 6.6
_+0.2 _+0.2 + 0.2 + 0.2 _+0.3 _+1.9 _+0.3 -+0.4
+ 0.2 _+0.3 _+0.4 _+0.3 +0.3 _+0.2 _+0.3 -+0.7
333 TABLE IV EFFECT OF ANTIOXIDANT INTAKE ON THE LEVEL OF TOTAL GLUTATHIONE IN HOUSEFLIES OF VARIOUS AGES
Group
Control (for 0.5%) 0.5% Ascorbate 0.5% f-Carotene 0.5% a-Tocopherol Control (for 2%) 2% Ascorbate 2% f-Carotene 2% a-Tocopherol
Glutathione concentration (~g/g body wt) 4-day
8-day
12-day
342 290 295 327 340 270 282 332
358 319 373 418 346 285 334 369
300 277 293 287 374 226 340 345
+5 + 12" _+9* _+ 11 _+4 _+ 1" + 9* -+ 10
_+9 _+2* _+5 _+6 _+2 _+3* -+ 2 _+6
_+6 _+8* _+6 _+8 _+11 -+4* -+ 11 +6
_+S.D. *P < 0.05.
Glutathione G l u t a t h i o n e was m e a s u r e d at 4-, 8-, and 12-days o f age (Table IV). A d m i n i s t r a t i o n o f ascorbate at 0.5% and 2% decreased glutathione level, fl-Carotene also appeared to decrease glutathione levels in y o u n g e r flies at b o t h c o n c e n t r a t i o n s used. et-Tocopherol had n o e f f e c t on g l u t a t h i o n e c o n c e n t r a t i o n .
Inorganic peroxides C o n c e n t r a t i o n o f inorganic peroxides 0-I202) was c o m p a r e d in 12-day-old flies (Table V). A s c o r b a t e a d m i n i s t r a t i o n decreased inorganic p e r o x i d e c o n c e n t r a t i o n in p r o p o r t i o n TABLE V EFFECT OF EXOGENOUS ANTIOXIDANTS ON CONCENTRATION OF INORGANIC PEROXIDES AND CHLOROFORM-SOLUBLE FLUORESCENT MATERIAL (SFM) IN 12-DAY-OLD MALE HOUSEFLIES
Group
Inorganic peroxides (l~g/gbody wt}
SFM (Units/rag body wt)
Control (for 0.5%) 0.5% Ascorbate 0.5% #-Carotene 0.5% a-Toeopherol Control (for 2%) 2% Ascorbate 2% #-Carotene 2% a-Toeopheml
19.8 11.6 17.0 22.3 30.1 9.8 39.8 36.1
0.020 0.024 0.022 0.021 0.023 0.018 0.016 0.025
+ S.D. *P < 0.05.
+ 0.3 _+0.8* + 1.4 + 0.5 _+0.9 + 0.9* + 1.3" _+1.5*
_+0.002 + 0.004 _+0.004 _+0.002 _+0.002 _+0.004* _+0.001" _+0.005
334 to dose./I-Carotene had no effect at 0.5%, but increased the organic peroxide level when administered at a concentration of 2%. On the other hand, a-tocopherol increased inorganic peroxide concentration in proportion to dose.
Soluble fluorescent material (SFM) The concentration of SFM in chloroform extracts of tissues was compared at 12 days of age (Table V). SFM level was unaffected by the 0.5% concentration of antioxidants. A concentration of 2% ascorbate and/3-carotene decreased SFM concentration while atocopherol had no appreciable effect. DISCUSSION In order to evaluate the possible involvement of free radicals in the aging process, it is imperative to recognize both the overlapping and the compensational nature of various endogenous antioxidants. For example, superoxide dismutase and glutathione are both effective scavengers of superoxide; catalase and glutathione can independently eliminate HzO2 [14]. GSH and ascorbate can both donate reducing equivalents to oxidants and can also react with a-tocopherol radical [14,15]. It should also be recognized that various enzymic and non-enzymic antioxidants are compartmentalized in specific intracellular domains, e.g. glutathione and ascorbate are present in the aqueous phase, whereas, t3carotene and a-tocopherol exist in the hydrocarbon core of the membranes [ 15]. Results of this study indicate that the average life-span of flies is not lengthened by antioxidant administration and relatively high intake of ascorbate and a-tocopherol may indeed be toxic. The most pronounced effects on mortality were produced by ascorbate which also depressed SOD activity and glutathione concentration in the body. Inorganic peroxide (H2Oz) concentration was lower in ascorbate-fed flies than in the control, possibly due to an increase in intracellular reducing equivalents. The life-shortening effect of ascorbate may have resulted from an unfavorable alteration in cellular redox state. Since inorganic peroxides and SFM were both lowered in the ascorbate-administered insects, it would seem improbable that the deleterious effects resulted from ascorbatedependent iron-catalyzed free radical reactions [ 16]. Present results deafly indicate the compensatory effects of exogenous antioxidants on endogenous antioxidants, as hypothesized by Cutler [4,5]. Administration ofascorbate, which has an overlapping function with glutathione and SOD was found to depress cellular levels of both. Effects of/I-carotene and a-tocophcrol were less pronounced, but they also tended to decrease SOD activity. In addition, ~carotene decreased ghitathione concentration. Although these lipid-soluble antioxidants did not appear to affect catalase activity, inorganic peroxide levels were elevated by an unknown mechanism in treated flies. Chloroform-soluble fluorescent material (SFM) is widely believed to be arLend-product of free radical-induced oxidation of polyunsaturated fatty acids and other macromolecules [17]. SFM has been shown to progressively accumulate with age in a variety of organisms [ 18,19]. Results of this study indicate that at low concentrations exogenous antioxidants
335 have little effect on SFM; higher doses tend to decrease SFM accumulation without beneficial effect on life-span. Blackett and Hall [20] have also observed this phenomenon in rats. Results o f our previous studies which examined the effects o f experimentally-induced oxidative stress on endogenous antioxidants also support the compensational hypothesis [21]. F o r example, inhibition o f SOD [22] and catalase [9] in the houseflies caused an increase in the level o f glutathione and depression o f metabolic rate. This suggests that organisms adapt to the imposed oxidative stress b y modulation o f endogenous antioxidants and b y decreasing the rate o f oxidative metabolism, which is a major source o f oxygen radicals. In conclusion, results o f this study neither support nor negate the postulates o f the free radical theory o f aging. However, they do indicate, that due to the compensational nature o f antioxidant defense mechanisms, the use o f a high antioxidant regime to test the predictions o f the free radical theory o f aging is invalid and may in fact be misleading. ACKNOWLEDGEMENTS This study was supported b y a grant from the Glenn F o u n d a t i o n for Medical Research. REFERENCES 1 D.H. Harman, Aging: A theory based on free radical and radiation chemistry. J. GerontoL, 11 (1980) 298-300. 2 D.H. Harman, The free radical theory of aging. In W.A. Pryor (ed.), Free Radicals in Biology, Vol. V, Academic Press, New York, 1982, pp. 255-275. 3 A.K. Balin, Testing the free radical theory of aging. In R.C. Adelman and G.C. Roth (eds.), Testing the Theories o/Aging, CRC Press, Boca Raton, Florida, 1982, pp. 137-182. 4 R.G. Cutler, Evolutionary biology of aging and longevity in mammalian species. In J.E. Johnson Jr. (ed.), Aging and Cell Function, Plenum Press, New York, 1984, pp. 1-147. 5 R.G. Cutler, Antioxidants, aging, and longevity. In W.A. Pryor (ed.), b3"eeRadicals in Biology, Vol. VI, Academic Press, New York, 1984, pp. 371-437. 6 D.H. Harman, Free radical theory of aging: Effects of free radical reaction inhibitors on the mortality rate of male LAF, mice. J. Gerontol., 23 (1968) 475-482. 7 R.R. Kohn, Effect of antioxidants on life-span of C57BL mice. J. GerontoL, 26 (1971) 378-380. 8 G.A. Sacher, Life table modifications and life prolongation. In C.E. Finch and L. Hay/lick (eds.), Handbook o/the Biology o/Aging, Van Nostrand, New York, 1977, pp. 582-638. 9 R.G. Allen, 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. Biochem. J., 216 (1983) 503-506. 10 E. Bernt and H.U. Bersmeyer, Inorganic peroxides. In H.U. Bergmeyer (ed.), Methods of Enzymatic Analysis, Vol. IV, Academic Press, New York, 1976. pp. 2246-2248. 11 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) 502-522. 12 H. Luck, Catalase. In H.U. Bergmeyer (ed.), Methods o f Enzymatic Analysis, Academic Press, New York, 1965, pp. 885-894. 13 H.P. Misra and I. Fridovieh, Superoxide dismutase; "positive" spectrophotometric assays. Anal Biochem., 79 (1976) 553-560.
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