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Effects of ambient temperature on free radical generation, antioxidant defenses and life span in the adult housefly, Musca domestica
ExperimentalGerontology,Vol. 22, pp. 59-65, 1987
0531-5565/87 $3.00 + .00 Copyright © 1987 Pergamon Journals Ltd
Printed in the USA. All rights reserved.
EFFECTS OF AMBIENT TEMPERATURE ON FREE RADICAL GENERATION, ANTIOXIDANT DEFENSES AND LIFE SPAN IN THE ADULT HOUSEFLY, MUSCA DOMESTICA
K.J. FARMER and R.S. SOHAL Department of Biology, Southern Methodist University, Dallas, Texas 75275 Abstract-Life spans of poikilotherms like the housefly are shortened by elevation of
ambient temperature. The objective of this study was to examine the possible involvement of active oxygen species in temperature induced life-shortening of the adult male housefly. Effects of varied ambient temperature, 20°C and 28°C, on life span, cyanideresistant respiration, HzO2 concentration, superoxide dismutase (SOD) and catalase activities and glutathione (GSH) concentration were examined. Average life span of flies raised at 28°C was about 52% lower than those raised at 20°C. Rate of cyanide-resistant respiration, an indicator of oxygen free radical generation, was higher in flies raised at 28°C, whereas steady-state concentration of H202 was decreased at this temperature. Catalase activity and GSH concentration were lower at 28°C while SOD activity was unaffected by the ambient temperature. Results of this study suggest that life-shortening effects of elevated ambient temperature may be due, in part, to increased oxidative stress. Key Words: free radicals, aging, metabolic rate, temperature, glutathione, insects
INTRODUCTION TrmRE IS a large body of evidence indicating the existence of an inverse relationship between metabolic rate and life span (for review, see Sohal and Allen, 1986). Within viable limits, experimental regimes that decrease metabolic rate invariably extend life span of poikilotherms, and vice versa. Variation in ambient temperature has been frequently employed to vary metabolic rate of poikilotherms (for review, see Sohal, 1976, 1984, 1986). Because an inverse relationship also seems to exist between basal metabolic rate and life span of homeotherms (Sacher, 1977), it can be argued that an understanding of the mechanism underlying the relationship between rate of metabolism and life span may shed light on the nature of the aging process. It is now well known that active oxygen species are produced during the course of normal aerobic metabolism, with superoxide (O[) and hydrogen peroxide (H202) being the
(Received 7 August 1986, Accepted 24 September 1986)
59
60
K.J. FARMER AND R.S. SOHAL
initial products (Chance et al., 1979). Life-shortening effects of elevated ambient temperature have been postulated to be due to increased generation of oxygen free radicals, resulting in oxidative stress (Fleming et al., 1982; Sohal et al., 1985). Indirect evidence for increased free radical damage at elevated temperatures is provided by the increase in n-pentane exhalation and concentration of thiobarbituric acid (TBA) - reactants in houseflies reared at relatively higher temperatures (Sohal et al., 1985); these substances are endproducts of lipid peroxidation and are considered to be indicative of free radical induced lipid peroxidation. More direct evidence of increased generation of oxygen free radicals at higher ambient temperatures has not as yet been presented. Cyanide prevents the tetravalent reduction of molecular oxygen to water by inactivating cytochrome oxidase; hence, any oxygen consumed in the presence of inhibiting amounts of cyanide is assumed to be univalently reduced by sequential additions of electrons (Hassan and Fridovich, 1979). The rate of cyanide-resistant respiration has been shown to be related to the rate of oxygen free radical and hydroperoxide generation in cells (Freeman et al., 1982). The primary enzymatic defenses against O~ and H202, the initial products of univalent oxygen reduction, are provided by superoxide dismutase (SOD), which reduces 02 to H202, and by catalase, which converts H202 to H20. The main nonenzymatic protection is provided by glutathione (GSH), which can directly reduce H202, lipid peroxides and disulfides (for review, see Halliwell, 1981). Glutathione peroxidase, which eliminates H202 is absent in insects (Smith and Shrift, 1979). The general objective of this study was to elucidate the mechanism by which increased ambient temperature shortens the life span of poikilotherms. This study examines the relationship between ambient temperature, generation of intermediates of oxygen reduction and levels of antioxidant defenses. An increase in generation of oxygen free radicals with increased temperature would support the suggestion that metabolic rate affects life span through the mechanism of oxygen free radical generation. MATERIALS AND METHODS Rearing o f insects Eggs were obtained from 7- to 10-day old houseflies (Cambridge strain) and placed on moist CSMA fly larval medium obtained from the Ralston Purina Co., Richmond IN. After emergence from pupae, flies were housed in 1 ft 3 cages, 200 males/cage and maintained at either 28°C or 20°C. All flies were fed sucrose and water. Experimental procedures Procedures for some of the biochemical assays employed in this study have been previously reported in detail (Sohal et al., 1984a; Allen et al., 1984) and will only be briefly described here. SOD activity was measured by the positive method suggested by Misra and Fridovich (1976). Catalase activity was determined by the method of Luck (1965). The level of total glutathione was measured by a modification of the method described by Tietze (1969). Concentration of H202 was measured by the method of Bernt and Bergmeyer (1976). Protein was determined by the method of Lowry et al. (1951). Rates of total and CN--resistant respiration were measured as the amount of oxygen consumed in the presence or absence of lmM potassium cyanide, using a Clark-type oxy-
TEMPERATURE, FREE RADICALSAND AGING
61
gen electrode. Whole-body homogenates were prepared by homogenizing 12-15 flies, weighing about 0.15gm, in 1.0 ml 50mM potassium phosphate buffer, pH 7.1, containing 0. lmM MgSO4. Rate of total respiration was measured in both groups at 25°C by adding 0.3 ml homogenate to 2.7 ml air-saturated 50mM potassium phosphate buffer, pH 7.1, containing 0.1mM MgSO4 and 5.0mM succinate, and recording the change in percent oxygen concentration during a two minute time span. The rate of CN--resistant respiration was determined in the same manner except that 0.33 ml of 10mM KCN was added and the change in oxygen concentration was determined during an 8 min time period. RESULTS The mortality characteristics of houseflies, kept at 20 ° and 28°C, are presented as survivorship curves in Fig. 1. As expected, both the average as well as the maximum life span was considerably longer at the cooler temperature; the average life span being 21.9 + 6.2 (S.D.) days at 28°C and 45.4 ± 13.3 days at 20°C. Total and CN--resistant respiration were compared in whole body homogenates of 8-day old flies at 25°C (Table 1). The rate of total respiration was higher in homogenates of flies housed at 28 ° than those housed at 20°C. The percent of respiration that was insensitive to lmM cyanide was also greater in the former than the latter. The activities of total and cyanide-insensitive superoxide dismutase (SOD) were measured in 3-, 8- and 15-day old flies (Table 2). No significant differences were found in the activity of either cyanide-insensitive or cyanide-sensitive enzyme between the groups reared at the two different temperatures. Catalase activity was measured at 3, 8 and 15 days of age (Table 2). At all three ages, the activity was 15 to 33% lower in flies kept at 28 ° than those kept at 20°C. The levels of H202 (Table 1), compared in 8-day old flies, were approximately 20% higher in tissues of flies kept at 20 ° as compared to those at 28°C. Glutathione concentrations were measured at 3, 8 and 15 days of age (Table 2). Houseflies kept at 28°C had 12 to 24% lower levels of glutathione at all comparable ages than those housed at 20°C.
100
• ......o.o.O..ooo
oo Oo "o °°co
711
o
i .~
so
~
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28°c
\
,
"
2o ° c
\ k
\° °°ooo
°~o
Oo o-~ o
° • e--__...~
0
1;
2'0
3;
40
5'0
6'0
?lO
Age (days)
FIG. I. Survivorship curves of male houseflies, 200 flies/l cu.-ft cage, reared at 20°C and 28°C.
62
K.J. FARMER AND R.S. SOHAL
TABLE 1. TOTAL AND CN--RESISTANT RESPIRATION' AND LEVELS OF H 2 0 2 IN 8-DAY OLD HOUSEFLIES AT DIFFERENT AMBIENT TEMPERATURE t
Temperature Parameter Total Respiration (nmol O2/min/mg protein) CN--resistant Respiration (°70 of total) H202 (ttg/g wet weight)
20 ° C
28 ° C
18.7 ± 2.3
19.4 ± 1.6
3.4070 26.9 ± 3.5
5.2070 20.4 ± 3.6
1Rates of respiration of both groups were measured in vitro at 25oC.
DISCUSSION Results of this study indicate that increase in ambient temperature stimulates both the total in vitro respiration as well as the percent respiration that is insensitive to CN- inhibition. Levels of antioxidants, such as glutathione concentration and catalase activity, are decreased at higher temperature while SOD activity remains unaffected. The results indicate that the rate of cyanide-resistant respiration, when measured at the same temperature, is higher in flies aged at the higher temperature, suggesting that the potential for free radical generation is higher in these flies. However, the expected increase, at the higher temperature, in the concentration of H202, a product of free radical generation in vivo, did not occur. Since the levels of catalase activity and GSH concentration in flies kept at 28°C are 33°70 and 24°70 lower, respectively, than those kept at 20°C, it would be expected that the former would contain higher concentration of H202 than the latter. To the contrary, H202 levels were about 20°70 lower in flies housed at 28°C compared to those at 20°C. This apparent discrepancy between O~ generation in vitro and H202 levels in vivo may be attributable to the following processes: TABLE 2. ACTIVITIES OF SUPEROXIDE DISMUTASE (SOD) AND CATALASE AND CONCENTRATION OF GLUTATHIONE (GSH) IN HOUSEFLIES OF DIFFERENT AGES REARED AT 2 0 ° C AND 2 8 ° C
Age 3 days A ntioxidant
20 ° C
8 days 28 ° C
20 ° C
15 d a y s 28 ° C
20 ° C
28 ° C
SOD a
Total activity CN'-insensitive activity Catalase a GSH b
aunits/mg protein 4- S.D. b/tg/g wet weight ± S.D.
11.4 5.9 36.9 669
-*± ± ±
0.6 0.3 3.2 32
14.7 6.8 25.6 532
± ± ± ±
2.7 1.1 1.4 79
15.9 7.8 23.4 709
-¢~± ±
1.4 0.4 1.3 114
16.7 8.1 15.6 500
± ± ± ±
2.2 1.5 1.4 152
15.3 7.9 17.9 519
± ± ± ±
1.0 0.2 1.8 36
17.0 8.5 12.8 421
± ± ± ±
1.4 0.4 0.9 84
TEMPERATURE,FREERADICALSANDAGING
63
1. Since the rates of chemical reactions, including enzyme activity, increase with temperature within physiological limits, it is possible that lower concentrations of H202 at the higher temperature may be due to increased cataiase activity and GSH reactivity. 2. It is also possible that H202 may be eliminated at higher temperatures more rapidly because of its increased reactivity with other molecules, such as Fe 2÷ in the Fenton Reaction (Halliwell and Gutteridge, 1984). 3. Another possibility is that sources of H202 generation may vary at different temperatures. H202 is also produced in cells by reactions other than those involving oxygen reduction by the univalent pathway in the mitochondria. For example, in peroxisomes, enzymes catalyzing amino acid and fatty acid oxidation produce H202 as a by-product (for references, see Masters and Holmes, 1977). It is possible that, at lower temperatures, these alternate sources may contribute relatively greater amounts of H202. Previous studies have shown that houseflies and other insects are under greater oxidative stress at higher temperatures, as evidenced by the increase in the rate of n-pentane production (Sohal et al., 1985), level of TBA-reactants (Sohal et al., 1981) and rate of lipofuscin accumulation (Miquel et al., 1974, 1976; Sheldahl and Tappel, 1974; McArthur and Sohal, 1982), all of which are associated with lipid peroxidation. In the present study, it was found that CN--resistant respiration is increased in flies at 28°C as compared to 20°C while SOD activity remains unaffected, which would tend to increase O~ concentration in cells. Such an increase in the concentration of O~ may increase the generation of hydroxyl radical (OH.), which is thought to be primarily responsible for the initiation of lipid peroxidation reactions (Halliwell, 1981), due to reaction between O[ and H202. If indeed accelerated H~O2 elimination is due to its increased interaction with O[ in the above manner, then this mechanism could account for both the oxidative damage as well as lowered H202 levels observed at the higher temperature. The reasons for the decrease in catalase activity and GSH concentration at elevated temperatures can not be precisely understood on the basis of this study. It is, however, possible that increased O~ generation at elevated temperatures may be responsible for inactivation of catalase (Kono and Fridovich, 1982). The decrease in GSH levels may be attributable to its increased oxidation at the elevated temperature due to the enhanced rate of free radical generation. Results of this study again emphasize the complexity of the temperature effects on the metabolism of poikilotherms. For example, in contrast to the present study, increase in metabolic rate of the houseflies, induced by elevation of physicai activity, resulted in augmentation rather than depression of H202 and GSH levels (Sohal et al., 1984b). It was previously pointed out that alterations in ambient temperature affect, unequally, a variety of processes other than the rate of metabolism (Sohal, 1976, 1986). For instance, the rate of protein synthesis in many poikilotherms is lower at relatively higher temperatures (for review, see Prosser, 1973). In conclusion, the results of this study suggest that elevated temperature may shorten the life span of houseflies by an increase in the rate of free radical generation and a decrease in the level of antioxidant defenses. Acknowledgments--This research was supported by grants from the Glenn Foundation for Medical Research. Technical assistance of Mrs. Izabella Leznicki is gratefully acknowledged.
64
K.J. FARMERANDR.S. SOHAL REFERENCES
Allen, R.G., Farmer, K.J., Newton, R.K. and Sohal, R.S. Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, eatalase, glutathione reductase, inorganic peroxides and glutathione in the adult housefly. Comp. Biochem. Physiol. 78C, 283-288, 1984. Bernt, E. and Bergmeyer, H.U. Inorganic peroxides. In: Methods of Enzymatic Analysis, Bergmeyer, H.U., (Editor), pp. 2246-2248, Academic Press, New York, 1976. Chance, B., Sies, H. and Boveris, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-603, 1979. Fleming, J.E., Miquel, J., Cottrell, S.F., Yengoyan, L.S. and Economos, A.C. Is cell aging caused by respirationdependent injury to the mitochondrial genome? Gerontol. 28, 44-53, 1982. Freeman, B.A., Topolosky, M.K. and Crapo, J.D. Hyperoxia increases oxygen radical production in rat lung homogenates. Arch. Biochem. Biophys. 216, 477-484, 1982. Halliwell, B. Free radicals, oxygen toxicity and ageing. In: Age Pigments, Sohal, R.S., (Editor), pp. 1-62, Elsevier/North Holland, Amsterdam, 1981. HaUiwen, B. and Gutteridge, J.M.C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1-14, 1984. Hassan, H.M. and Fridovich, I. Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch. Biochem. Biophys. 196, 385-395, 1979. Kono, Y. and Fridovich, I. Superoxide radical inhibits eatalase. J. Biol. Chem. 257, 5751-5754, 1982. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and RandaU, R.I. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275, 1951. Luck, H. Catalase, In: Methods of Enzymatic Analysis, Bergmeyer, H., (Editor), pp. 885-894, Academic Press, New York, 1965. Masters, C. and Holmes, R. Peroxisomes: new aspects of cell physiology and biochemistry, Physiol. Rev. 57, 816-882, 1977. McArthur, M.C. and Sohal, R.S. Relationship between metabolic rate, aging, lipid peroxidation and fluorescent age pigment in the milkweed bug, Oncopeltusfasciatus (Hemiptera), J. Gerontol. 37, 268-274, 1982. Miquel, J., Tappel, A., Dillard, C., Herman, M. and Bensch, K. Fluorescent products and lysosomal components in aging Drosophila melanogaster. J. Gerontol. 29, 622-637, 1974. Miquel, J., Lundgren, P.R., Benseh, K.J. and Atlan, H. Effect of temperature on the life span, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Dev. 5, 347-370, 1976. Misra, H.P. and Fridovich, I. Superoxide dismutase: "positive" spectrophotometric assays. Analyt. Biochem. 79, 553-560, 1976. Prosser, C.L. Comparative Animal Physiology, 3rd ed., ch. 3., Saunders, Philadelphia, 1973. Sacher, G.A. Life table modification and life prolongation. In: The Biology of Aging, Finch, C.E. and Hayflick, L., (Editors), pp. 582-638, Van Nostrand Reinhold, New York, 1977. Sheldahl, J.A. and Tappel, A.L. Fluorescent products from aging Drosophila melanogaster: an indication of free radical lipid peroxidation damage. Exp. Gerontol. 9, 33-41, 1974. Smith, J. and Shrift, A. Phylogenetic distribution of glutathione peroxidase. Comp. Biochem. Physiol. 63B, 39-44, 1979. Sohal, R.S. Metabolic rate and life span. In: Interdisciplinary Topics in Gerontology, Cutler, R.G., (Editor), Vol. 9, pp. 25-40, S. Karger, Basel, 1976. Sohal, R.S. Metabolic rate, free radicals and aging. In: Free Radicals in Molecular Biology, Aging, and Disease, Armstrong, D., Sohal, R.S., Cutler, R.G. and Slater, T.F., (Editors), pp. 119-127, Raven Press, New York, 1984. Sohal, R.S. The rate of living theory: a contemporary interpretation. In: Insect Aging, Collatz, K.G. and Sohal, R.S., (Editors), pp. 23-44, Springer-Verlag, Berlin, 1986. Sohal, R.S. and Allen, R.G. Relationship between oxygen metabolism, aging, and development. Adv. Free Rad. Biol. Med. 2, 117-160, 1986. Sohal, R.S., Donato, H. and Biehl, E.R. Effect of age and metabolic rate on lipid peroxidation in the housefly, Musca domestica. Mech. Ageing Dev. 16, 159-167, 1981. Sohal, R.S., Farmer, K.J., Allen, R.G. and Cohen, N.R. 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, 185-195, 1984a. Sohal, R.S., Allen, R.G., Farmer, K.J. and Procter, J. Effect of physical activity on superoxide dismutase,
TEMPERATURE,FREERADICALSANDAGING
65
catalase, inorganic peroxides and glutathione in the housefly, Musca domestica. Mech. Ageing Dev. 26, 75-81, 1984b. Sohal, R.S., Muller, A., Koletzko, B. and Sies, H. Effect of age and ambient temperature on n-pentane production in adult housefly, Musca domestica. Mech. Ageing Dev. 29, 317-326, 1985. Tietze, F. Enzymic method for the quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Analyt. Biochem. 27, 502-522, 1969.
0531-5565/87 $3.00 + .00 Copyright © 1987 Pergamon Journals Ltd
Printed in the USA. All rights reserved.
EFFECTS OF AMBIENT TEMPERATURE ON FREE RADICAL GENERATION, ANTIOXIDANT DEFENSES AND LIFE SPAN IN THE ADULT HOUSEFLY, MUSCA DOMESTICA
K.J. FARMER and R.S. SOHAL Department of Biology, Southern Methodist University, Dallas, Texas 75275 Abstract-Life spans of poikilotherms like the housefly are shortened by elevation of
ambient temperature. The objective of this study was to examine the possible involvement of active oxygen species in temperature induced life-shortening of the adult male housefly. Effects of varied ambient temperature, 20°C and 28°C, on life span, cyanideresistant respiration, HzO2 concentration, superoxide dismutase (SOD) and catalase activities and glutathione (GSH) concentration were examined. Average life span of flies raised at 28°C was about 52% lower than those raised at 20°C. Rate of cyanide-resistant respiration, an indicator of oxygen free radical generation, was higher in flies raised at 28°C, whereas steady-state concentration of H202 was decreased at this temperature. Catalase activity and GSH concentration were lower at 28°C while SOD activity was unaffected by the ambient temperature. Results of this study suggest that life-shortening effects of elevated ambient temperature may be due, in part, to increased oxidative stress. Key Words: free radicals, aging, metabolic rate, temperature, glutathione, insects
INTRODUCTION TrmRE IS a large body of evidence indicating the existence of an inverse relationship between metabolic rate and life span (for review, see Sohal and Allen, 1986). Within viable limits, experimental regimes that decrease metabolic rate invariably extend life span of poikilotherms, and vice versa. Variation in ambient temperature has been frequently employed to vary metabolic rate of poikilotherms (for review, see Sohal, 1976, 1984, 1986). Because an inverse relationship also seems to exist between basal metabolic rate and life span of homeotherms (Sacher, 1977), it can be argued that an understanding of the mechanism underlying the relationship between rate of metabolism and life span may shed light on the nature of the aging process. It is now well known that active oxygen species are produced during the course of normal aerobic metabolism, with superoxide (O[) and hydrogen peroxide (H202) being the
(Received 7 August 1986, Accepted 24 September 1986)
59
60
K.J. FARMER AND R.S. SOHAL
initial products (Chance et al., 1979). Life-shortening effects of elevated ambient temperature have been postulated to be due to increased generation of oxygen free radicals, resulting in oxidative stress (Fleming et al., 1982; Sohal et al., 1985). Indirect evidence for increased free radical damage at elevated temperatures is provided by the increase in n-pentane exhalation and concentration of thiobarbituric acid (TBA) - reactants in houseflies reared at relatively higher temperatures (Sohal et al., 1985); these substances are endproducts of lipid peroxidation and are considered to be indicative of free radical induced lipid peroxidation. More direct evidence of increased generation of oxygen free radicals at higher ambient temperatures has not as yet been presented. Cyanide prevents the tetravalent reduction of molecular oxygen to water by inactivating cytochrome oxidase; hence, any oxygen consumed in the presence of inhibiting amounts of cyanide is assumed to be univalently reduced by sequential additions of electrons (Hassan and Fridovich, 1979). The rate of cyanide-resistant respiration has been shown to be related to the rate of oxygen free radical and hydroperoxide generation in cells (Freeman et al., 1982). The primary enzymatic defenses against O~ and H202, the initial products of univalent oxygen reduction, are provided by superoxide dismutase (SOD), which reduces 02 to H202, and by catalase, which converts H202 to H20. The main nonenzymatic protection is provided by glutathione (GSH), which can directly reduce H202, lipid peroxides and disulfides (for review, see Halliwell, 1981). Glutathione peroxidase, which eliminates H202 is absent in insects (Smith and Shrift, 1979). The general objective of this study was to elucidate the mechanism by which increased ambient temperature shortens the life span of poikilotherms. This study examines the relationship between ambient temperature, generation of intermediates of oxygen reduction and levels of antioxidant defenses. An increase in generation of oxygen free radicals with increased temperature would support the suggestion that metabolic rate affects life span through the mechanism of oxygen free radical generation. MATERIALS AND METHODS Rearing o f insects Eggs were obtained from 7- to 10-day old houseflies (Cambridge strain) and placed on moist CSMA fly larval medium obtained from the Ralston Purina Co., Richmond IN. After emergence from pupae, flies were housed in 1 ft 3 cages, 200 males/cage and maintained at either 28°C or 20°C. All flies were fed sucrose and water. Experimental procedures Procedures for some of the biochemical assays employed in this study have been previously reported in detail (Sohal et al., 1984a; Allen et al., 1984) and will only be briefly described here. SOD activity was measured by the positive method suggested by Misra and Fridovich (1976). Catalase activity was determined by the method of Luck (1965). The level of total glutathione was measured by a modification of the method described by Tietze (1969). Concentration of H202 was measured by the method of Bernt and Bergmeyer (1976). Protein was determined by the method of Lowry et al. (1951). Rates of total and CN--resistant respiration were measured as the amount of oxygen consumed in the presence or absence of lmM potassium cyanide, using a Clark-type oxy-
TEMPERATURE, FREE RADICALSAND AGING
61
gen electrode. Whole-body homogenates were prepared by homogenizing 12-15 flies, weighing about 0.15gm, in 1.0 ml 50mM potassium phosphate buffer, pH 7.1, containing 0. lmM MgSO4. Rate of total respiration was measured in both groups at 25°C by adding 0.3 ml homogenate to 2.7 ml air-saturated 50mM potassium phosphate buffer, pH 7.1, containing 0.1mM MgSO4 and 5.0mM succinate, and recording the change in percent oxygen concentration during a two minute time span. The rate of CN--resistant respiration was determined in the same manner except that 0.33 ml of 10mM KCN was added and the change in oxygen concentration was determined during an 8 min time period. RESULTS The mortality characteristics of houseflies, kept at 20 ° and 28°C, are presented as survivorship curves in Fig. 1. As expected, both the average as well as the maximum life span was considerably longer at the cooler temperature; the average life span being 21.9 + 6.2 (S.D.) days at 28°C and 45.4 ± 13.3 days at 20°C. Total and CN--resistant respiration were compared in whole body homogenates of 8-day old flies at 25°C (Table 1). The rate of total respiration was higher in homogenates of flies housed at 28 ° than those housed at 20°C. The percent of respiration that was insensitive to lmM cyanide was also greater in the former than the latter. The activities of total and cyanide-insensitive superoxide dismutase (SOD) were measured in 3-, 8- and 15-day old flies (Table 2). No significant differences were found in the activity of either cyanide-insensitive or cyanide-sensitive enzyme between the groups reared at the two different temperatures. Catalase activity was measured at 3, 8 and 15 days of age (Table 2). At all three ages, the activity was 15 to 33% lower in flies kept at 28 ° than those kept at 20°C. The levels of H202 (Table 1), compared in 8-day old flies, were approximately 20% higher in tissues of flies kept at 20 ° as compared to those at 28°C. Glutathione concentrations were measured at 3, 8 and 15 days of age (Table 2). Houseflies kept at 28°C had 12 to 24% lower levels of glutathione at all comparable ages than those housed at 20°C.
100
• ......o.o.O..ooo
oo Oo "o °°co
711
o
i .~
so
~
\
Oooo \o
28°c
\
,
"
2o ° c
\ k
\° °°ooo
°~o
Oo o-~ o
° • e--__...~
0
1;
2'0
3;
40
5'0
6'0
?lO
Age (days)
FIG. I. Survivorship curves of male houseflies, 200 flies/l cu.-ft cage, reared at 20°C and 28°C.
62
K.J. FARMER AND R.S. SOHAL
TABLE 1. TOTAL AND CN--RESISTANT RESPIRATION' AND LEVELS OF H 2 0 2 IN 8-DAY OLD HOUSEFLIES AT DIFFERENT AMBIENT TEMPERATURE t
Temperature Parameter Total Respiration (nmol O2/min/mg protein) CN--resistant Respiration (°70 of total) H202 (ttg/g wet weight)
20 ° C
28 ° C
18.7 ± 2.3
19.4 ± 1.6
3.4070 26.9 ± 3.5
5.2070 20.4 ± 3.6
1Rates of respiration of both groups were measured in vitro at 25oC.
DISCUSSION Results of this study indicate that increase in ambient temperature stimulates both the total in vitro respiration as well as the percent respiration that is insensitive to CN- inhibition. Levels of antioxidants, such as glutathione concentration and catalase activity, are decreased at higher temperature while SOD activity remains unaffected. The results indicate that the rate of cyanide-resistant respiration, when measured at the same temperature, is higher in flies aged at the higher temperature, suggesting that the potential for free radical generation is higher in these flies. However, the expected increase, at the higher temperature, in the concentration of H202, a product of free radical generation in vivo, did not occur. Since the levels of catalase activity and GSH concentration in flies kept at 28°C are 33°70 and 24°70 lower, respectively, than those kept at 20°C, it would be expected that the former would contain higher concentration of H202 than the latter. To the contrary, H202 levels were about 20°70 lower in flies housed at 28°C compared to those at 20°C. This apparent discrepancy between O~ generation in vitro and H202 levels in vivo may be attributable to the following processes: TABLE 2. ACTIVITIES OF SUPEROXIDE DISMUTASE (SOD) AND CATALASE AND CONCENTRATION OF GLUTATHIONE (GSH) IN HOUSEFLIES OF DIFFERENT AGES REARED AT 2 0 ° C AND 2 8 ° C
Age 3 days A ntioxidant
20 ° C
8 days 28 ° C
20 ° C
15 d a y s 28 ° C
20 ° C
28 ° C
SOD a
Total activity CN'-insensitive activity Catalase a GSH b
aunits/mg protein 4- S.D. b/tg/g wet weight ± S.D.
11.4 5.9 36.9 669
-*± ± ±
0.6 0.3 3.2 32
14.7 6.8 25.6 532
± ± ± ±
2.7 1.1 1.4 79
15.9 7.8 23.4 709
-¢~± ±
1.4 0.4 1.3 114
16.7 8.1 15.6 500
± ± ± ±
2.2 1.5 1.4 152
15.3 7.9 17.9 519
± ± ± ±
1.0 0.2 1.8 36
17.0 8.5 12.8 421
± ± ± ±
1.4 0.4 0.9 84
TEMPERATURE,FREERADICALSANDAGING
63
1. Since the rates of chemical reactions, including enzyme activity, increase with temperature within physiological limits, it is possible that lower concentrations of H202 at the higher temperature may be due to increased cataiase activity and GSH reactivity. 2. It is also possible that H202 may be eliminated at higher temperatures more rapidly because of its increased reactivity with other molecules, such as Fe 2÷ in the Fenton Reaction (Halliwell and Gutteridge, 1984). 3. Another possibility is that sources of H202 generation may vary at different temperatures. H202 is also produced in cells by reactions other than those involving oxygen reduction by the univalent pathway in the mitochondria. For example, in peroxisomes, enzymes catalyzing amino acid and fatty acid oxidation produce H202 as a by-product (for references, see Masters and Holmes, 1977). It is possible that, at lower temperatures, these alternate sources may contribute relatively greater amounts of H202. Previous studies have shown that houseflies and other insects are under greater oxidative stress at higher temperatures, as evidenced by the increase in the rate of n-pentane production (Sohal et al., 1985), level of TBA-reactants (Sohal et al., 1981) and rate of lipofuscin accumulation (Miquel et al., 1974, 1976; Sheldahl and Tappel, 1974; McArthur and Sohal, 1982), all of which are associated with lipid peroxidation. In the present study, it was found that CN--resistant respiration is increased in flies at 28°C as compared to 20°C while SOD activity remains unaffected, which would tend to increase O~ concentration in cells. Such an increase in the concentration of O~ may increase the generation of hydroxyl radical (OH.), which is thought to be primarily responsible for the initiation of lipid peroxidation reactions (Halliwell, 1981), due to reaction between O[ and H202. If indeed accelerated H~O2 elimination is due to its increased interaction with O[ in the above manner, then this mechanism could account for both the oxidative damage as well as lowered H202 levels observed at the higher temperature. The reasons for the decrease in catalase activity and GSH concentration at elevated temperatures can not be precisely understood on the basis of this study. It is, however, possible that increased O~ generation at elevated temperatures may be responsible for inactivation of catalase (Kono and Fridovich, 1982). The decrease in GSH levels may be attributable to its increased oxidation at the elevated temperature due to the enhanced rate of free radical generation. Results of this study again emphasize the complexity of the temperature effects on the metabolism of poikilotherms. For example, in contrast to the present study, increase in metabolic rate of the houseflies, induced by elevation of physicai activity, resulted in augmentation rather than depression of H202 and GSH levels (Sohal et al., 1984b). It was previously pointed out that alterations in ambient temperature affect, unequally, a variety of processes other than the rate of metabolism (Sohal, 1976, 1986). For instance, the rate of protein synthesis in many poikilotherms is lower at relatively higher temperatures (for review, see Prosser, 1973). In conclusion, the results of this study suggest that elevated temperature may shorten the life span of houseflies by an increase in the rate of free radical generation and a decrease in the level of antioxidant defenses. Acknowledgments--This research was supported by grants from the Glenn Foundation for Medical Research. Technical assistance of Mrs. Izabella Leznicki is gratefully acknowledged.
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Allen, R.G., Farmer, K.J., Newton, R.K. and Sohal, R.S. Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, eatalase, glutathione reductase, inorganic peroxides and glutathione in the adult housefly. Comp. Biochem. Physiol. 78C, 283-288, 1984. Bernt, E. and Bergmeyer, H.U. Inorganic peroxides. In: Methods of Enzymatic Analysis, Bergmeyer, H.U., (Editor), pp. 2246-2248, Academic Press, New York, 1976. Chance, B., Sies, H. and Boveris, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-603, 1979. Fleming, J.E., Miquel, J., Cottrell, S.F., Yengoyan, L.S. and Economos, A.C. Is cell aging caused by respirationdependent injury to the mitochondrial genome? Gerontol. 28, 44-53, 1982. Freeman, B.A., Topolosky, M.K. and Crapo, J.D. Hyperoxia increases oxygen radical production in rat lung homogenates. Arch. Biochem. Biophys. 216, 477-484, 1982. Halliwell, B. Free radicals, oxygen toxicity and ageing. In: Age Pigments, Sohal, R.S., (Editor), pp. 1-62, Elsevier/North Holland, Amsterdam, 1981. HaUiwen, B. and Gutteridge, J.M.C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1-14, 1984. Hassan, H.M. and Fridovich, I. Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch. Biochem. Biophys. 196, 385-395, 1979. Kono, Y. and Fridovich, I. Superoxide radical inhibits eatalase. J. Biol. Chem. 257, 5751-5754, 1982. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and RandaU, R.I. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275, 1951. Luck, H. Catalase, In: Methods of Enzymatic Analysis, Bergmeyer, H., (Editor), pp. 885-894, Academic Press, New York, 1965. Masters, C. and Holmes, R. Peroxisomes: new aspects of cell physiology and biochemistry, Physiol. Rev. 57, 816-882, 1977. McArthur, M.C. and Sohal, R.S. Relationship between metabolic rate, aging, lipid peroxidation and fluorescent age pigment in the milkweed bug, Oncopeltusfasciatus (Hemiptera), J. Gerontol. 37, 268-274, 1982. Miquel, J., Tappel, A., Dillard, C., Herman, M. and Bensch, K. Fluorescent products and lysosomal components in aging Drosophila melanogaster. J. Gerontol. 29, 622-637, 1974. Miquel, J., Lundgren, P.R., Benseh, K.J. and Atlan, H. Effect of temperature on the life span, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Dev. 5, 347-370, 1976. Misra, H.P. and Fridovich, I. Superoxide dismutase: "positive" spectrophotometric assays. Analyt. Biochem. 79, 553-560, 1976. Prosser, C.L. Comparative Animal Physiology, 3rd ed., ch. 3., Saunders, Philadelphia, 1973. Sacher, G.A. Life table modification and life prolongation. In: The Biology of Aging, Finch, C.E. and Hayflick, L., (Editors), pp. 582-638, Van Nostrand Reinhold, New York, 1977. Sheldahl, J.A. and Tappel, A.L. Fluorescent products from aging Drosophila melanogaster: an indication of free radical lipid peroxidation damage. Exp. Gerontol. 9, 33-41, 1974. Smith, J. and Shrift, A. Phylogenetic distribution of glutathione peroxidase. Comp. Biochem. Physiol. 63B, 39-44, 1979. Sohal, R.S. Metabolic rate and life span. In: Interdisciplinary Topics in Gerontology, Cutler, R.G., (Editor), Vol. 9, pp. 25-40, S. Karger, Basel, 1976. Sohal, R.S. Metabolic rate, free radicals and aging. In: Free Radicals in Molecular Biology, Aging, and Disease, Armstrong, D., Sohal, R.S., Cutler, R.G. and Slater, T.F., (Editors), pp. 119-127, Raven Press, New York, 1984. Sohal, R.S. The rate of living theory: a contemporary interpretation. In: Insect Aging, Collatz, K.G. and Sohal, R.S., (Editors), pp. 23-44, Springer-Verlag, Berlin, 1986. Sohal, R.S. and Allen, R.G. Relationship between oxygen metabolism, aging, and development. Adv. Free Rad. Biol. Med. 2, 117-160, 1986. Sohal, R.S., Donato, H. and Biehl, E.R. Effect of age and metabolic rate on lipid peroxidation in the housefly, Musca domestica. Mech. Ageing Dev. 16, 159-167, 1981. Sohal, R.S., Farmer, K.J., Allen, R.G. and Cohen, N.R. 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, 185-195, 1984a. Sohal, R.S., Allen, R.G., Farmer, K.J. and Procter, J. Effect of physical activity on superoxide dismutase,
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catalase, inorganic peroxides and glutathione in the housefly, Musca domestica. Mech. Ageing Dev. 26, 75-81, 1984b. Sohal, R.S., Muller, A., Koletzko, B. and Sies, H. Effect of age and ambient temperature on n-pentane production in adult housefly, Musca domestica. Mech. Ageing Dev. 29, 317-326, 1985. Tietze, F. Enzymic method for the quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Analyt. Biochem. 27, 502-522, 1969.