Journal of Gerontology 1976, Vol. 31, No. 4, 405-408
Superoxide Dismutase in the Rat and Mouse as a Function of Age and Longevity1 Superoxide dismutase was assayed in extracts of a variety of tissues in relatively short-lived (A/J) and long-lived (LP/J) male mice. At 9 mo of age the brains and lungs of LP/J mice contained more of this activity than did those of A/J mice. No significant differences were seen in the other tissues investigated. It was also found that specific activities of total superoxide dismutase in extracts of the brain and liver of male CD Sprague-Dawley rats did not diminish during aging; however, the cyanide-insensitive superoxide dismutase, which reflects the mitochondrially localized enzyme and which constitutes only a fraction of total activity, did diminish somewhat with age in liver, but not in brain.
HE superoxide radical is a commonT place intermediate in the biological reduction of oxygen. Superoxide dismutases are enzymes which provide the basic defense against the potentially cytotoxic reactivities of this radical. Work in this field has been reviewed (Fridovich, 1972, 1974, 1975). It has been suggested that free radicals, by virtue of their chemical reactivity, damage living cells and that the irreversible portion of this damage is a contributing cause in senescence (Harman, 1956, 1971). We may now consider the possibility that superoxide radicals and the reactive species that they engender are involved in reactions which lead to irreversible damage of cell function. In this case senescence may be, partially at least, an expression of oxygen toxicity. Thus, superoxide dismutases may act to slow aging processes. The report by Miquel, Lundgren, and Bensch (1975), that exposure of Drosophila to elevated levels of oxygen results in an apparent increase in the rate of senescence, gives some support to this idea. It has been reported that the activities of several enzymes decline with age (Erlanger & Gershon, 1970; Gershon & Gershon, 1973; Reiss & Rothstein, 1975). If superoxide dismutase activity exhibited a similar decline, this could lead to a lowering of protection against superoxide-induced damage and a conThis work was supported in full by Research Grant GM-10287 from NIMH, Bethesda. 'Dept. of Biochemistry, Duke Univ. Medical Center, Durham, NC 27710.
comitant acceleration of age-dependent changes. Superoxide dismutase levels were therefore investigated as a function of age. In 1958 Grahn found a positive correlation between radiation resistance and mean life-span in various mouse strains. Recent work has suggested an important role for superoxide dismutase in radiation protection (Petkau, Chelack, Pleskach,,Meeker, & Brady, 1975). It seemed important, in this light, to compare the superoxide dismutase levels of several tissues in relatively long and short-lived strains of mice in the hope of finding differences that might correlate with their apparent aging rate. MATERIALS AND METHODS
Methionine was purchased from General Biochemicals, riboflavin from Eastman Organic Chemicals, and nitroblue tetrazolium, xanthine, and cytochrome c type III from Sigma Chemical. Dialysis tubing was purchased from Union Carbide and was cleaned by sequential heating at 70C in solutions of 1.0 M KC1, 0.1 M EDTA, and then 3 changes of water, and was stored in ethanol. Sodium cyanide was from Baker Chemical, and solutions of this reagent were kept cold and prepared fresh daily. Xanthine oxidase was isolated from cream as described by Waud, Brady, Wiley, and Rajagopalan (1975). The copper and zinccontaining superoxide dismutase was isolated from bovine erythrocytes (McCord & Fridovich, 1969) while the manganese-contain405
Downloaded from http://geronj.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Edwin Welles Kellogg, III, BS,2 and Irwin Fridovich, PhD2
406
KELLOGG AND FRIDOVICH
& Fridovich, 1973). The latter activity was therefore measured by performing the photochemical assays in the presence of 2 x 10 3 M cyanide. Under the conditions used, the cytochrome c reduction assay for superoxide dismutase was less sensitive, by a factor of approximately five, than was the photochemical assay. However the photochemical assay for superoxide dismutase is more liable to error due to chemical interference. For this reason all samples used in this assay were dialyzed. The protein content of all extracts was assayed by the biuret test (Gornall, Bardawill, & David, 1949). In the CD rats possible variability due to turbidity effects seen in older fatty livers was eliminated by using corrective controls prepared by the omission of CuSO4 from the reagent. RESULTS
Comparison of long-lived (LP/J) and shortlived (A/J) mouse strains. — At about 9 mo of age both strains had comparable levels of superoxide dismutase in all organs save brain and lung. In these tissues the longer-lived LP/J strain exhibited significantly higher specific activities. These results are presented in Table 1. Of the organs tested, heart and lung had the highest activities, while spleen had the lowest. Age-related changes in superoxide dismutase were assessed by comparing extracts of tissues from animals at different periods of life-span. Fig. 1A demonstrates that the LP/J mice maintained a slightly higher level of superoxide dismutase in brain than did the A/J from 2 to 12 mo of age. In contrast figure IB demonstrates Table 1. SOD Tissue Levels in Long- and Short-Lived Male Mice at 9 Mo. of Age. SOD Units mf $"' Protein" A/J'
LP/J'
Brain'
12.1 + 1.1
Heart
19.4±1.6
I5.9±1.O»" 17.4±1.1
Kidneys
14.8 ± 1.9
15.8 + 1.3
Liver
15.0 ±0.7
14.3 ±1.1
Lungs''
17.1 ±4.4
24.5 ±5.7*
Spleen
4.51±0.43
4.17 ±0.51
• Determined by cytochrome c method. ' 7 d animals - A/J age = 252 ± 14 days; LP/J age = 238 ± 14 days. Mean life-span for A/J d = 490 ± 18.4; LP/J = 748 ± 18.6. Both strains are reported to die of nonspecific causes (Storer, 1966). ' Dialyzed before assay. ' Corrected for blood contamination. • • • p < 0.001 students 1 tailed t-test. •p
Superoxide Dismutase in the Rat and Mouse as a Function of Age and Longevity1 Superoxide dismutase was assayed in extracts of a variety of tissues in relatively short-lived (A/J) and long-lived (LP/J) male mice. At 9 mo of age the brains and lungs of LP/J mice contained more of this activity than did those of A/J mice. No significant differences were seen in the other tissues investigated. It was also found that specific activities of total superoxide dismutase in extracts of the brain and liver of male CD Sprague-Dawley rats did not diminish during aging; however, the cyanide-insensitive superoxide dismutase, which reflects the mitochondrially localized enzyme and which constitutes only a fraction of total activity, did diminish somewhat with age in liver, but not in brain.
HE superoxide radical is a commonT place intermediate in the biological reduction of oxygen. Superoxide dismutases are enzymes which provide the basic defense against the potentially cytotoxic reactivities of this radical. Work in this field has been reviewed (Fridovich, 1972, 1974, 1975). It has been suggested that free radicals, by virtue of their chemical reactivity, damage living cells and that the irreversible portion of this damage is a contributing cause in senescence (Harman, 1956, 1971). We may now consider the possibility that superoxide radicals and the reactive species that they engender are involved in reactions which lead to irreversible damage of cell function. In this case senescence may be, partially at least, an expression of oxygen toxicity. Thus, superoxide dismutases may act to slow aging processes. The report by Miquel, Lundgren, and Bensch (1975), that exposure of Drosophila to elevated levels of oxygen results in an apparent increase in the rate of senescence, gives some support to this idea. It has been reported that the activities of several enzymes decline with age (Erlanger & Gershon, 1970; Gershon & Gershon, 1973; Reiss & Rothstein, 1975). If superoxide dismutase activity exhibited a similar decline, this could lead to a lowering of protection against superoxide-induced damage and a conThis work was supported in full by Research Grant GM-10287 from NIMH, Bethesda. 'Dept. of Biochemistry, Duke Univ. Medical Center, Durham, NC 27710.
comitant acceleration of age-dependent changes. Superoxide dismutase levels were therefore investigated as a function of age. In 1958 Grahn found a positive correlation between radiation resistance and mean life-span in various mouse strains. Recent work has suggested an important role for superoxide dismutase in radiation protection (Petkau, Chelack, Pleskach,,Meeker, & Brady, 1975). It seemed important, in this light, to compare the superoxide dismutase levels of several tissues in relatively long and short-lived strains of mice in the hope of finding differences that might correlate with their apparent aging rate. MATERIALS AND METHODS
Methionine was purchased from General Biochemicals, riboflavin from Eastman Organic Chemicals, and nitroblue tetrazolium, xanthine, and cytochrome c type III from Sigma Chemical. Dialysis tubing was purchased from Union Carbide and was cleaned by sequential heating at 70C in solutions of 1.0 M KC1, 0.1 M EDTA, and then 3 changes of water, and was stored in ethanol. Sodium cyanide was from Baker Chemical, and solutions of this reagent were kept cold and prepared fresh daily. Xanthine oxidase was isolated from cream as described by Waud, Brady, Wiley, and Rajagopalan (1975). The copper and zinccontaining superoxide dismutase was isolated from bovine erythrocytes (McCord & Fridovich, 1969) while the manganese-contain405
Downloaded from http://geronj.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 29, 2015
Edwin Welles Kellogg, III, BS,2 and Irwin Fridovich, PhD2
406
KELLOGG AND FRIDOVICH
& Fridovich, 1973). The latter activity was therefore measured by performing the photochemical assays in the presence of 2 x 10 3 M cyanide. Under the conditions used, the cytochrome c reduction assay for superoxide dismutase was less sensitive, by a factor of approximately five, than was the photochemical assay. However the photochemical assay for superoxide dismutase is more liable to error due to chemical interference. For this reason all samples used in this assay were dialyzed. The protein content of all extracts was assayed by the biuret test (Gornall, Bardawill, & David, 1949). In the CD rats possible variability due to turbidity effects seen in older fatty livers was eliminated by using corrective controls prepared by the omission of CuSO4 from the reagent. RESULTS
Comparison of long-lived (LP/J) and shortlived (A/J) mouse strains. — At about 9 mo of age both strains had comparable levels of superoxide dismutase in all organs save brain and lung. In these tissues the longer-lived LP/J strain exhibited significantly higher specific activities. These results are presented in Table 1. Of the organs tested, heart and lung had the highest activities, while spleen had the lowest. Age-related changes in superoxide dismutase were assessed by comparing extracts of tissues from animals at different periods of life-span. Fig. 1A demonstrates that the LP/J mice maintained a slightly higher level of superoxide dismutase in brain than did the A/J from 2 to 12 mo of age. In contrast figure IB demonstrates Table 1. SOD Tissue Levels in Long- and Short-Lived Male Mice at 9 Mo. of Age. SOD Units mf $"' Protein" A/J'
LP/J'
Brain'
12.1 + 1.1
Heart
19.4±1.6
I5.9±1.O»" 17.4±1.1
Kidneys
14.8 ± 1.9
15.8 + 1.3
Liver
15.0 ±0.7
14.3 ±1.1
Lungs''
17.1 ±4.4
24.5 ±5.7*
Spleen
4.51±0.43
4.17 ±0.51
• Determined by cytochrome c method. ' 7 d animals - A/J age = 252 ± 14 days; LP/J age = 238 ± 14 days. Mean life-span for A/J d = 490 ± 18.4; LP/J = 748 ± 18.6. Both strains are reported to die of nonspecific causes (Storer, 1966). ' Dialyzed before assay. ' Corrected for blood contamination. • • • p < 0.001 students 1 tailed t-test. •p
