BRIEF COMMU NICATION
Superoxide Dismutase Specific Activities in Cultured Human Diploid Cells of Various Donor Ages MICHAEL R. DUNCAN, ROBERT T. DELL'ORCO AND K. D. KIRK Biomedical Division, The Samuel Roberts Noble Foundation, lnc., Ardmore, Oklahoma 73401
ABSTRACT It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespans. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult > newborn > fetal. The possible relationship between these findings and the free radical theory of aging is discussed. The free radical theory of aging attributes senescence to the accumulation of free radical-induced damage (Harman, '56). Evidence has been obtained that chain reactions propagated by free radicals can result in altered membrane structure due to peroxidation of unsaturated lipid components (Tappel, '65, '73). Accumulation with age of such alterations could result in impairment of a variety of membrane functions. In this regard, increasing anomalies in cell and organelle membrane function with age have been reported (Gordon, '71). In addition, deposits of fluorescent material, thought to be products of unsaturated lipid peroxidation similar to age pigment, have been observed in senescent WI38 cells (Deamer and Gonzales, '74) and thus provided further evidence for increasing free radical damage with age. In biological systems oxygen can generate free radicals. Superoxide dismutase (SOD) protects cells against 0;-induced damage by dismutation of the radical (McCord and Fridovich, '60). Several investigations attempting to relate the functional loss of this enzyme with senescence have resulted in conflicting results. Reiss and Gershon ('76a,b) reported that the specific activity of cytoplasmic SOD from rat and mouse liver declined with age. In contrast, Kellogg and Fridovich ('76) reported no change in SOD specific activity with age in rat brain or liver. Yamanaka and Deamer ('74), using WI-38 cells, detected .
J. CELL.
PHYSIOL. (1979)98: 437-442.
no alteration in SOD activity with increasing population doubling level (PDL) of the cultures; and recently i t was reported that SOD activity increases with age in rat and rabbits, as well as in certain human tissues (Yam et al., '78; Autor et al., '76). In experiments described here, SOD specific activity was measured in established human diploid cell lines of various donor ages, as well as within individual cell lines as they accumulated population doublings. The results indicated that SOD specific activity increased as both donor age and in vitro age increased. MATERIALS AND METHODS
Cell culture All cell lines used in these experiments were human diploid fibroblast-like cells. IMR-91 obtained from fetal lung and GM-275 from adult skin were purchased from the Institute for Medical Research, Camden, New Jersey. The IMR-91 cultures had a characteristic in vitro lifespan of 46 % 4 population doublings (PD), while the GM-275 cultures achieved 24 2 PD. FeSin, from fetal skin, was purchased from the A.T.C.C. repository. The CF-3 line was originated in our laboratory from newborn foreskin material. The characteristic lifespans for CF-3 and FeSin were 60 % 5 PD and 40 PD, respectively. Cells cultured in McCoy's medium 7a (Kruse et al., '69) supplemented with 10%
*
Received Aug. 29, '78.Accepted Oct. 11, '78.
437
438
M. R. DUNCAN, R. T. DELL‘ORCO AND K. D. KIRK
fetal bovine serum were fed with fresh medium three times per week and achieved confluency during this time. The macro-method described by Barile (‘73) was used t o determine that the cultures were free of detectable mycoplasma contamination. Cell homogenate preparation Cultures were assayed for SOD activity after they had become confluent to minimize the possibility of fluctuation in activity after transfer (Yamanaka and Deamer, ’74). Cell sheets were washed three times with 10 ml cold phosphate buffered saline (PBS). The cells were scraped into cold PBS after exposure to 0.05% pronase and washed three times by centrifugation and resuspension. The final pellet was resuspended in 1.0 ml PBS, and the cells were disrupted by sonication a t 50 watts for 20 seconds with a Model 185D Heat Systems-Ultrasonics, Inc., sonifier. The 30,000 x g supernatant of this sonicate was used in the SOD assay. Gel electrophoresis Acrylamide gel electrophoresis was performed according to Davis (‘64). One hundred microliters of the crude SOD preparation containing approximately 50 pg protein was applied to each gel. A current of 1.25 mA per gel was applied for 30 minutes and was then increased to 2.5 mA per gel until the bromophenol blue marker dye was completely through the gels. SOD activity bands were identified according to the method of Beauchamp and Fridovich (‘71).
SOD assay SOD activity was assayed according to its ability to inhibit the autoxidation of epinephrine as described by Misra and Fridovich (‘72). The stock epinephrine solution contained M adrenochrome. The rate of autoxidation was determined by monitoring the increase in absorbance a t 480 nm for 3.5 minutes. Each sample was assayed three times and the activities averaged. One unit of SOD activity was arbitrarily defined as the amount of enzyme that gave a 50% reduction in the rate of epinephrine autoxidation. Samples were diluted to contain approximately 200 pg/ml protein and 0.1 ml of this dilution was used in each assay. Protein content was determined by the method of Lowry e t al. (‘51).All specific activities are expressed as units of SOD activity per milligram of protein.
Fig. 1 Photograph of acrylamide gels stained to demonstrate SOD activity. Gels stained dark blue while bands of activity remained clear. (a) Control gel showing two distinct bands of activity. b) Identical preparation as figure l a except that the second staining solution contained 1.0 mM NaCN showing inhibition of fastest migrating band. Each cell line gave the illustrated results. RESULTS
Gel electrophoresis
Weisiger and Fridovich (‘73) demonstrated two distinct SOD proteins in chicken liver, one of cytoplasmic origin and one associated with mitochondria. We analyzed the SOD proteins from all the cell lines using acrylamide gel electrophoresis. Each cell line was found to contain two bands of SOD activity (fig. la). Further, when 1.0 mM sodium cyanide was added to the second staining solution, the faster migrating activity was inhibited (fig. lb), identifying it as the cytoplasmic form of the enzyme (Weisiger and Fridovich, ’73). The specific activity values given in the remainder of this report represent the total SOD activity in cell sonicates with no attempt to distinguish between cytoplasmic or mitochondria1 origin.
SOD specific activity as a function of PDL Figure 2 illustrates the results of SOD specific activity measurements taken throughout the lifespan of the CF-3 cell line. The only significant variation appeared to occur early in the lifespan. This observation was confirmed by the statistical treatment of the data which is presented in table 1.The specific activity increased after PDL20 and remained elevated
439
SOD ACTIVITY IN HUMAN DIPLOID CELLS
. * .
I
. .
10
I
30 40 50 Population Doubling Level Fig. 2 SOD specific activity a8 a function of PDL in CF-3 cells. 10
20
TABLE 1
SODspecific activities duringthe in vitro lifespan of CF-dcells (newborn donor) PDL
Number of experiments
SOD specific activity '
10-20 21-40 41-60
8 16 11
2822 4222 45%2
' Expressed as mean t S.E.M. *Significantly different from PDL 10-20 (p
< 0.001).
TABLE 2
SODspecific activities during the in vitro lifespans of IMR-91 and FeSin cells (fetal donors) Cell line
Number of SOD experiments specific activity '
PDL
IMR-91
22-35 36-50
11 11
2 9 22 4223
FeSin
12-26 21-31
11 9
2923 3922
' Expres~edas mean t S.E.M. *Significantly different from IMR-91 PDL 22-35 (p < 0.005). Significantly different from FeSin PDL 12-26 (p < 0 025).
TABLE 3
SODspecific activities duringthe in vitro lifespan of GM-275cells (adult donor) PDL
Number of experiments
SOD specific activity
11-16 17-24
13 11
5322 5122
I
60
during the final two-thirds of the lifespan. The variation in SOD activity for cultures of identical PDL seen in figure 2 is apparently due to imprecision in the assay procedure. However, this assay procedure is reportedly more sensitive than others previously described (Misra and Fridovich, '72). Cells from two fetal and one adult donor were tested also; however, it should be noted that these cell lines were available a t PDLs that allowed the examination of only the final two-thirds of their in vitro lifespans. Any fluctuations occurring early in their lifespans were not revealed in these experiments. The results from the two fetal donors are given in table 2. The specific activities increased after PDL35 and 26 for IMR-91 and FeSin, respectively, and remained elevated during the final one-third of their lifespans. In contrast to this, the SOD specific activity in cells from the adult donor was constant during the final twothirds of their lifespans (table 3).
SOD specific activity as a function of donor age Finally, SOD specific activities from cells of various donor ages were compared. The values listed in table 4 represent the means of SOD specific activities obtained from experiments conducted during the final two-thirds of the in vitro lifespan of each cell line. These data show that the mean activities from the various cell lines increased as the age of the donor increased.
~~
~~
~
' Expressed as mean
f
S.E.M.
DISCUSSION
Recent reports indicated that increased SOD specific activity may be age related in a
440
M. R. DUNCAN, R. T. DELL’ORCO AND K. D. KIRK TABLE 4
SODspecific activities from cell lines of various donor ages SOD
Cell line
Number of experiments
specific activity
FeSin IMR-91 CF-3 GM-275
20 22 33 24
34*2 3622 44*2l 52*2
I Expressed as mean 2 S.E.M. 2Significantly different from FeSin (p < 0.001), IMR-91 (p < 0.005),and GM-275 (p < 0.001). ‘Significantly different from FeSin (p < 0.001), IMR-91 (p < 0.001). and CF-3 (p < 0.001).
variety of mammalian systems. Yam et al. (‘78) found that pulmonary SOD activity in rats increased with age from birth through 80 days. Autor et al. (‘76) reported similar results with rats and rabbits, with low values in fetal animals, and maximum values in adults. This relationship was shown by these authors to exist also in human blood as well as in human lung tissue obtained from autopsy material. Our data indicate that SOD specific activity in cultured human diploid cells also may be age-related. The relationship was demonstrated in two ways. First, within the cell lines tested activity in cells from fetal donors was significantly higher in the final one-third of the in vitro lifespan, whereas in cells from a newborn donor the activity was higher in the final two-thirds of the lifespan (tables 1, 2). Secondly, the SOD specific activity from cell lines of various donor ages increased as the age of the donor increased (table 4). Our results concerning cell lines from fetal donors disagree with a report by Yamanaka and Deamer (‘74). These investigators found no age-related alterations in SOD activity in WI-38 cells (embryonic donor) studied from approximately PDL20-50. It is possible that variation exists among cell lines of fetal or embryonic donors and that studies very early in the lifespan of WI-38 cultures might reveal alterations in SOD activity. Another explanation for the differing results may be related to the assay systems used. The technique used here has been reported to be more sensitive than that used previously (Misra and Fridovich, ’72) and yielded values that were less variable than those reported by Yamanaka and Deamer (‘74). Additionally, the alteration in SOD specific activity observed with CF-3 cells parallels other age-related changes occurring in this cell line. Previously reported
studies from our laboratory have indicated that several biochemical parameters become altered after these cells complete the first onethird of their characteristic in vitro lifespan. These age-related changes include an increased rate of protein turnover (Dell’Orco and Guthrie, ’76), the appearance of significant levels of heat labile enzyme (Duncan et al., ’77), and a n elevation in the level of DNA repair synthesis (Dell’Orco and Whittle, ’78). Our data suggest at least two possible conclusions. First, the superoxide radical may not play a significant role in in vitro aging. Provided that exposure and susceptibility to the radical remain constant, it appeared that aged cells were a t least as well protected as younger cells. This seems unlikely, however, since available evidence indicates that free radicalinduced damage accumulates with age (Gordon, ’71; Deamer and Gonzales, ’74). Second, aged cells might be attempting to respond to increasing free radical exposure or damage. In this regard other authors have shown that certain mammalian tissues respond to hyperoxia by increasing their SOD specific activities and suggested that this ability may become less responsive with age (Crapo and Tierney, ’74; Autor et al., ’76). It is interesting to note, however, that in these studies (Autor et al., ’76) the “induced” level of activity from young tissue was not as great as the “constitutive” activity from tissue of aged organisms. The meaning of these data and their relationship to the free radical theory of aging are presently unclear. Experiments are in progress in our laboratory to determine whether cultured human cells will increase their SOD specific activities in response to an exogenous agent, methyl viologen, which has been reported to increase the intracellular level of the superoxide radical (Hassan and Fridovich, ’77). Agerelated alterations in the nature and/or the magnitude of the response to this challenge may provide a better insight into the effect of free radical induced damage on cellular senescence. LITERATURE CITED Autor, A. P., L. Frank and R. J. Roberts 1976 Developmental characteristics of pulmonary superoxide dismutase: Relationship to idiopathic respiratory distress syndrome. Pediat. Res., 10: 154-158. Barile, M. 1973 Mycoplasma contamination of cell cultures: Incidence, source, prevention, and problems of elimination. In: Tissue Culture - Methods and Applications. P. F. Kruse, Jr. and M. K. Patterson, Jr., eds. Academic Press, New York, pp. 729-735. Beauchamp, C., and I. Fridovich 1971 Superoxide dismu-
SOD ACTIVITY IN HUMAN DIPLOID CELLS tase: Improved assays and assay applicable to acrylamide gels. Anal. Biochem., 44: 276-287. Crapo, J. D., and D. F. Tierney 1974 Superoxide dismutase and pulmonary oxygen toxicity. Am. J. Physiol., 226: 1401-1407. Davis, B. J. 1964 Disc electrophoresis. 11. Method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121: 404-427. Deamer, D. W., and 3. Gonzales 1974 Autofluorescent structures in cultured WI-38 cells. Arch. Biochem. Biophys., 165: 421-426. Dell'Orco, R. T., and P. L. Guthrie 1976 Altered protein metabolism in arrested populations of aging human diploid fibroblasts. Mech. Ageing Develop., 5: 399-407. Dell'Orco, R. T., and W. L. Whittle 1978 Unscheduled DNA synthesis in confluent and mitotically arrested populations of aging human diploid fibroblasts. Mech. Ageing Develop., 8: 269-279. Duncan, M. R., R. T. Dell'Orco and P. L. Guthrie 1977 Relationship of heat labile glucose-6-phosphate dehydrogenase and multiple molecular forms of the enzyme in senescent human fibroblasts. J. Cell. Physiol., 93: 49-56. Gordon, P. 1971 Molecular approaches to the drug enhancement of deteriorated functioning in the aged. In: Advances in Gerontological Research. Vol. 3. B. L. Strehler, ed. Academic Press, New York and London, pp. 199-248. Harman, D. 1956 Aging: A theory based on free radicals and radiation chemistry. J. Gerontol., 11: 298-300. Hassan, H. M., and L. Fridovich 1977 Regulation of synthesis of superoxide dismutase inEscherichia coli: Induction by methyl viologen. J. Biol. Chem., 252: 7667-7672. Kellogg, E. W., 111, and I. Fridovich 1976 Superoxide dismutase in the rat and mouse a s a function of age and longevity. J. Gerontol., 31: 405-408.
44 1
Kruse, P. F., Jr., W. L. Whittle and E. Miedema 1969 Mitotic and non-mitotic multiple-layered perfusion cultures. J. Cell Biol., 42: 113-121. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin phenol reagent. J. Uiol. Chem., 193: 265-275. McCord, J. M., and I. Eidovich 1969 Superoxide dismutase: An enzymatic function for erythrocuprein. J. Biol. Chem., 244: 6049-6055. Misra, H. P., and I. Fridovich 1972 The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J . Biol. Chem., 247: 3170-3175. Reiss, U., and D. Gershon 1976a Comparison of cytoplasmic superoxide dismutase in liver, heart and brain of aging rats and mice. Biochem. Biophys. Res. Commun., 73: 255-262. 1976b Rat-liver superoxide dismutase: Purification and age-related modifications. Eur. J. Biochem., 63: 617-623. Tappel, A. L. 1965 Free-radical lipid peroxidation damage and its inhibition by vitamin E and selenium. Fed. ROC., 24: 73-78, 1973 Lipid peroxidation damage to cell components. Fed. Proc., 32: 1870-1874. Weisiger, R. A., and I. Fridovich 1973 Superoxide dismutase: Organelle specificity. J. Biol. Chem., 248: 3582-3592. Yam, J., L. Frank and R. J. Roberts 1978 Age-related development of pulmonary antioxidant enzymes in the rat. Proc. SOC.Exp. Biol. Med., 157: 293-296. Yamanaka, N., and D. Deamer 1974 Superoxide dismutase activity in WI-38 cell cultures: Effects of age, trypsinization and SV-40 transformation. Physiol. Chem. Physics, 6: 95-106.
Superoxide Dismutase Specific Activities in Cultured Human Diploid Cells of Various Donor Ages MICHAEL R. DUNCAN, ROBERT T. DELL'ORCO AND K. D. KIRK Biomedical Division, The Samuel Roberts Noble Foundation, lnc., Ardmore, Oklahoma 73401
ABSTRACT It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespans. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult > newborn > fetal. The possible relationship between these findings and the free radical theory of aging is discussed. The free radical theory of aging attributes senescence to the accumulation of free radical-induced damage (Harman, '56). Evidence has been obtained that chain reactions propagated by free radicals can result in altered membrane structure due to peroxidation of unsaturated lipid components (Tappel, '65, '73). Accumulation with age of such alterations could result in impairment of a variety of membrane functions. In this regard, increasing anomalies in cell and organelle membrane function with age have been reported (Gordon, '71). In addition, deposits of fluorescent material, thought to be products of unsaturated lipid peroxidation similar to age pigment, have been observed in senescent WI38 cells (Deamer and Gonzales, '74) and thus provided further evidence for increasing free radical damage with age. In biological systems oxygen can generate free radicals. Superoxide dismutase (SOD) protects cells against 0;-induced damage by dismutation of the radical (McCord and Fridovich, '60). Several investigations attempting to relate the functional loss of this enzyme with senescence have resulted in conflicting results. Reiss and Gershon ('76a,b) reported that the specific activity of cytoplasmic SOD from rat and mouse liver declined with age. In contrast, Kellogg and Fridovich ('76) reported no change in SOD specific activity with age in rat brain or liver. Yamanaka and Deamer ('74), using WI-38 cells, detected .
J. CELL.
PHYSIOL. (1979)98: 437-442.
no alteration in SOD activity with increasing population doubling level (PDL) of the cultures; and recently i t was reported that SOD activity increases with age in rat and rabbits, as well as in certain human tissues (Yam et al., '78; Autor et al., '76). In experiments described here, SOD specific activity was measured in established human diploid cell lines of various donor ages, as well as within individual cell lines as they accumulated population doublings. The results indicated that SOD specific activity increased as both donor age and in vitro age increased. MATERIALS AND METHODS
Cell culture All cell lines used in these experiments were human diploid fibroblast-like cells. IMR-91 obtained from fetal lung and GM-275 from adult skin were purchased from the Institute for Medical Research, Camden, New Jersey. The IMR-91 cultures had a characteristic in vitro lifespan of 46 % 4 population doublings (PD), while the GM-275 cultures achieved 24 2 PD. FeSin, from fetal skin, was purchased from the A.T.C.C. repository. The CF-3 line was originated in our laboratory from newborn foreskin material. The characteristic lifespans for CF-3 and FeSin were 60 % 5 PD and 40 PD, respectively. Cells cultured in McCoy's medium 7a (Kruse et al., '69) supplemented with 10%
*
Received Aug. 29, '78.Accepted Oct. 11, '78.
437
438
M. R. DUNCAN, R. T. DELL‘ORCO AND K. D. KIRK
fetal bovine serum were fed with fresh medium three times per week and achieved confluency during this time. The macro-method described by Barile (‘73) was used t o determine that the cultures were free of detectable mycoplasma contamination. Cell homogenate preparation Cultures were assayed for SOD activity after they had become confluent to minimize the possibility of fluctuation in activity after transfer (Yamanaka and Deamer, ’74). Cell sheets were washed three times with 10 ml cold phosphate buffered saline (PBS). The cells were scraped into cold PBS after exposure to 0.05% pronase and washed three times by centrifugation and resuspension. The final pellet was resuspended in 1.0 ml PBS, and the cells were disrupted by sonication a t 50 watts for 20 seconds with a Model 185D Heat Systems-Ultrasonics, Inc., sonifier. The 30,000 x g supernatant of this sonicate was used in the SOD assay. Gel electrophoresis Acrylamide gel electrophoresis was performed according to Davis (‘64). One hundred microliters of the crude SOD preparation containing approximately 50 pg protein was applied to each gel. A current of 1.25 mA per gel was applied for 30 minutes and was then increased to 2.5 mA per gel until the bromophenol blue marker dye was completely through the gels. SOD activity bands were identified according to the method of Beauchamp and Fridovich (‘71).
SOD assay SOD activity was assayed according to its ability to inhibit the autoxidation of epinephrine as described by Misra and Fridovich (‘72). The stock epinephrine solution contained M adrenochrome. The rate of autoxidation was determined by monitoring the increase in absorbance a t 480 nm for 3.5 minutes. Each sample was assayed three times and the activities averaged. One unit of SOD activity was arbitrarily defined as the amount of enzyme that gave a 50% reduction in the rate of epinephrine autoxidation. Samples were diluted to contain approximately 200 pg/ml protein and 0.1 ml of this dilution was used in each assay. Protein content was determined by the method of Lowry e t al. (‘51).All specific activities are expressed as units of SOD activity per milligram of protein.
Fig. 1 Photograph of acrylamide gels stained to demonstrate SOD activity. Gels stained dark blue while bands of activity remained clear. (a) Control gel showing two distinct bands of activity. b) Identical preparation as figure l a except that the second staining solution contained 1.0 mM NaCN showing inhibition of fastest migrating band. Each cell line gave the illustrated results. RESULTS
Gel electrophoresis
Weisiger and Fridovich (‘73) demonstrated two distinct SOD proteins in chicken liver, one of cytoplasmic origin and one associated with mitochondria. We analyzed the SOD proteins from all the cell lines using acrylamide gel electrophoresis. Each cell line was found to contain two bands of SOD activity (fig. la). Further, when 1.0 mM sodium cyanide was added to the second staining solution, the faster migrating activity was inhibited (fig. lb), identifying it as the cytoplasmic form of the enzyme (Weisiger and Fridovich, ’73). The specific activity values given in the remainder of this report represent the total SOD activity in cell sonicates with no attempt to distinguish between cytoplasmic or mitochondria1 origin.
SOD specific activity as a function of PDL Figure 2 illustrates the results of SOD specific activity measurements taken throughout the lifespan of the CF-3 cell line. The only significant variation appeared to occur early in the lifespan. This observation was confirmed by the statistical treatment of the data which is presented in table 1.The specific activity increased after PDL20 and remained elevated
439
SOD ACTIVITY IN HUMAN DIPLOID CELLS
. * .
I
. .
10
I
30 40 50 Population Doubling Level Fig. 2 SOD specific activity a8 a function of PDL in CF-3 cells. 10
20
TABLE 1
SODspecific activities duringthe in vitro lifespan of CF-dcells (newborn donor) PDL
Number of experiments
SOD specific activity '
10-20 21-40 41-60
8 16 11
2822 4222 45%2
' Expressed as mean t S.E.M. *Significantly different from PDL 10-20 (p
< 0.001).
TABLE 2
SODspecific activities during the in vitro lifespans of IMR-91 and FeSin cells (fetal donors) Cell line
Number of SOD experiments specific activity '
PDL
IMR-91
22-35 36-50
11 11
2 9 22 4223
FeSin
12-26 21-31
11 9
2923 3922
' Expres~edas mean t S.E.M. *Significantly different from IMR-91 PDL 22-35 (p < 0.005). Significantly different from FeSin PDL 12-26 (p < 0 025).
TABLE 3
SODspecific activities duringthe in vitro lifespan of GM-275cells (adult donor) PDL
Number of experiments
SOD specific activity
11-16 17-24
13 11
5322 5122
I
60
during the final two-thirds of the lifespan. The variation in SOD activity for cultures of identical PDL seen in figure 2 is apparently due to imprecision in the assay procedure. However, this assay procedure is reportedly more sensitive than others previously described (Misra and Fridovich, '72). Cells from two fetal and one adult donor were tested also; however, it should be noted that these cell lines were available a t PDLs that allowed the examination of only the final two-thirds of their in vitro lifespans. Any fluctuations occurring early in their lifespans were not revealed in these experiments. The results from the two fetal donors are given in table 2. The specific activities increased after PDL35 and 26 for IMR-91 and FeSin, respectively, and remained elevated during the final one-third of their lifespans. In contrast to this, the SOD specific activity in cells from the adult donor was constant during the final twothirds of their lifespans (table 3).
SOD specific activity as a function of donor age Finally, SOD specific activities from cells of various donor ages were compared. The values listed in table 4 represent the means of SOD specific activities obtained from experiments conducted during the final two-thirds of the in vitro lifespan of each cell line. These data show that the mean activities from the various cell lines increased as the age of the donor increased.
~~
~~
~
' Expressed as mean
f
S.E.M.
DISCUSSION
Recent reports indicated that increased SOD specific activity may be age related in a
440
M. R. DUNCAN, R. T. DELL’ORCO AND K. D. KIRK TABLE 4
SODspecific activities from cell lines of various donor ages SOD
Cell line
Number of experiments
specific activity
FeSin IMR-91 CF-3 GM-275
20 22 33 24
34*2 3622 44*2l 52*2
I Expressed as mean 2 S.E.M. 2Significantly different from FeSin (p < 0.001), IMR-91 (p < 0.005),and GM-275 (p < 0.001). ‘Significantly different from FeSin (p < 0.001), IMR-91 (p < 0.001). and CF-3 (p < 0.001).
variety of mammalian systems. Yam et al. (‘78) found that pulmonary SOD activity in rats increased with age from birth through 80 days. Autor et al. (‘76) reported similar results with rats and rabbits, with low values in fetal animals, and maximum values in adults. This relationship was shown by these authors to exist also in human blood as well as in human lung tissue obtained from autopsy material. Our data indicate that SOD specific activity in cultured human diploid cells also may be age-related. The relationship was demonstrated in two ways. First, within the cell lines tested activity in cells from fetal donors was significantly higher in the final one-third of the in vitro lifespan, whereas in cells from a newborn donor the activity was higher in the final two-thirds of the lifespan (tables 1, 2). Secondly, the SOD specific activity from cell lines of various donor ages increased as the age of the donor increased (table 4). Our results concerning cell lines from fetal donors disagree with a report by Yamanaka and Deamer (‘74). These investigators found no age-related alterations in SOD activity in WI-38 cells (embryonic donor) studied from approximately PDL20-50. It is possible that variation exists among cell lines of fetal or embryonic donors and that studies very early in the lifespan of WI-38 cultures might reveal alterations in SOD activity. Another explanation for the differing results may be related to the assay systems used. The technique used here has been reported to be more sensitive than that used previously (Misra and Fridovich, ’72) and yielded values that were less variable than those reported by Yamanaka and Deamer (‘74). Additionally, the alteration in SOD specific activity observed with CF-3 cells parallels other age-related changes occurring in this cell line. Previously reported
studies from our laboratory have indicated that several biochemical parameters become altered after these cells complete the first onethird of their characteristic in vitro lifespan. These age-related changes include an increased rate of protein turnover (Dell’Orco and Guthrie, ’76), the appearance of significant levels of heat labile enzyme (Duncan et al., ’77), and a n elevation in the level of DNA repair synthesis (Dell’Orco and Whittle, ’78). Our data suggest at least two possible conclusions. First, the superoxide radical may not play a significant role in in vitro aging. Provided that exposure and susceptibility to the radical remain constant, it appeared that aged cells were a t least as well protected as younger cells. This seems unlikely, however, since available evidence indicates that free radicalinduced damage accumulates with age (Gordon, ’71; Deamer and Gonzales, ’74). Second, aged cells might be attempting to respond to increasing free radical exposure or damage. In this regard other authors have shown that certain mammalian tissues respond to hyperoxia by increasing their SOD specific activities and suggested that this ability may become less responsive with age (Crapo and Tierney, ’74; Autor et al., ’76). It is interesting to note, however, that in these studies (Autor et al., ’76) the “induced” level of activity from young tissue was not as great as the “constitutive” activity from tissue of aged organisms. The meaning of these data and their relationship to the free radical theory of aging are presently unclear. Experiments are in progress in our laboratory to determine whether cultured human cells will increase their SOD specific activities in response to an exogenous agent, methyl viologen, which has been reported to increase the intracellular level of the superoxide radical (Hassan and Fridovich, ’77). Agerelated alterations in the nature and/or the magnitude of the response to this challenge may provide a better insight into the effect of free radical induced damage on cellular senescence. LITERATURE CITED Autor, A. P., L. Frank and R. J. Roberts 1976 Developmental characteristics of pulmonary superoxide dismutase: Relationship to idiopathic respiratory distress syndrome. Pediat. Res., 10: 154-158. Barile, M. 1973 Mycoplasma contamination of cell cultures: Incidence, source, prevention, and problems of elimination. In: Tissue Culture - Methods and Applications. P. F. Kruse, Jr. and M. K. Patterson, Jr., eds. Academic Press, New York, pp. 729-735. Beauchamp, C., and I. Fridovich 1971 Superoxide dismu-
SOD ACTIVITY IN HUMAN DIPLOID CELLS tase: Improved assays and assay applicable to acrylamide gels. Anal. Biochem., 44: 276-287. Crapo, J. D., and D. F. Tierney 1974 Superoxide dismutase and pulmonary oxygen toxicity. Am. J. Physiol., 226: 1401-1407. Davis, B. J. 1964 Disc electrophoresis. 11. Method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121: 404-427. Deamer, D. W., and 3. Gonzales 1974 Autofluorescent structures in cultured WI-38 cells. Arch. Biochem. Biophys., 165: 421-426. Dell'Orco, R. T., and P. L. Guthrie 1976 Altered protein metabolism in arrested populations of aging human diploid fibroblasts. Mech. Ageing Develop., 5: 399-407. Dell'Orco, R. T., and W. L. Whittle 1978 Unscheduled DNA synthesis in confluent and mitotically arrested populations of aging human diploid fibroblasts. Mech. Ageing Develop., 8: 269-279. Duncan, M. R., R. T. Dell'Orco and P. L. Guthrie 1977 Relationship of heat labile glucose-6-phosphate dehydrogenase and multiple molecular forms of the enzyme in senescent human fibroblasts. J. Cell. Physiol., 93: 49-56. Gordon, P. 1971 Molecular approaches to the drug enhancement of deteriorated functioning in the aged. In: Advances in Gerontological Research. Vol. 3. B. L. Strehler, ed. Academic Press, New York and London, pp. 199-248. Harman, D. 1956 Aging: A theory based on free radicals and radiation chemistry. J. Gerontol., 11: 298-300. Hassan, H. M., and L. Fridovich 1977 Regulation of synthesis of superoxide dismutase inEscherichia coli: Induction by methyl viologen. J. Biol. Chem., 252: 7667-7672. Kellogg, E. W., 111, and I. Fridovich 1976 Superoxide dismutase in the rat and mouse a s a function of age and longevity. J. Gerontol., 31: 405-408.
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