Me~hamsms of Ageing and Development, 60 (1991) 189--198
189
Elsevier Scientific Pubhshers Ireland Ltd
H Y D R O G E N P E R O X I D E P R O D U C T I O N BY M I T O C H O N D R I A M A Y BE A BIOMARKER OF AGING
R S SOHAL Department of Btologwal Sctences, Southern Methodtst Umverstty, Dallas, Teras ( U S A )
(Reeewed December 26th, 1990) (Revision received March 3rd, 1991)
SUMMARY The hypothesis that rate of mtracellular prooxldant production is associated with the rate of aging was tested by comparing the rate of H202 generation by mltochond n a m houseflies of similar chronological but &fferent physiological ages Physiological age represents the hfe expectancy or 'nearness to death' Average and maximum life spans of files were extended 2-fold by the ehmmatlon of flying actwlty In ad&Uon, using senescence-related loss of flight ability as a phenotyplc marker of impending death, relatively short-lived and long-lived subpopulatlons of files were isolated from cohort populaUons Rate of H202 generation was measured fluorometrically in mltochondna from thoracic flight muscles using ot-glycerophosphate as a substrate and w~thout employing any respiratory mhlbltors as Is often the case m m a m m a h a n stu&es The rate of mltochondrlal H202 release was found to be associated with hfe expectancy or the physiological age of files rather than the chronological age At the same chronological age, mltochondrla from fl~es with a shorter hfe expectancy had a markedly higher rate of H202 generaUon than those with a longer hfe expectancy Results of this and some previous studies m this laboratory are mterpreted to suggest that the rate of prooxldant generation rather than the level of anUoxldant defenses may be a key correlate of the rate of agmg Key words Mltochondrla, Oxy-ra&cals, Aging, Free radicals, Oxygen, Muscle
INTRODUCTION It IS axiomatic that any causal hypothesis of aging should adequately explain, both the time-related physiological decline observed m lndwlduals, as well as mtra- and Correspondence to R S Sohal, Department of Biological Sciences, Southern Metho&st Umverslty, Dallas, TX 75275, U S A
0047-6374/91/$03 50 Printed and Pubhshed in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd
190
rater-species variations m life spans of ammals Indeed, considerable effort has been made to identify longevity determining factors and "blomarkers' of aging, but nothing that receives wide support has emerged [1] Oxidative stress, 1 e the balance between prooxldants and antloxldants [2], has been hypothesized to be one of the causal factors In the aging process [3,4] Presently, there are two schools of thought concerning the nature of the relationship between oxidative stress and aging In one view, the rate of aging is dependent on the level of antloxldant defenses whde in the alternative wew, at ~s dependent on rate of prooxldant generation The former v~ew has long been championed by Cutler and his co-workers [5,6], who initially reported a positive correlation between maximum hfe span potential (MLSP) of several mammahan species and the activity of superoxlde &smutase (SOD) dwlded by the metabohc rate of the organ [51 Rapid conversion of 0 2 - into H202 by SOD and the resultant low steady state concentration of 0 2 - were beheved to be longevity-determining factors Cutler [6] also reported a negative correlation between MLSP and actw~t~es of catalase and glutathlone perox~dase, which together eliminate H202, believed to be the precursor of the highly reactwe OH radical However, a positive correlation was found between MLSP and some low molecular weight antlOxldants such as urate, tocopherols and carotenolds, prompting Cutler to propose that antloxldant defenses are determinants of the rate of aging [6] For several reasons Cutler's hypothesis that variations in the levels of SOD and some low molecular weight antloxldants act as longevity determinants can not be sustained Firstly, relatively high levels of SOD and correspondmgly low levels of catalase and glutathlone peroxldase would almost certainly increase the Intracellular concentration of H,O~ as well as the hkehhood of OH radical generation, against which the low molecular weight antloxldants would prove to be a poor defense because of the extreme, and virtually &ffuslon dependent, reactivity of OH radicals [2] Secondly, overexpresslon of SOD in genetic transformants has been found to mcrease the level of oxidative stress in E colt [7] and does not prolong the hfe span of Drosphda [8] Thirdly, recent stu&es m this laboratory have indicated that the overall level of antloxldant defenses neither significantly declines with age in insects or mammals nor differs m the tissues of six different mammalian species, ranging between 3 5 and 30 years m hfe span [9--11] Fourthly, admimstration of low molecular weight antloxldants has no effect on maximum life span [4] Thus variations m antloxidant defenses cannot explain either mdlwdual aging or variations m MLSP of different species Since the level of oxidatwe stress tends to mcrease during aging as indicated by the m wvo exhalation of ethane and pentane [12--14], it is, alternatively, possible that the rate of prooxldant generation rather than the level of antloxldant defenses is a crucial factor in the aging process [9,11] Rates of mltochondrml 0 2 - and H~O2 generation were found to, both, increase with age in insects [15,16] and mammals [9] as well as mversely correspond to MLSP of six different mammalian species [ 17,18] The objective of the present study was to further explore the relationship between
191
proox~dant generation and aging by determmlng whether the rate of mltochondrlal H202 generation corresponded to mtra-specles variations in hfe span It was reasoned that if the rate o f H202 generation played a causal role in aging It should mversely correspond to the hfe expectancy of the ammals in a cohort population, i e lntra-specles variations in hfe span should be correlated to the rate of H202 generation The present study was conducted in adult houseflies which provide a fortuitous model for this investigation as the aging rate of files can be slowed down and maxlmum life span doubled by the ehmmatlon of flying actwlty In addition, subpopulatlons of files with relatively short or long hfe expectancy can be separated from a cohort population MATERIALS A N D METHODS
Rearing of files A stock of housefhes, originally obtained from the Zoology department of the Umverslty of Cambridge and maintained for about fifteen years m this laboratory, was used in the present study Larvae were fed on CSMA (Chemical Specialties Manufacturer's Association) fly larval diet Adults were fed on sucrose and water and kept at 25"C and relative humidity of 45--50%
Expertmental vartattons m phystcal actlvtty and hfe span After emergence from the pupae adult files were separated by sex and male files, which were solely used for biochemical studies, were placed under conditions of relatively high (HA) or low (LA) levels of physical activity H A files were housed in one-cubic-foot cages (0 027 m3), where they were able to fly, with 200 files per cage To achieve low levels of physical actwlty, lndwldual files were confined m 150 ml glass urine-specimen bottles fitted with a cardboard maze in such a fashion that files could walk but were unable to undertake flight due to restriction of space
Selection of relattvely short-and long-hved fltes Loss of the abdlty to fly is a umversal feature of senescence m housefiles and provldes a convenient phenotyplc characteristic to recogmze mdwlduals that have reached a point near the end of their hfe Fhghtless fhes were recognized as 'crawlers' as they were unable to fly away, when approached for capture, were separated from their cohorts, the 'tilers', that were still able to fly at 12 days of age Crawlers and tilers were then placed in separate l-cubic-foot cages
Isolanon of mttochondrla Mltochondrla were isolated from the thoraces of the flies according to the procedure of Nordln and Wood [19] as described previously [16] Briefly, the thoraces were separated from the body and placed in a chilled mortar containing buffer A
192 (0 1 ml/thorax), consisting of 154 m M KCl and 1 mM E G T A (pH 7 0), and pounded gently with a pestle into a brel The brel was filtered by suction through eight layers of cheese cloth and the filterate was centrifuged at 125 × g for 3 mm The pellet was discarded and the supernatant was centrifuged at 3000 × g for 8 mm and the pellet resuspended m buffer A
Measurement of 14,0: release by mttochondrta The rate of H202 released by mltochondrla was measured by the method of Hyslop and Sklar [20], which is based on the oxadatlon of p-hydroxyphenylacetate (PHPA) into a fluorescent form during the enzymatic reduction of H202 by horseradish peroxldase The reaction mixture consisted of buffer B (154 mM KCI, 5 mM potassium phosphate, 3 m M MgC12 and 0 1 mM EGTA, pH 7 4), 166/zg/ml PHPA, 83 umts of horseradish perox~dase/ml, 7 mM a-glycerophosphate and about 20/~g mltochondnal proteln/ml of the incubation m e d m m The rate of H202 released was monitored by following the increase in fluorescence at an excitation maximum of 317 nm and an emasslon maximum of 400 nm, using Perkln Elmer LS-5 spectrofluorometer Known concentrations of H202 were used to establish the standard concentration curve RESUL~g Two different approaches were taken to dlstlngmsh between the chronological age and the physlolog~cal age (or hfe expectancy) of files One was to experimentally alter the hfe span of fhes by varying the level of physical activity, and the other was to phenotyplcally select, from a cohort population, subpopulatlons with a relatively short or a long hfe expectancy, as described below
Vartatlons m l~e span mduced by alterattons m phy~tcal acttvlO' Previous studies in this laboratory have shown that metabohc rate of the files has an unambiguous effect on their longevity [14,21,22] Since houseflies are very active tilers and flying results in an up to 100-fold increase m the rate of oxygen consumption as compared to sitting state [22], manipulation of flying activity can be used as a means to control metabohc rate Mortahty characteristics of the houseflies, kept under conditions of relatively low (LA) and high (HA) levels of physical activity, are presented m Fig 1 The average and the maximum life spans of HA flies were 19 + 6 (S D ) days and 40 days, respectively, while those of LA flies were 44 ± 14 (S D ) days and 77 days Thus, the sohtary confinement of tileS under conditions preventing flight resulted in a virtual doubhng of the average and the maximum hfe spans
Selectton of short-hved and long-lived subpopulatlon~ [rom a ~ohort populatton Since all houseflies lose flying abdlty before death, fllghtlessness can be used as
193
100 rl
480
U) 0
÷ 60
O
÷
e." =:) 40
o
LA
+
HA
÷ ÷ ÷
20
wb= •D []
0
[]
!
2'0
0
,0
!
6'0
8O
AGE (days)
Fzg 1 •urvlv•rshlpcurves•fma•e••esk•ptunderc•ndm•ns•fre•atwe•yhlgh(HA)and••w(LA)••ve•s of physical acUvlty Under HA condmon, 200 files were housed per l-cubic-foot cage, whereas under LA c o n d m o n s fl]es were md]vldually c o n f n e d m small glass jars where they were unable to fly Presented data are based on the mortahty o f 200 HA flies and 46 LA flies
an indicator of senescence and impending death Fhghtless fhes, termed crawlers, can be easdy recognized in housefly populations, as these fires are unable to fly away when approached An increasing proportion of flies m an agmg population becomes flightless after about 10 days of age Survlvorshtp curves of crawlers and their cohorts that are sttll able to fly (termed fliers), separated from cohort populations at 12 days of age and placed m separate cages are presented m Fig 2 After isolation
1 O0 "
m
[] [] []
÷ 80" U) nO >
[] +
FLIERS CRAWLERS
%,
60 ÷
n(/1
[]
÷
D
13
40 ÷ ÷
o
189
Elsevier Scientific Pubhshers Ireland Ltd
H Y D R O G E N P E R O X I D E P R O D U C T I O N BY M I T O C H O N D R I A M A Y BE A BIOMARKER OF AGING
R S SOHAL Department of Btologwal Sctences, Southern Methodtst Umverstty, Dallas, Teras ( U S A )
(Reeewed December 26th, 1990) (Revision received March 3rd, 1991)
SUMMARY The hypothesis that rate of mtracellular prooxldant production is associated with the rate of aging was tested by comparing the rate of H202 generation by mltochond n a m houseflies of similar chronological but &fferent physiological ages Physiological age represents the hfe expectancy or 'nearness to death' Average and maximum life spans of files were extended 2-fold by the ehmmatlon of flying actwlty In ad&Uon, using senescence-related loss of flight ability as a phenotyplc marker of impending death, relatively short-lived and long-lived subpopulatlons of files were isolated from cohort populaUons Rate of H202 generation was measured fluorometrically in mltochondna from thoracic flight muscles using ot-glycerophosphate as a substrate and w~thout employing any respiratory mhlbltors as Is often the case m m a m m a h a n stu&es The rate of mltochondrlal H202 release was found to be associated with hfe expectancy or the physiological age of files rather than the chronological age At the same chronological age, mltochondrla from fl~es with a shorter hfe expectancy had a markedly higher rate of H202 generaUon than those with a longer hfe expectancy Results of this and some previous studies m this laboratory are mterpreted to suggest that the rate of prooxldant generation rather than the level of anUoxldant defenses may be a key correlate of the rate of agmg Key words Mltochondrla, Oxy-ra&cals, Aging, Free radicals, Oxygen, Muscle
INTRODUCTION It IS axiomatic that any causal hypothesis of aging should adequately explain, both the time-related physiological decline observed m lndwlduals, as well as mtra- and Correspondence to R S Sohal, Department of Biological Sciences, Southern Metho&st Umverslty, Dallas, TX 75275, U S A
0047-6374/91/$03 50 Printed and Pubhshed in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd
190
rater-species variations m life spans of ammals Indeed, considerable effort has been made to identify longevity determining factors and "blomarkers' of aging, but nothing that receives wide support has emerged [1] Oxidative stress, 1 e the balance between prooxldants and antloxldants [2], has been hypothesized to be one of the causal factors In the aging process [3,4] Presently, there are two schools of thought concerning the nature of the relationship between oxidative stress and aging In one view, the rate of aging is dependent on the level of antloxldant defenses whde in the alternative wew, at ~s dependent on rate of prooxldant generation The former v~ew has long been championed by Cutler and his co-workers [5,6], who initially reported a positive correlation between maximum hfe span potential (MLSP) of several mammahan species and the activity of superoxlde &smutase (SOD) dwlded by the metabohc rate of the organ [51 Rapid conversion of 0 2 - into H202 by SOD and the resultant low steady state concentration of 0 2 - were beheved to be longevity-determining factors Cutler [6] also reported a negative correlation between MLSP and actw~t~es of catalase and glutathlone perox~dase, which together eliminate H202, believed to be the precursor of the highly reactwe OH radical However, a positive correlation was found between MLSP and some low molecular weight antlOxldants such as urate, tocopherols and carotenolds, prompting Cutler to propose that antloxldant defenses are determinants of the rate of aging [6] For several reasons Cutler's hypothesis that variations in the levels of SOD and some low molecular weight antloxldants act as longevity determinants can not be sustained Firstly, relatively high levels of SOD and correspondmgly low levels of catalase and glutathlone peroxldase would almost certainly increase the Intracellular concentration of H,O~ as well as the hkehhood of OH radical generation, against which the low molecular weight antloxldants would prove to be a poor defense because of the extreme, and virtually &ffuslon dependent, reactivity of OH radicals [2] Secondly, overexpresslon of SOD in genetic transformants has been found to mcrease the level of oxidative stress in E colt [7] and does not prolong the hfe span of Drosphda [8] Thirdly, recent stu&es m this laboratory have indicated that the overall level of antloxldant defenses neither significantly declines with age in insects or mammals nor differs m the tissues of six different mammalian species, ranging between 3 5 and 30 years m hfe span [9--11] Fourthly, admimstration of low molecular weight antloxldants has no effect on maximum life span [4] Thus variations m antloxidant defenses cannot explain either mdlwdual aging or variations m MLSP of different species Since the level of oxidatwe stress tends to mcrease during aging as indicated by the m wvo exhalation of ethane and pentane [12--14], it is, alternatively, possible that the rate of prooxldant generation rather than the level of antloxldant defenses is a crucial factor in the aging process [9,11] Rates of mltochondrml 0 2 - and H~O2 generation were found to, both, increase with age in insects [15,16] and mammals [9] as well as mversely correspond to MLSP of six different mammalian species [ 17,18] The objective of the present study was to further explore the relationship between
191
proox~dant generation and aging by determmlng whether the rate of mltochondrlal H202 generation corresponded to mtra-specles variations in hfe span It was reasoned that if the rate o f H202 generation played a causal role in aging It should mversely correspond to the hfe expectancy of the ammals in a cohort population, i e lntra-specles variations in hfe span should be correlated to the rate of H202 generation The present study was conducted in adult houseflies which provide a fortuitous model for this investigation as the aging rate of files can be slowed down and maxlmum life span doubled by the ehmmatlon of flying actwlty In addition, subpopulatlons of files with relatively short or long hfe expectancy can be separated from a cohort population MATERIALS A N D METHODS
Rearing of files A stock of housefhes, originally obtained from the Zoology department of the Umverslty of Cambridge and maintained for about fifteen years m this laboratory, was used in the present study Larvae were fed on CSMA (Chemical Specialties Manufacturer's Association) fly larval diet Adults were fed on sucrose and water and kept at 25"C and relative humidity of 45--50%
Expertmental vartattons m phystcal actlvtty and hfe span After emergence from the pupae adult files were separated by sex and male files, which were solely used for biochemical studies, were placed under conditions of relatively high (HA) or low (LA) levels of physical activity H A files were housed in one-cubic-foot cages (0 027 m3), where they were able to fly, with 200 files per cage To achieve low levels of physical actwlty, lndwldual files were confined m 150 ml glass urine-specimen bottles fitted with a cardboard maze in such a fashion that files could walk but were unable to undertake flight due to restriction of space
Selection of relattvely short-and long-hved fltes Loss of the abdlty to fly is a umversal feature of senescence m housefiles and provldes a convenient phenotyplc characteristic to recogmze mdwlduals that have reached a point near the end of their hfe Fhghtless fhes were recognized as 'crawlers' as they were unable to fly away, when approached for capture, were separated from their cohorts, the 'tilers', that were still able to fly at 12 days of age Crawlers and tilers were then placed in separate l-cubic-foot cages
Isolanon of mttochondrla Mltochondrla were isolated from the thoraces of the flies according to the procedure of Nordln and Wood [19] as described previously [16] Briefly, the thoraces were separated from the body and placed in a chilled mortar containing buffer A
192 (0 1 ml/thorax), consisting of 154 m M KCl and 1 mM E G T A (pH 7 0), and pounded gently with a pestle into a brel The brel was filtered by suction through eight layers of cheese cloth and the filterate was centrifuged at 125 × g for 3 mm The pellet was discarded and the supernatant was centrifuged at 3000 × g for 8 mm and the pellet resuspended m buffer A
Measurement of 14,0: release by mttochondrta The rate of H202 released by mltochondrla was measured by the method of Hyslop and Sklar [20], which is based on the oxadatlon of p-hydroxyphenylacetate (PHPA) into a fluorescent form during the enzymatic reduction of H202 by horseradish peroxldase The reaction mixture consisted of buffer B (154 mM KCI, 5 mM potassium phosphate, 3 m M MgC12 and 0 1 mM EGTA, pH 7 4), 166/zg/ml PHPA, 83 umts of horseradish perox~dase/ml, 7 mM a-glycerophosphate and about 20/~g mltochondnal proteln/ml of the incubation m e d m m The rate of H202 released was monitored by following the increase in fluorescence at an excitation maximum of 317 nm and an emasslon maximum of 400 nm, using Perkln Elmer LS-5 spectrofluorometer Known concentrations of H202 were used to establish the standard concentration curve RESUL~g Two different approaches were taken to dlstlngmsh between the chronological age and the physlolog~cal age (or hfe expectancy) of files One was to experimentally alter the hfe span of fhes by varying the level of physical activity, and the other was to phenotyplcally select, from a cohort population, subpopulatlons with a relatively short or a long hfe expectancy, as described below
Vartatlons m l~e span mduced by alterattons m phy~tcal acttvlO' Previous studies in this laboratory have shown that metabohc rate of the files has an unambiguous effect on their longevity [14,21,22] Since houseflies are very active tilers and flying results in an up to 100-fold increase m the rate of oxygen consumption as compared to sitting state [22], manipulation of flying activity can be used as a means to control metabohc rate Mortahty characteristics of the houseflies, kept under conditions of relatively low (LA) and high (HA) levels of physical activity, are presented m Fig 1 The average and the maximum life spans of HA flies were 19 + 6 (S D ) days and 40 days, respectively, while those of LA flies were 44 ± 14 (S D ) days and 77 days Thus, the sohtary confinement of tileS under conditions preventing flight resulted in a virtual doubhng of the average and the maximum hfe spans
Selectton of short-hved and long-lived subpopulatlon~ [rom a ~ohort populatton Since all houseflies lose flying abdlty before death, fllghtlessness can be used as
193
100 rl
480
U) 0
÷ 60
O
÷
e." =:) 40
o
LA
+
HA
÷ ÷ ÷
20
wb= •D []
0
[]
!
2'0
0
,0
!
6'0
8O
AGE (days)
Fzg 1 •urvlv•rshlpcurves•fma•e••esk•ptunderc•ndm•ns•fre•atwe•yhlgh(HA)and••w(LA)••ve•s of physical acUvlty Under HA condmon, 200 files were housed per l-cubic-foot cage, whereas under LA c o n d m o n s fl]es were md]vldually c o n f n e d m small glass jars where they were unable to fly Presented data are based on the mortahty o f 200 HA flies and 46 LA flies
an indicator of senescence and impending death Fhghtless fhes, termed crawlers, can be easdy recognized in housefly populations, as these fires are unable to fly away when approached An increasing proportion of flies m an agmg population becomes flightless after about 10 days of age Survlvorshtp curves of crawlers and their cohorts that are sttll able to fly (termed fliers), separated from cohort populations at 12 days of age and placed m separate cages are presented m Fig 2 After isolation
1 O0 "
m
[] [] []
÷ 80" U) nO >
[] +
FLIERS CRAWLERS
%,
60 ÷
n(/1
[]
÷
D
13
40 ÷ ÷
o
