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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 56:B415-B425 (2001)
© 2001 The Gerontological Society of America

Direct Selection for Paraquat Resistance in Drosophila Results in a Different Extended Longevity Phenotype

John Vettrainoa, Steven Bucka and Robert Arkinga

a Department of Biological Sciences, Wayne State University, Detroit, Michigan

Robert Arking, Department of Biological Sciences, Wayne State University, Detroit, MI 48202 E-mail: rarking{at}biology.biosci.wayne.edu.

Decision Editor: John Faulkner, PhD


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
When normal-lived Ra strain Drosophila were indirectly selected for longevity, they gave rise to long-lived La strain animals with lower oxidized protein and lipid levels that were temporally coincident with higher antioxidant activities. We wanted to determine whether it was possible to create long-lived animals by a direct selection for increased antioxidant activities. Using the same Ra strain, we selected them over 24 generations for increased resistance to paraquat. Selection was successful: the paraquat-resistant flies had a fourfold increase in their LT50 (mean lethal time) values. Their extended longevity pattern differs from that of the La strain. The paraquat-resistant animals also have a lower level of antioxidant activity, an increased total P450 enzyme activity level, an altered pattern of energy metabolism, and a significantly lower developmental viability. We interpret these findings as suggesting that similar stress response phenotypes may be generated by different molecular mechanisms, some of which may generate very different types of extended longevity phenotypes.

THE free radical theory of aging postulates that oxidative stress-induced damage is a major causal factor leading to the loss of function and structure that is characteristic of the aging process (1). Much work has been done to test the implications of the theory and the interventions based on it, particularly as to their ability to decrease morbidity and increase longevity. Experiments in which various antioxidants have been fed to experimental animals proved disappointing in that the effects observed mostly affected the mean but not the maximum life span and were neither robust nor reproducible (2) (3). Much more successful have been the experiments using Drosophila in which longevity has been extended, either by the use of transgenic techniques to directly increase antioxidant activity (4) (5) (6) or else by selection experiments for extended longevity in which an increased antioxidant gene activity is strongly implicated as the causal mechanism underlying the increased longevity (7). In the latter study, we have demonstrated that the extended longevity characteristic of the genetically selected La strain certainly appears to be the direct outcome of a coordinate upregulation of a number of different genes whose products are known to function as antioxidants. This activation of the antioxidant defense system genes begins early, at days 5–9 of adult life, and is accompanied by a significantly decreased level of oxidative damage in the La adults (8). This lowered level of damage is believed to be causally related to the delayed onset of senescence in these animals (9). A thorough examination has revealed no other biochemical or stress factors that bear a strong correlation with strain longevity (10). Indeed, reverse selecting the long-lived La strain for shortened longevity results in a downregulation of the antioxidant defense system (ADS) activities to control levels (8). These data led us to conclude that the enhanced ADS activities played a major and probably causal role in bringing about the extended longevity.

These extended longevity animals were originally created by an indirect selection procedure in which animals were directly selected for delayed fecundity and indirectly for long life (11) (12). Only later was it determined that the selected animals were living longer as a result of enhanced antioxidant activities (7). Given our success with this indirect selection strategy, it seemed plausible that it should also be possible to select directly for enhanced antioxidant resistance and indirectly for long life. In view of our prior demonstration that the animals' resistance to exogenous paraquat is an excellent predictor of its antioxidant activity (13) and in view of the fact that the paraquat test is very robust and repeatable, we used the animals' resistance to exogenous paraquat as the direct selection device. The Ra strain was the original progenitor strain of our long-lived La strain (11) (12) and has been kept under nonselective conditions (14); it was used here as the progenitor of the desired oxidative resistant strain, which we will henceforth term the "paraquat-resistant strain," or PQR strain.

Paraquat has been used before in investigations into the genetics of sensitivity to this agent. The prior experiments used paraquat as a screening agent to identify genes involved in ADS activities (15) (16) (17). To the best of our knowledge, there have been no prior reported experiments involving a direct selection for paraquat resistance.

We now report the creation of strains highly resistant to paraquat. The strains have a significant increase in their early adult survivability and thereby exhibit a modest but significant increase in their mean and maximum longevities. We interpret their pattern of extended longevity as being qualitatively different from that observed in our long-lived La strain. The selected PQR animals show a nonsignificant decrease in their ADS activities relative to the progenitor Ra strain as measured by multiple tests; thus an enhanced ADS activity cannot be the mechanism responsible for their elevated early survival nor for their significant resistance to paraquat. They appear, however, to have a significant increase in their total cytochrome P450 (P450) enzyme content. These data suggest that similar stress-resistant phenotypes may be brought about by quite different molecular mechanisms, which themselves may have qualitatively different effects on the observed pattern of extended longevity.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Paraquat Resistance Selection Protocol
The flies of the Ra parental strain (12) were less than 8 hours old at the time of collection to ensure virginity, and they were sexed. Ten groups of 105 flies of each sex and one mixed-sex bottle of controls were set up on new media and held until they were 5 days of age. Nalgene 250-ml screw-cap jars were used for the test chambers (Nalg Nunc International, Apogent, Portsmouth, NH). A 1.5-in. (~3.8 cm) hole was cut into the bottom of each jar and plugged with a sponge stopper to allow for air circulation. Nine Whatman #2 7.0-cm filter disks were put into each of the lids of the screw-cap jars (Whatman, Maidstone, Kent, England); the lids were screwed on and sealed with parafilm. Then 7 ml of 10mM paraquat (PQ) in 1% sucrose was applied to the filters of each of the experimental containers, but only 7 ml of 1% sucrose was put into the control container. The parental flies were transferred into the test jars and the time noted. Observations of the number of dead animals in each test jar were made approximately every 3 hours. At LT50 (that time when half the flies in each container are dead), the flies were removed and put on food. They were designated as PQR1-1, which stands for paraquat-resistant strain derived from the Ra strain, generation one, replicate one. They were allowed a few days to recover before being allowed to reproduce the next generation. The five Ra parental bottles were used again to create the PQR1-2 and PQR1-3 replicate lines. Thus we ran three independent replicate lines with 10 dependent assays per line per generation. As resistance increased and the time to reach LT50 became longer, it was felt necessary to put the flies into fresh experimental containers on the fourth day so as to prevent even subtle desiccation or starvation effects. Control animals showed no significant mortality during the course of the selection protocol.

In the eleventh generation, the third replicate line (PQR11-3) was changed from selection at 5 days of age to selection at 15 days of age. In the nineteenth generation, the second replicate line (PQR19-2) was changed from selection on 10mM PQ to selection on 50mM PQ. PQR19-1 and PQR19-3 were not selected on exogenous PQ for logistical reasons, but the selection was resumed in the next generation. The paraquat selection pressure was later removed from all but those flies selected at 50mM PQ for practical reasons. The phenotype appears to be reasonably stable. Selection on the 50mM PQ line was continued through generation 58 inclusive, and sporadically thereafter.

Longevity Test Protocol
Flies were put at a density of 100–105 flies in standard culture bottles containing our sucrose–yeast media (12). They were transferred to new media three times per week, and the number of dead flies was counted.

Enzyme Assays
Protein extraction..-- The fly protein was extracted by using the procedure of Orr and Sohal (4) but modified slightly by the addition of a protein-denaturation step in which the sample was treated twice with 1.6 vol of chloroform:ethanol (1:1). After vortexing, the sample was spun for 5 minutes at 3900 rpm at 4°C. The extract was placed at -20°C overnight and any precipitate was removed by centrifugation at 3900 rpm in a microcentrifuge. Protein concentration was determined by using the BioRad miniprotocol as described (BioRad, Oakland, CA).

Antioxidant enzyme activity assays..-- The superoxide dismutase activity was measured in whole animals by using the protocol of Misra and Fridovich (18) as modified by Lee and colleagues (19), and as further modified slightly by us (8). All samples were measured in triplicate and expressed as the specific activity per milligram of protein.

The catalase enzyme activity was measured in whole animals by using the protocol of Goth (20). All samples were measured in triplicate and expressed as the specific activity per milligram of protein.

An alternative measurement of catalase activity (21) is its inhibition with 3-amino-1,2,4-triazole (3AT). This compound is known to bind irreversibly to the catalase protein and inhibit its activity. Thus measuring the animals' resistance to PQ after a standardized course of 3AT treatment gives an approximate measure of the catalase activity. Seven milliliters of a solution containing 5mM 3AT in 1% sucrose was applied to the nine Whatman filters in our standard test containers. Between 100 and 105 virgin flies that had been sexed and aged were placed into each container. Control animals were treated identically, except they were only exposed to a 1% sucrose solution. The animals were exposed for 6 hours and then tested for their resistance to 10mM PQ as described in our standard PQ assay.

Another measurement of catalase activity is the "fizz test," in which the bubbles produced by catalase breakdown of H2O2 are visually estimated (22). It is strictly a qualitative test. A microtiter plate was filled with one fly per well, and each was ground by using a homogenizer and 20 µl of 0.1M Tris-PO4 and 1% Triton-X 100, pH 7.0. To this was added 25 µl of 30% H2O2, and the extract was assayed for level of fizzing by visually estimating the approximate number of bubbles released per unit of time.

Difference spectra quantitative determination of cytochrome P450 content..-- We used the CO difference spectra method of Omura and Sato (23) as modified by Estabrook and Werringloer (24) and Rutten and colleagues (25) to quantitatively measure the cytochrome P450 content of the Ra, La, and PQR animals. The frozen flies (~0.15 g) were suspended in 1.5 ml of MKEG buffer, that is, 0.1M MOPS (3-[N-morpholino] propanesulfonic acid), 0.1M KCl, 1mM ethylenediamine tetraacetic acid, 20% glycerol (25), and homogenized with a Teflon homogenizer in a 5-ml Pyrex tube. The homogenate was spun at 9000x g for 20 minutes to pellet debris. The aqueous supernatant was removed while avoiding the lipid layer, and this was respun twice or until no visible pellet or lipid layer was visible. A 500-µl aliquot of the low-speed supernatant was spun twice at 105,000x g (54,000 rpm) by using the TLA100.1 rotor for the Beckman Optima TL Ultracentrifuge (Beckman Coulter, Inc, Fullerton, CA). The pellet was rinsed twice with buffer before resuspension. After the second high-speed spin and resuspension, the extract was spun at low speed for 1 minute to pellet any unsuspended debris. Protein content was measured with the BioRad assay. CO was bubbled into the microfuge tubes containing the microsomal suspension for 1 minute each. Dithionite (0.1852 g/350 µl sample) was added to each microsomal suspension; the sample was vortexed and then spun for 7 minutes at low speed to clear the bubbles. Seven minutes after each sample was vortexed, a spectral scan using a 1-nm slit width that included 450 and 490 nm was run by using a Beckman DU640 spectrophotometer. The P450 concentration per milligram of protein was calculated by using the absorbances at 450 and 490 nm, the molar extinction coefficient of 99 mM-1 cm-1, and the protein concentration of the microsomal suspension.

Piperonyl butoxide assay..-- Piperonyl butoxide (PBO) was obtained from Aldrich Chemical (Sigma-Aldrich Corporation, St. Louis, MO; cat. 29.110-2). It is a thick oil and is not miscible with aqueous solutions. We modified the technique of Frank and Fogleman (26) by suspending 1 g of finely ground yeast into 5.33 ml of ethyl ether and adding an appropriate volume of PBO. The mix was then stirred and the ether was allowed to evaporate in a fume hood overnight. When no trace of ether could be detected, 5.33 ml of H2O was added, the mix was stirred, and then 1 ml of the slurry was put on top of an agar base in a standard food bottle and allowed to dry. Thus the flies' only source of food was the PBO-laced yeast paste. A known number of same-age mixed-sex animals were put into the bottle, and their survival was monitored until all had died. Controls were treated in an identical manner save that no PBO was added to the ether-treated yeast.

Metabolic Enzyme Activity Assay
For determination of the 17 metabolic enzymes listed in Table 5 , Table 5 - to 7-day-old flies were collected at the same time from both the Ra and PQR strains, and they were processed and assayed as a group as described elsewhere (8). The protein content of each sample was determined with the dye-binding assay (kit 500-0002, BioRad Labs, Richmond, CA). The data from the three replicates of each sample were tallied and enzyme activity was expressed as the mean ± SEM absorbance units (µg protein/min).


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  		Table 5. Effect of Selection for PQ Resistance on Enzymes Involved in Energy Metabolism

 
Developmental Viability
Developmental viability, or the mean number of fertilized eggs that eventually yielded viable adults, was assayed as previously described (27).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Results of Selection Over 24 Generations
Selection for the ability to resist exogenous PQ was carried out on three replicate lines for a period of 24 generations. The selection procedure was quite stringent. Briefly, the procedure involved collecting at 5 days of age approximately 500 each virgin males and females (1000 total) from each replicate line and exposing them in same-sex groups of 100 each to a solution of 10mM PQ in 1% sucrose administered under a set of standard conditions. The treatment was maintained until 50% of each group had died. This LT50 time was noted, and the survivors were collected and allowed to mate with each other so as to give rise to the next generation. Thus all parents of each generation were selected for resistance to exogenous PQ and all offspring were derived only from survivors. Fig. 1 shows the results of this selection for the first replicate line (PQR-1) through the first 24 generations. It may be seen that the LT50 increased about fourfold, from ~44 hours to ~180 hours. The selection response was similar in all three selection lines, as can be seen from the variances. No response was observed in the absence of selection (i.e., the Ra parental strain).



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  		Figure 1. The response of successive generations of paraquat-resistant (PQR) animals to selection for survival on 10mM paraquat (PQ) at 5 days of age, as indicated by the obvious increase in the mean (± SEM) hours necessary to kill 50% of the exposed animals (LT50) as described in the text. Each point is based on the results of 10 replicates of ~105 flies each.

 
In generation 11, the third replicate line (PQR-3) was shifted from selection at 5 days of age to selection at 15 days of age. At this age the LT50 of the Ra parental line is approximately 20 hours or less. Selection was effective at this later age as well, increasing from ~72 hours to ~102 hours over the course of the next ten generations ( Fig. 2).



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  		Figure 2. At generation 11 of Fig. 1, an aliquot of paraquat-resistant flies was subjected to selection for survival on 10mM paraquat at 15 days of age. Each point is the mean (± SEM) of 10 replicates of ~105 flies each; LT50 = mean lethal time.

 
By generation 18, the LT50 had increased so much that measuring it was both inconvenient and potentially inaccurate because of the necessity of replenishing the PQ–sugar solution. Accordingly, the selection protocol was modified so that the second replicate line, at the age of 5 days, was exposed to an increased dose of 50mM PQ ( Fig. 3). In this line, the LT50 sharply increased from ~72 hours to ~107 hours over a period of 10 generations. Although this value is less than the LT50 value obtained for PQR29-1, the fivefold higher PQ concentration used ensures that this is in fact a real increase in resistance. The use of this high dose allowed us to establish an empirical limit to the strain's PQ resistance.



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  		Figure 3. At generation 19 of Fig. 1, an aliquot of paraquat-resistant flies was subjected to selection for survival on 50mM paraquat at 5 days of age. Each point is the mean (± SEM) of 10 replicates of ~105 flies each. LT50 = mean lethal time.

 
The high levels of PQ resistance brought about by selection are not restricted to the age of selection. Testing the strains for PQ resistance at different times during their adult lives shows that there is a significant increase in PQ resistance throughout the entire life span ( Fig. 4). Although the peak values of the PQ resistance occur at 5 days and decrease at later ages, they never attain the low levels of the nonselected control. They resemble the PQ curves that are characteristic of the selected long-lived strains (10).



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  		Figure 4. The increase in paraquat resistance (PQR) extends throughout the entire adult life span. LT50 = mean lethal time.

 
Effect of PQ Resistance on Longevity
The effect of endogenous PQ resistance on longevity was assayed by measuring the life span of the selected PQR strains, the normal-lived progenitor Ra strain, and the long-lived La strain. The data for the F5, F7, F16, and F23 of selection line one (i.e., PQR-1) as well as the F23 of line two and the F20 of line three is shown in Fig. 5. A Kaplan–Meier survival test showed that both replicates of the PQR F23 lines had survival curves that were significantly different than those of the Ra control strain (log-rank test = 24.76, df = 1, p < .00005). A significant increase in both the mean and median life span occurred, from 39.01/40.00 days for the Ra strain, respectively, to 47.27/48.00 days for the F23 of the PQR line two strain. These changes are significant (Kolmogorov–Smirnov test, T = 3.047, p < .01). Similar median life spans are noted for lines one and three as well (not shown). There is a comparable increase in the maximum (LT95) life spans, increasing from ~56 days in the Ra strain to ~63 days in the PQR F23 strain, or approximately a 12.5% increase. Note that the comparable mean and maximum life span values for the long-lived La strain are 80 and 97 days, respectively, or approximately a 105% and 73% increase, respectively. Although both the PQR and La strains live significantly longer than do the Ra control strains, their extended longevity phenotypes seem to be qualitatively different from each other.



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  		Figure 5. Effects of selection for paraquat (PQ) resistance (10mM, 5 days) on longevity. Measurements were made on both the long-lived La and normal-lived Ra strains, as well as on the indicated generations of the selected PQ-resistant (PQR) strain. An increase in early (1–20 day) survival relative to the La strain was noted by PQR F5 and continued thereafter. This resulted in a modest increase in mean and median life spans but not in maximum life spans.

 
The increased median life span of the PQR strain appears to be due to a significant improvement in the early survival of the animals (Table 1 ). By the age of 20 days, between 12% and 15% of the Ra strain have died, compared with ~6% of the La strain, and less than 3–5% of the PQR strains. This effect happened early in the course of selection, because even the F5 showed a significant increase relative to the progenitor Ra strain. Note that this increased early survival increased from one generation to the next ( Fig. 5), suggesting that it is a consequence of the selection regime.


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  		Table 1. Effects of Selection for PQ Resistance on the Adult Survival Characteristics of the Population

 
Involvement of the Antioxidant Defense System
The ADS contains a large number of different enzymes as well as nonenzymatic free radical scavengers. There is no one test that will adequately test the entire system. We decided to assay the level of two enzymes, copper–zinc superoxide dismutase (CuZn SOD) and catalase (Cat), in the 5-day-old La, Ra, and PQR animals. Table 2 lists the results of several different assays. The results of the CuZn SOD enzyme activity tests ( Fig. 6) show that significant differences exist between the three strains (one-way analysis of variance, 13 df, F = 14.891, p = .001) such that there is a large difference between the La strain and the other two strains (Scheffe, p = .008), but there is no significant difference between the Ra and PQR strains in this regard (Scheffe, p = .637). The Cat activity levels ( Fig. 7) between the three strains are not significantly different from one another (one-way analysis of variance, F = 2.770, 13 df, p = .106) even though the PQR animals have a 20% lower specific activity than the Ra strain.


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  		Table 2. Summary of Relative Antioxidant Enzyme Activities/Levels in PQR and Control Strains

 


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  		Figure 6. The effect of selection for paraquat resistance (PQR) on antioxidant enzyme activity. The mean (± SEM) copper–zinc superoxide dismutase (SOD) enzyme activity levels in 5-day-old animals of the indicated strains is shown. There is a significant difference between the La and the other two strains but not between the Ra and PQR strains (see text for details).

 


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  		Figure 7. The effect of selection for paraquat resistance (PQR) on antioxidant enzyme activity. The mean (± SEM) catalase (Cat) enzyme activity levels in 5-day-old animals of the indicated strains is shown. There is no significant difference between the three strains (see text for details).

 
When subjected to the rough screening of the fizz test (28), the differences between the three strains are minor and not easily seen with the naked eye (Table 2 ). However, when the flies were pretreated with 3AT (a specific inhibitor of Cat enzyme activity), the PQR animals had a lower level of Cat enzyme activity than did the Ra or La animals (Table 2 ). This suggested that perhaps the fizz test was too crude to detect the Cat activity differences until a constant amount of 3AT irreversibly inhibited different proportions of the enzyme in the three strains, thus making the difference clear. Even though the PQR animals have less Cat activity than do the other strains, they still rely in part on Cat to counter the effects of the PQ, because prefeeding 3AT to these animals results in a significant lowering of their resistance to PQ ( Fig. 8; Kolmogorov–Smirnov test, T = 2.750, p < .01).



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  		Figure 8. The effect of prefeeding 3-amino-1,2,4-triazole (3AT), a known catalase inhibitor, on the ability of the paraquat (PQ)-resistant strain to survive 10mM PQ at 5 days. There is a significant difference between the two experimental curves, suggesting that catalase makes a minor contribution to PQ resistance (see text for details).

 
Involvement of the P450 System
We used the CO difference spectra method (as described earlier) to determine the content of P450 in microsomal supernatants of our PQR strain relative to its normal progenitor R strain. We were able to quantitatively estimate the P450 content of each strain; our values are consistent with those in the Drosophila literature (29) and are shown in Table 3 . These data suggest that the PQR strain animals have significantly higher P450 levels than those of the normal-lived progenitor Ra strain or the long-lived La strain ({chi}2 = 116.7, 2 df, p < .001). This assay measures the overall enzyme content; it does not allow us to determine whether specific P450 enzymes are elevated, decreased, or left unchanged.


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  		Table 3. PQR Animals Have a Higher P450 Content Than Control Strains

 
PBO is a nonspecific inhibitor of the P450 enzymes (30). If the PQR animals really do have significantly higher P450 enzyme activity levels as suggested by the data of Table 3 , then one would expect the PQR animals to be significantly more resistant to the toxic effects of PBO than are either the Ra or the La strains. The results of this experiment, shown in Fig. 9, suggest that the PQR animals are proportionately more resistant to PBO than are the Ra animals from which they were derived. The data also suggest that the increased resistance of the La strain to PBO may be due in part to some effect of its higher ADS levels and/or to some unknown increased activity of a specific P450 enzyme(s) that did not, however, detectably alter the overall P450 content.



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  		Figure 9. The ability of the Ra, La, and paraquat-resistant (PQR) strains to survive the lethal effects of piperonyl butoxide (PBO), a known P450 inhibitor. The Ra animals were tested with 5.54mM PBO and the La and PQR strains were tested with 16.62mM PBO. Note the large survival difference between the PQR and the other strains, which suggests that the P450 enzymes make a major contribution to PQ resistance.

 
Finally, we point out that there is a reciprocal relationship between the ability of the animal to mobilize its ADS resources and its P450 resources (Table 4 ). It appears as if an increase in one protective system is balanced by a loss in the other protective system. The two systems are not independent of one another but are linked together somehow.


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  		Table 4. Reciprocal Mobilization of ADS and P450 System

 
Involvement of Other Enzymes
Table 5 shows the changes in specific activity measured in 17 different enzymes assayed in both the Ra and PQR strains. All but three of the measured enzymes showed significant changes in specific activity. It is apparent that the PQR animal has an altered energy metabolism relative to its progenitor Ra strain.

Effects of Selection on Developmental Viability
Table 6 shows the results of two independent developmental viability measurements on the PQR strain. It may be seen that the Ra progenitor strain has an average overall developmental viability of ~84%, whereas the PQR strain has an average overall viability of ~66%. These values are significantly different from each other, but there is no significant difference between the two PQR samples (see table).


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  		Table 6. Effects of Selection on Overall Developmental Viability

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Selection and PQ Resistance
The data show that it is possible to use direct selection to create Drosophila strains that are significantly resistant to PQ. Twenty-four generations of selection led to a fourfold increase in the mean LT50 value (our measure of PQ resistance) in the PQR strains relative to the nonselected Ra control ( Fig. 1). Significant increases in PQ resistance were obtained with selection by using either 5-day-old or 15-day-old adults ( Fig. 2), although the greater absolute and relative increase was obtained with the younger adults ( Fig. 4). This pattern was fully consistent with the lifetime pattern of PQ resistance ( Fig. 4), in which the 5-day-old adults showed the highest LT50 values. This pattern appears identical to the PQ resistance pattern exhibited by our selected long-lived La strain (10) and suggests that the early peak in PQ resistance must reflect some important characteristic of young adults. Note, however, that as selection proceeded, the LT50 value of older adults exceeded the LT50 value of younger adults in the preceding cohort ( Fig. 4), indicating that real physiological changes are taking place at all adult ages. Interestingly, subjecting the young adults to selection with a high PQ dose (50mM) led to a sudden and large increase in PQ resistance; this resistance level decreased and then slowly regained this high initial value by the end of selection ( Fig. 3). The performance of each of the replicate lines probably represents the maximum response of the genome to that particular selection pressure inasmuch as our reliance on mostly using virgin animals ensured that all offspring were descended only from resistant parents.

PQ Resistance and Longevity
The 21% increase in the mean and median longevity appears to arise from the fact that the PQR strains display an enhanced early survival, relative to their normal-lived Ra control strain (Table 1 ). In fact, the early survival rate of the PQR strain is comparable with that of our long-lived La strain, although this latter strain has a significantly longer mean and maximum longevity ( Fig. 5 and Table 1 ). This finding suggests that enhanced early survival is separable from an increase in the maximum longevity, and that different processes may be responsible for each of these separable components.

There is a smaller increase (~12%) in the maximum (LT95) life-span values. We interpret these data as suggesting that the increased longevity observed as a result of this direct selection for PQ resistance stems mostly from the increased early survival but is not associated with any mechanistic effects on the maximum longevity. The increase in the latter is most likely an indirect outcome of the increased early survival.

PQ Resistance and Antioxidant Activity
The increase in paraquat resistance is not due to an increased activity of the antioxidant enzymes. Both CuZn SOD and Cat show substantial but nonsignificant decreases in their enzyme activities and protein levels in the PQR strain relative to the normal-lived Ra control strain (Table 2 ; Fig. 6 and Fig. 7). As expected on the basis of our previous work (7), the long-lived La strain animals had significantly higher levels of CuZn SOD enzyme activity, whereas the essentially normal levels of Cat activity in the La strain are consistent with a more extensive analysis (8). Therefore, although direct selection for late fecundity leads to high antioxidant activity and high PQ resistance as well as increased longevity in the La strain (7) (8) (12), direct selection of the same Ra progenitor strain for high PQ resistance does not lead to high antioxidant activity and yields a different pattern of extended longevity.

This does not, however, mean that the ADS is not involved in this PQR resistance. Pretreatment of the animals with a potent Cat inhibitor (3AT) followed by exposure to PQ led to a reduced survival time on PQ when compared with flies not given the inhibitor ( Fig. 8). Catalase, at least, is playing some role in the mechanisms underlying PQ resistance, or else we would not have observed a 12-hour difference in the LT50 values between the 3AT-treated and 3AT-untreated PQR animals.

PQ Resistance and P450 Enzymes
The data of Table 3 suggest that the PQR animals have significantly higher P450 activity levels than do either the La or Ra strains. This observation offers a clue to the biochemical mechanisms underlying the observed PQ resistance. There are no direct reports of P450 metabolism of PQ. However, it seems to us that the potential involvement of the cytochrome P450 system may be very plausible, given its role in detoxification in general and in conveying resistance to common insecticides in particular. This is especially true because the P450 system has been known to convey resistance to isoquinoline alkaloids (31), which are very structurally similar to PQ. Given the broad substrate specificity of the P450 enzyme system held responsible for this activity (32), this clue merits further investigation.

PQ Resistance, Energy Metabolism, and Developmental Viability
On the basis of the enzyme activity data presented in Table 5 , it appears as if the net effects of the changes noted might be (1), a partial compensatory increase in resistance to oxidative stress; (2), an alteration in the levels of NAD(P)H, either nicotinamide adenine dinucleotide phosphate or reduced nicotinamide adenine dinucleotide, production; (3), an increased sensitivity of adenosine monophosphate (AMP)-dependent enzyme regulatory mechanisms; and (4), a possible shift in energy pathways. The reasons supporting this statement are as follows.

The large increase in xanthine dehydrogenase (XDH) activity results in the increased production of urate and reduced nicotinamide adenine dinucleotide (NADH). Urate can function as a strong reducing agent. Its ability to reduce oxidants may compensate in part for the decreased activity levels of CuZn SOD and Cat (Table 2 ; Fig. 6 and Fig. 7). Hayes and colleagues (33) showed that some P450 enzymes generate increased reactive oxygen species (ROS) as a by-product of their specific enzyme reactions, and that the induction of many P450 enzymes is accompanied by the co-induction of glutathione-S-transferases (GSTs). Increased levels of GSTs would eventually result in transiently increased levels of glutathione (GSH), which can be converted to conjugates and eliminated, thereby protecting the cell against the extra ROS generated by the P450 enzymes.

However, the resulting oxidized glutathione (GSSG) requires NADH for recycling. This could come in part from the increased XDH activity noted above. In addition, the NADH would be needed to act as a cofactor in several reactions listed in Table 5 , such as that with NADH diaphorase, glycerol-3-phosphate dehydrogenase, and others.

It has long been known that many enzymes regulate their activity by means of the binding of AMP. An increase in adenylate kinase activity (Table 5 ) may indicate the increased sensitivity or activity of such AMP-dependent metabolic reactions.

Clark (34) showed that quantitative changes in a number of metabolic enzymes are highly correlated with changes in viability on different types of media. The selection for PQR led not only to an increase in P450 content and a decrease in CuZn SOD content, but also to concomitant changes in energy metabolism and probably to the observed changes in developmental viability as shown in Table 6 . Similar effects on energy metabolism and viability were observed as a result of selection for longevity exerted on the Ra strain (8) (27). Because different selection regimes cause similar decreases in energy metabolism and Darwinian fitness, we conclude that the Ra animal's metabolism is at an optimal or "mini–max" value. Changes in gene expression brought about by selection will almost always result in a less than optimal value and thus result in a decrease in fitness. It may be this fact that underlies the observation that animals with very high levels of resistance to oxidative stress, paraquat, and so on are not normally seen in the wild in the absence of selection.

Longevity and Stress-Resistance Mechanisms
An interesting observation flows from the comparison of the relative mobilization of the ADS and P450 system in animals subjected to two different selection regimes. As the data in Table 4 suggest, there appears to be a reciprocal relationship between the two different types of defense systems. Subjecting animals to a direct selection for delayed fecundity had the apparent effect of significantly increasing the CuZn SOD enzyme protein and activity levels by 75% while simultaneously decreasing the overall P450 activity levels by 7%. Conversely, directly selecting animals of the same strain for increased PQ resistance apparently led to a 105% increase in P450 enzyme content and a 15% decrease in CuZn SOD enzyme protein and activity levels. It appears as if an increase in one protective system is balanced by a loss in the other protective system, although more data are needed to convert this from a suggestion to a fact. One implication of this observation is that the two systems are not independent of one another but are linked together somehow. If this hypothesis is confirmed, then it might suggest that the animals' response to selection might depend upon the nature of these linkages and that certain responses might be prohibited. Simultaneous increases in both the ADS and P450 system, for example, may not be normally possible. Presumably these linkages represent the summed vector of the regulatory genes modulating each defense system. We have already isolated and identified a number of trans-acting regulatory genes on chromosome 2 that significantly alter the expression of the ADS genes located on chromosome 3 (9). It will be interesting to determine if these ADS regulatory genes also regulate the expression of the P450 system and, if so, whether they regulate the two systems in a reciprocal manner or not.

However, the most interesting observation flows from the fact that the directly selected PQR animals are very resistant to PQ but have a qualitatively different pattern of extended longevity than do our indirectly selected La animals (8). Our ADS-dependent La strain shows an increased early survival and a delayed onset of senescence, the net effect of which is to extend longevity in the early, middle, and late adult stages. The (presumably) P450-dependent PQR stain shows an increased early survival but shows no sign of delaying the onset of senescence. The net effect is to extend longevity in the early adult stages but not in the later life stages. Increasing early survival is not the same as delaying the onset of senescence in midlife. Different molecular mechanisms will enhance longevity in different ways.

PQ toxicity stems from the fact that it is a ROS generator in the cell (35). Cells can manifest two different adaptive responses to deal with this xenobiotic. One response is to detoxify the PQ molecules by metabolizing them with the appropriate P450 enzyme(s). The other response is to neutralize the ROS formed in the cell by means of the induction of ADS enzymes such as CuZn SOD and catalase. It seems as if these two different mechanisms will each give rise to a different extended longevity phenotype.

The detoxification response is carried out by cytochrome P450 enzymes. These terminal electron acceptors receive electrons from NADP by means of flavoprotein P450 reductase. The P450 heme proteins bind molecular oxygen when reduced, one molecule of which is incorporated into the substrate and one molecule into water in a two-electron reduction process (36). This begins the detoxification of the xenobiotic. Many of the P450 enzymes induced by xenobiotics also exhibit antioxidant functions and can protect the cell against both ROS and the toxic by-products generated by the interaction of free radicals with macromolecules (33). These antioxidant functions seem to be carried out by means of the secondary induction of GST and/or NAD(P)H quinone oxidoreductases, that is, NAD(P)H diaphorase (Table 5 ; (33), (37)). Note that NADP diaphorase is significantly increased in PQR animals while NADH diaphorase is significantly decreased (Table 5 ), suggesting that this mechanism is involved in the PQR animals. There is no evidence that CuZn SOD and/or Cat are involved in this P450-based antioxidant protection.

The second response, neutralization of the ROS molecules, does seem to rely on CuZn SOD, Cat, and other ADS enzymes (35). We have presented evidence elsewhere (8) showing that the extended longevity of our La strain animals depends on high levels of at least these two enzymes. In fact, all Drosophila strains, transgenic lines, and mutants that have been demonstrated to have an enhanced antioxidant resistance based on significantly increased levels of CuZn SOD and/or Cat have also been shown to express a significantly increased longevity (4) (5) (6) (7) (8). In addition, it has been shown that processes that reduce the leakiness of the mitochondria to ROS are also associated with extended longevity (38) (39) (40). Thus prevention of ROS generation and/or scavenging of ROS once formed are both effective mechanisms of prolonging longevity.

Multiple experiments have demonstrated that there exists a genetic basis for various forms of stress resistance (41). These experiments have also demonstrated that selection for one form of stress resistance often yields a correlated resistance to other stressors as well (42) (43). It is known that mutants affecting the daf pathway in Caenorhabditis elegans can extend longevity and have multiple physiological effects, including an upregulation of the CuZn SOD (44) and Cat (45) enzyme levels. Despite the ubiquitous involvement of the ADS enzymes in extended longevity, it was assumed that a generalized stress resistance was the major mechanism underlying extended longevity (42) (46). However, the data showing that Drosophila strains selected for resistance to one stress do not always express an extended longevity phenotype suggest that this cannot be entirely correct (47). It is also known that selected animals often have a reduced resistance to some common stressors (this report; (27), (48)). Taken together, these facts suggest that a normal-lived animal may have a generalized ability to resist many if not all stressors, whereas the long-lived animal may have become genetically specialized in ways that decrease its general ability to handle stress while increasing its ability to resist some smaller number of specific stressors. This conclusion is consistent with the data we report in this paper.

Stability of the Control Strain
One conclusion of this study is that different experimental procedures of selecting for a stress-resistant phenotype will engage different molecular responses on the part of the organism. This conclusion rests on the assumption that the range of genetic diversity present in the Ra strain at the time of the original La indirect selection experiments is still present in that strain approximately 15 years later, when we began these PQR direct selection experiments. If the diversity has inadvertently decreased, then the failure to produce an La-type extended longevity might solely be due to the loss of the required genetic potential from the Ra line. We do not believe this objection holds in this case, for the following reasons.

First, we deliberately maintain our Ra strain in such a way so as to minimize selection effects and maximize the retention of genetic variability (12).

Second, loss of genetic diversity should be indicated by a decreased effectiveness of selection. The generation of our initial La strain from the Ra strain, begun in 1980, took 25 generations to accomplish with 72% of the effect being realized by the F13 (12). One year later, we again used the Ra strain to create a replicate La strain (2La) with similar kinetics. Several years later, a replicate short-lived strain (2Ea) was generated from the Ra strain, again with similar kinetics. The PQR selection, begun in 1995, was accomplished in 24 generations with 64% of the effect being realized by the F12 ( Fig. 1). Thus, over a 15-year period, the Ra strain yielded four successful selection strains with no indication of a reduced genetic diversity.

Third, by contrast, the generation of our reverse-selected RevLa strain from the selected La strain illustrates the kinetics of selection when practiced on a strain that, by definition, has lost significant genetic diversity. The generation of the RevLa strain took more than 65 generations and is still not complete (8). This process likely depends on the selective screening of new mutations rather than the uncovering of existing genetic diversity, and the kinetics are correspondingly slowed.

Thus the available evidence supports our assumption that the Ra strain has not lost significant genetic diversity over the past decades, and that the organism can deal with the same stressor by mustering different molecular mechanisms, depending on the mode of stressor presentation, which may have quite different physiological and phenotypic effects.

Conclusions
We interpret our data presented here as suggesting that similar forms of stress resistance may involve different molecular mechanisms that can then give rise to different patterns of extended longevity. These different extended longevity phenotypes have to be identified and understood if we are to effectively understand the aging processes. Some of the recent discussions concerning the effectiveness of different antiaging interventions may have their origins in this phenomenon. We plan to further investigate this topic elsewhere (Arking, in preparation).


   Acknowledgments
 
This research was supported in part by National Institutes of Health Grant AG08884 to R. Arking.

We thank Mr. Paul Shabash, David Mancini, S. Vongpunsawad, and Elliott Feldman for their assistance during various phases of this project. We also thank two anonymous reviewers for their constructive suggestions.

Received September 28, 2000

Accepted April 10, 2001


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Harman D, 1956. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 298–300:
  2. Comfort A, 1971. Effect of ethoxyquin on the longevity of C3H mice. Nature. 229:254-255. [Medline]
  3. Yu BP, 1995. A new outlook for anti-oxidant defense system. Aging (Milano) 7: (5) 338-339. [Medline]
  4. Orr WC, Sohal RS, 1994. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263: (5150) 1128-1130. [Abstract/Free Full Text]
  5. Parkes TL, Elia AJ, Dickinson D, Hilliker AJ, Phillips JP, Boulianne GL, 1998. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat Genet. 19:171-174. [Medline]
  6. Sun J, Tower J, 1999. FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster flies. Mol Cell Biol. 19: (1) 216-228. [Abstract/Free Full Text]
  7. Dudas SP, Arking R, 1995. A coordinate upregulation of antioxidant gene activities is associated with the delayed onset of senescence in a long-lived strain of Drosophila. J Gerontol Biol Sci. 50A:B117-B127. [Abstract]
  8. Arking R, Burde V, Graves K, et al. 2000. Forward and reverse selection for longevity in Drosophila is characterized by alteration of antioxidant gene expression and oxidative damage patterns. Exp Gerontol. 35:167-185. [Medline]
  9. Arking R. Gene expression and regulation in the extended longevity phenotypes of Drosophila. Ann NY Acad Sci. In press.
  10. Force AG, Staples T, Soliman S, Arking R, 1995. Comparative biochemical and stress analysis of genetically selected Drosophila strains with different longevities. Dev Genet. 17: (4) 340-351. [Medline]
  11. Luckinbill LS, Arking R, Clare MJ, Cirocco WC, Buck S, 1984. Selection for delayed senescence in Drosophila melanogaster. Evolution. 38:996-1004.
  12. Arking R, 1987. Successful selection for increased longevity in Drosophila: analysis of the survival data and presentation of a hypothesis on the genetic regulation of longevity. Exp Gerontol. 22:199-220. [Medline]
  13. Arking R, Buck S, Berrios A, Dwyer S, Baker GT, III 1991. Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila. Develop Genet. 12:362-370. [Medline]
  14. Arking R, Buck S, 1995. Selection for increased longevity in Drosophila melanogaster: a reply to Lints. Gerontology. 41:69-76.
  15. Humphreys JM, Hilliker AJ, Phillips JP, 1993. Paraquat selection identifies X-linked oxygen defense genes in Drosophila melanogaster. Genome. 36:162-165. [Medline]
  16. Humphreys JM, Duyf B, Joiner M-LS, Phillips JP, Hilliker AJ, 1996. Genetic analysis of oxygen defense mechanisms in Drosophila melanogaster and identification of a novel behavioral mutant with a Shaker phenotype. Genome. 39:749-757. [Medline]
  17. Wang JW, Humphreys JM, Phillips JP, Hilliker AJ, Wu C-F, 2000. A novel leg-shaking Drosophila mutant defective in a voltage-gated K+ current and hypersensitive to reactive oxygen species. J Neurosci. 20:5958-5964. [Abstract/Free Full Text]
  18. Misra HP, Fridovich I, 1972. The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 247:3170-3175. [Abstract/Free Full Text]
  19. Lee YM, Ayala FJ, Misra HP, 1981. Purification and properties of superoxide dismutases from Drosophila melanogaster. J Biol Chem. 256:8506-8509. [Abstract/Free Full Text]
  20. Goth L, 1991. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta. 196:143-152. [Medline]
  21. Lubinsky S, Bewley GC, 1979. Genetics of catalase in Drosophila melanogaster: rates of synthesis and degradation of the enzyme in flies aneuploid and euploid for the structural gene. Genetics 91:723-742. [Abstract/Free Full Text]
  22. Mackay WJ, Bewley GC, 1989. The genetics of catalase in Drosophila melanogaster: isolation and characterization of acatalasemic mutants. Genetics 122:643-652. [Abstract/Free Full Text]
  23. Omura T, Sato R, 1964. The carbon monoxide-binding pigment of liver microsomes: I. Evidence for its hemoprotein nature. J Biol Chem. 239:2370-2378. [Free Full Text]
  24. Estabrook RW, Werringloer J, 1978. The measurement of difference spectra: application to the cytochromes of microsomes. Meth Enzymol. 52:89-96. [Medline]
  25. Rutten AAJJL, Folke HE, Catsburg JF, et al. 1987. Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes. Reinvestigation of assay conditions. Arch Toxicol. 61:27-33. [Medline]
  26. Frank MR, Fogleman JC, 1992. Involvement of cytochrome P450 in host-plant utilization by Sonoran desert Drosophila. Proc Natl Acad Sci USA. 89:11,998-12,002. [Abstract/Free Full Text]
  27. Buck S, Vettraino J, Force AG, Arking R, 2000. Extended longevity in Drosophila is consistently associated with a decrease in developmental viability. J Gerontol Biol Sci. 55A:B292-B301. [Abstract/Free Full Text]
  28. Mackay WJ, Bewley GC, 1989. The genetics of catalase in Drosphila melanogaster: isolation and characterization of acatalasemic mutants. Genetics 122:643-652.
  29. Danielson PB, Fogelman JC, 1994. Inhibition of cytochrome P450 enzymes by the eye pigments in D. mojavensis. Drosophila Inf Serv. 75:90-91.
  30. Miranda CL, Henderson MC, Buhler DR, 1998. Evaluation of chemicals as inhibitors of trout cytochrome P450s. Toxicol Appl Pharmacol. 148:237-244. [Medline]
  31. Danielson PB, 1996. Cytochrome P450-mediated resistance to isoquinoline alkaloids and susceptibility to synthetic insecticides in Drosophila. Pestic Biochem Physiol. 55:172-179.
  32. Fogleman JC, Danielson PB, MacIntyre RJ, 1998. The molecular basis of adaptation in Drosophila: the role of cytochrome P4505. Evol Biol. 30:15-77.
  33. Hayes JD, Ellis EM, Newal GE, Harrison DJ, Manson MM, 1999. Cellular response to cancer chemopreventive agents: contribution of the antioxidant response element to the adaptive response to oxidative and chemical stress. Downes CP, Wolfe CR, Loane DP, , ed.Cellular Responses to Stress 141-168. Princeton University Press, Princeton, NJ.
  34. Clark AG, 1989. Causes and consequences of variation in energy storage in Drosophila. Genetics. 123:131-144. [Abstract/Free Full Text]
  35. Halliwell B, Gutteridge JMC, 1989. Free Radicals in Biology and Medicine 2nd ed. Clarendon Press, Oxford.
  36. Wolf CR, Smith G, Smith AG, Brown K, Henderson CJ, 1999. Adaptive response to environmental chemicals. Biochem Soc Symp. 64:129-140. [Medline]
  37. Hayes JD, Pulford DJ, 1995. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 30: (6) 445-600. [Medline]
  38. Herrero A, Barja G, 1997. Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon. Mech Ageing Dev. 98:95-111. [Medline]
  39. Sohal RS, Sohal BH, Orr WC, 1995. Mitochondrial superoxide and hydrogen peroxide generation, protein oxidative damage and longevity in different species of flies. Free Rad Biol Med. 19:499-504. [Medline]
  40. Schwarze SR, Weindruch R, Aiken J, 1998. Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila. Free Rad Biol Med. 25:740-747. [Medline]
  41. Minois N, 2000. Longevity and aging: beneficial effects of exposure to mild stress. Biogerontology. 1:15-29. [Medline]
  42. Parsons P, 1995. Inherited stress resistance and longevity: a stress theory of aging. Heredity. 75:216-221.
  43. Rose MR, Vu LN, Park SU, Graves JL, Jr 1992. Selection on stress resistance increases longevity in Drosophila melanogaster. Exp Gerontol. 27:241-250. [Medline]
  44. Larson PL, 1993. Aging and resistance to oxidative damage in Caenorhabditis elegans. Proc Natl Acad Sci USA. 90:8905-8909. [Abstract/Free Full Text]
  45. Taub J, Lau JF, Ma C, Hahn JH, Rothblatt J, Chalfie M, 1999. A cytosolic catalase is needed to extend adult life span in C. elegans daf-C and clk-1 mutants. Nature. 399:162-166. [Medline]
  46. Johnson TE, Lithgow GJ, Murakami S, 1996. Hypothesis: interventions that increase the response to stress offer the potential for effective life prolongation and increased health. J Gerontol Biol Sci. 51A:B392-B395. [Abstract]
  47. Harshman LG, Moore KM, Sty MS, Magwire MM, 1999. Stress resistance and longevity in selected lines of Drosophila melanogaster. Neurobiol Aging. 20:521-529. [Medline]
  48. Kuether K, Arking R, 1999. Drosophila selected for extended longevity are more sensitive to heat shock. Age. 22:175-180.



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