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(The FASEB Journal. 1999;13:1733-1742.)
© 1999 FASEB

Overexpression of glutathione reductase extends survival in transgenic Drosophila melanogaster under hyperoxia but not normoxia

ROBIN J. MOCKETT, RAJINDAR S. SOHAL and WILLIAM C. ORR1

Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA

1Correspondence: Department of Biological Sciences, 220 Fondren Science Building, Southern Methodist University, Dallas, TX 75275 USA. E-mail: borr{at}mail.smu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of this study was to test the hypothesis that overexpression of glutathione reductase in transgenic Drosophila melanogaster increases resistance to oxidative stress and retards the aging process. Transgenic flies were generated by microinjection and subsequent mobilization of a P element construct containing the genomic glutathione reductase gene of Drosophila, with 4 kb upstream and 1.5 kb downstream of the coding region. Transgenic animals stably overexpressed glutathione reductase by up to 100% throughout adult life and under continuous exposure to 100% oxygen or air. Under hyperoxic conditions, overexpressors had increased longevity, decreased accrual of protein carbonyls, and dramatically increased survival rates after recovery from a semi-lethal dose of 100% oxygen. Under normoxic conditions, overexpression of glutathione reductase had no effect on longevity, protein carbonyl content, reduced glutathione, or glutathione disulfide content, although the total consumption of oxygen was slightly decreased. Glutathione reductase activity does not appear to be a rate-limiting factor in anti-aging defenses under normoxic conditions, but it may become a limiting factor when the level of oxidative stress is elevated.—Mockett, R. J., Sohal, R. S., Orr, W. C. Overexpression of glutathione reductase extends survival in transgenic Drosophila melanogaster under hyperoxia but not normoxia.


Key Words: aging • oxidative stress • reduced glutathione • free radicals • antioxidant defenses


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE OXIDATIVE STRESS hypothesis posits that aging is caused, at least in part, by an imbalance between prooxidant production, antioxidant defenses, and repair processes. This imbalance is postulated to result in the steady accrual of oxidative molecular damage, which may underlie senescence-associated physiological attrition and increasing rates of mortality (1 2 3) . The hypothesis is supported by an extensive body of data correlating aging with increased rates of prooxidant production and accrual of oxidative damage to lipids, proteins, and nucleic acids (1 2 3) . Natural and experimental variations in longevity, within and between species of multiple phyla, are also correlated with levels of oxidative stress and accumulation of molecular damage.

Reactive oxygen species (ROS),2 formed as by-products of mitochondrial oxidative phosphorylation, are believed to be a major source of oxidative stress in vivo. In insects, due to the absence of oxygen-binding proteins in the blood vascular system, oxygen is carried directly to the cells via a tracheolar network, which exposes insect tissues to four- to fivefold higher concentrations of oxygen than in animals with blood hemoglobin (4) . The rate of mitochondrial ROS production has been shown to be directly proportional to the ambient oxygen concentration (5 , 6) . Thus, among experimental sources of oxidative stress, hyperoxia may be a particularly relevant model for testing the role of oxidative stress in aging of insects.

In Drosophila, the oxidative stress hypothesis has been tested in transgenic animals overexpressing enzymatic antioxidants. Overexpression of a single Drosophila antioxidative enzyme, such as Cu-Zn superoxide dismutase (Cu-Zn SOD) or catalase, using the native gene and flanking regulatory sequences, has generally had little effect on the aging process (7 , 8) . By contrast, expression of human Cu-Zn SOD in Drosophila motor neurons or temporally restricted elevation of Drosophila Cu-Zn SOD, using the FLP recombinase binary transgenic system, both extended survival times up to ~40% (9 , 10) . Tandem elevation of Drosophila Cu-Zn SOD and catalase, controlled by the native promoters, also led to increased longevity associated with slower accumulation of oxidative damage products and physiological attrition, and increased resistance to experimental oxidative stress (11 , 12) .

One limitation of the existing information about antioxidative enzyme overexpression in transgenic flies is that it is restricted to only two enzymes: Cu-Zn SOD and catalase. At present, no information is available on the effects of bolstering small molecular weight antioxidants by the transgenic approach. Glutathione is among the most abundant and ubiquitous small molecular weight antioxidants (13) . The production and metabolism of glutathione are controlled by a number of well-characterized enzymes (14) , making the supply of glutathione a potential target for modulation by the transgenic approach. Earlier studies showed an age-related decrease in the GSH/GSSG ratio (15) , whereas feeding Drosophila the glutathione precursor N-acetylcysteine increased longevity up to 26% (16) . Reversal of an age-related decrease in glutathione content by feeding the cysteine precursor magnesium thiazolidine-4-carboxylic acid increased the median life span up to 38% in the mosquito (17) . For these reasons, the previously-characterized glutathione reductase (GR) gene (18) was selected for overexpression. Overexpression of GR was predicted to increase the supply of reduced glutathione (GSH) and prevent accumulation of glutathione disulfide (GSSG), leading to life span extension and increased resistance to experimental oxidative stress. Results of this study support the prediction concerning experimental stress in the form of exposure to 100% oxygen, but the supply of glutathione and life span were unchanged in the absence of experimental oxidative stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Creation of transgenic lines
A pCaSpeR vector with a SacII linker incorporated between the EcoRI and PstI sites was provided by M. Candas (Southern Methodist University). A 10 kb SacII fragment containing the genomic sequence encoding GR, its 2.5 kb intron and ~4 kb upstream and 1.5 kb downstream from the coding domain, was subcloned into this SacII site. `Experimental' (E) lines were generated by P element-mediated transformation with this construct, followed by transgene mobilization using the strain y w; CyO/Sp; Sb{Delta} (2–3)/TM6,Ubx, which possesses endogenous P element transposase activity. `Control' (C) lines were created by transformation with the unmodified pCaSpeR vector. The Drosophila strains used for transformation and chromosomal mapping, the microinjection procedure, and establishment of transgenic lines were performed as described in detail previously (8 , 19) .

The presence of transgenes was verified by Southern analysis using 5–10 µg DNA samples digested with StuI (E lines), EcoRI (C lines), or SspI (C lines). After electrophoresis through 1.0% agarose and transfer to nitrocellulose, the fly DNA was hybridized with digoxigenin-labeled probes complementary to either a 2.8 kb NotI/SacII fragment at the 3' end of the genomic GR clone (E lines) or a 1.7 kb ClaI/PvuII region in pCaSpeR (C lines).

Animal husbandry, life span determination, and experimental oxidative stress
For all experiments, male flies, heterozygous with respect to the appropriate transgene, were generated by outcrossing homozygous stocks with the parental y w strain. The flies were housed in groups of 25 under constant light at 24.5 ± 1.0°C and transferred to fresh containers every 24–48 h prior to death or killing for biochemical assays. The medium contained cornmeal, yeast, sugar and agar, with methylparaben as mold inhibitor. Treatment with 100% oxygen or 10 mM paraquat commenced 7–13 days post-eclosion and was performed as described (7) .

Activity of glutathione reductase
GR activity was measured by monitoring the rate of production of 5-thio-2-nitrobenzoic acid (TNB) from 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) at 412 nm, which is coupled with the GR reaction, as described previously (20) . Homogenates (5%) were centrifuged 10 min at 8400 x g; assays were performed on crude supernatants as described, except that no glutathione disulfide was added to the reaction mixture. The blank with no NADPH in the mixture was subtracted from the rate with 0.1 mM NADPH (final concentration) for each sample. Activity was calculated using an extinction coefficient of 13,600 M-1 cm-1, based on the change in absorbance 2.0–4.8 min after simultaneous mixing of control and experimental samples and blanks.

The assay for glutathione reductase in the absence of exogenous GSSG, using absence of exogenous NADPH as a blank, produced maximum activity, whereas exogenous GSSG had a slight inhibitory effect. Efforts to make the rate dependent on the addition of exogenous GSSG were unsuccessful, possibly because the reduction of DTNB requires only catalytic amounts of reduced glutathione or GSSG (21 , 22) . The assay was validated with the GR inhibitor, ethacrynic acid. Excess TNB production, above the level of the blank, was fully inhibitable by ethacrynic acid. For both E and parental y w lines, the percent inhibition as a function of ethacrynic acid concentration closely approximated the published result for purified bovine GR (23; results not shown). The blank rate, which was attributed to the reaction of DTNB with protein sulfhydryl groups (24) , was comparable in all lines and was not inhibited by ethacrynic acid.

Quantitation of glutathione
GSH and GSSG were measured by briefly chilling 25 flies on ice, preparing 5% (w/v) homogenates in 5% metaphosphoric acid, centrifuging 15 min at 12 000 x g, and injecting 20 µl supernatant into a Waters Associates high-performance liquid chromatography (HPLC) system. The HPLC method was adapted from Lakritz et al. (25) . The mobile phase was 50 mM sodium phosphate, pH 2.7, 0.05 mM octanesulfonic acid, 2% acetonitrile; the flow rate was 1.0 ml/min. Separation was effected in 14.0 min with a reverse phase C18 column (25.0 x 0.46 cm, 5 µM, Supelco). Dual electrochemical detection was performed with analytical cell settings of +400 mV and +1125 mV, using an ESA Coulochem II detector. The guard cell, upstream of the injector, was set at +1400 mV; the conditioning cell, downstream of the column, was set at +525 mV. Peak areas were quantified and compared with peak areas obtained with pure GSH and GSSG standards.

Protein carbonyl content
Protein carbonyl content was measured on a Waters Associates HPLC system. Samples were prepared by the method of Levine et al. (26) , essentially as described (27) , except that aprotinin was omitted and the homogenate was not filtered through gauze. The final pellet was resuspended in sodium phosphate, pH 6.8, 3% sodium dodecyl sulfate (SDS) and recentrifuged 5 min at 3000 x g before injection onto a Zorbax GF-250 column (Rockland Technologies Inc., Chadds Ford, Pa.). Absorbance was detected simultaneously for 14.0 min at 365 nm with a Waters Associates Model 440 Absorbance Detector and at 280 nm with a Pharmacia LKB optical unit UV-1. The mobile phase was 200 mM sodium phosphate, pH 6.5, 1% SDS, with a flow rate of 2.0 ml/min. Calculations were performed by comparison of peak areas, using unoxidized 1 mg/ml bovine serum albumin (BSA) and oxidized BSA containing 11.3 nmol protein carbonyl per milligram (supplied by A. Lass) as standards.

Biochemical assays
Superoxide dismutase activity was measured using the bathocuproine disulfonate disodium salt-nitroblue tetrazolium assay of Spitz and Oberley (28) , with slight modifications. Samples were prepared by homogenizing 20 flies in 2 ml of 50 mM phosphate buffer, pH 7.8, and obtaining supernatants after 10 min centrifugation at 8400 x g.

Catalase activity was measured by homogenizing five flies in 0.5 ml Triton X-100 (0.1%), centrifuging 5 min at 3000 x g, recentrifuging the supernatant 10 min at 16,400 x g, then monitoring consumption of 12.5 mM hydrogen peroxide in 67 mM phosphate buffer, pH 7.0, at 240 nm. Activity was calculated using an extinction coefficient of 0.043 mM-1 cm-1.

Animal physiology
The rate of oxygen consumption was measured with a differential Gilson respirometer, as described (12) , except that a 30 min equilibration with open stopcock was permitted.

Negative geotaxis was measured under constant light in a room with no windows. Flies were chilled 1 min on ice, then confined individually in plastic pipettes (i.d. 5 mm) with the tips removed and enclosed with Parafilm. After 10 min recovery, each fly was tapped to the bottom of the pipette and the maximum height obtained after 12 s was recorded. After all flies in a group of 20 had been monitored, the measurement process was repeated three more times and the maximum height obtained in four attempts was converted to an average walking speed (cm/s) for each fly.

Statistical analysis
Kaplan-Meier analyses of survival curves, with stratified log rank tests, were performed using SYSTAT 7.0 SURVIVAL software. Analyses of variance and Student's t tests were performed using Microsoft EXCEL software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of transgenic lines
Microinjection of the modified pCaSpeR vector containing a 10 kb SacII genomic fragment encoding Drosophila GR (Fig. 1 ) yielded six independent transgenic lines. Transgene mobilization, by passage through the {Delta}(2–3) background, generated an additional 41 lines. Southern analysis demonstrated that 24 lines possessed single transgene insertions. These insertions were mapped to either chromosome II or chromosome III, as were insertions from 10 control lines. Viable stocks, homozygous with respect to transgenes, were obtained from 10 control (C) and 15 experimental (E) lines.



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  		Figure 1. pCaSpeR-SacII-GR construct. A 10 kb fragment (broad arc) containing the entire Drosophila glutathione reductase (GR) coding sequence (exons, solid arcs with arrowheads; noncoding sequences, open arcs) was subcloned into the SacII site of a modified pCaSpeR vector (narrow arc). A partial restriction map is shown, indicating some of the sites used for diagnostic digests prior to microinjection of the construct into Drosophila embryos.

Overexpression of glutathione reductase
All control lines had GR activity comparable to the y w parental line, within a 15% range. Heterozygous E lines had activity 0.6–85.3% greater than the maximum C line, and several E lines had ~100% higher activity than typical C lines (Fig. 2 A, B). The elevation of GR activity was maintained for at least 3 days during exposure to 100% oxygen (Fig. 2C ) and was stable throughout adult life (10–57 days; Fig. 2D ).



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  		Figure 2. Activity of glutathione reductase under normoxic and hyperoxic conditions. A) Experimental (E) lines were generated by transgene mobilization and GR lines were generated by microinjection. All flies were males, heterozygous for GR transgenes, aged 10–11 days. B) Control (C) lines were generated by microinjection of y w flies with a pCaSpeR vector containing no insertion. Activity was measured by monitoring TNB production from DTNB at 412 nm. The blank reaction was performed in the absence of NADPH. Results are mean ± SD of four determinations and are representative of two separate experiments. C) Activity was measured as a function of duration of exposure to 100% oxygen in young flies (12 days) from lines E4 (filled circles) and C2 (open circles). D) Activity was measured as a function of age in lines E4, E6, and E11 (filled symbols) and C2, C5, and C9 (open symbols).

Effects of glutathione reductase overexpression on resistance to hyperoxia
Life spans were determined in three E and three C lines, under continuous exposure to 100% oxygen (Fig. 3 A). The mean survival times were 4.45 days for the E lines, and 3.72 days for the C lines, a difference of 20% (P<0.001). Among individual E lines, the mean survival time was increased 7–25% in relation to the strongest C line. The maximum survival time was 6.0 days for all lines.



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  		Figure 3. Life span of GR overexpressors and control strains exposed to 100% oxygen. A) Results are survival data for three experimental (E) lines (filled symbols) and three control (C) lines (open symbols) under continuous exposure to 100% oxygen. Pooled survivorship data are also shown (inset). B) Results are survival data for the same lines after exposure to 100% oxygen for 75 h, beginning 7 days post-eclosion (arrows indicate the beginning and end of exposure to 100% oxygen). Pooled data are also shown (inset).

The ability of flies to recover from near-lethal hyperoxia was tested by exposing the same lines to 100% oxygen for 3.1 days, after which the surviving animals were maintained under normoxic conditions for the remainder of the life span (Fig. 3B ). The 3.1-day oxygen exposure killed 10–15% of the flies from the three C lines, and 5–8% from the three E lines. A large number of flies, critically injured by the hyperoxic exposure, died within 7 days after the return to normoxic conditions, and all of the flies showed overt signs of injury. The subpopulation surviving 7 days of recovery comprised 66–84% of the initial population of E flies and 38–52% of the C population. Within this subpopulation, both the median (57–72 days for C lines and 68–75 days for E lines) and maximum life spans (107–108 days) were virtually identical to those of unexposed flies (see Fig. 7 ). In the total population of exposed flies, the median survival times were 66 days for E lines and 14 days for C lines (including 7 days prior to oxygen exposure), a difference of 370% (P<0.001). The median survival times for individual E lines were 250–278% longer than those of the strongest C line.



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  		Figure 7. Life span of glutathione reductase overexpressor and control strains under normoxic conditions. Male flies (150) were housed in groups of 25 under constant light at 24.5 ± 1.0°C and transferred to fresh food vials every 24–48 h. Results were pooled from 12 experimental lines (filled circles; n=1784 flies) and 10 control lines (open circles; n=1489 flies), and are representative of 3 independent experiments.

The content of GSH and GSSG was measured in one E line and one C line after exposure to 100% oxygen for varying lengths of time. No depletion (and no compensatory elevation) of the GSH pool was observed in either the E or C line (Fig. 4 A). The content of GSSG remained quite low and variable between individual cohorts (Fig. 4B ). Analysis of variance showed no significant effect of line or duration of oxygen exposure, but linear regression analysis showed a significant increase of GSSG content only in the C line (P<0.01), with no corresponding increase in the E line (P>0.1).



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  		Figure 4. Effect of hyperoxia on content of reduced glutathione and glutathione disulfide. Live flies (25) were chilled on ice; 5% homogenates were made in 5% metaphosphoric acid and centrifuged 15 min at 12,000 x g, then 20 µl supernatant was separated by HPLC on a C18 reverse-phase column, followed by dual electrochemical detection at +400 mV and +1125 mV. Results for GSH (A) and GSSG (B) for lines E4 (filled circles) and C2 (open circles) are mean ± SE of 6–7 independent samples.

Protein carbonyl content, used as an index of protein oxidative damage, was significantly increased after exposure to 100% oxygen for 3.2 days (P<0.0001), with a significant interaction between oxygen exposure and E vs. C line (Fig. 5 A, B; P=0.02). On average, carbonyl content was increased 11.2% in E lines and 34.2% in C lines during the exposure to 100% oxygen. One-tailed t tests showed no significant differences between unexposed E and C lines or exposed and unexposed E flies, but C lines exposed to oxygen had greater carbonyl content than exposed E lines (P<0.05) orunexposed C lines (P<0.005).



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  		Figure 5. Protein carbonyl content and walking speed after exposure to 100% oxygen. Carbonyl content was measured by HPLC after derivatization with DNPH, as described previously (27) . The protein was resuspended in sodium phosphate, pH 6.8, 3% SDS, and centrifuged 5 min at 3000 x g, then 80 µl supernatant was sepa-rated by HPLC on a Zorbax GF-250 column, with simultaneous UV detection at 280 nm and 365 nm. A) Results for each line are mean ± SE of 8 determinations without (stippled bars) or immediately after (open bars) exposure to 100% oxygen for 77 h. All flies were 11 days of age. B) Pooled results are mean ± SD for the lines shown in panel A. The asterisk indicates a significant difference in carbonyl content in comparison with oxygen-exposed experimental flies (P<0.05) and unexposed controls (P<0.005). C) Walking speed in negative geotaxis experiments was measured 2 days after recovery from oxygen exposures of varying durations. All flies were 14 days of age. The distance traversed in 12 s was determined four times for each fly from lines E4 (stippled bars) and C2 (open bars). The maximum distance was recorded and expressed as speed per second. Results are mean ± SE for 15 flies in each group and are representative of two independent experiments.

Oxygen exposures of increasing duration had a progressive, deleterious effect on walking speed in negative geotaxis experiments (Fig. 5C ). The speed of walking was impaired more rapidly in control flies (P=0.014), and the impairment persisted throughout the life span when recovery was permitted.

Other sources of experimental stress
Longevity was studied upon exposure to paraquat, an alternative source of oxidative stress, and starvation conditions, a severe nonoxidative experimental stress. There was no effect of GR overexpression on mean, median, or maximum life span during paraquat exposure (Fig. 6 A). Experimental lines experienced an 18% decrease in mean life span and up to 25% decrease in maximum survival time under starvation conditions, relative to controls (Fig. 6B ). Longevity was also examined in response to increases in the ambient temperature. Temperature elevation from 24.5°C to 29.5°C decreased median and maximum longevities by 34 to 46%, but pooled results for 14 E and 11 C lines (including the parental y w line) showed a maximum difference of 2 days for each percentile of survivorship (Fig. 6C ). Temperature elevation to 37°C killed all of the flies within 13 h, with no net difference between E and C lines.



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  		Figure 6. Life spans of flies exposed to experimental stress. A) Paraquat. Pooled survivorship data are shown for groups of 100 flies from three experimental (E) and three control (C) lines fed 1% sucrose, 10 mM paraquat beginning 13 days post-eclosion. B) Starvation. Pooled survival data are shown for three E and three C lines, placed in empty vials sealed with moistened rayon plugs beginning 11 days post-eclosion. C) Heat stress. Average survivorship is shown for 14 GR overexpressor lines and 11 control lines, 100 flies per line, housed at 29.5 ± 1.0°C beginning 4 days post-eclosion. For all panels, filled circles represent data from experimental lines and open circles represent data from control lines.

Effects of glutathione reductase overexpression on longevity, aging, oxidative stress and oxidative damage
Life spans were determined in 12 E and 10 C lines (Fig. 7 ). Although there were some differences among individual lines and cohorts, there was no net difference between E and C lines, collectively, at any point on the survivorship curve.

The content of reduced glutathione decreased by 5–15% (Fig. 8 A) and glutathione disulfide increased two- to fivefold (Fig. 8B ) during aging (P<0.001). GSSG represented less than 1% of total GSH content in young animals, rising to a maximum of 3.3% in very old flies. The elevation of GR activity had no significant effect on the age-related changes in GSH and GSSG content in whole body homogenates.



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  		Figure 8. Content of reduced glutathione and glutathione disulfide as a function of age. Measurements were made as described in the legend of Fig. 4 . Results for GSH (A) and GSSG (B) for each line are mean ± SE for 2–3 samples, with 1–2 injections per sample (3–5 injections). Stippled bars represent experimental lines (E4, E6, and E11) and open bars represent control lines (C2, C5, and C9). Pooled results for all experimental (filled circles) and control lines (open circles) are shown as overlaid line graphs (mean ± SD).

Protein carbonyl content increased by ~50% during adult life (Fig. 9 ). Overexpression of GR had no effect on the initial level or the rate of increase in carbonyl content under normoxic conditions.



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  		Figure 9. Protein carbonyl content in young (10–14 days) and old (57–62 days) Drosophila. Carbonyl content was measured as described in the legend of Fig. 5 . A) Carbonyl content in young (stippled bars) and old flies (open bars) of 3 E and 3 C lines. Results are mean ± SE of 6–14 injections. B) Pooled results (mean ± SD) for groups shown in panel A.

Activities of Cu-Zn SOD, Mn SOD (manganese-containing superoxide dismutase), and catalase were determined in order to detect compensation for GR overexpression at the biochemical level. There was no change in total SOD activity either between E and C lines or as a function of age (Table 1 ). Also, there was no change in Mn SOD and, by extension, no difference in Cu-Zn SOD activity, measured as the difference between total SOD and Mn SOD activities. Similarly, there was no difference in catalase activity, either between E and C lines or between young and old animals (Table 1) .


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  		Table 1. Comparison of antioxidant enzyme activities between young and old flies of experimental (E) and control (C) linesa

Potential compensatory changes at the physiological level were tested by measuring maximum exercise performance in negative geotaxis experiments and normal metabolic rate reflected by oxygen consumption without exercise. Maximum exercise capability was found to decrease linearly during adult life (Fig. 10 A), and GR overexpression had no effect on the rate of this decrease. Oxygen consumption, by contrast, initially increased and subsequently reached a plateau or decreased slightly (Fig. 10B ). The initial increase was more pronounced in the E lines, which consumed significantly less oxygen than C lines at 10–11 days, the youngest age tested (P<0.05). At later ages (19–52 days), there was no significant difference in oxygen consumption between E and C lines, but analysis of variance showed significant effects of both age (P<0.0001) and E vs. C line (P<0.05) on oxygen consumption.



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  		Figure 10. Physiological effects of glutathione reductase overexpression. A) Walking speed in negative geotaxis experiments was measured as described in the legend of Fig. 5C , except that groups of 20 flies were used. Each data point represents the mean ± SE of a total of six E (filled circles) or five C (open circles) groups of 20 flies from the three lines used in earlier experiments. B) Oxygen consumption was measured as a function of age in a Gilson respirometer, using groups of 25 flies housed in 20 ml flasks. Results are mean ± SE of 12–18 groups from three E lines (filled circles) and three C lines (open circles).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The principal finding of this study is that glutathione reductase overexpression increases survivorship in Drosophila melanogaster under hyperoxic but not normoxic conditions. The increased resistance to hyperoxia is associated with marginally slower accumulation of the GR substrate, GSSG, and with slower accrual of oxidative molecular damage in the form of protein carbonyl content. The lack of change in longevity under `standard' normoxic conditions is associated with a lack of attenuation of age-related increases in protein carbonyl and GSSG content and decrease in GSH content. There were no compensatory changes in the activities of other antioxidative enzymes measured in this study, no physiological compensation in the form of increased activity or metabolic rate, and no increase in resistance to experimental stress conditions other than hyperoxia.

Several previous studies have provided suggestive evidence for an important role of the glutathione system in resistance to hyperoxia. Administration of glutathione to rats and mice extended their survivorship under hyperoxia, whereas GSH depletion with diethyl maleate decreased survival times of rats exposed to hyperbaric hyperoxia (29) . An important role for GR in resistance to hyperoxia was indicated by decreased survival times of BALB/c mice exposed to 85% oxygen after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea at doses that decreased GR activity by ~50% (30) . Results of the current study show for the first time that stable overexpression of GR by nearly 100% significantly increases the survival time of Drosophila under continuous exposure to 100% oxygen. The increased resistance to hyperoxia is accompanied by decreased mortality in the period after a semi-lethal oxygen exposure and by less rapid impairment of physiological function. These effects appear to be due to the action of GR itself, rather than the steady-state amount of GSH, which remained stable throughout the exposure to 100% oxygen. This finding is consistent with the predictions of Moore et al. (31) , who found that in Escherichia coli the capacity for GSH synthesis was a more important determinant of radiation resistance than the level of GSH at the onset of experimental stress. In Drosophila, the increased capacity for rapid conversion of GSSG to GSH may be the reason for the enhanced resistance of GR overexpressors to oxygen toxicity.

Altogether, the results of this study do not contradict the idea that oxidative stress is a causal factor in aging under normoxic conditions, but neither do they provide strong support for the idea. According to the oxidative stress hypothesis, increased life span and decreased oxidative damage should be observed only when oxidative stress is decreased. The apparent lack of change in the level of oxidative stress under normoxic conditions means that GR overexpression failed to provide a direct test of this hypothesis. By contrast, the results provide direct evidence for oxidative stress as a causal factor in oxygen toxicity. This outcome is quite novel, since previous studies with transgenic flies showed either a beneficial effect on both normal aging and resistance to experimental oxidative stress (12 , 32) or no effect in either case (7 , 8) , whereas this study shows differential effects depending on environmental conditions.

One interpretation of these findings is that hyperoxia and normal aging kill the flies by a different mechanism, with the former but not the latter mechanism involving oxidative stress attenuated by GR overexpression. An alternative explanation is that oxidative stress kills the flies by a similar mechanism under both normoxic and hyperoxic conditions, but the rate of exposure to oxidative stress is dramatically different in each case. In support of this hypothesis, the flies live ~15 times longer under normoxic as compared with hyperoxic conditions. The magnitude of the decrease in GSH:GSSG ratio and increase in protein oxidative damage in control flies is almost as large in a 3-day interval under hyperoxic conditions as it is in a 50-day interval under normoxia. Thus, the rate of change in these parameters is ~15 times faster under hyperoxic conditions, a result consistent with the longevity data. Previous studies have shown that glutathione reductase activity may be a rate-limiting component in the response to severe oxidative stress imposed by exposing fibroblasts to high concentrations of tert-butyl hydroperoxide (33) , but not in the response to mild oxidative stress, when the extent of prooxidant production is much smaller. Similarly, the rate of prooxidant production in Drosophila control lines may be sufficiently high for GR activity to become a limiting component of the defensive response under hyperoxic but not normoxic conditions.

There is considerable evidence that overexpression of antioxidative enzymes under normoxic conditions can have a beneficial effect on longevity in Drosophila (9 10 11 12) . For instance, simultaneous overexpression of Cu-Zn SOD and catalase increased median and maximum life spans, in association with decreased levels of oxidative stress and slower accrual of oxidative damage. Similar effects may result from simultaneous overexpression of GR and other antioxidative enzymes, such as Cu-Zn SOD and catalase. Based on the hypothesis that GSH and SOD work in tandem—channeling unpaired electrons from various radicals to oxygen, with concomitant oxidation of GSH to GSSG, then detoxifying the resulting superoxide radical (34) —the simultaneous overexpression of all three enzymes would be predicted to strengthen every step in a pathway terminating free radical chain reactions in vivo.

Conversely, the age-related loss of GSH and gain of GSSG are quite modest in magnitude and are not attenuated by overexpression of GR. One explanation for the latter finding is that the age-related change in the cellular GSH:GSSG ratio may be regulated, as is normally observed in the secretory pathway, where an unusually high proportion of GSSG is believed to facilitate protein folding (35) . If the existing ratio of GSH:GSSG under normoxic conditions represents an evolutionary optimum, the overexpression of GR may impose a mild challenge to homeostasis. This suggestion is consistent with the findings that GR overexpressors have decreased resistance to starvation conditions and lower oxygen consumption at young ages, representing a decrease in metabolic potential since it is not offset by increased longevity or by higher oxygen consumption at later ages.

The results of this study and many previous studies lead to the suggestion that prooxidant production causes oxidative molecular damage, which accumulates and is a causal factor in aging, but antioxidant defenses provide only limited protection against this process. Below a certain threshold rate of prooxidant production, as in normal aging, the accrual of oxidative damage is relatively insensitive to changes in antioxidant levels, possibly because these oxidants react with specific targets in their immediate vicinity. Thus, these oxidants will usually encounter a target molecule rather than an antioxidant, almost regardless of the ambient concentration of antioxidants. When oxidant production is widespread, as in the case of severe, experimental oxidative stress, overexpression of antioxidants may provide a beneficial effect because an increased fraction of the oxidants encounter antioxidants instead of target molecules. Consistent with this idea, overexpression of glutathione reductase in the present study slowed the accrual of oxidative damage and prolonged survival under hyperoxic but not normoxic conditions.


   ACKNOWLEDGMENTS
 
This work was supported by grants RO1 AG7657 and RO1 AG15122 from the National Institute on Aging, National Institutes of Health. The authors wish to thank J. J. Benes, L. K. Kwong, A. Lass, J. B. McGowan, and B. H. Sohal for their expert technical assistance.


   FOOTNOTES
 
2 Abbreviations: ROS, reactive oxygen species; Cu-Zn SOD, copper- and zinc-containing superoxide dismutase; Mn SOD, manganese-containing superoxide dismutase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, glutathione disulfide; E, experimental line; C, control line; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); TNB, 5-thio-2-nitrobenzoic acid; HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin.

Received for publication March 12, 1999. Accepted for publication May 15, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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