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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: aging • oxidative stress • reduced glutathione • free radicals • antioxidant defenses
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) yielded six independent transgenic lines. Transgene mobilization, by
passage through the (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.
|
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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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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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.
|
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).
|
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).
|
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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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.
|
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.
|
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)
.
|
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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 12, 1999. Accepted for publication May 15, 1999.
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REFERENCES |
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