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The Effects of Vitamin Supplementation on Drosophila Life Span Under Normoxia and Under Oxidative Stress

¹ Department of Biology, York University, Toronto, Ontario, Canada.
² Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.

Address correspondence to Arthur J. Hilliker, PhD, Department of Biology, York University, Toronto, Ontario, M3J 1P3, Canada. E-mail: hilliker{at}yorku.ca

	Abstract

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Vitamin A (retinol), vitamin C (ascorbic acid), and vitamin E (-tocopherol) are each thought to play an important role in the aging process. Here, we investigated the effects of these vitamins on Drosophila melanogaster life span under different oxidative stress conditions. Among the vitamins tested, -tocopherol exhibited the strongest antioxidant activity, extending average and maximum life span for wild-type flies under hyperoxia and for Cu/Zn superoxide dismutase-deficient (SOD1-deficient) flies under normoxia. Retinol supplementation extended life span of SOD1-deficient flies under normoxia, and ascorbic acid supplementation extended life span of wild-type flies under normoxia. However, both retinol and ascorbic acid exhibited a strong prooxidant activity under hyperoxia and shortened life span. Furthermore, ascorbic acid supplementation enhanced the toxic effects of iron, with the iron pool significantly increased in adult whole-body extracts. Taken together, our results document antioxidant and prooxidant contributions of vitamins to D. melanogaster life-span determination under normoxia and under oxidative stress.

Key Words: Vitamin supplementation—-Tocopherol—Drosophila melanogaster—Oxidative stress—Normoxia

Dietary antioxidants are known to play an important role in maintaining oxidative balance, with the combination of diet-derived antioxidants and the endogenous antioxidants resulting in a highly effective defense network against oxidative damage (8). Ascorbic acid (vitamin C), -tocopherol (vitamin E), and retinol (vitamin A) have long been considered as chain-breaking antioxidants, which are capable of scavenging chain-carrying radicals and consequently interrupt the oxidative chain reactions (9–11). By preventing the free radical–mediated damage to cellular compartments, chain-breaking antioxidants are thought to play a key role in delaying the aging process. In the present study, we investigated the effects of antioxidant vitamins on Drosophila melanogaster life span under normal and oxidative stress conditions. The relatively short life span of this model organism, the ability to control the genetic background and the environmental conditions, and, finally, the availability of antioxidant-deficient lines should further facilitate the understanding of the potential role of vitamins in the aging process. Previous studies were mainly focused on the effects of vitamins on D. melanogaster life span under normal conditions. The present study adds new information to the pool of data regarding the antioxidant effects of vitamins on life span under oxidative stress conditions and clarifies the controversial results from some of the previous vitamin studies.

	METHODS

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Fly Stocks and Maintenance
Unless specified otherwise, wild-type rosy+5 flies were used throughout all experiments. The rosy+5 strain was obtained from the laboratory of Arthur Chovnick at the University of Connecticut. For RNA interference studies, flies carrying the UAS-SOD1-IR (12,13) transgene were crossed to flies from da^G32Gal4 driver stock (FlyBase: P[GAL4-da.G32]). The F1 progenies carrying both the GAL4 driver and the UAS-transgene experienced silencing of the superoxide dismutase (SOD)1 gene.

Life-span studies were carried out at 25°C on a 12-hour light/dark cycle, with a total of 100 newly eclosed males (initially 20 per vial) tested for each longevity experiment. Survivors were transferred to fresh vials every 2 days.

Culture Medium
One liter of stock medium consisted of 100 g of sugar, 50 g of dry yeast, 18 g of agar, 8 g of sodium potassium tartrate tetrahydrate, 1 g of potassium phosphate monobasic, 0.5 g of sodium chloride, 0.5 g of calcium chloride dihydrate, 0.5 g of magnesium chloride hexahydrate, 0.5 g of iron (III) sulfate hydrate, 5 mL of propanoic acid, and 2 g of tegosept (2 g of tegosept dissolved in 20 mL of 95% ethanol). It was necessary for us to add tegosept to the culture medium, or vitamin-supplemented media were quickly contaminated with fungus and/or bacteria.

Vitamin-supplemented foods were prepared by mixing and dissolving various amounts of vitamins into the normal culture medium. For fat-soluble vitamins, retinol, and -tocopherol, the food mixture was shaken vigorously to ensure a uniform distribution of fat particles in the culture medium. Iron-supplemented medium (20 mM) was prepared by addition of iron (III) sulfate hydrate into the culture medium. The selected iron concentration had no significant effect on adult feeding behavior (Bahadorani S, Hilliker AJ, unpublished observations, 2006).

Gustatory Assay
For the adult gustatory assay, we performed a similar protocol to what has been described elsewhere (14). Newly-eclosed flies were reared on normal food for 3–5 days and, thereafter, starved for 20 hours on 3MM Whatman paper soaked with distilled water. After this treatment, starved flies (15 females per glass vial) were transferred onto vitamin-supplemented medium with 0.2% sulforhodamine B sodium salt (Acid-Red) for 2 hours. (For control flies, culture medium was only supplied with 0.2% Acid-Red but not vitamins.) After 2 hours of feeding, flies were anesthetized, and the degree of abdomen redness was blind-scored using a subjective grading scale ranging from grade 0 (colorless abdomen) to grade 5 (fully red abdomen). Abdomen redness was used as an indicator of the amount of food taken by the insect.

Hyperoxia Assay
For the hyperoxia experiment, newly-eclosed males were collected and aged on vitamin-supplemented medium for 10 days and were subsequently exposed to 100% oxygen bubbled into water in a sealed chamber. A total of 100 adult males (initially 20 flies per vial) were tested for each survival curve, with survivors transferred to fresh food vials every 2 days. Control flies were fed with normal food throughout the entire experiment.

Antioxidant Capacity Assay
Total antioxidant capacity of flies was measured using the trolox antioxidant assay kit from Cayman Chemical (Ann Arbor, MI), following the manufacturer's protocol. The assay measures the ability of antioxidants in a sample to inhibit oxidation of 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS^R) by metmyoglobin. Prior to the experiment, flies were fed on normal or vitamin-supplemented medium (retinol at 1000 IU/mL, -tocopherol at 25 IU/mL, or ascorbic acid at 100 mM) for 5 days. Thereafter, a total of 10 flies were homogenized in 200 µL of 5 mM potassium phosphate (pH 7.4) containing 0.9% sodium chloride and 0.1% glucose (assay buffer). Samples were centrifuged at 10,000 g for 15 minutes at 4°C, and the supernatant (2 µg protein/µL) was diluted 1:40 with assay buffer and analyzed for total antioxidant capacity.

SOD Activity Assay
SOD activity was measured using the nitroblue tetrazolium (NBT) in-gel assay as described previously (12). The signal intensity of the bands was quantified using Scion Image software (Scion Corp., Frederick, MD) and was expressed as ratios normalized to that of the da^G32Gal4 control band.

Iron-Content Measurements
Newly-eclosed adult males were fed on iron- and vitamin-supplemented media for 10 days and subsequently dried overnight at 65°C. For each sample, a total of 30 flies were digested in 150 µL of 65% nitric acid in a microwave oven and diluted (1:60) in distilled water. Whole-body iron content in each sample was measured at 248.33 nm using an atomic absorption spectrophotometer (AAnalyst 200; Perkin Elmer, Boston, MA) as described previously (15).

Statistical Analysis
All data are expressed as mean ± standard error of the mean (SEM), and the significance of the difference between means was determined using the one-way analysis of variance (ANOVA) statistical test (significant level: *p <.05; very significant level: **p <.01). For longevity assays, the significance (significant level: *p <.01; very significant level: **p <.00001) between survival curves was analyzed using the Kaplan–Meier log-rank statistical test (SAS version 9.1.3; SAS Institute Inc., Cary, NC).

	RESULTS

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Feeding Assay
Feeding behavior and the nutritional constituents of a culture medium are two important factors in life-span determination (16). To ensure that adult life span is not affected with an altered feeding behavior, we tested the adult gustatory preference on each vitamin-supplemented medium. As Figure 1 illustrates, none of the selected vitamins inflict a behavioral change in adult flies, indicating that any changes in the life span are attributed to the supplemented vitamins rather than an altered feeding behavior due to their presence.

View larger version (40K): [in this window] [in a new window]   Figure 1. Adult feeding behavior was not significantly affected (one-way analysis of variance, p >.05) by vitamin supplementation. Each bar represents the mean ± standard error of the mean for a sample of at least 60 flies

View larger version (26K): [in this window] [in a new window]   Figure 2. Effects of vitamin supplementation on wild-type adult life span under normoxia. A, B, and C, Survival curve for wild-type male flies fed vitamin-supplemented media. Ascorbic acid supplementation increased the average life span at 20 mM concentration but enhanced mortality at 100 mM concentration. D, Longevity curves for a replicate experiment for ascorbic acid. The significance of the difference between survival curves (normal food vs vitamin-supplemented medium) was analyzed using the Kaplan–Meier log-rank statistical test (*p <.01; **p <.00001)

View larger version (19K): [in this window] [in a new window]   Figure 3. Supplementation with -tocopherol (25 IU/mL) increased resistance to hyperoxia, whereas that with retinol (1000 IU/mL) or ascorbic acid (100 mM) enhanced an earlier mortality relative to the control group. The hyperoxia experiment was repeated once, and the results were pooled. The significance of the difference between survival curves (normal food vs vitamin-supplemented medium) was analyzed using the Kaplan–Meier log-rank statistical test (*p <.01; **p <.00001)

View larger version (30K): [in this window] [in a new window]   Figure 4. Supplementation with -tocopherol and retinol partially rescued the short life span of superoxide dismutase (SOD)1-deficient flies. A, SOD activity of SOD1-deficient flies (w1; UAS-SOD1-IR/+; daG32Gal4/+) and of control flies (w1; +; daG32Gal4/+) was measured using the nitroblue tetrazolium (NBT) in-gel assay. Bar diagrams illustrate the relative SOD activity of SOD1-deficient flies normalized to that of the daG32Gal4 control flies. B, C, and D, Survival curve for SOD1-deficient flies fed on retinol, -tocopherol, and ascorbic acid, respectively. E, Longevity curves for a replicate experiment for -tocopherol and retinol. For retinol, 1000 IU/mL was the highest concentration that we could incorporate into the culture medium without altering adult feeding behavior. The significance of the difference between survival curves (normal food vs vitamin-supplemented medium) was analyzed using the Kaplan–Meier log-rank statistical test (*p <.01; **p <.00001)

View larger version (27K): [in this window] [in a new window]   Figure 5. Ascorbic acid and -tocopherol supplementation significantly increased the total antioxidant capacity of superoxide dismutase (SOD)1-deficient flies as assayed by trolox equivalents antioxidant assay. The assay was performed twice with duplicate samples. Bars report the mean ± standard error of the mean of antioxidant capacity in undiluted samples (2 µg protein/µL) as calculated from 1:40 diluted samples (see Methods). The significance of the difference between means (normal food vs vitamin-supplemented medium) was assessed using the one-way analysis of variance statistical test (*p <.05)

View larger version (26K): [in this window] [in a new window]   Figure 6. Ascorbic acid supplementation enhanced an earlier mortality relative to controls in the presence of iron (III) sulfate hydrate. A, B, and C, Longevity curve for wild-type flies fed on iron-supplemented medium in the presence of vitamins. D, Longevity curves for a replicate experiment for ascorbic acid in the presence of iron. The significance of the difference between survival curves (iron-supplemented food vs iron and vitamin supplemented medium) was analyzed using the Kaplan–Meier log-rank statistical test (*p <.01; **p <.00001)

View larger version (46K): [in this window] [in a new window]   Figure 7. Ascorbic acid supplementation increased the iron pool as assessed in adult whole-body extracts. All data are expressed as mean ± standard error of the mean, and the significance of the difference between means was assessed using the one-way analysis of variance statistical test (*p <.05; **p <.01)

	DISCUSSION

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-Tocopherol

Through this reaction, -tocopherol (-TOH) transfers a hydrogen atom with a single electron to the peroxyl radical (LOO•) and becomes an -tocopheroxyl radical (-TO•), which is a fairly stable radical due to delocalization of the unpaired electron throughout its aromatic ring (20).

In contrast to mammals, D. melanogaster retains very little vitamin E from the medium (21), perhaps due to its low biological needs for this dietary antioxidant. Our data illustrate that -tocopherol supplementation has no visible effect on life span under normal laboratory conditions but significantly extends average and maximum life span under oxidative stress conditions (Figures 2–4). In addition, we illustrate that -tocopherol supplementation significantly increases the total antioxidant capacity of SOD1-deficient flies, whereas its contribution to total antioxidant capacity of wild-type flies is negligible (Figure 5). Other Drosophila studies revealed that vitamin E supplementation has no effect on life span of wild-type flies under normoxia (22), but it rescues phenotypes of Drosophila models of human neurodegenerative disorders (23–25) and protects flies from the acute oxidative stress of paraquat (26). From these observations, we suggest that there is a sufficient amount of antioxidants in wild-type flies such that dietary supplementation with vitamin E has no effect on longevity. However, under oxygen stress or in genetically compromised backgrounds, -tocopherol supplementation significantly strengthens the antioxidant defenses available for oxidative stress, and, consequently, extends life span. It is worth noting that -tocopherol supplementation does not protect adult flies from iron toxicity (Figure 6A), indicating that life-extending capacity of vitamin E may be limited depending on the nature of the oxidative damage.

Other studies with Caenorhabditis elegans and mice illustrate that vitamin E supplementation significantly extends life span (27–29). In contrast, there were cases where -tocopherol supplementation had no visible effect on life span (22,30). It was suggested by Harrington and Harley (29) that vitamin E extends life span in C. elegans partly by delaying development or timing of reproduction. From these observations, we speculate that, similar to D. melanogaster, C. elegans and mice have sufficient endogenous antioxidants that -tocopherol supplementation has no significant effect on life span, and, consequently, any changes in life span may be attributed to an altered behavior or to the presence of a source of oxidative stress (i.e., due to the experimental conditions or the genetic background).

Retinol
Retinol is a fat-soluble, chain-breaking antioxidant with a capacity to combine with peroxyl radicals and prevent lipid peroxidation. The antioxidant activity of retinol is conferred by delocalization of the unpaired radical over its polyene chain (9,20,31). Growing evidence, however, suggests that vitamin A is a relatively poor antioxidant [reviewed in (32)]. Our results illustrate that retinol exhibits some antioxidant activities and extends life span in SOD1-deficient flies (Figure 4); in contrast, retinol exhibits a strong prooxidant activity under hyperoxia and enhances mortality (Figure 3). Another Drosophila study revealed that vitamin A supplementation can increase the median life span at a certain range of concentrations and at relatively low concentrations, vitamin A inhibited lipid peroxidation but stimulated peroxidation when presented at high doses (33). These results support those of others, suggesting that vitamin A is a weak antioxidant that may act as a prooxidant under certain conditions [reviewed in (34)]. As for the mechanism, we speculate that high oxygen concentration oxidizes retinol under hyperoxia, where the oxidized retinol converts lipid to lipid peroxide in the following reaction:

Although this mechanism is speculative, it explains the switch from an antioxidative to a prooxidative activity at high oxygen concentrations. Taken together, these observations caution against therapeutic application of vitamin A as a life-extending antioxidant because the balance between antioxidant and prooxidant activities of retinol may shift in favor of the latter and, consequently, shorten life span. Indeed, a recent meta-analysis of vitamin A trials supported our conclusion, suggesting that vitamin A supplementation as an antioxidant associates with an increased risk of all-cause mortality [reviewed in (35)].

Ascorbic Acid
Ascorbic acid is regarded as the most important water-soluble antioxidant in mammalian cells with a particular function in protecting DNA and low-density lipoproteins (LDL) from oxidative damage (36,37). Ascorbate (AscH^–) efficiently quenches free radicals (X^.) and yields ascorbate radical (Asc^.–) through the following reaction:

Contrary to the highly reactive free radicals, the ascorbate radical is very stable with the unpaired electron delocalized over the conjugated tricarbonyl system (10). Nevertheless, vitamin C supplementation only delays oxidation before the initiation of the process but will further enhance oxidation when added to minimally oxidized LDL (38).

Dietary studies illustrate that vitamin C supplementation significantly improves the average life span in mice but shortens that of guinea pigs (39,40). We demonstrate that 20 mM ascorbic acid supplementation significantly extends the average life span under normoxia, whereas at 100 mM concentration vitamin C shortens life span (Figure 2). The beneficial effects of ascorbic acid on life span may be attributed to its antioxidant activities (Figure 5); at high concentrations, in contrast, ascorbic acid may enhance mortality due to its cytotoxic and prooxidant activities [reviewed in (41)]. It is worth noting that although 20 mM ascorbic acid supplementation increased the average life span of wild-type flies, it had no significant effect on the short life span of Cu/Zn SOD-deficient flies (Figure 4). This observation is contrary to an earlier study in which ascorbate supplementation restored the short life span of Cu/Zn SOD-deficient yeast (42). From these observations, we speculate that ascorbic acid distribution in D. melanogaster is probably tissue-specific and, as a consequence, may not be able to restore the essential function of SOD in certain critical tissues. This conclusion is supported with the observation that ascorbic acid supplementation restores the total antioxidant capacity of SOD1-deficient flies (Figure 5) but not the life span.

Finally, we illustrate that ascorbic acid supplementation shortens life span under hyperoxia (Figure 3) or in the presence of iron (Figure 6). The prooxidant activity of ascorbic acid under hyperoxia may be attributed to an enhanced oxidation of LDL (38). In the presence of iron, in contrast, the toxic effects may be attributed to both an increased absorption and/or retention of iron (Figure 7) and the prooxidant activity of vitamin C against this metal (43). The prooxidant activity of ascorbic acid takes place through the conversion of ferric iron to the more toxic ferrous form, which is more readily absorbed than ferric iron and may act as a Fenton catalyst in the Haber–Weiss generation of extremely toxic hydroxyl radicals from superoxide () and hydrogen peroxide (H₂O₂) (10,43–45). These observations caution against the clinical application of ascorbic acid as an antioxidant because the prooxidant activity of this vitamin as well as an enhanced absorption of metal ions may promote hydroxyl radical generation and cause oxidative damage. In fact, a recent meta-analysis of vitamin C trials indicates that ascorbic acid supplementation may not be of benefit for prevention and/or treatment of cancer [reviewed in (46)].

	Acknowledgments

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This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to AJH).

	Footnotes

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Decision Editor: Huber R. Warner, PhD

Received May 10, 2007

Accepted September 18, 2007

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