Vitamin C and lifespan in model organisms

Food and Chemical Toxicology 58 (2013) 255–263

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Vitamin C and lifespan in model organisms K. Pallauf a,⇑,1, J.K. Bendall b,1, C. Scheiermann a, K. Watschinger b, J. Hoffmann c, T. Roeder c, G. Rimbach a a

Institute of Human Nutrition and Food Science, Christian-Albrechts-University Kiel, Hermann-Rodewald-Straße 6-8, D-24118 Kiel, Germany Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX39DU, UK c Zoological Institute, Zoophysiology II, Christian-Albrechts-University Kiel, Olshausenstraße 40, D-24098 Kiel, Germany b

a r t i c l e

i n f o

Article history: Received 14 March 2013 Accepted 25 April 2013 Available online 1 May 2013 Keywords: Ascorbic acid Longevity Antioxidant Vitamin supplementation Ageing

a b s t r a c t The process of ageing has been repeatedly associated with increasing oxidative damage which has led to the hypothesis that reducing oxidative stress through antioxidant dietary factors may prolong lifespan. Ascorbic acid is an essential antioxidant in human diets and is widely used for supplementation. However, it is rather unclear if and to what extent ascorbic acid may affect lifespan in humans and model organisms. In our review of literature on vitamin C supplementation and its effect on lifespan in different model organisms we found that some studies suggest an increase in lifespan, other studies failed to observe any beneficial effect of vitamin C on longevity and some studies even reported a decrease in lifespan following vitamin C supplementation. Of the 14 studies included in our analysis, three were carried out in worms, four in flies and seven in rodents. The discrepancies between the studies may be related to species-specific differences, the concentration of vitamin C administered, the duration of supplementation and whether vitamin C was used alone or as part of a combined antioxidant diet. Potential underlying mechanisms through which vitamin C may influence lifespan and differences amongst species regarding the capacity to produce vitamin C endogenously are discussed. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Vitamin C, or ascorbic acid, is one of the quantitatively most important water soluble antioxidants. Ever since the free radical theory of ageing was put forward by Harman almost 50 years ago (Harman, 1956) there has been speculation on the possible role of ascorbic acid in preventing age-related oxidative damage. In some primates including humans, and a few other species such as guinea pigs and bats ascorbic acid is an essential nutrient due to a lack of biosynthetic capacity. Therefore, humans and these animals depend on their diet as a source of vitamin C to prevent scurvy and maintain general health (Birney et al., 1976; Linster and Van Schaftingen, 2007). Vitamin C is acquired primarily through the consumption of fruit, vegetables, fortified drinks and cereals, as well as supplements.

Abbreviations: ApoE, apolipoprotein E; DOG, 2-deoxy-D-glucose; GLO, L-gulonolactone oxidase; HFHC, high fat/high cholesterol; Nrf2, nuclear factor like 2; ODS, Osteogenic Disorder Shionogi; PGC, peroxisome proliferator-activated receptor gamma coactivator; ROS, reactive oxygen species; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescenceaccelerated mouse-resistant 1; SOD, superoxide dismutase. ⇑ Corresponding author. Tel.: +49 431 8805312. E-mail address: [email protected] (K. Pallauf). 1 These authors contributed equally to this work. 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.04.046

Ascorbic acid has been shown to be important for the synthesis of catecholamines (Bornstein et al., 2003; Deana et al., 1975), carnitine (Hulse et al., 1978) and collagen (Van Robertson and Schwartz, 1953), the impaired production of which leads to the symptoms associated with scurvy. The health-promoting effects of vitamin C can be attributed to its biological functions as a cofactor for a number of enzymes, such as hydroxylases involved in collagen synthesis, and as a water-soluble antioxidant. Ascorbic acid donates two electrons from a double bond between the second and third carbons of the 6-carbon molecule. It is hypothesised that by donating its electrons vitamin C prevents proteins and other cellular compounds from being oxidized. In view of this, there is increasing speculation on whether increasing our dietary intake of vitamin C (and other micronutrients) is able to delay the ageing process and promote increased longevity (Ames, 1998). Indeed, administration of other antioxidants and overexpression of antioxidative enzymes, such as superoxide dismutase (SOD), catalase and glutathione reductase, have been shown to increase the lifespan of animals, although the results are not always consistent. For example, overexpression of SOD (Parkes et al., 1998) or SOD with catalase (Li et al., 2007) increased lifespan in some strains of Drosophila. Further, vitamin E administration (Bahadorani et al., 2008) and overexpression of glutathione reductase (Mockett et al., 1999) have been shown to extend the lifespan of Drosophila during hyperoxia but not during normoxia. We have recently

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reviewed the data on vitamin E and longevity in model organisms and found varying study outcomes (Ernst et al., 2013). Clinical trials to determine the effect of antioxidant supplementation, including vitamin C, on health status in humans have been largely disappointing and have yielded inconsistent results (Traber and Stevens, 2011). However, most research on ascorbic acid and ageing has been carried out in model organisms, ranging from nematodes to guinea pigs. This review focuses on current literature dealing with the effect of vitamin C on lifespan in model organisms, encompassing 14 studies with nematodes, flies and rodents. 2. Vitamin C and lifespan in C. elegans The nematode, Caenorhabditis elegans, is a key model organism and one of the most studied organisms to date. The main reasons why C. elegans are so commonly used as an in vivo model organism are because they are easy to handle in the laboratory and their cell lineages and anatomical developmental stages are well described and characterised. Furthermore, the genome of C. elegans has now been fully sequenced and a detailed map of the genome is available. Many mutant strains have been generated and the resulting phenotypes have identified the function of a number of individual genes (Surco-Laos et al., 2011). Mutant strains are readily available and information on these mutants has been deposited on websites such as http://www.wormbase.org. C. elegans has a mean lifespan of between 12 and 18 days and a generation time of 3 days (Gems and Riddle, 2000). They can be easily grown on agar plates or in multi-well plates in large numbers when fed on a diet of Escherichia coli (E. coli). Importantly, the worms have many cell types that are similar to those of humans including neurons, muscle cells, gut and excretory cells. Above all, we share many conserved genes (60–80% homology) and cellular mechanisms (SurcoLaos et al., 2011). The on-going accumulation of data on C. elegans is beginning to demonstrate that most of the important biological processes have remained essentially unchanged during evolution. Worms suffer from many of the diseases that afflict humans, including neurodegeneration, infectious diseases, disorders of physiological control and ageing. There are three studies which show an influence of vitamin C on the lifespan of C. elegans (Bakaev and Bakaeva, 2011; Schulz et al., 2007; Shibamura et al., 2009). Two of the three studies reported that ascorbic acid supplementation prolonged lifespan (Bakaev and Bakaeva, 2011; Shibamura et al., 2009), but only up to a certain concentration above which toxic effects could be observed (Bakaev and Bakaeva, 2011). Bakaev and Bakaeva (2011) also found that the positive effect of ascorbic acid on the lifespan of these nematodes was dependent on the time point at which treatment was initiated. The study by Shibamura et al. (2009) indicated a lifespan-increasing effect of vitamin C treatment, but only if given in combination with liposomes to aid uptake. In contrast, Schulz et al. (2007) reported no beneficial effect of vitamin C on the lifespan of C. elegans. 2.1. Effect of glucose restriction and ascorbic acid on the lifespan of C. elegans Schulz et al. (2007) investigated the effects of glucose restriction and vitamin C on the lifespan of C. elegans. To study the potential role of impaired glucose metabolism, they exposed nematodes to 2-deoxy-D-glucose (DOG) which cannot be completely metabolised and therefore leads to a specific blockade of glucose metabolism and glycolysis. Wild-type ‘Bristol N2’ C. elegans were transferred immediately after hatching in group sizes of 100–150 to nutrition media (E. coli) containing the treatment. The three treatment groups received either 5 mM DOG, 5 mM ascorbic acid or DOG plus ascorbic acid (both 5 mM) (Schulz et al., 2007).

Treatment with DOG significantly increased the lifespan of C. elegans. However, a combination of both DOG and ascorbic acid had no effect on the maximum or mean lifespan. Furthermore, ascorbic acid alone had no beneficial effect on longevity. From these studies, it appears that glucose restriction alone is sufficient to increase the lifespan of C. elegans and that supplementing with ascorbic acid or combining glucose restriction with ascorbic acid has no significant effect. The positive effects of DOG could be a result of hormesis i.e. that glucose restriction increases reactive oxygen species (ROS) formation leading to an induction of stress resistance (Masoro, 2007). 2.2. Liposomal ingestion and delivery of ascorbic acid and the lifespan of C. elegans The majority of studies using C. elegans incorporate the test compounds, including ascorbic acid, into the growth media. However, actual levels of uptake are difficult to assess since they depend on the rates of ingestion into the intestine and absorption from the intestinal lumen. Furthermore, C. elegans feed by taking up liquid containing suspended particles (bacteria) and then spitting out much of the liquid whilst retaining the particles, making the ingestion of solutions, such as ascorbic acid, very inefficient (Avery and Thomas, 1997). Shibamura et al. (2009) used liposomes (tiny vesicles composed of phospholipids) to deliver chemicals including ascorbic acid successfully and more efficiently into the intestines of C. elegans. Three day old worms were divided into groups of 25 worms which received 25 ll liposomes with 60, 120 or 240 lg ascorbic acid. One control group received liposomes incubated with water and a second control group received 120 lg ascorbic acid in the nutrition media alone in order to assess the effects of liposomal delivery. It was found that the longevity of C. elegans receiving ascorbic acid in the presence of liposomes was significantly increased compared to both the liposome control and also the non-liposome ascorbic acid control. However, the dose of ascorbic acid had little effect on mean lifespan in that all three doses (60, 120 and 240 lg) caused a similarly significant rightward shift of the survival curve. Nevertheless, ascorbic acid did dosedependently influence maximum lifespan, with the 240 lg group demonstrating the greatest maximum survival time. Importantly, in accordance with conventional studies on ascorbic acid administration in the media, Schulz et al. (2007) and Shibamura et al. (2009) found no significant life-prolonging benefit of 120 lg ascorbic acid when given in the media alone, further demonstrating the success of liposomal delivery in these nematodes. 2.3. The influence of ascorbic acid on the different life stages of C. elegans Bakaev and Bakaeva (2011) also investigated the influence of ascorbic acid on the lifespan of C. elegans. Ascorbic acid was given in the media at different concentrations and at different stages in the nematode life cycle. In the first experiment, ascorbic acid was administered on day 3 of life. At concentrations between 0.001 and 0.1 mg/ml (0.0057 mM and 0.57 mM, respectively), ascorbic acid in the media significantly increased both the mean and maximum lifespan of C. elegans. However, at concentrations of 0.0001 mg/ml or less and 1 mg/ml or higher, ascorbic acid had no significant effect on longevity. They also investigated the effects of administering ascorbic acid between days 3 and 10 of life, a time period encompassing the reproductive phase of C. elegans. For all seven doses of ascorbic acid (ranging between 0.0001 and 100 mg/ml, 0.00057 mM and 570 mM, respectively), no significant effects on mean lifespan were observed compared to the control group. The third tier of the study examined the effect of ascorbic acid supplementation from the

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K. Pallauf et al. / Food and Chemical Toxicology 58 (2013) 255–263 Table 1 Results of studies investigating the effects of vitamin C on the lifespan of flies in chronological order – data not given. Study

Species

n

Form of vitamin C

Dose

Massie et al. (1976)

Drosophila melanogaster (Oregon R)

125

Ascorbic acid

Males 1 mM 10 mM 50 mM Females 1 mM 10 mM 50 mM

Sohal et al. (1985)

Musca domestica

800

Ascorbate

0.5% 2.0%

Massie et al. (1991)

Drosophila melanogaster (Oregon R, Swedish C)

700

Ascorbic acid

Oregon R 10 mM 100 mM

Start of dosing

Duration of dosing

After hatching

Lifelong

–

Lifelong

–

Mean (days)

Lifespan (%)

2.5 0.5 2.0

5.4 1.1 4.3

+1.0 3.5 6.5

+2.0 6.9 12.8

3.0 10.8

13.9 52.2

+0.9 4.8

+1.5 8.2

+2.5 +2.5 0.1

+5.5 +5.5 0.2

Lifelong Swedish C 1 mM 10 mM 100 mM Bahadorani et al. (2008)

Drosophila melanogaster (Wild-type rosy + 5)

900

Ascorbic acid

10th day of life onwards, a time period comprising the post-reproductive or ageing phase in the life of C. elegans. During this phase, ascorbic acid supplementation had a significant negative effect on mean and maximum lifespan, with both being shortened. 3. Vitamin C and the lifespan of flies The fruit fly Drosophila melanogaster is one of the most extensively characterised experimental organisms. Furthermore, Drosophila is a complex multi-cellular organism in which many aspects of development and behaviour parallel those in humans. These combined advantages have permitted research in Drosophila to make seminal contributions to the understanding of fundamental biological processes, including the process of ageing. These flies have a relatively short lifespan of 2–3 months and are easy and cheap to house. Another big advantage of using Drosophila as model organisms is that, like C. elegans, their genome sequence has been fully characterised (Helfand and Inouye, 2003). Similar to the online database http://www.wormbase.org, information on Drosophila genes and mutants can be found at http:// www.flybase.org. The influence of vitamin C on the lifespan of flies was analysed in four separate studies; three studies used D. melanogaster (Bahadorani et al., 2008; Massie et al., 1976, 1991) and the fourth used Musca domestica (Sohal et al., 1985). These studies yielded conflicting results; Bahadorani et al. (2008) demonstrated that ascorbic acid prolonged longevity whilst (Massie et al., 1976, 1991) and Sohal et al. (1985) reported a decrease in lifespan following vitamin C treatment in flies. The results from the different studies are summarised in Table 1. 3.1. Ascorbic acid, its oxidation products and the longevity of Drosophila To study the potential life-prolonging effects of ascorbic acid in Drosophila, Massie et al. (1976) used flies of the Oregon R strain maintained on media containing either water (control) or ascorbic acid at concentrations ranging between 1 and 50 mM. In their first experiment, the media was changed weekly for the duration of life

0.02 mM 0.2 mM 2 mM 20 mM 100 mM

1 day After hatching

Lifelong

Not significant Not significant Not significant Increased Decreased

and the mean lifespan of male flies was reduced by all concentrations of vitamin C employed (1 mM, 10 mM and 50 mM) by up to 5.4%. A concentration of 1 mM vitamin C modestly increased the mean lifespan of female flies although higher concentrations (10 mM and 50 mM) diminished lifespan as in males by 6.9% and 12.8% respectively. This led the authors to question whether an accumulation of the oxidation products of vitamin C could potentially be responsible for the observed decrease in lifespan. Consequently, in a further experiment the media was changed daily and the mean lifespan of flies was found to be similar to controls for concentrations of 0.1 mM and 10 mM ascorbic acid (Massie et al., 1976). Only the highest concentration of ascorbic acid (100 mM) caused a reduction in lifespan (by 5%). Furthermore, when the flies were maintained on the media supplemented with the oxidation product of ascorbic acid (dehydro-ascorbic acid; 1 mM and 10 mM) the mean lifespan of the flies remained unchanged indicating that the reduction in lifespan observed in the first experiments is a direct result of the ascorbic acid rather than a consequence of its oxidation products. In the third experiment, the nutrition media was changed twice daily (Massie et al., 1976). However, vitamin C still did not trigger a beneficial effect and the mean lifespan of the flies was significantly decreased by all three concentrations of ascorbic acid used by up to 8.7% compared to the controls.

3.2. Influence of ascorbate on the lifespan of the housefly Musca domestica Sohal et al. (1985) examined the effect of ascorbate on the lifespan of the housefly M. domestica. Male adult flies (200 per group) received either sucrose as their nutrition media (controls) or sucrose with 0.5% or 2% ascorbate. Although 0.5% ascorbate did not significantly influence the mean lifespan of the flies (18.6 ± 5.4 days versus 21.6 ± 7.5 days for the control group), 2% ascorbate led to a significant decrease in the mean lifespan by approximately half (to 9.9 ± 3.8 days, p < 0.05). Further studies showed that 0.5% ascorbate did not change the metabolic rate of the houseflies whereas a concentration of 2% ascorbate was sufficient to significantly decrease their metabolic rate.

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3.3. Ascorbic acid and lifespan of two Drosophila strains Massie et al. (1991) also investigated the effects of ascorbic acid on the lifespan of two Drosophila strains, Oregon R and Swedish C. A concentration of 10 mM ascorbic acid was found to modestly increase the mean lifespan of Oregon R flies (to 59.7 days compared to 58.8 days for the controls, not significantly different). In contrast, a concentration of 100 mM ascorbic acid led to a significant decrease in the average lifespan of Oregon R flies to 54.0 days (p < 0.01). Control Swedish C flies had a shorter average lifespan than control Oregon R flies (45.2 days versus 58.8 days). Both 1 and 10 mM ascorbic acid increased the mean lifespan of the Swedish C strain by 2.5 days, representing a change of 5.5%. The higher concentration of 100 mM ascorbic acid had no effect on longevity compared to the control group. The authors also demonstrated that vitamin C content decreased with ageing in both strains, however newly emerged Swedish C flies had higher initial ascorbic acid levels compared to counterpart Oregon R flies (0.078 lg/fly versus 0.058 lg/ fly)(Massie et al., 1991). However, the rate of decline in ascorbic acid levels during ageing was markedly higher in Swedish C flies (70.4%) compared to a decline of only 19.9% in Oregon R flies which exhibited a longer overall lifespan. Based on this data, it was hypothesised that ascorbic acid supplementation in Swedish C flies would thus increase longevity by counteracting this marked decline associated with ageing. However, no beneficial effect of ascorbic acid supplementation was observed. Instead, 100 mM ascorbic acid had the effect of significantly decreasing, rather than prolonging, longevity in Oregon R flies. 3.4. Wild-type Drosophila and ascorbic acid supplementation The study of Bahadorani and Hilliker (2008) investigated the effects of different vitamins, including ascorbic acid, on the lifespan of wild type (rosy+) Drosophila. Flies were supplemented with 0 (control), 0.02, 0.2, 2, 20 or 100 mM ascorbic acid in the nutrition media. The experiment was repeated with concentrations of 0, 20 and 100 mM ascorbic acid to corroborate the initial findings. The authors found that, at concentrations of 0.02, 0.2 and 2 mM, ascorbic acid had no significant effect on the lifespan of the Drosophila compared to the control group. Only when a dose of 20 mM ascorbic acid was included in the media did the lifespan significantly increase (p < 0.01). However, 100 mM ascorbic acid led to a very marked reduction in lifespan by 30 days (p < 0.00001). 4. Vitamin C and the lifespan of rodents Rodents (including mice, rats and guinea pigs) are commonly used as model organisms in research, having the distinct advantage over other model organisms of being mammals, like humans. Approximately 98% of all rodent genes have a human homologue (Copeland et al., 2002). Since rodents only require a small space, are very resilient, and have a relatively short lifespan compared to other mammals (2–3 years for a mouse) they can be kept relatively easily in the laboratory and at low cost. Furthermore, rodents reproduce relatively quickly and yield a high number of offspring. As such, they have become the model organisms of choice not only for experimental investigations in molecular genetics but also for ageing research in mammals. The influence of vitamin C on the lifespan of rodents has been explored in seven different studies. Five of these studies used mice as their model organism (Bezlepkin et al., 1996; Massie et al., 1984; Selman et al., 2006; Tappel et al., 1973; Veurink et al., 2003), one

was performed in rats (Holloszy, 1998) and one in guinea pigs (Davies et al., 1977). Some of the studies showed a significant prolongation of lifespan following vitamin C supplementation (Bezlepkin et al., 1996; Massie et al., 1984; Veurink et al., 2003), whilst others showed no beneficial effect of the vitamin (Holloszy, 1998; Selman et al., 2006; Tappel et al., 1973). Davies et al. (1977) reported a shortening of lifespan following vitamin C treatment. The principal findings of the studies are summarised in Table 2. 4.1. The influence of ascorbic acid on lifespan in mice Tappel et al. (1973) investigated the effect of combining antioxidants and nutrients on lipid peroxidation and ageing in male CD-1 mice. Nine month old mice were allocated to three groups; one group (of 100 mice) received a control diet and the other two groups (50 mice each) received a diet enriched either in antioxidants or other nutrients. All mice were maintained on their respective diets until 1.9 years of age which represents old age in mice. The results demonstrated that none of the test diets had a significant effect on the lifespan of the mice compared to the control group. Body weights were also unaffected by all three diets. Another study was performed by Massie et al. (1984) who examined the influence of 1% ascorbic acid (equivalent to 1.43 mg/kg body weight) in the drinking water of male 37 days old C57BL/6 J mice. They reported that vitamin C supplementation significantly increased the mean lifespan of mice by 8.6% (p < 0.05), although the maximum lifespan remained unchanged. Mice in the ascorbic acid group also had a reduced body weight up to 26 months of age (by 6.5%). Whilst they found that 1% ascorbic acid had no effect on copper levels in heart, liver, kidneys and brain, a 2% dose caused a 20% reduction in cardiac copper content leading the authors to conclude that higher doses of ascorbic acid may exert toxic effects and result in a reduced longevity (Massie et al., 1984). Bezlepkin et al. (1996) explored whether the age at which antioxidant supplementation is initiated has an impact on the experimental outcome. Male C57BL/6J mice were fed a diet containing a combination of antioxidants (b-carotene, a-tocopherol, sodium selenite, zinc and ascorbic acid (50 mg/kg)). Animals which were given the diet from the ages of 2 or 9 months had a significantly prolonged mean lifespan compared to controls (110 or 87 days, respectively). The maximal lifespan was also elevated; by 108 days for the mice fed from 2 months and by 86 days for those fed from 9 months. In contrast, when the antioxidant diet was introduced at a relatively old age (16 and 23 months), lifespan remained unchanged. The effect of antioxidant supplementation on longevity has also been studied in apolipoprotein E (ApoE)-deficient mice by Veurink et al. (2003). ApoE knockout mice are widely used as an animal model for atherosclerosis and are well known to have elevated ROS production and oxidative stress. Therefore, a potential antiageing benefit of an antioxidant therapy, should it exist, might be easier to demonstrate in these mice. Female mice (6–8 weeks old) were fed on a high fat/high cholesterol (HFHC) diet with or without a combination of antioxidants (doses based on recommended doses in humans; 37.8 mg vitamin E acetate, 3.8 mg gingko biloba, 3.8 mg pycnogenol and 37.8 mg ascorbyl palmitate) for 3 months. Mice were then placed on standard chow for 3 months before resuming their test diets for a further 3 months. As expected, the HFHC diet caused a marked increase in mortality compared to the chow diet. However, longevity was significantly prolonged in animals fed the HFHC diet supplemented with antioxidants. At 100, 200 and 270 days, the percentage survival was 100%, 89% and 55% respectively in the antioxidant-fed group compared to 66%, 55% and 22% respectively in untreated animals during the corresponding period. The increase in survival observed in

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K. Pallauf et al. / Food and Chemical Toxicology 58 (2013) 255–263 Table 2 Results of studies investigating the effect of vitamin C on the lifespan of rodents (in chronological order). n.s. – not significant, – data not given. Study

Species

n

Form of vitamin C

Dose

Start time of treatment

Treatment duration

Mean lifespan (days)

Maximum lifespan (days)

Tappel et al. (1973)

Mouse (CD-1)

200

Ascorbic acid

920 mg/kg*

9 months

Until 1.9 years old

n.s.

n.s.

Davies et al. (1977)

Guinea pigs (Dunkin Hartley)

50

Ascorbic acid

5 mg/kg#

864 5 weeks

Lifelong

1%e

*

– 783

1%e (1.43 mg/kg#)

37 days

Lifelong

+8.6%

+28 (2.9%)

Ascorbic acid

50 mg/kg#

2 months 9 months 16 months 23 months

Lifelong

+110 § (16.4%) +87§ (13.1%) +31§ (4.2%) +15§ (1.9%)

+108 (11.4%) +86 (9.5%) +25 (2.7%) 8 ( 0.8%)

192

Ascorbic acid

2500 mg/kg*

3 months

Lifelong

Sedentary: +17 (2.0%) Active: +14 (1.5%)

–

ApoE / mouse (C57BL/6J)

18

Ascorbyl-palmitate

37.8 mg/kg*

6–8 weeks

2  3 months with 3 months standard diet in between

–

–

Mouse (C57BL/6J)

41

Ascorbyl-2-polyphosphate

180 mg/kg*

4 months

Lifelong

n.s.

–

Massie et al. (1984)

Mouse (C57BL/6J)

24

L-ascorbic

Bezlepkin et al. (1996)

Mouse (C57BL/6J)

606

Holloszy (1998)

Rat (Long Evans)

Veurink et al. (2003) Selman et al. (2006)

acid

–

mg/kg Diet. mg/kg Rodent body weight. Added to drinking water. Median lifespan.

# e §

the antioxidant-supplemented HFHC-fed animals compared with their counterpart controls correlated with a significant reduction in inclusion bodies in the hippocampal region of their brains and a reduction in DNA fragmentation. Selman et al. (2006) also investigated the effect of vitamin C supplementation on oxidative stress and lifespan in mice. In this study, a life-long cold exposure regime was employed since cold has been shown to increase metabolic rate (Selman et al., 2002) and oxidative stress (Selman et al., 2002; Venditti et al., 2004), therefore enhancing the likelihood of detecting a beneficial impact of antioxidant treatment as in the ApoE animals (Veurink et al., 2003). Lifespan was measured in both control and vitamin C (ascorbyl-2-polyphosphate, 180 mg/kg body weight)-supplemented 12 weeks old, female C57BL/6 J mice maintained in the cold (7 ± 2 °C). Despite a significant increase in hepatic ascorbic acid levels in vitamin C-supplemented mice compared to controls, there was no treatment effect on body mass, resting metabolism, hepatic lipid peroxidation and hepatocyte or lymphocyte oxidative DNA damage. Furthermore, there was no extension of the lifespan or reduction in the mortality rate of cold-exposed mice given lifelong vitamin C supplementation compared to matched mice fed on a control diet. However, the cold exposure did not result in the expected decrease in lifespan in control animals due to elevated oxidative metabolism and consequently the potential to detect an effect of vitamin C in these studies was unlikely to have been enhanced. Importantly, Selman et al. (2006) did, however, observe significant decreases in the expression of key antioxidant genes connected with free-radical scavenging and repair following 18 months of vitamin C supplementation, including SOD2, catalase and glutathione peroxidase. It could be that vitamin C acts as an antioxidant but has also simultaneously a negative impact on the endogenous protection system, counteracting any potential beneficial effect. 4.2. Ascorbic acid and the lifespan of rats Based on the findings that food restriction increases maximal lifespan in rodents, Holloszy (1998) set out to explore whether

an antioxidant diet was able to increase longevity in exercised rats which do not show an increase in lifespan despite having a similar calorific deficit, hypothesising that exercise-induced oxidative stress may mask this effect. Three month old Long Evans rats were assigned to four different groups; two groups performed voluntary wheel running and two were sedentary. One running and one sedentary group were fed on a diet supplemented with antioxidants whilst the other two groups were fed the same diet without supplementation. The supplementation was comprised of 2.5 g ascorbic acid, 2 g a-tocopherol, 0.1 g b-carotene and 20 mg menadione sodium bisulphite complex. Serum ascorbate levels reached 1.05 ± 0.11 mg/dl in the antioxidant diet groups compared to 0.61 ± 0.08 mg/dl in the control groups (p < 0.01) confirming efficient uptake. Whilst substantiating that voluntary wheel running modestly increased mean lifespan (p < 0.05), Holloszy (1998) was unable to show an additional beneficial effect on longevity by antioxidant supplementation. In actual fact, the antioxidant and free radical-scavenging diet had no effect on the longevity of either wheel running or sedentary rats. The ineffectiveness of the antioxidant diet in prolonging the longevity of the wheel runners argues against the hypothesis that exercise-associated oxidative stress prevents a life-extending effect of a calorific deficit manifesting itself in exercising animals. 4.3. Ascorbic acid and the lifespan of guinea pigs The majority of studies investigating the effects of ascorbic acid on ageing have used mice and rats as experimental animals. However, unlike humans, these small rodents are capable of synthesising ascorbic acid themselves. Akin to humans, guinea pigs are unable to biosynthesise ascorbic acid making it an essential nutrient. The study by Davies et al. (1977) subjected 5 weeks old male Dunkin Hartley guinea pigs either to a diet containing 5 mg/kg body weight ascorbic acid or to drinking water containing 1% ascorbic acid. They reported no difference in lifespan between the two groups, however the results are difficult to evaluate since

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no control group was included. Nevertheless, when analysing the deaths of the first six animals in each group, the 5 mg/kg-treated group had a 44% longer lifespan than the animals given 1% ascorbic acid in their drinking water.

contain vitamin C without it being consumed through their diet (Massie et al., 1991). However, in the case of C. elegans evidence for endogenous ascorbate synthesis is missing (Adler et al., 2011) and it remains unclear whether and to what extent the studied organisms need exogenous vitamin C to prevent ascorbic acid deficiency. It is possible that low dose vitamin C supplementation prolongs lifespan by preventing deficiencies in catecholamine, carnitine- or collagen synthesis in humans or organisms with limited endogenous synthesis rather than by slowing down the ageing process through mechanisms related to antioxidant defence. In humans, the recommended daily allowance for vitamin C intake varies from organization to organization and is based on the amount that was found to prevent scurvy. The United States Department of Agriculture (http://www.iom.edu) recommends 90 mg/day for men and 75 mg/day for women. However, there are data that suggest a higher intake of vitamin C of around 120–200 mg/day is necessary to reduce a vitamin C deficiency-related health risk (Carr and Frei, 1999). Although this amount of vitamin C is easily consumed with a diet containing fruit and vegetables, many adults and children even in the Western world do not eat sufficient quantities of these foodstuffs to ingest the recommended amount of vitamin C (Ames, 1998). In contrast, even under high supplementation, blood plasma levels would not rise above a certain level in healthy volunteers and excess vitamin C was excreted. This led the authors to hypothesise that vitamin C has no additional value if consumed at high doses, nevertheless they promote increasing the recommended daily allowance to 200 mg/day (Levine et al., 1996).

5. Discussion Oxidative stress is suggested as being central to the ageing process, with endogenous antioxidant defence and repair mechanisms in place to minimise damage. However, supplementation trials with antioxidants in animal models have had limited success. The present review comprehensively examines the literature on vitamin C and lifespan in model organisms including nematodes, flies and laboratory rodents. Of the 14 studies included in our analysis, some studies reported an increase in longevity following vitamin C supplementation whilst others failed to show a beneficial effect (Massie et al., 1976, 1991; Schulz et al., 2007; Sohal et al., 1985) or demonstrated a reduction in lifespan as a result of vitamin C treatment (Bakaev and Bakaeva, 2011; Holloszy, 1998; Selman et al., 2006; Tappel et al., 1973). Human epidemiological data have been similarly disappointing, leading some even to question whether vitamin C has pro-oxidant properties in vivo (Carr and Frei, 1999; Levine et al., 2011; Podmore et al., 1998). 5.1. In humans health benefits from vitamin C supplementation could be related to preventing deficiency As mentioned in the introduction, humans, some other primates, bats and guinea pigs lack the L-gulonolactone oxidase (GLO) which is necessary for ascorbic acid synthesis due to mutations in the encoding gene (Burns, 1957) (see Fig. 1 and Table 3). There are also rodents with genetic defects that affect the ascorbic synthesis or transport (Yu and Schellhorn, 2013). Similar to humans, the Osteogenic Disorder Shionogi (ODS) rat lacks GLO and thus needs to consume ascorbic acid with its diet (Mizushima et al., 1984). Additionally, there are knockout mice for ascorbic acid uptake transporters (Sotiriou et al., 2002) and synthesis including GLO (Maeda et al., 2000) and regucalcin (Kondo et al., 2006). These models have been mostly used to study vitamin C deficiency rather than supplementation. However, in terms of ascorbic acid necessity, models with impaired vitamin C synthesis might resemble humans somewhat better than animals that synthesise the vitamin endogenously. Although the enzymes contributing to ascorbic acid synthesis in Drosophila have not been identified in detail, flies were found to

H

H OH H

OH

UDP-glucose 6-dehydrogenase …

OH

HO H

H

O

UTP-glucose-1-phosphate uridylyltransferase

OH

O

Whilst the influence of vitamin C on animal lifespan varied, in general a decrease in lifespan was observed with higher concentrations of ascorbic acid and lower concentrations were more likely to increase lifespan (Bahadorani and Hilliker, 2008; Massie et al., 1984, 1991; Sohal et al., 1985; Veurink et al., 2003). The method of delivery was also shown to have an impact on the efficacy of vitamin C to prolong life. The conventional method of dosing C. elegans is to supplement the media. However, this is likely to lead to inefficient ingestion into the intestine and therefore inefficient absorption since nematodes filter out particles from the media and emit the liquid containing the ascorbic acid. Shibamura et al. (2009) elegantly demonstrated that when ascorbic acid is successfully and measurably delivered by liposomes, ascorbic acid Glucuronate reductase

OH

H OH H

O

OH OH

-

O

OH H

OH

OH

HO

OH

D-glucose

5.2. Different doses and forms of vitamin C at different life stages of the model organisms

OH

OH

D-glucuronic acid

OH

L-gulonate Gluconolactonase

O

OH

O

O

OH

O

OH HO

OH

L-ascorbic acid

L-Gulonolactone oxidase

O

OH

O

OH

OH O

OH

2-keto-L-gulonolactone

HO

OH

L-gulonolactone

Fig. 1. Ascorbic acid synthesis in vertebrates. Ascorbic acid can be synthesised in most vertebrates from D-glucose or D-galactose (not depicted). In a first step involving various enzymes, D-glucose is oxidized to D-glucoronic acid which is then reduced to L-gulonate before forming the 1,4-lactone. The last step in which the L-gulonolactone oxidase oxidizes the L-gulonolactone to become the keto form of ascorbic acid is dysfunctional in humans, some other primates and guinea pigs due to the lack of gulonolactone oxidase. Thus, these animals and humans depend on consuming vitamin C with their diet (modified from Drouin et al., 2011).

1.1.3.8. ?*

#

sgl ? rgn/smp-30

? Gulo LGO/GLO oxidase L-gulonolactone

RC/GNL

Gulo

Ugdh Akr1a1/Akr1a4 Rgn;Synonym:Smp30

Ugdh Akr1a1/Alr Rgn;Synonym:Smp30

#

UGDH AKR1A1 RGN;Synonym: SMP30 –

Ugp2 Ugp2 UGP2;Synonym:UGP1

UDPGP/ UGPase UDPGDH

UTP-glucose-1-phosphate uridylyltransferase/UDP-glucose pyrophosphorylase UDP-glucose 6-dehydrogenase Alcohol dehydrogenase [NADP (+)]/glucuronate reductasee Regucalcin/gluconolactonase

There may be no vitamin C synthesis in C. elegans. According to http://www.ncbi.nlm.nih.gov/homologene/. The glucoronate reductase seems to be identical with the alcohol dehydrogenase [NADP (+)] (http://enzyme.expasy.org; http://www.uniprot.org), therefore the genes encoding the alcohol dehydrogenase [NADP (+)] are given. Further sources http://www.chem.qmul.ac.uk/iubmb/enzyme/, Drouin et al. (2011), Linster and Van Schaftingen (2007). #

e

*

1.1.1.22. 1.1.1.19. (=?) 1.1.1.2. 3.1.1.17. sqv-4 ?* ?*

2.7.7.9. K08E3.5(ORF)* UGP

#

Enzyme commission (EC) number C. elegans gene Drosophila melanogaster gene Rat gene Mouse gene Human gene Short name Recommended name/alternative name

Table 3 Some of the enzymes that participate in the vertebral ascorbic acid synthesis and the corresponding genes in humans, mice, rats, flies and worms.

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significantly increased both the mean and maximum lifespan of C. elegans. They also showed that conventional supplementation in the media with the same dose had no such effect (Shibamura et al., 2009). There were also different routes of supplementation in the rodent studies; via drinking water or food (Selman et al., 2006; Tappel et al., 1973). Two studies confirmed efficient absorption in the rodent studies (Holloszy, 1998; Selman et al., 2006). One cannot rule out the possibility that a null effect in the studies using C. elegans in particular, is only a result of a failure to adequately dose these animals. Other discrepancies between the studies included the form of ascorbic acid employed (ascorbic acid, ascorbyl palmitate or ascorbyl-2-polyphosphate were all used in the studies described in this review), and whether it was administered alone or in conjunction with other antioxidants or micronutrients. The age at which ascorbic acid supplementation is initiated was also shown to influence the effect of the antioxidant on lifespan. Only when lifelong vitamin C supplementation was initiated in the early stages of life was a beneficial effect on longevity observed in both C. elegans (Bakaev and Bakaeva, 2011) and mice (Bezlepkin et al., 1996). When treatment commenced at a late phase of life in C. elegans, lifespan was actually diminished (Bakaev and Bakaeva, 2011). 5.3. Possible pro-oxidant activity of vitamin C Three of the studies included in this review also employed model organisms with higher basal levels of ROS production, potentially making a beneficial effect on lifespan easier to detect (Holloszy, 1998; Selman et al., 2006; Veurink et al., 2003). Importantly, while Selman et al. (2006) failed to show a life-extending effect of ascorbyl-2-polyphosphate in mice exposed to lifelong cold they found that the expression of key antioxidant genes was reduced in these mice. Therefore, it may be that vitamin C does successfully scavenge ROS but this may not be translated into a reduction in damage and lifespan enhancement because of compensatory effects on endogenous scavenging and repair systems, either directly (Podmore et al., 1998) or via systems that sense reduced radical production. A recent research article questioned whether lifespan extension in C. elegans by compounds that seem to work as antioxidants in vitro is, in fact, caused by antioxidant activity in vivo since the authors could not find the in vitro and in vivo antioxidant capacities to correlate (Pun et al., 2010). Interestingly, in a study using the senescence-accelerated mouse prone 8 (SAMP8) as a model for accelerated ageing, vitamin C liver content was higher in SAMP8 mice as compared to the senescence-accelerated mouse-resistant 1 (SAMR1) strain which exhibits a normal ageing phenotype (Bayram et al., 2012). As tocopherol levels were also elevated in ageing mice it seems that higher antioxidant tissue content does not necessarily lead to less oxidative damage at higher age. In humans, it was shown that 500 mg vitamin C led to oxidation of lymphocyte DNA (Podmore et al., 1998). By acting as a pro-oxidant in vivo, vitamin C could be causing rather than inhibiting detrimental biological effects on macromolecules (Carr and Frei, 1999). The pro-oxidant potential of vitamin C seems especially relevant in humans with elevated iron storage and it is possible that these individuals benefit from low vitamin C intake. Free iron can lead to hydroxyl radical formation and consequently, tissue damage (Young et al., 1994). In a cell culture study, it was shown that ascorbate sensitised the cells to H2O2-induced damage and that iron ions further increased the oxidative damage (Riviere et al., 2006). Additionally, vitamin C was shown to inhibit autophagic degradation of the iron storing ferritin, thereby increasing intracellular iron levels. Of interest, autophagy is induced by ROS (ScherzShouval et al., 2007) and there is increasing evidence that low

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K. Pallauf et al. / Food and Chemical Toxicology 58 (2013) 255–263

Exercise

Caloric restriction

Plant bioactives HO OH

OH HO

OH O

H

O

OH OH

H2O2

O

._

.

Low level ROS production

O2

HO

Activation of antioxidant defence mechanisms and improvement of insulin sensitivity through induction of Nrf2, PGC1α and PPARγ Fig. 2. Indirect action of vitamin C on the activation of antioxidant defence mechanisms. Through its radical scavenging properties ascorbic acid might neutralize low levels of reactive oxygen species that are produced upon exercise, caloric restriction and consumption of certain plant bioactives such as resveratrol or quercetin. This low level ROS production is believed to induce mechanisms that reduce oxidative stress and improve insulin sensitivity through transcriptional events. Ascorbic acid has been shown to inhibit this activation (Ristow et al., 2009; Wagner et al., 2011).

levels of ROS production may be advantageous for longevity (Ristow and Schmeisser, 2011). In this case vitamin C would antagonize the beneficial effect of low doses of ROS on redox-responsive transcription factors such as nuclear factor like 2 (Nrf2) as shown by Wagner et al. (2011). Also during exercise, low levels of ROS are produced which in turn activate ROS-sensitive transcription of genes involved in antioxidant defence and insulin sensitivity (Ristow et al., 2009). These studies are consistent with the hormesis concept that describes how low levels of a stimulus (such as ROS in this case) can have a beneficial effect on antioxidant status within an organism while high levels seem detrimental. Additionally, it has been hypothesised that diet-related health-promoting measures, such as dietary restriction or consumption of polyphenols may reduce overall antioxidant damage through promoting low levels of ROS production (Ahmad et al., 2003; Schulz et al., 2007). Therefore, vitamin C could negatively interfere with antioxidant defence mechanisms by removing their trigger. In the context of vitamin C potentially having a lifespan-reducing effect, it is also interesting that Ristow et al. (2009) demonstrated that vitamin C inhibited the exercise-induced up-regulation of peroxisome proliferator-activated receptor gamma coactivator (PGC) 1alpha transcription. This protein is important for the regulation of mitochondrial biogenesis and might promote longevity (Clark et al., 2011; Ristow et al., 2009; Rodgers et al., 2005) (see Fig. 2). A study comparing cigarette smoking ApoE4 carriers with nonsmoking ApoE4 carriers found that vitamin C supplementation reduced inflammatory gene expression in smokers (Majewicz et al., 2005). Whether this effect on gene expression following vitamin C supplementation was indirectly caused by scavenging of pro-inflammatory molecules or whether vitamin C influences transcription directly, remains unclear. Nonetheless, it seems that certain populations such as cigarette smoking ApoE4 carriers could benefit more from vitamin C supplementation than others and that the influence of vitamin C on gene expression could be advantageous in some cases. However, in a randomized, double blind study to investigate the effect of antioxidant vitamin supplementation on cardiovascular disease, ascorbic acid neither increased nor reduced the incidence of cardiovascular events. Similar findings resulted from a

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