The effects of exogenous antioxidants on lifespan and oxidative stress resistance in Drosophila melanogaster

Mechanisms of Ageing and Development 127 (2006) 356–370 www.elsevier.com/locate/mechagedev

The effects of exogenous antioxidants on lifespan and oxidative stress resistance in Drosophila melanogaster Tapiwanashe Magwere a, Melanie West a, Kumars Riyahi a, Michael P. Murphy b, Robin A.J. Smith c, Linda Partridge a,* a

Centre for Research on Ageing, University College London, Department of Biology, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom b MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, United Kingdom c University of Otago, Department of Chemistry, University of Otago, Dunedin, New Zealand Received 7 October 2005; received in revised form 7 December 2005; accepted 12 December 2005 Available online 25 January 2006

Abstract We used the fruit fly Drosophila melanogaster to test the effects of feeding the superoxide dismutase (SOD) mimetic drugs Euk-8 and -134 and the mitochondria-targeted mitoquinone (MitoQ) on lifespan and oxidative stress resistance of wild type and SOD-deficient flies. Our results reaffirm the findings by other workers that exogenous antioxidant can rescue pathology associated with compromised defences to oxidative stress, but fail to extend the lifespan of normal, wild type animals. All three drugs showed a dose-dependent increase in toxicity in wild type flies, an effect that was exacerbated in the presence of the redox-cycling drug paraquat. However, important findings from this study were that in SOD-deficient flies, where the antioxidant drugs increased lifespan, the effects were sex-specific and, for either sex, the effects were also variable depending on (1) the stage of development from which the drugs were given, and (2) the magnitude of the dose. These findings place significant constraints on the role of oxidative stress in normal ageing. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Antioxidants; Oxidative stress; Lifespan; Drosophila; Mitoquinone; Superoxide dismutase

1. Introduction According to the free radical theory, ageing results from the accumulation of oxidative damage to cellular macromolecules, ultimately leading to the death of the organism (Harman, 1956; Sohal et al., 2000). The validity of this theory has been tested experimentally through manipulation of the production of reactive oxygen species (ROS) by mitochondria (Bevilacqua et al., 2004; Criscuolo et al., 2005; Sanz et al., 2004) and of systems that detoxify and defend against ROS (Huang et al., 1999; Kirby et al., 2002; Orr and Sohal, 1994; Phillips et al., 1989). Defence against ROS has been manipulated both by misexpression of antioxidant defence genes (Kirby et al., 2002; Lebovitz et al., 1996; Orr and Sohal, 1994; Parkes et al., 1998; Phillips et al., 1989) and through administration of antioxidant drugs to different organisms including worms (Keaney and

* Corresponding author. Tel.: +44 2076792983; fax: +44 2076797096. E-mail address: [email protected] (L. Partridge). 0047-6374/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2005.12.009

Gems, 2003; Keaney et al., 2004; Melov et al., 2000; Sampayo et al., 2003), houseflies (Bayne and Sohal, 2002) and mice (Kohn, 1971; Melov et al., 2001). While these studies have provided some evidence that is consistent with a role of oxidative damage in ageing, the issue is still shrouded in controversy because: (1) experiments involving manipulation of antioxidant defences have produced inconsistent and variable effects (Phillips et al., 2000), (2) no single antioxidant has yet been shown to consistently increase lifespan in a broad range of long-lived strains of multiple species (Bayne and Sohal, 2002), (3) no extension of lifespan associated with elevation of defence mechanisms has yet been unequivocally demonstrated to be caused by reduced ROS-induced damage rather than, for instance, by an effect on cellular-signalling mechanisms (Dugan and Quick, 2005). Gene knockouts of enzymes involved in antioxidant defence, such as the copper–zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), are characterised by deleterious effects in a number of animal species. In mice (Lebovitz et al., 1996), and also in Drosophila

T. Magwere et al. / Mechanisms of Ageing and Development 127 (2006) 356–370

(Duttaroy et al., 2003), deletion of the MnSOD gene (Sod2) severely limits lifespan; and RNA interference-mediated silencing of this gene in Drosophila also causes extremely short lifespan and elevated endogenous oxidative stress (Kirby et al., 2002). The CuZnSOD gene null (Sod1 null) mutation in Drosophila was also shown to decrease lifespan by 80% and cause extreme paraquat sensitivity (Phillips et al., 1989). Such manipulations, involving deletion and/or silencing of antioxidant genes, show that they are required for achieving normal lifespan. However, because they also introduce other novel pathologies that may be life limiting (Huang et al., 1999; Lebovitz et al., 1996; Li et al., 1995; Melov et al., 1999; Van Remmen et al., 2003), they do not provide clear support for a causal role of oxidative damage in normal ageing. Furthermore, in mice it was shown that lifelong reduction in MnSOD activity resulted in higher levels of oxidative damage to DNA and higher incidence of tumours, but did not accelerate ageing, suggesting that accrual of additional oxidative damage did not accelerate ageing (Van Remmen et al., 2003). Other studies have examined the effects of over-expression of genes encoding components of the machinery of defence against ROS. While targeted over-expression of CuZnSOD in motor neurons was shown to extend lifespan by about 40% in Drosophila (Parkes et al., 1998; Phillips et al., 2000), other studies involving over-expression of this enzyme in the same organism have shown that lifespan-extension may either be tissue-specific (Landis and Tower, 2005) or can be strongly influenced by genetic background (Spencer et al., 2003). Induced over-expression of mitochondrial MnSOD was shown to significantly increase lifespan in transgenic lines of D. melanogaster (Sun et al., 2002) and the increase in lifespan was found to be proportional to the increase in MnSOD enzyme activity (Sun et al., 2004). However, while simultaneous overexpression of MnSOD and CuZnSOD had partially additive effects on increasing lifespan (Sun et al., 2004), simultaneous over-expression of MnSOD and the H2O2-removal enzyme catalase had no additional benefit on increasing lifespan (Sun et al., 2002). Perhaps the clearest evidence in favour of the free radical theory of ageing is the finding that over-expression of catalase targeted to the mitochondria could extend the lifespan of mice (Schriner et al., 2005). However, from this study (Schriner et al., 2005) it is difficult to attribute the increase in lifespan in the mouse to the reductions in oxidative damage, for two main reasons: (1) the results were obtained in early backcrossed generations and should be replicated in later generations to rule out the contribution of founder-line genes to the increased longevity (Dugan and Quick, 2005), and (2) reactive oxygen species such as H2O2 are involved in intracellular signalling, and the possible contribution of altered signalling pathways as a result of over-expressing catalase in the mitochondrion cannot be ruled out. Natural or synthetic antioxidant compounds have so far failed to show consistent effects in increasing maximum lifespan of wild type animals from different species (Bayne and Sohal, 2002; Keaney and Gems, 2003; Melov et al., 2000). In C. elegans, where increases in lifespan after administration of

357

superoxide dismutase/catalase mimetics (Euk-8 and -134) were noted (Melov et al., 2000), these findings could not be independently confirmed by other workers (Keaney and Gems, 2003), leading to the conclusion that the effects of these drugs could be culture-condition specific (Keaney and Gems, 2003). In other species, Euk-8 and -134 were found to have no effect on lifespan and failed to prevent the age-dependent accumulation of protein carbonyls in house flies, suggesting that the effects of these drugs could also be species-specific (Bayne and Sohal, 2002). These two drugs were, however, successful in improving survival under paraquat-induced stress (Keaney et al., 2004; Sampayo et al., 2003) or heat stress (Sampayo et al., 2003) in C. elegans, but decreased lifespan under hyperoxic conditions in M. domestica (Bayne and Sohal, 2002). In mice, Euk-8 and -134 were shown to extend the lifespan and rescue the spongiform encephalopathy of Sod2 nullizygous mice (Melov et al., 2001), perhaps reaffirming the notion that these drugs can ameliorate conditions where lifespan is limited by some underlying pathology or stress, but do not retard ageing. The corollary to the free radical/oxidative damage theory is the mitochondrial theory of ageing, which proposes that mitochondria play a crucial role in the ageing process by virtue of being the major intracellular generator and target of ROS. A new class of mitochondria-targeted antioxidants has been developed (Dhanasekaran et al., 2005; Kelso et al., 2001; Smith et al., 1999) that has been shown to protect mammalian cells from oxidative damage and H2O2-induced apoptosis (Kelso et al., 2002) as well as to protect against ROS-induced inactivation of key mitochondrial enzymes (Dhanasekaran et al., 2005). Mitoquinone (MitoQ) is one mitochondriatargeted antioxidant (James et al., 2005; Smith et al., 1999) that has been shown to protect against cardiac ischemia-reperfusion injury in rats (Adlam et al., 2005). The chemical structure of MitoQ consists of a ubiquinone molecule attached to a triphenyl-phosphonium lipophilic cation (TPP) and it acts as an antioxidant through its reduced ubiquinol form that can block lipid peroxidation (James et al., 2005). The charge of the TPP moiety is shielded by large hydrophobic groups that facilitate its easy passage through bio-membranes and the positive charge on the whole molecule facilitates its membrane-potentialdependent accumulation by the mitochondrion (James et al., 2005). The simple molecule methyltriphenyl-phosphonium (TPMP) contains the same TPP moiety as MitoQ but does not have any antioxidant function, and can therefore act as a control in experiments involving MitoQ to distinguish antioxidant effects from other biological effects of the accumulation of lipophilic cations in vivo. Mitochondria-targeted antioxidants appear more attractive to ageing research because they can be both administered and withdrawn as well as administered to cells and whole organisms, making investigation of their effects much easier compared to transgenic modifications. The use of antioxidant compounds provides an easy and convenient way of testing the validity of the free radical theory using model organisms such as Drosophila. The organism is easier to culture and manipulate under laboratory conditions than are mammals. Administration of these drugs can be done

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easily by adding the solubilised compounds to the food medium thus ensuring their uptake by the flies. In this study we used the superoxide dismutase/catalase mimetics (Euk-8 and -134, SCMs) as well as the mitochondria-targeted drug MitoQ, to test the free radical/oxidative damage theory of ageing in Drosophila. The aim was to see if their antioxidant action could be detected in vivo and whether this could slow down ageing. Our specific objectives were 3-fold: first, to test whether these antioxidants could rescue the extremely short lifespan of mutant flies lacking cytosolic CuZnSOD (Sodn1 null) (Phillips et al., 1989) or whose mitochondrial MnSOD expression had been suppressed via RNA interference (Kirby et al., 2002); second, to test whether these drugs could protect these flies against paraquat-induced stress as observed in other species (Keaney et al., 2004; Sampayo et al., 2003); third, to test whether these antioxidant drugs could increase the lifespan of adult wild type D. melanogaster. Our results show that, while these drugs can act as antioxidants and rescue pathology associated with elevated oxidative stress, they do not extend lifespan in normal, wild type flies.

kept at 25 8C and 60% humidity on a 12:12 h light:dark cycle throughout the experimental period.

2.3. Antioxidant drugs and their addition to fly food EUK-8 (manganese N,N0 -bis(salicylidene)ethylenediamine chloride) and EUK-134 (manganese 3-methoxy N,N0 -bis(salicylidene)ethylenediamine chloride) were supplied by Eukarion Inc. (Bedford, Massachusetts) or were donated as a gift by Dr. Martyn A. Sharpe (Institute of Neurology, London, UK) and Dr. Richard G. Faragher (School of Pharmacy and Biomolecular Science, University of Brighton). MitoQ (mitoquinone) was produced in the laboratory of Professor Robin A.J. Smith (University of Otago, New Zealand). TPMP (methyltriphenylphosphonium) was from Sigma. Stock solutions of Euk-8 and -134 were prepared by dissolving the compounds in deionised water. Those of MitoQ and TPMP were dissolved in absolute ethanol just prior to food preparation. TPMP was included to act as control for the MitoQ experiments; it has no antioxidant function of its own and is the moiety that targets the MitoQ to the mitochondria (Kelso et al., 2001). Food media with drug concentrations ranging from 0 to 10 mM were prepared once every week and stored in the dark at 4 8C. Paraquat (methyl viologen) was obtained from Sigma Chemical Company (St. Louis, MO., USA) and working concentrations were prepared by dilution with deionised water.

2.4. Testing whether the antioxidants could rescue the short lifespan of SOD-deficient flies

2. Materials and methods 2.1. Fly stocks The wild type flies used were from the Dahomey base stock (Chapman and Partridge, 1996). The Dahomey stock is outbred and maintained in large population cages in our laboratory at University College London. The Sodn1 stock (Sod[n1] red[1]/TM3, Sb[1] Ser[1]) was obtained from the Bloomington Drosophila Stock Center (stock number 4015). Experimental progeny (Sodn1 homozygous null flies, Sodn1(
/
)) were generated by crossing heterozygous Sodn1(
/TM3) virgin females to heterozygous Sodn1(
/TM3) males. The Sodn1 mutants have shortened lifespan and extreme paraquat sensitivity as described (Phillips, 1988; Phillips et al., 1989). Homozygotes (Sodn1(
/
)) are infertile and, although viable as larvae, have an elevated death rate during eclosion, attributed to the reduced capacity of embryos, larvae, and pupae to adequately protect the developing preimaginal cells from superoxideinduced damage (Phillips et al., 1989). The daGal4/MnSOD-RNAi experimental flies (UAS-Sod2IR15/+; daG32Gal4/+) were generated by crossing heterozygous Sod2IR15 virgin females [w; UAS-Sod2IR15/SM5; +/+] obtained from the laboratory of Dr. John P. Phillips (University of Guelph, Ontario, Canada) to homozygous daG32Gal4 driver males [w; +/+; daG32/daG32] kindly provided by Dr. Maria E. Giannakou (University College London). The experimental daGal4/MnSOD-RNAi progeny were flies whose expression of mitochondrial MnSOD had been suppressed by RNA interference using the binary Gal4-UAS system (Kirby et al., 2002) and, similar to the Sodn1(
/
) flies, have very short lifespan and increased paraquat sensitivity.

There are three well-characterised superoxide dismutases in Drosophila melanogaster which are the cystosolic CuZnSOD (Sod1), the mitochondrial MnSOD (Sod2), and an extracellular CuZnSOD (Sod3) (Landis and Tower, 2005). The Sodn1 mutation affects the cytosolic CuZnSOD as previously described (Phillips, 1988). The purpose of these experiments was to test whether Euk-8, Euk-134, and MitoQ were acting as antioxidants in vivo and whether or not they could rescue the short lifespan of homozygous SOD null (Sodn1(
/
)) (Phillips et al., 1989) or daGal4/MnSOD-RNAi (Kirby et al., 2002) flies. First, the drugs were tested for antioxidant effect by determining their ability to rescue the reduced pre-adult viability of the Sodn1(
/
) flies (Phillips et al., 1989). If the drugs rescued the pathology of the Sodn1(
/
) flies, then their proportion among the emerging flies would increase (antioxidant effects) or, if the drugs were toxic, then the absolute numbers of both genotypes of flies emerging from the drug cultures would decline with drug dose over some range of concentrations. Further experiments were carried out to determine whether the antioxidant drugs could rescue the short lifespan of either the Sodn1(
/
) or daGal4/ MnSOD-RNAi flies. The drugs were administered to the flies starting from the larval stage (larval exposure) or only during adulthood (adult exposure). Experimental flies were generated from parental crosses as described earlier. After eclosion from pupae, experimental flies were sorted into female and male groups and allocated to 20 ml vials containing the drug and food medium at a density of 10 flies per vial (20 flies per vial for the daGal4/MnSOD-RNAi flies). The flies were transferred to fresh food once after every 2 days and deaths were scored daily.

2.2. Experimental setup

2.5. Testing whether the drugs protect against paraquat-induced oxidative stress

Larval exposure experiments were those in which drug administration started from the larval stage and continued throughout adulthood. The experiments were set up by first collecting eggs from the parental crosses on grape agar plates seeded with live yeast during a 6-h period and allowing them to hatch for 24 h at 25 8C. Groups of 50 first instar larvae from the grape agar plates were isolated using a mounted needle under the microscope and placed in 20 ml vials of SY (1% agar, 10% sucrose–yeast, 2.5% nipagin) or ASG (1% agar, 8.5% sucrose, 2% yeast, 6% maize meal and 2.5% nipagin) food medium containing the drug, at a density of 50 larvae/vial. Control larvae were cultured in parallel in food vials containing only drug vehicle. For adult exposure experiments, larvae were similarly cultured on food medium containing only drug vehicle as for control, and adult flies were exposed to the drug after eclosion. All flies were

Doses of the antioxidant drugs ranging from 0.05 to 1.0 mM were used to test whether they could protect flies against paraquat-induced oxidative stress. These drug concentrations were chosen on the basis that they did not exhibit any severe larval toxicity and therefore did not affect fly development. In the first of two experimental cohorts for each drug, flies were cultured from larvae on food medium containing drug as described earlier while, in the second cohort, flies were fed the drugs only after eclosion. Flies were transferred to SY medium plus drug (ASG medium for the daGal4/MnSOD-RNAi flies) containing either 15 mM paraquat (wild type Dahomeys) or 0.5 mM paraquat (Sodn1(
/
) and daGal4/MnSOD-RNAi flies) 24 h after eclosion. A milder dose of paraquat was chosen for the SOD-deficient flies because of their extreme sensitivity to the compound, allowing lifespan effects to be assayed over a larger number of days

T. Magwere et al. / Mechanisms of Ageing and Development 127 (2006) 356–370 compared to higher doses that could kill the flies in a matter of hours. Control flies for each genotype were simultaneously placed on food containing paraquat and the drug vehicle. The flies were transferred to fresh vials of paraquat and drug every other day and deaths were scored daily.

2.6. Testing the effects of drugs on larval development and lifespan of adult wild type flies EUK-8, EUK-134, and MitoQ were tested to determine whether they could increase the lifespan of adult wild type Dahomey flies when given from the larval stage or only during adulthood. The flies were cultured as described above and experiments were carried out in vials containing 20 flies each. Transfer to fresh food was carried out once every 2 days and deaths were scored daily.

2.7. Statistical analyses The Kaplan–Meier method was utilised to generate survival curves for all survival experiments using the JMPTM Statistics program (SAS Institute Inc., Cary, NC). Differences in survival between treatment groups were compared using the non-parametric Log-Rank test, where p < 0.05 indicates a significant difference between experimental and control groups. The x2-test (two-way contingency table) was used to determine whether the proportions of heterozygous and homozygous Sodn1 flies emerging from drug-treated food were significantly different from those emerging from non-drug-treated (control) food. The t-test was used to determine whether the numbers of adult flies eclosing at each drug concentration were significantly different from control; a value of p < 0.05 was considered statistically significant.

3. Results 3.1. Effects of antioxidant drugs on larval viability of SOD-deficient and wild type flies The drugs (Euk-8 and MitoQ) were first tested to determine whether they could rescue the pre-adult viability of Sodn1(
/
)

359

flies. The Sodn1(
/
) flies are characterised by inability to protect against ROS-induced damage during early development (Phillips et al., 1989) and many die during the process of eclosion. The two questions we needed to answer therefore were as follows: (1) does the absolute number of heterozygous or homozygous flies decrease over a range of drug concentrations? If it does, this would indicate that the drugs were toxic. (2) Does the proportion of Sodn1(
/
) flies among the eclosing adults increase, decrease, or stay the same, with increasing drug concentration? If the proportion of Sodn1(
/
) flies increases then this would be evidence of protective effects; or if it decreases, this would be an indication of toxic effects. The t-test was used to determine whether the absolute numbers of heterozygous or homozygous flies decreased significantly over a range of drug concentrations (answer to question 1), and twoway contingency tables (2  2 x2-test) were used to test whether the proportion of Sodn1(
/
) flies at each drug concentration different from that of control (answer to question 2). We first looked at the absolute numbers of the two Sodn1 genotypes eclosing from the drug cultures to determine whether the drug was protective or toxic at any dose for either genotype (Table 1). The absolute numbers of heterozygous and homozygous flies eclosing from the Euk-8 cultures (Table 1: Trials 1 and 2) were not significantly affected by concentrations of up to 1 mM. The numbers of both genotypes however decreased significantly ( p < 0.001 versus controls) for Euk-8 concentrations above 2.0 mM, indicating that the drug was toxic at high concentrations. The results for MitoQ (Table 1) show that the absolute numbers of both heterozygous and homozygous flies were also significantly affected by increasing concentrations of MitoQ. The numbers of heterozygous flies

Table 1 The absolute numbers of Sodn1(
/TM3) and Sodn1(
/
) flies emerging from larval treatments with Euk-8 and MitoQ Drug

Sodn1(
/TM3) a

Sodn1(
/
)

Number

( p vs. Control 1)

Euk-8 Trial 1 Control 1 Control 2 0.025 mM 0.05 mM 0.5 mM

27  5 26  3 27  4 28  6 29  2

– 0.6442 0.4614 0.7086 0.8730

0.6442 – 0.6189 0.8237 0.9803

Trial 2 Control 1 Control 2 1.0 mM 2.0 mM 3.0 mM

22  6 22  3 22  4 22  5 14  3

0.5173 0.4836 0.4841 0.0010*

0.5173 – 0.5000 0.5000 <0.0001*

27  5 24  3 23  5 26  4 26  3 19  4

– 0.9209 0.0470 0.3422 0.2340 0.0009*

0.9209 – 0.2787 0.8677 0.8626 0.0037*

MitoQ Control 1 Control 2 0.05 mM 0.1 mM 1.0 mM 2.5 mM a *

( p vs. Control 2)

Numbera

82 93 93 83 73

( p vs. Control 1)

( p vs. Control 2)

– 0.0737 0.8618 0.5695 0.3697

0.0737 – 0.4184 0.1080 0.0539

62 74 73 21 1  0.7

– 0.1845 0.6888 <0.0001* <0.0001*

0.1845 – 0.3009 0.0009* 0.0002*

52 52 62 32 0 0

– 0.5000 0.6622 0.0372* <0.0001* <0.0001*

0.5000 – 0.6984 0.0182* <0.0001* <0.0001*

This is the number of adult flies obtained from the drug cultures out of 50 larvae. Values are expressed as expressed as mean  S.D. for N = 10 replicates. Significantly different from Control 1 or 2 with p-value as shown.

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T. Magwere et al. / Mechanisms of Ageing and Development 127 (2006) 356–370

Table 2 The proportions of Sodn1(
/TM3) and Sodn1(
/
) flies emerging from larval treatment with Euk-8 and MitoQ Treatment

Number of adults

x2( p vs. Control 1)

x2( p vs. Control 2)

Sodn1(
/TM3)

Sodn1(
/
)

Euk-8 Trial 1 Control 1 Control 2 0.025 mM 0.05 mM 0.5 mM

269 262 269 283 290

76 89 99 78 72

– 1.0648(0.3021) 2.2833 (0.1307) 0.0184(0.8920) 0.4885 (0.4846)

1.0648(0.3021) – 0.2224 (0.6372) 1.3936(0.2378) 3.0463 (0.0809)

Trial 2 a Control 1 Control 2 1.0 mM 2.0 mM 3.0 mM

224 222 231 223 140

75 74 66 19 6

– 0.0005 (0.9812) 0.6755(0.4111) 27.666 (0.0000)* 28.984 (0.0000)*

0.0005 (0.9812) – 0.6343 (0.4258) 27.385 (0.0000)* 28.785 (0.0000)*

273 246 238 262 257 197

51 51 57 31 0 0

– 0.2311 (0.6307) 1.3750(0.2410) 3.5557(0.0593) 44.346 (0.0000)* 34.374 (0.0000)*

0.2311 (0.6307) – 0.4588 (0.4982) 5.3551 (0.0207)* 48.606 (0.0000)* 37.723 (0.0000)*

MitoQ Control 1 Control 2 0.05 mM 0.1 mM 1.0 mM 2.5 mM a *

Sodn1(
/TM3) parental stock out-crossed once to wild type Dahomey. Value significantly different from control at p-value shown.

decreased significantly for 2.5 mM MitoQ ( p = 0.0009 and 0.0037 versus Control 1 and 2, respectively); whereas the numbers of homozygous flies were depleted severely at concentrations of 0.1 mM and higher ( p < 0.04 versus both controls) indicating a higher degree of toxicity. Taken together, the results for both Euk-8 and MitoQ show that for both genotypes, there was no noticeable effect of the drug up to a threshold dosage above which the drug was toxic. The threshold dose at which the drugs were toxic was always lower for the homozygous larvae (Table 1). We then carried out further experiments using the Sodn1 flies to determine whether there was differential toxicity of the drugs on the two genotypes of flies (Table 2). The results for Euk-8 Trials 1 and 2 (Table 2) show that the proportion of Sodn1(
/
) flies among the eclosing flies was not affected by drug concentration up to a concentration of 1 mM (x2 = 0.6755, p = 0.4111 versus Control 1 and x2 = 0.6343, p = 0.4258 versus Control 2). However, the proportion of Sodn1(
/
) flies decreased significantly for concentrations above 2 mM Euk-8 (Table 1: x2 > 27.000, p = 0.0000 versus both Control 1 and 2) indicating that the drug was toxic to Sodn1(
/
) larvae. MitoQ was also very toxic to Sodn1(
/
) larvae and the proportion of this genotype decreased significantly for concentrations as low as 0.1 mM (x2 = 5.3551, p = 0.0207 versus Control 2); and virtually no Sodn1(
/
) flies emerged at concentrations above 1.0 mM. In summary, neither Euk-8 nor MitoQ appeared able to significantly improve the pre-adult viability of the two genotypes and, at all doses at which any toxic effects were apparent, the drug was more toxic to the homozygous larvae (Table 2). The effects of Euk-8 and MitoQ on the pre-adult development of daGal4/MnSOD-RNAi larvae were also tested

to assess their toxicity. The daGal4/MnSOD-RNAi flies have no distinguishable larval phenotype (Kirby et al., 2002); nevertheless larval toxicity studies were still carried out to ascertain whether the drugs could affect the total number of flies and/or the relative proportions of female and male flies emerging from the drug cultures. This information could be useful in revealing sex-specific effects of the drugs. The total numbers of adult daGal4/MnSOD-RNAi flies as well as the numbers of each sex were counted from the drug cultures and the results are shown in Figs. 1 and 2. Euk-8 was not significantly toxic to daGal4/ MnSOD-RNAi larvae at concentrations up to 1 mM (Fig. 1), and the relative proportions of male and female flies were not altered. Similarly, MitoQ did not exhibit any larval toxicity

Fig. 1. The effect of Euk-8 on development of daGal4/MnSOD-RNAi larvae. The numbers of adult flies on the vertical axis also represent the number of larvae that developed to adulthood out of 50 larvae placed in 20 ml vials of ASG food containing Euk-8. Control larvae were placed on food containing only drug vehicle. Each bar is mean  S.D. for N = 10 separate experiments.

T. Magwere et al. / Mechanisms of Ageing and Development 127 (2006) 356–370

Fig. 2. The effect of MitoQ and TPMP on development of daGal4/MnSODRNAi larvae. The numbers of adult flies on the vertical axis also represent the number of larvae that developed to adulthood out of 50 larvae placed in 20 ml vials of ASG food containing the drugs. Control larvae were placed on food containing only drug vehicle. (a) Total numbers of flies; (b) numbers of female flies; (c) numbers of male flies. Each bar is mean  S.D. for N = 10 separate experiments. *Number of females flies significantly different from MitoQ ( p < 0.05) and Control ( p < 0.0001).

(Fig. 2a–c) and it also did not affect the relative numbers of male and females flies over a 1000-fold drug concentration. However, TPMP showed significant toxicity at 1 mM concentration as total number of adult flies decreased by close to 50% (Fig. 2a: p < 0.05 versus MitoQ and p = 0.0001 versus ethanol Control). TPMP also appeared to significantly affect the development of only female flies (Fig. 2b: p < 0.0001 versus both MitoQ and Control) but had no significant effects on male flies (Fig. 2c). These results suggest that MitoQ itself did not have any severe effects on larval development up to concentrations of 1 mM, and that both MitoQ and Euk-8 could be used to assay for lifespan effects up to this concentration. MitoQ was also tested for toxicity to larvae of wild type D. melanogaster and the effects were compared to those of TPMP

361

Fig. 3. The effect of MitoQ and TPMP on development of wild type D. melanogaster larvae. The numbers of adult flies on the vertical axis also represent the number of larvae that developed to adulthood out of 50 larvae placed in 20 ml vials of 10% SY food containing the drugs. Control larvae were placed on SY food containing only drug vehicle. (a) Total numbers of flies; (b) numbers of female flies; (c) numbers of male flies. Each bar is mean  S.D. for N = 3–5 separate experiments. *Number of female flies significantly different from Control ( p < 0.0001).

and ethanol Control (Fig. 3). About 80% of larvae emerged as adult flies at all drug concentrations of either MitoQ or TPMP (Fig. 3a). However, sex-specific effects of TPMP were seen in females (Fig. 3b) where the numbers of female flies were significantly reduced ( p < 0.05) compared to those from control conditions, and also relative to the numbers of male flies (Fig. 3c). The sex-specific toxic effects of TPMP to wild type D. melanogaster larvae were similar to those observed earlier for daGal4/MnSOD-RNAi larvae. 3.2. Effects of antioxidant drugs on adult lifespan of SODdeficient flies To test whether Euk-8 or MitoQ could rescue the short lifespan of the daGal4/MnSOD-RNAi flies, flies were fed the

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T. Magwere et al. / Mechanisms of Ageing and Development 127 (2006) 356–370

Fig. 4. Survival curves for female and male daGal4/MnSOD-RNAi flies fed Euk-8 from larval and adult stages. The numbers of flies in each experiment were: larval exposures — females: Control (76), 0.01 mM (100), 0.1 mM (97), 1.0 mM (91); males: Control (86), 0.01 mM (98), 0.1 mM (100), 1.0 mM (96); adult exposures — females: Control (98), 0.1 mM (96), 1 mM (96), and 10.0 mM (99); males: Control (100), 0.1 mM (95), 1.0 mM (99), and 10.0 mM (99).

drugs throughout life starting from either the larval (developmental) stage or from adulthood and the results are presented in Figs. 4–6. For female daGal4/MnSOD-RNAi flies fed Euk-8 starting from the larval stage (Fig. 4) low drug concentrations had no significant effect on lifespan; however median lifespan increased significantly at 1 mM Euk-8 (10 days: 95% confidence interval 10–11; x2 = 16.54, p < 0.0001) compared to control (8 days: 7–9). The effects of feeding Euk-8 from larvae were however different for male flies: all doses of Euk-8 had no significant effect on male lifespan from 0.01 up to 1 mM (Fig. 4). When Euk-8 was fed to adult daGal4/MnSOD-RNAi flies (Fig. 4), female lifespan was significantly increased at 0.1 mM (8 days: 8–9; x2 = 11.43, p < 0.0007) and 1 mM (8 days: 8–9; x2 = 16.14, p < 0.0001), but decreased dramatically when the dose was increased to 10 mM (3 days: 3–3; x2 = 92.88, p = 0.0001) compared to control (8 days: 7–8).

For adult male daGal4/MnSOD-RNAi flies, lifespan showed only a dose-dependent decrease with an increase in Euk-8 concentration up to 10 mM (3 days: 2–3; x2 = 135.73, p < 0.0001) compared to control (4 days: 4, 4). Taken together these results indicate that Euk-8 increased the lifespan of only female daGal4/MnSOD-RNAi flies at particular concentrations above which the drug became toxic. The effects of Euk-8 on lifespan also differed between flies fed from eclosion and those fed from the larval stage of development. The effects of feeding MitoQ and TPMP on adult lifespan of daGal4/MnSOD-RNAi flies are shown in Figs. 5 and 6. Female flies fed MitoQ from larvae showed significant dose-dependent increases in lifespan as drug doses were increased, an effect that was also mirrored in males (Fig. 5). Median lifespan was significantly higher for females fed MitoQ from larvae at 0.01 mM (8 days: 7–8; x2 = 14.42, p < 0.0001), 0.1 mM (8 days: 7–8; x2 = 4.47, p = 0.03), and 1.0 mM (11 days: 10–11;

Fig. 5. Survival curves for female and male daGal4/MnSOD-RNAi flies fed MitoQ from larval and adult stages. The numbers of flies in each experiment were: larval exposures — females: Control (97), 0.01 mM (99), 0.1 mM (98), 1.0 mM (97); males: Control (93), 0.01 mM (92), 0.1 mM (75), 1.0 mM (68); adult exposures — females: Control (100), 0.1 mM (80), 1 mM (60), and 10.0 mM (60); males: Control (100), 0.1 mM (80), 1.0 mM (80), and 10.0 mM (80).

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Fig. 6. Survival curves for female and male daGal4/MnSOD-RNAi flies fed TPMP from larval and adult stages. The numbers of flies in each experiment were: larval exposures — females: Control (96), 0.01 mM (96), 0.1 mM (94), 1.0 mM (20); males: Control (92), 0.01 mM (92), 0.1 mM (93), 1.0 mM (72); adult exposures — females: Control (96), 0.1 mM (100), 1 mM (99), and 10.0 mM (98); males: Control (86), 0.1 mM (93), 1.0 mM (91), and 10.0 mM (100).

x2 = 105, p < 0.0001) compared to control median lifespan of 7 days (6–7). For male flies, median lifespan was significantly higher for 0.01 mM MitoQ (5 days: 5-5; x2 = 8.84, p = 0.003) and for 1.0 mM (6 days: 5–7; x2 = 17.77, p < 0.0001) compared to controls (5 days: 4–5). The effects of TPMP on fly lifespan when fed from larvae (Fig. 6) were identical to those of MitoQ, in that increasing the drug dose significantly increased the lifespan of either female or male flies. Median lifespan increased for female flies fed 0.1 mM TPMP (8 days: 7–8; x2 = 11.61, p = 0.0007) and 1.0 mM TPMP (8 days: 7–10; x2 = 11.61, p = 0.0007) compared to control lifespan (7 days: 6–7) (Fig. 6). Since TPMP does not have antioxidant function, the similarities between the effects of TPMP and MitoQ imply that the increase in lifespan shown for MitoQ were not due to its antioxidant effects. When fed to daGal4/MnSOD-RNAi flies from adulthood, MitoQ had different effects on lifespans of female and male flies (Fig. 5). The highest dose (10 mM MitoQ) increased female lifespan by 25% (10 days: 95% confidence interval 9–11 versus control 8 days: 7–9; x2 = 32.20, p < 0.0001). However when equivalent doses of MitoQ were fed to male daGal4/ MnSOD-RNAi adults (Fig. 5), toxic effects were evident as median lifespan was significantly decreased by 0.1 mM (4 days: 4–5; x2 = 19.88, p < 0.0001) and 1.0 mM MitoQ (4 days: 4-4; x2 = 13.80, p = 0.0002). TPMP given to daGal4/MnSODRNAi adult females (Fig. 6) significantly increased median lifespan at an intermediate dose of 1.0 mM (9 days: 8–10; x2 = 5.40, p = 0.02), but the high dose of 10 mM TPMP dramatically decreased their median lifespan by nearly 40% (5 days: 5–6; x2 = 74.43, p < 0.0001) compared to controls (8 days: 8–9). When fed to male daGal4/MnSOD-RNAi flies, doses of 0.01 mM and 1.0 mM TPMP had no significant effect on lifespan (Fig. 6) but the highest dose of 10 mM significantly decreased lifespan (4 days: 4–4; x2 = 26.75, p < 0.0001) versus control (5 days: 5–5). In summary, only MitoQ increased the lifespan of female daGal4/MnSOD-RNAi flies whereas TPMP was toxic. These results suggest that MitoQ may have an antioxidant effect when fed to adult female, but not male flies.

Furthermore, the results again show that there was an intermediate dose of MitoQ that was sufficient to improve survival of the female flies above which toxic effects were seen. 4. Effects of antioxidant drugs on paraquat sensitivity of wild type and SOD-deficient flies To test whether the antioxidant drugs could rescue the paraquat sensitivity of wild type D. melanogaster and SODdeficient mutants, paraquat was given to the flies mixed in the food containing the antioxidant drugs. Paraquat exerts toxicity through a redox-cycling mechanism that produces superoxide and from this other ROS in vivo (Bus and Gibson, 1984), and has been used successfully by other workers to induce oxidative stress in different organisms (Keaney et al., 2004; Phillips et al., 1989). 4.1. Wild type Dahomeys The effects of Euk-8 and -134 on paraquat sensitivity of the wild type Dahomey and Sodn1(
/
) flies are shown in Table 3. Adult Dahomey flies cultured as larvae on 0.05 mM EUK-8 were not protected from the harmful effects of the radicals generated by 15 mM paraquat (Table 3). The median lifespan of flies reared on 0.05 mM Euk-8 from larvae was significantly reduced compared to controls ( p < 0.0001 compared to both Control 1 and 2). A higher dose of 0.25 mM Euk-8 also did not protect the flies whether fed from larval stage or during adulthood (Table 3). It can be seen further that Dahomey flies given Euk-8 were also significantly more vulnerable to the effects of paraquat ( p < 0.0001 compared to Control 1 and 2 for both groups). Similar doses of Euk-134 also did not protect wild type flies from the harmful effects of paraquat (Table 3) irrespective of whether the drug was fed to the flies from larval stage or from adulthood. As seen for Euk-8, the high dose of Euk-134 decreased the survival of drug-fed flies compared to controls ( p < 0.0001). In summary, neither Euk-8 nor Euk-134 could rescue the paraquat sensitivity of wild type flies and each

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Table 3 Effect of Euk-8 and -134 on lifespan of wild type Dahomeys and Sodn1(
/
) flies stressed with paraquat Strain (life stage/ gender)a

Treatment, mM salen manganese compoundb

Mean lifespan  S.E.

Maximum lifespan

Nc

p* (treatment vs. Control 1)

p* (treatment vs. Control 2)

EUK-8 Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [A/f] Dahomey [A/f] Dahomey [A/f] Sodn1(
/
)[L/f] Sodn1(
/
)[L/f] Sodn1(
/
)[L/f] Sodn1(
/
)[L/f] Sodn1(
/
)[L/f] Sodn1(
/
)[L/f] Sodn1(
/
)[A/f] Sodn1(
/
)[A/f] Sodn1(
/
)[A/f]

CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY

0 (Control 1) 0 mM (Control 2) 0.05 0 (Control 1) 0 (Control 2) 0.25 0 (Control 1) 0 (Control 2) 0.25 0 (Control 1) 0 (Control 2) 0.05 0 (Control 1) 0 (Control 2) 0.25 0 (Control 1) 0 (Control 2) 0.25

13.2  0.3 13.4  0.3 11.0  0.2 14.2  0.4 13.6  0.4 7.9  0.05 13.3  0.4 12.5  0.3 10.8  0.1 1.9  0.1 2.0  0.1 2.8  0.1 1.9  0.1 2.0  0.1 2.8  0.1 1.9  0.1 2.0  0.1 1.9  0.1

21 21 14 20 22 8 25 25 16 4 3 6 4 3 7 4 3 4

120 124 111 118 119 120 102 123 124 113 118 119 113 118 120 113 118 114

– 0.8401 <0.0001 – 0.5061 <0.0001 – 0.1333 <0.0001 – 0.4818 <0.0001 – 0.4818 <0.0001 – 0.4818 0.9079

0.8401 – <0.0001 0.5061 – <0.0001 0.1333 – <0.0001 0.4818 – <0.0001 0.4818 – <0.0001 0.4818 – 0.3905

EUK-134 Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [L/f] Dahomey [A/f] Dahomey [A/f] Dahomey [A/f] Sodn1(
/
) [Lf] Sodn1(
/
) [L/f] Sodn1(
/
) [L/f] Sodn1(
/
) [L/f] Sodn1(
/
) [L/f] Sodn1(
/
) [L/f] Sodn1(
/
) [A/f] Sodn1(
/
) [A/f] Sodn1(
/
) [A/f]

CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY CSY

0 (Control 0 (Control 0.05 0 (Control 0 (Control 0.25 0 (Control 0 (Control 0.25 0 (Control 0 (Control 0.05 0 (Control 0 (Control 0.25 0 (Control 0 (Control 0.25

16  0.5 17.3  0.4 17.3  0.4 16  0.5 17.3  0.4 9  0.3 16  0.5 17.3  0.4 10.4  0.5 1.9  0.1 2.0  0.1 2.4  0.1 1.9  0.1 2.0  0.1 2.8  0.1 1.9  0.1 2.0  0.1 1.9  0.1

23 25 21 23 25 14 23 25 21 4 3 5 4 3 7 4 3 4

120 121 115 120 121 124 120 121 123 113 118 110 113 118 115 113 118 120

– 0.0504 0.1954 – 0.0504 <0.0001 – 0.0504 <0.0001 – 0.4818 <0.0001 – 0.4818 <0.0001 – 0.4818 0.8463

0.0504 – 0.6281 0.0504 – <0.0001 0.0504 – <0.0001 0.4818 – 0.0008 0.4818 – <0.0001 0.4818 – 0.357

1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2)

a

wt: wild type; A: adults; L: larvae; f: female. Drugs were added to concentrated sugar-yeast (CSY) food media. The concentrations of paraquat in the food media were 15 mM for the Dahomeys and 0.5 mM forthe Sodn1(
/
) flies. c N: number of deaths scored. * p: probability of survival of Euk-8 or Euk-134-treated flies being the same as untreated controls (log-rank test, p < 0.05). b

drug appeared to act synergistically with paraquat to decrease lifespan. 4.2. Homozygous Sodn1(
/
) flies The effects of Euk-8 and -134 on the paraquat sensitivity of Sodn1(
/
) flies (Table 3) were very different from wild type Dahomey flies. Sodn1(
/
) flies reared as larvae and maintained on food containing 0.05 mM Euk-8 throughout adulthood had significantly increased survival compared to controls ( p < 0.0001) following exposure to 0.5 mM paraquat. This was also true for flies reared and kept on 0.25 mM Euk-8 (Table 3, p < 0.0001). However, flies that had been fed 0.25 mM Euk-8 as adults only were not protected from the effects of paraquat, showing survivorship similar to that of the

controls. In summary, both Euk-8 and -134 improved the tolerance of Sodn1(
/
) flies to paraquat toxicity. Furthermore, the developmental stage at which the antioxidant drug was given seemed important, as significant resistance to paraquat only occurred in Sodn1(
/
) flies given the superoxide dismutase/catalase mimetics (SCMs) from larval stage. 4.3. daGal4/MnSOD-RNAi flies In experiments involving the SCMs, we determined the effects of only Euk-8 since both Euk-8 and -134 have been demonstrated to exhibit similar SOD activities (Doctrow et al., 2002). The daGal4/MnSOD-RNAi flies were very sensitive to the toxic effects of paraquat and survival was dramatically reduced to 2–5 days (Fig. 7) compared to a normal average of

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365

Fig. 7. Survivorship under 0.5 mM paraquat of female and male daGal4/MnSOD-RNAi flies fed 1 mM Euk-8, 1 mM MitoQ, and 1 mM TPMP from larval and adult stages. For all graphs, open triangles = Larval exposure; open squares = adult exposure; open circles = controls. The numbers of flies in each experiment were: Euk-8 — females: Control (120), larval treatment (112), and adult treatment (102); males: Control (94), larval treatment (113), and adult treatment (99); MitoQ — females: Control (117), larval treatment (129), and adult treatment (115); males: Control (120), larval treatment (135), and adult treatment (117); TPMP — females: Control (109), larval treatment (45), and adult treatment (97); males: Control (108), larval treatment (54), and adult treatment (116).

about 7–8 days for control flies (compare lifespan with Figs. 4– 6). For female daGal4/MnSOD-RNAi flies fed Euk-8 only as adults (Fig. 7) the sensitivity to paraquat, survival (median lifespan 2 days: 95% CI 2–3 and was not significantly different from that of controls (2 days: 2–2). However, females fed 1 mM Euk-8 from the larval stage had significantly higher median survival to paraquat toxicity compared to the other two groups (4 days: 2–4, x2 = 17.3, p < 0.0001 versus control; and x2 = 11.42, p = 0.0007 versus adult exposure). Male daGal4/ MnSOD-RNAi flies also showed a similar pattern and indeed sensitivity to paraquat was neither improved nor worsened by Euk-8 given as adults (2 days: 2–2) compared to controls (2 days: 2–3). However, resistance to paraquat toxicity improved significantly in flies fed 1 mM Euk-8 from larval stage (4 days: 2–4; x2 = 21.26, p < 0.0001) compared to adult exposure. Taken together, these results indicate that Euk-8 enhanced the survival of daGal4/MnSOD-RNAi flies under paraquat challenge; however as for the Sodn1(
/
) mutants, significant protection was only achieved when the drug was given to the flies starting from the larval stage. MitoQ was more protective to daGal4/MnSOD-RNAi flies against paraquat stress when fed from larval stage compared to when the drug was fed only from adulthood (Fig. 7). Female daGal4/MnSOD-RNAi flies fed 1 mM MitoQ from larval stage had significantly enhanced paraquat survival (8 days: 8–11) compared to either control (4 days: 2–6; x2 = 112.10, p < 0.0001) or adult exposure (5 days: 3–5; x2 = 120.0, p < 0.0001). Male flies fed MitoQ from larval stage also had significantly higher median survival under paraquat challenge (5 days: 5-5) compared to either control (5 days: 4–5; x2 = 14.62, p = 0.0001) or adult exposure (4 days: 3–4; x2 = 18.15, p < 0.0001). The paraquat sensitivity of either

female or males flies fed MitoQ from adulthood did not significantly differ from that of the controls (Fig. 7). Treatment with TPMP (Fig. 7) from the larval stage was also more protective to daGal4/MnSOD-RNAi flies compared to when flies were only fed TPMP during adulthood. Females fed 1 mM TPMP from larval stage had more than twice the survival of controls under 0.5 mM paraquat challenge (3 days: 3–4 for control versus 8 days: 8–11 for 1 mM TPMP; x2 = 62.45, p < 0.0001). For male flies fed TPMP from larvae (Fig. 7), survival time under 0.5 mM paraquat was 5 days (5–8) versus control 4 days (3–4), x2 = 37.68, p < 0.0001). The sensitivity to paraquat of female or male flies fed TPMP only as adults did not differ significantly from that of controls. In summary, MitoQ and TPMP both increased paraquat survival of daGal4/ MnSOD-RNAi flies but only when given from larvae. However, the similarities between the effects of MitoQ and TPMP may suggest that the observed effects may not have been due to antioxidant action but through alternative mechanisms. 5. Effects of drugs on lifespan of wild type flies Euk-8, Euk-134, and MitoQ were fed to wild type Dahomey flies to find out whether they could increase lifespan and their effects are shown in Table 4. Euk-8 (Trials 1–4), showed a dosedependent increase in toxicity as drug concentration was increased; and these toxic effects were evident regardless of whether the drug was fed from larval stage or from adulthood (Table 4). The toxicity of Euk-8 was similar in both once mated (Trials 1–4) and virgin (Trial 5) flies; and changing the ambient temperature (from 25 to 27 8C) did not alter the severity of the toxicity. The effects of Euk-134 (Table 4) were similar to those of Euk-8. From these results it can be concluded that the two

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Table 4 Effect of Euk-8 and -134 on wild type Dahomey lifespan Strain (life stage/ gender)a

Treatment, mM salen-manganese compound

Mean lifespan  S.E.

Maximum lifespan

Nb

p* (treatment vs. Control 1)

EUK-8 Trial 1 (27 8C) wt [A/f] wt [A/f] wt [A/f] wt [A/f]

CSY CSY CSY CSY

0 mM 0.001 mM 0.01 mM 0.1 mM

20.0  0.5 18.7  0.6 18.9  0.7 19.7  0.6

44 41 44 41

124 127 136 129

– 0.1981 0.2296 0.8911

Trial 2 (27 8C) wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f]

CSY CSY CSY CSY CSY CSY

0 mM (Control 1) 0 mM (Control 2) 0.05 mM 0.5 mM 5 mM 10 mM

19.0  0.9 21.0  0.9 17.9  0.8 12.2  0.6 10.2  0.4 7.7  0.2

43 46 43 38 25 25

116 114 116 117 114 116

– 0.1702 0.459 <0.0001 <0.0001 <0.0001

0.1702 – 0.0252 <0.0001 <0.0001 <0.0001

Trial 3 (25 8C) wt [L/f] wt [L/f] wt [L/f] wt [L/f] wt [L/f] wt [L/f]

CSY CSY CSY CSY CSY CSY

0 mM (Control 1) 0 mM (Control 2) 0.05 mM 0.5 mMc 5 mMc 10 mMc

15.2  0.9 12.2  0.6 12.7  0.6 – – –

55 50 50 – – –

131 145 141 – – –

– 0.006 0.0386 – – –

0.006 – 0.4441 – – –

Trial 4 (25 8C) wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f]

CSY CSY CSY CSY CSY CSY CSY

0 mM (Control 1) 0 mM (Control 2) 0.01 mM 0.025 mM 0.05 mM 0.1 mM 0.25 mM

41.6  0.6 40.3  0.6 38.1  0.7 39.6  0.6 39.8  0.7 39.2  0.6 39.8  0.6

60 56 58 60 60 60 53

196 188 185 194 192 198 186

– 0.0194 <0.0001 0.0161 0.1627 <0.0001 0.001

0.0194 – 0.0366 0.6945 0.4116 0.0836 0.3185

Trial 5 (Virgin females, 25 8C) wt [A/f] CSY 0 mM (Control 1) wt [A/f] CSY 0 mM (Control 2) wt [A/f] CSY 0.05 mM wt [A/f] CSY 0.5 mM wt [A/f] CSY 5.0 mM

48.9  0.4 49.0  0.2 46.8  0.3 44.5  0.2 4.0  0.5

66 62 61 59 5

171 168 172 181 199

– 0.626 0.0062 <0.0001 <0.0001

0.626 – 0.0010 <0.0001 <0.0001

EUK-134 Trial 1 (27 8C) wt [A/f] wt [A/f] wt [A/f] wt [A/f]

CSY CSY CSY CSY

0 mM 0.0001 mM 0.001 mM 0.05 mM

20.0  0.5 18.9  0.6 20.0  0.6 19.2  0.6

44 37 37 44

124 134 131 128

0.3925 0.7541 0.7541 0.5081

Trial 2 (25 8C) wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f] wt [A/f]

CSY CSY CSY CSY CSY CSY CSY

0 mM (Control 1) 0 mM (Control 2) 0.025 mM 0.05 mM 0.1 mM 0.25 mM 0.5 mM

44.0  0.7 43.0  0.7 39.9  0.6 42.7  0.6 43.2  0.8 40.1  0.8 38.8  0.9

65 63 52 58 65 60 65

187 188 193 195 194 190 191

– 0.2961 <0.0001 0.007 0.9373 0.0003 <0.0001

a b c *

p* (treatment vs. Control 2)

– – – –

– – – – 0.2961 – <0.0001 0.0544 0.2846 0.007 0.001

wt: wild type; A: adults; L: larvae; f: female; CSY: concentrated sugar-yeast fly food. N: number of deaths scored. Only 27 adults emerged on 0.5 mM EUK-8; no adults emerged at 5 mM and10 mM EUK-8. p: Probability of survival of antioxidant-treated flies being the same as untreated controls (log-rank test, p < 0.05).

drugs did not extend the lifespan of adult wild type flies, but reduced it in a dose-dependent manner. MitoQ produced a dose-dependent decrease in lifespan of wild type D. melanogaster when fed during the larval stage or during adulthood (Fig. 8). Lifespan was significantly greater for

female larval exposure controls at 62 days (upper–lower 95% confidence interval: 55–65) and decreased to 58 days: 54–62 (x2 = 4.77, p = 0.029) for 0.01 mM MitoQ; 54 days: 51–58 (x2 = 4.46, p = 0.035) for 0.1 mM MitoQ; 44 days: 37–44 (x2 = 64.76, p < 0.0001) for 1 mM MitoQ. For female flies fed

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Fig. 8. Survival curves for female and male Dahomey flies fed the drug MitoQ from larval and adult stages as described in the text. The numbers of flies in each experiment were: larval exposure — females: Control (85), 0.01 mM (39), 0.1 mM (58), 1.0 mM (42); males: Control (55), 0.01 mM (80), 0.1 mM (87), 1.0 mM (88); adult exposure — females: Control (172), 0.001 mM (187), 0.01 mM (182), 0.1 mM (192), and 1.0 mM (176); males: Control (188), 0.001 mM (190), 0.01 mM (182), 0.1 mM (178), and 1.0 mM (177).

MitoQ from adulthood, median lifespan was significantly decreased for 1 mM MitoQ (44 days: 37–45; x2 = 36.34, p < 0.0001) compared to control (54 days: 52–58). Male flies were less responsive to the effects of MitoQ and the statistical significance for the changes in median lifespan were less compared to females. In males treated with MitoQ from the larval period, there were no significant effects of any dose on lifespan (Fig. 8). Lifespan was however significantly lowered for the adult exposure males by 1 mM MitoQ (45 days: 44–46; x2 = 4.79, p = 0.03) compared to control (51 days: 47–52). TPMP fed from the larval stage (Fig. 9) had no significant effect on the flies at lower doses but the toxicity increased

dramatically when drug concentrations were increased to 1 mM. Lifespan for female flies decreased significantly for 1 mM TPMP (52 days: 53–52; x2 = 13.24, p = 0.0003) compared to control lifespan (58 days: 58–62). Male flies were also sensitive to the toxic effects of TPMP (Fig. 9) and lifespan was reduced significantly by 0.1 mM TPMP (42 days: 35–52; x2 = 18.54, p < 0.0001) compared to controls (52 days: 52–52). The effects of MitoQ on lifespan of adult D. melanogaster were very similar to those of TPMP, indicating that both drugs were toxic to flies and the developmental stage from which the drugs were given to the flies did not alter this toxicity. In summary, these results clearly showed that none of the antioxidant drugs could increase the lifespan of adult wild type flies when given from either larval or adult stages of development. In fact, each drug showed dose-dependent increase in toxicity with increasing drug concentration. 6. Discussion

Fig. 9. Survival curves for female and male Dahomey flies fed the drug TPMP from larval stage. The numbers of flies in each experiment were: females: Control (86), 0.01 mM (86), 0.1 mM (85), 1.0 mM (44); males: Control (90), 0.01 mM (95), 0.1 mM (95), 1.0 mM (62).

The major findings of this study are that the SCMs (Euk-8 and -134) and the mitochondria-targeted MitoQ neither increased the lifespan nor rescued the paraquat sensitivity of wild type D. melanogaster; however, the three drugs did significantly increase the lifespan of SOD-deficient flies and improved their tolerance to paraquat stress. Where the antioxidant drugs were shown to significantly increase lifespan in SOD-deficient flies, the effects were sometimes sex-specific and, for either sex, the effects were also variable depending on the stage of development from which the drugs were administered and the magnitude of the drug dose given. The ubiquity of superoxide dismutases in organisms ranging from bacteria (Hassan, 1989), to plants (Alscher et al., 2002), and animals (Fridovich, 1989) is strong support for the damaging role of ROS to life, against which all organisms need protection. And indeed, abrogation of the normal function of SOD through genetic manipulations in organisms such as mice

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(Lebovitz et al., 1996) and fruit flies (Kirby et al., 2002; Phillips et al., 1989) has deleterious effects. Taken in conjunction with the postulates of the free radical theory of ageing, this evidence suggests that increasing the activity and/or concentration of antioxidant defences in vivo might prolong lifespan and/or confer improved resistance to oxidative stress. Yet our results from this study, and those from others (Bayne and Sohal, 2002; Keaney and Gems, 2003) have shown that attempts to increase the activity of this enzyme in vivo through administration of superoxide dismutase/catalase mimetics (SCMs) to wild type animals has life-shortening effects. So far the only evidence that these drugs increase lifespan in wild type animals has come from C. elegans (Melov et al., 2000) but these results have since not been independently reproduced by other workers (Keaney and Gems, 2003). The toxicity shown by the antioxidant drugs to wild type D. melanogaster could imply that antioxidant defences are optimal in this species under normal conditions and any increase in antioxidant activity through administration of drugs may be detrimental possibly through either (1) disruption of cellular-signalling pathways that depend on cellular redox status or (2) toxic effects through interaction with other enzyme systems and/or a disturbance in the intracellular homeostasis. Results from this study, however, do concur with findings by other workers that additional defences may extend lifespan in organisms with compromised antioxidant defences (Melov et al., 2001), or may be needed for survival under stressful conditions (Keaney et al., 2004; Sampayo et al., 2003). Even in these two scenarios it has been demonstrated that the developmental stage at which the drugs were given to the animals may also be important. For example in C. elegans pre-adult exposure to SCMs conferred a greater degree of stress resistance compared to when the drugs were given only during adulthood (Sampayo et al., 2003). In this study we have also shown that drugs given during the larval stage had greater effects on lifespan and paraquat resistance of SOD-deficient flies. However, what is not immediately clear from our studies is whether the enhanced stress resistance through larval exposure was due to antioxidant action of the drugs and not through some other mechanism. A case in point is the similarities in the paraquat resistance of daGal4/MnSOD-RNAi flies given MitoQ and a drug that has no antioxidant function TPMP (Fig. 7). These data suggest that TPMP may not be a suitable control for comparing the effects of MitoQ in vivo. TPMP has severe female-specific larval toxicity in flies (Fig. 2), and there is a greater likelihood of flies that survive this pre-adult challenge becoming more resistant to further stress through either hormetic effects or improved detoxification of paraquat. Foreign compounds are known to induce the activity of drug metabolising enzymes via the xenobiotic responsive elements (Rushmore and Kong, 2002) resulting in increased detoxification of foreign compounds, and it is possible that compounds such as TPMP may have similar effects in vivo. Microarray studies may be useful in identifying which genes are upregulated in flies in response to treatment with compounds such as TPMP. One intriguing observation from our studies was that administration of the antioxidant drugs to mutant (Sodn1(
/
)

or daGal4/MnSOD-RNAi) flies improved their survival, but could not restore lifespan to that of wild type flies. This may suggest that mutations that disrupt normal antioxidant enzymes do not just limit lifespan through oxidative damage, but possibly through introduction of other pathologies that may also be life limiting. A caveat, however, is that the bioavailability of these drugs when given to flies in food is not known; and the feeding rates of the flies given the different drug concentrations were not determined. It has also not yet been assessed whether antioxidant activities in the mutant flies following administration of the drugs could be augmented to the same levels as in wild type flies. In studies where overexpression of either CuZnSOD or MnSOD extended lifespan in flies (Parkes et al., 1998; Sun et al., 2002, 2004), it has been suggested that tissue-specificity of over-expression may be critical for lifespan extension (Landis and Tower, 2005). In this study either the exogenous antioxidants administered may not have raised antioxidant activity to the requisite level, or may not have done so in the appropriate cellular compartment to confer full protection from ROS. In previous studies we have shown that males and females of the same species may be as different from each other as are different species in terms of their response to interventions that affect lifespan (Magwere et al., 2004). This observation has also come up in this study, where a similar dose of the antioxidant drugs showed sex-specific effects on lifespan (see Figs. 4, 5 and 8). This underscores the importance of characterising sexspecific effects of certain interventions, which may be useful in identifying the different mechanisms through which the sexes age. In this study, the sex-specific effects of the antioxidant drugs may be explained in terms of one or all of three possible ways: (1) differences in feeding habits between males and females; more significant effects of the drugs were seen in females possibly because generally they eat more than males, but feeding assays still need to be done to fully confirm this; (2) differences in metabolic rates between males and females, suggesting that levels of oxidative damage may also differ as the sexes age. Perhaps in support of this supposition is the work from Vina and colleagues (2003) who showed that mitochondria from female animals produced significantly less ROS and had lower levels of oxidative damage to mitochondrial DNA than males (Vina et al., 2003); (3) possible physiological differences in the response to changes in intracellular redox status between males and females. Summing up our current results with those in the literature, it can be concluded that testing the effects of exogenous antioxidants in different wild type long-lived organisms so far has failed to provide unequivocal support for the free radical theory of ageing. A common feature in Drosophila and some of the organisms tested to date is that the outcome of experiments testing the biological effects of administered antioxidant drugs may ultimately depend on four different parameters all of which may not be mutually exclusive: (1) the animals species used; (2) the gender of the model organism; (3) the developmental stage from which the drugs are given; (4) the magnitude of the dose. While exogenous antioxidant drugs can help animals that are compromised in antioxidant defence, this

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study has shown that these effects may not be entirely due to their antioxidant properties. Acknowledgements The authors thank the Biotechnological and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), and the Wellcome Trust for funding. Special thanks go to Dr. John P. Philips (University of Guelph, Ontario, Canada) for donating the UAS-Sod2IR15 fly stock; to Dr. Martyn A. Sharpe (Institute of Neurology, London, UK) and Dr. Richard G. Faragher (School of Pharmacy and Biomolecular Science, University of Brighton) for donating the Euk-8 and 134 drugs; and to Dr. Maria E. Giannakou (University College London) for providing the daG32Gal4 driver stock. The authors would also like to thank David Gems, Matthew D. W. Piper, Janne Toivonen, and Jake Jacobson (all University College London) for useful comments on the manuscript; Pedro Martinez-Diaz for technical assistance. We also thank the anonymous reviewers for their insightful and constructive comments. References Adlam, V.J., Harrison, J.C., Porteous, C.M., James, A.M., Smith, R.A.J., Murphy, M.P., Sammut, I.A., 2005. Targeting an antioxidant to mitochondria decreases cardiac ischemia-reperfusion injury. FASEB J. 19, 1088– 1095. Alscher, R.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 53, 1331–1341. Bayne, A.C., Sohal, R.S., 2002. Effects of superoxide dismutase/catalase mimetics on lifespan and oxidative stress resistance in the housefly Musca domestica. Free Radical Biol. Med. 32, 1229–1234. Bevilacqua, L., Ramsey, J.J., Hagopian, K., Weindruch, R., Harper, M.E., 2004. Effects of short- and medium-term calorie restriction on muscle mitochondrial proton leak and reactive oxygen species production. Am. J. Physiol. Endocrinol. Metab. 286, E852–E861. Bus, J.S., Gibson, J.E., 1984. Paraquat: model for oxidant-initiated toxicity. Environ. Health Perspect. 55, 37–46. Chapman, T., Partridge, L., 1996. Female fitness in Drosophila melanogaster: an interaction between the effect of nutrition and of encounter rate with males. Proc. R. Soc. Lond. B: Biol. Sci. 263, 755–759. Criscuolo, F., Gonzalez-Barroso Mdel, M., Le Maho, Y., Ricquier, D., Bouillaud, F., 2005. Avian uncoupling protein expressed in yeast mitochondria prevents endogenous free radical damage. Proc. Biol. Sci. 272, 803–810. Dhanasekaran, A., Kotamraju, S., Karunakaran, C., Kalivendi, S.V., Thomas, S., Joseph, J., Kalyanaraman, B., 2005. Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide. Free Radical Biol Med 39, 567–583. Doctrow, S.R., Huffman, K., Marcus, C.B., Tocco, G., Malfroy, E., Adinolfi, C.A., Kruk, H., Baker, K., Lazarowych, N., Mascarenhas, J., Malfroy, B., 2002. Salen–manganese complexes as catalytic scavengers of hydrogen peroxide and cytoprotective agents: structure–activity relationship studies. J. Med. Chem. 45, 4549–4558. Dugan, L.L., Quick, K.L., 2005. Reactive oxygen species and aging: evolving questions. Sci. Aging Knowl. Environ. e20. Duttaroy, A., Paul, A., Kundu, M., Belton, A., 2003. A Sod2 null mutation confers severely reduced adult lifespan in Drosophila. Genetics 165, 2295– 2299. Fridovich, I., 1989. Superoxide dismutases. An adaptation to a paramagnetic gas. J. Biol. Chem. 264, 7761–7764. Harman, D., 1956. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300.

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