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Extension of Life-Span with Superoxide Dismutase/Catalase Mimetics

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Science  01 Sep 2000:
Vol. 289, Issue 5484, pp. 1567-1569
DOI: 10.1126/science.289.5484.1567

Abstract

We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems ofCaenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wild-type worms increased their mean life-span by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their life-span (a 67 percent increase). It appears that oxidative stress is a major determinant of life-span and that it can be counteracted by pharmacological intervention.

Reactive oxygen species (ROS) primarily arise as by-products of normal metabolic activities and are thought to influence the etiology of age-related diseases (1, 2). If ROS contribute to aging, then the aging process would be slowed if either the production of ROS were reduced or the endogenous antioxidant or repair activities were increased. Indeed, genetic mutations and manipulations that confer resistance to oxidative stress also cause extended life-span (3–8); however, dietary antioxidant supplements have been shown to have very limited effects on longevity (9).

We were prompted to test the oxygen radical theory of aging by the development of synthetic catalytic compounds that ameliorate oxidative stress in several disease models (10–15) and partially rescue mice that are mutant for mitochondrial superoxide dismutase (SOD) (16). We tested the effect of two mimetics, EUK-8 and EUK-134, on life-span in Caenorhabditis elegans. In vitro, these compounds exhibit both SOD- and catalase-like activities (they are SOD/catalase mimetics). EUK-134 is an analog of EUK-8, with increased catalase activity and equivalent SOD activity (13).

We treated synchronous adult hermaphrodite populations of worms in S medium (17) with various concentrations of mimetics and maintained those populations concurrently with control populations of untreated wild-type worms and, in some experiments, of long-lived age-1(hx546) mutant worms (18). The adult worms were transferred each day to new medium during the worms' reproductive period and every 3 days thereafter. In every experiment, treatment of wild-type worms with SOD/catalase mimetics significantly increased mean and maximum life-span (n = 14 comparisons, Table 1). Treatment of wild-type worms with 0.05 mM EUK-134 resulted in an increase in mean life-span of 54% (P < 0.0001; Fig. 1, A and B, and Table 1). Higher concentrations of EUK-134 and various concentrations of EUK-8 also extended life-span (P < 0.0001; Table 1). No overall dose response was observed. These compounds probably enter the worm through ingestion alone. As the worms age, they feed less, and consequently there is likely to be an increasing limitation on the amount of drug taken up.

Figure 1

Kaplan-Meier survival curve (±SE) of wild-type (wt) and mev-1(kn1) adult worms treated with SOD/catalase mimetics. Synchronously aging hermaphrodite worms were cultured in S medium with Escherichia coli as a food source (17). Worms were scored as dead when they failed to respond to repeated touching with a platinum wire pick. (A) Mean life-span (±SEM) in days of strain N2 (wild-type) = 24 ± 1 (solid squares); of strain TJ1052 [age-1(hx546)] = 38 ± 2 (circles); and of strain N2 (wild-type) treated with 0.05 mM EUK-134 = 31 ± 3 (open squares). (B) Mean life-span (±SEM) in days of strain N2 (wild-type) = 24 ± 1 (squares); of strain TJ1052 [age-1(hx546)] = 41 ± 3 (circles); and of strain N2 (wild-type) treated with 0.5 mM EUK-134 = 37 ± 2 (open squares). (C) Mean life-span (±SEM) in days of strain N2 (wild-type) = 24 ± 2 (squares), n = 7 worms; of strainmev-1(kn1) = 15 ± 1 (solid triangles),n = 19 worms; and of strain mev-1(kn1)treated with 0.5 mM EUK-134 = 25 ± 2, n = 16 worms. Very similar results were obtained in independent experiments.

Table 1

Effect of SOD/catalase mimetics on wild-type C. elegans life-span. Five experiments are shown.N + is the number of worms after censoring. wt, wild type.

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We took a genetic approach to determining the mode of action of the mimetics. We examined the effects of EUK-134 on the life-span of a mutant worm strain that exhibits accelerated aging. Mutation of themev-1 gene, encoding the cytochrome b subunit of succinate dehydrogenase (complex II) of the electron transport chain, results in an elevated accumulation of oxidative damage during aging, an increased sensitivity to oxygen, and a life-span shortened by 37% (P < 0.0001; Fig. 1C) (19, 20). Treatment with 0.5 mM EUK-134 restored a normal life-span to themev-1(kn1) mutants by increasing their life-span by 67% (P < 0.0001; Fig. 1C). These results are consistent with amelioration of an endogenous and chronic oxidative stress.

Genetic manipulation of the life-span is generally associated with pleiotropic effects on life history traits such as reduced fertility and altered growth rate (21). Consequently, we tested whether the SOD/catalase mimetics affected such traits. Treatment of wild-type worms with mimetics did not significantly alter either hermaphrodite self-fertility or body size measured at two stages during development (Fig. 2, A through F). These results suggest that the mimetic-associated life-span increase is not due to a nonspecific reduction in metabolism. This is consistent with the observation that the treated worms did not appear moribund until close to the end of the extended life-span.

Figure 2

Growth and fertility of wild-type worms treated with SOD/catalase mimetics. (A) Mean area (±SD) of second-stage larvae (L2) worms (22) cultured under the conditions indicated. Untreated, n = 27 worms; treated with 0.05 mM EUK-8, n = 13 worms; treated with 0.5 mM EUK-8, n = 20 worms; treated with 0.05 mM EUK-134, n = 12 worms; treated with 0.5 mM EUK-134,n = 12 worms. No significant differences in area were detected. (B) Mean area (±SD) of fourth-stage larvae (L4) worms cultured under the conditions indicated. Untreated,n = 20 worms; treated with 0.05 mM EUK-8,n = 22 worms; treated with 0.5 mM EUK-8,n = 15 worms; treated with 0.05 mM EUK-134,n = 11 worms; treated with 0.5 mM EUK-134,n = 23 worms. No significant differences in area were detected. (C) Daily fertility (±SD) of untreated wild-type worms (23). (D) Daily fertility (±SD) of wild-type worms treated from hatching with 0.5 mM EUK-8. (E) Daily fertility (±SD) of wild-type worms treated from hatching with 0.5 mM EUK-134. (F) Total fertilities (±SD) for untreated, EUK-8–treated, and EUK-134–treated worms. Untreated worms exhibited a mean total fertility (±SEM) of 76 ± 14 offspring (n = 6 worms). Worms treated with EUK-8 had a mean total fertility of 97 ± 12 offspring (n = 6 worms). Worms treated with EUK-134 had a mean fertility of 76 ± 16 offspring (n = 6 worms). Comparisons were made of total fertilities and for each day throughout the fertile period, but no significant differences were observed between treatments (as assessed with Student's ttest).

We demonstrated that treatment of adult hermaphrodite worms with SOD/catalase mimetics causes a large increase in life-span. In addition, these compounds rescue the life-span defect of themev-1(kn1) mutation. We propose that EUK-8 and EUK-134 extend life-span by augmenting natural antioxidant defenses without having any overt effects on other traits. These results suggest that endogenous oxidative stress is a major determinant of the rate of aging.

  • * Present address: Aventis Pharma, London Road, Holmes Chapel, Crewe, Cheshire, CW4 8BE, UK.

  • Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

  • To whom correspondence should be addressed. E-mail: Gordon.Lithgow{at}man.ac.uk

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