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. 2010 Aug 24;107(34):15123-8.
doi: 10.1073/pnas.1004432107. Epub 2010 Aug 9.

Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity

Ana Mesquita  1 , Martin Weinberger, Alexandra Silva, Belém Sampaio-Marques, Bruno Almeida, Cecília Leão, Vítor Costa, Fernando Rodrigues, William C Burhans, Paula Ludovico
Affiliations

Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity

Ana Mesquita et al. Proc Natl Acad Sci U S A. .

Abstract

The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in Saccharomyces cerevisiae, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Caloric restriction (CR) or inactivation of catalases extends Saccharomyces cerevisiae chronological lifespan by increasing intracellular levels of H2O2. Survival of (A) wild-type (BY4742) cells, (C) Δcta1 cells, (E) wild-type cells in the absence or presence of the catalase inhibitor 10 mM 3-amino-1,2,4-triazole (3AT), and (G) wild-type cells transformed with an empty vector or a plasmid that overexpresses CTA1 (“MET-CTA1”). (I) Effects of ectopic exposure of wild-type cells to indicated concentrations of H2O2. Cell viability was measured at 2- to 3-d intervals beginning the day cultures achieved stationary phase (day 0) and is expressed as % survival compared with survival at day 0 (100%). Percentage of cells exhibiting high levels of intracellular ROS detected by FACS measurements of fluorescence of the probe dihydrorhodamine 123 (DHR) in (B) wild-type cells, (D) Δcta1 cells, (F) wild-type cells in the absence or presence of 3AT, and (H) wild-type cells transformed with an empty vector or a plasmid that overexpresses CTA1 (“MET-CTA1”). Three to five biological replicas of each experiment were performed. Survival and DHR positive cell values are mean ± SD or mean ± SEM, respectively, in all experiments. Statistical significance (*P < 0.05) was determined by Student's t-test.
Fig. 2.
Fig. 2.
The longevity-promoting effects of high intracellular H2O2 levels induced by CR or inactivation of catalases are accompanied by a reduction in the chronological age-dependent accumulation of superoxide anions. (A) FACS measurements of superoxide anions using the probe dihydroethidium (DHE) in parallel with measurements of H2O2 using dihydrorhodamine 123 (DHR) in wild-type (gray histograms) and Δcta1 (green histograms) cells at day 0 and day 3 of stationary phase. Bar graphs indicate mean ± SD fluorescence/cell (arbitrary units) measured in 25,000 cells/sample in three independent experiments. (B) FACS measurements of superoxide anions (DHE) and H2O2 (DHR) in wild-type cells in the absence (gray histograms) or presence (green histograms) of 10 mM 3-amino-1,2,4-triazole (3AT) at day 0 of stationary phase. Bar graphs indicate mean ± SD fluorescence/cell (arbitrary units) as described above. Statistical significance (*P < 0.05) was determined by Student's t-test.
Fig. 3.
Fig. 3.
Induction of superoxide dismutase activity by intracellular H2O2. (A) In situ determination of superoxide dismutase activity in stationary phase wild-type and Δcta1 cells measured as previously described. MnSOD (Sod2p) activity was assessed in the presence of 2 mM potassium cyanide, which inhibits Sod1p, but not Sod2p activity (Fig. S7B). (B) Quantification of fold increases in Sod1p and Sod2p activity under indicated conditions in wild-type and Δcta1 cells. Sod1p and Sod2p activity at each time point was normalized to activity in wild-type cells under non-CR conditions (2% glucose). (C) Quantification of fold increases in Sod1p and Sod2p activity in wild-type cells after ectopic exposure to 1 mM H2O2. Sod1p and Sod2p activity at each time point was normalized to activity in wild-type cells under non-CR conditions (2% glucose). Values indicate mean ± SEM from three independent experiments. Statistical significance (*P < 0.05) was determined by Student's t-test.
Fig. 4.
Fig. 4.
Effects of increased H2O2 induced by CR or by inactivation of CTA1 on oxidative damage to macromolecules. (A) Oxidative damage was assessed by measuring levels of oxidized proteins (carbonyls) in stationary phase wild-type and Δcta1 cells under non-CR and CR conditions. Levels of carbonyls were normalized at each time point to wild-type cell values under non-CR conditions (2% glucose). (B) Oxidative damage to proteins and lipids measured as autofluorescence of stationary phase wild-type and Δcta1 cells under non-CR and CR conditions. Histograms are representative of data collected at day 3. Values indicate mean ± SEM from three independent experiments. Statistical significance (*P < 0.05) was determined by Student's t-test.
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