Age Related Changes in NAD+ Metabolism Figure 7. Reduced NAD+ levels contributes to reduced Sirt1 activity in the liver, heart, kidney and lung with aging. (A) Reduced Sirt1 activity was observed in the rat liver, heart, kidney and lung after 12 months of age using a flourometry. All values are means 6 S.E from tissue obtained from eight different rats for each age group. Significance *p,0.01 compared to 3 month old rats. (B) Western blotting for Sirt1 in (i) liver, (ii) heart, (iii) kidney, and (iv) lung with aging using anti-Sirt1 antibody. The blots shown are representative tracings of an experiment done eight times. Graphs are mean 6 S.E brains from tissue obtained from eight different rats for each age group. Each bar of the quantification graph represents the corresponding band for each age group. Significance *p,0.01 compared to 3 month old rats. (C) Immunodetection for Sirt1 in the liver, heart, kidney and lung from 3 month, 12 month and 24 month old rats. Sirt1 (red) and DAPI (blue). Higher immunoreactivity for Sirt1 was observed in 12 and 24 month old rats compared to 3-month old rats. doi:10.1371/journal.pone.0019194.g007 damage occurring between 12 and 24 months (Fig. 4) (p,0.01). Immunohistofluorochemistry (Fig. 4C) confirmed the increase in phospho-H2AX-Serine139. PARP Activation & Poly(ADP-ribose) polymers To demonstrate a direct link between DNA damage and poly- ADP-ribosylation, we measured PARP activity in nuclei from the liver, heart, kidney and lung of aging rats. Our results show a significant (p,0.01) up-regulation of PARP activity in all organs by 12 months of age (Fig. 5A). This is consistent with increased formation of poly(ADP-ribose) nuclear protein in the liver (Fig. 5B), heart (Fig. 5C), kidney (Fig. 5D),and lung (Fig. 5E), using western blot and immunohistofluorochemistry (Fig. 5F). Intracellular NAD+(H) Levels As NAD+ is the sole substrate for PARP we assessed the impact of PARP activation on intracellular NAD+ content in the same aging rat tissue. We found that increased PARP activity was associated with a significant (p,0.01) reduction in the NAD+ content by 24 months of age in each of the organs investigated (Fig. 6A). This is in line with a significant increase in NADH (Fig. 6B) (p,0.01) and a significant decline in the NAD+:NADH ratio (Fig. 6C) (p,0.01). Most of the decline in intracellular NAD+ content was observed after 12 months of age, and appears to be more noteworthy in the heart. Sirt1 deacetylase Activity (but not protein levels) Declines with Aging Sirt1 is localised in the nucleus [27]. Therefore, both Sirt1 protein level and activity were measured in the liver, heart, kidney and lung of aging rats. Aging induced a significant decline in Sirt1 activity in the liver (p,0.01), heart (p,0.01), kidney (p,0.01), and lung (p,0.01) (Fig. 7A) consistent with decreased substrate (NAD+) availability (Fig. 6). In contrast, we observed that aging significantly (p,0.01) increased the amount of Sirt1 protein in the nucleus in all organs examined in this study, using both western blotting (Fig. 7B–E) and immunohistofluorochemistry (Fig. 7F). Increased p53 Acetylation in Aging Brain To confirm the effect of decreased NAD+ levels and Sirt1 function on the acetylation status of p53, we measured both acetylated and total p53 protein levels using western blotting. As shown in Figure 8, we found a significant age-dependent increase in acetylated p53 expression in the liver (p,0.01), heart (p,0.01), kidney (p,0.01), and lung (p,0.01). However, no change was observed in total p53 content between young and aged rats in all organs investigated. Impaired Mitochondrial Respiratory Chain Activity with Aging Although mitochondrial production of ROS has been shown to increase with advancing age [46], changes in the mitochondrial redox status with age in the heart, lung, liver and kidney has not, to our knowledge, been evaluated. We measured the activities of the mitochondrial respiratory complexes as shown in Figure 9A– D. The activity of all complexes decreased after 12 months of age, and reached statistical significance at 24 months in the liver (p,0.01), heart (p,0.01), kidney (p,0.01) and lung (p,0.01). Fig. 8E shows the ATP levels determined in all organs during the aging process. ATP levels significantly (p,0.01) declined in the heart, lung, liver and kidney after 12 months of age. This is in line with increased PARP activation and decline in cellular NAD+ content during the aging process. Discussion There is growing consensus around the hypothesis that oxidatively damage proteins, lipids, and nucleic acids play a key role in the aging process. Oxidative stress occurs in several degenerative diseases including neurodegenerative disease (e.g. Alzheimer’s, Parkinson’s), rheumatoid arthritis, atherosclerosis, diabetes, and cardiovascular disease [47]. This study is the first to investigate the impact of age associated changes in oxidative stress levels on both intracellular NAD+ levels and activity of the ‘longevity’ enzyme Sirt1. This study is also the first to report changes in o- and m- tryosine (specific markers of protein oxidation) in aging heart, lung, liver and kidney. In this report we provide consistent evidence of accumulating oxidative damage to tissue proteins, lipids and nuclear DNA with age. Reactive oxygen species (ROS) are byproducts of normal cellular physiology [48]. A delicate balance between ROS generation and detoxification is maintained by several cellular antioxidant defense systems [49,50]. These comprise antioxidant enzymes such as SOD1, catalase, glutathione peroxidase; and endogenous non-enzymatically acting compounds, such as vitamins A, C and E [9]. Nevertheless, various endogenous and exogenous triggers may stimulate the overproduction and accumulation of ROS leading to oxidative damage [9]. While oxidative stress increases with aging, it is yet unclear at which age free-radicals may act to initiate the senescence process. Young and aged rat models are relatively homogenous in their gene expressions in the various organs. This may be in contrast to young adult and aged human populations which may demonstrate much greater heterogeneity. Unlike aged rodents, humans may diverge in their rates of aging, as they translate from middle age to old age. In this study at middle age (i.e.12 months), female wistar rats already exhibit higher levels of oxidative stress induced damage to proteins, lipids, and DNA, with a lower total antioxidant efficiency than young adult animals of 3 months of age. Our results suggest an age- dependent increase in oxidative stress in these organs that appears to accelerate after middle age (i.e.12 months). These data support the existence of a progressive disequilibrium between the formation of ROS and the antioxidant protective mechanisms during the process of normal aging [9] in rats that may be applicable to the human population. Apart from increased levels of protein oxidation and lipid peroxidation, DNA is also susceptible to oxidative damage by ROS [7]. We assessed DNA damage by measuring the PLoS ONE | www.plosone.org 12 April 2011 | Volume 6 | Issue 4 | e19194