Age Related Changes in NAD+ Metabolism Figure 9. Oxidative stress-mediated reduction in mitochondrial respiratory chain activity in the liver, heart, kidney and lung with age. Reduced (A) complex I, (B) complex II, (C) complex III, and (D) complex IV, activities at 24 months of age. 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. doi:10.1371/journal.pone.0019194.g009 [73]. While p53 has been shown to be bind to PARP and be poly(ADP-ribosylated), an effect which enhances their pro- apoptotic activity, Sirt1-dependent deacetylation inhibits their apoptotic activity [55]. These results suggest that changes in intracellular NAD+ levels may regulate the post-translational acetylation of p53, thus providing further evidence of the association between NAD+ levels, oxidative DNA damage- mediated PARP activation, and reduced Sirt1 deacetylase activity [55]. One factor which may sensitise cells to increased DNA damage is impaired mitochondrial function [74]. The mito- chondrial electron transport system can trigger the formation of superoxide leading to increased production of H2O2 by superoxide dismutase [49,50]. Reduced electron flow through the mitochondrial respiratory chain, particularly through the inhibition of complex I or complex III, favours the enhanced production of superoxide and H2O2 [75]. Together, with the age-dependent increase in oxidative stress and decline in NAD+ and ATP content, we found a tendency to the reduction in the activity of the respiratory complexes with age in all organs. Sipos et al. (2003) showed that mitochondrial formation of H2O2 due to complex I inhibition is more clinically relevant than ROS production due to inhibition of complex III and IV in situ [76]. Using the same experimental paradigm, Rodriguez et al. (2008) reported similar mitochon- drial oxidative failure in the diaphragm of senescent prone mice [49]. It has also been demonstrated that an increased NADH:NAD+ ratio, favours ROS generation by the catalytic activity of a-Ketoglutarate dehydrogenase (a KGDH) [77]. The dependence on ROS production by a KGDH on the intracellular NADH content may play a crucial role in promoting the accumulation of free radicals in aging. When the reoxidation of NADH is impaired due to reduced complex I activity, the NADH:NAD+ ratio increases, thus reducing the activity of several NAD+ dependent dehydrogenases [77]. Moreover, NADH is also able to promote the formation of H2O2 in the presence of iron [78]. Given the observations in the present study, increased ROS production in aging may be a consequence of impaired mitochondrial respiratory function, and may, at least in part, be attributed to increased NADH:NAD+ ratio inducing free radical production via aKGDH and Fenton chemistry. Overall, our results show that oxidative stress increased with age, (appearing to accelerate more rapidly after mid life), in association with reduced antioxidant defence mechanisms, and mitochondrial dysfunction; data consistent with the predictions of the free-radical theory of aging. The current study, however, provides new insight into the potential mechanisms of aging involved in response to oxidative damage, particularly focusing on NAD+ metabolism and the roles of PARP and Sirt1 in the heart, lung, liver, and kidney (Fig. 10). Adequate NAD+ concentrations may therefore be an important longevity assur- ance factor. Therefore, we hypothesize that strategies targeted toward maintaining adequate NAD+ content during the aging process may prove a novel and potentially effective mechanism for retarding oxidative stress mediated cell degeneration, and age associated disorders. Figure 10. Schematic representation for the association between oxidative stress, PARP-mediated and decline in NAD+ content, and NAD+ dependent functions with aging. Oxidative stress to DNA activates PARP leading to poly(ADP-ribosylation) of proteins in a reaction which consumes NAD + . Depletion of cellular NAD+ stores attenuates the activity of Sirt1 deacetylase leading to hyperacetylation of p53, and consequently tilting the balance to cell death via an apoptotic mechanism. doi:10.1371/journal.pone.0019194.g010 PLoS ONE | www.plosone.org 16 April 2011 | Volume 6 | Issue 4 | e19194