is facilitated by Poly(ADP-ribose) polymerase-1 (PARP) [12,13,14]. PARP is an abundant protein modifying nuclear enzyme involved in DNA repair. The enzymatic activity of PARP is strongly activated in cells in response to treatment with ROS such as H2O2 [15]. Activation of PARP leads to the transfer of ADP-ribose moieties from NAD+, to the target protein [16]. Since PARP uses NAD+ as the only endogenous substrate for poly-ADP-ribosylation, PARP activity is dependent on the amount of NAD+ available, and may act as a nuclear energy sensor. Under physiological conditions, mild activation of PARP can regulate several cellular processes, including DNA repair, cell cycle progression, cell survival, chromatin remodel- ing, and genomic stability [13,17]. However, overactivation of PARP can repress genomic transcription and reduce cell survival. NAD+, in addition to being a substrate for PARP, also serves as an important redox carrier to power oxidative phosphorylation and ATP production [18]. Depletion of NAD+ following PARP hyperactivation has been shown to deplete intracellular ATP stores leading to the release of apoptosis- inducing factors (AIF) and consequent cell death due to energy restriction [19]. PARP activation has been implicated in the pathogenesis of hypertension, atherosclerosis, lung injury, haemorrhagic shock, and diabetic cardiovascular and kidney complications [20,21,22,23,24]. In these diseases, the oxidant- mediated endothelial cell injury is dependent on PARP activation, and can be attenuated by pharmacological PARP inhibitors [25,26]. Therefore, tight regulation of PARP activity is crucial to prevent the development of several age-related pathological disorders. In addition to its role in PARP activity, another essential factor that is greatly affected by changes in intracellular NAD+ levels is the class III histone deacetylases known as sirtuins, or silent information regulator of gene transcription [27]. Gene silencing by this family of enzymes has been correlated directly with longer lifespan in yeast and worms [28]. In yeast, sir2 plays a critical role in transcriptional silencing and maintenance of genomic stability [29,30]. Sirt1 is the human homolog of sir2 and appears to be involved in several physiological functions including the control of gene expression, cell cycle regulation, apoptosis, DNA repair, metabolism, and aging [31,32,33]. Sirt1 can deacetylate numerous proteins such as tumor suppressor protein, p53, which modulates various genes that control damaged DNA [34]. The deacetylase activity of SIRT proteins is dependent on the intracellular NAD+ content [27]. They catalyse a unique reaction that releases nicotinamide, acetyl ADP-ribose (AADPR), and the deacetylated substrate [27]. Impaired SIRT1 activity due to PARP mediated NAD+ depletion allows increased activity of several apoptotic effectors such as p53, therefore sensitising cells to apoptosis. Adequate NAD+ levels are therefore critical to maintaining Sirt1 activity Age Related Changes in NAD+ Metabolism which can delay apoptosis and provide vulnerable cells with additional time to repair even after repeated exposure to oxidative stress [35]. Though a number of studies have demonstrated elevated levels of oxidative stress associated damage in aged tissue, to our knowledge, no study has yet reported on changes in NAD+ levels during the aging process. In this study, we have characterized and quantified changes in NAD+ metabolism in the liver, heart, kidney and lung from female wistar rats aged from 3 to 24 months, spanning life stages from young adulthood to old age [36]. We quantified the levels of oxidative stress (in the form of protein carbonyls, lipid peroxidation, and oxidative DNA damage), total antioxidant and NAD+ levels and PARP, Sirt1 and mitochondrial activities in these tissues at different stages of life. Our results suggest that oxidative stress induced NAD+ depletion could play a significant role in the aging process, by compromising, energy production, DNA repair and genomic surveillance. Materials and Methods Reagents and Chemicals Phosphate buffer solution (PBS) was from Invitrogen (Mel- bourne, Australia). Nicotinamide, bicine, b-nicotinamide adenine dinucleotide reduced form (b-NADH), 3-[-4,5-di- methylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), alcohol dehydrogenase (ADH), sodium pyruvate, TRIS, c- globulins, N-(1-naphthyl) ethylenediamine dihydrochloride, EGTA, EDTA, tricarboxylic acid (TCA), Hepes, proteinase K, Percoll, mannitol, 1,-dithio DL-threitol (DTT), KCN, decylubiquinone, succinate, antimycin, rotenone, cytochrome c, and sodium borohydride were obtained from Sigma-Aldrich (Castle-Hill, Australia). Phenazine methosulfate (PMS) was obtained from ICN Biochemicals (Ohio, USA). Bradford reagent was obtained from BioRad (Hercules, CA, USA). DAPI and polyclonal antibodies (pAb) for b-actin and all chemicals used for Western blots (unless otherwise stated) were obtained from Sigma-Aldrich (Castle-Hill, Australia). Polyclonal antibod- ies for detection of (E)-4- Hydroxynonenal, Sirt1, phospho- H2AX-ser139 were obtained from Alexis Biochemicals (San Diego, CA, USA). Monoclonal antibody for the detection of Poly(ADP-ribose) was purchased from Alexis Biochemicals (San Diego, CA, USA). Polyclonal antibody for the detection of acetylated p53 was obtained from Abcam (Cambridge, UK). Monoclonal antibody to total p53 was obtained from Millipore (Melbourne, Australia). Alexa 488- or Alexa 594-conjugated anti-mouse IgG or anti-rabbit were purchased from Invitrogen. All commercial antibodies were used at the concentrations recommended by the manufacturer. Table 1. List of primary antibodies used. Antibody (E)-4-Hydroxynonenal Phospho-H2AX-Serine139 Poly(ADP-ribose) (10H) Sirt1 Acetyl K386 p53 Oncogene doi:10.1371/journal.pone.0019194.t001 Type Polyclonal Polyclonal Monoclonal Polyclonal Polyclonal Monoclonal Antigen Free HNE (E)-4-hydroxynonenal Phosphorylated H2AX at Serine 139 Poly(ADP-ribose) Sirt1 Acetylated p53 at lysine 386 Total p53 oncogene Dilution Source 1:1000 1:1000 1:400 1:2000 1:1000 1:1000 Alexis Biochemicals Alexis Biochemicals Alexis Biochemicals Alexis Biochemicals Abcam Millipore PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e19194