Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats Nady Braidy1, Gilles J. Guillemin1,2, Hussein Mansour3, Tailoi Chan-Ling 3 , Anne Poljak4, Ross Grant1,5* 1 Department of Pharmacology, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia, 2 St Vincent’s Centre for Applied Medical Research, Sydney, Australia, 3 Retinal and Developmental Neurobiology Lab, Discipline of Anatomy and Histology, School of Medical Sciences, University of Sydney, Australia, 4 Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, Australia, 5 Australasian Research Institute, Sydney Adventist Hospital, Sydney, Australia Abstract The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I–IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor. Citation: Braidy N, Guillemin GJ, Mansour H, Chan-Ling T, Poljak A, et al. (2011) Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats. PLoS ONE 6(4): e19194. doi:10.1371/journal.pone.0019194 Editor: Aimin Xu, University of Hong Kong, China Received October 6, 2010; Accepted March 29, 2011; Published April 26, 2011 Copyright: 2011 Braidy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to Professor Tailoi Chan-Ling from the National Health and Medical Research Council of Australia, and to Dr. Gilles J. Guillemin and Professor Tailoi Chan-Ling from the Rebecca Cooper Medical Research Foundation (Sydney, Australia). Nady Braidy is the recipient of an Australian Postgraduate Award at the University of New South Wales. Hussein Mansour is the recipient of a University of Sydney Medical Foundation/Bluesand Research Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: r.grant@unsw.edu.au Introduction Multiple degenerative processes are implicated in natural senescence. As aging is associated with progressive decline in organ function, elucidating the complex pathways controlling the rate of aging is of significant clinical importance [1]. An important mechanism contributing to aging is oxidative stress. The ‘‘free- radical theory of aging’’, initially proposed by Harman (1956) suggests that oxidative damage occurs with advanced aging due to an imbalance between free radical and reactive species (ROS) production, and cellular antioxidant defense mechanisms [2]. Elevated levels of intracellular ROS through hydrogen peroxide treatment, or deficiency of ROS scavenging enzymes such as superoxide dismutase (SOD1) knockdown, has been shown to induce premature senescence and reduce cellular life span [3,4,5]. The mitochondria, represents the main producer of cellular ROS in the human body, and approximately 1–2% of the oxygen molecules consumed during normal respiration are converted into highly reactive superoxide anion, which is rapidly dismutated to H2O2 by the superoxide dismutases [6]. Other pathways and events able to produce ROS include peroxisomal metabolism, enzymatic synthesis of nitric oxide, phagocytic leukocytes, heat, ultraviolet (UV) light, therapeutic drugs, and ionizing radiation [7]. Intracellular ROS, due to their high reactivity, can interact with a spectrum of biological molecules, leading to the oxidation of several macromolecules, such as protein, lipids, and nucleic acids [8]. As a result, vital functions, such as energy production, maintenance of plasma membrane potential, and cellular ionic homeostasis may be impaired in the early stage of oxidative stress [8]. Excessive oxidative insult may also stimulate secondary events leading to cell death via an apoptotic mechanism [9]. A major factor associated with age-related diseases is the increase of oxidative DNA damage [7]. It is estimated that at least 5000 single- stranded DNA breaks occur during a single cell cycle as a result of ROS production [10,11]. Approximately 1% of these DNA breaks are converted into double-stranded DNA breaks, primarily during DNA replication. Accumulation of unrepaired DNA damage induced by ROS can lead to arrest or induction of transcription, induction of signal transduction pathways, replication errors and genomic instability [10,11]. These molecular changes are observed in both cancer and aging, and this supports the notion that chronic oxidative damage to DNA might trigger cancer and promote aging [10,11]. The removal of oxidative DNA damage through repair of DNA single strand breaks by DNA base excision repair, PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19194