Age Related Changes in NAD+ Metabolism Figure 6. Increased PARP activation alters pyridine nucleotide metabolism with aging. (A) NAD+ content and (B) NADH content were determined in the rat liver, heart, kidney and lung with aging using a spectrophotometric assay. 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. (C) NAD+:NADH ratio was determined as the total NAD+ content divided by total NADH levels. 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.g006 containing 250 mM sucrose; 50 mM potassium-phosphate; 1 mM KCN; 50 mM decylubiquinone; 0.8 mM antimycin, pH 7.4. The reaction was started following the addition of NADH (100 mM). The activity of NADH CoQ oxidoreductase was measured as the rate of oxidation of NADH at 340 nm using the Model 680XR microplate reader (BioRad, Hercules). The activity of complex I was expressed as nmol oxidised NADH/ min/mg protein. The activity of complex II was measured in buffer solution containing submitochondrial fractions (0.03 mg/ ml); 100 mM potassium-phosphate; 20 mM succinate; 0.8 mM antimycin; 50 mM rotenone; 2 mM KCN; 50 mM DCIP, pH 7.4. The reaction was initiated by the addition of 50 mM decylubi- quinone. The activity of succinate:DCIP oxireductase was measured as the rate of reduction of 2, 6-DCIP at 600 nm with 520 nm as reference wavelength. The activity of complex II was expressed in nmol reduced DCIP/min/mg protein. The activity of complex III was measured in buffer solution containing submitochondrial fractions (0.03 mg/ml); 35 mM potassium- phosphate; 5 mM MgCl2; 2.5 mg/ml BSA; 50 mM rotenone; 1.8 mM KCN; 2 mM decylubiquinone, pH 7.5. The reaction was initiated by the addition of 125 mM cytochrome c. The activity of ubiquinol:cytochrome c reductase was measured as the rate of reduction of cytochrome c at 550 nm with 580 nm as the reference wavelength. The activity of complex III was expressed in nmol reduced cytochrome c/min/mg protein. The activity of complex IV was measured in a buffer solution containing submitochondrial fractions (0.1 mg/ml); 50 mM potassium- phosphate, pH 6.8. The reaction was started by the addition of 75 mM cytochrome c previously reduced with sodium borohy- dride and measuring the absorbance at 550 nm. The activity of cytochrome c oxidase was expressed as nmol oxidised cytochrome c/min/mg protein. Immunoflourescence Staining for 4-Hydroxynonenal, Phospho-H2AX-Serine139, Poly(ADP)-ribose, and Sirt1 Liver, heart, kidney and lung whole mounts were immersion- fixed with ice cold 70% ethanol for 20 mins followed by 4% paraformaldehyde (PFA), pH 7.4 for 10 min at 4uC. Immuno- staining was established on brain whole mount. Fixed tissue were permeabilised with 1% Triton-X 100 in DPBS for 30 min at room temperature and non-specific binding was blocked with 1% BSA in DPBS for 2 h before application of primary antibodies. All specific primary mAb or pAb antibodies were diluted in 1% BSA in PBS and incubated overnight at 4uC. Tissue whole mounts were washed three times with DPBS and incubated for 1 h at 37uC with the appropriate labeled secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG coupled to Alexa 488 or Alexa 594). Nuclear staining was performed using DAPI at 1 mg/ml for 5 min at room temperature. The slides were washed 3 times in DPBS at room temperature and coverslips were mounted on glass slides with Flouromount-G and were examined with an Olympus BX60 fluorescence microscope fitted with a SensiCam digital camera. Titration series and conjugate experimental controls were performed on both primary and secondary antibodies to optimise for low background noise, maximal stain sensitivity, avoidance of non-specific staining and cross-reactivity. Bradford Protein Assay for the Quantification of Total Protein DNA damage, total antioxidant capacity, PARP and Sirt1 activities, NAD+ concentration, and mitochondrial function were adjusted for variations in protein number using the Bradford protein assay described by Bradford [43]. Data Analysis Results obtained are presented as the means 6 the standard error of measurement (SEM) of at least eight animals per age group analysed in duplicate. One way analysis of variance (ANOVA) and post hoc Tukey’s multiple comparison tests were used to determine statistical significance between treatment groups. Differences between treatment groups were considered significant if p was less than 0.05 (p,0.05). Results Age-related Increase in Protein Carbonyl Formation Hydroxyl radical attack on the amino acid phenylalanine generates o-, m- and p- tyrosine were detected using gas chromatography/mass spectrometry (GC/MS) [44,45]. Figure 1 shows the age-related changes in o- (Fig. 1A) and m- tyrosine (Fig. 1B) levels in the liver, heart, kidney and lung. Our results show a significant increase in both o- and m- tyrosine formation with age, with the highest rate of increase occurring after 12 months of age in all organs (p,0.01). 4-HNE as a Biomarker for Lipid Peroxidation in Aging Peroxidative damage to cellular lipid constituents was deter- mined by using 4-HNE as a marker of lipid peroxidation. Increased 4-HNE protein expression using western blotting was observed in the liver (p,0.01) (Fig. 2A), heart (p,0.01) (Fig. 2B), kidney (p,0.01) (Fig. 2C) and lung (p,0.01) (Fig. 2D), consistent with an increase in protein carbonyl formation (Fig. 1).Using immunohistofluorochemistry, we stained frozen sections for 4- HNE (Fig. 2E). Two age related changes are clearly visible: (1) There is an increase in 4-HNE expression consistent with the previous western blot studies (Fig. 2A–D). (2) Aged tissue appears to demonstrate significantly lower cell volume, indicative of extensive cell loss occurring during the aging process. Total Antioxidant Capacity Declines with Aging To investigate changes in endogenous antioxidant function age, we measured total antioxidant capacity in-situ using the Trolox assay. We observed a significant reduction in total antioxidant activity by 12 months of age in all organs with the most rapid decline occurring between 12 and 24 months (p,0.01) (Fig. 3). DNA Damage Increases with Aging The aging process is also considered to cause changes in the DNA repair process leading to increased vulnerability to DNA damage [7]. We assessed DNA damage by measuring the phosphorylation of H2AX at Serine139 residue using a flouro- metric assay. Increased DNA damage was observed in all aged organs assessed in this study by 12 months of age with accelerated PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e19194