The main objective of this review is to provide an appraisal of the current status of the relationship between energy intake and the life span of animals. The concept that a reduction in food intake, or caloric restriction (CR), retards the aging process, delays the age-associated decline in physiological fitness, and extends the life span of organisms of diverse phylogenetic groups is one of the leading paradigms in gerontology. However, emerging evidence disputes some of the primary tenets of this conception. One disparity is that the CR-related increase in longevity is not universal and may not even be shared among different strains of the same species. A further misgiving is that the control animals, fed ad libitum (AL), become overweight and prone to early onset of diseases and death, and thus may not be the ideal control animals for studies concerned with comparisons of longevity. Reexamination of body weight and longevity data from a study involving over 60,000 mice and rats, conducted by a National Institute on Aging-sponsored project, suggests that CR-related increase in life span of specific genotypes is directly related to the gain in body weight under the AL feeding regimen. Additionally, CR in mammals and “dietary restriction” in organisms such as Drosophila are dissimilar phenomena, albeit they are often presented to be the very same. The latter involves a reduction in yeast rather than caloric intake, which is inconsistent with the notion of a common, conserved mechanism of CR action in different species. Although specific mechanisms by which CR affects longevity are not well understood, existing evidence supports the view that CR increases the life span of those particular genotypes that develop energy imbalance owing to AL feeding. In such groups, CR lowers body temperature, rate of metabolism, and oxidant production and retards the age-related pro-oxidizing shift in the redox state.

The phenotypic effects of under- and over-expression of CcO (cytochrome c oxidase) regulatory subunits IV and Vb were examined in Drosophila melanogaster in order to test further the hypothesis that suppression of the activities of mitochondrial ETC (electron-transport chain) oxidoreductases retards the aging process and extends lifespan. Underexpression of both CcO subunits, induced by RNAi, resulted in decreases in the respective mRNA and protein levels, CcO holoenzyme activity, rate of mitochondrial respiration, walking speed and the lifespan of fruitflies. Overexpression of CcO IV or Vb in young fruitflies increased the amount of mRNA, but had no effect on the protein level or CcO catalytic activity. On the other hand, in older fruitflies, overexpression of CcO Vb, but not CcO IV, elevated the mRNA and protein amounts as well as the CcO holoenzyme activity, thereby preventing the typical age-related decline in CcO activity. Nevertheless, lifespans of the fruitflies overexpressing CcO IV or Vb were neither extended nor shortened. Our results demonstrate that: (i) the suppression of CcO function exerts deleterious rather than benign effects on fitness and survival, and (ii) the structure/function of CcO, an ETC oxidoreductase, can be ‘re-engineered’ in vivo.

Significance: The main objective of this review was to provide an exposition of investigations, conducted in Drosophila melanogaster, on the role of reactive oxygen species and redox state in the aging process. While early transgenic studies did not clearly support the validity of the oxidative stress hypothesis of aging, predicated on the accumulation of structural damage, they spawned a broader search for redox-related effects that might impact the aging process. Recent Advances: Initial evidence implicating the thiol redox state as a possible causative factor in aging has been obtained in Drosophila. Overexpression of genes, such as GCL, G6PD, Prx2, and Prx5, which are involved in the maintenance of thiol redox homeostasis, has strong positive effects on longevity. Further, the depletion of peroxiredoxin activity in the mitochondria through the double knockdown of Prx5 and Prx3 not only results in a redox crisis but also elicits a rapid aging phenotype. Critical Issues: Herein, we summarize the present status of knowledge about the main components of the machinery controlling thiol redox homeostasis and describe how age-related redox fluctuations might impact aging more acutely through disruption of the redox-sensitive signaling mechanisms rather than via the simple accumulation of structural damage. Future Directions: Based on these initial insights into the plausible impact of redox fluctuations on redox signaling, future studies should focus on the pathways that have been explicitly implicated in aging, such as insulin signaling, TOR, and JNK/FOXO, with particular attention to elements that are redox sensitive.

Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F2-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F2-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and the control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies.

The hypothesis that life-span extension by caloric restriction (CR) is contingent upon the attenuation of macromolecular oxidative damage was tested in two different strains of mice: the C57BL/6, whose life span is extended by CR, and the DBA/2, in which CR has relatively minor or no impact on longevity. Mice were fed ad libitum (AL) or restricted to 40% lesser food, starting at 4 months of age. Protein damage was measured as protein-linked adducts of 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) in skeletal muscle mitochondria at 6 and 23 months of age. Protein-HNE and -MDA content increased with age in C57BL/6 mice and CR significantly attenuated these augmentations. Metalloprotease 1, NADP-dependent mitochondrial malic enzyme (isoform 2) and citrate synthase were identified by mass spectroscopy to contain HNE/MDA adducts. DBA/2 mice exhibited little effect of age or CR on protein HNE/MDA content in skeletal muscle mitochondria. In contrast, protein-HNE levels in liver mitochondria showed a significant increase with age in AL-fed mice of both strains, and CR caused significant attenuation of this damage. Overall, results indicated that the age-related increase in protein oxidative damage and its abatement by CR are genotype- and tissue-specific, and not a universal phenomenon.

The main objective of this review is to examine the role of endogenous reactive oxygen/nitrogen species (ROS) in the aging process. Until relatively recently, ROS were considered to be potentially toxic by-products of aerobic metabolism, which, if not eliminated, may inflict structural damage on various macromolecules. Accrual of such damage over time was postulated to be responsible for the physiological deterioration in the postreproductive phase of life and eventually the death of the organism. This “structural damage-based oxidative stress” hypothesis has received support from the age-associated increases in the rate of ROS production and the steady-state amounts of oxidized macromolecules; however, there are increasing indications that structural damage alone is insufficient to satisfactorily explain the age-associated functional losses. The level of oxidative damage accrued during aging often does not match the magnitude of functional losses. Although experimental augmentation of antioxidant defenses tends to enhance resistance to induced oxidative stress, such manipulations are generally ineffective in the extension of life span of long-lived strains of animals. More recently, in a major conceptual shift, ROS have been found to be physiologically vital for signal transduction, gene regulation, and redox regulation, among others, implying that their complete elimination would be harmful. An alternative notion, advocated here, termed the “redox stress hypothesis,” proposes that aging-associated functional losses are primarily caused by a progressive pro-oxidizing shift in the redox state of the cells, which leads to the overoxidation of redox-sensitive protein thiols and the consequent disruption of the redox-regulated signaling mechanisms.

The hypothesis that the life-extending effect of caloric restriction (CR) is associated with an attenuation of the age-related pro-oxidant shift in the thiol redox state was tested employing a novel experimental design. Amounts of GSH, GSSG, and protein mixed disulfides (Pr-SSG) in the skeletal muscle and liver were compared between two strains of mice that have similar life spans when fed ad libitum (AL), but different life spans under the standard CR regimen. The life span of one strain, C57BL/6, is extended under CR, whereas it remains unaffected in the other strain, DBA/2. Mice were fed AL or 40% less food starting at 4months and compared at 6 and 24months of age. The amounts of GSSG and Pr-SSG increased and the GSH:GSSG ratios decreased with age in both strains of AL-fed mice. CR prevented these age-related changes in the C57BL/6, whose life span is extended by CR, but not in the DBA/2 mice, in which it remains unaffected. CR enhanced the activity of glutamate–cysteine ligase in the C57BL/6, but not in the DBA/2 mice. The results suggest that longevity extension by CR may be associated with the attenuation of age-related pro-oxidizing shifts in the thiol redox state.

Drosophila mitochondria contain two peroxidases peroxiredoxin 3 (dPrx3) and peroxiredoxin 5 (dPrx5) which together constitute the sole known intramitochondrial mechanism for the catalytic removal of hydrogen and organic peroxides dPrx3 exists exclusively within mitochondria whereas dPrx5 is also present in some other intracellular compartments Levels of these two peroxiredoxins were genetically manipulated singly and together in D melanogaster for the purpose of understanding their respective functions Underexpression of dPrx3 by 90-95% had no discernable effect on life span under normal or oxidative stress conditions the dPrx5 null flies were previously reported to exhibit a 10% shortening of mean life span and an increase in sensitivity to oxidative stress Flies underexpressing both dPrx3 and dPrx5 showed an 80% decrease in life span a severe disruption in thiol homeostasis and a massive induction of apoptosis in the muscle and digestive system tissues The early mortality in flies underexpressing both peroxiredoxins was partially offset by overexpression of thioredoxin reductase but not mitochondrion-targeted catalase These results suggest that mitochondrial peroxiredoxins confer specific protection for thioredoxin/glutathione systems play a critical role in the maintenance of global thiol homeostasis and prevent the age-associated apoptosis and premature death (C) 2010 Elsevier Inc All rights reserved

The simultaneous overexpression of multiple copies of Mn superoxide dismutase (SOD) and ectopic catalase (mtCat) transgenes in the mitochondria of the fruit fly, Drosophila melanogaster, was shown previously to diminish the life span. The hypothesis tested in this study was that this effect was due primarily to the presence of one or the other transgene. An alternative hypothesis was that both transgenes have additive, negative effects. Crosses were performed between five pairs of transgenic lines containing single-copy insertions of mtCat, Mn SOD, or P element vector control transgenes at unique loci, and the life spans of progeny containing two mtCat, Mn SOD, or vector insertions were determined. Increasing amounts of mitochondrial catalase activity tended to be associated with decreases in mean life span. Overexpression of two copies of the genomic Mn SOD transgene had no effect on life span. The results do not support the hypothesis that enhanced mitochondrial SOD or catalase activity promotes longevity in flies.

The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the agerelated decline in mitochondrial respiratory activity and an increase in H2O2 generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology. (C) 2010 Elsevier Inc. All rights reserved.

This study examined the protein targets of nitration and the consequent impact on protein function in rat kidney mitochondria at 4, 13, 19, and 24 months of age. Succinyl-CoA transferase (SCOT), a rate-limiting enzyme in the degradation of ketone bodies, was the most intensely reactive protein against anti-3-nitrotyrosine antibody in rat kidney mitochondria. However, subsequent mass spectrometric and amino acid analyses of purified SCOT indicated that tryptophan 372, rather than a tyrosine residue, was the actual site of simultaneous additions of nitro and hydroxy groups. This finding suggests that identification of nitrated tyrosine residues based solely on reactivity with anti-3-nitrotyrosine antibody can be potentially misleading. Between 4 and 24 months of age, the amounts of SCOT protein and catalytic activity, expressed per milligram of mitochondrial proteins, decreased by 55 and 45%, respectively. SCOT, and particularly its nitrated carboxy-terminal region, was relatively more susceptible to in vitro proteolysis than other randomly selected kidney mitochondrial proteins. The age-related decreases in SCOT protein amount and catalytic activity were prevented by a relatively long-term 40% reduction in the amount of food intake. Loss of SCOT protein in the aged rats may attenuate the capacity of kidney mitochondria to utilize ketone bodies for energy production.

Coenzyme Q10 (CoQ10) is widely consumed as a dietary supplement to enhance bioenergetic capacity and to ameliorate the debilitative effects of the aging process or certain pathological conditions. Our main purpose in this study was to determine whether CoQ10 intake does indeed attenuate the age-associated losses in motor, sensory, and cognitive functions or decrease the rate of mortality in mice. Mice were fed a control nonpurified diet or that diet containing 0.68 mg/g (low dosage) or 2.6 mg/g (high dosage) CoQ10, starting at 4 mo of age, and were tested for sensory, motor, and cognitive function at 7, 15, and 25 mo of age. Amounts of the ubiquinols CoQ9H2 and CoQ10H2 measured in a parallel study were augmented in the cerebral cortex but not in any other region of the brain. Intake of the low-CoQ10 diet did not affect ageassociated decrements in muscle strength, balance, coordinated running, or learning/memory, whereas intake at the higher amount increased spontaneous activity, worsened the age-related losses in acuity to auditory and shock stimuli, and impaired the spatial learning/memory of oldmice. The CoQ10 diets did not affect survivorship of mice through 25mo of age. Our results suggest that prolonged intake of CoQ10 in low amounts has no discernable impact on cognitive andmotor functions whereas intake at higher amounts exacerbates cognitive and sensory impairments encountered in old mice. These findings do not support the notion that CoQ10 is a fitness-enhancing or an “antiaging” substance under normal physiological conditions. J. Nutr. 139: 1926–1932, 2009.

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm). In Drosophila, GCLc, but not GCLm, contains a nuclear localization signal (NLS). Drosophila S2 cells, constitutively expressing regular GCLc protein or expressing GCLc protein with a mutated NLS motif, were generated by transfection. In quiescent S2 cells, GCLc is aggregated in the perinuclear cytosol and the nucleus, whereas GLCm resides solely in the cytosol. In actively proliferating S2 cells, expressing the normal NLS motif, GCLc migrates from the perinuclear cytoplasm into the nucleus, and the nuclear GSH level becomes elevated; in contrast, in proliferating cells, expressing the mutated NLS motif, neither does the GCLc migrate into the nucleus nor does the nuclear GSH amount rise. In S2 cells expressing wild type GCLc, perturbation of cellular redox state by exposure to cadmium resulted in the migration of GCLc into the nucleus but not in cells expressing GCLc with the mutated NLS motif. Overall, results indicated that GSH biosynthesis in the nucleus is associated with migration of only the GCLc subunit from the cytoplasm into the nucleus, and this migration requires the presence of an intact NLS.

In this study, our main objective was to determine whether energy restriction (ER) affects the rate of oxygen consumption of mice transiently or lastingly and whether metabolic rate plays a role in the ER-related extension of life span. We compared rates of resting oxygen consumption between C57BL/6 mice, whose life span is prolonged by ER, and the DBA/2 mice where it is not, at 6 and 23 mo of age, following 40% ER for 2 and 19 mo, respectively. Mice of the 2 strains that consumed food ad libitum (AL) had a similar body mass at the age of 4 mo and consumed similar amounts of food throughout the experiment; however, the body weight subsequently significantly increased (20%) in the C57BL/6 mice but did not increase significantly in the DBA/2 mice. The resting rate of oxygen consumption was normalized as per g body weight, lean body mass, organ weight, and per mouse. The resting rate of oxygen consumption at 6 mo was significantly higher in AL DBA/2 mice than the AL C57BL/6 mice for all of the criteria except organ weight. A similar difference in AL mice of the 2 strains was present at 23 mo when resting oxygen consumption was normalized to body weight. Resting oxygen consumption was lowered by ER in both age groups of each strain according to all 4 criteria used for normalization, except body weight in the C57BL/6 mice. The effect of ER on resting oxygen consumption was thus neither transient nor age or strain dependent. Our results suggest that ER-induced extension of life span occurs in the mouse genotype in which there is a positive imbalance between energy intake and energy expenditure.

Peroxiredoxin 5 is a distinct isoform of the peroxiredoxin gene family. The antioxidative and anti-apoptotic functions of peroxiredoxin 5 have been extensively demonstrated in cell culture experiments. In the present paper, we provide the first functional analysis of peroxiredoxin 5 in a multicellular organism, Drosophila melanogaster. Similar to its mammalian, yeast or human counterparts, dPrx5 (Drosophila peroxiredoxin 5) is expressed in several cellular compartments, including the cytosol, nucleus and the mitochondrion. Global overexpression of dPrx5 in flies increased resistance to oxidative stress and extended their life span by up to 30% under normal conditions. The dprx5(-/-) null flies were comparatively more susceptible to oxidative stress, had higher incidence of apoptosis, and a shortened life span. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis revealed that the dprx5(-/-) null mutant had discernible tissue-specific apoptotic patterns, similar to those observed in control flies exposed to paraquat. In addition, apoptosis was particularly notable in oenocytes. During development the dPrx5 levels co-varied with ecdysone pulses, suggesting inter-relationship between ecdystreroids and dPrx5 expression. The importance of dPrx5 for development was further underscored by the embryonic lethal phenotype of progeny derived from the dprx5(-/-) null mutant. Results from the present study suggest that the antioxidant and anti-apoptotic activities of dPrx5 play a critical role in development and aging of the fly.

Coenzyme Q(10) (CoQ(10)) is widely consumed as a dietary supplement to enhance bioenergetic capacity and to ameliorate the debilitative effects of the aging process or certain pathological conditions. Our main purpose in this study was to determine whether CoQ(10) intake does indeed attenuate the age-associated losses in motor, sensory, and cognitive functions or decrease the rate of mortality in mice. Mice were fed a control nonpurified diet or that diet containing 0.68 mg/g (low dosage) or 2.6 mg/g (high dosage) CoQ(10), starting at 4 mo of age, and were tested for sensory, motor, and cognitive function at 7, 15, and 25 mo of age. Amounts of the ubiquinols CoQ(9)H(2) and CoQ(10)H(2) measured in a parallel study were augmented in the cerebral cortex but not in any other region of the brain. Intake of the low-CoQ(10) diet did not affect age-associated decrements in muscle strength, balance, coordinated running, or learning/memory, whereas intake at the higher amount increased spontaneous activity, worsened the age-related losses in acuity to auditory and shock stimuli, and impaired the spatial learning/memory of old mice. The CoQ(10) diets did not affect survivorship of mice through 25 mo of age. Our results suggest that prolonged intake of CoQ(10) in low amounts has no discernable impact on cognitive and motor functions whereas intake at higher amounts exacerbates cognitive and sensory impairments encountered in old mice. These findings do not support the notion that CoQ(10) is a fitness-enhancing or an "antiaging" substance under normal physiological conditions.

The conduction process as well as the unipolar resistive switching behavior of Au∕HfO2∕TiN metal-insulator-metal structures were investigated for future nonvolatile memory applications. With current-voltage measurements performed at different temperatures (200–400K), the Poole–Frenkel effect as conduction process was identified. In particular, we extracted a trap energy level at ϕt=0.35±0.05eV below the HfO2 conduction band to which a microscopic origin is tentatively assigned. From current-voltage measurements of Au∕HfO2∕TiN structures, low-power (as low as 120μW) resistive switching was observed. The required forming process is shown to be an energy-induced phenomenon. The characteristics include electric pulse-induced resistive switching by applying pulses up to 100μs and a retention time upon continuous nondestructive readout of more than 104s.

The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4). The mean life spans of G6PD overexpressor flies were extended, in comparison with driver and responder controls, as follows: armadillo-GAL4 (up to 38%), Tubulin-GAL4 (up to 29%), C23-GAL4 (up to 27%), da-GAL4 (up to 24%), D42-GAL4 (up to 18%), and Appl-GAL4 (up to 16%). The G6PD enzymatic activity was increased, as were the levels of NADPH, NADH, and the GSH/GSSG ratio. Resistance to experimental oxidative stress was enhanced. Furthermore, metabolic rates and fertility were essentially the same in G6PD overexpressors and control flies. Collectively, the results demonstrate that enhancement of the NADPH biosynthetic capability can extend the life span of a relatively long-lived strain of flies, which supports the oxidative stress hypothesis of aging.

Nitration and oxidation of tyrosine, tryptophan, and methionine residues in proteins are potential markers of their interaction with peroxynitrite. This chapter describes the procedure for the detection of these nitro-oxidative modifications by tandem mass spectrometry. The peptide YGDLANWMIPGK, shown to contain a nitrohydroxytryptophan in the mitochondrial enzyme succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT) in vivo, was synthesized and exposed to peroxynitrite in order to test whether an identical tryptophan derivative could be generated in vitro. Data show that the occurrence of specific fragmented ions corresponding to the oxidation of methionine, nitration of tyrosine, and nitration/oxidation of tryptophan residues can be used to identify the sites of the nitration and oxidation of proteins in vitro and in vivo. It is also demonstrated that a nitrohydroxy addition to the tryptophan, similar to that present in SCOT in vivo, can be produced in vitro.