Shoichi Tahara

THE EFFECT OF EXERCISE TRAINING ON OXIDATIVE DAMAGE OF LIPIDS, PROTEINS, AND DNA IN RAT SKELETAL MUSCLE: EVIDENCE FOR BENEFICIAL OUTCOMES

Moderate daily exercise is known to be beneficial to health, reducing risks of a number of age-related disorders. Molecular mechanisms that bring about these effects are not clear. In contrast, it has been claimed that some types of prolonged physical exertion are detrimental to health because active oxygen species are generated excessively by enhanced oxygen consumption. Using two age groups of rats, young (4 week) and middle aged (14 months), we investigated the effects of long-term swimming training on the oxidative status of phospholipids, proteins, and DNA. The concentration of thiobarbituric acid reactive substances and 4-hydroxynonenal protein adducts did not differ in the gastrocnemius muscle between exercised and nonexercised animals in the two age groups. The extent of carbonylation in a protein of molecular weight around 29 KDa and the amount of 8-hydroxydeoxyguanosine in nuclear DNA were smaller (p<.05) in the exercised rats than in the sedentary animals. Activities of DT-diaphorase (C1: 29.3+/-1.9; C2: 36.1+/-2.6; E1: 27.2+/-1.3; C2: 33.4+/-2.9 nmol/mg protein) and proteasome, a major proteolytic enzyme for oxidatively modified proteins were significantly higher in the exercised animals of both age groups (p<.05). The adaptive response against oxidative stress induced by moderate endurance exercise constitutes a beneficial effect of exercise.

SOD1 (Copper/Zinc Superoxide Dismutase) Deficiency Drives Amyloid β Protein Oligomerization and Memory Loss in Mouse Model of Alzheimer Disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282–11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of Nϵ-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.

The effects of moderate-, strenuous- and over-training on oxidative stress markers, DNA repair, and memory, in rat brain

We have tested the hypothesis that training with moderate- (MT), strenuous- (ST), or over- (OT) load can cause alterations in memory, lipid peroxidation, protein oxidation, DNA damage, activity of 8-oxoG-DNA glycosylase (OGG1) and brain-derived neurotrophic factor (BDNF), in rat brain. Rat memory was assessed by a passive avoidance test and the ST and OT group demonstrated improved memory. The content of BDNF was increased only in the OT group. The oxidative damage of lipids and DNA, as measured by thiobarbituric acid reactive substances (TBARS), and 8-hydroxydeoxyguanosine (8-OHdG), did not change significantly with exercise. Similarly, the activity of DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), was not altered with exercise training. On the other hand, the content of reactive carbonyl derivatives (RCDs) decreased in all groups and the decrease reached significance levels in the ST and OT groups. The activity of the proteasome complex increased in the brain of OT. The findings of this study imply that over-training does not induce oxidative stress in the brain and does not cause loss of memory. The improved memory was associated with enhanced BDNF content.

Regular exercise reduces 8-oxodG in the nuclear and mitochondrial DNA and modulates the DNA repair activity in the liver of old rats

Exercise is often said to increase the generation of reactive oxygen species that are potentially harmful. On the other hand, regular exercise has various health benefits even late in life. The specific aim of this study was to explore effects of regular exercise on oxidative status of DNA in aged animals. We report that 2 months of regular treadmill running of aged rats (21 month old) significantly reduced 8-oxodG content to the level of young adult animals (11 month old) in both nuclear and mitochondrial DNA of the liver. The mitochondrial DNA showed 10-fold higher content of the oxidative lesion than the nuclear DNA. The levels in old animals were 2- and 1.5-fold higher than that in young adults for the nucleus and mitochondria, respectively. The activity of the repair enzyme OGG1 was upregulated significantly in the nucleus but not in mitochondria by the exercise. To our knowledge, this is the first report demonstrating that regular exercise can reduce significantly oxidative damage to both the nuclear and mitochondrial DNA. We suggest that the apparent beneficial outcomes in reducing the DNA damage by regular exercise can be interpreted in terms of hormetic effect by moderate oxidative stress and potential adaptation to stronger stresses.

Single bout of exercise eliminates the immobilization-induced oxidative stress in rat brain

We were interested in the effects of immobilization (IM), a single bout of exercise (E) and immobilization followed by exercise (EIM) on memory and oxidative damage of macromolecules in hippocampus of rat brain. Eight hours of IM resulted in impairment of passive avoidance test (memory retrieval deficit) and increased latency to start locomotion in an open-field test. Two hours of swimming did not significantly alter the memory retrieval deficit and latency, while the EIM group had longer latency and similar memory than control and E groups. The oxidative damage of lipids, proteins and nuclear DNA increased significantly in IM group and no increase was observed in E and EIM animals. The activity of proteasome was not altered in any groups. The activity of glutamine synthetase (GS) was decreased in IM group (P < 0.05), this down regulation was not observed in E and EIM groups. These data suggest that oxidative damage of macromolecules is associated with impaired cognitive function. Single bout of exercise after immobilization eliminates the oxidative damage of macromolecules and normalizes memory function, probably by its ability to restore the activity level of GS and eliminate the consequences of immobilization-induced prolonged efflux of glutamate.

Oxidative stress in skeletal muscle causes severe disturbance of exercise activity without muscle atrophy.

The increase in reactive oxygen species (ROS) levels that occurs during intense exercise has been proposed to be one of the major causes of muscle fatigue. In addition, the accumulation of cellular damage due to ROS is widely regarded to be one of the factors triggering age-related pathological conditions in skeletal muscle. To investigate the pathological significance of oxidative stress in skeletal muscle, we generated skeletal muscle-specific manganese superoxide dismutase-deficient (muscle-Sod2(-/-)) mice. The mutant mice showed severe disturbances in exercise activity, but no atrophic changes in their skeletal muscles. In histological and histochemical analyses, the mutant mice showed centralized nuclei in their muscle fibers and selective loss of enzymatic activity in mitochondrial respiratory chain complexes. In addition, the mutant mice displayed increased oxidative damage and reduced ATP content in their muscle tissue. Furthermore, a single administration of the antioxidant EUK-8 significantly improved exercise activity and increased the cellular ATP level in skeletal muscle. These results imply that the superoxide anions generated in mitochondria play a pivotal role in the progression of exercise intolerance.

Age‐related increase of superoxide generation in the brains of mammals and birds

Oxidative stress, an imbalance between endogenous levels of oxygen radicals and antioxidative defense, increases with aging. However, it is not clear which of these two factors is the more critical. To clarify the production of oxygen radicals increases with age, we examined oxygen radical‐dependent chemiluminescent signals in ex vivo brain slices using a novel photonic imaging method. The chemiluminescent intensity was significantly decreased by the membrane permeable superoxide dismutase (SOD)/catalase mimic, but not by Cu,Zn‐SOD. Inhibitors for complex I, III, and IV of the mitochondrial electron transport chain transiently enhanced the chemiluminescent signal. The superoxide‐dependent chemiluminescent intensity in senescence accelerated mouse (SAM) brain tissues increases with age. Moreover, the slope of the age‐dependent increase was steeper in SAMP10, a strain characterized by a short lifespan and atrophy in the frontal cerebral cortex, than the senescence‐resistant strain SAMR1, which has a longer lifespan. An increase in chemiluminescence with age was also observed in C57/BL6 mice, Wistar rats, and pigeons, although levels of chemiluminescence were lower in the pigeons than murines. The rate of age‐related increases of superoxide‐dependent chemiluminescence was inversely related to the maximum lifespan of the animals. The activity of superoxide dismutase was unchanged during the aging process in the brain. This suggested that superoxide production itself may increase with age. We speculated that reactive oxygen may be a signal to determine the aging process.

Formation of 8-oxo-2'-deoxyguanosine in the DNA of human diploid fibroblasts by treatment with linoleic acid hydroperoxide and ferric ion.

Lipid peroxides are suggested to be related to the occurrence of a variety of diseases including cancer and atherosclerosis. We examined whether lipid peroxides cause oxidative damage to DNA in intact cells. Linoleic acid hydroperoxide (LOOH) and ferric chloride were used at concentrations at which separate treatment had no effect on the formation of 8-oxo-2′-deoxyguanosine (8-oxodG) in DNA or the survival rate of cultured human diploid fibroblasts, TIG-7. The amount of 8-oxodG in the cellular DNA increased significantly when TIG-7 cells were treated concurrently with LOOH and ferric chloride. In a LOOH concentration-dependent manner 8-oxodG was formed. However, no significant induction of the activities of superoxide dismutases, catalase, or glutathione peroxidase was observed under these conditions. The formation of 8-oxodG by lipid hydroperoxides seems to be due to the generation of reactive species other than superoxide radicals and hydrogen peroxide. These results indicate that some species formed during the reaction of lipid hydroperoxides with ferric ion can cause oxidative damage to DNA.

Accumulation of 8-Oxo-2’-Deoxyguanosine (as a Biomarker of Oxidative DNA Damage) in the Tissues of Aged Hamsters and Change in Antioxidant Enzyme Activities after Single Administration of N-Nitrosobis(2-Oxopropyl) Amine

Background: It has been reported that DNA oxidative damage accumulates with age. Two reasons for this phenomenon are the decline in the antioxidant system and the decline in the repair system. It is not clear which of these is the main reason. Objective: To study whether the decline in antioxidant enzyme activities causes the accumulation of DNA oxidative damage, an experimental study was performed with hamsters. Methods: Seventy-four female Syrian golden hamsters were divided into 2 groups: a young group (28 hamsters), and an aged group (46 hamsters). The hamsters in the aged group were kept in our laboratory until they were 18 months old. The levels of 8-oxo-2′-deoxyguanosine (8-oxodG) and the activities of antioxidant enzymes, i.e. catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), were measured in both groups. Furthermore, the same parameters were measured in the pancreas and liver following administration of N-nitrosobis(2-oxopropyl)amine (BOP), an inducer of oxidative stress in the hamster pancreas. Results: In the mid brain, cerebellum, lung, heart, spleen and kidney, the 8-oxodG contents in aged hamsters were significantly higher than those in young hamsters. GPx activity decreased with age in the lung, liver and kidney, whereas SOD activity increased in the lung and liver but decreased in the kidney of aged animals. Catalase activity increased in the cerebrum, heart, pancreas and kidney but decreased in the lung and spleen of aged hamsters. When the pancreatic levels of 8-oxodG and antioxidant enzymes were measured after BOP administration, there was no clear-cut relation between the changes in those levels. Conclusions: From these results the increase in 8-oxodG contents in aged hamsters does not seem to be related to the antioxidant system but rather to a possible decline in the repair system against oxidative damage.

Age-related increase of reactive oxygen generation in the brains of mammals and birds: Is reactive oxygen a signaling molecule to determine the aging process and life span?

Since Harman proposed the “free‐radical theory of aging”, oxidative stress has been postulated to be a major causal factor of senescence. The accumulation of oxidative stress‐induced oxidatively modified macromolecules, including protein, DNA and lipid, were found in tissues during the aging process; however, it is not necessarily clear which factor is more critical, an increase in endogenous reactive oxygen and/or a decrease in anti‐oxidative defense, to the age‐related increase in oxidative damage. To clarify the increasing production of reactive oxygen with age, we examined reactive oxygen‐dependent chemiluminescent (CL) signals in ex vivo brain slices prepared from different‐aged animal brains during hypoxia–reoxygenation treatment using a novel photonic imaging method. The CL signal was intensified during reoxygenation. The signals in SAMP10 (short‐life strain) and SAMR1 (control) brain slices increased with aging. The slope of the increase of CL intensity with age in P10 was steeper than in R1. Age‐dependent increase of CL intensity was also observed in C57BL/6 mice, Wistar rats and pigeons; however, superoxide dismutase (SOD) activity in the brain did not change with age. These results suggest that reactive oxygen production itself increased with aging. The rate of age‐related increases of CL intensity was inversely related to the maximum lifespan of animals. We speculate that reactive oxygen might be a signaling molecule and its levels in tissue might determine the aging process and lifespan. Decelerating age‐related increases of reactive oxygen production are expected to be a potent strategy for anti‐aging interventions. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S10–S24.