Relationship between levels of oxidative DNA damage, lipid peroxidation and mitochondrial membrane potential in young and old F344 rats

The extent of in vivo oxidative damage has been known to be cumulative over the period of the life of mammals. Our hypothesis is that there should be a positive correlation between the levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) in major rat tissues. We also investigated whether increased level of oxidative stress causes a decrease in the mitochondrial membrane potential of peripheral lymphocytes of old rats using the MitoTracker Red fluorochrome. Our results show positive correlations between 8OHdG and 8-iso-PGF(2alpha) for liver, brain and kidney measured by HPLC-UV-ECD (electrochemical detector) and EIA methods, respectively. However, heart tissues show a negative correlation. The mitochondrial membrane potential of old rat lymphocytes records significant decrease compared with the young lymphocytes. Based on our results, we conclude that in ageing studies, specific tissues need to be examined in order to measure the localised DNA damage and lipid peroxidation as different tissues display different extent of oxidative damage. We believe this approach of using combined markers is useful to verify the true efficacy of health intervention studies in animals and humans. In addition, the isoprostane assay can be further developed looking at lipid peroxidation as a potential marker in ageing studies.     

Effect of hyperthyroidism and hypothyroidism on rat red blood cell insulin receptors and catecholamines: relationship with cellular ageing

Insulin receptor activity and its relationship with catecholamines in rat young, middle aged and old red blood cells were investigated in experimental hypothyroidism and hyperthyroidism. In control animals, a loss of insulin receptor activity was found with cellular ageing and increased levels of norepinephrine, epinephrine and glycosylated hemoglobin. There was down regulation of insulin receptors together with alterations in membrane bound catecholamines in thyroid hormones imbalances. These results suggest that loss of insulin receptor in cellular ageing is probably part of a more generalised alteration and rat serves as an excellent model in defining the role of thyroid hormones in carbohydrate tolerance.     

Aging-Related Changes in Proteasomal Activity and Activity of Neutral Proteinases in Brain Tissues of the Rats

We compared the proteasomal activity and activity of neutral proteinases in tissues of the neocortex and cerebellum in old (18 months) and young mature (5 months) rats. We found that, in homogenates of the tissues obtained from brains of old animals, the chymotrypsin-like activity of the proteasome complex in the cortex increased by 50% as compared with the control, while in the cerebellum such an activity remained practically unchanged. Peptidylglutamyl peptide hydrolase proteasomal activity increased on average by 72% in the cortex and by 14% in the cerebellum. Protamine-splitting activity, which is indicative of the activity of neutral proteinases, dropped insignificantly in the cortex and cerebellum (by 16.4 and 15.3%, respectively). The data obtained allow us to suppose that aging-related changes in brain cells result from disturbances of the functional connections between lysosomal and proteasomal proteolysis.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 11–14, January–February, 2005.     

Posttranslational deamidation of crystalline lens proteins during animal aging

The amide content of total proteins and protein fractions (alpha-, beta-, gamma-cristallins and albuminoid) from cortex and nuclear lens zones of cattle has been investigated. The amide content in proteins of cortex and nuclear lens of young animals (1,5-2 years old) is the same. The decrease of the amide content in the proteins of nuclear lenz zone of old animals (6-12 years old) is due to fraction of readily hydrolysed amides. The data obtained prove the posttranslational desamidation of lens proteins at ageing. beta-Cristallines--the proteins with the highest amides content is exposed to posttranslational desamidation most of all.     

Physiological ageing of red blood cells and changes in membrane carbohydrates

Evidence is presented to indicate a generalized role for the terminal sialic acid residues of circulating erythrocytes. After reinjection into their donors, neuraminidase-treated human, rabbit, rat and dog erythrocytes were promptly removed from the circulation : intect erythrocytes, previously incubated under the same conditions but without neuraminidase, were removed after a significantly longer period. The neuraminidase-treated erythrocytes were cleared by the liver and in a little part by the spleen. Old and young human, rabbit, rat erythrocytes contained different quantities of stromal sialic acid, significantly lowered on the old cells. But the half-life of old intact rabbit erythrocytes is sigificantly shorter than that of neuraminidase-treated young erythrocytes with a similar minidase-treated young erythrocytes with a similar sialic acid content. Indeed sialic acid is not the only carbohydrate component of the membrane that is decreased during erythrocyte ageing, the others membranous sugars are decreased too. Theses changes in the carbohydrate moity could have a role in the clearance of the erythrocytes.     

Influence of Age on Arsenic-Induced Oxidative Stress in Rat

Influence of age on arsenic-induced (0.05, 0.1, and 0.2 lethal dose to 50?% population (LD₅₀) given intraperitoneally) oxidative stress was investigated in young, adult, and old rats at days?7 and 14 post-exposure. A significant dose-dependent effect of arsenic on biochemical variables suggestive of oxidative stress was noted at day?7 following exposure in old rats. The parameters which were significantly altered include an increased reactive oxygen species, thiobarbituric acid reactive substances (TBARS), catalase activity accompanied by a decreased glutathione level. At day?14 following arsenic exposure (0.05 and 0.1 LD₅₀ dose), we observed a significant oxidative injury as evident from significant depletion of superoxide dismutase (SOD) and catalase activities in blood and tissues in addition to more pronounced accumulation of arsenic in blood and tissues. Interestingly, the toxicity was pronounced in young and old rats compared with adult rats. Accumulation of arsenic found to be more prominent in old rats compared with young and adult, which might be due to impaired metabolism with ageing. We conclude that young and old animals are more vulnerable to the arsenic-induced oxidative injury which is comparable with arsenic accumulation in blood and tissues and duration of exposure.     

A pilot study on alterations of brain chromosomal protein phosphorylation by steroids during ageing.

The modulatory effects of the steroid hormones estradiol-17 beta and hydrocortisone on phosphorylation of chromosomal proteins of the cerebral hemispheres (CH) of young and old rats was studied in vitro. The results indicate that the extent of phosphorylation decreases in both histone and non-histone-chromosomal (NHC) protein fractions as the age proceeds. Besides, both the hormones stimulate phosphorylation in young as well as old rats. However the pattern of stimulation shown by estradiol differs from that by hydrocortisone. In addition the extent of stimulation by these hormones is significantly reduced in old rats. Modulation of phosphorylation as provoked by these two hormones seems to play an important role in differential gene expression during ageing.     

Opposite pathobiochemical fate of pyruvate kinase and adenylate kinase in aged rat skeletal muscle as revealed by proteomic DIGE analysis

Sarcopenia is the drastic loss of skeletal muscle mass and strength during ageing. In order to better understand the molecular pathogenesis of age-related muscle wasting, we have performed a DIGE analysis of young adult versus old rat skeletal muscle. Proteomic profiling revealed that out of 2493 separated 2-D spots, 69 proteins exhibited a drastically changed expression. Age-dependent alterations in protein abundance indicated dramatic changes in metabolism, contractile activity, myofibrillar remodelling and stress response. In contrast to decreased levels of pyruvate kinase (PK), enolase and phosphofructokinase, the mitochondrial ATP synthase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and adenylate kinase (AK) were increased in senescent fibres. Higher expression levels of myoglobin and fatty acid binding-protein indicated a shift to more aerobic-oxidative metabolism in a slower-twitching aged fibre population. The drastic increase in alphaB-crystallin and myotilin demonstrated substantial filament remodelling during ageing. An immunoblotting survey of selected muscle proteins confirmed the pathobiochemical transition process in aged muscle metabolism. The proteomic analysis of aged muscle has identified a large cohort of new biomarkers of sarcopenia including opposite changes in PK and AK, which might be useful for the design of improved diagnostic procedures and/or therapeutic strategies to counteract ageing-induced muscle degeneration.     

Energy metabolism in the liver of young and old rats during starvation

The concentration of key intermediate products of energy metabolism is determined in the liver of young and old rats under normal conditions and 24h after fasting. A decrease in the stationary concentrations of glucose, glucose-6-phosphate, ATP, ADP and AMP and an increase in the concentrations of lactate, glutamate, alpha-glycerophosphate and Pi were found in the liver of rats in ageing. The carbohydrate metabolism response to fasting is also disturbed. The total content of adenine nucleotides it the rat liver during ageing is 25-30% lower. The deficit of adenine nucleotides is not associated with the activation of AMP-desaminase; it may result from both physiological and functional disturbances in the ageing liver.     

Effect of hyperglycemia and hyperinsulinemia on rat red blood cell insulin receptors and catecholamines: relationship with cellular ageing

Insulin receptor activity and its relationship with catecholamines in rat young, middle aged and old red blood cells were investigated in short term (4-6) weeks and long term (6-8 months) hyperglycemia and hyperinsulinemia. Loss of insulin receptor activity is linear with cellular ageing and norepinephrine and epinephrine levels increase with age together with levels of glycosylated hemoglobin in control animals and this correlation is altered in hyperglycemia and hyperinsulinemia. These results suggest that loss of insulin receptor in cellular ageing is probably part of a more generalised alteration which is possibly brought about by glycosylation.     

Changes in nonpolar aldehydes in bean cotyledons during ageing

Ageing of plant organs is accompanied by an increased production of free radicals what results in membrane lipid peroxidation. Non-polar aldehydes originating from this process interact with the cellular material to form the fluorescent end-products, lipofuscin-like pigments (LFP). Their formation was studied both qualitatively and quantitatively in ageing of bean cotyledons. The concentration of lipofuscin-like pigments increased 9-fold in 14-d-old (senescent) cotyledons in relation to 8-d-old (young) cotyledons. HPLC fractionation patterns indicate changes in their composition during ageing. The LFP increase in old cotyledons was accompanied by elevated levels of non-polar aldehydes that increased during ageing to 167 %. The composition of aldehydes was studied by mass spectrometry. The most abundant fraction in both young and old cotyledon was represented by C12 aldehydes, which comprised both saturated and unsaturated species. We have observed differences in abundances of individual aldehydes between the young and the old cotyledons that might explain the differences in the composition of lipofuscin-like pigments. These results support the involvement of free radicals in plant ageing; however, it is suggested that plant aldehydic products of lipid peroxidation differ from those found in animals.     

Evaluation of different methods detecting intracellular generation of free radicals

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn²⁺. All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.     

Understanding tenderness variability and ageing changes in buffalo meat: biochemical,ultrastructural and proteome characterization

Understanding of biological impact of proteome profile on meat quality is vital for developing different approaches to improve meat quality. Present study was conducted to unravel the differences in biochemical, ultrastructural and proteome profile of longissimus dorsi muscle between buffaloes (Bubalus bubalis) of different age groups (young v. old). Higher (P<0.05) myofibrillar and total protein extractability, muscle fibre diameter, and Warner-Bratzler shear force (WBSF) values was observed in old buffalo meat relative to meat from young buffaloes. Scanning electron microscopy photographs revealed reduced fibre size with increased inter-myofibrillar space in young compared with old buffalo meat. Transmission electron microscopy results revealed longer sarcomeres in young buffalo meat relative to meat from old buffaloes. Proteomic characterization using two-dimensional gel electrophoresis (2DE) found 93 differentially expressed proteins between old and young buffalo meat. Proteome analysis using 2DE revealed 191 and 95 differentially expressed protein spots after 6 days of ageing in young and old buffalo meat, respectively. The matrix assisted laser desorption ionization time-of flight/time-of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of selected gel spots helped in identifying molecular markers of tenderness mainly consisting of structural proteins. Protein biomarkers identified in the present study have the potential to differentiate meat from young and old buffaloes and pave the way for optimizing strategies for improved buffalo meat quality.     

Purification and characterization of thiol proteinase inhibitor from rat liver

A thiol proteinase inhibitor was purified from rat liver by essentially the same procedure as reported previously (Kominami, E., Wakamatsu, N., and Katunuma, N. (1981) Biochem. Biophys. Res. Commun. 99, 568-575), but without heat treatment. The purified inhibitor appears homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor has Mr = 12,500 and contains 50.5% of polar amino acid residues, 9.3% aromatic amino acids, and no tryptophan. The presence of 2 half-cystines/molecule and the absence of free thiol groups indicate that the inhibitor possesses one disulfide bridges. The inhibitor inhibits cathepsin H by forming an enzyme-inhibitor complex in a molar ratio of 1:1. It inhibits most thiol proteinases such as cathepsin H, L, B, and C, papain, and ficin, but not calcium-activated neutral proteinase or serine proteinases or carboxyl proteinases. The inhibitor was found in various rat tissues. Immunological diffusion analysis with anti-liver thiol proteinase inhibitor serum indicated that the rat liver inhibitor is immunologically identical with the inhibitors from other rat tissues. On subcellular fractionation of rat liver, the thiol proteinase inhibitor was recovered in the cytosol fraction.     

Age-related increase in xanthine oxidase activity in human plasma and rat tissues

This study assessed the role of xanthine oxidase in vascular ageing. A positive correlation between xanthine oxidase activity and age was found in human plasma. Similar results were found in rat plasma. Xanthine oxidase expression and activity in homogenates from the aortic wall were significantly higher in samples from old rats than in their young counterparts (p < 0.01). In rat skeletal muscle homogenates both xanthine oxidase expression and activity showed a similar age-related profile. Superoxide production by xanthine oxidase in aortic rings was higher in aged rats. Uric acid, the final product of xanthine oxidase has been proposed as a risk factor for coronary heart disease and an independent marker of worse prognosis in patients with moderate-to-severe chronic heart failure. These results give a possible explanation for this correlation and underscore the role of xanthine oxidase in ageing.     

Distribution of high mobility group proteins in different tissues of rats during aging

The distribution of high mobility group (HMG) proteins has been studied in the liver, brain, kidney, lung, spleen, testis, thymus, and heart of young (19 weeks) and old (118 weeks) rats. These proteins were extracted with perchloric acid, fractionated by CM-Sephadex column chromatography, and analysed by acetic acid-urea polyacrylamide slab gel electrophoresis. As compared with that in young rats, the level of total HMG proteins in the old increased in liver and lung, decreased in thymus, heart, brain, and kidney, and remained unchanged in spleen and testis. In particular, the levels of HMG 1 and 2 were maximum in the thymus of young rats and dropped drastically in the old. However, the amount of HMG 17 was high in the spleen of both young and old rats, though it was comparatively higher in the former. Such age-dependent variation in the level of HMG proteins of different tissues denotes indirectly differences in the functional state of chromatin, and in growth and activity of cells, during aging.     

The effect of one year's swimming exercise on oxidant stress and antioxidant capacity in aged rats

The effect of exercise on oxidant stress and on alterations in antioxidant defense in elderly has been investigated extensively. However, the impact of regularly performed long-term physical activity starting from adulthood and prolonged up to the old age is not yet clear. We have investigated the changes in the activities of antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) - and lipid peroxidation in various tissues of rats which had performed (old-trained) or had not performed (old-control) regular swimming exercise for one year. These animals were compared with young-sedentary rats. Increased lipid peroxidation was observed with ageing in all tissues (heart, liver, kidney, striated muscle) and swimming had no additional effect on this elevation of lipid peroxidation. Heart and striated muscle SOD activites, and striated muscle CAT activity increased as a consequence of ageing, whereas kidney and liver CAT activities, as well as GPx activities in kidney, liver, lung and heart were significantly decreased compared to young controls. Lung and heart SOD, liver CAT activities as well as GPx activities in liver, lung and heart were increased significantly in rats which performed exercise during ageing, compared to the old-control group. These findings suggest that lifelong exercise can improve the antioxidant defense in many tissues without constituting any additional oxidant stress.     

Effects of Ferrofluid and Phytoalexin Spirobrassinin on Thioflavin-T-Based Fluorescence in Cerebrospinal Fluid of the Elderly and Multiple Sclerosis Patients

It is well known that misfolded peptides/proteins can play a role in processes of normal ageing and in the pathogenesis of many diseases including Alzheimer’s disease. Previously, we evaluated samples of cerebrospinal fluid from patients with Alzheimer’s disease and multiple sclerosis by means of thioflavin-T-based fluorescence. We observed attenuated effects of magnetite nanoparticles operated via anti-aggregation actions on peptides/proteins from patients with Alzheimer’s disease but not from those with multiple sclerosis when compared to age-related controls. In this study, we have evaluated the in vitro effects of anti-aggregation operating ferrofluid and phytoalexin spirobrassinin in the cerebrospinal fluid of patients with multiple sclerosis and Alzheimer’s disease. We have found significant differences in native fluorescence (λ excitation = 440 nm, λ emission = 485 nm) of samples among particular groups (young controls < multiple sclerosis, Alzheimer’s disease < old controls). Differences among groups were observed also in thioflavin-T-based fluorescence (young controls = multiple sclerosis < Alzheimer’s disease < old controls) and the most marked change from native to thioflavin-T-based fluorescence was found in young controls (28–40 years old people). Both ferrofluid and spirobrassinin evoked drops in thioflavin-T-based fluorescence; however, ferrofluid was more efficient in old controls (54–75 years old people) and spirobrassinin in multiple sclerosis patients, both compared to young controls. The results are discussed especially in relation to aggregated peptides/proteins and liposoluble fluorescent products of lipid peroxidation. Based on the significant effect of spirobrassinin in vitro, we suggest that spirobrassinin may be of therapeutic value in multiple sclerosis.