Relationship between antioxidant potential and oxidative damage to lipids, proteins and DNA in aged rats

Oxidative stress has been implicated to play a major role in aging and age-related diseases. In the present study, we investigated the effects of aging on the total antioxidant capacity, uric acid, lipid peroxidation, total sulfhydryl group content and damage to DNA in adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. The antioxidant capacity, determined by phycoerythrin-based TRAP method (total peroxyl radical-trapping potential) was significantly decreased in the plasma and myocardium of old and senescent rats, whereas plasma level of uric acid was elevated in 26-month-old rats. Age-related decline in plasma and heart antioxidant capacity was accompanied by a significant loss in total sulfhydryl group content, increased lipid peroxidation and higher DNA damage in lymphocytes. Correlations between TRAP and oxidative damage to lipids, proteins and DNA suggest that the decline in antioxidant status may play an important role in age-related accumulation of cell damage caused by reactive oxygen species.     

Age-related alterations in oxidatively damaged proteins of mouse skeletal muscle mitochondrial electron transport chain complexes

Age-associated mitochondrial dysfunction is a major source of reactive oxygen species (ROS) and oxidative modification to proteins. Mitochondrial electron transport chain (ETC) complexes I and III are the sites of ROS production and we hypothesize that proteins of the ETC complexes are primary targets of ROS-mediated modification which impairs their structure and function. The pectoralis, primarily an aerobic red muscle, and quadriceps, primarily an anaerobic white muscle, have different rates of respiration and oxygen-carrying capacity, and hence, different rates of ROS production. This raises the question of whether these muscles exhibit different levels of oxidative protein modification. Our studies reveal that the pectoralis shows a dramatic age-related decline in almost all complex activities that correlates with increased oxidative modification. Similar complex proteins were modified in the quadriceps, at a significantly lower level with less change in enzyme and ETC coupling function. We postulate that mitochondrial ROS causes damage to specific ETC subunits which increases with age and leads to further mitochondrial dysfunction. We conclude that physiological characteristics of the pectoralis vs quadriceps may play a role in age-associated rate of mitochondrial dysfunction and in the decline in tissue function.     

Hippocampal mitochondrial dysfunction in rat aging

Hippocampus mitochondrial dysfunction with impaired electron transfer and increased oxidative damage was observed upon rat aging. Hippocampal mitochondria of aged (12 mo) and senescent (20 mo) rats showed, compared with young (4 mo) rats, marked decreases in the rate of state 3 respiration with NAD-dependent substrates (32-51%) and in the activities of mitochondrial complexes I (57-73%) and IV (33-54%). The activity of mitochondrial nitric oxide synthase was also decreased, 53-66%, with age. These losses in enzymatic activity were more marked in the hippocampus than in brain cortex or in whole brain. The histochemical assay of mitochondrial complex IV in the hippocampus showed decreased staining upon aging. Oxidative damage, determined as the mitochondrial content of thiobarbituric-acid reactive substances (TBARS) and protein carbonyls, increased in aged and senescent hippocampus (66-74% in TBARS and 48-96% in carbonyls). A significant statistical correlation was observed between mitochondrial oxidative damage and enzymatic activity. Mitochondrial dysfunction with shortage of energy supply is considered a likely cause of dysfunction in aged hippocampus.     

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Mitochondrially generated ROS increase with age and are a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS, which may impair normal complex activity. This study of heart mitochondria from young, middle-aged, and old mice reveals that there is an age-related decline in complex I and V activity that correlates with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of the aged mouse heart.     

Mitochondrial Decay in the Brains of Old Rats: Ameliorating Effect of Alpha-Lipoic Acid and Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-carnitine

To investigate the mitochondrial decay and oxidative damage resulting from aging, the activities/kinetics of the mitochondrial complexes were examined in the brains of young and old rats as well as in old rats fed R-α-lipoic acid plus acetyl-l-carnitine (LA/ALC). The brain mitochondria of old rats, compared with young rats, had significantly decreased endogenous antioxidants and superoxide dismutase activity; more oxidative damage to lipids and proteins; and decreased activities of complex I, IV and V. Complex I showed a decrease in binding affinity (increase in K_m) for substrates. Feeding LA/ALC to old rats partially restored age-associated mitochondrial dysfunction to the levels of the young rats. These results indicate that oxidative mitochondrial decay plays an important role in brain aging and that a combination of nutrients targeting mitochondria, such as LA/ALC, could ameliorate mitochondrial decay through preventing mitochondrial oxidative damage. Special issue article in honor of Dr. Akitane Mori.     

Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.     

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats.

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.     

Mitochondrial Ageing and the Beneficial Role of α-Lipoic Acid

Oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the ageing process. Mitochondria are known to be a rich source for the production of free radicals and, consequently, mitochondrial components are susceptible to lipid peroxidation (LPO) that decreases respiratory activity. In the present investigation, we have evaluated mitochondrial LPO, 8-oxo-dG, oxidized glutathione, reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and electron transport chain (ETC) complex activities in the brain of young versus aged rats. In aged rats, the contents of LPO, oxidized glutathione and 8-oxo-dG were high whereas reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities were found to be low. Lipoic acid administration to aged rats reduced the levels of mitochondrial LPO, 8-oxo-dG and oxidized glutathione and enhanced reduced glutathione, ATP, lipoic acid and ETC complex activities. In young rats lipoic acid administration showed only minimal lowering the levels of LPO, 8-oxo-dG and oxidized glutathione and slight increase in the levels of reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities. These findings suggest that the dithiol, lipoic acid, provides protection against age-related oxidative damage in the mitochondria of aged rats.     

Oxidatively damaged proteins of heart mitochondrial electron transport complexes

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.     

Effect of Aging on the Metabolism of Pyruvate and 3-Hydroxybutyrate in Nonsynaptic and Synaptic Mitochondria from Rat Brain

Abstract: Age-dependent changes in the oxidative metabolism in nonsynaptic and synaptic mitochondria from brains of 3, 12, and 24-month-old rats were investigated. When pyruvate and malate were used in conjunction as substrates, a significant reduction in State 3 respiration was observed in both mitochondrial populations from 12-and 24-month-old rats compared with 3-month-old animals. A similar age-dependent reduction in the oxidation of [1-¹¹C]pyruvate was also observed in nonsynaptic and synaptic mitochondria from senescent rats. Pyruvate dehydrogenase complex activity (both active and total) was, however, not decreased in the two mitochondrial populations from brains of 3, 12, and 24-month-old rats. When DL-3-hydroxybutyrate plus malate were used as substrates, a decrease in State 3 respiration was observed only in synaptic mitochondria from 24-month-old rats compared with 3- month-old animals. Similarly, an age-dependent reduction in the oxidation of 3-hydroxy[3-¹¹C]butyrate was also observed only in synaptic mitochondria from 12-and 24-month-old rats. However, a significant reduction in the activities of ketone body-metabolizing enzymes, namely, 3-hydroxybutyrate dehydrogenase, 3-ketoacid CoA transferase, and acetoacetyl-CoA thiolase was observed in both mitochondrlal populations from 12- and 24-month-old rats compared with 3 month-old animals. These findings show that specific alterations in oxidative metabolism occur in nonsynaptic and synaptic mitochondria from aging rats. The data also suggest that in addition to alterations in enzyme activities, permeability of anions (e.g. pyruvate) across the inner mitochondrial membrane may be altered in nonsynaptic and synaptic mitochondria from senescent animals.     

Mitochondrial Dysfunction and Electron Transport Chain Complex Defect in a Rat Model of Tenofovir Disoproxil Fumarate Nephrotoxicity

The long‐term use of tenofovir, a commonly used anti‐HIV drug, can result in renal damage. The mechanism of tenofovir disoproxil fumarate (TDF) nephrotoxicity is not clear, although it has been shown to target proximal tubular mitochondria. In the present study, the effects of chronic TDF treatment on the proximal tubular function, renal mitochondrial function, and the activities of the electron transport chain (ETC) complexes were studied in rats. Damage to proximal tubular mitochondria and proximal tubular dysfunction was observed. The impaired mitochondrial function such as the respiratory control ratio, 2‐(4,5‐dimethyl‐2‐thiazolyl)‐3,5‐diphenyl‐2H‐tetrazolium bromide (MTT) reduction, and mitochondrial swelling was observed. The activities of the electron chain complexes I, II, IV, and V were decreased by 46%, 20%, 26%, and 21%, respectively, in the TDF‐treated rat kidneys. It is suggested that TDF induced proximal tubular mitochondrial dysfunction and ETC defects may impair ATP production, resulting in proximal tubular damage and dysfunction.     

Aging alters the functional expression of enzymatic and non-enzymatic anti-oxidant defense systems in testicular rat Leydig cells

In aged rats, trophic hormone-stimulated testosterone secretion by isolated Leydig cells is greatly reduced. The current studies were initiated to establish a functional link between excess oxidative stress and the age-related decline in steroidogenesis. Highly purified Leydig cell preparations obtained from 5-month (young mature) and 24-month (old) Sprague-Dawley rats were employed to measure and compare levels of lipid peroxidation, non-enzymatic (alpha-tocopherol, ascorbic acid, and reduced/oxidized glutathione) and enzymatic (Cu, Zn-superoxide dismutase, Cu, Zn-SOD; Mn-superoxide dismutase, Mn-SOD; glutathione peroxidase-1, GPX-1, and catalase, CAT) anti-oxidants. The extent of lipid peroxidation (oxidative damage) in isolated membrane fractions was quantified by measuring the content of thiobarbituric acid-reactive substances (TBARS) under basal conditions, or in the presence of non-enzymatic or enzymatic pro-oxidants. Membrane preparations isolated from Leydig cells from old rats exhibited two- to three-fold enhancement of basal TBARS formation. However, aging had no significant effect on TBARS formation in response to either non-enzymatic or enzymatic pro-oxidants. Among the non-enzymatic anti-oxidants, the levels of reduced glutathione were drastically reduced during aging, while levels of alpha-tocopherol and ascorbic acid remained unchanged. Both steady-state mRNA levels and catalytic activities of Cu, Zn-SOD, Mn-SOD, and GPX-1 were also significantly lower in Leydig cells from 24-month-old rats as compared with 5-month-old control rats. In contrast, neither mRNA levels nor enzyme activity of catalase was sensitive to aging. From these data we conclude that aging is accompanied by reduced expression of key enzymatic and non-enzymatic anti-oxidants in Leydig cells leading to excessive oxidative stress and enhanced oxidative damage (lipid peroxidation). It is postulated that such excessive oxidative insult may contribute to the observed age-related decline in testosterone secretion by testicular Leydig cells.     

Time-course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging

The study of aging is critical for a better understanding of many age-related diseases. The free radical theory of aging, one of the prominent aging hypotheses, holds that during aging, increasing reactive oxygen species in mitochondria causes mutations in the mitochondrial DNA and damages mitochondrial components, resulting in senescence. Understanding a mitochondrial gene expression profile and its relationship to mitochondrial function becomes an important step in understanding aging. The objective of the present study was to determine mRNA expression of mitochondrial-encoded genes in brain slices from C57BL6 mice at four ages (2, 12, 18, and 24 months) and to determine how these altered mitochondrial genes influence age-related changes, including oxidative damage and cytochrome c in apoptosis. Using northern blot analysis, in situ hybridization, and immunofluorescence analyses, we analyzed changes in the expression of mitochondrial RNA encoding the mitochondrial genes, oxidative damage marker, 8-hydroxyguanosine (8-OHG), and cytochrome c in brain slices from the cortex of C57BL6 mice at each of the four ages. Our northern blot analysis revealed an increased expression of mitochondrial-encoded genes in complexes I, III, IV, and V of the respiratory chain in 12- and 18-month-old C57BL6 mice compared to 2-month-old mice, suggesting a compensatory mechanism that allows the production of proteins involved in the electron transport chain. In contrast to the up-regulation of mitochondrial genes in 12- and 18-month-old C57BL6 mice, mRNA expression in 24-month-old C57BL6 mice was decreased, suggesting that compensation maintained by the up-regulated genes cannot be sustained and that the down-regulation of expression results in the later stage of aging. Our in situ hybridization analyses of mitochondrial genes from the hippocampus and the cortex revealed that mitochondrial genes were over-expressed, suggesting that these brain areas are critical for mitochondrial functions. Our immunofluorescence analysis of 8-OHG and cytochrome c revealed increased 8-OHG and cytochrome c in 12-month-old C57BL6 mice, suggesting that age-related mitochondrial oxidative damage and apoptosis are associated with mitochondrial dysfunction. Our double-labeling analysis of in situ hybridization of ATPase 6 and our immunofluorescence analysis of 8-OHG suggest that specific neuronal populations undergo oxidative damage. Further, double-labeling analysis of in situ hybridization of ATPase 6 and immunofluorescence analysis of cytochrome c suggest cytochrome c release is related to mitochondrial dysfunction in the aging C57BL6 mouse brain. This study also suggests that these mitochondrial gene expression changes may relate to the role of mitochondrial dysfunction, oxidative damage, and cytochrome c in aging and in age-related diseases such as Alzheimer's disease and Parkinson's disease.     

Age-Dependent Impairment of Mitochondrial Function in Primate Brain

Abstract: It has been hypothesized that some of the functional impairments associated with aging are the result of increasing oxidative damage to mitochondrial DNA that produces defects in oxidative phosphorylation. To test this hypothesis, we examined the enzymes that catalyze oxidative phosphorylation in crude mitochondrial preparations from frontoparietal cortex of 20 rhesus monkeys (5-34 years old). Samples were assayed for complex I, complex II-III, complex IV, complex V, and citrate synthase activities. When enzyme activities were corrected for citrate synthase activities (to account for variable degrees of mitochondrial enrichment), linear regression analysis demonstrated a significant negative correlation of the activities of complex I (p < 0.002) and complex IV (p < 0.03) with age but no significant change in complex II-III or complex V activities. Relative to animals 6.9 ± 0.9 years old (n = 7), the citrate synthase-corrected activity of complex I was reduced by 17% in animals 22.5 ± 0.9 years old (n = 6) (p < 0.05) and by 22% in animals 30.7 ± 0.9 years old (n = 7) (p < 0.01). Similar age-related reductions in the activities of complexes I and IV were obtained when enzyme activities were corrected for complex II-III activity. These findings show an age-associated progressive impairment of mitochondrial complex I and complex IV activities in cerebral cortices of primates.     

Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria

Mitochondrial-derived oxidative injury contributes to cellular aging as well as to reperfusion-induced tissue damage. While the aging-heart suffers greater tissue damage following ischemia and reperfusion than the adult heart, the occurrence of aging-related alterations in mitochondrial oxidative metabolism in the elderly heart has remained uncertain. We determined if aging altered oxidative metabolism in either of the two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) that reside beneath the plasma membrane or interfibrillar mitochondria (IFM) located between the myofibrils. SSM and IFM were isolated from 6-month adult and 24- and 28-month elderly Fischer 344 rat hearts. Aging-related alterations were limited to IFM, while SSM remained unaffected. Aging decreased the rate of oxidative phosphorylation in IFM, including when stimulated by electron donors specific for cytochrome oxidase. Cytochrome oxidase enzyme activity was decreased in IFM from aging hearts, while activity in SSM remained similar to adult controls. These findings allow future studies of aging-related decrements in oxidative function to focus upon IFM, while SSM provide an inherent control group of mitochondria that are free of aging-related alterations in oxidative function. The selective alteration of IFM during aging raises the possibility that the consequences of aging-induced mitochondrial dysfunction will be enhanced in specific subcellular regions of the senescent myocyte.     

Endoplasmic reticulum stress-mediated mitochondrial dysfunction in aged hearts

Aging impairs the mitochondrial electron transport chain (ETC), especially in interfibrillar mitochondria (IFM). Mitochondria are in close contact with the endoplasmic reticulum (ER). Induction of ER stress leads to ETC injury in adult heart mitochondria. We asked if ER stress contributes to the mitochondrial dysfunction during aging. Subsarcolemmal mitochondria (SSM) and IFM were isolated from 3, 18, and 24 mo. C57Bl/6 mouse hearts. ER stress progressively increased with age, especially in 24 mo. mice that manifest mitochondrial dysfunction. OXPHOS was decreased in 24 mo. IFM oxidizing complex I and complex IV substrates. Proteomic analysis showed that the content of multiple complex I subunits was decreased in IFM from 24 mo. hearts, but remained unchanged in in 18 mo. IFM without a decrease in OXPHOS. Feeding 24 mo. old mice with 4-phenylbutyrate (4-PBA) for two weeks attenuated the ER stress and improved mitochondrial function. These results indicate that ER stress contributes to the mitochondrial dysfunction in aged hearts. Attenuation of ER stress is a potential approach to improve mitochondrial function in aged hearts.     

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart.     

Rat brain and liver mitochondria develop oxidative stress and lose enzymatic activities on aging

The mitochondrial mass of rat brain and liver remained unchanged on aging in young adults, old adults, and senescent animals (28, 60, and 92 wk of age); the values were 15-17 and 29-31 mg protein/g for brain and liver, respectively. The whole aging process was associated with an increased content of the oxidation products, thiobarbituric acid-reactive substances and protein carbonyls, by 61-69% in brain and 36-45% in liver, respectively. The activities of critical enzymes for mitochondrial function, mitochondrial nitric oxide synthase, Mn-superoxide dismutase, complex I, and complex IV, decreased progressively during aging with activity losses of 73, 37, 29, and 28%, respectively, in the brain and 47, 46, 30, and 24% in the liver of senescent rats compared with young adults. Brain mitochondria isolated from aged rats showed increased mitochondrial fragility, as assayed by mitochondrial marker enzyme activities in the postmitochondrial supernatant, and increased volume and water permeability, as assayed by light scattering. Liver mitochondria isolated from young and old rats did not show differences in fragility and water permeability. A subpopulation of brain mitochondria with increased size and fragility was differentiated in aging rats, whereas liver showed a homogeneous mitochondrial population.     

Calorie restriction: A new therapeutic intervention for age-related dry eye disease in rats

A decrease in lacrimal gland secretory function is closely related to aging and leads to an increased prevalence of dry eye syndrome. Since calorie restriction (CR) is considered to prevent functional decline of various organs due to aging, we hypothesized that CR could prevent age-related lacrimal dysfunction. Six-month-old male Fischer 344 rats were randomly divided into ad libitum (AL) and CR (−35%) groups. After 6 months of CR, tear function was examined under conscious state. After euthanasia, lacrimal glands were subjected to histological examination, tear protein secretion stimulation test with Carbachol, and assessment of oxidative stress with 8-hydroxy-2 deoxyguanosine (8-OHdG) and 4-hydroxynonenal (HNE) antibodies. CR significantly improved tear volume and tended to increase tear protein secretion volume after stimulation with Carbachol compared to AL. The acinar unit density was significantly higher in the CR rats compared to AL rats. Lacrimal glands in the CR rats showed a lesser degree of interstitial fibrosis. CR reduced the concentration of 8-OHdG and the extent of staining with HNE in the lacrimal gland, compared to AL. Furthermore, our electron microscopic observations showed that mitochondrial structure of the lacrimal gland obtained from the middle-aged CR rats was preserved in comparison to the AL rats. Collectively, these results demonstrate for the first time that CR may attenuate oxidative stress related damage in the lacrimal gland with preservation of lacrimal gland functions. Although molecular mechanism(s) by which CR maintains lacrimal gland function remains to be resolved, CR might provide a novel therapeutic strategy for treating dry eye syndrome.     

Age-related Oxidative Modifications of Proteins and Lipids in Rat Brain

Oxidants have been shown to play a major role in ageing and ageing-related neurodegenerative diseases. In the present study, we investigated the effect of ageing on oxidative damage to lipids and proteins in brain homogenate, mitochondria and synaptosomes of adult (6-month-old), old (15-month-old), and senescent (26-month-old) Wistar rats. There was a significant increase in thiobarbituric acid-reactive substances and conjugated dienes in homogenates, which indicate increased lipid peroxidation (LPO). Oxidative modifications of homogenate proteins were demonstrated by a loss of sulfhydryl content, accumulation of dityrosines and formation of protein conjugates with LPO-end products. Increase in protein conjugates with LPO-end products and a decrease in SH groups were observed also in mitochondria and synaptosomes, but dityrosine content was elevated only in synaptosomes. Protein surface hydrophobicity, measured by fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS), was increased only in homogenate. These results suggest that besides mitochondria and synaptosomes other cellular compartments are oxidatively modified during brain ageing.