Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Selectivity of protein oxidative damage during aging in Drosophila melanogaster.

The purpose of the present study was to determine whether oxidation of various proteins during the aging process occurs selectively or randomly, and whether the same proteins are damaged in different species. Protein oxidative damage to the proteins, present in the matrix of mitochondria in the flight muscles of Drosophila melanogaster and manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl-group antibodies. Aconitase was found to be the only protein in the mitochondrial matrix that exhibited an age-associated increase in carbonylation. The accrual of oxidative damage was accompanied by an approx. 50% loss in aconitase activity. An increase in ambient temperature, which elevates the rate of metabolism and shortens the life span of flies, caused an elevation in the amount of aconitase carbonylation and an accelerated loss in its activity. Exposure to 100% ambient oxygen showed that aconitase was highly susceptible to undergo oxidative damage and loss of activity under oxidative stress. Administration of fluoroacetate, a competitive inhibitor of aconitase activity, resulted in a dose-dependent decrease in the life span of the flies. Results of the present study demonstrate that protein oxidative damage during aging is a selective phenomenon, and might constitute a mechanism by which oxidative stress causes age-associated losses in specific biochemical functions.

The peroxiredoxin gene family in Drosophila melanogaster.

Five peroxiredoxin genes have been identified in Drosophila melanogaster on the basis of a genome-wide search. Three of the genes (DPx-4156, DPx-4783, and DPx-5037) fall into the 2-Cys subgroup, while the other two (DPx-2540 and DPx-6005) belong to the 1-Cys subgroup. Using cDNAs, all five were expressed in E. coli and the purified recombinant proteins were shown to reduce H(2)O(2) in the presence of dithiothreitol. The three 2-Cys Prx were also shown to be active in the thioredoxin system and were, consequently, classified as thioredoxin peroxidases. Antisera raised against the DPx-4783 recombinant protein crossreacted with all family members and recognized protein species of the predicted sizes (22-27 kD). All five family members, when individually overexpressed in Drosophila S2 cells, conferred some resistance to H(2)O(2) treatment, as measured by cell viability. Functional diversification of the Drosophila peroxiredoxin family members was suggested by two lines of evidence: (i) the patterns of mRNA accumulation varied for the different genes during development and (ii) recombinant proteins fused to an epitope tag and overexpressed in Drosophila cells, differed in subcellular localizations--three proteins occurred in the cytosol, one was localized to the mitochondria, and one was found to be secreted.

Antioxidant status and stress resistance in long- and short-lived lines of Drosophila melanogaster.

The purpose of this study was to understand the nature of the biochemical and physiological variations between genetically different lines of Drosophila melanogaster. Selection for early or delayed reproduction has given rise to lines with substantial and heritable differences in longevity. The hypotheses tested were that either: (i) a compensatory slowing of metabolism, (ii) increased antioxidative enzyme activities, or (iii) elevated resistance to stressful conditions underlie these differences in longevity. The metabolic rate, metabolic potential (i.e. total amount of oxygen consumed during average lifespan) and speed of walking were all greater in long-lived than in short-lived flies, but there was no enhancement of antioxidant defenses. In fact, catalase activity was significantly lower in the long-lived flies. Long life was largely maintained under heat stress and starvation conditions, and was maintained to a lesser extent upon exposure to paraquat, a superoxide radical generator. In contrast, the 'short-lived' flies had a longer lifespan under cold stress and hyperoxia, also an inducer of radical generation. These results contradict the first two hypotheses and suggest that alleles underlying either long or short life are linked with enhanced resistance to specific kinds of stress, which may account for the preservation of these alleles in the parental population.

Analysis of oxidative modification of proteins.

Protein oxidation has been implicated in a variety of degenerative diseases as well as in the aging process. This unit describes techniques for the quantification of various protein oxidation products, including protein carbonyls, loss of protein thiol groups, dityrosine and nitrotyrosine, and isoaspartate formation. Such oxidatively modified products may also be used as biomarkers for the assessment of oxidative stress during aging and/or disease.

Prevention of flight activity prolongs the life span of the housefly, Musca domestica, and attenuates the age-associated oxidative damamge to specific mitochondrial proteins.

The purpose of this study was to explore the mechanisms by which oxidative stress affects the aging process. The hypothesis that the rate of accumulation of oxidative damage to specific mitochondrial proteins is linked to the life expectancy of animals was tested in the housefly. The rate of oxygen consumption and life expectancy of the flies were experimentally altered by confining the flies in small jars, where they were unable to fly. Prevention of flight activity decreased the rate of oxygen utilization of flies and almost tripled their life span as compared to those permitted to fly. Rate of mitochondrial H(2)O(2) generation at various ages was lower in the low activity flies than in the high activity flies. Oxidative damage to mitochondrial proteins, adenine nucelotide translocase, and aconitase, detected as carbonyl modifications, was attenuated; and the loss in their functional activity occurring with age was retarded in the long-lived low activity flies as compared to the short-lived high activity flies. The two proteins were previously identified to be the only mitochondrial proteins exhibiting age-related increases in carbonylation. Results support the hypothesis that accrual of oxidative damage to specific protein targets and the consequent loss of their function may constitute a mechanism by which oxidative stress controls the aging process.

Reversible effects of long-term caloric restriction on protein oxidative damage.

The age-associated increase in oxidative damage in ad libitum-fed mice is attenuated in mice fed calorically restricted (CR) diets. The objective of this study was to determine if this effect results from a slowing of age-related accumulation of oxidative damage, or from a reversible decrease of oxidative damage by caloric restriction. To address these possibilities, crossover studies were conducted in C57BL/6 mice aged 15 to 22 months that had been maintained, after 4 months of age, on ad libitum (AL) or a 60% of AL caloric regimen. One half of the mice in these groups were switched to the opposite regimen of caloric intake for periods up to 6 weeks, and protein oxidative damage (measured as carbonyl concentration and loss of sulfhydryl content) was measured in homogenates of brain and heart. In AL-fed mice, the protein carbonyl content increased with age, whereas the sulfhydryl content decreased. Old mice maintained continuously under CR had reduced levels of protein oxidative damage when compared with the old mice fed AL. The effects of chronic CR on the carbonyl content of the whole brain and the sulfhydryl content of the heart were fully reversible within 3-6 weeks following reinstatement of AL feeding. The effect of chronic CR on the sulfhydryl content of the brain cortex was only partially reversible. The introduction of CR for 6 weeks in the old mice resulted in a reduction of protein oxidative damage (as indicated by whole brain carbonyl content and cortex sulfhydryl), although this effect was not equivalent to that of CR from 4 months of age. The introduction of CR did not affect the sulfhydryl content of the heart. Overall, the current findings indicate that changes in the level of caloric intake may reversibly affect the concentration of oxidized proteins and sufhydryl content. In addition, chronic restriction of caloric intake also retards the age-associated accumulation of oxidative damage. The magnitude of the reversible and chronic effects appears to be dependent upon the tissue examined and the nature of the oxidative alteration.

Effects of aging and hyperoxia on oxidative damage to cytochrome c in the housefly, Musca domestica.

Cytochrome c is a component of the mitochondrial electron transport chain, where it transfers electrons from ubiquinol-cytochrome c reductase to cytochrome c oxidase. Autoxidation of some of the components of the electron transport chain is the main source of intracellular O(2)(-*)/H(2)O(2) production in aerobic organisms. Because cytochrome c is located on the outer surface of the inner mitochondrial membrane, it is likely to be constantly exposed to H(2)O(2), secreted by mitochondria into the cytosol. The specific objective of this study was to determine whether cytochrome c in the flight muscle mitochondria of the housefly is oxidatively damaged during aging and/or under severe oxidative stress induced by exposure of flies to 100% oxygen. Results of two independent methods, namely tritiated borohydride labeling for determining carbonylation and mass spectral analysis for the measurement of molecular mass, indicated that neither the carbonyl level nor the molecular mass of cytochrome c was affected by aging or hyperoxia. Thus, either cytochrome c is resistant to oxidative damage in vivo or the oxidized cytochrome c is promptly degraded. These findings also support the concept that protein oxidative damage during aging and under oxidative stress is selective.

Current issues concerning the role of oxidative stress in aging: a perspective.

The main tenet of the oxidative stress hypothesis of aging is that accrual of molecular oxidative damage is the principal causal factor in the senescence-related loss of ability to maintain homeostasis. This hypothesis has garnered a considerable amount of supportive correlational evidence, which is now being extended experimentally in transgenic Drosophila over-expressing antioxidative defense enzymes. Some of these studies have reported extensions of life span, while others have not. Interpretation of life spans in poikilotherms is complicated by a number of factors, including the interrelationship between metabolic rate and longevity. The life spans of poikilotherms can be extended multi-fold by reducing the metabolic rate but without affecting the metabolic potential, i.e., the total amount of energy expended during life. A hypometabolic state in poikilotherms also enhances stress resistance and activities of antioxidative enzymes. It is emphasized that extension of life span without simultaneously increasing metabolic potential is of questionable biological significance.

Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl(4) poisoning?

Antioxidants in the blood plasma of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. For this initial study an animal model of CCl(4) poisoning was studied. The time (2, 7, and 16 h) and dose (120 and 1200 mg/kg, intraperitoneally)-dependent effects of CCl(4) on plasma levels of alpha-tocopherol, coenzyme Q (CoQ), ascorbic acid, glutathione (GSH and GSSG), uric acid, and total antioxidant capacity were investigated to determine whether the oxidative effects of CCl(4) would result in losses of antioxidants from plasma. Concentrations of alpha-tocopherol and CoQ were decreased in CCl(4)-treated rats. Because of concomitant decreases in cholesterol and triglycerides, it was impossible to dissociate oxidation of alpha-tocopherol and the loss of CoQ from generalized lipid changes, due to liver damage. Ascorbic acid levels were higher with treatment at the earliest time point; the ratio of GSH to GSSG generally declined, and uric acid remained unchanged. Total antioxidant capacity showed no significant change except for 16 h after the high dose, when it was increased. These results suggest that plasma changes caused by liver malfunction and rupture of liver cells together with a decrease in plasma lipids do not permit an unambiguous interpretation of the results and impede detection of any potential changes in the antioxidant status of the plasma.

Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse.

The purpose of the present study was to examine the role of mitochondria in the aging process by determining whether the activities of various electron transport chain oxidoreductases are deleteriously affected during aging and whether the hypothesized age-related alterations in different tissues follow a common pattern. Activities of respiratory complexes I, II, III, and IV were measured in mitochondria isolated from brain, heart, skeletal muscle, liver, and kidney of young (3.5 months), adult (12-14 months), and old (28-30 months) C57BL/6 mice. Activities of some individual complexes were decreased in old animals, but no common pattern can be discerned among various tissues. In general, activities of the complexes were more adversely affected in tissues such as brain, heart, and skeletal muscle, whose parenchyma is composed of postmitotic cells, than those in the liver and kidney, which are composed of slowly dividing cells. The main feature of age-related potentially dysfunctional alterations in tissues was the development of a shift in activity ratios among different complexes, such that it would tend to hinder the ability of mitochondria to effectively transfer electrons down the respiratory chain and thus adversely affect oxidative phosphorylation and/or autooxidizability of the respiratory components.

Effect of coenzyme Q(10) and alpha-tocopherol content of mitochondria on the production of superoxide anion radicals.

The effects of coenzyme Q(10) (CoQ(10)) and alpha-tocopherol on the rate of mitochondrial superoxide anion radical (O2(./-)) generation were examined in skeletal muscle, liver, and kidney of 24-month-old mice. Mice were orally administered alpha-tocopherol (200 mg.kg(-1).day(-1)) alone, CoQ(10) (123 mg.kg(-1).day(-1)) alone, or the two together for 13 wk. Administration of alpha-tocopherol resulted in an approximately sevenfold elevation of mitochondrial alpha-tocopherol content. Intake of CoQ(10) alone caused an approximately fivefold increase in CoQ content (CoQ(9) and/or CoQ(10)) and alpha-tocopherol of mitochondria. The rate of (O2(./-)) generation by submitochondrial particles (SMPs) was inversely related to their alpha-tocopherol content but unrelated to CoQ content. Experimental in vitro augmentation of SMPs with varying amounts of alpha-tocopherol caused an up to approximately 50% decrease in the rate of O2(./-) generation. Similar in vitro augmentations of SMPs with CoQ(10) had previously been found to have no effect on the rate of O2(./-) generation The CoQ(10)-induced elevation of alpha-tocopherol in the present study was inferred to be due to a 'sparing/regeneration' by CoQ. Results indicate the involvement of alpha-tocopherol in the elimination of mitochondrially generated O2(./-)

Decreased mitochondrial hydrogen peroxide release in transgenic Drosophila melanogaster expressing intramitochondrial catalase.

The objective of this study was to develop strategies for manipulating oxidative stress transgenically in a multicellular organism. Ectopic catalase was introduced into the mitochondrial matrix, which is the main intracellular site of H2O2 formation and where catalase is normally absent. Transgenic Drosophila melanogaster were generated by microinjection of a P element construct, containing the genomic catalase sequence of Drosophila, with the mitochondrial leader sequence of ornithine aminotransferase inserted upstream of the coding region. Total catalase activities in whole-body homogenates of 10-day-old flies from four transgenic lines were approximately 30-160% higher than those from the parental and four vector-only control lines. Expression of catalase in the mitochondrial matrix was confirmed by immunoblotting and catalase activity assays. Mitochondrial release of H2O2 was decreased by approximately 90% in the transgenic lines when compared to levels in vector-only controls. This in vivo system provides a novel model for examining the functional significance of decreased mitochondrial H2O2 release.

Overexpression of Mn-containing superoxide dismutase in transgenic Drosophila melanogaster.

The general objective of this study was to examine the role of mitochondria in the aging process. Two alternative hypotheses were tested: (i) that overexpression of Mn superoxide dismutase (Mn SOD) in the mitochondria of Drosophila melanogaster would slow the accrual of oxidative damage and prolong survival or (ii) that there is an evolved optimum level of superoxide anion radical, such that overexpression of Mn SOD would have deleterious or neutral effects. Microinjection and mobilization of a transgene, which contained a 9-kb genomic sequence encoding Mn SOD, produced 15 experimental lines overexpressing Mn SOD by 5-116% relative to the parental y w strain. Comparisons between these lines and control lines containing inserted vector sequences alone indicated that the mean longevity of the experimental lines was decreased by 4-5% relative to controls. There were no compensatory changes in the metabolic rate, level of physical activity, or the levels of other antioxidants, namely Cu-Zn SOD, catalase, and glutathione. There were no differences between groups in rates of mitochondrial hydrogen peroxide release, protein oxidative damage, or resistance to 100% oxygen or starvation conditions. The experimental lines had a marginally increased resistance to moderate heat stress. These results are consistent with the existence of an optimum level of Mn SOD activity which minimizes oxidative stress. The naturally evolved level of Mn SOD activity in Drosophila appears to be near the optimum required under normal conditions, although the optimum may be shifted to a higher level under more stressful conditions.

Overexpression of glutathione reductase extends survival in transgenic Drosophila melanogaster under hyperoxia but not normoxia.

The purpose of this study was to test the hypothesis that overexpression of glutathione reductase in transgenic Drosophila melanogaster increases resistance to oxidative stress and retards the aging process. Transgenic flies were generated by microinjection and subsequent mobilization of a P element construct containing the genomic glutathione reductase gene of Drosophila, with 4 kb upstream and 1.5 kb downstream of the coding region. Transgenic animals stably overexpressed glutathione reductase by up to 100% throughout adult life and under continuous exposure to 100% oxygen or air. Under hyperoxic conditions, overexpressors had increased longevity, decreased accrual of protein carbonyls, and dramatically increased survival rates after recovery from a semi-lethal dose of 100% oxygen. Under normoxic conditions, overexpression of glutathione reductase had no effect on longevity, protein carbonyl content, reduced glutathione, or glutathione disulfide content, although the total consumption of oxygen was slightly decreased. Glutathione reductase activity does not appear to be a rate-limiting factor in anti-aging defenses under normoxic conditions, but it may become a limiting factor when the level of oxidative stress is elevated.

Effect of age and caloric restriction on bleomycin-chelatable and nonheme iron in different tissues of C57BL/6 mice.

The objective of this study was to test the hypothesis that the widely observed age-associated increase in the amounts of macromolecular oxidative damage is due to an elevation in the availability of redox-active iron, that is believed to catalyze the scission of H2O2 to generate the highly reactive hydroxyl radical. Concentrations of bleomycin-chelatable iron and nonheme iron were measured in various tissues and different regions of the brain of mice fed on ad libitum (AL) or a calorically restricted (to 60% of AL) diet at different ages. The concentrations of these two pools of iron varied markedly as a function of tissue, age, and caloric intake. There was no consistent ratio between the amounts of nonheme and the bleomycin-chelatable iron pools across these conditions. Nonheme iron concentration increased with age in the liver, kidney, heart, striatum, hippocampus, midbrain and cerebellum of AL animals, whereas bleomycin-chelatable iron increased significantly with age only in the liver. Amounts of both nonheme and bleomycin-chelatable iron remained unaltered during aging in the cerebral cortex and hindbrain of AL mice. Caloric restriction had no effect on iron concentration in the brain or heart, but caused a marked increase in the concentration of both bleomycin-chelatable and nonheme iron in the liver and the kidney. The results do not support the hypothesis that accumulation of oxidative damage with age, or its attenuation by CR, are associated with corresponding variations in redox-active iron.

Comparisons of coenzyme Q bound to mitochondrial membrane proteins among different mammalian species.

The objective of this study was to elucidate the mechanisms that govern the variations in the rates of mitochondrial superoxide anion radical (O2-*) generation in different species. The amounts of coenzyme Q (CoQ) associated with mitochondrial membrane proteins were compared in five different mammalian species, namely mouse, rat, rabbit, pig, and cow. Micelles of cardiac mitochondria were prepared using Triton X-100 or deoxycholate (DOC) as detergents, and the micelles containing mitochondrial proteins were sedimented by sucrose density ultracentrifugation. The amount of CoQ present in both types of micelles varied in different species, whereas alpha-tocopherol, another lipoidal molecule in mitochondrial membranes, could not be detected in the micelles of any of these species. The amounts of CoQ bound to mitochondrial proteins in DOC micelles were higher in those mammalian species where CoQ10 was the predominant CoQ homologue, and the amounts were found to be inversely correlated with the rate of mitochondrial 02-* generation among different species. Results also indicated that mitochondrial CoQ exists in at least two distinct pools, one of which is associated with the membrane proteins. The degree of association between CoQ and membrane proteins appears to be a factor determining the rate of mitochondrial O2-* generation.

Effects of coenzyme Q10 and alpha-tocopherol administration on their tissue levels in the mouse: elevation of mitochondrial alpha-tocopherol by coenzyme Q10.

Coenzyme Q (CoQ) was previously demonstrated in vitro to indirectly act as an antioxidant in respiring mitochondria by regenerating alpha-tocopherol from its phenoxyl radical. The objective of this study was to determine whether CoQ has a similar sparing effect on alpha-tocopherol in vivo. Mice were administered CoQ10 (123 mg/kg/day) alone, or alpha-tocopherol (200 mg/kg/day) alone, or both, for 13 weeks, after which the amounts of CoQ10, CoQ9 and alpha-tocopherol were determined by HPLC in the serum as well as homogenates and mitochondria of liver, kidney, heart, upper hindlimb skeletal muscle and brain. Administration of CoQ10 and alpha-tocopherol, alone or together, increased the corresponding levels of CoQ10 and alpha-tocopherol in the serum. Supplementation with CoQ10 also elevated the amounts of the predominant homologue CoQ9 in the serum and the mitochondria. A notable effect of CoQ10 intake was the enhancement of alpha-tocopherol in mitochondria. alpha-Tocopherol administration resulted in an elevation of alpha-tocopherol content in the homogenates of nearly all tissues and their mitochondria. Results of this study thus indicate that relatively long-term administration of CoQ10 or alpha-tocopherol can result in an elevation of their concentrations in the tissues of the mouse. More importantly, CoQ10 intake has a sparing effect on alpha-tocopherol in mitochondria in vivo.

Mitochondrial coenzyme Q content and aging.

The main objective of this study was to determine the nature of the relationship between aging and mitochondrial coenzyme Q (CoQ) content. Mitochondria in the heart, skeletal muscle, kidney and brain of the mouse varied in both the amount of total CoQ (CoQ9 + CoQ10) content as well as in the ratio of the CoQ9 to CoQ10. CoQ content declined with age only in the skeletal muscle. Caloric restriction (CR) resulted in an increase in the amount of CoQ9 in skeletal muscle mitochondria. This effect was partially reversible upon termination of the caloric restriction regimen. Results suggest that a decrease in mitochondrial CoQ content is an integral aspect of aging in skeletal muscle.

Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria.

The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories--a regimen that increases life span by approximately 30-40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.