Peter Voss

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.

Clinical oxidation parameters of aging

Aging is a complex progressive physiological alteration of the organism which ultimately leads to death. During the whole life a human being is confronted with oxidative stress. To measure how this oxidative stress is developing during the aging process and how it changes the cellular metabolism several substances have been pronounced as biomarkers including lipid peroxidation (LPO) products, protein oxidation products, antioxidative acting enzymes, minerals, vitamins, glutathione, flavonoids, bilirubin and uric acid (UA). But none of them could develop to the leading one which is accepted by the whole scientific community to determine the life expectancy of the individual person or biological age or age-related health status. Further there are many conflicting data about the changes of each single biomarker during the aging process. There are so many different influences acting on the concentration or activity of single substances or single enzymes that it is not possible to measure only one clinical marker and determine how healthy an individual is or to predict the life expectancy of the corresponding person. Therefore, always a set or pattern of clinical biomarkers should be used to determine the oxidation status of the person. This set should include at least one marker for the LPO, the protein oxidation and the total antioxidative status and ideally also one for DNA damages.

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 μmol/l produced tyrosine nitration and inactivation of GS, whereas 50 μmol/l peroxynitrite additionally increased S‐nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (−)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 μmol/l). Further, a putative “denitrase” activity restored the activity of peroxynitrite (5 μmol/l)‐treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions.

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation.

Inverse correlation of protein oxidation and proteasome activity in liver and lung.

Several studies have demonstrated that proteasome activity decreases whereas protein oxidation increases with aging in various tissues. However, no studies are available correlating both parameters directly comparing different tissues of one organism. Therefore, we determined whether there is an age-related change in proteasome activity and protein oxidation in heart, lung, liver, kidney and skeletal muscle samples of 6-, 10-, 18- and 26-month-old rats. There was a significant age-related increase in protein carbonyls at 18 and 26 months compared to young rats. Thereby, protein carbonyl formation was rather due to a general than a specific protein carbonylation as shown by immunblot studies. The highest increase in protein carbonyl formation was found in liver, lung and kidney samples. Proteasome activity decreased significantly with age in lung and liver samples. Proteasome activity in liver and lung decreased by factor five compared to young rats. Strong correlations between proteasome activity and protein oxidation were found in liver and lung, whereas in other tissues only a trend was found. These results demonstrate that the increase in protein oxidation and the decline in proteasome activity are correlating. Further studies are needed to determine the mechanisms which cause organ-specific aging-rates and their consequences.

Ferritin oxidation and proteasomal degradation: protection by antioxidants.

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimers, Parkinsons and Huntigtons diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H2O2 induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation. Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin.

Irradiation of GAPDH: a model for environmentally induced protein damage.

Abstract Environmental factors, including sunlight, are able to induce severe oxidative protein damage. The modified proteins are either repaired, degraded or escape from degradation and aggregate. In the present study we tested the effect of different sunlight components such as UV-A, UV-B, and infrared radiation on protein oxidation in vitro. We chose glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a model enzyme and analyzed the irradiation-induced enzyme activity loss, fragmentation and aggregation, and quantified various oxidative amino acid modifications. Since γ-irradiation was used in numerous studies before, we used it for comparative purposes. Infrared radiation was unable to damage GAPDH in the dose range tested (0–1000 J/cm2). UV-A led to a decrease in free thiol content, which was connected with a loss in enzyme activity, while only at very high doses could moderate protein aggregation and fragmentation be observed. UV-B (0–2 J/cm2) and γ-irradiation (0–500 Gy) led to a dose-dependent increase in protein modification. Interestingly, UV-B acted on specific amino acids, such as arginine, proline, and tyrosine, whereas γ-irradiation acted more randomly. The possibility of using the amino acid oxidation pattern as a biomarker of the source of damage is discussed.

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary.The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.

Protective effect of antioxidants against sarcoplasmic reticulum (SR) oxidation by Fenton reaction, however without prevention of Ca-pump activity

The Ca(2+)-ATPase of the sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle was oxidized by Fe2+/H2O2/ascorbic acid (AA), a system which generates HO(.) radicals according to the Fenton reaction: (Fe2(+)+H2O2-->HO(.)+OH(-)+Fe(3+)) under conditions similar to the pathological state of inflammation. Under these conditions, when hydroxyl-radicals and/or ferryl-radicals are generated, a 50% decrease of the SERCA activity was observed, a significant decrease of SH groups and an increase of protein carbonyl groups and lipid peroxidation were identified. Two new bands, time dependent in density, appeared in the SERCA protein electrophoresis after incubation with the Fenton system (at approximately 50 and 75kDa), probably due to structural changes as supported also by trypsin digestion. Immunoblotting of DNPH derivatized protein bound carbonyls detected a time dependent increase after incubation of SERCA with the Fenton system. Trolox and the pyridoindole stobadine (50microM) protected SR against oxidation induced via the Fenton system by preventing SH group oxidation and lipid peroxidation. Pycnogenol((R)) and EGb761 (40microg/ml) protected SERCA in addition against protein bound carbonyl formation. In spite of the antioxidant effects, trolox and stobadine were not able to prevent a decrease in the SERCA Ca(2+)-ATPase activity. Pycnogenol and EGb761 even enhanced the decrease of the Ca(2+)-ATPase activity induced by the Fenton system, probably by secondary oxidative reactions.

Postanoxic damage of microglial cells is mediated by xanthine oxidase and cyclooxygenase

Brain ischemia and the following reperfusion are important causes for brain damage and leading causes of brain morbidity and human mortality. Numerous observations exist describing the neuronal damage during ischemia/reperfusion, but the outcome of such conditions towards glial cells still remains to be elucidated. Microglia are resident macrophages in the brain. In this study, we investigated the anoxia/reoxygenation caused damage to a microglial cell line via determination of energy metabolism, free radical production by dichlorofluorescein fluorescence and nitric oxide production by Griess reagent. Consequences of oxidant production were determined by measurements of protein oxidation and lipid peroxidation, as well. By using site-specific antioxidants and inhibitors of various oxidant-producing pathways, we identified major sources of free radical production in the postanoxic microglial cells. The protective influences of these compounds were tested by measurements of cell viability and apoptosis. Although, numerous free radical generating systems may contribute to the postanoxic microglial cell damage, the xanthine oxidase- and the cyclooxygenase-mediated oxidant production seems to be of major importance.