Kelvin J.A. Davies

Free radicals and tissue damage produced by exercise

Summary We report a two- to three-fold increase in free radical (R • ) concentrations of muscle and liver following exercise to exhaustion. Exhaustive exercise also resulted in decreased mitochondrial respiratory control, loss of sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) integrity, and increased levels of lipid peroxidation products. Free radical concentrations, lipid peroxidation, and SR, ER, and mitochondrial damage were similar in exercise exhausted control animals and non-exercised vitamin E deficient animals, suggesting the possibility of a common R • dependent damage process. In agreement with previous work showing that exercise endurance capacity is largely determined by the functional mitochondrial content of muscle (1–4), vitamin E deficient animals endurance was 40% lower than that of controls. The results suggest that R • induced damage may provide a stimulus to the mitochondrial biogenesis which results from endurance training.

Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations

The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results.

Degradation of oxidized proteins in mammalian cells.

Protein oxidation in vivo is a natural consequence of aerobic life. Oxygen radicals and other activated oxygen species generated as by‐products of cellular metabolism or from environmental sources cause modifications to the amino acids of proteins that generally result in loss of protein function/enzymatic activity. Oxidatively modified proteins can undergo direct chemical fragmentation or can form large aggregates due to covalent cross‐linking reactions and increased surface hydrophobicity. Mammalian cells exhibit only limited direct repair mechanisms and most oxidized proteins undergo selective proteolysis. The proteasome appears to be largely responsible for the degradation of soluble intracellular proteins. In most cells, oxidized proteins are cleaved in an ATP‐and ubiquitin‐independent pathway by the 20 S “core” proteasome. The proteasome complex recognizes hydrophobic amino acid residues, aromatic residues, and bulky aliphatic residues that are exposed during the oxidative rearrangement of secondary and tertiary protein structure: increased surface hydrophobicity is a feature common to all oxidized proteins so far tested. The recognition of such (normally shielded) hydrophobic residues is the suggested mechanism by which proteasome catalyzes the selective removal of oxidatively modified cell proteins. By minimizing protein aggregation and cross‐linking and by removing potentially toxic protein fragments, proteasome plays a key role in the overall antioxidant defenses that minimize the ravages of aging and disease.—Grune, T., Reinheckel, T., Davies, K. J. A. Degradation of oxidized proteins in mammalian cells. FASEB J. 11, 526–534 (1997)

Oxidative Stress, Antioxidant Defenses, and Damage Removal, Repair, and Replacement Systems

Oxidative stress is an unavoidable consequence of life in an oxygen‐rich atmosphere. Oxygen radicals and other activated oxygen species are generated as by‐products of aerobic metabolism and exposure to various natural and synthetic toxicants. The “Oxygen Paradox” is that oxygen is dangerous to the very life‐forms for which it has become an essential component of energy production. The first defense against oxygen toxicity is the sharp gradient of oxygen tension, seen in all mammals, from the environmental level of 20% to a tissue concentration of only 3‐4% oxygen. These relatively low tissue levels of oxygen prevent most oxidative damage from ever occurring. Cells, tissues, organs, and organisms utilize multiple layers of antioxidant defenses and damage removal, and replacement or repair systems in order to cope with the remaining stress and damage that oxygen engenders. The enzymes comprising many of these protective systems are inducible under conditions of oxidative stress adaptation, in which the expression of over 40 mammalian genes is upregulated. Mitotic cells have the additional defensive ability of entering a transient growth‐arrested state (in the first stages of adaptation) in which DNA is protected by histone proteins, energy is conserved by diminished expression of nonessential genes, and the expression of shock and stress proteins is greatly increased. Failure to fully cope with an oxidative stress can switch mitotic cells into a permanent growth‐arrested, senescence‐like state in which they may survive for long periods. Faced with even more severe oxidative stress, or the declining protective enzymes and adaptive capacity associated with aging, cells may “sacrifice themselves” by apoptosis, which protects surrounding healthy tissue from further damage. Only under the most severe oxidative stress conditions will cells undergo a necrotic death, which exposes surrounding tissues to the further vicissitudes of an inflammatory immune response. This remarkable array of systems for defense; damage removal, replacement, and repair; adaptation; growth modulation; and apoptosis make it possible for us to enjoy life in an oxygen‐rich environment.

Calcium and oxidative stress: from cell signaling to cell death

Reactive oxygen and nitrogen species can be used as a messengers in normal cell functions. However, at oxidative stress levels they can disrupt normal physiological pathways and cause cell death. Such a switch is largely mediated through Ca(2+) signaling. Oxidative stress causes Ca(2+) influx into the cytoplasm from the extracellular environment and from the endoplasmic reticulum or sarcoplasmic reticulum (ER/SR) through the cell membrane and the ER/SR channels, respectively. Rising Ca(2+) concentration in the cytoplasm causes Ca(2+) influx into mitochondria and nuclei. In mitochondria Ca(2+) accelerates and disrupts normal metabolism leading to cell death. In nuclei Ca(2+) modulates gene transcription and nucleases that control cell apoptosis. Both in nuclei and cytoplasm Ca(2+) can regulate phosphorylation/dephosphorylation of proteins and can modulate signal transduction pathways as a result. Since oxidative stress is associated with many diseases and the aging process, understanding how oxidants alter Ca(2+) signaling can help to understand process of aging and disease, and may lead to new strategies for their prevention.

Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism

Mitochondrial aconitase is sensitive to oxidative inactivation and can aggregate and accumulate in many age-related disorders. Here we report that Lon protease, an ATP-stimulated mitochondrial matrix protein, selectively recognizes and degrades the oxidized, hydrophobic form of aconitase after mild oxidative modification, but that severe oxidation results in aconitase aggregation, which makes it a poor substrate for Lon. Similarly, a morpholino oligodeoxynucleotide directed against the lon gene markedly decreases the amount of Lon protein, Lon activity and aconitase degradation in WI-38 VA-13 human lung fibroblasts and causes accumulation of oxidatively modified aconitase. The ATP-stimulated Lon protease may be an essential defence against the stress of life in an oxygen environment. By recognizing minor oxidative changes to protein structure and rapidly degrading the mildly modified protein, Lon protease may prevent extensive oxidation, aggregation and accumulation of aconitase, which could otherwise compromise mitochondrial function and cellular viability. Aconitase is probably only one of many mitochondrial matrix proteins that are preferentially degraded by Lon protease after oxidative modification.

The Broad Spectrum of Responses to Oxidants in Proliferating Cells: A New Paradigm for Oxidative Stress

Proliferating mammalian cells exhibit a broad spectrum of responses to oxidative stress, depending on the stress level encountered. Very low levels of hydrogen peroxide, e.g., 3 to 15 mu M, or 0.1 to 0.5 mu mol/107 cells, cause a significant mitogenic response, 25% to 45% growth stimulation. Greater concentrations of H2O2, 120 to 150 mu M, or 2 to 5 mu mol/107 cells, cause a temporary growth arrest that appears to protect cells from excess energy use and DNA damage. After 4 6 h of temporary growth arrest, many cells will exhibit up to a 40‐fold transient adaptive response in which genes for oxidant protection and damage repair are preferentially expressed. After 18 h of H2O2 adaptation (including the 4‐6h of temporary growth arrest) cells exhibit maximal protection against oxidative stress. The H2O2 originally added is metabolized within 30‐40min, and if no more is added the cells will gradually de‐adapt, so that by 36h after the initial H2O2 stimulus they have returned to their original level of H2O2 sensitivity. At H2O2 concentrations of 250 to 400 mu M, or 9 to 14 mu mol/107 cells, mammalian fibroblasts are not able to adapt but instead enter a permanently growth‐arrested state in which they appear to perform most normal cell functions but never divide again. This state of permanent growth arrest has often been confused with cell death in toxicity studies relying solely on cell proliferation assays as measures of viability. If the oxidative stress level is further increased to 0.5 to 1.0 mM H2O2, or 15 to 30 mu mol/107 cells, apoptosis results. This oxidative stressinduced apoptosis involves nuclear condensation, loss of mitochon/drial transmembrane potential, degradation/down‐regulation of mitochondrial mRNAs and rRNAs, and degradation laddering of both nuclear and mitochondrial DNA. At very high H2O2 concentrations of 5.0 to 10.0 mM, or 150 to 300 mu mol/107 cells and above, cell membranes disintegrate, proteins and nucleic acids denature, and necrosis swiftly follows. Cultured cells grown in 20% oxygen are essentially preadapted or preselected to survive under conditions of oxidative stress. If cells are instead grown in 3% oxygen, much closer to physiological cellular levels, they are more sensitive to an oxidative challenge but exhibit far less accumulated oxidant damage. This broad spectrum of cellular responses to oxidant stress, depending on the amount of oxidant applied and the concentration of oxygen in the cell culture system, provides for a new paradigm of cellular oxidative stress responses.

Biochemical adaptation of mitochondria, muscle, and whole-animal respiration to endurance training.

The experimental intervention of exercise training has been used to study mitochondrial biosynthesis, and the physiologic integration of subcellular, cellular, and whole-animal energetics. Gross mitochondrial composition was unchanged in rat muscle by a 10-week program of endurance treadmill running. The mitochondrial concentration of iron-sulfur clusters, cytochromes, flavoprotein, dehydrogenases, oxidases, and membrane protein and lipid, as well as the ratios of each component to the others, maintained constant proportions. The mitochondrial content of muscle, however, increased by approximately 100% as did absolute tissue oxidative capacity. The soluble portions of mitochondria maintained a constant total protein content and mass, relative to the membrane fraction, despite adaptive changes in the specific activities of some citric acid-cycle enzymes. Mitochondria from endurance-trained muscles generated normal transmembrane potentials, ADP/O ratios, and respiratory control ratios. Muscle oxidase activity was highly correlated (r = 0.92) with endurance capacity, which increased 403%. Whole-animal maximal O2 consumption (VO2max), however, increased only 14% and was a relatively poor predictor of endurance. Thus, mitochondrial factors, rather than VO2max, must play an important role in dictating the limits of endurance activity. Conversely, VO2max was strongly related to the maximal intensity of work which could be attained aerobically (r = 0.82). Comparison of O2 consumption at the mitochondrial, muscle, and whole-animal levels revealed that maximal muscle oxidase activity was not an absolute limitation to VO2max: It is concluded that other factors intervene to control the percentage of muscle O2 consumption capacity which may be utilized during exercise.

PROTEIN, LIPID AND DNA REPAIR SYSTEMS IN OXIDATIVE STRESS : THE FREE-RADICAL THEORY OF AGING REVISITED

Aerobic organisms are constantly exposed to oxygen radicals and related oxidants. The antioxidant compounds and enzymes they have evolved remove most of the potentially damaging radicals/oxidants; however, damage to cellular proteins, lipids, nucleic acids and carbohydrates can be observed even under normal physiological conditions. Re-reduction of cellular components (direct repair) may be important for some biomolecules. In most cases studied to date, however, enzymatic degradation (by proteases, lipases, nucleases) appears to release damaged elements for excretion and conserve undamaged components for reutilization (indirect repair). In addition, the removal of damaged components appears to prevent or diminish the potential cytotoxicity of oxidized macromolecules. Several studies have reported an accumulation of oxidatively damaged cellular components with age (e.g., cataract formation, lipofuscin). Such reports are evidence that oxidant damage is one of several factors which contribute to the aging process, and provide at least partial support for the free-radical theory of aging. Studies of age-related changes in the activities, or levels of antioxidant enzymes and antioxidant compounds, however, have not provided complete understanding of the putative role of free radicals/oxidants in the aging process. In this review, we present the hypothesis that decreased activities or constitutive levels of oxidant repair enzymes may contribute to a progressive accumulation of oxidant damage with aging. Furthermore, the ability to mount an effective response to oxidative stress (induction of oxidant stress genes and proteins) may decline with age, thus predisposing older cells and organisms to oxidant damage.

HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise☆

Exercise causes heat shock (muscle temperatures of up to 45 degrees C, core temperatures of up to 44 degrees C) and oxidative stress (generation of O2- and H2O2), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32P]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30-60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O2- generation with increasing temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)