The paradox of aerobic life, or the 'Oxygen Paradox', is that higher eukaryotic aerobic organisms cannot exist without oxygen, yet oxygen is inherently dangerous to their existence. This 'dark side' of oxygen relates directly to the fact that each oxygen atom has one unpaired electron in its outer valence shell, and molecular oxygen has two unpaired electrons. Thus atomic oxygen is a free radical and molecular oxygen is a (free) bi-radical. Concerted tetravalent reduction of oxygen by the mitochondrial electron-transport chain, to produce water, is considered to be a relatively safe process; however, the univalent reduction of oxygen generates reactive intermediates. The reductive environment of the cellular milieu provides ample opportunities for oxygen to undergo unscheduled univalent reduction. Thus the superoxide anion radical, hydrogen peroxide and the extremely reactive hydroxyl radical are common products of life in an aerobic environment, and these agents appear to be responsible for oxygen toxicity. To survive in such an unfriendly oxygen environment, living organisms generate--or garner from their surroundings--a variety of water- and lipid-soluble antioxidant compounds. Additionally, a series of antioxidant enzymes, whose role is to intercept and inactivate reactive oxygen intermediates, is synthesized by all known aerobic organisms. Although extremely important, the antioxidant enzymes and compounds are not completely effective in preventing oxidative damage. To deal with the damage that does still occur, a series of damage removal/repair enzymes, for proteins, lipids and DNA, is synthesized. Finally, since oxidative stress levels may vary from time to time, organisms are able to adapt to such fluctuating stresses by inducing the synthesis of antioxidant enzymes and damage removal/repair enzymes. In a perfect world the story would end here; unfortunately, biology is seldom so precise. The reality appears to be that, despite the valiant antioxidant and repair mechanisms described above, oxidative damage remains an inescapable outcome of aerobic existence. In recent years oxidative stress has been implicated in a wide variety of degenerative processes, diseases and syndromes, including the following: mutagenesis, cell transformation and cancer; atherosclerosis, arteriosclerosis, heart attacks, strokes and ischaemia/reperfusion injury; chronic inflammatory diseases, such as rheumatoid arthritis, lupus erythematosus and psoriatic arthritis; acute inflammatory problems, such as wound healing; photo-oxidative stresses to the eye, such as cataract; central-nervous-system disorders, such as certain forms of familial amyotrophic lateral sclerosis, certain glutathione peroxidase-linked adolescent seizures, Parkinson's disease and Alzheimer's dementia; and a wide variety of age-related disorders, perhaps even including factors underlying the aging process itself. Some of these oxidation-linked diseases or disorders can be exacerbated, perhaps even initiated, by numerous environmental pro-oxidants and/or pro-oxid...

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.

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.

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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 microM, or 0.1 to 0.5 micromol/10(7) cells, cause a significant mitogenic response, 25% to 45 % growth stimulation. Greater concentrations of H2O2, 120 to 150 microM, or 2 to 5 micromol/10(7) 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 4-6 h of temporary growth arrest) cells exhibit maximal protection against oxidative stress. The H2O2 originally added is metabolized within 30-40 min, and if no more is added the cells will gradually de-adapt, so that by 36 h after the initial H2O2 stimulus they have returned to their original level of H2O2 sensitivity. At H2O2 concentrations of 250 to 400 microM, or 9 to 14 micromol/10(7) 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 micromol/10(7) cells, apoptosis results. This oxidative stress-induced apoptosis involves nuclear condensation, loss of mitochondrial 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 micromol/10(7) 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.

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.

The 26S proteasome system is involved in eliminating various proteins, including ubiquitinated misfolded/unfolded proteins, and its inhibition results in cellular accumulation of protein aggregates. Intramuscle-fiber ubiquitinated multiprotein-aggregates are characteristic of sporadic inclusion-body myositis (s-IBM) muscle fibers. Two major types of aggregates exist, containing either amyloid-beta (Abeta) or phosphorylated tau (p-tau). We have now asked whether abnormalities of the 26S proteasome contribute to s-IBM pathogenesis and whether the multiprotein aggregates have features of aggresomes. Using cultured human muscle fibers we also studied the effect of amyloid-beta precursor protein (AbetaPP) overexpression on proteasome function and the influence of proteasome inhibition on aggresome formation. We report that in s-IBM muscle biopsies 26S proteasome subunits were immunodetected in the gamma-tubulin-associated aggresomes, which also contained Abeta, p-tau, ubiquitin, and HSP70. In addition, a) expression of proteasome subunits was greatly increased, b) the 20Salpha proteasome subunit co-immunoprecipitated with AbetaPP/Abeta, and c) the three major proteasomal proteolytic activities were reduced. In cultured muscle fibers, AbetaPP-overexpressing fibers displayed diminished proteasomal proteolytic activities, and addition of proteasome inhibitor strikingly increased aggresome formation. Accordingly, proteasome dysfunction in s-IBM muscle fibers may play a role in accumulation of misfolded, potentially cytotoxic proteins and may be induced by increased intracellular AbetaPP/Abeta.

Oxidized and cross-linked proteins tend to accumulate in aging cells. Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross-linked proteins has been demonstrated in vitro. We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the accompanying paper, ref 1 ) of human BJ fibroblasts. During proliferative senescence, we found a marked decline in all proteasome activities (trypsin-like activity, chymotrypsin-like activity, and peptidyl-glutamyl-hydrolyzing activity) and in lysosomal cathepsin activity. Despite the loss of proteasome activity, there was no concomitant change in cellular levels of actual proteasome protein (immunoassays) or in the steady-state levels of mRNAs for essential proteasome subunits. The decline in proteasome activities and lysosomal cathepsin activities was accompanied by dramatic increases in the accumulation of oxidized and cross-linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxidatively damaged proteins generated by an acute, experimentally applied oxidative stress. Thus, oxidized and cross-linked proteins accumulated rapidly in cells of higher proliferation stages. Our data are consistent with the hypothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross-linked proteins during proliferative senescence until late proliferation stages, when so much proteasome activity has been lost that oxidized proteins accumulate at ever-increasing rates. Lysosomes attempt to deal with the accumulating oxidized and cross-linked proteins, but declining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulation of oxidized and cross-linked proteins in aging, is further explored during postmitotic senescence in the accompanying paper (1).