The oxidative hypothesis of senescence
M Gilca, I Stoian, V Atanasiu, B Virgolici
Department of Biochemistry, Faculty of Medicine, "Carol Davila" University of Medicine and Pharmacy, 8, Eroilor Sanitari, 76241 Bucharest, Romania
Department of Biochemistry, Faculty of Medicine, «DQ»Carol Davila«DQ» University of Medicine and Pharmacy, 8, Eroilor Sanitari, 76241 Bucharest, Romania
The oxidative hypothesis of senescence, since its origin in 1956, has garnered significant evidence and growing support among scientists for the notion that free radicals play an important role in ageing, either as «DQ»damaging«DQ» molecules or as signaling molecules. Age-increasing oxidative injuries induced by free radicals, higher susceptibility to oxidative stress in short-lived organisms, genetic manipulations that alter both oxidative resistance and longevity and the anti-ageing effect of caloric restriction and intermittent fasting are a few examples of accepted scientific facts that support the oxidative theory of senescence. Though not completely understood due to the complex «DQ»network«DQ» of redox regulatory systems, the implication of oxidative stress in the ageing process is now well documented. Moreover, it is compatible with other current ageing theories (e.g., those implicating the mitochondrial damage/mitochondrial-lysosomal axis, stress-induced premature senescence, biological «DQ»garbage«DQ» accumulation, etc).
This review is intended to summarize and critically discuss the redox mechanisms involved during the ageing process: sources of oxidant agents in ageing (mitochondrial -electron transport chain, nitric oxide synthase reaction- and non-mitochondrial- Fenton reaction, microsomal cytochrome P450 enzymes, peroxisomal β -oxidation and respiratory burst of phagocytic cells), antioxidant changes in ageing (enzymatic- superoxide dismutase, glutathione-reductase, glutathion peroxidase, catalase- and non-enzymatic glutathione, ascorbate, urate, bilirubine, melatonin, tocopherols, carotenoids, ubiquinol), alteration of oxidative damage repairing mechanisms and the role of free radicals as signaling molecules in ageing.
|How to cite this article:|
Gilca M, Stoian I, Atanasiu V, Virgolici B. The oxidative hypothesis of senescence.J Postgrad Med 2007;53:207-213
|How to cite this URL:|
Gilca M, Stoian I, Atanasiu V, Virgolici B. The oxidative hypothesis of senescence. J Postgrad Med [serial online] 2007 [cited 2019 Jun 19 ];53:207-213
Available from: http://www.jpgmonline.com/text.asp?2007/53/3/207/33869
Two principal types of ageing theories have been developed: theories of "accidental" ageing produced by "errors" represented by random deleterious mechanisms that induce progressive damage of various levels; and theories of "programmed" ageing induced by the collection of by-products of gene action selected to enhance reproductive fitness.  Even if there are no genes that specifically evolved to induce senescence, scientists estimated that allelic variation or mutation at up to 7000 relevant genes might modulate patterns of ageing in man.  Certain polymorphisms (antagonistic pleiotropy) might underlie common or "public" mechanisms of ageing, while rare mutations lead to uncommon or "private" mechanisms of ageing. , These two theories are not mutually exclusive, especially when oxidative stress is considered. The oxidative theory of aging was first advanced in 1956 by Harman: free radicals, normally produced in the organisms, react with cellular constituents and initiate the age-associated changes. ,
A Medline search (years 1966 through December 2006, using search terms senescence, ageing, oxidative stress and free radicals), supplemented by subsequent reference searches of retrieved articles and hand search of Free Radicals Biology and Medicine 1989-2006 identified studies relevant to the oxidative hypothesis of ageing.
Age-increasing oxidative damages induced by free radicals or higher susceptibility to oxidative stress in short-lived organisms are mentioned by supporters of both types of theories:
a. Oxygen-reactive species play a key role in age-related sporadic degenerative diseases (e.g., Alzheimer's disease, atherosclerosis, diabetes) or various specific components of what scientists refer to as the senescent phenotype (e.g., tissue atrophies, etc.). 
b. Resistance to oxidative stress is a common trait of long-lived genetic variants of non-mammalian and mammalian organisms. , Genetic variations in antioxidant defense genes (e.g. prion protein gene PRNP) were found to have important influences on the trajectory of normal ageing.  This fits with the oxidative hypothesis of senescence.
Unfortunately, extensive research on the relationship between polymorphisms likely to be responsible for the common mechanism of ageing and resistance to oxidative stress has been neglected. Therefore, the paucity of data does not allow us to yet conclude that the oxidative theory supports the theory of programmed ageing.
However, the most recent studies strongly support the idea that oxidative stress is a significant marker of senescence, being established in different species. This age-related oxidative stress is generated by a combination of increased production of free radicals and other oxidant agents, decreased antioxidant levels and impaired repair of oxidative damages. Oxidant agents, including reactive oxygen species (ROS) and reactive nitrogen species (RNS) (e.g., nitric oxide, NO) are recognized to play a dual role as both malefic and beneficial species, being sometimes compared with fire, which is dangerous, but nonetheless useful to humans.  Reactive oxygen species and RNS have many crucial biological functions as signaling molecules in growth, apoptosis, neurotransmission, etc. To control these reactive species with "two faces", cells evolved complex and critical regulatory mechanisms which become disrupted with age. Senescence is just one example of pathophysiological implications of redox dysregulation.  The initiation of ageing is marked by a shift from redox regulation to redox dysregulation.  Why this shift takes place is not yet clear.
This review focuses on how alterations in oxidants, protective agents and repair machinery during the lifespan may offer insights into the possible scenarios of biological ageing.
Definition of Ageing
Ageing is an extremely complex, multifactorial process and represents the gradual deterioration in function that occurs after maturity and leads to disability or death. A broader perspective includes all biological processes occurring within the organism from the beginning of life (fertilization) until death. A more recent and precise definition identifies ageing with the inability of the organism to respond to stress and to maintain homeostatic regulation when given a challenge, thereby decreasing the capacity of the organism to survive detrimental changes occurring with time during postmaturational life. 
Main Sources of Oxidant Agents in Ageing
There are two main types of oxidant sources: mitochondrial sources (which play the principal role in ageing) and non-mitochondrial sources (which play different and sometimes specific roles, especially in the pathogenesis of age-related diseases).
Mitochondrial sources are represented by the electron transport chain and the nitric oxide synthase reaction.
Mitochondria seem to be the principal source of endogenous oxidants implicated in ageing. The rate of respiration is responsible for the rate of generation of reactive oxygen species (ROS), this characteristic being consistent with the observation that the higher metabolic rates an organism has, the shorter maximum lifespan potential it presents.  One-electron reduction of O 2 to form the superoxide anion (O 2· - ) and dismutation of O 2· - to yield hydrogen peroxide (H 2 O 2 ) occurs during mitochondrial respiration.
Mitochondria are also involved in the generation of nitric oxide (NO·) via the nitric oxide synthase (NOS) reaction. O 2· - and NO· react to form another oxidant, peroxynitrite (ONOO - ), which represents a potential source for the more powerful and aggressive hydroxi radical (·OH).
Mitochondria possess superoxide dimutase (mSOD) that rapidly scavenges O 2· - . Some researchers believe that mSOD prevents the accumulation of O 2· - , while others argue that mSOD increases the rate of O 2· - generation by accelerating the removal of this radical by dismutation to H 2 O 2 . ,
Dysfunctional mitochondria accumulate during ageing due to the oxidative damage of mitochondrial macromolecules.  The role of mitochondrial oxidative damage in ageing and Alzheimer's disease (AD) has important implications for therapeutics: mitochondrial antioxidant therapy has been found to be one of the most efficacious methods in reducing pathological changes in the brain tissues in AD animal model studies. 
The theory of mitochondrial ageing predicts that the mitochondrial DNA (mtDNA) proximity to the cell's major source of free radicals renders it very susceptible to oxidative insults and thereby increases the rate of mtDNA mutations, leading to an aggravation of the aerobic respiration dysfunction (mtDNA encodes proteins of the respiratory chain). The consequent decrease of electron transfer leads to further production of ROS, thus establishing a vicious circle of oxidative stress and energetic decline, which is suspected to be one of the principal causes of ageing. 
Mitochondrial impairments lead also to the activation of nuclear genes. This signaling pathway from the mitochondrion to the nucleus, named the retrograde response, seems to influence cell division, stress resistance and, eventually, ageing rate and lifespan, at least in fungal models. 
Caloric restriction leads to reduced production of mitochondrial ROS and thus to a reduction in mitochondrial oxidative stress, which may be responsible for an increase in the lifespan. 
However, most of the available data supporting the mitochondrial theory of ageing are merely correlative and therefore do not exclude the possibility that ROS production and mtDNA mutations are effects rather than driving forces of ageing. Moreover, there is recent controversial evidence that mitochondrial mutations do not limit the lifespan of wild-type mice, specific point mutations may not accumulate with ageing in the mouse mitochondrial DNA control region and mitochondrial ROS production might not be affected in mtDNA mutator mice displaying the ageing phenotype. ,,
The H 2 O 2 -degrading Fenton reaction is catalyzed by free iron bivalent ions and leads to the generation of ·OH. Recent studies localized the Fenton reaction at the endoplasmic reticulum or perinuclear, but not at mitochondria or other compartments.  Sources of H 2 O 2 could be mitochondria (superoxide dismutase reaction), peroxisomes (acyl-CoA oxidase reaction) and amyloid b of senile plaques (superoxide dismutase-like reactions).  H 2 O 2 that escapes antioxidant machinery, such as glutathione peroxidase and catalase, might be converted nonenzymatically in a perinuclear-localized Fenton reaction and act as an RNA- or DNA-damaging agent. A recent study showed that the Fenton reaction is involved in the oxidation of ribosomal RNA in tissue brain obtained at autopsy from confirmed cases of Alzheimer's disease.  Therefore, it may play a special role in the context of ageing, also taking into account that the body's content of iron increases with age. ,
Microsomal cytochrome P450 enzymes
Microsomes contain the cytochrome P450 enzymes, which catalyze univalent oxidation or reduction of xenobiotic compounds (e.g., drugs); simultaneously O 2· - is generated.  Direct studies on drug-metabolizing capacity in elderly humans are scant.  However, several laboratories have concluded that ageing induces a decline in concentrations of cyt P450 monoxygenases in senescent animals, while starvation, which has a well-known lifespan-prolonging effect, increases the expression of the different proteins of the cytochromes P450 family.  The highest upregulated gene among the 20.000 genes analyzed before and after 24h and 48h starvation, is cytochrome P450 4A14 (Cyp4a14).  Beyond the secondary production of O 2· - , the members of the cyt P450 family are involved in resistance to oxidative stress, thereby increasing longevity.
The age-induced decline of cyt P450 enzymes could contribute to the high incidence of adverse drug reactions and toxicities reported in older people, as well as decreased antioxidant protection, but cannot account for a higher ROS production during ageing. 
Respiratory burst of phagocytic cells
The respiratory burst of phagocytic cells is a source of O 2· - via the NADPH oxidase reaction during inflammatory or infectious conditions. According to the theory of stress-induced premature senescence (SIPS), sublethal doses of different stressor agents (H 2 O 2 , hyperoxia, tertbuthylhidroperoxide, UV) lead to the exhaustion of the replicative potential of the proliferative normal cell types and the accumulation of senescent cells, which might be responsible for the creation of a micro-inflammatory state and the activation of phagocytic cells, thereby participating in tissue ageing.  Franceschi has shown that in the healthy elderly and the centenarians, the proinflammatory status is elevated and IL-6 plasma levels correlate with risk of death. 
Short-term fasting (80h) in healthy human subjects has induced a decrease of the stimulatory index of leukocytes activated with opsonized zymosan, phytohemagglutinin P and concanavalin A.  Recent animal studies have also shown that short-term repeated fasting is effective in the prolongation of lifespan or protects against age-related diseases. ,,, It is a reasonable assumption that the anti-ageing effect of intermittent fasting might be also due to the reversal of the age-related proinflammatory state.
Peroxisomal β-oxidation of fatty acids generates H 2 O 2 . The peroxisomes also contain catalase that decomposes H 2 O 2 and thus prevents local acumulation of this toxic compound. As cells age, the ability of peroxisomes to maintain this balance between peroxisomal pro-oxidants and antioxidants is gradually compromised. Senescent peroxisomes produce an increasing amount of ROS, not by increasing the H 2 O 2 synthesis, which is, on the contrary, probably decreased, but through an inefficient import of catalase (peroxisomes import enzymes post-translationally from cytosol). ,
Peroxisome proliferators, which increase the number of peroxisomes and the activity of enzymes involved in the β-oxidation of fatty acids, also cause oxidative damage.  PPAR protects against the oxidative damage associated with ageing, possibly by preventing the accumulation of oxidized fatty acids.
Antioxidant Agents in Ageing
Antioxidants are classified in two main groups: enzymatic antioxidant agents and non- enzymatic antioxidant agents. Both of them are modified during the process of ageing.
Enzymatic antioxidant agents
Several studies indicate that age induces different patterns of antioxidant enzymatic activities (superoxide dismutase SOD, glutathione peroxidase GSH-Px, glutathione reductase GR, glutathione-S-transpherase GSH-S-T, MSRA- methionine sulfoxide reductase) expressions. Some discrepancy exists in the described activities of the same enzyme [Table 1].
Most of the studies on the genetic manipulation of antioxidant enzyme genes support the oxidative theory of ageing [Table 2].
Non-enzymatic antioxidant agents
Hydrophilic non-enzymatic antioxidants are radical scavengers (e.g. glutathione (GSH), ascorbate, urate and bilirubin). Reduced glutathione, reduced glutathione/total glutathione ratio and ascorbate decline slightly in plasma or in lymphocytes with age. ,
The altered levels of hydrophilic antioxidants have also been correlated with various age-related diseases (e.g. the decline of melatonin in aged individuals has been suggested to contribute to the development of neurodegenerative disease). 
There are contradictory results concerning the effects of age on serum urate, which was either increased in aged women, but not in men or unchanged in centenarian subjects when compared with healthy adults. , Total bilirubin was significantly reduced in aged people. 
Lipophilic non-enzymatic antioxidants are radical scavengers such as tocopherols, carotenoids, ubiquinol and flavonoids.
In one human study, the plasma alpha and total tocopherol concentrations did not change significantly with age. However, the plasma gamma tocopherol, platelet alpha, gamma tocopherol, total tocopherol concentrations and the platelet-to-plasma ratios of tocopherol concentrations decreased significantly with age.  Also, decreased levels of coenzyme Q10 in rat and human tissues have been reported during ageing. 
Repair of Oxidative Damages in Ageing
There are several types of repairing mechanisms depending on the nature of the oxidized target.  These mechanisms are mainly based either on regenerating the slightly oxidized macromolecules keeping critical chemical groups in their reduced forms or on degrading defective highly oxidized macromolecules into low-molecular-mass compounds (that are then removed or re-utilized for building up new biological structures).
Some enzymes play several protective roles, simultaneously acting as an antioxidant enzyme that scavenges ROS and as a repair enzyme that eliminates damages (e.g. GSH-Px/GR, methionine sulfoxide reductase).
Many of these essential maintenance and repair systems become deficient in senescent cells, thus a high amount of biological "garbage" is accumulated (e.g. intralysosomal accumulation of lipofuscin). , Age-related oxidative changes are most prominent in non-proliferating cells, such as neurons and cardiac myocites, because there is a lack of dilution effect of damaged structures through cell division. 
The DNA repair ability correlates with species-specific lifespan, being necessary but not sufficient for longevity. 
There is an age-related decline in proteasome peptidase activities and proteasome content in different tissues (e.g., rat liver, human epidermis), which leads to accumulation of oxidatively modified proteins.  The total amount of oxidatively modified proteins for an 80-year-old human is estimated to be up to 50%. 
Among protein-bound aminoacids, cysteine and methionine residues are particularly sensitive to oxidation to disulfide bridges and methionine derivatives sulfoxides, but these minor oxidative damages are readily repaired by the action of thioredoxin reductase and methionine sulfoxide reductase (MSRA).  According to several studies, MSRA seems to be a regulator of antioxidant defense, oxidative stress-response gene expression and ageing in mammals. ,
Oxidative Damages in Ageing
All the biologically active molecules are susceptible to suffering oxidative damages and thus failing to accomplish their native roles.  A huge diversity of negative effects results from this indiscriminate oxidation. Elevated levels of oxidized lipids, DNA, proteins and glycoxidation macromolecules are found in aged organisms. ,,
The hydroxil radical oxidizes DNA, leading to the formation of adducts 8-oxo-7,8 dihydro-2'-deoxyguanosine (oxo 8 dG) in susceptible 5-GC-3'. The frequency of oxidative DNA adducts increases by as much as twofold with age in different species and tissues. 
Mitochondrial DNA is more vulnerable to oxidative damages than nuclear DNA because it is not protected by histones and mitochondria are the primary sites of ROS generation. , This leads to mutations of mitochondrial DNA, involving the genes coding for respiratory chain proteins and also may disturb the division of mitochondria, resulting in their enlargement. Larger mitochondria are less autophagocytosed (by lysosomes which are overloaded with lipofuscin) and undergo further oxidative damage. Briefly, the mitochondrial-lysosomal axis theory of ageing sustains that the accumulation of dysfunctional mitochondria and lysosomes leads irreversibly to cell death. 
Proteins isolated from aged individuals exhibit a higher carbonyl content, which estimates the overall extent of protein oxidation (e.g. "old" ceruloplasmin versus "young" ceruloplasmin contains 0.6mol versus 0.2 mol of carbonyl/mol of protein).  In spite of these age-induced oxidative changes, some proteins do not lose their biological function (e.g. ceruloplasmin oxidase activity is not altered in older individuals). 
Reactive oxygen species as mediators of cellular senescence
Reactive oxygen species also play the role of signaling molecules in cellular senescence and the age-related increase in mithocondrial-triggered apoptosis. ,,, However, the molecular mechanism of signaling remains obscure in certain cases (e.g. superoxide signaling). A new signaling hypothesis based on the frequently forgotten "super"-nucleophilic properties of superoxide anion has been recently proposed.  Free radicals induce alterations in gene expression (e.g. p53, HSP70, Bcl family genes). Regulation errors of the signaling cascade may also be responsible for the development of ageing. ,,
Seladin-1, a protein whose expression is down-regulated in Alzheimer's disease, protects cells from oxidative stress.  Reactive oxygen species induce redistribution of seladin-1 from cytosol to the nucleus, where it physically binds to p53 and leads to p53 accumulation, G1 arrest and replicative senescence. 
Another mechanism underlying cellular senescence is telomere attrition.  Telomeres are located at the end of chromosomes and each division is associated with a decrease in telomere length. It is already established that a high level of oxidative stress shortens telomeres and triggers the senescence. 
Controversial issues concerning oxydative hypothesis of senescence
"Premature" ageing without oxidative stress hypersensitivity. The oxidative hypothesis of senescence does not explain "premature" ageing. Neither accelerated nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models, which are mainly associated with accelerated fibroblast senescence.  Gene actions that alter oxidative stress resistance, but not lifespan. Genetic manipulations that increases CuZn-SOD activity and, thus, resistance to oxidative stress, have only a slight, if any, effect on maximum lifespan in several species.  Simultaneous overexpression of MnSOD and mitochondrial CAT in transgenic Drosophila induced a paradoxical decrease of lifespan, which might signify that only physiological levels of O 2· - /H 2 O 2 (neither excess or deficit) promote normal ageing. 
No association between A16V, C47T MnSOD or C262T CAT polymorphisms and age-related mortality or phenotypes was found in humans. However, genotype AA MnSOD was associated with an immunosenescence profile and DNA damage, while TT CAT was associated with improved physical functioning. ,,
In another study, P66(shc-/-) mice exhibited prolonged lifespan and increased resistance to oxidative stress, but unexpectedly, centenarians showed the highest basal levels of p66(shc) when compared with young people and the elderly. Lack of predictability. Some critics of the oxidative theory claim that the failure of antioxidant interventions to stop or reverse the aging process and to quell the current pandemic of age-related diseases (e.g. cardiovascular disease) brings the oxidative hypothesis into question.  Nevertheless, since ageing is a complex dysregulation of many redox systems, single antioxidant administration should not necessarily be expected to influence the ageing process. Complex multiple antioxidant interventions or complete dietary changes might be more successful in this respect.Lack of direct cause-and-effect evidence. Although a growing body of evidence points towards the implication of redox dysregulation as an important determinant of ageing, a direct cause-and-effect relationship between the accumulation of oxidatively mediated damage and ageing has not been clearly established. 
In one way or another, oxidative stress is mentioned in many theoretical and experimental scenarios of ageing. The life trajectory seems to be parallel with the oxidative stress resistance in most cases. Therefore, revealing all the aspects of redox alterations during senescence may be a key for controlling the rate of ageing and the longevity potential of the organisms. Nevertheless, the main question is still without answer: which of the mechanisms (oxidative damages, garbage catastrophe, mutation accumulation, antagonistic pleiotropy, etc.) proposed by scientists to cause senescence do cause ageing in the natural population? The answer might be more complex than we expect and probably a more integrative approach should be adopted to solve the dilemma.
Viasan Grant from the Ministry of Education and Science, Romania, project "New aspects concerning redox systems involved in ageing and potential therapeutic implications". The authors would particularly like to thank Dr. Ralph Miller (Canadian Commission's Research ex-Director) for his comments and support during the revision process.
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