“Evidence for and against the role of oxidative stress in ageing.”
Ageing is defined as the progressive accumulation of changes, characterised by changes over time in physiological function and increased probability of pathological diseases and death.(1)It is of the general consensus that ageing begins with molecular damage that leads to cell, tissue, and eventual organ dysfunction.(1) Many theories have been put forward to explain ageing process. One of the most examined theory is the oxidative stress theory of ageing. This idea was first proposed by Denham Harman in 1956 as the free radical theory of ageing, suggesting that oxygen free radicals are related to ageing process.(2)In 1972, he published a refinement of his theory by highlighting mitochondria as most of reactive oxygen species (ROS) are produced here.(3) This theory postulates that oxidative stress is caused by an imbalance between prooxidants and antioxidants, leading to accumulation of oxidative damage to the cellular macromoleculecules over time. Under this framework, theoretically a long-lived organism should have a reduced oxidative stress.
A free radical is a molecule that has one or more unpaired valence electrons on its outer shell. As such, it is unstable and is highly reactive in order seek electrons to pair. Reactive Oxygen Species (ROS) include free radicals which contain oxygen, such as superoxide anion (•O2) and hydroxyl radical (•OH), and nonradical molecules that contribute to their formation, such as hydrogen peroxide (H2O2). In an effort to stabilize its bond, ROS can take away an electron from a target molecule in a process called oxidation, leading to a chain reaction which produce even more free radicals and causing deterioration to the targets. These targets can be DNA, protein, and lipid.(2,4)
ROS is produced by several processes in the body. It is a by-product of a normal metabolism. It is also produced in other enzymatic reactions such as xanthine oxidase, cytochrome P45, secretion of activated leukocytes, etc.(1) However, it is mainly produced by mitochondria, most of which are generated from the electron transport system as they consume aproximately 85% of the oxygen utilized in the cell.(4)
The production of ROS in mitochondria happens during oxidative phosphorylation occuring in the inner mitochondrial membrane, specificially in complexes I and III.(1,5) In this process, ROS are continuously produced in the mitochondrial electron transport chain where O2 is reduced to water H2O2. Unfortunately, this system is imperfect, causing electrons to leak from the electron transport chain to form •O2– instead. •O2– is short-lived and cannnot pass through the membrane. However, it can be converted to hydrogen peroxide (H2O2) either spontaneously or catalyzed by superoxide dismutase (SOD). H2O2 is membrane permeable and relatively stable, thus able to pass to the cytosol and damage other components. It has been estimated that about 2% of oxygen consumed in the mitochondria is turned into superoxide.(6)•OH, the most reactive ROS, is produced from Haber Weiss reaction, Fenton reaction, and reaction between hypochlorous acid and •O2–.(1)
Antioxidants function to limit the oxidative damage. There are three different mechanisms in which they may work: inhibiting the generation of ROS, scavenging the free radicals in an enzymatic way, and elevating the endogenous antioxidant defences.(1) Antioxidants consist of enzymatic and non-enzymatic types. The main enzymatic antioxidants are superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX). SOD catalyse the dismutation of superoxide to hydrogen peroxide. It is thought to be the primary antioxidant as it limits the further generation of free radicals.(1) SOD1 is located in the mitochondrial intermembrane space, SOD2 is located in the mitochondrial matrix, while SOD3 is located extracellularly.(7) Catalase works by catalysing the conversion hydrogen peroxide to water. It is mainly found in the peroxisomes, but it also found in lesser amount in mitochondria.(1) GPX functions in the same way, but it requires glutathione as a cofactor. GPX also prevent lipid peroxidation, thus maintaining the structure and function of biological membranes. It is located in mitochondria and cytosol.(1)
Non-enzymatic antioxidant, also known as small molecule antioxidants, are especially important extracellularly where enzymatic antioxidants are not present.(1) It consists of lipid-soluble and water-soluble components.(1) The lipid-soluble antioxidants are present in cellular membranes and lipoproteins while water-soluble antioxidants are present in aqueous fluid such as in the blood.(1)
Generation and of ROS and its metabolism by antioxidants are summarized in Figure 1.
Figure 1. Generation of ROS and metabolism by antioxidants. von Zglinicki T. Aging at the molecular level. Netherlands: Kluwer Academic Publisher; 2003.
As mentioned before, oxidative stress can cause damage to DNA, protein, and lipids. DNA lesions are divided into five types: oxidised purines, oxidised pyrimidines, abasic sites, single-strand breaks, and double strands breaks.(1)The most important repair mechanism for DNA is base excision repair (BER) (Figure 2).(1)BER begins with DNA glycosylase that recognizes the modified base and subsequently removes it, forming an abasic (AP) site.(1) This abasic site can be acted upon by two types of enzyme: AP lyase that cleaves the DNA strand 3’ or AP endonuclease that hydrolises the phosphodiester bond 5’.(1) Many glycosylase has an intrinsic lyase activity and is thus bifunctional.(1) The choice between these two is based on what damage is going to be repaired.(1) Removal of oxidised purines and pyrimides in involve DNA glycolylase, while repair of abasic sites is primarily acted on by AP endonucleases.(1) Action of AP lyase or AP endonuclease results in the formation of one-nucleotide gap, which may be further extended by Flap Endonuclease I (FEN1) and later the gap will be filled by DNA polymerase.(1) Single strand repair protein complex includes of POLB, XRCC1, ADPRT, LIG3, and PNK while double strand protein repair occurs by homologous recombination of RAD50/51/52/54 or nonhomologous end rejoining involving DNA-dependent protein kinase, KU70, KU80, LIG4, etc.(8) Repair of mitochondrial DNA adepends on the repair proteins being transported into the mitochondria and through BER mechanism.(1) Nucleotide excision repair (NER) also exist to repair bulky DNA lesions. It is shown that DNA in aged liver cells of mice contain a higher amount of 8-Oxo-2’-deoxyguanosine (oxo8dG) (Figure 3).(9)
Figure 2. Base Excision Repair. von Zglinicki T. Aging at the molecular level. Netherlands: Kluwer Academic Publisher; 2003.
Figure 3. Comparison of oxidative DNA damage from livers of young and old rats. Ames BN, Shigenaga MK, Hagen TM. Mitochondrial decay in aging. BBA – Molecular Basis of Disease. 1995;1271(1):165-70.
There are several ways in which oxidative pathways can damage protein: oxidation of the the protein backbone, formation of protein cross-linkages, oxidation of amino acid side chains, and protein fragmentation.(1) Indirect damage can also occur by ROS oxidising lipids and carbohydrates to form derivatives that can form protein carbonyl adducts or oxidising glycated proteins.(1) Repair of oxidative protein damage can occur directly or indirectly.(1) One of the most important process in direct repair systems is the re-reduction of oxidised sulfhydryl groups.(1) Another process that can involves methionine sulfoxide reductase (MsrA) which can regenerate methionine residues.(1) The indirect repair system consists of two steps: the recognition, removal, and degradation of the damaged protein molecule, followed by de novo synthesis of the removed protein.(1)
Damaged proteins are recognized by chaperones that can either repair or degrade the damaged protein.(10) However, as cells get older, there is a chaperone overload, thus causing the damaged proteins to accumulate intracellularly.(10)The two major proteolytic system involved in intracellular protein turnover are the lysosomal system and the ubiquitin-proteasome system (UPS).(10) The lysosomal system is associated with autophagy which consists macroautophagy, microautophagy, and chaperone-mediated autophagy.(10) Each of these types differ in the way in which they deliver substrates to the lysosomes, the type of substrates, and their regulation.(10)UPS consists of two steps: ubiquitylation and degradation.(10)Attachment ubiquitin molecules is the “tagging” of the damaged protein for proteasome to recognize and degrade.(10)
Oxidative lipid damage results in decreasing fluidity of cellular membranes with age.(11)This change is associated with changes in the composition of lipid membrane.(12) In the liver microsomal and mitochondrial membrane isolated from rodents, there is a decline in the amount of linoleic acid accompanied by an increase in the amount of long chain polyunsaturated fatty acids (PUFA), which are more sensitive to oxidative reactions.(12)Phospholipids which contain a high amount of PUFA make them prime target of oxidation, resulting in lipid peroxides.(1) MDA is a marker for lipid peroxidation, and it is shown that malondialdehyde accumulation is higher in aged liver and brain tissues (Figure 4).(9)Oxidatively damaged lipids are repaired by phospolipase A2.(4) Phospholipase A2 is located in the inner mitochondrial membrane and increase in activity when there is an increased oxidant production.(13)It catalyzes the removal of oxidised lipid in the membrane.(4)
Figure 4. Comparison of malondialdehyde accumulation in young (3 months) and old (26-31) rats. Ames BN, Shigenaga MK, Hagen TM. Mitochondrial decay in aging. BBA – Molecular Basis of Disease. 1995;1271(1):165-70.
In addition to mitochondria being the major producer of ROS, mitochondrial DNA (mtDNA) is also close in proximity to ROS.(14) Thus, mtDNA is more prone to oxidative damage than the nuclear DNA, as also shown in Figure 3.(4) As mitochondria is both the producer and the target of ROS, the mitochondrial theory of ageing postulates that the oxidative damage generated produce the most damage to mitochondrial macromolecules, including its mtDNA, proteins, and lipids.(14) Therefore, this damage leads to impaired oxidative phosphorylation and protein synthesis machinery, creating an even more imperfect system which will lead to further accumulation of ROS.(14) This is called the vicious cycle hypothesis.(14) If this continues, eventually the cell will be deprived of energy and eventually die.(14)
In trying to prove oxidative stress theory, most literatures try to prove the notion that long-lived animals has reduced oxidative damage or increased oxidative resistance. These interventions include caloric restriction (CR), genetic mutation, and pharmacological intervention such as metformin.
CR, an intervention consisting of reducing caloric intake without causing malnutrition, is one of the most examined intervention and is the most robust. For more than 80 years, it has produced reproducible results that it can increase lifespan up to 50% and decelerate the onset of age-associated pathologic and biologic changes in rodent and primate models.(15)This increase in lifespan is associated with reduced oxidative damage, shown by reduced levels of oxidised protein, lipid, and DNA, decreased production of ROS from the mitochondria, and increased oxidative resistance compared to rodents that are fed ad libitum.(7)Studies of CR in humans support the notion that CR remains the cornerstone to a healthy ageing, prevention of obesity and its complications, and is able to decrease mortality rate.(16)For example, in World War I and II, the CR imposed on them managed to reduce mortality rates by around 30% compared to pre-war level.(17) A study by Civitarese et al.(18)regarding CR in humans confirmed that it is accompanied by a decline of oxidative stress markers, consistent with the animal models.
As antioxidants can limit oxidative damage, theoretically organisms with long life-span should have an increased concentration of antioxidants to provide protection to oxidative resistance. However, several models of antioxidant knockdown and transgenic mice have not supported this concept.(7)It does, however, increase healthspan, the period in which an organism remain free of disease.(7)For example, a mice knockdown model of SOD2+/-, despite showing increased oxidative damage markes, have no change in lifespan compared to control mice, but has an increased incidence of cancer.(19)Transgenic mice studies provide results that antioxidants can reduce oxidative damage accumulation and provide resistance to oxidative, but the majority of the studies produce no effect on the lifespan, thus putting the oxidative damage theory to doubt.(7)An example of this is a transgenic mice that overexpress SOD1 and SOD2, shown to have increased resistance to superoxide toxicity but no effect in lifespan.(20)In fact, from this same study, out of 18 genetic manipulations they have done, only the deletion of SOD1 gene had an effect on lifespan.(20) Another potential explanation to this gap in theory is associated with the environment.(7)The environment contributes in ageing in which it differs in the amount of stress. In an environment with minimal stress, oxidative damage will play minimal role thus no enhanced antioxidant defense is needed.(7) In terms of these mice models, the environment is translated to the husbandry, as proven by the contradicting results of study between Moskovitz et al.(21)and Salmon et al.(22) Moskovitz et al.(21) observed a shorter lifespan in MsrA-/- mice while Salmon et al.(22) observed no change, suggesting that the mice used by Moskovitz et al.(21) may be kept at a less optimal husbandry.
A drug that has been most intensely examined as a geroprotective agent is metformin. Metformin is an insulin sensitizer widely used as a drug for diabetes type II. A meta-analysis shows that metformin can increase lifespan indepeendent of diabetes, as shown by it being able to reduce all-mortality cause, especially related to age-related diseases.(23)
Ageing has been closely associated such as sarcopenia, Alzheimer’s disease (AD), several types of cancer, cardiovascular diseases, etc. Several genetic mice models of AD have been developed. One of the most commonly used is the one that overexpresses a mutation of amyloid precursor protein (APP TG).(24) Reduction of SOD2 in these mice is shown to accelerate AD-like pathology such as amyloid deposition and nerodegeneration, conferring a positive association between decrease oxidative defense and AD.(25) In a similar manner, reduction of GPX4 also increase amyloid plaque burden and increases amyloid-β deposition.(26) Overexpression of SOD1 can increase lifespan of these mice.(27) However, this extension does not change the pathology of AD, thus it is thought that it either only protect against AD side effect or just give an oxidative stress protection in general which improves health.(7)As opposed to AD, there is a significant association between increased oxidative protein damage with lowered grip strength among older women.(28)
Nevertheless, a study in human oxidative stress marker in age-related disease support the oxidative stress theory. In a study by Schottker et al.(29)examining derivatives of reactive oxygen metabolite (D-ROM) levels and total thiol levels (TTL) in a population-based cohort from four countries, these serum markers are all strongly associated all-cause and cardiovascular mortality.(29) D-ROM levels even have an additional strong association with cancer mortality.(29)
In conclusion, researches regarding oxidative stress have produced conflicting results. In regards to oxidative stress theory itself, it can be tested either by modulating the oxidative stress or the antioxidant defenses. Interventions supporting the role of decreased oxidative stress, such as CR and metformin, have produced supporting results towards the oxidative stress theory and is now still being investigated more intensively, which is good especially since these interventions can be applied to humans with seemingly favorable results. Interventions regarding genetic modification of antioxidant defenses, however, seems to point towards changes in healthspan rather in lifespan. Therefore, I think further research in this area should focus more on healthspan. Moreover, this conflicting results is probably due to the presence of more factors at play. Oxidative stress theory is only one example of the single-cause molecular theories. It is highly unlikely that each of the molecular theories work alone and only by understanding all these theories can ageing be fully understood. In addition, I personally think that this theory need more clinical studies in humans as the majority are now done in animal models. However, I realize that in some cases it may be impossible to do. For example, examination of antioxidant defences which are done by genetic alteration is of course impossible to be done in human. Therefore, I think as of now, the most promising oxidative damage modification is CR as it has been proven over and over again to be able to increase lifespan and delay age-related conditions.
1. von Zglinicki T. Aging at the molecular level. Netherlands: Kluwer Academic Publisher; 2003.
2. Harman D. Aging: a theory based on free radical and radiation chemistry. Journal of gerontology. 1956;11(3):298-300.
3. Harman D. The Biologic Clock: The Mitochondria? Journal of the American Geriatrics Society. 1972;20(4):145-7.
4. Ames BN, Shigenaga MK, Hagen TM. Mitochondrial decay in aging. BBA – Molecular Basis of Disease. 1995;1271(1):165-70.
5. Nakamura S, Takamura T, Matsuzawa-Nagata N, Takayama H, Misu H, Noda H, et al. Palmitate induces insulin resistance in H4IIEC3 hepatocytes through reactive oxygen species produced by mitochondria. Journal of Biological Chemistry. 2009;284(22):14809-18.
6. Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochemical Journal. 1973;134(3):707-16.
7. Salmon AB, Richardson A, Pérez VI. Update on the oxidative stress theory of aging: Does oxidative stress play a role in aging or healthy aging? Free Radical Biology and Medicine. 2010;48(5):642-55.
8. Whitehouse CJ, Taylor RM, Thistlethwaite A, Zhang H, Karimi-Busheri F, Lasko DD, et al. XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair. Cell. 2001;104(1):107-17.
9. Fraga CG, Shigenaga MK, Park JW, Degan P, Ames BN. Oxidative damage to DNA during aging: 8-Hydroxy-2′-deoxyguanosine in rat organ DNA and urine. Proceedings of the National Academy of Sciences of the United States of America. 1990;87(12):4533-7.
10. Martinez-Vicente M, Sovak G, Cuervo AM. Protein degradation and aging. Experimental Gerontology. 2005;40(8-9):622-33.
11. Huber LA, Xu QB, Jürgens G, Böck G, Bühler E, Fred Gey K, et al. Correlation of lymphocyte lipid composition membrane microviscosity and mitogen response in the aged. European Journal of Immunology. 1991;21(11):2761-5.
12. Laganiere S, Yu BP. Modulation of membrane phospholipid fatty acid composition by age and food restriction. Gerontology. 1993;39(1):7-18.
13. Hatch GM, Vance DE, Wilton DC. Rat liver mitochondrial phospholipase A2 is an endotoxin-stimulated membrane-associated enzyme of Kupffer cells which is released during liver perfusion. Biochemical Journal. 1993;293(1):143-50.
14. Cui H, Kong Y, Zhang H. Oxidative stress, mitochondrial dysfunction, and aging. Journal of Signal Transduction. 2011;2012(646353):1-13.
15. Weindruch R. The retardation of aging by caloric restriction: Studies in rodents and primates. Toxicologic Pathology. 1996;24(6):742-5.
16. Most J, Tosti V, Redman LM, Fontana L. Calorie restriction in humans: An update. Ageing Research Reviews. 2017;39:36-45.
17. Hindhede M. The effect of food restriction during war on mortality in copenhagen. Journal of the American Medical Association. 1920;74(6):381-2.
18. Civitarese AE, Carling S, Heilbronn LK, Hulver MH, Ukropcova B, Deutsch WA, et al. Calorie restriction increases muscle mitochondrial biogenesis in healthy humans. PLoS Medicine. 2007;4(3):485-94.
19. Van Remmen H, Salvador C, Yang H, Huang TT, Epstein CJ, Richardson A. Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse. Archives of Biochemistry and Biophysics. 1999;363(1):91-7.
20. Pérez VI, Bokov A, Remmen HV, Mele J, Ran Q, Ikeno Y, et al. Is the oxidative stress theory of aging dead? Biochimica et Biophysica Acta – General Subjects. 2009;1790(10):1005-14.
21. Moskovitz J, Bar-Noy S, Williams WM, Requena J, Berlett BS, Stadtman ER. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(23):12920-5.
22. Salmon AB, Pérez VI, Bokov A, Jernigan A, Kim G, Zhao H, et al. Lack of methionine sulfoxide reductase A in mice increases sensitivity to oxidative stress but does not diminish life span. FASEB Journal. 2009;23(10):3601-8.
23. Campbell JM, Bellman SM, Stephenson MD, Lisy K. Metformin reduces all-cause mortality and diseases of ageing independent of its effect on diabetes control: A systematic review and meta-analysis. Ageing Research Reviews. 2017;40:31-44.
24. Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, et al. Correlative memory deficits, Aβ elevation, and amyloid plaques in transgenic mice. Science. 1996;274(5284):99-102.
25. Esposito L, Raber J, Kekonius L, Yan F, Yu GQ, Bien-Ly N, et al. Reduction in mitochondrial superoxide dismutase modulates Alzheimer’s disease-like pathology and accelerates the onset of behavioral changes in human amyloid precursor protein transgenic mice. Journal of Neuroscience. 2006;26(19):5167-79.
26. Chen L, Na R, Gu M, Richardson A, Ran Q. Lipid peroxidation up-regulates BACE1 expression in vivo: A possible early event of amyloidogenesis in Alzheimer’s disease. Journal of Neurochemistry. 2008;107(1):197-207.
27. Borg J, Chereul E. Differential MRI patterns of brain atrophy in double or single transgenic mice for APP and/or SOD. Journal of Neuroscience Research. 2008;86(15):3275-84.
28. Howard C, Ferrucci L, Sun K, Fried LP, Walston J, Varadhan R, et al. Oxidative protein damage is associated with poor grip strength among older women living in the community. Journal of Applied Physiology. 2007;103(1):17-20.
29. Schöttker B, Brenner H, Jansen EHJM, Gardiner J, Peasey A, Kubínová R, et al. Evidence for the free radical/oxidative stress theory of ageing from the CHANCES consortium: A meta-analysis of individual participant data. BMC Medicine. 2015;13(1).