Beneficial and Detrimental Effects of Reactive Oxygen Species on Lifespan: A Comprehensive Review of Comparative and Experimental Studies

1Aging can be described as the gradual loss of fitness due to detrimental changes occurring at the cell and molecular level over time. It is characterized by dysregulation of cellular processes, accumulation of damaged materials and toxins, altered gene expression, and poor immune and stress responses. While the changes that occur during the aging process have been well described, the mechanisms underlying aging remain poorly understood, and have thus been the subject of much research. One of the most widely accepted theories of aging, called the free radical theory of aging (FRTA), proposes that oxidative damage caused by reactive oxygen species (ROS) is the primary cause of aging. While there have been many studies examining the relationship between ROS and aging, this is still an area of much debate. In this work, we review comparative studies and studies involving experimental modulation that have investigated the role of ROS in aging across species. Overall, these studies demonstrate that the relationship between ROS and aging is complex, in that ROS can both increase or decrease lifespan depending on the experimental conditions.

2Reactive oxygen species are highly reactive, oxygen containing molecules that are the result of an incomplete reduction of molecular oxygen in the cell. ROS can be free radicals, or molecules that have the capacity to generate free radicals. Free radicals consist of atoms or molecules with an unpaired electron in their outer shell causing them to be unstable and highly reactive, or in other words, prone to “stealing” electrons from other molecules. Free radicals are highly reactive and are therefore generally short-lived and often unable to leave the subcellular location where they are generated without first becoming reduced. Examples of ROS that are free radicals include the superoxide, hydroxyl (HO•), peroxyl, hydroperoxyl, and alkoxyl radicals (RO•). ROS that are not free radicals do not have unpaired electrons, and are often less reactive, thus allowing them to leave the subcellular location where they are generated as well as pass through membranes. Examples of ROS that are not free radicals include hydrogen peroxide (H2O2), the hydroxide ion (OH–) and organic peroxides (ROOH).

3Despite being less reactive, ROS that are not free radicals are still incompletely reduced and thus can undergo redox reactions to produce free radicals as a result. For example, if hydrogen peroxide encounters a reduced transition metal ion such as ferrous iron (Fe2+) or cuprous copper (Cu+), the Fenton reaction will occur (Figure 1), resulting in the production of the hydroxyl radical which acts as a potent oxidant. The ability for ROS that are not free radicals, such as hydrogen peroxide, to move within the cell becomes important when considering the redox state for cell signaling in various subcellular locations as well as antioxidant distribution within the cell.

4In addition to ROS, organisms also generate reactive nitrogen species (RNS). Nitric oxide (NO) is produced from L-arginine by the enzyme nitric oxide synthase of which there are three forms: endothelial (eNOS), inducible (iNOS) and neuronal (nNOS). Nitric oxide can then react with superoxide to produce peroxynitrite (ONOO–), which can directly damage cellular components, or further react to generate other types of RNS. As with ROS, there are both free radical forms of RNS [nitric oxide, nitric dioxide (NO2⋅), nitrate radical (NO3⋅)] and non-radical forms [peroxynitrite, nitrous acid (HNO2), nitrite (NO2−), nitrosyl cation (NO+), nitroxyl anion (NO−), peroxynitrous acid (ONOOH), dinitrogen trioxide (N2O3)]. Also similar to ROS, RNS can cause cellular damage called nitrosative damage, but also have functional roles in cellular signaling and pathogen defense. Little is known about the effects of RNS on longevity.

5The tendency for free radicals to steal electrons in order to stabilize themselves can be problematic in cells, where they may cause oxidative damage to macromolecules such as DNA, proteins and lipids. When DNA is exposed to ROS, guanine is modified to 8-oxoguanine (Figure 2A), allowing it to pair with cytosine and adenine. This mutation can occur in both nuclear and mitochondrial DNA and can give rise to double stranded breaks (DSBs) in the DNA, leading to genomic instability. Proteins can be damaged when amino acid side chains and backbones, especially in thiol-containing cysteine and methionine residues, are oxidized by ROS (Figure 2B); this can result in structural changes to the protein that may lead to loss of function or be used for ROS-mediated signaling. Additionally, the exposure of lipids to ROS results in lipid peroxidation (Figure 2C), which gives rise to cell membrane damage and generates reactive by-products which can further damage the cell.

6All of these types of oxidative damage have been shown to increase with advancing age including DNA damage, protein carbonylation, lipid peroxidation and damage to mitochondrial DNA. As a result, these outcome measures have been proposed as biomarkers of aging [reviewed elsewhere e.g.].

7The FRTA proposes that aging is caused by the accumulation of molecular damage caused by ROS that are generated by normal metabolism. The logic of the FRTA stems from the idea that ROS have the capacity to be highly damaging to cell components, as well as the idea that the mechanism of aging is likely to be an intrinsic process given that aging always occurs, regardless of differences in environment. The FRTA developed into the mitochondrial FRTA (MFRTA) which became a popular way to explain why the rate of aging and the maximum lifespan varies so significantly among species. The MFRTA specifies that the ROS that cause aging are produced by the mitochondria, and that the lifespan of an organism thereby depends on their rate of oxygen consumption by the mitochondrial respiratory chain, which is believed to be the main process that produces ROS. The theory fit with the idea that the mechanism of aging is intrinsic, given the constant endogenous production of ROS as a byproduct of a process essential to all known aerobic organisms.

8Initially proposed by and revised in, the FRTA has since been supported by observations on the association between ROS, oxidative damage and longevity. These observations include: (1) higher levels of ROS generation with increasing age; (2) higher levels of oxidative damage with increasing age; (3) gradual increase in mitochondrial dysfunction with age; (4) an increase in ROS generation upon inhibition of components of the electron transport chain; and (5) high levels of oxidative stress in several age-related diseases.

9In addition to support for the FRTA, observations refuting the theory have also accumulated, with recent findings ultimately leading to the formation of new models for the relationship between ROS, redox signaling, oxidative damage and lifespan. Newer models for the role of ROS in the cell suggest that a mild elevation in ROS can be beneficial to an organism, perhaps through the activation of cellular stress response signaling pathways, while very low and very high levels can be detrimental to organisms. These models incorporate the importance of the maintenance of adequate ROS levels for redox signaling components (ex. thiol-containing proteins) which may rely on ROS-mediated oxidation in order to activate desired cell survival pathways.

10Mitochondrial respiration is a major source of ROS within the cell. As electrons are transferred between the complexes of the electron transport chain, some of these electrons can “leak” out to react directly with oxygen to form superoxide (Figures 1A and 3). The majority of electron leakage is thought to occur as electrons are passed from Complex I or Complex II to Complex III via ubiquinone.

11There are also important non-mitochondrial sources of ROS in the cell. For example, immune cells attack pathogens in the body by releasing ROS extracellularly or into the phagolysosome, a strategy termed the respiratory burst (Figure 3). The ROS used in this attack are generated by the membrane-bound phagocyte NADPH oxidase as well as the granule-localized myeloperoxidase. NADPH oxidase uses the electrons donated from NADPH to convert molecular oxygen to a superoxide anion (Figure 1B), while myeloperoxidase (MPO) converts hydrogen peroxide to hypochlorous acid (Figure 1D). Importantly, NADPH oxidase homologs (NOX enzymes; Figure 3) expressed in other cell types also play important roles in the generation of ROS for signal transduction in pathways for growth, survival and apoptosis.

12In fact, there are many ROS-generating enzymes that play essential roles in various physiological systems (Figure 3). Some examples include: the cytochrome P450 (CYP) family of enzymes, which produce ROS in the process of the detoxification and excretion of xenobiotics; xanthine oxidoreductase (XOR), which produces superoxide anions during the break down of purines to uric acid; superoxide dismutase (SOD), which converts superoxide to hydrogen peroxide and oxygen (Figure 1C); and monoamine oxidase, which breaks down the neurotransmitter dopamine after signaling occurs and produces hydrogen peroxide in neurons in the process.

13Finally, an additional source of ROS comes from β-oxidation of fatty acids in the peroxisome, producing hydrogen peroxide which can then cross the plasma membrane of the peroxisome, to reach the cytoplasm (Figure 3). Along with NADPH oxidase-generated ROS, peroxisome-generated ROS are thought to contribute to regulation of the activity of NF-kB and mTORC1 which both play important roles in cell survival in response to stressors.

14Reactive oxygen species levels in the cell can be seen as a balance between levels of ROS generation and levels of ROS detoxification by antioxidants. Maintaining homeostatic levels of ROS as a result of this balance is important both for preventing oxidative damage as well as maintaining an appropriate redox environment for normal signaling pathways within the cell. Thus, just as ROS detoxification is required to avoid increases in ROS levels, antioxidant activity must be regulated to avoid over-detoxification and low levels of ROS. In response to an imbalanced redox environment, gene expression of antioxidant enzymes is triggered. Antioxidants scavenge ROS through various reactions which facilitate the reduction of ROS to less reactive and more stable forms. Enzymatic antioxidants are localized to various subcellular locations in order to be in proximity to sites of ROS generation. Non-enzymatic antioxidants can be endogenous or supplied by dietary nutrients.

15The superoxide dismutase (SOD) enzyme converts the highly reactive superoxide anion to a less reactive hydrogen peroxide. The enzyme catalyzes the transfer of electrons through two redox reactions using transition metals in its active site. In the first reaction, superoxide is oxidized to molecular oxygen, leaving the transition metal in the active site reduced; in the second reaction, superoxide is reduced to hydrogen peroxide and the transition metal in the active site returns to its oxidized form (Figure 4A). Humans have three different forms of SOD (Figure 5): SOD1 is located in the cytosol and its active site is Cu/Zn; SOD2 is located in the mitochondria and its active site is Mn; and SOD3 is located extracellularly with a Cu/Zn active site.

16Catalase (CAT) catalyzes the conversion of hydrogen peroxide to water and molecular oxygen (Figure 4B) and thus plays an important role in dealing with hydrogen peroxide-producing reactions, including that of the SODs. Its active site is composed of heme groups which facilitate the transfer of electrons. Catalase is primarily located in the peroxisomes (Figure 5), where hydrogen peroxide production can be high due to β-oxidation.

17Glutathione peroxidase (GPx) functions in conjunction with catalase to reduce hydrogen peroxide levels in the cell. GPx is located in the cytoplasm and mitochondria, while catalase is mainly in peroxisomes. While catalase mainly detoxifies hydrogen peroxide, GPx is also able to detoxify various peroxides, notably lipid peroxides. In the reaction to reduce hydrogen peroxide (Figure 4C), the selenocysteine active site of GPx becomes oxidized. In order to return the active site to its functional state, two glutathione (GSH) molecules are needed, resulting in the production of glutathione disulfide (GS-SG). Given that GSH is essential to maintain the activity of GPx, GS-SG must be reduced back to two molecules of GSH; this is catalyzed by glutathione reductase (GR), and uses electrons donated by NADPH. The GPx enzymes act in multiple subcellular compartments (Figure 5): GPX1 and GPX2 deal with hydrogen peroxide and organic peroxides in the cytosol, GPX3 acts as an extracellular antioxidant and GPX4, which can metabolize phospholipid hydroperoxides, is located in cell membranes and in mitochondria.

18The glutaredoxin (Grx) and thioredoxin (Trx) family of antioxidant enzymes act to protect thiol-containing proteins by repairing damage caused by exposure to oxidants. This system catalyzes the transfer of electrons to the disulfide bonds of target proteins, in order to convert the target protein back to its reduced form. This is done through a series of reactions (Figures 6A,B), which begin with the reduction of the target residue, and oxidation of the Grx or Trx active site, which consist of cysteine residues. Each enzyme then needs to be reduced back to its active form: Grx is reduced by two GSH molecules, which are in turn oxidized to glutathione disulfide while thioredoxin is reduced by an enzyme called thioredoxin reductase (TrxR). TrxR then harnesses electrons donated by NADPH molecules to reduce its own thiol-containing active site back to its active state while glutathione disulfide is reduced back to two GSH molecules by the GR, which also harnesses electrons from NADPH. Importantly, both enzymes are recognized as playing important roles in redox signaling pathways within the cell, especially given their abundance throughout the cell (Figure 5). In mammalian cells, for example, TRX1 and GRX1 are both located in the cytosol, nucleus and can be secreted, TRX2 and GRX2 are located in the mitochondria, GRX3 is also located in the cytosol and nucleus and GRX5 is in the mitochondria.

19The family of peroxiredoxin (Prx) enzymes are cysteine-containing enzymes that reduce peroxides, including organic peroxides (Figure 6C). Several Prx enzymes are located throughout the cell (Figure 5): in mammals, PRDX1 and PRDX2 are located in the cytosol and nucleus, PRDX3 in the mitochondria, PRDX4 in the endoplasmic reticulum, PRDX5 in peroxisomes, mitochondria and cytosol, and PRDX6 in the cytosol. Once the cysteine-containing active site reduces the target peroxide, Prx enzymes are returned to their active state when reduced by either Trx or GSH.

20Glutathione S-transferases (GSTs) have been linked to various stress responses and are thought to help GPx in attenuating lipid peroxidation by reducing fatty acid hydroperoxides. The enzyme does so by binding GSH and catalyzing the transfer of electrons to the target lipid, its active site consisting of a serine residue. In addition to preventing lipid peroxidation, GSTs may also help detoxify the end products of lipid peroxidation, which can be very harmful to the cell. There are many GST isozymes, most of which are in the cytosol, microsomes and plasma membrane (Figure 5).

21Appropriate glutathione (GSH) levels are essential for the activity of the GPx and Grx systems, where GSH acts to return each enzyme back to its active state. In addition, GSH itself can act as an antioxidant using the sulfhydryl group to donate electrons in order to reduce and detoxify ROS. Levels of active GSH can be restored either by converting oxidized GSH back to its reduced state by GR and NADPH, or by dietary supplementation. GSH can be added to oxidized cysteine residues in proteins through the process of S-glutathionylation. Because this process is reversible and can modify protein function, glutathionylation can be used in intracellular signaling. Interestingly, the levels of glutathione can be increased by the endogenously-produced signaling molecule hydrogen sulfide (H2S), which has also been shown to increase lifespan.

22Not only is N-acetyl cysteine (NAC) a precursor molecule in the formation of GSH, it also has its own thiol group, allowing it to participate in redox reactions and donate its electrons for the detoxification of ROS and the protection of sulfhydryl-containing proteins from oxidative damage. NAC levels are maintained by the consumption of cysteine in high protein foods.

23Vitamin C (ascorbic acid) is a water soluble antioxidant that is present both intracellularly and extracellularly. It acts by scavenging oxygen free radicals in aqueous environments, and by providing a line of defense against oxidation of cholesterols. Vitamin E (alpha-, beta-, gamma-, delta-tocopherols, and alpha-, beta-, gamma-, delta-tocotrienols) is lipid soluble and present in cell membrane and lipoproteins, working to inhibit lipid peroxidation as a result of oxidative stress. Vitamin C can help convert Vitamin E back to it reduced form.

24While antioxidant systems work to balance ROS levels and prevent oxidative damage, the cell also employs various repair systems to reverse damaging oxidation of cell components. As described above, glutaredoxins and thioredoxins play an important role in the reduction of thiol-containing proteins. Additional enzymes work alongside thioredoxins such as thioredoxin-related protein of 14 kDa and methionine sulfide reductase to reduce cysteine and methionine residues, respectively, both of which are amino acids that are most susceptible to oxidation by ROS. DNA damage repair mechanisms can be trigged by redox sensors which activate the expression of enzymes mediating base excision repair (BER), nucleotide excision repair (NER) and mismatch repair. The common DNA oxidation product, 8-oxoguanine, is repaired by 8-oxoguanine glycosylase (OGG1) via base excision.

25In addition to mechanisms which work to reverse oxidative damage, quality control systems which prevent the over-accumulation of oxidatively damaged molecules and organelles have been described. Irreversibly oxidized proteins are targeted by unfolded protein response machinery and can be degraded via the proteasome in the cytosol or by proteases in the mitochondria. Furthermore, oxidative stress may trigger autophagy of peroxisomes (pexophagy) and mitochondria (mitophagy) allowing for the removal of dysfunctional and ROS-generating organelles, followed by the synthesis of new undamaged organelles.

26While the short lifespan and high reactivity of ROS make them difficult to measure accurately, especially in vivo, a number of methods to measure ROS directly have been developed [for more comprehensive reviews of approaches to measure ROS see]. Many of these approaches rely on molecules that emit fluorescence after they have been oxidized by ROS. Among the most utilized of these molecules are 2′,7′-dichlorodihydrofluorescein diacetate (DCF), dihydroethidium (DHE), and MitoSOX, which are believed to measure overall cellular ROS, cellular superoxide levels, and mitochondrial superoxide levels, respectively. Similarly, Amplex Red has been used extensively to measure hydrogen peroxide levels. However, there is much disagreement about the sensitivity and specificity of these approaches especially given the fact that these molecules can also participate in non-specific redox reactions, producing a different but similarly fluorescent adduct that can be hard to distinguish from the fluorescent products derived from reactions with ROS. Furthermore, these molecules are membrane permeable and can leak out of the cell, posing a problem when measuring cellular ROS in tissues.

27Given the difficulties in directly measuring ROS levels, investigators often instead quantify ROS indirectly, either by assessing oxidative damage to cell components, or measuring survival under conditions of oxidative stress. Levels of oxidative damage have most commonly been assessed by measuring the levels of 8-oxoguanine residues in DNA, carbonylation of protein, or levels of malondialdehyde (MDA), an end product of lipid peroxidation (Figure 2). Increased levels of oxidative damage are generally interpreted to indicate increased levels of ROS; however, this outcome could also result from decreased levels of antioxidant or repair enzymes. To quantify resistance to oxidative stress, animals can be treated with ROS-generating compounds such as paraquat, juglone, and menadione. Decreased resistance to oxidative stress may be an indication of elevated baseline levels of ROS; however, increased levels of ROS can also cause upregulation of antioxidant enzymes, and thus resistance to oxidative stress must be interpreted with great care.

28More recently, genetically-encoded redox sensors have been developed to measure ROS in vivo without the spatial and temporal restrictions encountered with other ROS detection methods. These sensors involve the expression of a redox-sensitive protein that emits fluorescence after it encounters ROS. These sensors are minimally invasive, can be stably expressed in specific tissues, can be targeted to specific sub-cellular locations, and can be used for real-time ROS detection in vivo. Given that an increasing body of work shows that the specific conditions (e.g., timing, sub-cellular location, and levels) determine whether ROS have beneficial or detrimental effects, genetically-encoded redox sensors are expected to provide a highly useful tool for future studies on the role of ROS in aging and disease.

29In order to study the relationship between ROS and aging, many groups have utilized a comparative biology approach in which the levels of ROS, or ROS-related outcomes, are measured across species with differing lifespans. In examining the relationship between lifespan and either ROS levels, antioxidant levels or oxidative damage, most studies have demonstrated a negative correlation. While this comparative data is generally supportive of the FRTA, exceptions and contradictions have also been observed.

30Across species with varying lifespan, data collected from comparative studies shows a negative correlation between the rate of mtROS production and lifespan (Table 1). Comparing the superoxide and hydrogen peroxide generation rates across seven mammalian species revealed an inverse relationship between ROS production rates and lifespan. Furthermore, a similar study comparing five different species of dipteran flies supported these findings, showing that longer lifespan correlates with relatively low levels of ROS generation. In these studies, measurement of superoxide production was carried out in submitochondrial particles by looking at the reduction of cytochrome c due to its interaction with superoxide, while hydrogen peroxide generation was measured fluorometrically by monitoring the oxidation of p-hydroxyphenylacetate (PHPA) coupled to the enzymatic reduction of H2O2 by horseradish peroxidase.

31Despite seemingly supportive evidence from these studies, it was found that metabolic rate also correlated with lifespan. In order to isolate which factor is responsible for the observed results, the levels of mtROS production were compared between species with the same metabolic rate but different lifespans. While pigeons and rats are similar in size and have the same metabolic rate, pigeons live 35 years whereas rats only live 4 years. When superoxide and hydrogen peroxide generation rates where studied in the brain, heart, and kidney of these two animals, it was found that the pigeon had a significantly lower ROS generation rate, again supporting a negative relationship between ROS production and lifespan.

32While many studies have observed the same inverse relationship between lifespan and the rate of ROS generation, not all of these studies took into account possible confounding variables such as the effects of body mass and non-independence of different species due to phylogenetic relatedness. To address these potential shortcomings, one study examined the rates of hydrogen peroxide generation by heart mitochondria isolated from pairs or groups of two classes of vertebrate homeotherms with similar body mass, but very different lifespan. As with previous studies, the results showed a negative correlation between the rate of H2O2 generation and lifespan.

33Although data from comparative studies supports the idea that lower rates of mtROS generation correlate with a greater lifespan across species, there are certain exceptions. For example, Heterocephalus glabe (the naked mole-rat) is the longest living rodent species known to date, with an lifespan upward of 30 years, yet it produces similar levels of mtROS to mice, who have a lifespan of less than 4 years. This exception may result from the fact that naked mole-rats also have increased antioxidant defenses, which may compensate for their elevated levels of ROS. This highlights the importance of not considering ROS levels in a vacuum, as it is a fine balance between ROS production and ROS detoxification.

34As with levels of ROS, multiple comparative studies have examined the relationship between antioxidants and lifespan. The results from these studies are somewhat variable in that some studies show a negative correlation while others show a positive correlation, or no correlation (Table 2). To some extent the results are dependent on the specific antioxidant being measured and which tissue it is being measured in.

35In a study examining antioxidant enzyme activity in six mammalian species, it was found that superoxide dismutase and catalase activity were positively correlated with lifespan, while glutathione concentration was negatively correlated with longevity. A subsequent study examining antioxidant levels in liver tissue found that catalase, glutathione peroxidase and glutathione levels are negatively correlated with lifespan, while superoxide dismutase and glutathione reductase were not correlated with longevity. Comparison of superoxide dismutase, catalase, glutathione peroxidases, and glutathione reductase between various amphibia, mammal and bird species in lung tissue revealed a negative correlation with lifespan. In brain, heart and liver tissue of mammalian and avian species, CuZnSOD, glutathione peroxidase and glutathione reductase show no correlation with lifespan in any of the three tissues. MnSOD and catalase were observed to be positively correlated with lifespan, however, only in brain tissue.

36Analysis of a magnitude of studies, encompassing a large variety of vertebrate species and comparing antioxidant levels in tissues from multiple organs, suggests that there is an overall negative correlation between antioxidants and lifespan. In a meta-analysis of 78 correlations between endogenous antioxidants and longevity, it was found that 72 were negative, one was positive and the rest were insignificant. While on the surface a negative correlation between antioxidant levels and lifespan appears to contradict the FRTA because high levels of antioxidants should result in low levels of ROS, another possible interpretation is that the levels of antioxidants are regulated in response to the levels of ROS that are present, such that high levels of antioxidants are in fact an indication of high levels of ROS generation. These two possibilities can be distinguished by measuring oxidative damage, which is determined by the balance between ROS and antioxidants.

37Oxidative damage results when the levels of ROS generated exceed a cell’s antioxidant capacity. In examining the relationship between oxidative damage and lifespan, comparative studies have generally observed a negative correlation (Table 3).

38Reactive oxygen species can oxidatively damage many biomolecules such as DNA, protein and lipids (Figure 2). The two biomolecules which show the greatest correlation with lifespan are membrane fatty acids and mtDNA. mtDNA is located in close proximity to the site of mtROS production and is therefore highly susceptible to damage during aging, leading to large deletions over time. This accumulation of oxidative damage during the aging process implicated mtDNA as a potential regulator of lifespan. In support of this idea, oxidative damage to mtDNA in the heart and brain, estimated by levels of 8-hydroxy-2-deoxyguanosine (8-oxodG), negatively correlates with lifespan in mammals and birds. Additionally, despite extensive oxidative damage occurring to nuclear DNA (nDNA), the same correlation with lifespan is not observed. Since the rate of repair of 8-oxodG is rapid in both the mitochondria and the nucleus, increased 8-oxodG levels in the mitochondria are due to higher rates of oxidative attack, not an inefficient repair mechanism.

39Studies looking at oxidative damage to mtDNA in bivalves and other marine species have also shown a negative correlation with lifespan. The longest living non-colonial animal, Arctica islandica, has a lifespan of up to 400 years and provides an excellent opportunity to study aging in distant taxonomic groups. Studies using A. islandica find low rates of ROS generation, low levels of oxidative damage to DNA and a lower mitochondrial membrane peroxidation index compared to short-lived bivalves in the same subclass – Heteroconchia. Additionally, comparison between A. islandica and a shorter lived bivalve from the same subclass, Mercenaria mercenaria (lifespan of 106 years), revealed increased oxidative stress resistance associated with the species’ heightened longevity. Previous correlations between oxidative damage to mtDNA and lifespan, found in mammals and avian species, have further been corroborated in Sebastes (rockfish).

40The degree of fatty acid unsaturation in mitochondrial and cellular membranes is another parameter negatively correlated with both lifespan and oxidative stress. Unsaturated fatty acids possess extremely unstable electrons near the double bonds, making them highly susceptible to oxidative damage – a sensitivity that increases exponentially as a function of the number of double bonds per molecule. Studies have shown that the degree of fatty acid unsaturation is lower in long-lived animals than in short-lived. This adaptation in long-lived animals may be advantageous as it decreases their sensitivity to lipid peroxidation, in turn protecting other molecules from lipoxidation-derived damage.

41A study in Drosophila melanogaster found that flies that developed under larval crowding had a shorter lifespan and a greater proportion of unsaturated fatty acids. They found a significant negative correlation between lifespan and the peroxidation index. Similar results have been found in studies comparing unsaturated fatty acid content and lipid peroxidation levels between mammalian and avian species. Despite high metabolic rates, birds, on average, have an increased lifespan compared to mammals of a similar size. Fatty acid unsaturation and lipid peroxidation are shown to be lower in parakeets and canaries compared to the mouse, protecting their tissues and organelles against free radical-induced lipid peroxidation and possibly contributing to their slower rate of aging. Similar results are obtained in studies comparing liver mitochondria of rats and pigeons and mammals. The increasing evidence supporting a causal role for membrane fatty acid composition has led to an expansion of the “oxidative stress” theory of aging, described by some as the “membrane pacemaker” theory of aging.

42Bats have been shown to live much longer than is predicted by their size and metabolic rates. While these mammals can live up to 40 years, similarly sized mice rarely live longer than 4 years. In comparing oxidative damage between bats and mice, it was found that bats have decreased protein carbonylation. Bats also show decreased ROS production compared to the much shorter lived mice and shrews, though only as a ratio of oxygen consumption.

43Despite strong evidence demonstrating a negative correlation between oxidative damage and lifespan, there are certain exceptions. The naked mole-rat produces high levels of mtROS and accumulates significant levels of oxidative damage and yet has a lifespan 10-fold greater than that of a similarly sized laboratory mouse. In fact, naked mole rates accrue greater oxidative damage to lipids (2-fold), mtDNA (2-fold), nDNA (8-fold) and proteins (1.5- to 2-fold) than physiologically age-matched mice. Similarly, long-lived strains of Drosophila, vampire bats and birds have also been observed to have high levels of oxidative damage.

44While data from comparative studies provides a useful starting point for understanding the relationship between ROS and aging, these studies are only able to demonstrate correlation, not causation. In order to determine the extent to which ROS contribute to aging, it is necessary to experimentally manipulate the levels of ROS and measure the resulting effect on lifespan. In these experimental studies the levels of ROS have been increased indirectly by knocking out genes encoding antioxidant enzymes, or directly through exposure to a ROS-generating compound. Similarly, ROS levels have been decreased by overexpressing antioxidant enzymes, or treatment with an antioxidant compound. While these types of experiments allow researchers to make conclusions about the causative role of ROS in lifespan, these studies have only been performed in a limited number of model organisms, all of which are relatively short-lived. Nonetheless, experiments involving the manipulation of ROS levels have been important in demonstrating that ROS can have beneficial or detrimental effects on longevity.

45In order to examine the effect of ROS on lifespan, researchers have generated transgenic and knockout animals that express increased or decreased levels of antioxidant enzymes and then quantified the resulting effect on lifespan. While increasing or decreasing levels of antioxidants would be predicted to decrease or increase ROS levels, it is important to measure the resulting effect on ROS levels to make firm conclusions about ROS and aging. In the case of disrupting antioxidant genes, it is possible that redundancy or a compensatory upregulation of other antioxidant genes leads to little or no change in ROS levels. In the case of overexpression of antioxidant genes, it is possible that the normal level of antioxidant gene expression is already in excess of the amount of ROS being produced such that increasing antioxidant levels has no effect.

46The effect of increasing or decreasing the levels of SOD on lifespan has been examined in yeast, worms, flies and mice. Overexpression of either cytoplasmic or mitochondrial SOD resulted in increased lifespan in yeast, worms and flies (Table 4). Interestingly, however, the increase in lifespan does not seem to be attributable to decreased oxidative damage, at least in worms, as worms overexpressing sod-1 show increased levels of protein oxidation and hydrogen peroxide in vivo, as well as increased sensitivity to paraquat compared to wild-type worms. In addition, the results in Drosophila are dependent on the strain background as overexpression of cytoplasmic SOD with catalase alone or in combination with mitochondrial SOD failed to increase lifespan in a relatively long-lived strain background. In contrast to the other model organisms, overexpression of cytoplasmic SOD, mitochondrial SOD or cytoplasmic and mitochondrial SOD together in mice has no effect on longevity.

47Deletion of cytoplasmic SOD decreases lifespan in yeast, worms, flies and mice (Table 5). Disruption of mitochondrial SOD also decreases lifespan in yeast and flies. In contrast, deletion of the mitochondrial sod gene, sod-2, results in a significant increase in lifespan in Caenorhabditis elegans. In mice, it has not been possible to examine the effect of knocking out Sod2 on adult lifespan as Sod2 knockout mice die early in development. However, the need for Sod2 in early development can now be circumvented using an inducible Sod2 knockdown mouse model. Examination of heterozygous Sod2+/− mice reveals normal longevity despite increased levels of oxidative damage.

48In contrast to what was observed with SOD, overexpression of catalase resulted in decreased lifespan in both yeast and flies (Table 4), despite the fact that catalase overexpression increased resistance to oxidative stress. In worms, overexpression of all three catalase genes combined (ctl-1, ctl-2, and ctl-3) did not affect lifespan but resulted in a paradoxical increase in oxidative damage to proteins. In mice, overexpression of catalase in the peroxisome, where catalase is normally found, did not affect longevity, while overexpression of catalase targeted to the mitochondria did increase lifespan.

49Simultaneous overexpression of cytosolic SOD and catalase was initially reported to extend lifespan in Drosophila, but when these experiments were replicated, there was no significant change in lifespan. Similar to the latter experiment, simultaneous overexpression of cytoplasmic SOD and catalase in mice did not affect lifespan. Simultaneous overexpression of mitochondrial SOD and catalase resulted in decreased lifespan in Drosophila.

50Inactivation of catalase in yeast resulted in increased lifespan, despite also increasing oxidative damage (Table 5). In C. elegans, deletion of cytoplasmic catalase has no effect on lifespan, while loss of peroxisomal catalase decreases lifespan. In Drosophila, complete loss of catalase activity does not affect longevity. Finally, disruption of catalase in mice results in decreased lifespan.

51There have been only a limited number of studies examining the effects of glutathione peroxidase modulation on lifespan. Deletion of gpx2 in yeast results in decreased lifespan. In C. elegans, simultaneous deletion of four intestinal gpx genes decreases lifespan, despite having no effect on resistance to oxidative stress (Table 5). In mice, deletion of Gpx1, one of the most abundant glutathione peroxidases, increases oxidative damage but does not significantly affect longevity, even when combined with a heterozygous mutation in Sod2. Interestingly, heterozygous mice that have a 50% reduction in Gpx4 levels have increased lifespan compared to wild-type mice. Finally, it has been shown that disruption of Gpx7 in mice results in decreased lifespan, which is associated with increased oxidative stress. Interestingly, GPX7 lacks enzymatic activity and instead acts as a stress sensor.

52Single copy overexpression of the yeast cytosolic peroxiredoxin gene, tsa1, is sufficient to increase lifespan (Table 4). Overexpression of either Prx2(Jafrac1) or Prx5 in flies also increases resistance to oxidative stress and lifespan. A moderate overexpression of Prx4 reduces oxidative damage and extends lifespan in flies, but high levels of expression shortens longevity.

53Deletion of the yeast cytosolic peroxiredoxin, tsa1, decreases lifespan (Table 5). While loss of Tsa1 also increases sensitivity to hydrogen peroxide, this does not contribute to the shortening of lifespan, which instead is attributed to the role of Tsa1 as an inhibitor of nutrient signaling via protein kinase A. Others have reported that disruption of all five yeast peroxiredoxin genes does not decrease lifespan. In C. elegans, deletion of the cytoplasmic peroxiredoxin, prdx-2, results in increased sensitivity to hydrogen peroxide and decreased lifespan. Interestingly, expression of PRDX-2 in the intestine of prdx-2 deletion mutants eliminates sensitivity to hydrogen peroxide but fails to restore lifespan suggesting that the decrease in lifespan may not be attributable to the increase in sensitivity to oxidative stress. As with prdx-2, knockdown of mitochondrial peroxiredoxin, prdx-3, in C. elegans using RNAi resulted in decreased lifespan. In Drosophila, deletion of Prx5 or Prx3 and Prx5 together increases susceptibility to oxidative stress and decreases lifespan. Finally, deletion of Prx1 in mice increases ROS production and oxidative damage and decreases lifespan.

54In C. elegans, overexpression of the cytoplasmic thioredoxin gene trx-1 moderately increases lifespan (Table 4). Similarly, overexpression of TrxT in flies is also found to increase lifespan. Downregulation of thioredoxin-interacting protein (TXNIP), a negative regulator or Trx1, increases resistance to oxidative stress and extends lifespan, while overexpression decreases lifespan. While one group reported that overexpressing mitochondrial thioredoxin reductase significantly increases lifespan in flies, it has also been reported that overexpression of thioredoxin reductase in combination with CuZnSOD and catalase does not affect longevity. While an earlier study found that overexpression of human thioredoxin in mice resulted in decreased oxidative stress and increased lifespan, subsequent studies observed less beneficial or even detrimental effects. Overexpression of Trx1 in mice was found to only have significant effects on lifespan in male, but not female, mice, and only increased the earlier part of lifespan. Interestingly, simultaneous overexpression of Trx1 and Trx2 resulted in significantly decreased lifespan.

55Deletion of the gene encoding thioredoxin in yeast, trx1, slightly reduces chronological lifespan (Table 5). Deletion of the yeast cytosolic thioredoxin reductase, trr1, also reduces chronological lifespan and increases sensitivity to oxidative stress. In C. elegans, disruption of the cytoplasmic thioredoxin gene, trx-1, causes a significant decrease in lifespan, while deletion of the mitochondrial thioredoxin gene, trx-2 has no effect. In contrast, disruption of the cytoplasmic (trxr-1) or mitochondrial (trxr-2) thioredoxin reductase genes, or both together, did not affect lifespan at the normal growing temperature of 20°C. In flies, mutations affecting Trx2 shortens lifespan and increases sensitivity to oxidative stress. Unlike C. elegans, mutations that reduce thioredoxin reductase activity in Drosophila cause a severe reduction in adult lifespan. In mice, a 50% reduction in expression of the mitochondrial thioredoxin gene, Trx2, increases ROS production and oxidative damage and slightly reduces lifespan.

56Relatively few studies have examined the effect of glutaredoxins on longevity. Intake of recombinant buckwheat glutaredoxin in C. elegans increases lifespan and resistance to oxidative stress (Table 4). Deletion of either grx1 or grx2 in yeast shortens the chronological lifespan via increased ROS accumulation which subsequently activates the RAS/PKA signaling pathway and decreases stress resistance (Table 5). Disruption of the mitochondrial glutaredoxin gene, grx5, also decreases lifespan in yeast.

57Despite the fact that there are many different glutathione S-transferases (e.g., 57 in C. elegans), very few have been shown to influence lifespan, possibly due to functional redundancy with other glutathione S-transferases. In C. elegans, overexpression of gst-4 increases resistance to oxidative stress but does not increase lifespan, while overexpression of gst-10 decreased oxidative damage (4-HNE) and increased lifespan (Table 4). In Drosophila, overexpression of GstS1 significantly increases mean lifespan and resistance to oxidative stress.

58Decreasing the expression of either gst-5 or gst-10 causes decreased lifespan in C. elegans (Table 5). In contrast, deletion of gst-14 has the opposite effect of increasing lifespan, although its effects were only reported in mutants that have impaired mitochondrial function. In Drosophila, deletion of microsomal glutathione S-transferase was found to reduce lifespan. In mice, deletion of GstA4, a glutathione S-transferase important for detoxification of the lipid peroxidation product 4-hydroxynonenal (4-HNE), extends mean lifespan.

59In summary, genetic manipulations of antioxidant gene expression have varied effects on the lifespan of model organisms. While the general trend is that overexpression of antioxidant enzymes increases lifespan and disruption of antioxidant genes decreases lifespan, there are multiple examples in which no effect or the opposite effect is observed. The ability of antioxidant gene manipulation to significantly change lifespan is dependent on a number of factors including the species in which the manipulation is taking place, the specific antioxidant being altered, and the subcellular location in which the antioxidant is expressed.

60Treatment with antioxidant compounds is another approach to studying the relationship between ROS and lifespan. While this approach is an indirect way of modulating ROS levels, it can be applied to a wide range of species, including humans. Nonetheless, experiments using antioxidant compounds have generally been performed in short-lived species so that experiments can be completed in a feasible time frame. Because this approach is indirect, it is important to measure the effect of the antioxidant compound on the levels of ROS in order to fully interpret the results. In addition, when performing experiments with compounds, it is important to consider that most or all of these antioxidant compounds have other effects aside from their antioxidant activity that may contribute to or account for their effects on lifespan. Experiments using this approach have provided mixed results as to the effect of antioxidant supplementation on lifespan (Table 6).

61N-acetyl cysteine (NAC) is a scavenger of free radicals as well as a precursor of L-cysteine and a source of sulfhydryl groups. It derives most of its antioxidant properties as a cysteine precursor, promoting the synthesis of glutathione. Treatment of C. elegans with NAC shows a U-shaped dose-response curve in which low concentrations of NAC can increase lifespan, while higher concentrations decrease lifespan. A similar pattern is observed in Drosophila where lower concentrations of NAC can increase lifespan, while higher concentrations shorten lifespan. The effect of NAC treatment in mice is sex dependent as NAC was found to only increase lifespan in males, but not in females. It is important to note, however, that mice treated with NAC show decreases in body weight making it possible that the increase in lifespan is due to dietary restriction.

62Vitamin C is a hydrophilic antioxidant and a strong inhibitor of lipid peroxidation. Vitamin C treatment in yeast was found to increase survival in wild-type strains. Treatment of C. elegans with Vitamin C has also been shown to increase wild-type lifespan when delivered with liposomes or when the C. elegans cuticle is made more permeable with a bus-8 mutation. Vitamin C supplementation in Drosophila can extend the lifespan of wild-type flies, while high doses of Vitamin C can be toxic and shorten lifespan. Vitamin C treatment in rodents has provided somewhat mixed results. Treating lab mice with Vitamin C resulted in a slight increase in lifespan. In contrast, Vitamin C treatment shortened the lifespan of wild-derived voles. Additionally, Vitamin C treatment in guinea-pigs had no effect on lifespan.

63Vitamin E is a lipophilic antioxidant that protects membranes from oxidative damage as well as regulating signal transduction and gene expression. Vitamin E supplementation in yeast is observed to shorten replicative lifespan despite increasing the mean lifespan of four other single cell organisms. Multiple groups have shown that Vitamin E treatment can increase lifespan in C. elegans, but interestingly this treatment was found to have no effect on superoxide levels. While some groups have reported that Vitamin E treatment increases lifespan in Drosophila, others have observed little or no effect on longevity. Vitamin E treatment in mice is largely observed to have no significant effect on lifespan but was found to significantly increase lifespan in male mice in one study. In contrast, Vitamin E supplementation was found to decrease lifespan in wild-derived voles.

64A number of randomized clinical trials have examined the effects of different antioxidants in humans, including Vitamin C, Vitamin E, Vitamin A, beta-carotene and selenium. A meta-analysis of 78 trials that included a total of 296,707 individuals showed no beneficial effect of any of the antioxidants for human longevity in control or disease populations. In fact, treatment with Vitamin E or beta carotene resulted in a higher risk of all-cause mortality. While the average length of supplementation was 3 years, these results suggest that decreasing ROS levels may not be beneficial for lifespan in people.

65Another approach to examine the relationship between ROS and aging is to expose animals to compounds that act to generate ROS. Although this approach can be applied to any species, these experiments have only been performed in relatively short-lived species for practical reasons. While exposing animals to high levels of ROS-generating compounds is toxic, evidence from multiple species indicates that mildly increasing ROS levels with ROS-generating compounds can extend longevity (Table 7).

66One of the first experiments to show that compounds that increase ROS levels can extend longevity involved treating C. elegans with 2-deoxy-D-glucose. 2-deoxy-D-glucose acts as an inhibitor of glycolysis, leading to increased mitochondrial respiration, increased production of ROS and increased lifespan. Importantly, the increase in ROS levels was found to be required for the lifespan extension as the increase in longevity was prevented by treatment with antioxidants (NAC, Vitamin C or Vitamin E). Similarly, treatment of C. elegans with arsenite results in increased ROS and extended longevity, which is completely dependent on the increase in ROS, as treatment with antioxidants (NAC, BHA[butylated hydroxyanisole]) reverts lifespan to control. In C. elegans, inhibition of mitochondrial electron transport chain Complex I with rotenone causes an increase in mitochondrial ROS and extends lifespan. Metformin has also been shown to increase worm lifespan through a similar mechanism. It was found that metformin treatment led to an inhibition of Complex I, increased respiration, increased ROS and extended longevity, which was prevented by treatment with the antioxidant NAC. It has also been shown that treating worms with either juglone or paraquat, which act to directly increase superoxide levels primarily in the mitochondria, extends longevity.

67The ability of ROS-generating compounds to extend lifespan is not limited to C. elegans, as treating yeast with menadione, which acts to generate mitochondrial ROS through redox cycling, was also shown to increase lifespan. Interestingly, menadione did not increase lifespan in C. elegans. Similarly, treatment with paraquat or metformin failed to extend lifespan in Drosophila. The reason why these ROS-generating compounds increase lifespan in some species but not others is currently unclear. Since these compounds have the potential to be toxic at higher doses, it is possible that the doses used were simply too high, or that other experimental parameters were not optimal for the compound to extend lifespan in the species showing no effect. It is also possible that there are differences between species in the ways that ROS can extend longevity. For example, it was proposed that compounds that affect Complex I, such as paraquat and metformin, don’t increase lifespan in Drosophila because an intact Complex I may be necessary for lifespan extension in this organism.

68Importantly, ROS-generating compounds have also been shown to increase lifespan in mammals. Similar to 2-deoxy-D-glucose, D-glucosamine acts as an inhibitor of glycolysis leading to increased respiration and increased production of ROS. Treatment of worms or mice with D-glucosamine increases lifespan, and this increase in lifespan was shown to be dependent on elevated ROS as it is prevented by treatment with antioxidants (NAC, BHA).

69Genetic mutations that lead to increased levels of ROS have been shown to have variable effects on lifespan: they can decrease lifespan, increase lifespan or have no effect. In this section, we will focus on genetic mutants in which increased ROS causes extended longevity, providing examples from yeast, worms, flies and mice (Table 8).

70In yeast, it has been shown that disruption of genes encoding catalase (cta1 or ctt1) increases the levels of intracellular ROS (hydrogen peroxide) and increases oxidative damage (protein carbonylation), but also increases lifespan. This shows that increasing ROS can increase lifespan, and that oxidative damage can be experimentally dissociated from lifespan.

71In C. elegans, it has been shown that deletion of the mitochondrial sod gene, sod-2, increases lifespan, despite also resulting in increased oxidative damage. The increase in lifespan in sod-2 mutant worms is dependent on elevated ROS, as treatment with antioxidants (Vitamin C, α-lipoic acid, or epigallocatechin gallate) reverts their lifespan toward wild-type. Three different mutations that affect mitochondrial function in C. elegans, clk-1, nuo-6, and isp-1, have all been shown to extend longevity despite increasing levels of ROS. As with sod-2 mutants, treatment of these strains with antioxidants decreases their lifespan, again demonstrating that the elevation in ROS levels in these strains is required for their longevity. Decreasing insulin-IGF1 signaling has been shown to increase lifespan in multiple species and to be associated with longevity in humans. In C. elegans, disruption of this pathway with a mutation in the insulin-IGF1 receptor gene, daf-2, results in increased levels of ROS, which are required for their long lifespan. Similarly, inhibition of the germline has been shown to increase lifespan in C. elegans in a ROS-dependent manner: mutations in glp-1 prevent germline development, increase ROS levels and extend longevity.

72In Drosophila, expression of an alternative component of the mitochondrial electron transport chain called NDI1 causes increased production of ROS during mitochondrial electron transport. This increase in ROS is sufficient to extend the longevity of the fly, and is prevented by overexpression of catalase. Also in Drosophila, it has been shown that muscle-specific knockdown of a subunit of electron transport chain Complex I called ND75 results in increased levels of ROS and increased lifespan. Overexpression of either catalase or glutathione peroxidase reverted the lifespan of ND75-knockdown flies to wild-type, thereby indicating that the increase in ROS is required for their long lifespan.

73In mice, it has been shown that a heterozygous mutation in the mouse homolog of clk-1, Mclk1, results in increased levels of mitochondrial ROS, increased levels of oxidative damage and increased lifespan. Decreasing body temperature has been shown to increase ROS and to extend longevity in mice.

74Combined, the experiments described above demonstrate that genetic mutations that lead to increased levels of ROS can extend longevity in yeast, worms, flies and mice. Importantly, in most of these experiments the lifespan-extending effect of the mutation was prevented by treatment with antioxidants, thereby indicating that the elevated levels of ROS are required for the observed lifespan extension.

75While the reactive nature of ROS can be perceived as making them dangerous to cells, it is also possible for cells to harness this property to use ROS as an efficient tool for intracellular signaling. ROS have been shown to act as signaling molecules for a number of biological processes, including development and survival. ROS can oxidize cysteine residues within proteins leading to allosteric changes that alter that protein’s function. Those cysteine residues can subsequently be reduced by thioredoxin or glutaredoxin to restore their original function or be further oxidized in a manner that is irreversible. In this way, ROS can activate kinase signaling pathways through inactivation of phosphatases or turn off kinase signaling pathways by inactivating kinases. Similarly, ROS can act directly on effector proteins to mediate their effects, such as inhibiting cell death proteins to promote survival. ROS involved in intracellular signaling can be generated by enzymes such as NADPH oxidases. In fact, there are over 40 enzymes that generate hydrogen peroxide or superoxide in human cells. In the context of signaling, the ROS generated from the electron transport chain may be a cell’s way of communicating the status of the electron transport chain function to the rest of the cell.

76The ability for redox-signaling proteins to switch between oxidized and reduced states is essential for many of the signaling pathways mentioned. The redox environment in the cell, as determined by levels of ROS and antioxidants, determines the balance between proteins in reduced and oxidized states, also known as the redox poise of a protein. Maintaining an optimal state of redox poise is important not only for signaling molecules that respond to changes within the redox environment, but also for optimal transfer of electrons in the electron transport chain. This notion that both over-reduction and over-oxidation of cell components can be detrimental to cell function supports the hypothesis that optimal levels of ROS and not simply low levels of ROS are required to maintain homeostasis.

77The fact that ROS are involved in intracellular signaling and cause oxidative damage highlights the crucial importance of maintaining ROS at an optimal level, and provides a mechanism for how ROS act to extend longevity. There appears to be a “goldilocks” zone in which the beneficial effects of ROS-mediated signaling are maximized while minimizing the amount of oxidative damage caused by ROS.

78The FRTA provides a conceptual framework that is used to think about the relationship between ROS and aging, as well as specific predictions that can be experimentally tested. At its most basic level, the FRTA posits that aging is driven by the accumulation of ROS-induced macromolecular damage. If the FRTA is correct then (1) levels of oxidative damage should be negatively correlated with lifespan, (2) experimental manipulations that decrease oxidative damage should increase longevity, and (3) experimental manipulations that increase oxidative damage should shorten lifespan.

79Comparative studies generally suggest that the levels of ROS and oxidative damage are negatively correlated with lifespan (Table 1 and Table 3). While examples that do not follow this trend exist, such as the naked mole-rat, it is unclear if these examples are exceptions to the rule, or evidence that the rule is incorrect. The great strength of comparative studies is that they study a diverse set of species with lifespans ranging from days to hundreds of years. However, care must be taken when interpreting comparative data, as correlation cannot demonstrate causation. Thus, even though oxidative damage increases with age, this does not necessarily mean that it causes aging. It is also possible that different factors cause oxidative damage and aging but that both increase over time, or that the same factor causes both, but does so independently. In order to distinguish between these possibilities, it is necessary to experimentally modulate the proposed causative factor and measure the resulting effect on lifespan.

80A number of approaches have been utilized to experimentally modulate the levels of ROS or oxidative damage to determine the effect of the intervention on longevity. In general, interventions aimed at decreasing ROS levels, including overexpression of antioxidant enzymes or treatment with antioxidant compounds, have been shown to increase lifespan, though there are also examples where no effect or a decrease in lifespan is observed (Table 4). While theoretically these interventions should decrease oxidative damage, this is not always the case. In some cases, interventions aimed at decreasing ROS resulted in increased oxidative damage and still increased lifespan, thereby indicating that the underlying mechanism of lifespan extension with interventions that decrease ROS is not necessarily a reduction in oxidative damage. In general, interventions aimed at increasing ROS levels, primarily disruption of genes encoding antioxidant enzymes, have been shown to decrease lifespan (Table 5). Nonetheless, there are also examples in which deletion of genes encoding antioxidant enzymes has no effect or results in increased lifespan, despite increasing levels of oxidative damage.

81Despite the fact that the bulk of evidence suggests that ROS are detrimental for longevity, a number of experimental studies have demonstrated that ROS can also increase lifespan. This has been demonstrated in yeast, worms, flies and mice and has been shown through both genetic manipulations and compounds that increase ROS levels (Tables 7 and 8). These experiments highlight the fact that ROS can be beneficial or detrimental with respect to longevity. The effect of ROS on lifespan is determined by a number of factors including which type of ROS it is (e.g., superoxide and hydrogen peroxide), which subcellular compartment it is in (e.g., mitochondria and cytoplasm), which tissue it is in (e.g., neurons and muscle), when during the life cycle it is increased (e.g., development and adulthood) and how much the ROS levels are increased. The mechanisms by which ROS increase lifespan are likely mediated by their ability to participate in ROS-mediated signaling to activate genetic pathways that increase survival (e.g.,; ).

82There are two important caveats for experimental studies. First, genetic studies involving overexpression or deletion of antioxidant genes have only been performed in four well-characterized genetic model organisms: yeast, worms, flies and mice. It is unclear to what extent findings in these four species are reflective of the wide diversity of organisms across the tree of life. The advance of genome sequencing technologies and genetic engineering tools such as CRISPR-Cas9 will make it easier to manipulate gene expression in other organisms, thereby enabling researchers to determine if the results from these four organisms are representative of a wider range of species. Given that different outcomes have already been observed between these four species, it is uncertain if general patterns will emerge when studying a broader range of species.

83The second major caveat of experimental studies, both genetic and pharmacological, is the fact that these studies have primarily been performed in short-lived species. In order to test the effect of a gene or compound on the lifespan of an organism, practicalities make it challenging to perform experiments in species that live longer than 5 years, which is the length of a typical grant and roughly the duration of a doctoral program. Thus, one cannot exclude the possibility that ROS affect lifespan differently in short-lived and long-lived species.

84Perhaps one of the greatest limitations of both comparative and experimental studies examining the effect of ROS on lifespan is the need to measure ROS. Comparative studies require accurate measurement of ROS and oxidative damage to correlate with longevity. Experimental studies require precise measurement of ROS levels to determine whether the experimental manipulation is having the desired effect. However, the instability and reactivity of ROS make them extremely difficult to quantify. If they are not measured in vivo, in real-time, it is unclear whether the ROS levels being measured are truly reflective of the ROS levels in the organisms being tested, since ROS can react rapidly with cellular components, or be detoxified by antioxidants. Unfortunately, current methods to measure ROS are still limited by many confounding variables, such as limited specificity and sensitivity. As experimental studies have demonstrated that the effect of ROS on lifespan is dependent on many factors, current approaches to measure ROS may be too blunt to distinguish between lifespan-extending ROS and lifespan-shortening ROS. This may be especially true with respect to the spatial localization of ROS within a cell. For example, elevated superoxide in the mitochondria can extend longevity through mitochondria-nucleus signaling, while increasing superoxide in other parts of the cell can shorten lifespan.

85Many of the studies reported here provide a limited view of the redox balance occurring in the organism being studied. Oxidative stress is a balance between ROS production and ROS detoxification by antioxidants, and this balance may change over time. However, most studies only measure one type of ROS, at one time point, in one tissue. A challenge for the field moving forward is to be able to measure and take into account all of these different factors that can influence how ROS affects longevity in a way that is experimentally feasible.

86As a final note, it is important to consider the possible effects of observation bias. In cases where an experiment shows no effect on lifespan, it is unclear whether this finding is because the intervention does not affect lifespan, or because the experiment failed to demonstrate an effect of the intervention on lifespan. As a result, experiments in which the outcome shows that the intervention tested does not affect lifespan are difficult to interpret and are more likely not to be reported than experiments showing a positive or negative effect on lifespan.

87Despite extensive research, the role of ROS in aging remains incompletely understood. While comparative studies have demonstrated a negative relationship between both ROS levels and oxidative damage, and lifespan, these correlations do not necessarily imply causation, and exceptions to these relationships have been observed. In experimental studies, it has generally been observed that manipulations that increase ROS decrease lifespan, while manipulations that decrease ROS increase lifespan. However, there are several examples in which a manipulation’s effect on ROS levels or oxidative damage can be experimentally dissociated from its effect on lifespan. In addition, there are examples in which increasing ROS levels extends longevity, and in which decreasing ROS levels shortens lifespan. One of the greatest limitations to our understanding of the role of ROS in aging is our ability to measure ROS. There are many different types of ROS, and the exact tissue, subcellular location, timing and levels of ROS act to determine whether ROS increase or decrease lifespan. With the currently available tools to measure ROS and the present limitations of comparative and experimental studies, it may not be possible to generalize the relationship between ROS and aging across the tree of life.

88JV: conceptualization and supervision. HS and AT: investigation and writing – original draft. HS, AT, and JV: visualization and writing – review and editing. All authors contributed to the article and approved the submitted version.

89The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors JV.