Most popular discussions of antioxidants are based on an outdated view of free radicals as evil, toxic compounds, ones which cause chain reactions of destructive damage culminating in degenerative diseases and aging. Research in recent years has revealed that, in addition to cellular energy production, radicals play a crucial roles in many important physiological processes, including signal transduction, cell-cycle regulation, and immune function. Long-lived organisms, like humans, have developed very sophisticated enzymatic systems for controlling and utilizing radicals. These natural antioxidant defenses are much more effective than crude antioxidant supplements which have not been shown to be effective at preventing aging, or any degenerative disease. Some clinical trials of antioxidant supplementation have even found harmful effects. A number of such trials have been prematurely terminated for this reason. In fact, to date, despite decades of intense research, and thousands of studies, with extremely few exceptions, like radiation sickness, conclusive evidence has not been found that radical formation is a causative factor in the pathogenesis of diseases.
On the contrary, evidence indicates that, in most cases, radical formation results from, but does not cause disease processes.(ref, ref) Radical formation results from tissue injury, and is a necessary step in healing processes.(ref) As discussed in references cited below, radicals play a crucial role in developmental, metabolic, immunological, and other physiological functions. Without them we would not be able to produce energy, develop properly, repair injury; nor would we be able to destroy pathogens or infected and malignant cells. In rare cases, radicals may cause DNA damage possibly leading to cancer; however, on a regular, widespread basis, radicals are involved in the destruction of malignant cells, protecting us from cancer.
What are Free Radicals?
In general, the reactivity of an atom is determined by the electrons that have the most energy and move farthest from the nucleus. Pairs of electrons also tend to be more stable than single, unpaired electrons. Molecules that have unpaired electrons in their outer, valence shells are called “free radicals”. They may have an electrical charge, ions; but most are neutral. Molecules that give up, or “donate” an electron are said to be “oxidized”; while molecules that accept the electron are “reduced”. Such reduction-oxidation processes are called “redox” reactions. These reactions are essential to cellular energy production, and play a vital role in very important biological signaling pathways. Antioxidants are reducing agents. They can decrease oxidative damage, but can also interfere with vital biological processes. Maintenance of redox homeostasis (a balanced level of oxidative radicals) is necessary for proper cellular function.
ROS, RNS and RSS
The oxygen molecule (O2), despite having two unpaired electrons, is itself relatively stable. However, many oxygen-containing compounds, such as peroxides and superoxides, are highly reactive free radicals, collectively called “reactive oxygen species”. ROS are often byproducts of cellular energy production. Many, like superoxide, are actually produced by the body using specialized enzymes for specific purposes. Free radicals containing nitrogen are referred to as “reactive nitrogen species.” RNS result from the reaction of nitric oxide and superoxide to produce peroxynitrite, and related compounds. Both ROS and RNS are highly reactive, and can damage proteins, lipids and DNA. RNS-induced damage is sometimes referred to as “nitrosative stress, to distinguish it from “oxidative stress.”
Due to their destructive potential, superoxide and RNS are often produced by the body as a weapon to attack and destroy foreign pathogens. Superoxide production is tightly controlled by a highly regulated network of enzymes, see: Nox Family NADPH Oxidases. For more a more detailed overview of RNS, see: Nitric Oxide and Peroxynitrite in Health and Disease. Sulphur-containing radicals are referred to as “RSS” (reactive sulphur species).(ref) These result from the reaction of thiols with ROS. Both RNS and RSS result from reactions involving ROS. I will use the terms “ROS” and “OS” (oxidative stress) loosely, without differentiating between the effects caused by specific types of secondary radicals.
Radicals in Biology
Prior to their discovery in biological processes, radicals were already well-known for their reactive, and destructive power in other areas of chemistry. Oxidative injury caused by ionizing radiation was graphically demonstrated in Nagasaki and Hiroshima. The symptoms of oxidative damage resulting from exposure to nuclear radiation closely resembled the degenerative effects of aging. In 1954 oxygen toxicity was shown to be the result of radical formation.(ref) In 1956, Denham Harman published his seminal Free Radical Theory of Aging. In 1972, Harman identified the mitochondria as the primary source of cellular ROS generation. Much research focused on mitochondrial ROS (mtROS), in particular the possibility of mtROS “leakage” to other cellular compartments, and the effects of oxidative damage to sensitive mitochondrial DNA (mtDNA). Subsequent research has shown that mtROS “leakage” is much less than originally thought. “The physiological level of ROS emission from mitochondria is negligible (as discussed in this review) and unlikely to be of any significance except as a signal.” (ref) Experiments have also shown that oxidase overexpression lowering mtOS and mtDNA mutations, does not increase lifespan. Furthermore, dramatically increased mutations in mtDNA (500 fold) produce no signs of accelerated aging. (ref) Focus has shifted to other organelles such as the peroxisomes (ref, ref, ref, ref, ref) and lysosomes (ref, ref,). Due to the role of lysosomes in recycling mitochondria, some have gone so far as to rename the “Mitochondrial Free Radical Theory of Aging” the “The Mitochondrial–Lysosomal Axis Theory of Aging.” (ref, ref).
Given the recognized destructive potential of ROS and the ability of antioxidants to neutralize them, why has antioxidant supplementation failed to produce consistent, positive outcomes, in many cases, causing harm, even increasing oxidative stress? In order to understand the answers, we need to first understand the body’s natural antioxidant defense mechanisms, and the biological function of ROS in human physiology.
The Body’s Natural Antioxidant Defenses
Long-lived species have developed sophisticated mechanisms for dealing with ROS and utilizing them. Controversy of Free Radical Hypothesis: “To be protected from potentially harmful effects of ROS, aerobic organisms evolved several specialized mechanisms. To detoxify ROS, they use system of antioxidants, including specific antioxidative enzymes, e.g. superoxide dismutase, catalase, glutathione peroxidase. . .This system consists of mostly degradative yet also other enzymes such as proteases, peptidases, phospholipases, acyl transferases, endonucleases, exonucleases, polymerases, ligases, etc., to cleave and replace irreversibly damaged macromolecules (Elliott et al. 2000). Importantly, the systems are integrated, they work in concert and their actions may be closely interconnected (Sies 1993; Berry and Kohen 1999; Gate et al. 1999)”.
Superoxide Dismutase (SOD) catalyzes the reduction of superoxide into hydrogen peroxide and water. In mammals, there are three isoforms which function in distinct cellular compartments. SOD1 is found in the cytosol and mitochondrial intermembrane. SOD2 is located in the mitochondrial matrix; and SOD3 functions in the extracellular space. (ref)
Glutathione Peroxidase (Gpx) transforms peroxides, especially lipid hydroperoxides, into water and alcohol. Specialized GPx forms function in distinct cellular compartments in specific tissue types. “Analysis of the selenoproteome identified five glutathione peroxidases (GPxs) in mammals: cytosolic GPx (cGPx, GPx1), phospholipid hydroperoxide GPx (PHGPX, GPx4), plasma GPx (pGPX, GPx3), gastrointestinal GPx (GI-GPx, GPx2) and, in humans, GPx6, which is restricted to the olfactory system. GPxs reduce hydroperoxides to the corresponding alcohols by means of glutathione (GSH). They have long been considered to only act as antioxidant enzymes. Increasing evidence, however, suggests that nature has not created redundant GPxs just to detoxify hydroperoxides. cGPx clearly acts as an antioxidant, as convincingly demonstrated in GPx1-knockout mice. PHGPx specifically interferes with NF-kappaB activation by interleukin-1, reduces leukotriene and prostanoid biosynthesis, prevents COX-2 expression, and is indispensable for sperm maturation and embryogenesis. GI-GPx, which is not exclusively expressed in the gastrointestinal system, is upregulated in colon and skin cancers and in certain cultured cancer cells. GI-GPx is a target for Nrf2, and thus is part of the adaptive response by itself, while PHGPx might prevent cancer by interfering with inflammatory pathways. In conclusion, cGPx, PHGPx and GI-GPx have distinct roles, particularly in cellular defence mechanisms. Redox sensing and redox regulation of metabolic events have become attractive paradigms to unravel the specific and in part still enigmatic roles of GPxs.”(ref) In addition to these six GPxs, two additional isoforms have recently been identified, GPx7 and Gpx8, which appear to function in the endoplasmic reticulum, where they enable the “productive use” of peroxides for the oxidative folding of proteins.(ref)
Catalase (CAT) uses iron to reduce peroxides. Hundreds of different forms are widely distributed in animal, plant and fungi tissues. Some contain manganese, and some are bifunctional catalase-peroxidases.(ref)
In addition to these principal antioxidant enzymes, the secondary antioxidant enzymes, thioredoxin (ref), glutaredoxin (ref), and peroxiredoxin (ref) systems also aid in the control, and selective removal, of ROS. The activity of all innate antioxidant enzymes is highly selective. They function in specific cellular compartments, within specific tissues, in response to specific signaling pathways, to reduce specific radical types. The body is able to increase or decrease their activity in target locations, as needed, to maintain ideal redox homeostasis. Antioxidant enzymes cannot be taken orally; it would not be advisable to do so, even if possible. Experiments with IV administration have not produced favorable results. Plasma levels are not the key, since redox activity must be differentially modulated within specific cellular compartments in specific tissue types.(ref, ref)
Unlike innate antioxidant defensive enzyme systems, nutritional antioxidants are nonenzymatic, meaning that they are not enzymes which catalyze redox reactions directly affecting pro-oxidant substrates. For the most part, they work by breaking oxidative chains, either by accepting (or donating) electrons, thereby eliminating the unpaired electron. They are inferior to the body’s natural enzymatic antioxidants, because they cannot be activated selectively in response to the continually changing redox status of specific cellular compartments. Their activity is indiscriminate. Since ROS serve many important functions (discussed below), neutralizing them is not always beneficial. Furthermore, by interfering with the normal signaling pathways that activate the body’s natural enzymatic defenses, in many cases, exogenous antioxidants can actually increase oxidative stress (OS).
I should also mention that certain botanical phenolic compounds appear to work indirectly. Rather than interrupt oxidative chains by directly reducing pro-oxidants, they appear to decrease OS through a variety of signaling pathways, some of which may result in upregulation of the body’s innate enzymatic antioxidants.(ref) This is true for the so called “hormetic” botanicals including catechins, quercetin, and curcumin which are actually mild pro-oxidants, even though they indirectly decrease OS.(ref, ref)
“Hormesis” is the idea that regular exposure to small amounts of toxins, or other forms of biological stress have salutory effects, by activating defensive mechanisms. The blog entry Hormesis and age retardation introduces hormesis and its possible relevancy as an anti-aging strategy. How increased oxidative stress promotes longevity and metabolic health: “Recent evidence suggests that calorie restriction and specifically reduced glucose metabolism induces mitochondrial metabolism to extend life span. In conflict with Harman’s free radical theory of aging (FRTA), these effects may be due to increased formation of reactive oxygen species (ROS) within the mitochondria causing an adaptive response that culminates in subsequently increased stress resistance assumed to ultimately cause a long-term reduction of oxidative stress. This type of retrograde response has been named mitochondrial hormesis or mitohormesis, and may in addition be applicable to the health-promoting effects of physical exercise in humans and, hypothetically, impaired insulin/IGF-1-signaling in model organisms. Consistently, abrogation of this mitochondrial ROS signal by antioxidants impairs the lifespan-extending and health-promoting capabilities of glucose restriction and physical exercise, respectively. In summary, the findings discussed in this review indicate that ROS are essential signaling molecules which are required to promote health and longevity. Hence, the concept of mitohormesis provides a common mechanistic denominator for the physiological effects of physical exercise, reduced calorie uptake, glucose restriction, and possibly beyond.”
Extending life span by increasing oxidative stress: “This review aims to summarize published evidence that several longevity-promoting interventions may converge by causing an activation of mitochondrial oxygen consumption to promote increased formation of reactive oxygen species (ROS). These serve as molecular signals to exert downstream effects to ultimately induce endogenous defense mechanisms culminating in increased stress resistance and longevity, an adaptive response more specifically named mitochondrial hormesis or mitohormesis. Consistently, we here summarize findings that antioxidant supplements that prevent these ROS signals interfere with the health-promoting and life-span-extending capabilities of calorie restriction and physical exercise. Taken together and consistent with ample published evidence, the findings summarized here question Harman’s Free Radical Theory of Aging and rather suggest that ROS act as primarily as essential signaling molecules to promote metabolic health and longevity.”
There is evidence that hormesis is the result of epigenetic adaptations. Hormesis and epigenetics: “Recent experimental studies clearly indicate that environmental fluctuations can induce specific and predictable epigenetic-related molecular changes, and support the possibility of adaptive epigenetic phenomenon. The epigenetic adaptation processes implying alterations of gene expression to buffer the organism against environmental changes support adaptability to the expected life-course conditions. It appears likely that adaptive epigenetic rearrangements can occur not only during early developmental stages but also through the adulthood, and they can cause hormesis, a phenomenon in which adaptive responses to low doses of otherwise harmful conditions improve the functional ability of cells and organisms. In this review, several lines of evidence are presented that epigenetic mechanisms can be involved in hormesis-like responses.” The pendulum appears to be swinging. In place of antioxidants, some are even beginning to call hormetic OS a cure for aging. See:
The Paradox of Exercise
The fact that enzymatic antioxidants are produced in response to ROS may, in part, explain the so called “paradox of exercise”. Why else would an activity, which results in dramatically increased levels of toxic ROS, produce undisputed health benefits? This may also explain why most studies of antioxidant supplementation with exercise have shown little benefit. Some studies have even found a negative effect to antioxidant supplementation combined with exercise. Here are a few examples:
Antioxidants prevent health-promoting effects of physical exercise in humans: “Exercise increased parameters of insulin sensitivity (GIR and plasma adiponectin) only in the absence of antioxidants in both previously untrained (P < 0.001) and pretrained (P < 0.001) individuals. This was paralleled by increased expression of ROS-sensitive transcriptional regulators of insulin sensitivity and ROS defense capacity, peroxisome-proliferator-activated receptor gamma (PPARγ), and PPARγ coactivators PGC1α and PGC1β only in the absence of antioxidants (P < 0.001 for all). Molecular mediators of endogenous ROS defense (superoxide dismutases 1 and 2; glutathione peroxidase) were also induced by exercise, and this effect too was blocked by antioxidant supplementation. Consistent with the concept of mitohormesis [mitochondrial hormesis], exercise-induced oxidative stress ameliorates insulin resistance and causes an adaptive response promoting endogenous antioxidant defense capacity. Supplementation with antioxidants may preclude these health-promoting effects of exercise in humans.”
For a detailed discussion of the exercise-induced muscular effects of ROS, see Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production: “Many early studies investigating exercise and free radical production focused on the damaging effects of oxidants in muscle (e.g., lipid peroxidation). However, a new era in redox biology exists today with an ever-growing number of reports detailing the advantageous biological effects of free radicals. Indeed, it is now clear that ROS and RNS are involved in modulation of cell signaling pathways and the control of numerous redox-sensitive transcription factors. Furthermore, physiological levels of ROS are essential for optimal force production in skeletal muscle. Nonetheless, high levels of ROS promote skeletal muscle contractile dysfunction resulting in muscle fatigue.”
Many Biological Functions of ROS
ROS are not merely toxic compounds. In recent years, research has only just begun to reveal some of the important functions of ROS. Space does not permit detailed discussion of the many beneficial roles of ROS in human physiology; however, below is an unsystematic sampling of examples with references for those who may be interested.
ROS play an intimate role in the processes of cellular energy production. Without them human life would not be possible. For an interesting discussion of the crucial role of bioenergetics in the development of complex life see: Bioenergetics, the origins of complexity, and the ascent of man. For an explanation of the basics of biological energy production, see: Energy Generation.
Sex hormones are electron mediators. Testosterone, estrogen and progesterone (as well as the catecholamine, adrenaline) are able to both donate and accept electrons. This ability to mediate redox reactions enables cellular communication without binding to their receptors. Electron emission by hormones and Adrenaline: communication by electron emission
Destruction of Pathogens and Infected Cells
Dendritic, Phagocyte and T-cell Regulation
(As a side note, Q10 actually increases OS during pregnancy, while vitamin E reduces it, see: Effects of exogenous antioxidants on oxidative stress in pregnancy.)
Cell-Cycle – Apoptosis/Proliferation – Destruction of Malignant Cells
The redox state of a cell plays an important role in determining whether the cell survives and proliferates, or dies. Moderate amounts of free radicals tend to promote survival and poliferation, while high levels result in apoptosis (cellular death). See: Redox Regulation of Cell Survival and CELLULAR REDOX SYSTEMS
“Under physiologic conditions, the balance between production and elimination of ROS ensures the proper maintenance of cellular metabolism and other functions.”
Capsaicin fights cancer by inhibiting antioxidant defenses, increasing ROS
ROS suppress cancer genes, while antioxidant defenses increase tumorigenesis Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis See, also: Redox regulation in cancer
Cardiovascular – Endothelial Function – Nitric Oxide
Hypothalamic Appetite Regulation
Insulin Production and Function
Antioxidant Efficacy Studies
This will be the focus of a separate, follow-up blog entry.
Reasons Why Antioxidant Supplementation Does Not Work
- May not reach target compartments, i.e. the mitochondria.
- Inactivate natural defensive enzyme systems.
- Disrupt important signal transduction pathways.
- Disrupt normal redox homeostasis.
- May have adverse effects on gene expression.
Even if antioxidant supplements don’t work, accumulated oxidative damage could still be the cause of aging and/or other conditions. Transgenic and knockout mice studies alter the expression of genes regulating the production of antioxidant enzymes. As already mentioned, such studies increasing or decreasing mitochondrial antioxidants, which alternatively increased or decreased the amount of corresponding mtOS, had no effect on lifespan, and produced no visible signs of aging. Similarly, a study increasing the rate of mitochondrial mutations by 500 fold produced no signs of aging, and no change in lifespan. Based on all available current evidence, it is very unlikely that accumulated oxidative damage is the general cause of aging. It may play a causal role in the development of certain specific pathologies, though evidence for this remains inconclusive.
End of the free radical theory of aging?
Is the Oxidative Stress Theory of Aging Dead? “A direct experimental test of the Oxidative Stress Theory of Aging is to alter the level of oxidative stress/damage and determine how these alterations affect lifespan. Using DNA recombinant technology, investigators over the past fifteen years have studied the effect of altering the expression of various components of the antioxidant defense system on lifespan; these studies are described in the Discussion. Below, we bring together all of the lifespan data that our group has conducted on transgenic/knockout mice with alterations in a wide variety of genes involved in the antioxidant defense system. These data demonstrate that almost all alterations in the antioxidant system of mice have no effect on lifespan.”
Since the free radical theory of aging is still recognized as a central if not the most-central theory of aging in the longevity science community, and since antioxidant supplementation has been thought to be unquestionably beneficial, the main messages of this post may be shocking to some. However, a few earlier posts in this blog have telegraphed that all is not well with these tenants. These posts include The free radical theory of aging. Is it really a theory of aging?, The anti-antioxidant side of the story and Another possible negative for antioxidants.
Caution Interpreting Data
I realize that readers of this review are likely to reply with some of the numerous studies purporting to demonstrate the health benefits of antioxidant supplementation. I do not deny that, in certain cases, there may be some benefits. Since these are already widely-touted, I have intentionally focused on the possible adverse effects of nutritional antioxidants, and the many positive physiological functions of ROS, in order to present a more balanced view. I suggest that caution be exercised when interpreting data. The following points may seem obvious; but they remain common fallacies committed routinely by both laymen, and even researchers in the field.
- Correlation is not causation. The fact that aging or other conditions are associated with increased levels of OS does not show that OS causes those conditions.
- In vivo effects are often very diffferent from in vitro. The fact that a compound exhibits certain properties in the lab does not mean it will have the same effects in the complex environment of a living organism.
- Effects vary between species. The fact that a certain treatment increases the lifespan of a nematode, for example, does not imply that it will do the same for humans.
- More is not better. If a nutrient has a beneficial effect at a given dose, it does not follow that greater doses will produce a greater beneficial effect.
- Many nutritional antioxidants have important health benefits independent of their antioxidant activity. In certain cases, correcting nutritional deficits may produce benefits. It does not follow that the benefits are necessarily the result of antioxidant activity.
- Viable anti-aging therapies must be capable of increasing maximum human lifespan, not just mean lifespan.
What does the future hold for antioxidant research?
TThe Free Radical Theory of Aging appears to be dying, if not already dead. Nutritional antioxidant supplementation does not appear to be the key to improved improved health or longevity. Does this mean antioxidants are a dead-end? No. Pharmaceutical or nutraceutial antioxidant therapies remain promising. Given the fact that antioxidant defenses diminish (as do most biological processes) with age, upregulating these defenses through pharmaceutical or nutraceutical intervention may, one day, be part of a comprehensive anti-aging program. Such therapies may not dramatically increase lifespan; but they are likely to increase healthspan, and may prove to be an effective treatment for specific disorders. To be effective such therapies must selectively target the activation of compromised, or suboptimal antioxidant systems. Like all proteins, enzymatic antioxidants are the result of the expression of their genes. So, viable therapeutic approaches might seek to increase the expression of those genes. The Nrf2 ARE (Antioxidant Response Element) controls the expression of enzymatic antioxidants. Clinical trials are already underway for compounds targeting the Nrf2-ARE pathway.(ref) An alternate approach is to target the upstream or downstream effects of oxidative damage in specific cellular systems. As an example of the latter, Cause and Consequence discusses stategies for targeting KGDHC (α-ketoglutarate dehydrogenase complex), which plays a role in neurodegeneration. To be effective both approaches will require a detailed understanding of the molecular processes involved, rather than the indiscriminate ingestion of compounds thought to “reduce” evil ROS. Even highly selective approaches are likely to involve some trade-offs. The prevention of some degenerative conditions might come at the price of increased the risk of certain cancers, etc. Optimal health and longevity result from seeking a judicious balance. Very few physiological pathways are purely “evil”.
Trashing the time-worn oxidative damage theory of aging and re-examining the roles of antioxidant supplements involve major paradigm shifts for the aging sciences community. Much more can and should be said than can be comprised in this single blog entry. Both Dr. Giuliano and I expect to be contributing additional blog discussions relating to aspects of this paradigm shift.