goldy
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- Jan 17, 2011
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Most popular discussions of antioxidants are based on an outdated view of free radicals as evil, toxic compounds, which cause chain reactions of destructive damage culminating in degenerative diseases and aging.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 innate 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, no conclusive evidence has been found that radical formation is a causative factor in the pathogenesis of any illness. On the contrary, most evidence indicates that radical formation results from, but does not cause disease processes. 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, wide-spread 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 (oxidant-antioxidant balance) 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 actually 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 answer, we need to 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 hyroperoxides, 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.(ref) 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 can not 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, ref)
Nutritional Antioxidants
Unlike innate antioxidant 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 can not 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 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
“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. 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 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:
Stress to the Rescue: Is Hormesis a ‘Cure’ for Aging?
Hormesis Against Aging and Diseases
Nutritional Hormesis and Aging
Inflammatory modulation of exercise salience: using hormesis to return to a healthy lifestyle
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:
Ascorbic acid supplementation does not attenuate post-exercise muscle soreness following muscle-damaging exercise but may delay the recovery process.
Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance
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.”
Biological Functions of ROS
ROS are not merely toxic compounds. In recent years, research has only just begun to reveal some of the many important functions of ROS. Space does not permit detailed discussion of the numerous beneficial roles of ROS in human physiology; however, below is an unsystematic sampling of examples with references for those who may be interested.
Energy Production
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.
Signal Transduction.
Oxyl radicals, redox-sensitive signalling cascades and antioxidants
Hydrogen peroxide sensing and signaling
Reactive oxygen species in cell signaling
Thiol peroxidases mediate specific genome-wide regulation of gene expression
The redox regulation of thiol dependent signaling pathways in cancer
Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis
Oxidative stress and cell signalling
Membrane regulation: Involvement of plasma membrane redox systems in hormone action
Immune System
The many roles of NOX2 NADPH oxidase-derived ROS in immunity
Destruction of pathogens and infected cells
Innate Immunity
The superoxide-generating oxidase of phagocytic cells
Dendritic, Phagocyte and T-cell Regulation
ROS Level Defines Dendritic Cell Development
Developmental biology: A bad boy comes good
Induction of regulatory T cells by macrophages is dependent on production of ROS
Redox Imbalance in T Cell-Mediated Skin Diseases
Macrophages suppress arthritis development by producing ROS.
Autophagy regulation
Activation of antibacterial autophagy by NADPH oxidases
Regulation of autophagy by ROS: physiology and pathology.
Autophagy, reactive oxygen species and the fate of mammalian cells.
Tissue Healing
The general case for redox control of wound repair
Wound Healing Essentials: Let There Be Oxygen
Redox Signals in Wound Healing
NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair
Regulation of cell proliferation by NADPH oxidase-mediated signaling: potential roles in tissue repair, regenerative medicine
Hydrogen peroxide mediates rapid wound detection
ROS as essential mediators of cell adhesion
Development
Function of ROS during animal development
Redox control in mammalian embryo development
The Roles of Glutathione Peroxidases during Embryo Development
(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.)
Redox Homeostasis
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.”
REACTIVE OXYGEN SPECIES, CELLULAR REDOX SYSTEMS AND APOPTOSIS
Capsaicin fights cancer by inhibiting antioxidant defenses, increasing ROS: Role of Mitochondrial Electron Transport Chain Complexes in Capsaicin Mediated Oxidative Stress Leading to Apoptosis in Pancreatic Cancer Cells
ROS suppress cancer genes, while antioxidant defenses increase tumorigenesis: Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis
Redox regulation in cancer
Cardiovascular – Endothelial Function – Nitric Oxide
Mitochondrial ROS-mediated signaling in endothelial cells
Exercise and Endothelial Function
Metabolic
Hypothalamic Appetite Regulation: ROS sets melanocortin tone and feeding in diet-induced obesity
Insulin Production and Function
Insulin action is facilitated by insulin-stimulated ROS
ROS and uncoupling protein 2 in pancreatic β-cell function.
Thyroxine Synthesis
Association of Duoxes with Thyroid Peroxidase and Its Regulation in Thyrocytes
Dual oxidase, hydrogen peroxide and thyroid diseases
Neurological
Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species
Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis
Cerebral Blood Flow Regulation by Nitric Oxide: Recent Advances
Mitochondrial preconditioning: a potential neuroprotective strategy
Fuel utilization by hypothalamic neurons: roles for ROS
Hypoxia Response
ROS-dependent endothelin signaling
ROS facilitate oxygen sensing
Antioxidant Efficacy Studies
I started looking at the human clinical efficacy studies expecting to find more positive results. Since earlier studies had been encouraging, many of the researchers of the later, large-scale, clinical studies were surprised by their own results. The earlier positive research included in vitro studies, animal studies and even some small human clinical studies. Population studies have consistently shown a benefit to consuming a diet rich in naturally occurring antioxidants from fruits and vegetables.
One would expect that health-conscious individuals who intentionally take antioxidant supplements would also make other life-style choices conducive to good health. Despite this possible confounding effect, research on intentional supplement users has also been disappointing. It is not possible to draw general conclusions based on studies of small numbers of subjects. It is also important that the duration of the the study be reasonably long, since we are interested in long-term effects. Although a very good model for many conditions, there are reasons why animal studies involving short-lived species, like mice, which have a lifespan of only a few years, may not be applicable to humans with a lifespan of a hundred years. In general, short-lived species produce much higher levels of ROS than long-lived species. Does this fact prove the Free Radical Theory of Aging? Of course, not. Species don’t have longer lifespans because they generate fewer ROS; rather, they have evolved the ability to generate fewer ROS in accordance with their lifespan. Longer-lived organisms have lower metabolic rates, and more advanced innate defense mechanisms. So, it is not surprising that studies involving mice don’t translate directly to humans.
Most of the large-scale, double-blind, placebo-controlled studies that I have seen involving antioxidant supplementation show no benefit, or adverse effects. You can always criticize studies saying that the dose used was too small, or too large, or the duration was not long enough to show the benefits, etc. But one would expect to see some studies showing benefits. Adverse effects are also very difficult for proponents of antioxidant supplementation to explain. If the dose used was too small to show benefit, then why was it large enough to show statistically significant adverse effects? Their outdated view of antioxidants fails to explain why there would ever be any adverse effect (except due to toxic overdose). Meta-analyses are usually criticized by claiming that the selection criteria were biased in some way. If this were the case, then alternate selection criteria should produce distinct results. What alternate criteria should be used? More importantly, where are the clinical studies that would produce different meta results?
It would be good to review any large-scale human clinical data supporting antioxidant supplementation, if they exists, please post them. Most of the studies have focused on vitamins C and E. (Interest in beta-carotene subsided early on when it was shown to dramatically increase the risk of cancer.) So, supplement proponents can also say that they tell us little about ALA, Q10 and other compounds. Antioxidant supplements have become a big business with revenue in 10’s of billion of dollars annually. Unfortunately, I feel that supplement sellers have often been as bad about misrepresenting research, as the pharmaceutical companies. I should also add that antioxidant supplementation is without a doubt beneficial in certain specific situations, such as exposure to ionizing radiation, which generates abnormally high levels of ROS. This includes exposure from routine medical procedures, like radio-isotope stress tests, cancer treatments, and CAT scans. (Melatonin seems to be especially useful here. It is both a direct antioxidant, and an indirect one by activating antioxidant enzyme systems.) Despite the change in our understanding of antioxidants, I still believe in and take supplements, including ALA, NAC, even moderate doses of vitamin C and E, as well as a variety of botanical extracts; although I admit that my enthusiasm for them has subsided with a better understanding of the biology involved. Below is an unsystematic summary of some human studies:
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 (oxidant-antioxidant balance) 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 actually 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 answer, we need to 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 hyroperoxides, 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.(ref) 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 can not 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, ref)
Nutritional Antioxidants
Unlike innate antioxidant 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 can not 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 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
“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. 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 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:
Stress to the Rescue: Is Hormesis a ‘Cure’ for Aging?
Hormesis Against Aging and Diseases
Nutritional Hormesis and Aging
Inflammatory modulation of exercise salience: using hormesis to return to a healthy lifestyle
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:
Ascorbic acid supplementation does not attenuate post-exercise muscle soreness following muscle-damaging exercise but may delay the recovery process.
Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance
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.”
Biological Functions of ROS
ROS are not merely toxic compounds. In recent years, research has only just begun to reveal some of the many important functions of ROS. Space does not permit detailed discussion of the numerous beneficial roles of ROS in human physiology; however, below is an unsystematic sampling of examples with references for those who may be interested.
Energy Production
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.
Signal Transduction.
Oxyl radicals, redox-sensitive signalling cascades and antioxidants
Hydrogen peroxide sensing and signaling
Reactive oxygen species in cell signaling
Thiol peroxidases mediate specific genome-wide regulation of gene expression
The redox regulation of thiol dependent signaling pathways in cancer
Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis
Oxidative stress and cell signalling
Membrane regulation: Involvement of plasma membrane redox systems in hormone action
Immune System
The many roles of NOX2 NADPH oxidase-derived ROS in immunity
Destruction of pathogens and infected cells
Innate Immunity
The superoxide-generating oxidase of phagocytic cells
Dendritic, Phagocyte and T-cell Regulation
ROS Level Defines Dendritic Cell Development
Developmental biology: A bad boy comes good
Induction of regulatory T cells by macrophages is dependent on production of ROS
Redox Imbalance in T Cell-Mediated Skin Diseases
Macrophages suppress arthritis development by producing ROS.
Autophagy regulation
Activation of antibacterial autophagy by NADPH oxidases
Regulation of autophagy by ROS: physiology and pathology.
Autophagy, reactive oxygen species and the fate of mammalian cells.
Tissue Healing
The general case for redox control of wound repair
Wound Healing Essentials: Let There Be Oxygen
Redox Signals in Wound Healing
NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair
Regulation of cell proliferation by NADPH oxidase-mediated signaling: potential roles in tissue repair, regenerative medicine
Hydrogen peroxide mediates rapid wound detection
ROS as essential mediators of cell adhesion
Development
Function of ROS during animal development
Redox control in mammalian embryo development
The Roles of Glutathione Peroxidases during Embryo Development
(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.)
Redox Homeostasis
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.”
REACTIVE OXYGEN SPECIES, CELLULAR REDOX SYSTEMS AND APOPTOSIS
Capsaicin fights cancer by inhibiting antioxidant defenses, increasing ROS: Role of Mitochondrial Electron Transport Chain Complexes in Capsaicin Mediated Oxidative Stress Leading to Apoptosis in Pancreatic Cancer Cells
ROS suppress cancer genes, while antioxidant defenses increase tumorigenesis: Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis
Redox regulation in cancer
Cardiovascular – Endothelial Function – Nitric Oxide
Mitochondrial ROS-mediated signaling in endothelial cells
Exercise and Endothelial Function
Metabolic
Hypothalamic Appetite Regulation: ROS sets melanocortin tone and feeding in diet-induced obesity
Insulin Production and Function
Insulin action is facilitated by insulin-stimulated ROS
ROS and uncoupling protein 2 in pancreatic β-cell function.
Thyroxine Synthesis
Association of Duoxes with Thyroid Peroxidase and Its Regulation in Thyrocytes
Dual oxidase, hydrogen peroxide and thyroid diseases
Neurological
Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species
Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis
Cerebral Blood Flow Regulation by Nitric Oxide: Recent Advances
Mitochondrial preconditioning: a potential neuroprotective strategy
Fuel utilization by hypothalamic neurons: roles for ROS
Hypoxia Response
ROS-dependent endothelin signaling
ROS facilitate oxygen sensing
Antioxidant Efficacy Studies
I started looking at the human clinical efficacy studies expecting to find more positive results. Since earlier studies had been encouraging, many of the researchers of the later, large-scale, clinical studies were surprised by their own results. The earlier positive research included in vitro studies, animal studies and even some small human clinical studies. Population studies have consistently shown a benefit to consuming a diet rich in naturally occurring antioxidants from fruits and vegetables.
One would expect that health-conscious individuals who intentionally take antioxidant supplements would also make other life-style choices conducive to good health. Despite this possible confounding effect, research on intentional supplement users has also been disappointing. It is not possible to draw general conclusions based on studies of small numbers of subjects. It is also important that the duration of the the study be reasonably long, since we are interested in long-term effects. Although a very good model for many conditions, there are reasons why animal studies involving short-lived species, like mice, which have a lifespan of only a few years, may not be applicable to humans with a lifespan of a hundred years. In general, short-lived species produce much higher levels of ROS than long-lived species. Does this fact prove the Free Radical Theory of Aging? Of course, not. Species don’t have longer lifespans because they generate fewer ROS; rather, they have evolved the ability to generate fewer ROS in accordance with their lifespan. Longer-lived organisms have lower metabolic rates, and more advanced innate defense mechanisms. So, it is not surprising that studies involving mice don’t translate directly to humans.
Most of the large-scale, double-blind, placebo-controlled studies that I have seen involving antioxidant supplementation show no benefit, or adverse effects. You can always criticize studies saying that the dose used was too small, or too large, or the duration was not long enough to show the benefits, etc. But one would expect to see some studies showing benefits. Adverse effects are also very difficult for proponents of antioxidant supplementation to explain. If the dose used was too small to show benefit, then why was it large enough to show statistically significant adverse effects? Their outdated view of antioxidants fails to explain why there would ever be any adverse effect (except due to toxic overdose). Meta-analyses are usually criticized by claiming that the selection criteria were biased in some way. If this were the case, then alternate selection criteria should produce distinct results. What alternate criteria should be used? More importantly, where are the clinical studies that would produce different meta results?
It would be good to review any large-scale human clinical data supporting antioxidant supplementation, if they exists, please post them. Most of the studies have focused on vitamins C and E. (Interest in beta-carotene subsided early on when it was shown to dramatically increase the risk of cancer.) So, supplement proponents can also say that they tell us little about ALA, Q10 and other compounds. Antioxidant supplements have become a big business with revenue in 10’s of billion of dollars annually. Unfortunately, I feel that supplement sellers have often been as bad about misrepresenting research, as the pharmaceutical companies. I should also add that antioxidant supplementation is without a doubt beneficial in certain specific situations, such as exposure to ionizing radiation, which generates abnormally high levels of ROS. This includes exposure from routine medical procedures, like radio-isotope stress tests, cancer treatments, and CAT scans. (Melatonin seems to be especially useful here. It is both a direct antioxidant, and an indirect one by activating antioxidant enzyme systems.) Despite the change in our understanding of antioxidants, I still believe in and take supplements, including ALA, NAC, even moderate doses of vitamin C and E, as well as a variety of botanical extracts; although I admit that my enthusiasm for them has subsided with a better understanding of the biology involved. Below is an unsystematic summary of some human studies:
