The Master Regulator of Aging? – Redox, Glutatione and Cysteine, Part 1

By Dan Campagnoli

(Welcome Dan, as a researcher and writer for this blog.  Jim Watson and I agree that with what is laid out in this blog entry, you clearly win both of the prizes in our contest Aging science wager challenges and prizesopen to readers.  We are arranging for an appropriate award ceremony and for the award fund transfer.  Vince Giuliano)

More important than a cure for cancer.

A bold claim, however a recently published study showed that with major advances in cancer treatment or heart disease, a 51-year-old can expect to live about one more year.  A modest improvement in delaying aging would double this to two additional years — and those years are much more likely to be spent in good health. “Even a marginal success in slowing aging is going to have a huge impact on health and quality of life.” said co-author S. Jay Olshansky of the School of Public Health at the University of Illinois-Chicago. These sentiments are echoed by Calico, the new Google funded start-up, on it’s ambitious quest to reverse the ageing process and extend human life.  Google CEO Larry Page said “One of the things I thought was amazing is that if you solve cancer, you’d add about three years to people’s average life expectancy.  We think of solving cancer as this huge thing that’ll totally change the world. But when you really take a step back and look at it, yeah, there are many, many tragic cases of cancer, and it’s very, very sad, but in the aggregate, it’s not as big an advance as you might think(ref).”

But perhaps Dr Herbert Nagasawa has beaten them to the punch with the technology he’s lead the development of over his accomplished 40+ year long career in medicinal chemistry.  More on that in another post. Vincent Giuliano, and more recently James (Jim) Watson, have been assembling a wealth of knowledge connecting the latest publish research into the aging process in this blog at www.anti-agingfirewalls.com, an invaluable source of information and ideas. Early this year Jim proposed to Vince an intellectual wager to determine the most important “signaling mechanism” for longevity and the most important cellular adaptation mechanism.  The abridged version is:

1. Wager #1 – What is the most important signal?  ROS, nutritional substrates, or hypoxia?  [i.e.ROS signaling (via Nrf2) vs Nutritional & Hypoxic Signaling (via HIF-1a and SIRTs)] (I, Vince, think this refers to anti-aging interventions).

Proposal:   I propose that we make an “intellectual wager” for the lump sum of one dollar.  To allow time for the debate, the winner will be “paid up” at end of 2013.  The “wager” is on which is the most important “signaling mechanism” for longevity – “ROS signaling” [Vince’s bet] or “Nutrient/Oxygen signaling” [Jims bet].

1. Wager #2 – What is the most important cellular adaptation mechanism? (i.e. hormetic response).  (i.e. anti-oxidant response element upregulation, unfolded protein response, mitochondrial biogenesis, DNA repair mechansims, autophagy, etc.)

Proposal:   I propose that we make an “intellectual wager” for the lump sum of one dollar.  The question is what is the most important cellular adaptation mechanism? (i.e. hormetic response) that promotes health and longevity.  (i.e. anti-oxidant response element upregulation, unfolded protein response, mitochondrial biogenesis, DNA repair mechanisms, autophagy, etc.)  You say it is upregulating the AREs [Anti-oxidant Response Element] and I say it is upregulating autophagy.”

To throw my hat into the ring I’ve finally put together my own particular research on the subject, as laid out here.  If I had to summarize it to the most concise form possible I would say maintenance of cellular redox homeostasis is the most important longevity factor.  To reword my response for wager #1 my premise is that redox signalling is the most important. My answer for wager #2 would then have to be the response to maintain redox homeostasis, with ARE/EpRE upregulation being one primary factor. This ties tightly into ROS signaling via Nrf2, they are pieces of the same puzzle and I’ll attempt to tease apart some the subtle differences.  I see redox state, implying GSH levels (and it’s direct functions), to be the primary factor, with increasing Nrf2 expression as one of two ways to achieve it: 1. Increasing the enzymes, and 2. Increasing the substrates.  I’ll cover the subtleties I’ve found of Nrf2 expression and GSH synthesis in Part 2.

One of the first main clues which set me down the redox path a while ago when investigating cysteine supply for GSH synthesis was this conclusion from Oxidative stress and ageing: is ageing a cysteine deficiency syndrome? “In humans, an age-related oxidative shift in the ratio of reduced to oxidized glutathione, i.e. the glutathione redox status, has been demonstrated in whole blood, and peripheral blood mononuclear cells. … Responses of signalling cascades to changes in redox status (see §1b) are, therefore, not merely experimental artefacts. As the thiol/disulfide redox status shifts to more oxidative conditions in old age, there is inevitably a shift in the set points of physiological signals.”

On further investigation redox state appeared to be the most far reaching factor, the one with a finger in every pie, and of growing interest in the research arena.  Two quick indicative examples are this article’s title, Changing paradigms in thiology from antioxidant defense toward redox regulation. (Methods Enzymol. 2010), and the following from Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors (Antioxid Redox Signal. 2011 October): “Activation of gene transcription has for long been considered to be primarily, if not exclusively, regulated by cascades of protein phosphorylation and de-phosphorylation… During the nineties, though, a second area was recognized to be intimately related to transcriptional regulation, the ubiquitin/proteasome system… The multiple ways of redox regulations that became obvious over the last two decades lead us to presume that most, if not all, of the classical routes to transcriptional activation are modulated by redox processes or even critically depend on oxidant signals”

And, from Glutathione and apoptosis Free Radic Res. 2008 August: “Increasingly, we have witnessed a growing appreciation of the role of GSH in redox signalling beyond its traditionally recognized role as the main cellular antioxidant against oxidative challenge. The heightened interest in GSH in post-translational control of cellular processes has brought to the fore the versatility of this ubiquitious molecule that is present in millimolar concentrations in most cells and whose homeostasis is rigorously controlled by GSH redox enzymes and glutamate-cysteine ligase-driven GSH synthesis”

A definition of Redox

“Redox state is the energetic force for electron transfer, much as pH is a measure of the strength of proton transfer. I.e., redox state measures the ability of a compound to donate or receive electrons, just as pH is a measure of the ability of a compound to donate or receive protons. Technically, redox state, E, is the electromotive force in mV relative to the standard state of hydrogen as follows. An example is also shown for the most abundant intracellular reductant, glutathione:

where E_o is the standard reduction state at pH 7 (-264 mV for glutathione, R is the gas constant, T is temperature (^oK), n is the number of transferred electrons and F is Faraday’s constant.” (ref:  Epigenetic oxidative redox shift (EORS) theory of aging unifies the free radical and insulin signaling theories)

Redox = Glutathione?

I’ll be taking some liberty of alternating between referring to redox state and glutathione (GSH) levels, as GSH/GSSG is the major intra-cellular redox couple.  Partly because my initial research initially focused on glutathione before looking more closely at redox, and also because GSH has its own direct functions such as detoxification via the GST enzymes. Also, some of the authors of the articles referenced may not have been aware of the significance of GSH levels and redox.  I focus here on the many roles of redox and GSH.  And, in a second blog entry to follow, Part 2 of this series will be on the many factors of GSH synthesis and function, and practical interventions.

“Cellular redox state, or the balance between oxidation/reduction reactions, is collectively determined by the reduction potentials and reducing capacities of the redox couples, such as GSH/GSSG, NADPH/NADP⁺, NADH/NAD⁺, cysteine/cystine, thioredoxin (reduced)/thioredooxin (oxidized) and glutaredoxin (reduced)/glutaredoxin (oxidized). Nonetheless, the GSH/ GSSG couple is regarded as the primary arbiter of the tissue redox state because it is comparatively 2 to 4 orders of magnitude higher in abundance than the other redox couples and it is also metabolically linked to the less abundant redox couples via direct or indirect donations of reducing equivalents for the reduction of their oxidized forms(ref).”

GSH, More Powerful than ATP

Having always thought of ATP as the energy currency of the cell, interestingly the prior quote follows on to say: “The mitochondrial electron transport chain efficiently oxidizes NADH with molecular oxygen over numerous smaller steps to siphon about 98% of the energy as a proton and electrical gradient across the mitochondrial inner membrane.   We commonly think of ATP as a high energy molecular currency, but hydrolysis of the phosphate bond produces only -7.3 kcal/mol, compared to almost -60 kcal/mol of oxidized NADH. Since the reducing energies of NADH, NADPH and GSH are so large, they can provide the power for a large number of reduction reactions in the cell including glycolysis, ATP generation, disulfide bond formation in numerous enzymes, transporters, signaling molecules and transcription factors. The driving force for these reactions depends on the relative concentrations of the oxidized and reduced forms of each redox couple(ref).”

(image source Copywright Exp Gerontol. 2010 March; 45(3): 173–179.)

“Biological redox energy scale. The highly reduced energy levels of NADPH, GSH and NADH are used to power catabolism with molecular oxygen as the terminal electron acceptor. The step-wise electron transport in mitochondria is used to generate ATP, a lower denomination of energy currency.”

Not a one-size-fits-all

Redox state and glutathione levels are maintained at different levels in various parts of the cells and body. “GSH concentration in brain cells is about 400-times higher than in blood. … The mitochondrial GSH accounts for approximately 15% of cellular glutathione. In general the mitochondrial GSH:GSSG ratio is greater than that of the cytosol, resulting in a more reducing environment. One study of the GSH and GSSG levels in rat mitochondria reported that liver GSH concentration was 8.4 um and GSSG was 0.02 um, corresponding to approximately 250:1 GSH:GSSG ratio. However, in brain mitochondria the GSH was reported to be 5.5 um and GSSG 0.09 um, giving an approximately 50:1 GSH:GSSG ratio. … Unlike most of the cell (which maintains a 100:1 GSH:GSSG ratio), the endoplasmic reticulum has an unusually oxidative environment estimated at a 5:1 GSH:GSSG ratio” (ref) Tissue levels of GSH vary widely between different tissues, with the highest concentrations in the eye lens (~10 mM) and the lowest concentrations in fast-twitch skeletal muscles (~0.5 mM). (ref) “Although synthesized exclusively in the cytosol, GSH is distributed to different cellular compartments where it maintains distinct redox environments uniquely suited to the function of the organelle, be it protein folding in the endoplasmic reticulum or gene transcription in the nucleus.” (ref) And from a further reference below “stem cells reside in redox niches with low ROS levels”

Image source  Some key pathways regulated by reactive oxygen species (ROS)

Image source   Functions of GSH/GSSG

Dysregulation of redox state is more important and independent of (precedes) ROS damage

This is a central message of this blog entry and the key point in my response to the wager offer.

The “Redox stress hypothesis of Aging” publication proposes aging-associated functional losses are primarily caused by a progressive pro-oxidizing shift in the redox state of the cells, which leads to the over-oxidation of redox-sensitive protein thiols and the consequent disruption of the redox-regulated signaling mechanisms.  Another article on the topic, Epigenetic oxidative redox shift (EORS) theory of aging unifies the free radical and insulin signaling theories (Exp Gerontol. 2010 March) says: “In order to begin to resolve these paradoxes, I propose that an oxidized redox state is upstream of the commonly observed ROS damage. Certainly, ROS damage is affected by the balance of oxyradical generation and anti-oxidant defenses. Numerous sources of oxyradical generation have been documented, but less appreciation exists for the essential role that ROS or redox signaling plays in metabolism. The common impression that the mitochondrial electron transport chain is the major source of oxyradical generation often overlooks other sources in the cytoplasm and plasma membrane and an upstream oxidized redox state.”

Experimental evidence is cited in  Dual-energy precursor and nuclear erythroid-related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species.  “To determine whether glutathione (GSH) loss or increased reactive oxygen species (ROS) are more important to neuron loss, aging, and Alzheimer’s disease (AD), we stressed or boosted GSH levels in neurons isolated from aging 3xTg-AD neurons compared with those from age-matched nontransgenic (non-Tg) neurons. … Remarkably, the rate of neuron loss with ROS did not increase in old age and was the same for both genotypes, which indicates that cognitive deficits in the AD model were not caused by ROS. … These stress tests and neuroprotective treatments suggest that the redox environment is more important for neuron survival than ROS.”

From The redox stress hypothesis of aging “In summary, the presently accessible information suggests that although the steady-state amounts of macromolecular oxidative damage tend to increase with age, the molar ratios of oxidized:unoxidized macromolecules are very low. Furthermore, the oxidized macromolecules, rather than being stored, are generally rapidly eliminated and replaced via nascent biosynthesis. Arguably, the age-related functional losses would be expected to depend more upon the pool size of the parent unoxidized macromolecules rather than the amounts of the oxidized macromolecules, unless it could be demonstrated that the very presence of oxidized macromolecules was itself deleterious, analogous to dominant negative mutations. Because the physiological losses in the latter part of life are often quite severe and mortality increases exponentially, whereas the accrued amounts of macromolecular oxidative damage are relatively minuscule, the case for a possible causal association remains tenuous. Nonetheless, this ambiguity does not imply that ROS or oxidative stress do not play an important role in the aging process. Rather, the steady-state amounts of oxidative structural damage are not synonymous with oxidative stress nor are they a reliable indicator of functional losses.”

Under normal conditions hydrogen peroxide functions as a signaling molecule and its levels are maintained by  catalase and the peroxidase enzymes.  Dysregulation of redox leading to an over oxidized state can cause an increase in the generation of the highly reactive and more-damaging hydroxyl free radical (OH·) via the iron-catalyzed, Haber-Weiss- and Fenton-type reactions, and an increase in reactions causing irreversible bonds on redox-sensitive protein thiols.  A more detailed description of this process is here.  Furthermore another article finds there is an “irreversible consequence of nuclear GSH depletion” even after GSH levels are restored(ref).

Supporting Evidence

Vincent Guiliano has detailed his 14 major theories of aging in his treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY, I’ll use these to group together some the of the research showing the extensive role redox and glutathione play in cellular processes and health.

1. 1.    Oxidative Damage

Glutathione is often referred to as the “master anti-oxidant” in the published literature due to its high concentrations and unique versatility in neutralizing ROS.  GSH is able to react non-enzymatically with ROS such as superoxide, NO, hydroxyl radical, and peroxynitrite.  With the glutathione peroxidase (GPx) enzymes it is able to reduce lipid hydroperoxides to their corresponding alcohols and reduce hydrogen peroxide to water.

2. Cell DNA Damage (and cell proliferation regulation)

From Nuclear glutathione,. Biochim Biophys Acta. 2013 May: “The sequestration of GSH in the nucleus of proliferating animal and plant cells suggests that common redox mechanisms exist for DNA regulation in G1 and mitosis in all eukaryotes. We propose that glutathione acts as “redox sensor” at the onset of DNA synthesis with roles in maintaining the nuclear architecture by providing the appropriate redox environment for the DNA replication and safeguarding DNA integrity. In addition, nuclear GSH may be involved in epigenetic phenomena and in the control of nuclear protein degradation by nuclear proteasome. Moreover, by increasing the nuclear GSH pool and reducing disulfide bonds on nuclear proteins at the onset of cell proliferation, an appropriate redox environment is generated for the stimulation of chromatin decompaction.”

From The primary role of glutathione against nuclear DNA damage of striatum induced by permethrin in rats,. Neuroscience. 2010 Jun: “On the contrary, GSH played a crucial role on striatum since it was able to protect the cells against nuclear DNA damage induced by PERM. In conclusion our outcomes suggested that nuclear DNA damage of striatum cells was directly related to GSH depletion due to PERM insecticide.”

The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts relates: “We show here and in a previous report that nuclear glutathiolation changes during the cell cycle and that the depletion of nuclear GSH changes the pattern of nuclear glutathionylated proteins. The suggestion that reduced nuclear environment could protect oxidant sensitive proteins from oxidation could be confirmed by our results: there was less glutathionilated and more oxidised proteins when the nuclear GSH was depleted by DEM. However, after nuclear GSH increased (72 h) the glutathionylation of nuclear protiens in DEM treated cells reached the values of control, while the level of protein oxidation remained high. This reflects the irreversible consequence of nuclear GSH depletion early in the culture So, the presence of the high glutathione level in the nucleus appears to be a prerequisite for the start of the cell proliferation. Our findings are in line with several other studies aimed to elucidate the fine redox regulatory mechanisms that lie behind the correct cell cycle progression. Conour et al., suggested that the reduction of the intracellular environment as cells progress from G1 to G2/M phase, as shown in our study, may protect genomic DNA from oxidative damage upon brake down of the nuclear envelope. Indeed, one of the assertions in support of this premise derives from the study of oxidative stress related to genotoxicity, recently published by Green; oxidative DNA modifications displayed a negative linear correlation with nuclear GSH. This is of special importance considering the report of Menon et al on the necessity of the oxidative event in early G1 phase to allow G1-S transition. Even more, it has recently been postulated that the restriction of DNA synthesis to the reductive phase of the cycle in yeast may be an evolutionarily important mechanism for reducing oxidative damage to DNA during replication, which implies the common mechanism of the DNA protection during S phase in all eukaryotes.”

Glutathione S-transferase and DNA damage 

Glutathione S-transferase are a family of enzymes which catalyze the conjugation of the reduced form of glutathione, GSH which can damage DNA, to xenobiotic substrates for the purpose of detoxification(ref). While these two examples look at GST polymorphisms causing reduced GST levels, the ability to detoxify DNA-damaging compounds will be determined by both the concentrations of the enzyme and substrate, i.e. GST and GSH levels.

From Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies, Mutat Res. 2012 Aug: “Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these proteins in DNA damage processing via DNA damage signalling.”

And, from Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1. Mol Carcinog. 2012 Jul: “Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells… These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage”

3.  Mitochondrial Damage

From Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics Trends in Biochemical Sciences, October 2013: “In excess, ROS can be detrimental; however, at low concentrations oxyradicals are essential signaling molecules. Mitochondria thus use a battery of systems to finely control types and levels of ROS, including antioxidants. Several antioxidant systems depend on glutathione. Here, we review mitochondrial ROS homeostatic systems, including emerging knowledge about roles of glutathione in redox balance and the control of protein function by post-translational modification.”

From Mitochondrial glutathione, a key survival antioxidant, Antioxid Redox Signal. 2009 Nov: “Among the arsenal of antioxidants and detoxifying enzymes existing in mitochondria, mitochondrial glutathione (mGSH) emerges as the main line of defense for the maintenance of the appropriate mitochondrial redox environment to avoid or repair oxidative modifications leading to mitochondrial dysfunction and cell death. mGSH importance is based not only on its abundance, but also on its versatility to counteract hydrogen peroxide, lipid hydroperoxides, or xenobiotics, mainly as a cofactor of enzymes such as glutathione peroxidase or glutathione-S-transferase (GST). Many death-inducing stimuli interact with mitochondria, causing oxidative stress; in addition, numerous pathologies are characterized by a consistent decrease in mGSH levels, which may sensitize to additional insults. From the evaluation of mGSH influence on different pathologic settings such as hypoxia, ischemia/reperfusion injury, aging, liver diseases, and neurologic disorders, it is becoming evident that it has an important role in the pathophysiology and biomedical strategies aimed to boost mGSH levels.”

4.  Tissue Glycation

From Functional Consequences of Age-Dependent Changes in Glutathione Status in the Brain, Antioxid Redox Signal. 2013 Feb: “Changes in redox homeostasis can also potentiate the accumulation of advanced glycation endproducts, resulting in defects in protein processing and function as well as a further increase in inflammation.”

From Glutathione reverses early effects of glycation on myosin function: “It is concluded that glucose modifies myosin function in a dose-dependent manner and that glutathione reverses the effect of glucose on myosin function. The present results demonstrate that GSH reverses the formation of early glycation products. The restoration of motility by GSH after incubation with a reducing sugar implies a reversal of Schiff base formation. The present results suggest that GSH, in addition to its antioxidant function, could play an important role in preventing the progress of glycation of intracellular proteins.”

5.  Lipofuscin Accumulation

From Influence of intracellular glutathione concentration of lipofuscin accumulation in cultured neonatal rat cardiac myocytes: “The authors show that reduced [decreased] GSH level leads to a simultaneous increase in accumulation of lipofuscin in cardiac myocytes, possibly by increasing the level of cytosolic hydrogen peroxide.”

Nothing specific in this publication, but a good recent review of lipofuscin  published coincidentally in Redox Biology Lipofuscin: formation, effects and role of macroautophagy Redox Biol. 2013.

6.  Chronic or Excess Inflammation

Searching PubMed for redox, or glutathione, and inflammation returns a multitude of articles.  In particular Nuclear factor-kappaB (NF-kappab) keeps showing up as a redox sensitive transcription factor critical to immune and inflammatory response.

Image source: NF-kappaB and Nrf2, the Yin and Yang of the inflammatory response.

Another key player in “inflammaging” is NLRP3, which recent research pinpointed it as the specific sensor that activates inflammation with age.  And as detailed in Redox control of NLRP3 inflammasome activation in health and disease J Leukoc Biol. 2012 Nov, redox signaling is crucial mechanism of inflammasome activation and IL-1 processing and secretion.

7. Immune System Compromise

Glutathione is important for immune cells protect to protect themselves when they attack pathogens with ROS blasts.  Also many studies have shown, a number listed below, that high levels of glutathione inhibit entry and/or replication for many types of commonly known viruses. From Glutathione: A key player in autoimmunity Autoimmun Rev. 2009 Jul:  “Altered glutathione concentrations may play an important role in many autoimmune pathological conditions prevalently elicited, detrimed and maintained by inflammatory/immune response mediated by oxidative stress reactions. From Inhibition of influenza infection by glutathione: “Accumulating evidence suggests that cellular redox status plays an important role in regulating viral replication and infectivity…  Together, the data suggest that the thiol antioxidant GSH has an anti-influenza activity in vitro and in vivo.”  From Glutathione Inhibits HIV Replication by Acting at Late Stages of the Virus Life Cycle: ” Overall data suggest that GSH can interfere with late stages of virus replication.  This would be in agreement with data obtained in cells exposed to herpesvirus type 1 (a DNA virus) or to Sendai (an RNA virus), showing that the suppression of virus replication by GSH is related to the selective inhibition of envelope glycoproteins.”

Going on, from Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication: “Data suggest that exogenous GSH inhibits the replication of HSV-1 by interfering with very late stages of the virus life cycle, without affecting cellular metabolism.” From Natural Killer Cells, Glutathione, Cytokines, and Innate Immunity Against Mycobacterium tuberculosis: “Furthermore, NK cell functions are dependent on adequate levels of glutathione.  Our results strongly indicate that glutathione in combination with IL-2+IL-12 augments NK cell functions, leading to control M. tuberculosis infection.”

From Glutathione and infection: “In this review we describe how GSH works to modulate the behavior of many cells including the cells of the immune system, augmenting the innate and the adaptive immunity as well as conferring protection against microbial, viral and parasitic infections. This article unveils the direct antimicrobial effects of GSH in controlling Mycobacterium tuberculosis (M. tb) infection within macrophages. In addition, we summarize the effects of GSH in enhancing the functional activity of various immune cells such as natural killer (NK) cells and T cells resulting in inhibition in the growth of M. tb inside monocytes and macrophages. Most importantly we correlate the decreased GSH levels previously observed in individuals with pulmonary tuberculosis (TB) with an increase in the levels of pro-inflammatory cytokines which aid in the growth of M. tb.”

Inhibitory Effect of Glutathione on Oxidative Liver Injury Induced by Dengue Virus Serotype 2 Infections in Mice relates: “in vitro treatment of HepG2 cells with antioxidants such as GSH inhibited viral entry as well as the production of reactive oxygen species in HepG2 cells.”

8.  Neurological Degeneration

High levels of GSH in neurons and white matter, suggests astrocytes rather than neurons may be particularly vulnerable to oxidative stress (ref).  Also astrocytes appear to be susceptible to high extracellular glutamate from immunoexcitotoxicity etc, which reduces their ability to uptake cysteine for GSH synthesis.  I plan to cover this in Part 2.

From Dysregulation of Glutathione Homeostasis in Neurodegenerative Diseases  Nutrients. 2012 October:  “Over the past several decades the role of intracellular GSH status in neurodegenerative diseases has been studied intensively. Such research continues to provide mechanistic insights pertaining to the cellular dysfunctions of the neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Friedreich’s ataxia. Disruption in GSH homeostasis and modification of the enzymes that are dependent on GSH as a substrate have been linked to initiation and progression of the neurodegenerative diseases. The dysregulation of GSH and GSH-dependent enzymes induces a variety of cellular problems that can lead to mitochondrial dysfunction, accumulation of ROS/RNS damage, disruption of signaling pathways, protein aggregation, and ultimately cell death.”

From Functional Consequences of Age-Dependent Changes in Glutathione Status in the Brain.  Antioxid Redox Signal. 2013 Feb: “Decreases in GSH are also associated with microglial activation and endothelial dysfunction, both of which can contribute to impairments in brain function. Changes in redox homeostasis can also potentiate the accumulation of advanced glycation endproducts, resulting in defects in protein processing and function as well as a further increase in inflammation.”

From Role of a Redox-Based Methylation Switch in mRNA Life Cycle (Pre- and Post-Transcriptional Maturation) and Protein Turnover: Implications in Neurological Disorders Front Neurosci. 2012: “A “holonarchy” for synaptic plasticity can be imagined, beginning at mRNA synthesis, transcription, translation, protein turnover, methylation reactions, and at the highest level redox status serves as the central regulatory switch.” (See a definition for holonarchy here) 

From Glutathione and Parkinson’s disease: Is this the elephant in the room?  Biomed Pharmacother. 2008 Apr-May: “At least 2 decades have past since the demonstration of a 40% deficit in total glutathione (GSH) levels in the substantia nigra in patients with Parkinson’s disease (PD). The similar loss of GSH in the nigra in Incidental Lewy body disease, thought to be an early form of PD, indicates that this is one of the earliest derangements to occur in the pre-symptomatic stages of PD”  See also Impaired Glutathione Synthesis in Neurodegeneration Int. J. Mol. Sci. 2013

11.  Susceptibility to Cardiovascular Disease

In Vince’s treatise laying out the 14 Theories of Aging, this Section says: “The age-related remodeling appears to involve an imbalance between omega-3 and omega-6 fatty acids in these membranes as well as dysfunctional Ca2+ metabolism.”

There are a few articles which look at the Calcium aspect, for example, Redox regulation of cardiac calcium channels and transporters Cardiovascular Research 71 (2006): “Changes in the intracellular redox environment can affect many cellular processes, including the gating properties of ion channels and the activity of ion transporters. Because cardiac contraction is highly dependent on intracellular Ca2+ levels ([Ca2+]I ) and [Ca2+]I regulation, redox modification of Ca2+ channels and transporters has a profound effect on cardiac function.” Because levels of ROS and RNS can increase significantly after stimulation of specific signal transduction pathways or during certain pathological conditions of the heart (e.g. ischemia– reperfusion) the redox defense system is essential for the maintenance of cellular homeostasis. In many mammalian cells, including cardiomyocytes, glutathione is considered the major cytosolic redox buffer.”

According to Redox regulation of calcium release in skeletal and cardiac muscle Biol Res. 2002;35(2):183-93: “In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity.”

From Crosstalk between calcium and redox signaling: from molecular mechanisms to health implications. Antioxid Redox Signal. 2008 Jul: “Studies done many years ago established unequivocally the key role of calcium as a universal second messenger… Furthermore, it is becoming increasingly apparent that there are significant interactions between calcium and redox species, and that these interactions modify a variety of proteins that participate in signaling transduction pathways and in other fundamental cellular functions that determine cell life or death.”

12.  Telomere Shortening and Damage. 

Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells reports: reports: “These findings demonstrate a key role for glutathione-dependent redox homeostasis in the preservation of telomere function in endothelial cells”

Glutathione regulates telomerase activity in 3T3 fibroblasts relates: “Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity.”

13.  Programmed epigenetic changes

From Redox regulation of the epigenetic landscape in cancer: a role for metabolic reprogramming in remodeling the epigenome. Free Radic Biol Med. 2012 Dec:  “We further speculate that redox biology can change epigenetic events through oxidation of enzymes and alterations in metabolic cofactors that affect epigenetic events such as DNA methylation. Combined, these metabolic and redox changes serve as the foundation for altering the epigenotype of normal cells and creating the epigenetic progenitor of cancer.”  This item also relates to number 10 in Vince’s list, Susceptibility to cancers.

From The redox basis of epigenetic modifications: from mechanisms to functional consequences. Antioxid Redox Signal. 2011 Jul: “Recent research is revealing that redox metabolism is an increasingly important determinant of epigenetic control that may have significant ramifications in both human health and disease. Numerous characterized epigenetic marks, including histone methylation, acetylation, and ADP-ribosylation, as well as DNA methylation, have direct linkages to central metabolism through critical redox intermediates such as NAD(+), S-adenosyl methionine, and 2-oxoglutarate.”

From Blood glutathione redox status and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults. Epigenetics. 2013 Jul: “Our findings support the hypothesis that a more oxidized blood GSH redox status is associated with decreased global methylation of peripheral blood mononuclear cell DNA”

14.  Stem Cell Supply Chain Breakdown

From Redox homeostasis: the linchpin in stem cell self-renewal and differentiation January 2013: “Recently, a growing body of literature has shown that stem cells reside in redox niches with low ROS levels. The balance of Redox homeostasis facilitates stem cell self-renewal by an intricate network. Thus, to fully decipher the underlying molecular mechanisms involved in the maintenance of stem cell self-renewal, it is critical to address the important role of redox homeostasis in the regulation of self-renewal and differentiation of stem cells.”

And finally, one additional grouping;

Cell Cycle/Death – Apoptosis and post-mitotic cells (cell senescence)

From Apoptosis and glutathione: beyond an antioxidant Cell Death and Differentiation (2009): “GSH depletion was initially ascribed to its oxidation by RS generated during oxidative stress. However, it is now recognized that under more physiological stimulation of apoptosis, such as activation of death receptors, GSH depletion occurs as an active process involving its extrusion across the plasma membrane. This phenomenon has also been shown to precede oxidative stress generated by the accumulation of RS and to be necessary for the progression of apoptosis. Indeed, GSH depletion has been shown to induce or potentiate apoptosis, and excessive oxidative stress. Although the exact mechanisms involved in the regulation of apoptosis by GSH remain elusive, recent reports show that oxidative post-translational modifications in proteins regulated by GSH content such as glutathionylation (protein-SSG) and nitrosylation (protein-SNO) are important regulators of apoptosis.”

From Glutathione and apoptosis Free Radic Res. 2008 August: “With regards to apoptosis, the mitochondrial GSH redox status is emerging to be a central player. Our studies have provided new perspectives on the role of mitochondrial GSH in mitochondrial DNA integrity and cell survival and the availability of genetic approaches targeting mitochondrial GSH transporters offers new strategies for studying the importance of this redox compartment in apoptosis. The current understanding of protein S-glutathiolation in conjunction with protein S-nitrosation as important post-translational regulatory mechanisms has contributed to recent advances in apoptosis research.”

From Time line of redox events in aging postmitotic cells: “Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. [for recycling oxidized GSSG to reduced GSH]”

Follow up comments to this article are here: A new answer to old questions 

From Atmospheric oxygen accelerates the induction of a post-mitotic phenotype in human dermal fibroblasts: the key protective role of glutathione Differentiation 2000 Oct: “Increasing oxidative stress by addition of hydrogen peroxide or depletion of glutathione also induced a switch from a mitotic to a post-mitotic phenotype in these cells, whereas addition of the anti-oxidant N-acetylcysteine under atmospheric (20%) oxygen tension potently inhibited this process. In addition, a statistically significant correlation was observed between the magnitude of intracellular glutathione depletion and the reduction in the population of mitotic cells in this model. We propose that the switch from a mitotic to a post-mitotic phenotype represents a process of cellular ageing and that standard atmospheric oxygen tension imposes a substantial oxidative stress on dermal fibroblasts which accelerates this process in culture. The data also suggest that intracellular glutathione levels strongly influence the induction of a post-mitotic phenotype and that, by implication, depletion of glutathione may play a significant role in the progression of cellular ageing in human skin.”

Further Proof Required?

As clear from the above, redox regulation is being accorded a greater interest in the research community.  Still further research will be required to elucidate the exact roles and mechanisms redox plays.  In this article, published in Front Neurosci. 2012, the authors suggests a number of study designs which could provide further evidence, saying: “These and other such studies would allow researchers to test the underlying major hypothesis that redox state is the ultimate source of regulatory control over mRNA methylation, mRNA processing, protein synthesis, and protein turnover.”

While searching for further supporting evidence, I very recently came across an article which appears to be the most detailed research article supporting the redox stress argument, taking into account transgenic species, organism fitness and more.  This article aided me in putting together some of the previous points.  So for the curious reader I suggest taking a moment to pause here and read it in its entirety. The Redox Stress Hypothesis of Aging Free Radic Biol Med. 2012 February 1; 52(3): 539–555:  “An alternative notion, advocated here, termed “redox stress hypothesis,” proposes that aging-associated functional losses are primarily caused by a progressive pro-oxidizing shift in the redox state of the cells, which leads to the over-oxidation of redox-sensitive protein thiols and the consequent disruption of the redox-regulated signaling mechanisms. —  Presently, the balance of evidence seems to favor the view that the role of structural damage is relatively minor compared to that emanating from the disruptions of the redox-based molecular switches. We have termed this phenomenon “redox stress”, as there are indications of a disturbance in the thiol redox state in the post-reproductive phase of life. Furthermore, transgenic studies have shown that augmentation of reducing power, provided by NADPH and GSH, is the most effective currently known experimental manipulation for the prolongation of lifespan.”

Image source   “Diagrammatic representation of the redox stress hypothesis of aging. The key features of this hypothesis are that: most of the residual ROS are non-radical oxidants rather than free radicals; ROS are physiologically essential molecules involved in the regulation of protein activity …”

 How GSH protects redox-sensitive proteins from transient oxidative stress

As well as directly neutralizing the more damaging types of ROS which can cause irreversible protein oxidation, GSH also protects redox-sensitive proteins by reversible S-glutathionylation under oxidative stress.

From Impaired Glutathione Synthesis in Neurodegeneration Int. J. Mol. Sci. 2013 “GSH is the major thiol redox buffer to maintain intracellular redox homeostasis. Under oxidative stress conditions, GSH can lead to the reversible formation of mixed disulfides between protein thiol groups (S-glutathionylation) to prevent irreversible protein oxidation. S-glutathionylation is a reversible modification for restoring these protein functions when the intracellular redox state returns to normal after the insults. Disulfide bonds are also reduced back to thiol residues by the reaction with disulfide reductases, such as thioredoxin or glutaredoxin, which facilitate normal protein folding. Protein S-glutathionylation is an important adaptive cellular response to protect crucial protein functions in the cell.”

Also S-glutathionylation of key protein targets has now emerged as a physiologically important redox signaling mechanism.

The Redox Shift with Age

So now we know how important redox is, here is a quick look at how it changes with age.  Also health conditions, lifestyle choices and environmental conditions can accelerate the loss of GSH and oxidative shift in the redox state, for example: “We conclude that subjects with type 2 diabetes have decreased oxidant capacity, evidenced by reduced synthesis of glutathione, and they are under increased oxidative stress(ref).”

Epigenetic oxidative redox shift (EORS) theory of aging unifies the free radical and insulin signaling theories offers this diagram which summarizes the situation in the most basic terms:

Image source  Use it or lose it!  A basic message

Dean Jones at Emory University was the first to show that human plasma GSH/GSSG is controlled at a relatively constant redox state of -137 mV in 740 healthy adults through age 50. However, an oxidative shift of about 7 mV/decade occurs over the next two decades, followed by a further decline to -110 mV in the 70 to 85-year-old group.

The publication Oxidative stress and ageing: is ageing a cysteine deficiency syndrome? Relates “In humans, an age-related oxidative shift in the ratio of reduced to oxidized glutathione, i.e. the glutathione redox status, has been demonstrated in whole blood, and peripheral blood mononuclear cells. The mean plasma cysteine/cystine redox status of human subjects shows a significant oxidative shift between the third and the ninth decade of life. This age-related oxidative shift is accompanied by a decrease in the plasma glutathione level and a decrease in the ratio of reduced versus oxidized forms of plasma albumin and other thiol/disulfide redox couples.”

Extracellular Redox State: Refining the Definition of Oxidative Stress in Aging relates: “Plasma GSH/GSSG redox in humans becomes oxidized with age, in response to chemotherapy, as a consequence of cigarette smoking, and in association with common age-related diseases (e.g., type 2 diabetes, cardiovascular disease). However, the GSH/GSSG redox is not equilibrated with the larger plasma cysteine/cystine (Cys/CySS) pool, and the Cys/CySS redox varies with age in a pattern that is distinct from that of GSH/GSSG redox.”

Cysteine’s other roles

While cysteine is the critical functional amino, and least abundant, of the 3 amino acids required for glutathione synthesis, it also plays key roles in other enzymes and proteins, including glutathione peroxidase 1 (GPX1) According to Cysteine-Based Redox Switches in Enzymes Antioxid Redox Signal. 2011 March: “While cysteine residues often play critical roles in enzyme catalysis, they also act as redox switches in many enzymes, allowing for communication between the global or local cellular redox properties and enzymatic function.” Cysteine is also important for forming the proper 3D structure of proteins from the disulfide bonds between cysteine groups.

From Metal and redox modulation of cysteine protein function. Chem Biol. 2003 Aug: “In biological systems, the amino acid cysteine combines catalytic activity with an extensive redox chemistry and unique metal binding properties. The interdependency of these three aspects of the thiol group permits the redox regulation of proteins and metal binding, metal control of redox activity, and ligand control of metal-based enzyme catalysis. Cysteine proteins are therefore able to act as “redox switches,” to sense concentrations of oxidative stressors and unbound zinc ions in the cytosol, to provide a “storage facility” for excess metal ions, to control the activity of metalloproteins, and to take part in important regulatory and signaling pathways.”

The Future?

While there are simple nutritional strategies for maintaining glutathione levels and redox homeostasis which I will discuss in Part 2,  the increasing interest and research in redox biology is already showing promising therapeutic potential for chronic conditions and drug development.

From Site-Specific Antioxidative Therapy for Prevention of Atherosclerosis and Cardiovascular Disease “In addition to the improvement of lifestyle, recently emerging drugs that are effective in treating CVD have a property to eliminate ROS with a site-specific manner without interrupting favorable redox signaling, thereby ameliorating oxidative stress to endothelial cells.”

From Redox-directed cancer therapeutics: molecular mechanisms and opportunities Antioxid Redox Signal. 2009 Dec: “Redox dysregulation originating from metabolic alterations and dependence on mitogenic and survival signaling through reactive oxygen species represents a specific vulnerability of malignant cells that can be selectively targeted by redox chemotherapeutics. The impressive number of ongoing clinical trials that examine therapeutic performance of novel redox drugs in cancer patients demonstrates that redox chemotherapy has made the crucial transition from bench to bedside.”

From Discovery of a statistically significant and interpretable relationship between redox reactivity and lethality of drugs. Curr Drug Metab. 2013 Sep: “A statistically significant and interpretable relationship between electrophilicity as a redox reactivity indicator and LD50 as a lethality indicator of drugs was discovered, and this relationship could be interpreted by the action of the cytochrome P450. The drugs chosen in this study were Topoisomerase II inhibitor anticancer drugs, and the electrophilicity of drugs was obtained by quantum chemical calculation. Since the P450 detoxification mechanism is the catalytic oxidation of drug molecules, it may infer that the drug molecules being easily oxidized (low electrophilicity) will be weak in lethality in general. In addition, this relationship revealed two structural scaffolds for the anthracycline-based topoisomerase II inhibitors, and their lethality mechanisms are not totally the same. Such relationship can assist in designing new drugs that candidates possessing low electrophilicity are recommended for lowering of lethality, and moieties providing a large inductive effect can reduce the electrophilicity of the anthracycline-based topoisomerase II inhibitors.”

Further Reading: Teaching the basics of redox biology to medical and graduate students: Oxidants, antioxidants and disease mechanisms Redox Biol. 2013; 1(1): 244–257. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling Cell Signal. 2012 May; 24(5): 981–990.   

Part 2 is coming soon! – Factors Affecting Redox, GSH Levels and Function