As we know, GSH/GSSH ratio is a key factor to determine oxidative stress level in cell or tissue. But, can I induce oxidative stress by GSSG in cell? If I could, how does GSSG induce oxidative stress?
Dear Chih-Feng,
Please read the following:
1-https://www.google.co.il/?gfe_rd=cr&ei=4luRVtutNoqI8QeGnrXYDw&gws_rd=ssl#
In mammals, the intracellular synthesis of glutathione and its utilization is described by the g-glutamyl cycle, a concept that was put forth almost four decades ago 1. Reduced glutathione represents the centerpiece of the g-glutamyl cycle, involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission. While GSH may form numerous adducts in the human body2, the most abundant GSH derivative is represented by GSSG. Oxidation of intracellular GSH by oxidants such as hydrogen peroxide and organic peroxides generally leads to the formation of GSSG. GSH can be further oxidized to the sulfenic, sulfinic, and sulfonic acid derivatives via successive two-electron oxidations of the thiol group.
GSSG is mostly viewed as a byproduct of GSH generated following reaction of GSH with an oxidizing species. As a result, GSSG/GSH ratio in the tissue emerged as a frequently used biochemical measure of oxidative stress 3. Advances in the concept of redox signaling, and redefining of oxidative stress in that light 4, has led to rethinking of the significance of GSSG in the cell 5. It is now widely acknowledged that changes in the cellular reduced/oxidized glutathione ratio trigger signal transduction mechanisms influencing cell survival. GSSG is capable of causing protein S-glutathionylation or reversible formation of protein mixed disulfides (protein-SSG). Post-translational reversible S-glutathionylation is known to regulate signal transduction as well as activities of several redox sensitive thiol-proteins 6.
Studies with exogenous non-permeable GSSG have demonstrated that extracellular GSSG may trigger apoptosis by a redox-mediated p38 mitogen-activated protein kinase pathway 7. While this addresses the significance of extracellular GSSG, the specific properties of intracellular GSSG remain under veil. GSH is oxidized to GSSG within the cell and pumped to the extracellular compartment 8, 9. Intracellular compartment being the primary site of GSSG generation, the significance of this disulfide within the cell becomes an important issue to address. Studies examining the significance of intracellular GSSG are complicated by the lack of a specific approach that would only elevate intracellular GSSG levels. For example, exposure of cells to pathogen related chemicals or to direct oxidant insult does elevate cellular GSSG but activates numerous other aspects of cell signaling 10. While the study of GSSG driven reactions in a cell-free system is relatively straightforward in approach, the in vivo significance of such findings remains questionable 11. This work presents first evidence from the use of a microinjection approach to study the significance of GSSG within the cell. Previously, we have utilized this approach to differentially study the cytosolic and nuclear compartments of HT4 cells as well as of primary cortical neurons 12. The approach is powerful in instantly and selectively introducing agents into specific compartments of the cell. Results of this study provide the first evidence demonstrating that the specific elevation of GSSG within the cell may cause cell death. Our observation demonstrating loss of mitochondrial membrane potential without affecting cell membrane integrity argues in favor of an apoptotic fate. Disorders of the central nervous system are frequently associated with concomitant glutathione depletion and oxidation 13, 14. Our observation that cells with compromised GSH levels are substantially more sensitive to GSSG-induced death leads to the notion that GSSG may play a role in cell death under conditions of disease and aging. We note that in GSH-sufficient cells (experimental) 0.5 mM GSSG is lethal. In GSH-deficient cells, a condition that mimics oxidative stress situation, the threshold of lethality sharply goes down by 20 fold to 0.025 mM GSSG. Our estimates show that in HT4 neural cells, total GSH content is in the range of 13 mM (not shown). This is consistent with the literature reporting that under basal conditions, cellular GSH levels is in the tune of 10 mM15, 16. In response to oxidant insult, GSSG levels sharply go up and may represent up to 50% of the total GSH in the cell 17, 18. Oxidative stress depletes cellular reducing equivalents such NADPH 19 compromising GSSG reductase function.
Glutamate toxicity is a major contributor to pathological cell death within the nervous system and is known to be mediated by reactive oxygen species and GSH loss 20, 21. Glutamate-induced death of neural cells is known to be associated with GSH loss and oxidation 22, 23. Our previous studies have identified 12-lipoxygenase as a key mediator of glutamate-induced neural cell death 24. We and others have reported that 12-lipoxygenase deficient mice are protected against stroke dependent injury to the brain 24, 25. GSH depletion causes neural degeneration by activating the 12-Lox pathway26, 27. Observations in this study suggest a direct influence of GSSG on 12-lipoxygenase activation. Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by 12-lipoxygenase. Previously it has been demonstrated that the presence of 0.5-1.5 mM GSH in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-lipoxygenase pathway. It was therefore concluded that 12-HPETE peroxidase in the 12-lipoxygenase pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymatic breakdown of the primary 12-lipoxygenase product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-HETE 28. In a cell system, disulfides are known to be able to act as biological oxidants that oxidize the zinc-thiolate clusters in metallothionein with concomitant zinc release 29. Intracellular zinc release is known to cause 12-lipoxygenase activation and neurotoxicity 30. Studies with BSO-treated GSH-deficient cells highlighted the significance of microinjected free arachidonic acid in neurotoxicity. These observations are consistent with the literature reporting a central role of the free arachidonic acid mobilizing enzyme phospholipase A2 in neurotoxicity 31.
S-Glutathionylated proteins (PSSG) can result from thiol/disulfide exchange between protein thiols (PSH) and GSSG 32. Protein S-glutathionylation, the reversible binding of glutathione to low-pKa cysteinyl residues in PSH, is involved in the redox regulation of protein function. Several enzymes are known to undergo this post translational modification. Importantly, whether glutathionylation inhibits or augments protein function may vary depending on the individual case. For example, glutathionylation inhibits phosphofructokinase 33, NFkB 34, glyceraldehydes-3-phosphate dehydrogenase 35, protein kinase C-α 36, creatine kinase 4, as well as actin 37. In contrast, glutathionylation-dependent gain of protein function has been reported for microsomal glutathione S-transferase 38, HIV-1 protease-Cys67 39, and matrix metalloproteinases 40. Furthermore, specific electron transport proteins of the mitochondria are sensitive to S-glutathionylation 19, 41. Consistently, we noted that GSSG induced cell death was associated with loss of mitochondrial membrane potential. The presence of multiple cysteine residues in 12-lipoxygenase makes it susceptible to S-glutathionylation. Results of this study provide first evidence demonstrating that 12-lipoxygenase may be glutathionylated in glutamate challenged neural cells. The finding that both glutaredoxin-1 expression as well as delivery may protect cells against glutamate-induced neurotoxicity suggests that glutamate-induced glutathionylation is implicated in the cell death pathway. GSSG is a functionally active byproduct of GSH metabolism that, under appropriate conditions, may trigger cell death. S-glutathionylation seems to be a mechanism that favors 12-lipoxygenase but not the classical caspase-3 dependent 42 death pathway.
The GSSG reductase inhibitor BCNU, clinically known as carmustine, is a proven chemotherapeutic agent 43. Cytotoxicity is a widely recognized side-effect of BCNU 44. Because BCNU treatment is associated with GSSG accumulation in cells 45, the significance of GSSG in BCNU-induced cytotoxicity is of interest. Observations of this study support that both GSSG as well as BCNU-induced cell death follow the same 12-lipoxygenase dependent path suggesting the possibility that BCNU may cause cell death via GSSG. Affirmation of this hypothesis would warrant examining the significance of pro-GSSG strategies for cancer therapy. Major forms of cancer therapy including radiation therapy as well as chemotherapy rely on oxygen-centered free radicals for their action. Thus, these interventions cause overt oxidative insult associated with elevated levels of cellular and tissue GSSG 46, 47. GSSG generated within the cell is pumped out of the cell perhaps to avert cytotoxicity caused by GSSG accumulation. Approaches to selectively block the GSSG efflux mechanisms in cancer cells might be useful for cancer therapy. Findings of this study support that BSO sensitizes cells to GSSG-induced death. Indeed, BSO has been founds to be an useful adjunct for both radiation 48 as well as chemotherapy 49. Also, inhibition of GSSG efflux by inhibition of MRP1 enhanced BCNU-induced cytoxicity suggesting that GSSG extrusion play a role in neural sensitivity to GSSG.
Three-fourth of all stroke in humans occur in distributions of the middle cerebral artery 50. Therefore, MCAO represents a common approach to study stroke in small as well as large animals 12. Using a MCAO approach we were able to obtain just over 16% infarction of the ipsilateral hemisphere as assessed by MRI. Stroke caused 8-fold increased in GSSG levels in the affected brain tissue. This is consistent with the known incidence of oxidative stress in the stroke affected brain 51. Our observation that the stereotaxic injection of GSSG to the brain may cause lesion by a 12-lipoxygenase sensitive mechanism leads to question the significance of GSSG in numerous brain pathologies commonly associated with elevated levels of GSSG in the brain 52, 53.
Taken together, this work presents first evidence demonstrating that intracellular GSSG may trigger cell death. GSSG cytotoxicity is substantially enhanced under conditions of compromised cellular GSH levels as observed during a wide variety of disease conditions as well as aging 53, 54. BSO-assisted glutathione lowering approaches are known to be effective to facilitate both chemo- as well as radiation- therapies. Furthermore, BCNU-dependent arrest of GSSG reductase activity leads to elevation of cellular GSSG and has chemotherapeutic functions. Findings of this study lead to question the significance of GSSG in such processes. From the standpoint of novel therapeutic approaches, strategies directed at improving or arresting cellular GSSG clearance may be effective in minimizing oxidative stress related tissue injury or potentiating the killing of tumor cells, respectively.
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2- Toxicol In Vitro. 2014 Aug;28(5):1006-15. doi: 10.1016/j.tiv.2014.04.017. Epub 2014 May 6.
GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress. (see attached file).
Sentellas S1, Morales-Ibanez O2, Zanuy M2, Albertí JJ2.
Author information
Abstract
The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC-MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.
http://www.ncbi.nlm.nih.gov/pubmed/24809893
3-Scand J Med Sci Sports 2009 & 2009 John Wiley & Sons A/S
doi: 10.1111/j.1600-0838.2009.00987.x
Plasma antioxidant responses and oxidative stress following a soccer
game in elite female players
H. Andersson1 , A. Karlsen2 , R. Blomhoff2 , T. Raastad3 , F. Kadi1 1
School of Health and Medical Sciences, O¨rebro University, O¨rebro, Sweden, 2
Department of Nutrition, Institute of Basic Medical
Sciences, University of Oslo, Oslo, Norway, 3
Norwegian School of Sport Sciences, Oslo, Norway
Corresponding author: Fawzi Kadi, School of Health and Medical Sciences, O¨rebro University, 701 82 O¨rebro, Sweden. Tel:
146 1930 1160, Fax: 146 1930 3486, E-mail: [email protected]
Accepted for publication 18 May 2009
We aimed to investigate markers of oxidative stress and levels of endogenous and dietary antioxidants in 16 elite female soccer players in response to a 90-min game (average intensity 82 3% HRpeak). Blood samples were taken before, immediately and 21 h after the game. Plasmaoxidized glutathione, the ratio of reduced to oxidized glutathione (GSH:GSSG) and lipid peroxidation measured by d-ROMs were used as markers of oxidative stress. Plasma endogenous [uric acid, total glutathione (TGSH)] and dietary antioxidants (a-tocopherol, ascorbic acid, total
carotenoids and polyphenols) were analyzed using liquid chromatography and the Folin–Ciocalteu method. Exercise induced an acute increase (Po0.05) in GSSG, uric acid, TGSH, a-tocopherol, and ascorbic acid. In parallel, the GSH:GSSG ratio and polyphenols decreased (Po0.05). GSSG, GSH:GSSG ratio, uric acid, TGSH, and ascorbic acid returned to baseline at 21 h, while polyphenols and a-tocopherol remained altered. Total carotenoids increased above baseline only at 21 h (Po0.05). Lipid peroxidation, measured by d-ROMs, remained unchanged throughout the study. Thus, intermittent exercise in well-trained female
athletes induces a transient increase in GSSG and a decrease in the GSH:GSSG ratio, which is effectively balanced by the recruitment of both endogenous and dietary antioxidants, resulting in the absence of lipid peroxidation measured by d-ROMs.
Hoping this will be helpful,
Rafik
yes
The objectives of this study were to determine whether differences in the size and composition of coarse (2.5-10 micro m), fine (< 2.5 microm), and ultrafine (< 0.1 microm) particulate matter (PM) are related to their uptake in macrophages and epithelial cells and their ability to induce oxidative stress. The premise for this study is the increasing awareness that various PM components induce pulmonary inflammation through the generation of oxidative stress. Coarse, fine, and ultrafine particles (UFPs) were collected by ambient particle concentrators in the Los Angeles basin in California and used to study their chemical composition in parallel with assays for generation of reactive oxygen species (ROS) and ability to induce oxidative stress in macrophages and epithelial cells. UFPs were most potent toward inducing cellular heme oxygenase-1 (HO-1) expression and depleting intracellular glutathione. HO-1 expression, a sensitive marker for oxidative stress, is directly correlated with the high organic carbon and polycyclic aromatic hydrocarbon (PAH) content of UFPs. The dithiothreitol (DTT) assay, a quantitative measure of in vitro ROS formation, was correlated with PAH content and HO-1 expression. UFPs also had the highest ROS activity in the DTT assay. Because the small size of UFPs allows better tissue penetration, we used electron microscopy to study subcellular localization. UFPs and, to a lesser extent, fine particles, localize in mitochondria, where they induce major structural damage. This may contribute to oxidative stress. Our studies demonstrate that the increased biological potency of UFPs is related to the content of redox cycling organic chemicals and their ability to damage mitochondria.
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http://www.annualreviews.org/doi/abs/10.1146/annurev.arplant.55.031903.141701
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1241427/
Thanks for you guys answer my question. It is useful to me. Thanks a lot.
Dear Dr. Chih-Fengh Lien,
Although I do not want to discourage colleagues from writing extensive comments on research gate questions, I have to make some critical comments on the answer you received from Rafik Karaman. In particular, the first article added to his mail contains some misleading or wrong statements.
1. As is evident from the title of ref.1, Alton Meister introduced the gamma-Glu cycle as an amino acid transport system. This hypothesis turned out to be wrong, and I hate to see my old friend Alton Meister always quoted for one of the very few mistakes he made.
2. It is not “widely acknowledged that changes in the cellular reduced/oxidized glutathione ratio trigger signal transduction mechanisms influencing cell survival.” Ratios do not regulate anything. Glutathione-dependent redox regulation is achieved by bimolecular reactions of reduced glutathione with target proteins, less likely of GSSG with target proteins. In fact, the only proteins with reasonable affinity to GSSG I am aware of are glutathione reductase glutaredoxins and GSSG transporters.
3. The sentence:” GSSG is capable of causing protein S-glutathionylation or reversible formation of protein mixed disulfides (protein-SSG)” is correct in principle. However, it describes the least likely mechanism of mixed disulfide formation, because the GSSG levels are usually too low. There is increasing evidence that the preferred route is sulfenic acid formation in susceptible proteins followed by reaction with GSH.
4. It has been known for almost half a century that lipoxygenases in general are inhibited by GSH plus any kind of GSH peroxidase. The generally accepted interpretation of this phenomenon is that their catalytic iron has to be oxidized for activity, which is prevented by depletion of either GSH or glutathione peroxidase. There is no need to involve GSSG as an inhibitor of lipoxygenases and I am not aware of any experimental evidence supporting this concept.
5. In vitro experiments with exogenous GSSG are not likely very relevant. The extracellular concentrations of GSSG are in the micromolar range, it is degrade with a half life time around one minute and am not aware of any experiment demonstrating (patho)physiological consequences of GSSG variation within the physiological range.
There are some reasonable reviews on these topics available (written by me and by others), e. g. Brigelius-Flohé & Flohé, ARS 2011; Forman, FRBM 2997; Janssen-Heininger et al, FRBM 2008; Forman et al, Biochemistry 2010, Flohé, Free Rad Res 2016). Enjoy reading!
Best regards
Leopold Flohé
GSSG is the effect and not the cause of oxidative stress.
I think you can't induce oxidative stress by GSSG in cell.
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