Differential effects of the glycolysis inhibitor 2-deoxy-D-glucose on the activity of pro-apoptotic agents in metastatic melanoma cells, and induction of a cytoprotective autophagic response.

2-Deoxy-D-glucose (2DG) is a synthetic glucose analogue that inhibits glycolysis and blocks cancer cell growth. In this report, we evaluated the role of 2DG in the induction of cell death in human metastatic melanoma cells. We have also examined the effects of 2DG in combined treatments with four different pro-apoptotic agents: (i) Temozolomide (TMZ), a chemotherapic drug commonly used to treat metastatic melanoma, (ii) Pyrimethamine (Pyr), a pro-apoptotic antifolate drug recently reappraised in cancer therapy, (iii) Cisplatin (CisPt), a drug capable of directly binding to DNA ultimately triggering apoptosis of cancer cells and (iv) the kinase inhibitor Staurosporine (STS), a prototypical inducer of mitochondria-mediated apoptosis. We found that 2DG per se: (i) induced a cell cycle arrest in G(0) /G(1) , (ii) promoted autophagy, (iii) was ineffective in inducing apoptosis in association with the chemotherapic drug TMZ, whereas (iv) it was synergistic with CisPt and STS pro-apoptotic drugs through a mechanism involving changes of mitochondrial homeostasis. Conversely, (v) 2DG hindered the pro-apoptotic effects of Pyr via a mechanism involving either the block of cell cycle in G(0) /G(1) or the modification of the free radical production of the cell, i.e., decreasing the production of reactive oxygen species (ROS) and increasing the production of reactive nitrogen species (RNS). Moreover, a clear-cut autophagic response involving endoplasmic reticulum remodelling was detectable. Since autophagic cytoprotection has been suggested to contribute to the induction of chemoresistance, these results could provide useful clues as concerns the use of 2DG as anticancer agent in combinatory protocols.

Peroxynitrite signaling in human erythrocytes: synergistic role of hemoglobin oxidation and band 3 tyrosine phosphorylation.

Peroxynitrite crosses the red blood cell (RBC) membrane and reacts with hemoglobin (Hb) producing mainly metHb, which is reduced back to ferrousHb by NADH- and NADPH-dependent reductases. Peroxynitrite also induces band 3 (B3) tyrosine phosphorylation, a signaling pathway believed to activate glucose metabolism. This study was aimed to decipher the relationship between these two peroxynitrite-dependent processes. Peroxynitrite induced a burst of the hexose monophosphate shunt (HMS), revealed by NMR studies, and a burst of the glycolytic pathway, measured by lactate production. The HMS plays a prominent role in membrane signaling, as demonstrated by B3 phosphotyrosine inhibition by the glycolytic pathway inhibitor 2-deoxy-glucose (2DG) and activation by dehydroepiandrosterone (DHEA), an inhibitor of HMS. Peroxynitrite-induced B3 tyrosine phosphorylation was paralleled by the inhibition of membrane-associated phosphotyrosine phosphatase (PTP) activity, which was protected by 2DG but not DHEA. Interestingly, heme poisoning with CO inhibited peroxynitrite-dependent Hb oxidation and lactate production but did not affect PTP down regulation. These results suggest two distinct and concurrent effects of peroxynitrite: one mediated by Hb which, likely in its oxidized state, binds more strongly to B3, and another mediated by PTP-dependent B3 phosphorylation. Both effects are directed towards a surge in glucose utilization.

Overexpression of human NOX1 complex induces genome instability in mammalian cells.

The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because of their potential to damage macromolecules, including DNA. To investigate possible links between high ROS levels, oxidative DNA damage, and genomic instability in mammalian cells, we established a novel model of chronic oxidative stress by coexpressing the NADPH oxidase human (h) NOX1 gene together with its cofactors NOXO1 and NOXA1. Transfectants of mismatch repair (MMR)-proficient HeLa cells or MMR-defective Msh2(-/-) mouse embryo fibroblasts overexpressing the hNOX1 complex displayed increased intracellular ROS levels. In one HeLa clone in which ROS were particularly elevated, reactive nitrogen species were also increased and nitrated proteins were identified with an anti-3-nitrotyrosine antibody. Overexpression of the hNOX1 complex increased the steady-state levels of DNA 8-oxo-7,8-dihydroguanine and caused a threefold increase in the HPRT mutation rate in HeLa cells. In contrast, additional oxidatively generated damage did not affect the constitutive mutator phenotype of the Msh2(-/-) fibroblasts. Because no significant changes in the expression of several DNA repair enzymes for oxidative DNA damage were identified, we suggest that chronic oxidative stress can saturate the cell's DNA repair capacity and cause significant genomic instability.

Radical generation and alterations of erythrocyte integrity as bioindicators of diagnostic or prognostic value in COPD?

Chronic obstructive pulmonary disease (COPD) has recently been viewed as an inflammation-dependent systemic disease. Oxidative modifications in the pulmonary microenvironment can result in a number of functional changes in pulmonary tissue as well as in the blood. Studies have been carried out to detect whether oxidatively modified molecules or cells could be considered possible markers of the disease. We hypothesize here that new insights into COPD could come from enzymes involved in deliberate radical generation (i.e., Nox and NOS family enzymes) as well as from alterations of erythrocyte integrity and function, which could become bioindicators of diagnostic or prognostic value in the near future.

Red blood cells as a model to differentiate between direct and indirect oxidation pathways of peroxynitrite.

Red blood cells are the major physiological scavengers of reactive nitrogen species and have been proposed as real-time biomarkers of some vascular-related diseases. This chapter proposes that the erythrocyte is a suitable cell model for studying the modifications induced by peroxynitrite. Peroxynitrite decays both extra- and intracellularly as a function of cell density and CO(2) concentration, inducing the appearance of distinct cellular biomarkers, as well as the modulation of signaling and metabolism. Intracellular oxidations are due mostly to direct reactions of peroxynitrite with hemoglobin but also lead to the appearance of apoptotic biomarkers. Surface/membrane oxidations are due principally to indirect radical reactions generated by CO(2)-catalyzed peroxynitrite homolysis.

The microenvironment can shift erythrocytes from a friendly to a harmful behavior: pathogenetic implications for vascular diseases.

Erythrocytes are peculiar cells aimed at the delivery of oxygen and nitric oxide to the periphery and carbon dioxide to the lungs. In addition, they also exert, under physiological conditions, a scavenging activity towards reactive oxygen and nitrogen species often over-produced in morbidity states, e.g. in inflamed tissues. Their deformability is essential for their circulation, specifically in small blood vessels, and this is an important pre-requisite for such vascular "antioxidant" functions. On the other hand, if the erythrocyte undergoes changes in its redox status, i.e. is not capable of counteracting the pro-oxidant status of the microenvironment, it becomes a source of reactive species and, consequently, its typical structural and functional features are lost. More importantly, the oxidatively modified red cell increases its aggregability and adhesiveness to the endothelium and to other blood cells, thus contributing to vascular damage. In line with recent data from the literature, erythrocytes can be proposed as bioindicators of progression in chronic or acute diseases characterized, as a hallmark, by oxidative alterations.

Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes.

CO(2) changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO(3)(-) / ()NO(2) radical pair, and (ii) peroxynitrite diffusion distance is significantly reduced. For peroxynitrite generated extracellularly this last effect is particularly dramatic at low cell density because CO(3)(-) and ()NO(2) are short-lived and decay mostly in the extracellular space or at the cell surface/membrane. This study was aimed to distinguish between peroxynitrite-induced extra- and intracellular modifications of red blood cells (RBC). Our results show that at low cell density and in the presence of CO(2) peroxynitrite induced the oxidation of surface thiols, the formation of 3-nitrotyrosine and DMPO-RBC adducts, and the down-regulation of glycophorins A and C (biomarkers of senescence). Reactivation of glycolysis reversed only the oxidation of surface thiols. Without CO(2) peroxynitrite also induced the oxidation of hemoglobin and glutathione, the accumulation of lactate, a decrease in ATP, the clustering of band 3, the externalization of phosphatidylserine, and the activation of caspases 8 and 3 (biomarkers of apoptosis). The latter biomarkers were all reversed by reactivation of glycolysis. We hypothesize that cell senescence could (generally) be derived by irreversible radical-mediated oxidation of membrane targets, while the appearance of apoptotic biomarkers could be bolstered by oxidation of intracellular targets. These results suggest that, depending on extracellular homolysis or diffusion to the intracellular space, peroxynitrite prompts RBCs toward either senescence or apoptosis through different oxidation mechanisms.

Free hemoglobin: a dangerous signal for the immune system in patients with carotid atherosclerosis?

Atherosclerosis is a chronic inflammatory multifactorial disease in which immune responses are key pathogenetic factors. T cell-mediated immunity contributes to the initiation and progression of atherosclerotic disease, but the nature of antigens responsible for immune cell activation is still not completely elucidated. Convincing evidence supports a determinant role of autoimmune responses to self-structures in shaping the progression of the disease. Autoimmune responses may be directed against altered self-structures, such as oxidized low-density lipoproteins (LDL). Oxidative stress, increasingly reported in patients with atherosclerosis, is the major event causing protein structural modification, thus inducing the appearance of neo/cryptic epitopes on the molecule. Intraplaque hemorrhage, a common event in advanced lesions, causes the deposition of large amounts of hemoglobin (Hb). The pro-oxidative intraplaque microenvironment may induce structural changes in extra-erythrocytic free Hb, thus generating novel/cryptic autoantigenic epitopes. We demonstrated that an oxidized Hb preparation enriched in hemichromes expands IFN-gamma-secreting T lymphocytes in patients with advanced carotid atherosclerosis and enhances the phenotypical and functional maturation of human monocyte-derived dendritic cells induced by lipopolysaccharide (LPS). Overall, our findings suggest that oxidized forms of Hb could act as a dangerous signal for the immune system, thus contributing to the inflammatory process that takes place within the atherosclerotic plaque.

Salivary uric acid at the acidic pH of the stomach is the principal defense against nitrite-derived reactive species: sparing effects of chlorogenic acid and serum albumin.

A complex antioxidant system is present in human saliva, with uric acid being the most concentrated component. Ascorbic acid, present at low concentrations in saliva, is actively secreted into the gastric lumen. We report that ascorbic acid added to human saliva at pH 2 was consumed within a few minutes, regenerating HNO(2), whereas uric acid was consumed relatively slowly in a nitrite-dependent manner. The consumption of uric acid was (i) rapid under normoxic conditions and slower at low oxygen tensions, (ii) coupled to *NO release, (iii) linked to the decrease in nitrite consumption and in nitrate formation, and (iv) unaffected by the nitrosation catalyst thiocyanate. Both chlorogenic acid and bovine serum albumin, representative of a phenol- and a protein-rich meal, respectively, were able to spare uric acid, although chlorogenic acid increased, whereas bovine serum albumin inhibited, *NO release. We hypothesize that the major role of uric acid in saliva at pH 2 could be to preserve the stomach from the formation of toxic nitrogen species and that low levels of uric acid, together with ascorbic acid consumption, may contribute to the high occurrence of tumors at the gastroesophageal junction and cardia. The sparing effects of dietary compounds may therefore be an important not fully appreciated effect.

Redox control of red blood cell biology: the red blood cell as a target and source of prooxidant species.

Red blood cells may exert both an antioxidant and a prooxidant activity. The first is exerted in physiologic conditions, whereas the second can be detected in several human pathologies. These opposite characteristics can depend on the environmental milieu as well as on intrinsic alterations. Both these aspects are summarized in this brief review that takes into account the possible implications of redox-associated alterations of red blood cells in determining their function and fate.

Peroxynitrite induces senescence and apoptosis of red blood cells through the activation of aspartyl and cysteinyl proteases.

Changes in the oxidative status of erythrocytes can reduce cell lifetime, oxygen transport, and delivery capacity to peripheral tissues and have been associated with a plethora of human diseases. Among reactive oxygen and nitrogen species of importance in red blood cell (RBC) homeostasis, superoxide and nitric oxide radicals play a key role. In the present work, we evaluated subcellular effects induced by peroxynitrite, the product of the fast reaction between superoxide and nitric oxide. Peroxynitrite induced 1) oxidation of oxyhemoglobin to methemoglobin, 2) cytoskeleton rearrangement, 3) ultrastructural alterations, and 4) altered expression of band-3 and decreased expression of glycophorin A. With respect to control cells, this occurred in a significantly higher percentage of human RBC (approximately 40%). The presence of antioxidants inhibited these modifications. Furthermore, besides these senescence-associated changes, other important modifications, absent in control RBC and usually associated with apoptotic cell death, were detected in a small but significant subset of peroxynitrite-exposed RBC (approximately 7%). Active protease cathepsin E and mu-calpain increased; activation of caspase 2 and caspase 3 was detected; and phosphatidylserine externalization, an early marker of apoptosis, was observed. Conversely, inhibition of cathepsin E, mu-calpain, as well as caspase 2 and 3 by specific inhibitors resulted in a significant impairment of erythrocyte "apoptosis" Altogether, these results indicate that peroxynitrite, a milestone of redox-mediated damage in human pathology, can hijack human RBC toward senescence and apoptosis by a mechanism involving both cysteinyl and aspartyl proteases.

HIV-1 Nef regulates the release of superoxide anions from human macrophages.

The NADPH oxidase enzymatic complex participates in the oxidative burst by producing ROS (reactive oxygen species). Altered levels of ROS production may have pathogenetic implications due to the loss of some innate immune functions such as oxidative burst and phagocytosis. Considering that HIV-1 Nef protein plays a primary role in AIDS pathogenesis, by affecting the immune system, we sought to dissect possible effects of Nef on the release of superoxide anions. We show here that the inducible expression of Nef in human phagocytic cells modulates the superoxide release in a biphasic manner. In particular, an early Nef-induced increase of the superoxide release was followed by a dramatic decrease starting from 10 h after the Nef induction. This was observed whatever the presence of cell activators such as GM-CSF (granulocyte/macrophage colony-stimulating factor) or fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine). Whereas the early increase in superoxide release is probably the result of the already described Nef-dependent activation of PAK-2 (p21-activated kinase 2)-Rac2, we were interested in investigating the mechanisms underlying the late inhibition of superoxide release observed originally. In this regard, we individuated at least three independent requirements for the Nef-induced blockade of superoxide release: (i) the active protein synthesis; (ii) both the membrane localization and the interaction with endocytotic machinery of Nef; and (iii) the release of soluble factor(s). Moreover, we observed that IL-10 (interleukin-10) inhibits superoxide release, whereas its depletion restored NADPH oxidase activity. We propose that the cell membrane-to-lysosome Nef transit leads to the synthesis and release of soluble factor(s) and, among them, IL-10 might significantly contribute to the inhibition of NAPDH oxidase activity.

On-line EPR study of free radicals induced by peroxidase/H(2)O(2) in human low-density lipoprotein.

The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 degrees C and spin trapping techniques to study radical species formed during horseradish peroxidase/H(2)O(2)-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the alpha-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with beta-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1-10 microM RSV is able to accelerate alpha-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.

Apples increase nitric oxide production by human saliva at the acidic pH of the stomach: a new biological function for polyphenols with a catechol group?

Dietary inorganic nitrate is secreted in saliva and reduced to nitrite by bacterial flora. At the acidic pH of the stomach nitrite is present as nitrous acid in equilibrium with nitric oxide (*NO), and other nitrogen oxides with nitrating and nitrosating activity. *NO in the stomach exerts several beneficial effects, but nitrosating/nitrating species have been implicated as a possible cause of epithelial neoplasia at the gastroesophageal junction. We investigated the effects of apple extracts on *NO release by human saliva at pH 2. A water extract obtained from apple homogenate increased *NO release caused by acidification of saliva. Data show that polyphenols were responsible for this activity, with chlorogenic acid and (+)-catechin the most active and concentrated species. However, ferulic acid, a hydroxycinnamic acid with only one aromatic hydroxyl group, did not increase *NO release. Fructose, the most representative sugar in apples, was also inactive. Interestingly, ascorbic acid in saliva induced a SCN(-)-enhanced burst of *NO but, unlike apple, the release was transient. The simultaneous addition of ascorbic acid and apple extract caused a burst of *NO followed by the increased steady-state level characteristic of saliva containing apple extract. Chlorogenic acid and (+)-catechin, but not ferulic acid, formed o-semiquinone radicals and nitrated polyphenols, suggesting the scavenging of *NO(2) by o-semiquinones. Our results propose that some apple polyphenols not only inhibit nitrosation/nitration but also promote *NO bio-availabilty at the gastric level, a previously unappreciated function.

Peroxynitrite-dependent upregulation of SRC kinases in red blood cells: strategies to study the activation mechanisms.

Several studies have demonstrated that treatment of cells with oxidants, and in particular with peroxynitrite, may cause the upregulation of tyrosine phosphorylation signaling. In erythrocytes, peroxynitrite induces tyrosine phosphorylation of the major intrinsic membrane protein, band 3. A closer look at the enzymes involved revealed that the effect of peroxynitrite was due to the inhibition of phosphotyrosine phosphatases and/or to the activation of src kinases. The activity of src kinases is modulated not only by phosphatases and other kinases but also through redox modification of cysteine residues: Peroxynitrite can, thus, affect src kinase activity by means of direct and indirect mechanisms. In this chapter, we describe the different pathways leading to src kinase activation and the experimental procedures that can be performed to reveal the activation mechanism. The aim is to provide a more general strategy adaptable to different cell types and different oxidants.

Scavenging of reactive nitrogen species by oxygenated hemoglobin: globin radicals and nitrotyrosines distinguish nitrite from nitric oxide reaction.

The reaction of *NO and NO2- with hemoglobin (Hb) is of pivotal importance to blood vessel function. Both species show at least two different reactions with Fe2+ Hb: one with deoxygenated Hb, in which the biological properties of *NO are preserved, and another with oxygenated hemoglobin (oxyHb), in which both species are oxidizes to NO3-. In this study we compared the oxidative reactions of *NO and NO2- and, in particular, the radical intermediates formed during transformation to NO3-. The reaction of NO2- with oxyHb was accelerated at high heme concentrations and produced stoichiometric amounts of NO3-. Direct EPR and spin trapping studies showed that NO2-, but not *NO, induced the formation of globin Tyr-, Trp-, and Cys-centered radicals. MS studies provided evidence of the formation of approximately 2% nitrotyrosine in both the alpha and beta subunits, suggesting that *NO2 diffuses in part away from the heme and reacts with Tyr radicals. No nitrotyrosines were detected in the reaction of *NO with oxyHb. Collectively, these results indicate that NO2- reaction with oxyHb causes an oxidative challenge not observed with *NO. The differences in oxidation mechanisms of *NO and NO2- are discussed.

Peroxynitrite activates kinases of the src family and upregulates tyrosine phosphorylation signaling.

The hypothesis that peroxynitrite may act as a signaling molecule able to upregulate protein tyrosine phosphorylation is discussed. This article focuses on the mechanisms for activating kinases of the src family, an important class of nonreceptor tyrosine kinases implicated in the regulation of cell communication, proliferation, migration, differentiation, and survival. Recent in vitro findings show that in erythrocytes, synaptosomes, and cerebellar primary culture cells peroxynitrite is able to inhibit phosphatases and to activate different members of the src kinase family through different mechanisms involving cysteine-dependent and -independent processes. The ability of nitrotyrosine-containing peptides with SH2 binding affinity to activate src kinases is also discussed.

Reaction of peroxynitrite with hyaluronan and related saccharides.

The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although 1H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.

Phenolic antioxidants and the protection of low density lipoprotein from peroxynitrite-mediated oxidations at physiologic CO2.

Dietary phenolic antioxidants have been shown to prevent LDL modifications mediated by several physiologic oxidants including peroxynitrite. However, more recent data demonstrated that CO(2) affected the fate of peroxynitrite in biological fluids and significantly reduced peroxynitrite scavenging by polyphenols, raising doubts concerning their antioxidant activity. We found that the oxidation of LDL lipids mediated by peroxynitrite decreased in the presence of bicarbonate, while Trp oxidation and 3-nitroTyr formation increased, suggesting a redirection of peroxynitrite reactivity toward the protein moiety. We therefore evaluated the protective activity of some phenolic antioxidants (quercetin, oleuropein, resveratrol, (+)-catechin, (-)-epicatechin, tyrosol, alpha- and gamma-tocopherol, ascorbate) on peroxynitrite-mediated oxidation of LDL aromatic residues. Some of these phenols protected LDL Trp from oxidation better than ascorbate or alpha-tocopherol, although protection at 100 microM did not exceed 30-40%. However, the same phenolic antioxidants were more active in inhibiting 3-nitroTyr formation and those with a catechin structure provided significant protection (IC(50%) 40-50 microM). Red wine, a polyphenol-rich beverage, showed a protective effect comparable to that of the most active phenolic antioxidants. Direct EPR studies showed that bicarbonate significantly increased the peroxynitrite-dependent formation of O-semiquinone radicals in red wine, supporting the hypothesis that polyphenols are efficient scavengers of radicals formed by peroxynitrite/CO(2). Ascorbate was a poor inhibitor of peroxynitrite/CO(2)-induced LDL tyrosine nitration, but the simultaneous addition to the most active polyphenols halved their IC(50%). In conclusion, although cooperation with other antioxidants can further decrease the IC(50%) of polyphenolics, as demonstrated for ascorbate, their antioxidant activity appears to occur at concentrations at least 1 order of magnitude higher than their bioavailability.

Carbon monoxide signaling in human red blood cells: evidence for pentose phosphate pathway activation and protein deglutathionylation.

AIMS: The biochemistry underlying the physiological, adaptive, and toxic effects of carbon monoxide (CO) is linked to its affinity for reduced transition metals. We investigated CO signaling in the vasculature, where hemoglobin (Hb), the CO most important metal-containing carrier is highly concentrated inside red blood cells (RBCs). RESULTS: By combining NMR, MS, and spectrophotometric techniques, we found that CO treatment of whole blood increases the concentration of reduced glutathione (GSH) in RBC cytosol, which is linked to a significant Hb deglutathionylation. In addition, this process (i) does not activate glycolytic metabolism, (ii) boosts the pentose phosphate pathway (PPP), (iii) increases glutathione reductase activity, and (iv) decreases oxidized glutathione concentration. Moreover, GSH concentration was partially decreased in the presence of 2-deoxyglucose and the PPP antagonist dehydroepiandrosterone. Our MS results show for the first time that, besides Cys93, Hb glutathionylation occurs also at Cys112 of the ß-chain, providing a new potential GSH source hitherto unknown. INNOVATION: This work provides new insights on the signaling and antioxidant-boosting properties of CO in human blood, identifying Hb as a major source of GSH release and the PPP as a metabolic mechanism supporting Hb deglutathionylation. CONCLUSIONS: CO-dependent GSH increase is a new RBC process linking a redox-inactive molecule, CO, to GSH redox signaling. This mechanism may be involved in the adaptive responses aimed to counteract stress conditions in mammalian tissues.