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 β-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.

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 G0/G1, (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 G0/G1 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 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.

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.

Clenbuterol (CLB) is an antiasthmatic drug used also illegally as a lean muscle mass enhancer in both humans and animals. CLB and amine-related drugs in general are nitrosatable, thus raising concerns regarding possible genotoxic/carcinogenic activity. Oral administration of CLB raises the issue of its possible transformation by salivary nitrite at the acidic pH of gastric juice. In acidic human saliva CLB was rapidly transformed to the CLB arenediazonium ion. This suggests a reaction of CLB with salivary nitrite, as confirmed in aerobic HNO(2) solution by a drastic decrease in nitric oxide, nitrite, and nitrate. In human saliva, both glutathione and ascorbic acid were able to inhibit CLB arenediazonium formation and to react with preformed CLB arenediazonium. The effect of ascorbic acid is particularly pertinent because this vitamin is actively concentrated within the gastric juice. EPR spin trapping experiments showed that preformed CLB arenediazonium ion was reduced to the aryl radical by ascorbic acid, glutathione, and serum albumin, the major protein of saliva. As demonstrated by anti-CLB antibodies and MS, the CLB-albumin interaction leads to the formation of a covalent drug-protein adduct, with a preference for Tyr-rich regions. This study highlights the possible hazards associated with the use/abuse of this drug.

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.

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.

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.

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.

CO2 changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO3– / NO2 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 CO3– and NO2 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 CO2 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 CO2 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.

The epidemiology of clenbuterol food-borne intoxication outbreaks indicates a possible discrepancy between the severity and long duration of clinical symptoms, and the presumed dose ingested as parent compound residue. In this work, we explore the possibility that clenbuterol could undergo to a biological transformation, in presence of salivary nitrites, at gastric pH (< 3). Human salivary specimens were drawn before and after meal, accounting for the different physiological nitrite content (40 and 400 micromol L(-1), respectively, as average). Clenbuterol (10 micromol L(-1)) was then incubated within the pH range 2-6 and possible products monitored by liquid chromatography-mass spectrometry (LC-MS), drawing at regular intervals serial aliquots of the incubation mixture. With respect to controls, two differential peaks were noted along with a quantitative bio-transformation of the parent compound, at pH values < or = 3. Under pre-meal conditions, a 4 mono-nitro compound was identified as main metabolite, whereas under post-meal condition a second metabolite, showing a complete de-chlorination, along with the probable presence of three nitro groups on the aromatic ring, was revealed. The reaction was highly reproducible and the kinetics suggested the involvement of nitrogen-related free radicals. The results are discussed in the light of the possible formation of pharmacological active tissue-bound residues as cause of symptoms severity.

The occurrence of self- and xeno-cannibalism could be considered as two different aspects of the same well-regulated process. The formation of autophagosomes can represent a survival option for a cell in unfavorable conditions but it can also lead to cell demise. In fact, autophagy has been considered as an additional and clear-cut cell death pathway. We herein speculate that selfeating by autophagy could be paralleled by a cannibalistic behavior, e.g., by cell feeding of siblings, that can also become detrimental. This behavior in fact, once exacerbated, can also lead to cell death, probably bolstering intracellular oxidative imbalance. In this case, a survival option, such as self- and xeno-cannibalism, can be turned into a peculiar death option: cell death by feeding excess. Under this point of view, over-feeding cells are reminiscent of the frog in the Phedrus Fabula "Rana Rupta et Bos".

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.

Quinolinic acid (QA) has been shown to evoke neurotoxic events via NMDA receptor (NMDAR) overactivation and oxidative stress. NMDARs are particularly vulnerable to free radicals, which can modulate protein tyrosine kinase (PTK) and phosphotyrosine phosphatase (PTP) activities. The src family of tyrosine kinases are associated with the NMDAR complex and regulate NMDA channel function. Because QA is an NMDAR agonist as well as a pro-oxidant agent, we investigated whether it may affect the activity of PTKs and PTPs in vivo and in vitro. In synaptosomes prepared from striata dissected 15 min, 30 min or 15 days after bilateral injection of QA we observed modulation of the phosphotyrosine pattern; a significant decrease in PTP activity; and a sustained increase in c-src and lyn activity at 15 and 30 min after treatment with QA, followed by a decrease 2 weeks later. Striatal synaptosomes treated in vitro with QA showed time- and dose-dependent modulation of c-src and lyn kinase activities. Moreover, the nitric oxide synthase inhibitor N-G-nitro-L-arginine-methyl ester, the NMDAR antagonist D-2-amino-5-phosphonovaleric acid and pyruvate suppressed the QA-induced modulation of c-src activity. These findings suggest a novel feature of QA in regulating src kinase activity through the formation of reactive radical species and/or NMDAR overactivation.

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.

IUBMB LifeVolume 58, Issue 10 p. 611-613 ArticleFree Access Does CO2 modulate peroxynitrite specificity? Paolo Ascenzi, Paolo Ascenzi National Institute for Infectious Diseases IRCCS 'Lazzaro Spallanzani', Roma, ItalySearch for more papers by this authorAlessio Bocedi, Alessio Bocedi National Institute for Infectious Diseases IRCCS 'Lazzaro Spallanzani', Roma, ItalySearch for more papers by this authorPaolo Visca, Paolo Visca National Institute for Infectious Diseases IRCCS 'Lazzaro Spallanzani', Roma, ItalySearch for more papers by this authorMaurizio Minetti, Maurizio Minetti Department of Cell Biology and Neurosciences, National Institute of Health, Roma, ItalySearch for more papers by this authorEmilio Clementi, Emilio Clementi Stem Cell Research Institute, Hospital San Raffaele, and Department of Preclinical Sciences, University of Milano, Milano, ItalySearch for more papers by this author Paolo Ascenzi, Paolo Ascenzi National Institute for Infectious Diseases IRCCS 'Lazzaro Spallanzani', Roma, ItalySearch for more papers by this authorAlessio Bocedi, Alessio Bocedi National Institute for Infectious Diseases IRCCS 'Lazzaro Spallanzani', Roma, ItalySearch for more papers by this authorPaolo Visca, Paolo Visca National Institute for Infectious Diseases IRCCS 'Lazzaro Spallanzani', Roma, ItalySearch for more papers by this authorMaurizio Minetti, Maurizio Minetti Department of Cell Biology and Neurosciences, National Institute of Health, Roma, ItalySearch for more papers by this authorEmilio Clementi, Emilio Clementi Stem Cell Research Institute, Hospital San Raffaele, and Department of Preclinical Sciences, University of Milano, Milano, ItalySearch for more papers by this author First published: 03 January 2008 https://doi.org/10.1080/15216540600746344Citations: 20AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume58, Issue10October 2006Pages 611-613 RelatedInformation