Anion exchanger1 (AE1/SLC4A1) function is impaired in red blood cells from prediabetic subjects: Potential benefits of finger lime (Citrus australasica, Faustrime cultivar) juice extract.

Prediabetes is a risk state that defines a high chance of developing diabetes and cardiovascular disease. Oxidative stress mediated by hyperglycemia-induced production of reactive species could play a crucial role in this context. In the present study, we investigated whether the anion exchange capability mediated by AE1 (SLC4A1), which is sensitive to oxidative stress, was altered in human red blood cells (RBCs) obtained from prediabetic volunteers. In addition, we assessed the precise composition of bioactive compounds and the potential benefits of finger lime juice extract (Citrus australasica, Faustrime cultivar) in counteracting oxidative stress-related functional alterations. Human RBCs from normal and prediabetic volunteers were incubated with 50 µg/mL juice extract for 2 h at 25°C. Juice extract restored alterations of the anion exchange capability mediated by AE1 and prevented the structural rearrangements of AE1 and α/ß-spectrin in prediabetic RBCs. AE1 functional and structural alterations were not associated with an increase in lipid peroxidation or protein oxidation at the level of the plasma membrane. An increased production of intracellular ROS, which provoked the oxidation of hemoglobin to methemoglobin, both reverted by juice extract, was instead observed. Importantly, juice extract also induced a reduction in glycated hemoglobin levels in prediabetic RBCs. Finally, juice extract blunted the overactivation of the endogenous antioxidant enzymes catalase and superoxide dismutase and prevented glutathione depletion in prediabetic RBCs. These findings contribute to clarifying cellular and molecular mechanisms related to oxidative stress and glycation events that may influence RBC and systemic homeostasis in prediabetes, identify AE1 as a sensitive biomarker of RBC structural and function alterations in prediabetes and propose finger lime juice extract as a natural antioxidant for the treatment and/or prevention of the complications associated with the prediabetic condition.

Cellular and Molecular Mechanisms in Oxidative Stress-Related Diseases 2.0/3.0.

Oxidative stress is frequently described as the balance between the production of reactive species (including oxygen and nitrogen) in biological systems and the ability of the latter to defend itself through the sophisticated antioxidant machinery [...].

Oxidative Stress and Immune Response in Melanoma: Ion Channels as Targets of Therapy.

Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane or other cellular membranes and are targets of oxidative stress, which is particularly elevated in melanoma. At the same time, ion channels are crucial for normal and cancer cell physiology and are subject to multiple layers of regulation, and therefore represent promising targets for therapeutic intervention. In this review, we analyzed the effects of oxidative stress on ion channels on a molecular and cellular level and in the context of melanoma progression and immune evasion. The possible role of ion channels as targets of alternative therapeutic strategies in melanoma was discussed.

d-Galactose induced early aging in human erythrocytes: Role of band 3 protein.

Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO4²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

Cellular and Molecular Mechanisms in Oxidative Stress-Related Diseases.

The redox equilibrium is important in preserving the correct functionality of vital cellular functions [...].

Physiological or Pathological Molecular Alterations in Brain Aging.

Aging is a natural phenomenon that occurs due to a variety of loosely understood mechanisms [...].

Oxidation Stress as a Mechanism of Aging in Human Erythrocytes: Protective Effect of Quercetin.

Aging is a multi-factorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress (OS) as well as protein glycation. The aim of the present work was to verify the protective role of Quercetin (Q), a polyphenolic flavonoid compound, in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. The anion-exchange capability through the Band 3 protein (B3p) measured by the rate constant of the SO42- uptake, thiobarbituric acid reactive substances (TBARS) levels-a marker of lipid peroxidation-total sulfhydryl (-SH) groups, glycated hemoglobin (A1c), and a reduced glutathione/oxidized glutathione (GSH-GSSG) ratio were determined following the exposure of erythrocytes to 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µM Q. The results confirmed that d-Gal activated OS pathways in human erythrocytes, affecting both membrane lipids and proteins, as denoted by increased TBARS levels and decreased total sulfhydryl groups, respectively. In addition, d-Gal led to an acceleration of the rate constant of the SO42- uptake through the B3p. Both the alteration of the B3p function and oxidative damage have been improved by pre-treatment with Q, which preferentially ameliorated lipid peroxidation rather than protein oxidation. Moreover, Q prevented glycated A1c formation, while no protective effect on the endogenous antioxidant system (GSH-GSSG) was observed. These findings suggest that the B3p could be a novel potential target of antioxidant treatments to counteract aging-related disturbances. Further studies are needed to confirm the possible role of Q in pharmacological strategies against aging.

Antioxidant Activity of Quercetin in a H2O2-Induced Oxidative Stress Model in Red Blood Cells: Functional Role of Band 3 Protein.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 µM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42- uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 µM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

Band 3 protein function and oxidative stress in erythrocytes.

Band 3 protein (B3p), anion transporter, allows the HCO3- /Cl- exchange across plasma membrane and plays an important role for erythrocytes homeostasis. In addition, B3p is linked to proteins cytoskeleton, thus contributing to cell shape and deformability, essential to erythrocytes adjustment within narrowest capillaries. Taking into account that erythrocytes are a suitable cell model to investigate the response of the oxidative stress effects, B3p functions, and specifically anion exchange capability, determining the rate constant for SO42- uptake, has been considered. As, in the latter years, rising attention has been addressed to membrane transport system, and particularly to this protein, the present mini-review has been conceived to report the most recent knowledge about B3p, with specific regard to its functions in oxidative stress conditions, including oxidative stress-related diseases.

Physiological and Biochemical Changes in NRF2 Pathway in Aged Animals Subjected to Brain Injury.

BACKGROUND/AIMS: Oxidative stress plays a key role in aging, which in turn represents a substantial risk factor for brain injuries. The aim of the present study was to investigate the differences in physiological and biochemical changes in the brain during injury-related inflammation and oxidative stress, comparing young and old mice. METHODS: Young and old mice were subjected to focal cerebral ischemia induced by transient middle cerebral artery occlusion or to traumatic brain injury performed by a controlled cortical impactor. At the end of both experiments, mice were sacrificed 24h after injuries and brains were collected to perform biochemical analysis. RESULTS: In both ischemic stroke and traumatic brain injury, aging has not only led to damage-induced worsening of motor function and behavioural changes but also increased of infarct area compared to young animals. Moreover, aged mice show increased evidence of oxidative stress and reduced antioxidant capacity when compared to younger animals, as demonstrated by Nrf2-Keap1 signalling pathway and lower expression of antioxidant enzymes, such as HO-1, SOD-1 and GSH-Px. Additionally, brain tissues collected from elderly mice showed an increased IκB-α degradation into the cytoplasm and consequently NF-κB translocation into the nucleus, compared to young mice subjected to same injuries. The elderly mice showed significantly higher levels of iNOS and CoX-2 expression than the young mice, as well as higher levels of inflammatory cytokines such as TNFα, IL-1ß, and IL-6 after MCAO and TBI. CONCLUSION: Preserving and keeping the NRF-2 pathway active counteracts the onset of oxidative stress and consequent inflammation after ischemic and traumatic brain insult, particularly in the elderly. Not only that, NRF-2 pathway could represent a possible therapeutic target in the management of brain injuries.

Atrazine Inhalation Causes Neuroinflammation, Apoptosis and Accelerating Brain Aging.

BACKGROUND: exposure to environmental contaminants has been linked to an increased risk of neurological diseases and poor outcomes. Chemical name of Atrazine (ATR) is 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine, and it is the most commonly used broad-spectrum herbicide in agricultural crops. Several studies have demonstrated that ATR has the potential to be harmful to the brain's neuronal circuits. Until today nobody has explored the effect of ATR inhalation on young and aged mice. METHODS: young and aged mice were subject to 25 mg of ATR in a vehicle made with saline and 10% of Dimethyl sulfoxide (DMSO) every day for 28 days. At the end of experiment different behavioral test were made and brain was collected. RESULTS: exposure to ATR induced the same response in terms of behavioral alterations and motor and memory impairment in mice but in aged group was more marked. Additionally, in both young and aged mice ATR inhalations induced oxidative stress with impairment in physiological antioxidant response, lipid peroxidation, nuclear factor kappa-light-chain-enhancer of activated B cells (nf-κb) pathways activation with consequences of pro-inflammatory cytokines release and apoptosis. However, the older group was shown to be more sensitive to ATR inhalation. CONCLUSIONS: our results showed that aged mice were more susceptible compared to young mice to air pollutants exposure, put in place a minor physiologically response was seen when exposed to it.

Protective Role of Magnesium against Oxidative Stress on SO4= Uptake through Band 3 Protein in Human Erythrocytes.

BACKGROUND/AIMS: Magnesium, whose supplementation provides beneficial effects against oxidative stress-related conditions, has been here used to possibly protect Band 3 protein anion exchange capability and underlying signaling in an in vitro model of oxidative stress. METHODS: Whole blood samples pre-exposed to 10 mM MgCl2, were treated for 30 min with H2O2 (300 µM, 600 µM and 1 mM) chosen as oxidant molecule. In a separate protocol, NEM (0.5,1 and 2 mM), a phosphatase inhibitor and thiol-alkilant agent, has been also applied. The rate constant for SO4= uptake, accounting for Band 3 protein anion exchange capability, has been measured by a turbidimetric method, while intracellular reduced glutathione (GSH) levels and membrane -SH groups mostly belonging to Band 3 protein were spectrophotometrically quantified after reaction with DTNB (5,5'-dithiobis-(2-nitrobenzoic acid). Expression levels of Band 3 protein, phosporylated Tyrosine (P-Tyr) and tyrosine kinase (Syk) involved in signaling have been also measured. RESULTS: Our results show that Mg2+ prevented the reduction in the rate constant for SO4= uptake on H2O2-treated erythrocytes, not involving GSH levels and membrane -SH groups, unlike NEM, remaining both P-Tyr and Syk expression levels high. CONCLUSION: Hence, i) the measurement of the rate constant for SO4= uptake is a useful tool to evaluate Mg2+ protective effect; ii) the use of two different oxidant molecules shed light on Mg2+ effect which seems not to modulate phosphorylative pathways but would putatively stabilize membrane organization; iii) the use of Mg2+ in food supplementation can be reasonably supported to protect erythrocytes homeostasis in case of oxidative stress-related diseases.

Melatonin Protects Band 3 Protein in Human Erythrocytes against H2O2-Induced Oxidative Stress.

The beneficial effect of Melatonin (Mel), recognized as an anti-inflammatory and antioxidant compound, has been already proven to prevent oxidative stress-induced damage associated to lipid peroxidation. As previous studies modeled the impact of oxidative stress on Band 3 protein, an anion exchanger that is essential to erythrocytes homeostasis, by applying H2O2 at not hemolytic concentrations and not producing lipid peroxidation, the aim of the present work was to evaluate the possible antioxidant effect of pharmacological doses of Mel on Band 3 protein anion exchange capability. The experiments have been performed on human erythrocytes exposed to 300 µM H2O2-induced oxidative stress. To this end, oxidative damage has been verified by monitoring the rate constant for SO4= uptake through Band 3 protein. Expression levels of this protein Mel doses lower than 100 µM have also been excluded due to lipid peroxidation, Band 3 protein expression levels, and cell shape alterations, confirming a pro-oxidant action of Mel at certain doses. On the other hand, 100 µM Mel, not provoking lipid peroxidation, restored the rate constant for SO4= uptake, Band 3 protein expression levels, and H2O2-induced cell shape alterations. Such an effect was confirmed by abolishing the endogenous erythrocytes antioxidant system. Therefore, the present findings show the antioxidant power of Mel at pharmacological concentrations in an in vitro model of oxidative stress not associated to lipid peroxidation, thereby confirming Band 3 protein anion exchange capability measurement as a suitable model to prove the beneficial effect of Mel and support the use of this compound in oxidative stress-related diseases affecting Band 3 protein.

Neuronal-like differentiated SH-SY5Y cells adaptation to a mild and transient H2 O2 -induced oxidative stress.

Preconditioning (PC) is a cell adaptive response to oxidative stress and, with regard to neurons, can be considered as a neuroprotective strategy. The aim of the present study was to verify how neuronal-like differentiated SH-SY5Y cells adapt to a mild and transient H2 O2 -induced oxidative stress and, hence, whether may be considered as more sensitive cell model to study PC pathways. A first screening allowed to define H2 O2 concentrations for PC (10µM-50µM), applied before damage(100µM H2 O2 ). Cell viability measured 24 hours after 100µM H2 O2 -induced damage was ameliorated by 24-hour pre-exposure to low-concentration H2 O2 (10µM-30µM) with cell size as well restored. Markers for apoptosis (Bcl-2 and Bad), inflammation (iNOS), and redox system (MnSOD) were also determined, showing that, in cells pre-exposed to 10µM H2 O2 and then submitted to 100µM H2 O2 , Bcl-2 levels were higher, Bad and iNOS levels were lower than those observed in damaged cells, and MnSOD levels were unchanged. Such findings show that (1) neuronal-like differentiated SH-SY5Y cells are a suitable model to investigate PC response and more sensitive to the effect of a mild and transient H2 O2 -induced oxidative stress with respect to other neuronal cells; (2) 10µM H2 O2 -induced PC is mediated by apoptotic and inflammatory pathways, unlike antioxidant system; (3) such neuroprotective strategy and underlying signals proven in neuronal-like differentiated SH-SY5Y cells may contribute to understand in vivo PC mechanisms and to define a window for pharmacological intervention, namely, related to ischemic brain damage. SIGNIFICANCE OF THE STUDY: Neuronal-like differentiated SH-SY5Y cells are a suitable model to investigate PC, an endogenous neuroprotective response to a mild and transient H2 O2 -induced oxidative stress, elicited by 24-hour exposure to very low H2 O2 concentrations and mediated by both apoptotic and inflammatory pathways. This model reflects in vivo PC mechanisms occurring after brain trauma and provides novel information about pathways and time of protection useful for an appropriate pharmacological intervention.

Anion exchange through band 3 protein in canine leishmaniasis at different stages of disease.

Band 3 protein efficiency in mediating Cl-/HCO3- exchange through erythrocytes membrane is reduced by oxidative stress. The aim of the present study was to verify whether and how anion transport through band 3 protein may be useful in monitoring canine leishmaniasis (Leishmania infantum) development, a disease associated to membrane protein degradation and oxidative stress. To accomplish this aim, serological analysis to determine IFAT (immunofluorescence antibody test) titers against leishmaniasis has been performed and 1:160 and 1:540 titers, determined at diagnosis and after 6 months, were considered for experiments. Oxidative conditions have been assessed by estimating MDA (malondialdehyde) plasma levels, intracellular GSH (reduced glutathione) content, and membrane -SH groups. Band 3 protein anion exchange capability was evaluated by measuring the rate constant for SO4= uptake, and its expression levels, along with those of P-Tyr (phosphorylated tyrosine), involved in pathways underlying band 3 protein function, have been also determined. Our results show that, in infected dogs with 1:160 IFAT titer, high MDA plasma levels and oxidation of -SH groups are associated to increased P-Tyr expression levels, leading to a reduction in anion exchange capability throughout 6 months of diagnosis. On the other hand, infected dogs with 1:540 IFAT titer, exhibited oxidative conditions associated to an impaired anion exchange capability at diagnosis, were ameliorated after 6 months. Such findings suggest that (1) band 3 protein-mediated anion transport is reduced by oxidative conditions associated to leishmaniasis, putatively via phosphorylative pathways; (2) band 3 protein efficiency may account for canine leishmaniasis development; and (3) the assessment of band 3 protein function may represent an additional tool for canine leishmaniasis diagnosis and monitoring of its development, with potential application to humans, either in case of leishmaniasis or other oxidative-related pathologies.

SO4= uptake and catalase role in preconditioning after H2O2-induced oxidative stress in human erythrocytes.

Preconditioning (PC) is an adaptive response to a mild and transient oxidative stress, shown for the first time in myocardial cells and not described in erythrocytes so far. The possible adaptation of human erythrocytes to hydrogen peroxide (H2O2)-induced oxidative stress has been here verified by monitoring one of band 3 protein functions, i.e., Cl-/HCO3- exchange, through rate constant for SO4= uptake measurement. With this aim, erythrocytes were exposed to a mild and transient oxidative stress (30 min to either 10 or 100 µM H2O2), followed by a stronger oxidant condition (300- or, alternatively, 600-µM H2O2 treatment). SO4= uptake was measured by a turbidimetric method, and the possible role of catalase (CAT, significantly contributing to the anti-oxidant system in erythrocytes) in PC response has been verified by measuring the rate of H2O2 degradation. The preventive exposure of erythrocytes to 10 µM H2O2, and then to 300 µM H2O2, significantly ameliorated the rate constant for SO4= uptake with respect to 300 µM H2O2 alone, showing thus an adaptive response to oxidative stress. Our results show that (i) SO4= uptake measurement is a suitable model to monitor the effects of a mild and transient oxidative stress in human erythrocytes, (ii) band 3 protein anion exchange capability is retained after 10 µM H2O2 treatment, (iii) PC response induced by the 10 µM H2O2 pretreatment is clearly detected, and (iv) PC response, elicited by low-concentrated H2O2, is mediated by CAT enzyme and does not involve band 3 protein tyrosine phosphorylation pathways. Erythrocyte adaptation to a short-term oxidative stress may serve as a basis for future studies about the impact of more prolonged oxidative events, often associated to aging, drug consumption, chronic alcoholism, hyperglycemia, or neurodegenerative diseases.

Curcumin Protects -SH Groups and Sulphate Transport after Oxidative Damage in Human Erythrocytes.

BACKGROUND/AIMS: Erythrocytes, continuously exposed to oxygen pressure and toxic compounds, are sensitive to oxidative stress, namely acting on integral Band 3 protein, with consequences on cell membranes deformability and anion transport efficiency. The aim of the present investigation, conducted on human erythrocytes, is to verify whether curcumin (1 or 10µM), a natural compound with proved antioxidant properties, may counteract Band 3-mediated anion transport alterations due to oxidative stress. METHODS: Oxidative conditions were induced by exposure to, alternatively, either 2 mM N-ethylmaleimide (NEM) or pH-modified solutions (6.5 and 8.5). Rate constant for SO4(=) uptake and -SH groups estimation were measured to verify the effect of oxidative stress on anion transport efficiency and erythrocyte membranes. RESULTS: After the exposure of erythrocytes to, alternatively, NEM or pH-modified solutions, a significant decrease in both rate constant for SO4(=) uptake and -SH groups was observed, which was prevented by curcumin, with a dose-dependent effect. CONCLUSIONS: Our results show that: i) the decreased efficiency of anion transport may be due to changes in Band 3 protein structure caused by cysteine -SH groups oxidation, especially after exposure to NEM and pH 6.5; ii) 10 µM Curcumin is effective in protecting erythrocytes from oxidative stress events at level of cell membrane transport.

Heavy metals affect nematocysts discharge response and biological activity of crude venom in the jellyfish Pelagia noctiluca (Cnidaria, Scyphozoa).

BACKGROUND: Pollution of marine ecosystems and, specifically, heavy metals contamination may compromise the physiology of marine animals with events occurring on a cellular and molecular level. The present study focuses on the effect of short-term exposure to heavy metals like Zinc, Cadmium, Cobalt and Lanthanum (2-10 mM) on the homeostasis of Pelagia noctiluca (Cnidaria, Scyphozoa), a jellyfish abundant in the Mediterranean sea. This species possesses stinging organoids, termed nematocysts, whose discharge and concomitant delivery of venom underlie the survival of all Cnidaria. METHODS: Nematocysts discharge response, elicited by combined chemico-physical stimulation, was verified on excised oral arms exposed to heavy metals for 20 min. In addition, the hemolytic activity of toxins, contained in the crude venom extracted from nematocysts isolated from oral arms, was tested on human erythrocytes, in the presence of heavy metals or their mixture. RESULTS: Treatment with heavy metals significantly inhibited both nematocysts discharge response and hemolytic activity of crude venom, in a dose-dependent manner, not involving oxidative events, that was irreversible in the case of Lanthanum. CONCLUSION: Our findings show that the homeostasis of Pelagia noctiluca, in terms of nematocysts discharge capability and effectiveness of venom toxins, is dramatically and rapidly compromised by heavy metals and confirm that this jellyfish is eligible as a model for ecotoxicological investigations.

Pelagia noctiluca (Scyphozoa) crude venom injection elicits oxidative stress and inflammatory response in rats.

Cnidarian toxins represent a rich source of biologically active compounds. Since they may act via oxidative stress events, the aim of the present study was to verify whether crude venom, extracted from the jellyfish Pelagia noctiluca, elicits inflammation and oxidative stress processes, known to be mediated by Reactive Oxygen Species (ROS) production, in rats. In a first set of experiments, the animals were injected with crude venom (at three different doses 6, 30 and 60 µg/kg, suspended in saline solution, i.v.) to test the mortality and possible blood pressure changes. In a second set of experiments, to confirm that Pelagia noctiluca crude venom enhances ROS formation and may contribute to the pathophysiology of inflammation, crude venom-injected animals (30 µg/kg) were also treated with tempol, a powerful antioxidant (100 mg/kg i.p., 30 and 60 min after crude venom). Administration of tempol after crude venom challenge, caused a significant reduction of each parameter related to inflammation. The potential effect of Pelagia noctiluca crude venom in the systemic inflammation process has been here demonstrated, adding novel information about its biological activity.

Redox Homeostasis and Antioxidant Strategies in the Pathophysiology.

The molecular mechanisms underlying oxidative stress, and pathophysiological consequences in cell and tissue function, are frequently described as the imbalance between the production of reactive species and the ability to defend through sophisticated antioxidant machinery (Contributions 1-3) [...].