Mechanisms Underlying the Effects of Chloroquine on Red Blood Cells Metabolism.

Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug's ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 µM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

Catechins and Proanthocyanidins Involvement in Metabolic Syndrome.

Recent studies on natural antioxidant compounds have highlighted their potentiality against various pathological conditions. The present review aims to selectively evaluate the benefits of catechins and their polymeric structure on metabolic syndrome, a common disorder characterized by a cluster of three main risk factors: obesity, hypertension, and hyperglycemia. Patients with metabolic syndrome suffer chronic low inflammation state and oxidative stress both conditions effectively countered by flavanols and their polymers. The mechanism behind the activity of these molecules has been highlighted and correlated with the characteristic features present on their basic flavonoidic skelethon, as well as the efficient doses needed to perform their activity in both in vitro and in vivo studies. The amount of evidence provided in this review offers a starting point for flavanol dietary supplementation as a potential strategy to counteract several metabolic targets associated with metabolic syndrome and suggests a key role of albumin as flavanol-delivery system to the different target of action inside the organism.

Ferroptosis: Emerging Role in Diseases and Potential Implication of Bioactive Compounds.

Ferroptosis is a form of cell death that is distinguished from other types of death for its peculiar characteristics of death regulated by iron accumulation, increase in ROS, and lipid peroxidation. In the past few years, experimental evidence has correlated ferroptosis with various pathological processes including neurodegenerative and cardiovascular diseases. Ferroptosis also is involved in several types of cancer because it has been shown to induce tumor cell death. In particular, the pharmacological induction of ferroptosis, contributing to the inhibition of the proliferative process, provides new ideas for the pharmacological treatment of cancer. Emerging evidence suggests that certain mechanisms including the Xc- system, GPx4, and iron chelators play a key role in the regulation of ferroptosis and can be used to block the progression of many diseases. This review summarizes current knowledge on the mechanism of ferroptosis and the latest advances in its multiple regulatory pathways, underlining ferroptosis' involvement in the diseases. Finally, we focused on several types of ferroptosis inducers and inhibitors, evaluating their impact on the cell death principal targets to provide new perspectives in the treatment of the diseases and a potential pharmacological development of new clinical therapies.

Anti-Aggregative and Protective Effects of Vicenin-2 on Heat and Oxidative Stress-Induced Damage on Protein Structures.

Vicenin-2, a flavonoid categorized as a flavones subclass, exhibits a distinctive and uncommon C-glycosidic linkage. Emerging evidence challenges the notion that deglycosylation is not a prerequisite for the absorption of C-glycosyl flavonoid in the small intestine. Capitalizing on this experimental insight and considering its biological attributes, we conducted different assays to test the anti-aggregative and antioxidant capabilities of vicenin-2 on human serum albumin under stressful conditions. Within the concentration range of 0.1-25.0 µM, vicenin-2 effectively thwarted the heat-induced HSA fibrillation and aggregation of HSA. Furthermore, in this study, we have observed that vicenin-2 demonstrated protective effects against superoxide anion and hydroxyl radicals, but it did not provide defense against active chlorine. To elucidate the underlying mechanisms, behind this biological activity, various spectroscopy techniques were employed. UV-visible spectroscopy revealed an interaction between HSA and vicenin-2. This interaction involves the cinnamoyl system found in vicenin-2, with a peak of absorbance observed at around 338 nm. Further evidence of the interaction comes from circular dichroism spectrum, which shows that the formation of bimolecular complex causes a reduction in α-helix structures. Fluorescence and displacement investigations indicated modifications near Trp214, identifying Sudlow's site I, similarly to the primary binding site. Molecular modeling revealed that vicenin-2, in nonplanar conformation, generated hydrophobic interactions, Pi-pi stacking, and hydrogen bonds inside Sudlow's site I. These findings expand our understanding of how flavonoids bind to HSA, demonstrating the potential of the complex to counteract fibrillation and oxidative stress.

Implication of COVID-19 on Erythrocytes Functionality: Red Blood Cell Biochemical Implications and Morpho-Functional Aspects.

Several diseases (such as diabetes, cancer, and neurodegenerative disorders) affect the morpho-functional aspects of red blood cells, sometimes altering their normal metabolism. In this review, the hematological changes are evaluated, with particular focus on the morphology and metabolic aspects of erythrocytes. Changes in the functionality of such cells may, in fact, help provide important information about disease severity and progression. The viral infection causes significant damage to the blood cells that are altered in size, rigidity, and distribution width. Lower levels of hemoglobin and anemia have been reported in several studies, and an alteration in the concentration of antioxidant enzymes has been shown to promote a dangerous state of oxidative stress in red blood cells. Patients with severe COVID-19 showed an increase in hematological changes, indicating a progressive worsening as COVID-19 severity progressed. Therefore, monitored hematological alterations in patients with COVID-19 may play an important role in the management of the disease and prevent the risk of a severe course of the disease. Finally, monitored changes in erythrocytes and blood, in general, may be one of the causes of the condition known as Long COVID.

The Neuroprotective Potentiality of Flavonoids on Alzheimer's Disease.

Alzheimer's disease (AD), due to its spread, has become a global health priority, and is characterized by senile dementia and progressive disability. The main cause of AD and other neurodegenerations (Huntington, Parkinson, Amyotrophic Lateral Sclerosis) are aggregated protein accumulation and oxidative damage. Recent research on secondary metabolites of plants such as polyphenols demonstrated that they may slow the progression of AD. The flavonoids' mechanism of action in AD involved the inhibition of acetylcholinesterase, butyrylcholinesterase, Tau protein aggregation, ß-secretase, oxidative stress, inflammation, and apoptosis through modulation of signaling pathways which are implicated in cognitive and neuroprotective functions, such as ERK, PI3-kinase/Akt, NFKB, MAPKs, and endogenous antioxidant enzymatic systems. This review focuses on flavonoids and their role in AD, in terms of therapeutic potentiality for human health, antioxidant potential, and specific AD molecular targets.

Biotechnological Applications and Health-Promoting Properties of Flavonols: An Updated View.

Flavonols are a subclass of natural flavonoids characterized by a remarkable number of biotechnological applications and health-promoting properties. They attract researchers' attention due to many epidemiological studies supporting their usage. They are phytochemicals commonly present in our diet, being ubiquitous in the plant kingdom and, in particular, relatively very abundant in fruits and vegetables. All these aspects make flavonols candidates of choice for the valorization of products, based on the presence of a remarkable number of different chemical structures, each one characterized by specific chemical features capable of influencing biological targets inside the living organisms in very different manners. In this review, we analyzed the biochemical and physiological characteristics of flavonols focalizing our attention on the most promising compounds to shed some light on their increasing utilization in biotechnological applications in processing industries, as well as their suitable employment to improve the overall wellness of the humankind.

A new erythrocyte-based biochemical approach to predict the antiproliferative effects of heterocyclic scaffolds: The case of indolone.

BACKGROUND: The indole core is a key structural feature of many natural products and biomolecules with broad spectrum chemotherapeutic properties. Some of us have recently synthesized a library of biologically promising indolone-based compounds. The present study focuses on the effects of one of them, namely DPIT, on human erythrocytes. METHODS: We have examined the influence of DPIT on band 3 protein, intracellular ATP concentration and transport, caspase 3 activation, metabolic adaptation and membrane stability. RESULTS: Our study elucidates that DPIT, intercalated into the phospholipid bilayer, decreases the anion transport, the intracellular ATP concentration and the cytosolic pH, inducing a direct activation of caspase 3. CONCLUSIONS: Starting from the metabolic similarity between erythrocytes and cancer cells, we investigate how the metabolic derangements and membrane alterations induced by selected heterocycles could be related to the antiproliferative effects. GENERAL SIGNIFICANCE: Our work aims to propose a new model of study to predict the antiproliferative effects of heterocyclic scaffolds, pointing out that only one of the listed conditions would be unfavorable to the life cycle of neoplastic cells.

Molecular interactions of hemoglobin with resveratrol: potential protective antioxidant role and metabolic adaptations of the erythrocyte.

This article reports the role of resveratrol in the erythrocyte as a result of its interaction with hemoglobin and describes the effect of this interaction on the metabolism, the redox state, and the release of ATP. The drug crosses the erythrocyte membrane and binds to hemoglobin, altering its modulation and the release of ATP. Our study correlates the variation of the phosphorylation balance induced by resveratrol with the change in the intracellular concentration of ATP and with the decrease in ATP release from red blood cell and the consequent paracrine alteration on the vascular epithelium. Molecular docking calculations indicate larger specificity of binding for oxy-hemoglobin that correlates well with the stabilization of the R-quaternary structure and with the functional modulation of resveratrol on the protein. Finally, we locate a putative binding site at the central cavity of hemoglobin and characterize its key interacting residues with the drug. Computational results support the assumption that resveratrol may act as a protector agent against oxidative protein damage by interacting with hemoglobin.

Palytoxin induces functional changes of anion transport in red blood cells: metabolic impact.

Palytoxin (PTX) is classified as one of the most powerful marine biotoxins (of high molecular weight and no protein origin) because it is able to interact strongly with important cellular structures influencing their function in different biological processes. This study of the effects of PTX on red blood cells (RBC) extends the knowledge about its toxicity, which concerns not only the well-known action on Na(+)/K(+)-ATPase but also band 3 protein (B3 or AE1), the role of which is essential for anion transport and for the structure, function, and metabolic integrity of the erythrocyte. The effects of PTX on RBC can be summarized as follows: it alters the anionic flux and seriously compromises not only CO(2) transport but also the metabolic modulation centered on the oxy-deoxy cycle of hemoglobin; it stabilizes the plasma membrane by preventing lipid peroxidation; and its effect does not lead to activation of caspases 3 and 8. From what is reported in steps 2 and 3, and on the basis of the results obtained on hemolysis, methemoglobin levels, and phosphatase activity, an increase of the reducing power of the erythrocytes (RBC) in the presence of PTX clearly emerges. The results have enabled us to outline some metabolic adaptations induced in the RBC by PTX.

Resveratrol treatment induces redox stress in red blood cells: a possible role of caspase 3 in metabolism and anion transport.

Resveratrol, an important phytoalexine found in many plants, has been shown to be significantly effective in the treatment of several pathological conditions such as cancer, coronary heart disease and osteoarthritis. This study focuses on the effects of this drug on human red blood cells. In particular, we have examined the influence of resveratrol on Band 3, the anion exchanger protein, and hemoglobin as a function of the oxygenation-deoxygenation cycle. Moreover, special attention has been given to the metabolic changes imposed by caspase 3 activation. Resveratrol has proved to lower superoxide production, thereby decreasing heme-iron oxidation and saving the reducing power required for met-hemoglobin reduction. Oxygen binding experiments showed that resveratrol interacts with hemoglobin, shifting the T→R conformational transition towards the higher-affinity R state. This might contribute to altering the metabolic balance of the cell through an intensification of the pentose phosphate pathway. Moreover, at high oxygenation levels of the erythrocytic hemoglobin, resveratrol induces a significant activation of caspase 3, the action of which on Band 3 has a strong impact on cellular metabolism and anion transport.

Reviewing Biochemical Implications of Normal and Mutated Huntingtin in Huntington's Disease.

Huntingtin (Htt) is a multi-function protein of the brain. Normal Htt shows a common alpha-helical structure but conformational changes in the form with beta strands are the principal cause of Huntington's disease. Huntington's disease is a genetic neurological disorder caused by a repeated expansion of the CAG trinucleotide, causing instability in the N-terminal of the gene coding for the Huntingtin protein. The mutation leads to the abnormal expansion of the production of the polyglutamine tract (polyQ) resulting in the form of an unstable Huntingtin protein commonly referred to as mutant Huntingtin. Mutant Huntingtin is the cause of the complex neurological metabolic alteration of Huntington's disease, resulting in both the loss of all the functions of normal Huntingtin and the genesis of abnormal interactions due to the presence of this mutation. One of the problems arising from the misfolded Huntingtin is the increase in oxidative stress, which is common in many neurological diseases such as Alzheimer's, Parkinson's, Amyotrophic Lateral Sclerosis and Creutzfeldt-Jakob disease. In the last few years, the use of antioxidants had a strong incentive to find valid therapies for defence against neurodegenerations. Although further studies are needed, the use of antioxidant mixtures to counteract neuronal damages seems promising.

Influences of flavonoids on erythrocyte membrane and metabolic implication through anionic exchange modulation.

The antioxidative activity of some natural flavonoids was analyzed against the stable free radical 2,2-diphenyl-1-picryhydrazyl. The results indicate that the scavenging power of the tested flavonols is higher than that of the synthetic antioxidants butylated hydroxyanisole and butylated hydroxytoluene; instead, the flavanones show little activity, as indicated by efficient concentration (EC50) values. Flavonoid autoxidation and interaction with Fe2+ and hydrogen peroxide were tested using erythrocyte membranes as a model. The results show that some compounds, like hesperetin, evidence a pro-oxidant activity higher than the ascorbic acid/iron reference system. The compounds with strong oxidative capability do not only influence cellular redox balance but also activate caspase-3, producing lactate dehydrogenase release and enhancing anionic exchange at the level of band 3 protein.

Derangement of erythrocytic AE1 in beta-thalassemia by caspase 3: pathogenic mechanisms and implications in red blood cell senescence.

Considering its complex molecular pathophysiology, beta-thalassemia could be a good in vivo model to study some aspects related to erythrocyte functions with potential therapeutic implications not only within the frame of this particular hemoglobinopathy but also with respect to conditions in which the cellular milieu, altered by a deranged anion exchanger, could display a significant pathogenetic role (i.e., erythrocyte senescence, complications of red cell storage, renal tubular acidosis and some abnormal protein thesaurismosis). This work evaluates the anionic influx across band 3 protein in normal and beta-thalassemic red blood cells (RBCs) and ghosts. Since redox-mediated injury is an important pathway in the destruction of beta-thalassemic RBCs, we studied the anion transport and the activity of caspase 3 in the absence and presence of t-butylhydroperoxide in order to evaluate the effect of an increase of cellular oxidative stress. Interestingly, beta-thalassemic erythrocytes show a faster rate of anion exchange than normal RBCs and absence of any modulation mechanism of anion influx. These findings led us to formulate a hypothesis about the metabolic characteristics of beta-thalassemic erythrocytes, outlining that one of the main targets of caspase 3 in RBCs is the cytoplasmic domain of band 3 protein.

Influence of L-rhamnosyl-D-glucosyl derivatives on properties and biological interaction of flavonoids.

The anti-proliferative activity of hesperetin, hesperidin, neohesperidin and rutin was evaluated on human hepatoma cell lines (Hep G2) and correlated to their antioxidant activity. The results obtained showed strong anti-proliferative effects of hesperidin and neohesperidin, considerably higher than the other two additives. Hesperetin induced caspase-3 activation, release of LDH and endogenous accumulation of putrescine. Cell cycle distribution seems to indicate that the inhibitory effects of polyphenols on cell growth could be due to G0/G1 block, and activation of apoptotic pathway in the presence of hesperetin. Our results underline also that the glycone forms show reduced scavenging activity against DPPH, but present a remarkable inhibition of cell proliferation and low cytotoxicity.

Protective Effects of the Caffeine Against Neurodegenerative Diseases.

BACKGROUND: Recent studies and increased interest of the scientific community helped to clarify the neurological health property of caffeine, one of the pharmacologically active substances most consumed in the world. METHODS: This article is a review search to provide an overview on the current state of understanding neurobiochemical impact of caffeine, focusing on the ability of the drug to effectively counteract several neurodegenerative disorders such as Alzheimer's, Parkinson's, Huntington's diseases, Multiple sclerosis and Amyotrophic lateral sclerosis. RESULTS: Data collection shown in this review provide a significant therapeutic and prophylactic potentiality of caffeine which acts on human brain through several pathways because of its antioxidant activity combined with multiple molecular targets. However, the need to adjust the CF dosage to individuals, because some people are more sensitive to drugs than others, may constituted a limit to the CF effectiveness. CONCLUSION: What emerges from the complex of clinical and epidemiological studies is a significant CF potential impact against all neurological disorders. Although, further studies are needed to fully elucidate the several mechanisms of drug action which in part are still elusive.

New role for an old molecule: The 2,3-diphosphoglycerate case.

BACKGROUND: Aerobic organisms have to overcame the dangerous species derived from the unquestionable favorable effects due to the utilization of oxygen in the cellular respiration. 2,3-Diphosphoglycerate (DPG) could be one of the molecules able to perform different role inside the cells and (from the data obtained from our experimental work) may help cellular components, in particular hemoglobin, to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS). METHODS: Therefore, we have investigated the kinetic and antioxidant properties of this molecule against the main biological reactive species and the protective role of this molecules on hemoglobin treated with strong oxidant. RESULTS: DPG, at the physiological concentration is able to scavenge hydroxyl radical, peroxyl radical, cation radicals and to chelate iron in the reduced state. Moreover it is able to avoid oxidation of iron inside the hemoglobin following treatment with nitrite and tert-butyl hydroperoxide (t-BOOH). On the other side, it is not able to protect membrane components from oxidative burning. This different behavior towards radical species is probably linked to the polarity of the molecule and also the high levels of charged groups present on the surface of DPG, that avoid the possibility to act in an environment almost completely hydrophobic, as inside the membrane, where reactive species produce the main damages during the reactions of peroxidation. CONCLUSIONS: This is the first paper dealing with the potential role of DPG not only as a modulator of oxygen affinity in hemoglobin, but also as a scavenger of radicals.

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT.

Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0-3.0 M) on thermal unfolding of ornithine carbamoyltransferase (OCT) from Alopias vulpinus. Furthermore conformational changes in whole enzyme structure have been monitored by titration of SH-groups. OCT unfolding process follows an irreversible two-state mechanism with a first-order kinetic of denaturation, without breaking-point. NaCl shows very little stabilization effects at low concentration and its action become very important over 1.5 M concentration. The presence of 3.0 M NaCl completely avoids OCT unfolding at 60, 64 and 66 degrees C. Kinetic and thermodynamic parameters are strongly influenced by the presence of high NaCl concentration. Our experiments showed that NaCl stabilization process involved changes in preferential binding, in electrostatic and van der Waals interactions and exposure of buried site and SH-groups. During thermal denaturation, UV-vis and CD spectroscopy show that high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.

Neuroprotective effects of phloretin and its glycosylated derivative on rotenone-induced toxicity in human SH-SY5Y neuronal-like cells.

Phloretin and phlorizin are the two strong natural antioxidants whose biological and pharmacological applications are rapidly growing in different human pathological conditions. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells and evaluated by testing cell vitality, mitochondrial transmembrane potential and ROS production, antioxidant enzymes detection, activation of caspase 3, DNA damage, protein carbonylation, lipid peroxidation, and superoxide anion scavenging activity. Incubation of cells with rotenone caused cell death and significant increase in intracellular reactive oxygen species, activation of caspase 3, and variation in mitochondrial transmembrane potential. Although, rotenone exposure caused a significant increase of antioxidant enzymes, high levels of lipid peroxidation were also observed. Phloretin or phlorizin, at micromolar concentration, reduced rotenone-induced cell death by scavenging ability against superoxide anion radical, one of the main effectors of rotenone toxicity at level of mitochondrial respiratory chain complex I. Under our experimental conditions, a reduction of the intracellular ROS levels with consequent normalization of the aforementioned antioxidant enzymes occurred. Concomitantly, we observed the inhibition of caspase 3 activity and DNA damage. This study shows the promising neuroprotective ability of the two dihydrochalcones able to protect human differentiated neuroblastoma cells (commonly used as model of Parkinson's disease) from injury induced by rotenone, actively scavenging ROS, normalizing mitochondrial transmembrane potential and consequently avoiding energy depletion. © 2017 BioFactors, 43(4):549-557, 2017.

Short-Term Effects of Chlorpromazine on Oxidative Stress in Erythrocyte Functionality: Activation of Metabolism and Membrane Perturbation.

The purpose of this paper is to focus on the short-term effects of chlorpromazine on erythrocytes because it is reported that the drug, unstable in plasma but more stable in erythrocytes, interacts with erythrocyte membranes, membrane lipids, and hemoglobin. There is a rich literature about the side and therapeutic effects or complications due to chlorpromazine, but most of these studies explore the influence of long-term treatment. We think that evaluating the short-term effects of the drug may help to clarify the sequence of chlorpromazine molecular targets from which some long-term effects derive. Our results indicate that although the drug is primarily intercalated in the innermost side of the membrane, it does not influence band 3 anionic flux, lipid peroxidation, and protein carbonylation processes. On the other hand, it destabilizes and increases the autooxidation of haemoglobin, induces activation of caspase 3, and, markedly, influences the ATP and reduced glutathione levels, with subsequent exposure of phosphatidylserine at the erythrocyte surface. Overall our observations on the early stage of chlorpromazine influence on erythrocytes may contribute to better understanding of new and interesting characteristics of this compound improving knowledge of erythrocyte metabolism.