Novel Bioplastic Based on PVA Functionalized with Anthocyanins: Synthesis, Biochemical Properties and Food Applications.

Over the last ten years, researchers' efforts have aimed to replace the classic linear economy model with the circular economy model, favoring green chemical and industrial processes. From this point of view, biologically active molecules, coming from plants, flowers and biomass, are gaining considerable value. In this study, firstly we focus on the development of a green protocol to obtain the purification of anthocyanins from the flower of Callistemon citrinus, based on simulation and on response surface optimization methodology. After that, we utilize them to manufacture and add new properties to bioplastics belonging to class 3, based on modified polyvinyl alcohol (PVA) with increasing amounts from 0.10 to 1.00%. The new polymers are analyzed to monitor morphological changes, optical properties, mechanical properties and antioxidant and antimicrobial activities. Fourier transform infrared spectroscopy (FTIR) spectra of the new materials show the characteristic bands of the PVA alone and a modification of the band at around 1138 cm-1 and 1083 cm-1, showing an influence of the anthocyanins' addition on the sequence with crystalline and amorphous structures of the starting materials, as also shown by the results of the mechanical tests. These last showed an increase in thickening (from 29.92 µm to approx. 37 µm) and hydrophobicity with the concomitant increase in the added anthocyanins (change in wettability with water from 14° to 31°), decreasing the poor water/moisture resistance of PVA that decreases its strength and limits its application in food packaging, which makes the new materials ideal candidates for biodegradable packaging to extend the shelf-life of food. The functionalization also determines an increase in the opacity, from 2.46 to 3.42 T%/mm, the acquisition of antioxidant activity against 2,2-diphenyl-1-picrylhdrazyl and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals and, in the ferric reducing power assay, the antimicrobial (bactericidal) activity against different Staphylococcus aureus strains at the maximum tested concentration (1.00% of anthocyanins). On the whole, functionalization with anthocyanins results in the acquisition of new properties, making it suitable for food packaging purposes, as highlighted by a food fresh-keeping test.

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.

Brain Alteration Patterns in Children with Duchenne Muscular Dystrophy: A Machine Learning Approach to Magnetic Resonance Imaging.

Background: Duchenne Muscular Dystrophy (DMD) is a genetic disease in which lack of the dystrophin protein causes progressive muscular weakness, cardiomyopathy and respiratory insufficiency. DMD is often associated with other cognitive and behavioral impairments, however the correlation of abnormal dystrophin expression in the central nervous system with brain structure and functioning remains still unclear. Objective: To investigate brain involvement in patients with DMD through a multimodal and multivariate approach accounting for potential comorbidities. Methods: We acquired T1-weighted and Diffusion Tensor Imaging data from 18 patients with DMD and 18 age- and sex-matched controls with similar cognitive and behavioral profiles. Cortical thickness, structure volume, fractional anisotropy and mean diffusivity measures were used in a multivariate analysis performed using a Support Vector Machine classifier accounting for potential comorbidities in patients and controls. Results: the classification experiment significantly discriminates between the two populations (97.2% accuracy) and the forward model weights showed that DMD mostly affects the microstructural integrity of long fiber bundles, in particular in the cerebellar peduncles (bilaterally), in the posterior thalamic radiation (bilaterally), in the fornix and in the medial lemniscus (bilaterally). We also reported a reduced cortical thickness, mainly in the motor cortex, cingulate cortex, hippocampal area and insula. Conclusions: Our study identified a small pattern of alterations in the CNS likely associated with the DMD diagnosis.

Psychosocial resources and psychopathology among persons with neuromuscular disorders during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic substantially affected the lives of persons with inherited neuromuscular disorders (INMD), causing disruption in clinical and support services. While several studies have investigated mental health, distress and psychosocial resources in the general population during the pandemic, little is known about the experience of persons with INMD. METHODS: This study was aimed to fill this gap by jointly investigating both psychopathological symptoms and psychosocial resources - specifically, resilience and perceived social support - among persons with INMD during the pandemic, taking into account demographic and clinical factors. Between April and December 2020, 59 participants with INMD (aged 15-59, 71.2% M) completed a questionnaire collecting demographic and clinical data, the Multidimensional Scale of Perceived Social Support, the Resilience Scale for Adults, and the Achenbach System of Empirically Based Assessment. RESULTS: Overall, participants showed good levels of resilience and perceived social support. A minority of participants reported clinically relevant psychopathological symptoms, 28.81% for anxiety and depression. Most psychopathological symptoms were negatively correlated with resilience (-0.347 < r < - .420), but not significantly associated with social support. Consistent with previous studies, regression analyses highlighted that participants with Duchenne muscular dystrophy were more prone to report anxious and depressive symptoms (B = 1.748, p = .028, OR = 5.744), and participants with myotonic dystrophy, attention problems (B = 2.339, p = .006, OR = 10.376). Resilience emerged as a potential predictor of lower anxious-depressive symptoms (B=-1.264, p = .012, OR = 0.283). CONCLUSIONS: The findings suggest the importance to investigate psychosocial resources in addition to psychopathology among persons with INMD, and to design interventions supporting resilience as a protective factor for mental health promotion.

Hybrid Polyelectrolyte Nanocomplexes for Non-Viral Gene Delivery with Favorable Efficacy and Safety Profile.

The development of nanovectors for precise gene therapy is increasingly focusing on avoiding uncontrolled inflammation while still being able to effectively act on the target sites. Herein, we explore the use of non-viral hybrid polyelectrolyte nanocomplexes (hPECs) for gene delivery, which display good transfection efficacy coupled with non-inflammatory properties. Monodisperse hPECs were produced through a layer-by-layer self-assembling of biocompatible and biodegradable polymers. The resulting nanocomplexes had an inner core characterized by an EGFP-encoding plasmid DNA (pDNA) complexed with linear polyethyleneimine or protamine (PEI or PRM) stabilized with lecithin and poly(vinyl alcohol) (PVA) and an outer layer consisting of medium-molecular-weight chitosan (CH) combined with tripolyphosphate (TPP). PEI- and PRM-hPECs were able to efficiently protect the genetic cargo from nucleases and to perform a stimuli-responsive release of pDNA overtime, thus guaranteeing optimal transfection efficiency. Importantly, hPECs revealed a highly cytocompatible and a non-inflammatory profile in vitro. These results were further supported by evidence of the weak and unspecific interactions of serum proteins with both hPECs, thus confirming the antifouling properties of their outer shell. Therefore, these hPECs represent promising candidates for the development of effective, safe nanotools for gene delivery.

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.

Over three decades of natural history of limb girdle muscular dystrophy type R1/2A and R2/2B: Mathematical modelling of a multifactorial study.

We aimed to describe the natural history of Limb Girdle Muscular Dystrophy type 2A and 2B over more than three decades by considering muscular strength, motor, cardiac and respiratory function. 428 visits of nineteen 2A and twenty 2B patients were retrospectively analysed through a regression model to create the curves of evolution with disease duration of muscle strength (through Medical Research Council grading), motor function measure scale (D1, D2 and D3 domains) and cardio-pulmonary function tests. Clinically relevant muscular and motor function alterations occurred after the first decade of disease, while mild respiratory function alterations started after the second, with preserved cardiac function. Although type 2A showed relatively stronger distal lower limb muscles, while type 2B started with relatively stronger upper limb muscles, the corresponding motor functions were similar, becoming severely compromised after 25 years of disease. This was the longest retrospective study in types 2A and 2B. It defined curves of disease evolution not only from a neuromuscular, but also from functional, cardiac, and respiratory points of view, to be used to evaluate how the natural progression is changed by therapies. Due to slow disease progression, it was not possible to identify time sensitive endpoints.

Sensitivity of Neuroimaging Indicators in Monitoring the Effects of Interferon Gamma Treatment in Friedreich's Ataxia.

The identification of efficient markers of disease progression and response to possibly effective treatments is a key priority for slowly progressive, rare and neurodegenerative diseases, such as Friedreich's ataxia. Various imaging modalities have documented specific abnormalities in Friedreich's ataxia that could be tracked to provide useful indicators of efficacy in clinical trials. Advanced MRI imaging (diffusion tensor imaging, DTI; functional MRI, fMRI; and resting-state fMRI, rs-fMRI) and retinal imaging (optical coherence tomography, OCT) were tested longitudinally in a small group of Friedreich's ataxia patients participating in an open-label clinical trial testing the safety and the efficacy of 6-month treatment with interferon gamma. While the DTI indices documented the slow progression of fractional anisotropy loss, fMRI and rs-fMRI were significantly modified during and after treatment. The fMRI changes significantly correlated with the Scale for the Assessment and Rating of Ataxia, which is used to monitor clinical response. OCT documented the known thickness reduction of the retinal nerve fiber layer thickness, but there was no change over time. This pilot study provides indications for the potential utility of fMRI and rs-fMRI as ancillary measures in clinical trials for Friedreich's ataxia.

Diaphragm Involvement in Duchenne Muscular Dystrophy (DMD): An MRI Study.

BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by progressive weakness and wasting of skeletal, cardiac, and respiratory muscles, with consequent cardiopulmonary failure as the main cause of death. Reliable outcome measures able to demonstrate specific trends over disease progression are essential. PURPOSE: To investigate MRI as a noninvasive imaging modality to assess diaphragm impairment in DMD. In particular, we sought to correlate MRI measurement of diaphragm structure and function with pulmonary function tests and with the abdominal volumes (VAB ) measured by optoelectronic plethysmography, being an index of the action of the diaphragm. STUDY TYPE: Cross-sectional study. POPULATION: Twenty-six DMD patients (17.9 ± 6.2 years) and 12 age-matched controls (17.8 ± 5.9 years). FIELD STRENGTH/SEQUENCE: 3-Point gradient echo Dixon sequence at 3T. ASSESSMENT: Images were acquired in breath-hold at full-expiration (EXP) and full-inspiration (INSP). INSP and EXP lung volumes were segmented and the diaphragm surface was reconstructed as the bottom surface of the left and the right lung. The inspiratory and the expiratory diaphragm surfaces were aligned by a nonrigid iterative closest point algorithm. On MRI we measured: 1) craniocaudal diaphragmatic excursion; 2) diaphragm fatty infiltration. STATISTICAL TESTS: Three-parameter sigmoid regression, one-way analysis of variance (ANOVA), Spearman's correlation. RESULTS: In patients, diaphragm excursion decreased with age (r2 = 0.68, P < 0.0001) and fat fraction increased (r2 = 0.51, P = 0.0002). In healthy subjects, diaphragm excursion and fat fraction had no relationship with age. Diaphragm excursion decreased with decreasing FEV1 %pred (r = 0.78, P < 0.0001) and FVC %pred (r = 0.76, P < 0.0001) and correlated with VAB (r = 0.60, P = 0.0002). Fatty infiltration increased with decreasing FEV1 %pred (r = -0.88, P < 0.0001) and FVC %pred (r = -0.88, P < 0.0001). DATA CONCLUSION: The progressive structural and functional diaphragm impairment is highly related to pulmonary function tests and to VAB . The results suggest that MRI might represent a new and noninvasive tool for the functional and structural assessment of the diaphragm. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2020;51:461-471.

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.

Green olive leaf extract (OLE) provides cytoprotection in renal cells exposed to low doses of cadmium.

Cadmium (Cd) is a heavy and highly toxic metal that contaminates air, food and water. Cadmium accumulates in several organs altering normal functions. The kidney is the major organ at risk of damage from chronic exposure to cadmium as a contaminant in food and water. This study aims to investigate the beneficial effects of OLE in renal collecting duct MCD4 cells exposed to a low dose cadmium (1 µM). In MCD4 cells cadmium caused an increase in ROS production, as well as generation of lipid droplets and reduced cell viability. Moreover, cadmium exposure led to a remarkable increase in the frequency of micronuclei and DNA double-strand breaks, assessed using the alkaline comet assay. In addition, cadmium dramatically altered cell cytoskeleton architecture and caused S-glutathionylation of actin. Notably, all cadmium-induced cellular deregulations were prevented by co-treatment with OLE, possibly due to its antioxidant action and to the presence of bioactive phytocompounds. Indeed, OLE treatment attenuated Cd-induced actin S-glutathionylation, thereby stabilizing actin filaments. Taken together, these observations provide a novel insight into the biological action of OLE in renal cells and support the notion that OLE may serve as a potential adjuvant against cadmium-induced nephrotoxicity.

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.

Comparison between men and women of volume regulating hormones and aquaporin-2 excretion following graded central hypovolemia.

Central hypovolemia induced by orthostatic loading causes reno-vascular changes that can lead to orthostatic intolerance. In this study, we investigated volume regulating hormonal responses and reno-vascular changes in male and female subjects as they underwent central hypovolemia, induced by graded lower body negative pressure (LBNP). Aquaporin-2 (AQP2) excretion was measured as a biomarker for the renal system response to vasopressin. 37 young healthy subjects (n = 19 males; n = 18 females) were subjected to graded LBNP until - 40 mmHg LBNP. Under resting conditions, males had significantly higher copeptin (a stable peptide derived from vasopressin) levels compared with females. Adrenocorticotropin (ACTH), adrenomedullin (ADM), vasopressin (AVP) and brain natriuretic peptide (BNP) were not affected by our experimental protocol. Nevertheless, an analysis of ADM and BNP with the data normalized as percentages of the baseline value data showed an increase from baseline to 10 min after recovery in the males in ADM and in the females in BNP. Analysis of BNP and ADM raises the possibility of a preferential adaptive vascular response to central hypovolemia in males as shown by the normalized increase in ADM, whereas females showed a preferential renal response as shown by the normalized increase in BNP. Furthermore, our results suggest that there might be a difference between men and women in the copeptin response to alterations in orthostatic loading, simulated either using LBNP or during posture changes.

Multiparametric quantitative MRI assessment of thigh muscles in limb-girdle muscular dystrophy 2A and 2B.

INTRODUCTION: The aim of this study was to apply quantitative MRI (qMRI) to assess structural modifications in thigh muscles of subjects with limb girdle muscular dystrophy (LGMD) 2A and 2B with long disease duration. METHODS: Eleven LGMD2A, 9 LGMD2B patients and 11 healthy controls underwent a multi-parametric 3T MRI examination of the thigh. The protocol included structural T1-weighted images, DIXON sequences for fat fraction calculation, T2 values quantification and diffusion MRI. Region of interest analysis was performed on 4 different compartments (anterior compartment, posterior compartment, gracilis, sartorius). RESULTS: Patients showed high levels of fat infiltration as measured by DIXON sequences. Sartorius and anterior compartment were more infiltrated in LGMD2B than LGMD2A patients. T2 values were mildly reduced in both disorders. Correlations between clinical scores and qMRI were found. CONCLUSIONS: qMRI measures may help to quantify muscular degeneration, but careful interpretation is needed when fat infiltration is massive. Muscle Nerve 58: 550-558, 2018.

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70.

BACKGROUND/AIMS: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. METHODS: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. RESULTS: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. CONCLUSION: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.

Interleukin-13 increases pendrin abundance to the cell surface in bronchial NCI-H292 cells via Rho/actin signaling.

Interleukin-13 (IL13) is a major player in the development of airway hyperresponsiveness in several respiratory disorders. Emerging data suggest that an increased expression of pendrin in airway epithelia is associated with elevated airway hyperreactivity in asthma. Here, we investigate the effect of IL13 on pendrin localization and function using bronchiolar NCI-H292 cells. The data obtained revealed that IL13 increases the cell surface expression of pendrin. This effect was paralleled by a significant increase in the intracellular pH, possibly via indirect stimulation of NHE. IL13 effect on pendrin localization and intracellular pH was reversed by theophylline, a bronchodilator compound used to treat asthma. IL13 upregulated RhoA activity, a crucial protein controlling actin dynamics, via G-alpha-13. Specifically, IL13 stabilized actin cytoskeleton and promoted co-localization and a direct molecular interaction between pendrin and F-actin in the plasma membrane region. These effects were reversed following exposure of cells to theophylline. Selective inhibition of Rho kinase, a downstream effector of Rho, reduced the IL13-dependent cell surface expression of pendrin. Together, these data indicate that IL13 increases pendrin abundance to the cell surface via Rho/actin signaling, an effect reversed by theophylline.

Preliminary Study on the In vitro and In vivo Effects of Asparagopsis taxiformis Bioactive Phycoderivates on Teleosts.

Several compounds from marine organisms have been studied for their potential use in aquaculture. Among the red algae, Asparagopsis taxiformis is considered one of the most promising species for the production of bioactive metabolites with numerous proposed applications. Here, the in vitro antibacterial activity, the easy handling and the absence of adverse effects on marine fish species are reported. Depending on the seasonal period of sampling, ethanol extracts of A. taxiformis exhibited significantly different inhibitory activity against fish pathogenic bacteria. The extract obtained in late spring showed strong antibacterial activity against Aeromonas salmonicida subsp. salmonicida, Vibrio alginolyticus, and V. vulnificus, and moderate activity against Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida, V. harveyi and V. parahaemolyticus. Sea bass and gilthead sea bream were fed with pellets supplied with the alga and algal extracts. The absence of undesired effects on fish was demonstrated. Hematological and biochemical investigations allowed to confirm that the whole alga and its extracts could be proposed for a future application in aquaculture.

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.