Vanessa Faria Cortes

Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane

Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.

Differences of lipid membrane modulation and oxidative stress by digoxin and 21-benzylidene digoxin

&NA; Cardiotonic steroids (CTS) are compounds which bind to the Na,K‐ATPase, leading to its inhibition and in some cases initiating signaling cascades. Long utilized as a treatment for congestive heart disease, CTS have more recently been observed to inhibit proliferation and cause apoptosis in several cancer cell lines. A synthetic derivative of the CTS digoxin, called 21‐benzylidene digoxin (21‐BD), activates the Na,K‐ATPase rather than cause its inhibition, as its parent compound does. Here, the mechanism behind the unique effects of 21‐BD are further explored. In HeLa cancer cells, low (5 &mgr;M) and high (50 &mgr;M) doses of 21‐BD activated and inhibited the Na,K‐ATPase, respectively, without altering the membrane expression of the Na,K‐ATPase. While digoxin did not affect HeLa membrane cholesterol or phospholipid content, 50 &mgr;M 21‐BD increased both lipids via a mechanism reliant on an intact cell. Afterwards, the direct action of 21‐BD was evaluated on erythrocyte membranes; however, no effect was observed. As CTS may generate reactive oxygen species (ROS) which can affect plasma membrane fluidity and therefore Na,K‐ATPase activity, several markers involved in ROS generation were analyzed such as, lipid peroxidation (TBARS), reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD). GSH content and catalase activity were unaffected by digoxin or 21‐BD. Surprisingly, TBARS and SOD activity was decreased with digoxin and with 50 &mgr;M 21‐BD. Thus, 21‐BD and digoxin altered components involved in ROS generation and inhibition in a similar fashion. This study suggests alterations to the Na,K‐ATPase and membrane lipids by 21‐BD is not reliant on ROS generation.

Could Na,K-ATPase play a role in potassium leakage from irradiated erythrocytes?

BACKGROUND In developing countries, the access to red blood cell (RBC) irradiators is restricted. Thus, it is a common practice in blood banks to stock irradiated RBC units until they expire. The aim of this work is to elucidate the involvement of Na,K-ATPase in potassium leakage from prophylactically irradiated RBCs. METHODS Whole blood was collected from healthy donors, and blood concentrates were irradiated with 25Gy of γ-radiation within 24h of collection. At days 3, 5, 7, 9, 11, 14 and 28 post-irradiation, fractions were removed and centrifuged and Na,K-ATPase activity from ghost membranes was determined. RESULTS The inhibition of Na,K-ATPase activity in RBCs reached 12.6% by day 7 of storage and up to 50% by day 14 of storage. The addition of vitamin C prevented the irradiation-induced loss of Na,K-ATPase activity. The irradiation of RBCs provoked an increase in potassium plasma levels and a decrease in sodium plasma levels. The incubation of RBCs with ouabain did not change the sodium or potassium levels in the plasma, and the addition of vitamin C only partially prevented a decrease in sodium levels caused by irradiation. CONCLUSION Because the inhibition of Na,K-ATPase by ouabain did not cause potassium accumulation in the plasma, we conclude that the irradiation-induced inhibition of the pump is not a key factor driving this effect.

Performance of rapid diagnostic tests for detection of Hepatitis B and C markers in HIV infected patients

There is little information describing the influence of HIV infection upon the performance of rapid diagnostic tests (RDTs) for hepatitis B and C virus diagnosis. This study aims to evaluate the performance of RDTs for HBsAg and anti-HCV detection among HIV-infected individuals. A total of 362 HIV infected individuals were recruited from clinics between January 2013 to November 2014 in the southeast and northeast of Brazil. HBsAg and anti-HCV were detected using commercial EIAs and four RDTs: HBV (Vikia HBsAg® and Wama Imuno-Rapido HBV®) and HCV (Bioeasy Teste Rápido HCV® and Wama Imuno-Rapido HCV®). Reactive HBsAg and anti-HCV serum samples were tested for HBV DNA and HCV RNA. Sensitivity, specificity and kappa statistic were determined. Using EIA, HBsAg and anti-HCV were detected in 14 (3.9%) and 37 (10.2%) serum samples respectively. Using serum only, HBsAg RDTs demonstrated sensitivities and specificities above 92.0% and Kappa values above 89.0%. Anti-HCV RDTs demonstrated sensitivity and specificities above 82.0% and Kappa higher than 89.0%. Using whole blood samples, Vikia HBsAg® and Wama Imuno-Rapido HCV® showed sensitivity and specificity above 99.0% with Kappa of 66.4% and 100%, respectively. HIV viral load was higher among discordant results for anti-HCV RDT. RDTs demonstrated good performance in HIV infected individuals showing the usefulness of assays in this population.

Influência dos níveis de paratormônio em quedas entre idosos e adultos em hemodiálise

Objective: to evaluate whether the elderly with high levels of parathyroid hormone in hemodialysis and have more falls than adults under the same conditions. Method: it is a descriptive study, transverse, with a quantitative approach, performed in a nephrology ambulatory from a large hospital of the Central West of Minas Gerais and 80 patients participates in this study. Data were collected using a structured form and analysis of medical records of participants. Results: the falls occurred in 52.50% of the elderly patients and, although there was no significant association between PTH and fall, a considerable number of elderly patients with PTH above 200 pg / ml fell in the last year. Conclusion: future studies are needed to evaluate the intrinsic and extrinsic factors related to falls in the elderly, to prevent this event and ensure a better quality of life for the elderly

Cytotoxic and cytostatic effects of digitoxigenin monodigitoxoside (DGX) in human lung cancer cells and its link to Na,K-ATPase

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of β-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (-11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.

γ-Benzylidene digoxin derivatives synthesis and molecular modeling: Evaluation of anticancer and the Na,K-ATPase activity effect.

Cardiotonic steroids (CS), natural compounds with traditional use in cardiology, have been recently suggested to exert potent anticancer effects. However, the repertoire of molecules with Na,K-ATPase activity and anticancer properties is limited. This paper describes the synthesis of 6 new digoxin derivatives substituted (on the C17-butenolide) with γ-benzylidene group and their cytotoxic effect on human fibroblast (WI-26 VA4) and cancer (HeLa and RKO) cell lines as well as their effect on Na,K-ATPase activity and expression. As digoxin, compound BD-4 was almost 100-fold more potent than the other derivatives for cytotoxicity with the three types of cells used and was also the only one able to fully inhibit the Na,K-ATPase of HeLa cells after 24h treatment. No change in the Na,K-ATPase α1 isoform protein expression was detected. On the other hand it was 30-40 fold less potent for direct Na,K-ATPase inhibition, when compared to the most potent derivatives, BD-1 and BD-3, and digoxin. The data presented here demonstrated that the anticancer effect of digoxin derivatives substituted with γ-benzylidene were not related with their inhibition of Na,K-ATPase activity or alteration of its expression, suggesting that this classical molecular mechanism of CS is not involved in the cytotoxic effect of our derivatives.

Involvement of Src signaling in the synergistic effect between cisplatin and digoxin on cancer cell viability

Cisplatin and other platinum‐containing drugs have played a crucial role in anticancer treatments for over 30 years. However, treatment with cisplatin may cause serious side effects, such as myelosuppression, nausea, ototoxicity, nephrotoxicity, and cell resistance processes. In addition, cardiotonic steroids, particularly digoxin, have recently been suggested to exert potent anticancer effects. Therefore, it is possible that the combined treatment of HeLa cells with cisplatin and digoxin can ameliorate the cytotoxic effects and decrease the side effects of cisplatin. In this study, we demonstrated that the interaction between cisplatin and digoxin had a synergistic effect on cervical cancer cells and a significantly positive cytotoxic and antiproliferative effect on this cell line compared to the control and single cisplatin treatments. Although a decrease in the Na,K‐ATPase α1 subunit expression was observed in total extracts, its expression remains unchanged in the membrane, as does the Na,K‐ATPase activity. The antiproliferative effect of the synergistic treatment appears to depend on Src kinase activation, indicating the possible involvement of the Scr‐EGFR‐ERK1/2 pathway in the antitumor effect. The inhibition of ERK1/2 provoked the same synergism with 1 μM cisplatin as that observed with 1 nM digoxin plus 1 μM cisplatin but not with 1 nM digoxin. Pretreatment with PP2 during combined treatment abolished the synergistic effect on the antiproliferative activity. Cisplatin and digoxin are already used in the clinical setting; therefore, this study opens possibilities for future clinical trials of combined treatments to improve treatment outcomes with a lower incidence of toxicity and side effects.

Performance of point of care assays for hepatitis B and C viruses in chronic kidney disease patients

Aims Point of care testing (POCT) has been used for hepatitis B and C diagnosis in general population, but little is known about the influence of clinical conditions in the accuracy of these assays. This study aims to evaluate the performance of POCTs for detection of hepatitis B virus surface antigen (HBsAg) and antibodies to Hepatitis C Virus (anti-HCV) in Chronic Kidney Disease (CKD) patients. Methods A total of 286 subjects were included in this study. HBsAg and anti-HCV were detected using commercial EIAs and four POCTs: HBsAg (WAMA Imuno-Rápido HBsAg and VIKIA HBsAg) and anti-HCV (DOLES HCV teste rápido and WAMA Imuno-Rápido anti-HCV) in serum and whole blood. Results Using EIA, HBsAg and anti-HCV prevalence was 4.5% and 16.1% in CKD patients. HBsAg and anti-HCV POCTs had sensitivities from 92.3% to 100% and 84.8% to 89.1% while specificities were 99.3% to 100% and 99.2% to 99.6%, respectively. POCT using serum samples performed well compared with whole blood samples and true positive samples of POCTs had high optical density to cut-off (OD/CO) values compared with EIA. Conclusions This study demonstrates good performance of HBsAg and anti-HCV POCTs in CKD patients, especially in serum samples indicating low interference of this disease in the performance of these assays. POCTs could be an important tool for HBV and HCV screening in high-risk populations.

Ouabain attenuates oxidative stress and modulates lipid composition in hippocampus of rats in lipopolysaccharide-induced hypocampal neuroinflammation in rats: GARCIA et al.

Our study aimed to analyze the effect of ouabain (OUA) administration on lipopolysaccharide (LPS)‐induced changes in hippocampus of rats. Oxidative parameters were analyzed in Wistar rats after intraperitoneal injection of OUA (1.8 µg/kg), LPS (200 µg/kg), or OUA plus LPS or saline. To reach our goal, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), in addition to levels of reduced glutathione (GSH), protein carbonyl (PCO) and lipid peroxidation (LPO) were evaluated. We also analyzed the membrane lipid profile and some important lipids for the nervous system, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and sphingomyelin. The group that received only LPS showed increased oxidative stress, as evidenced by an increase in LPO (about twice), PCO (about three times) levels, and CAT activity (80%). Conversely, administration of LPS decreased GSH levels (55%), and GPx activity (30%), besides a reduction in the amount of PI (60%) and PC (45%). By other side, OUA alone increased the amount of PI (45%), PE (85%), and PC (70%). All harmful effects recorded were attenuated by OUA, suggesting a protective effect against LPS‐induced oxidative stress. The relevance of our results extends beyond changes in oxidative parameters induced by LPS, because nanomolar doses of OUA may be useful in neurodegenerative models. Other studies on other cardenolides and substances related issues, as well as the development of new molecules derived from OUA, could also be useful in general oxidative and/or cellular stress, a condition favoring the appearance of neuronal pathologies.