Permeability of human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a 64th Annual Meeting of the Biophysical Society, San Diego, CA. 2020Hydrogen peroxide (H2O2) and other reactive species are important physiological mediators in the vascular system. Enzymatic production of H2O2 is involved in regulating cell growth, proliferation and vasodilation. Whereas endothelial cells are important sources of H2O2, red blood cells (RBC) are considered the most important sinks of H2O2 in the vasculature. However, little is known about the permeability of their membrane to H2O2. The permeability coefficient of human RBC membranes to H2O2 was determined using the enzyme latency method, based on measuring the rate of H2O2 decomposition in lysed vs whole cells. If the passage through the membrane is the rate limiting step in H2O2 decomposition, then a difference is observed that can be used to calculate the permeability coefficient. Additional experiments were done to differentiate between simple diffusion through the lipid fraction and facilitated diffusion through protein channels. The lack of reported permeability coefficients for lipid membranes prompted us to do experiments with phospholipid-cholesterol liposome membranes that indicated that simple diffusion is a slow process. Determination of partition coefficients in different solvents mimicking different depths of the membrane indicate that the low permeability of lipid membranes to H2O2 is caused mainly by its very low solubility in the acyl region of the bilayer. The activation energy of permeation through RBC membranes suggested that protein channels were involved in facilitating H2O2 diffusion through the membrane. Inhibitors of hAQP3 and hAQP1 had no effect in H2O2 consumption rate, suggesting that other membrane proteins may be involved. Although the RBC membrane presents a significant barrier to H2O2 passage, especially in comparison with other solutes such as oxygen and nitric oxide, the permeability is still high enough to support the role of RBC as sinks of H2O2 in circulation.ANII: FMV_1_2019_15559

Permeability of lipid membranes to hydrogen peroxide

Resumen del Conference paper presentado a SfRBM 27th Annual Conferenc

Permeability of phospholipid membranes and human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a SfRBM 28th Annual ConferenceHydrogen peroxide (H2O2) is an oxygen-derived oxidant involved in multiple redox processes in the cell, ranging from physiological signaling pathways to oxidative damage reactions when it is found at higher concentrations. In the vascular system, H2O2 is metabolized mainly by red blood cells (RBC) due to their very efficient antioxidant systems and high membrane permeability. However, the information regarding H2O2 transport in the human RBC membrane is limited, as neither the exact value of the permeability coefficient (Pm) nor the permeation mechanisms are known. To explore whether H2O2 permeates through the lipid fraction or protein channels, we studied H2O2 solubility in organic solvents and its permeability in lipid membranes, in order to compare with the RBC membrane. Through measurements of partition constants, we found that H2O2 is 14 and 122000 times less soluble in octanol and hexadecane than in water, anticipating a large thermodynamic barrier to H2O2 permeation by lipid membranes. The Pm in phospholipid membranes of different compositions, determined using the catalase-latency method, varied from 4×10-4 to 5×10-3 cm s-1, at 37°C. On the other hand, in human RBC we determined a Pm of 1.6×10-3 cm s-1. After obtaining these results, we evaluated the potential role of aquaporins as H2O2 transporters by checking the effect of aquaporin inhibitors in H2O2 consumption by RBC, and also by studying H2O2 permeability in RBC devoid of either aquaporin 1 or aquaporin 3. Surprisingly, we could not detect any differences in H2O2 permeability in any case. Altogether, these results provide new information on lipid membrane permeability to H2O2 and a new value for the Pm in human RBC, which was previously unknown. Additionally, they indicate that H2O2 is not transported by aquaporins in human RBC membranes, suggesting simple diffusion or a still unidentified membrane protein as a more probable pathway.ANII: ANII: FMV_1_2019_15559

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformationANII: PD_NAC_2016_1_13332

Permeabilidad de membranas lipídicas al peróxido de hidrógeno

Agencia Nacional de Investigación e Innovación FCE_1_2017_1_136043Programa de Desarrollo de las Ciencias BásicasComisión Sectorial de Investigación Científica (CSIC

Informe final del proyecto: Estrategias para mejorar la manufactura, conservación y calidad de los preparados de hemocomponentes para transfusión

La transfusión de glóbulos rojos y de otros hemoderivados son una herramienta de extremo valor para la práctica clínica. Sin embargo, las reacciones adversas a la transfusión de hemo componentes son una complicación relativamente frecuente. La presencia de leucocitos y de los mediadores solubles generados por estas células en la bolsa de transfusión ha sido señalada como responsable de cambios estructurales y metabólicos en los glóbulos rojos durante el almacenamiento. El empleo de técnicas de leucoreducción en la Unión Europea y en parte de los Estados Unidos ha disminuido la incidencia de estas reacciones desfavorables. En nuestro país, de los 160.000 volúmenes de concentrados de glóbulos rojos trasfundidos al año, sólo un 15% se leucorreducen. Además, este proceso se realiza sobre preparados almacenados por un tiempo variable, lo que lleva a la acumulación de mediadores inflamatorios y la aparición de un fenotipo senescente en los eritrocitos. Este proyecto se origina a partir de la colaboración entre la Cátedra de Medicina Transfusional del Hospital de Clínicas, Facultad de Medicina e investigadores del Instituto de Química Biológica de la Facultad de Ciencias, con el fin de colaborar en la instauración de políticas sanitarias tendientes a mejorar la provisión de esta importante herramienta terapéutica. De los resultados obtenidos en el proyecto podemos concluir: 1. Que la leucorreducción es una herramienta efectiva para prevenir la acumulación de mediadores proinflamatorios en el preparado, 2. Que la remoción de leucocitos enlentece el desarrollo del fenotipo envejecido en los glóbulos rojos almacenados. 3. Que la presencia de lactato deshidrogenasa en el sobrenadante de los preparados es un índice sensible para evaluar el estado de los preparados para transfusión. 4. La financiación obtenida permitió además el dictado de dos cursos en el área, la realización de un simposio internacional y la formación de recursos humanos.Agencia Nacional de Investigación e Innovació