Testosterone exerts antiapoptotic effects against H2O2 in C2C12 skeletal muscle cells through the apoptotic intrinsic pathway

Experimental data indicate that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H2O2)-induced apoptosis in C2C12 muscle cells. Here we identified molecular events involved in the antiapoptotic effect of testosterone. At short times of exposure to H2O2 cells exhibit a defense response but at longer treatment times cells undergo apoptosis. Incubation with testosterone prior to H2O2 induces BAD inactivation, inhibition of poly (ADP-ribose) polymerase cleavage, and a decrease in BAX levels, and impedes the loss of mitochondrial membrane potential, suggesting that the hormone participates in the regulation of the apoptotic intrinsic pathway. Simultaneous treatment with testosterone, H2O2, and the androgen receptor (AR) antagonist, flutamide, reduces the effects of the hormone, pointing to a possible participation of the AR in the antiapoptotic effect. The data presented allow us to begin to elucidate the mechanism by which the hormone prevents apoptosis in skeletal muscle.Fil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentin

Testosterone induces up-regulation of mitochondrial gene expression in murine C2C12 skeletal muscle cells accompanied by an increase of nuclear respiratory factor-1 and its downstream effectors

The reduction in muscle mass and strength with age, sarcopenia, is a prevalent condition among the elderly, linked to skeletal muscle dysfunction and cell apoptosis. We demonstrated that testosterone protects against H2O2-induced apoptosis in C2C12 muscle cells. Here, we analyzed the effect of testosterone on mitochondrial gene expression in C2C12 skeletal muscle cells. We found that testosterone increases mRNA expression of genes encoded by mitochondrial DNA, such as NADPH dehydrogenase subunit 1 (ND1), subunit 4 (ND4), cytochrome b (CytB), cytochrome c oxidase subunit 1 (Cox1) and subunit 2 (Cox2) in C2C12. Additionally, the hormone induced the expression of the nuclear respiratory factors 1 and 2 (Nrf-1 and Nrf-2), the mitochondrial transcription factors A (Tfam) and B2 (TFB2M), and the optic atrophy 1 (OPA1). The simultaneous treatment with testosterone and the androgen receptor antagonist, Flutamide, reduced these effects. H2O2-oxidative stress induced treatment, significantly decreased mitochondrial gene expression. Computational analysis revealed that mitochondrial DNA contains specific sequences, which the androgen receptor could recognize and bind, probably taking place a direct regulation of mitochondrial transcription by the receptor. These findings indicate that androgen plays an important role in the regulation of mitochondrial transcription and biogenesis in skeletal muscle.Fil: Pronsato, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Non-polar extracts of Nicotiana glauca (Solanaceae) induce apoptosis in human rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is the most common soft-tissue tumour in children andadolescents. It originates in normal skeletal muscle from myogenic cells that have failed tofully differentiate, and it usually has a poor prognosis. Current RMS therapy has manyadverse effects. Hence, new treatments are needed. Various pharmacological properties,such as analgesic, antineoplastic, antimicrobial, and antiparasitic properties, have beendemonstrated in species of the Solanaceae family. We performed ethanolic extraction fromleaves of Nicotiana glauca (Solanaceae), and the extract was successively partitioned withn-hexane, chloroform, and ethyl acetate. We evaluated the effects of extracts on RMScells, and we found that the extracts trigger apoptosis. By bio-guided fractionation assays,we identified the apoptotic agents. Morphological assessment after apoptotic cell inductionof cultured cells, mitochondrial and nuclear morphology by Mitotracker, and 4,6-diamidino-2-phenylindole (DAPI) staining, respectively, were analysed in fluorescentmicroscopy. The capacity of the cells to migrate or proliferate was analysed by the Petitassay, followed by methylene blue staining. NMR and GC-MS spectrometry were used toidentify palmitic acid and scopoletin as the phytochemicals responsible for the observedeffects. These results indicate that these compounds are apoptotic inducers and they couldbe useful as chemotherapeutic agents against muscle tumours.Rabdomiossarcoma (RMS) é o tumor de tecidos moles mais comum em crianças e adolescentes. Ele se origina no músculo esquelético normal a partir de las células miogênicas que no conseguiram-se diferenciar completamente e pelo general tem um prognóstico ruim. A terapia atual com RMS tem muitos efeitos adversos e portanto, novos tratamentos são necessários. Várias propriedades farmacológicas, como propriedades analgésica, antineoplásica, antimicrobiana e antiparasitária, foram demostradas em espécies da família Solanaceae. Neste trabalho, foi realizada a extração etanólica das folhas de Nicotiana glauca (Solanaceae), eo extrato foi particionado sucessivamente com n-hexano, clorofórmio y acetato de etila. Avaliamos os efeitos dos extratos nas células RMS e descobrimos que os extratos desencadeiam apoptose. Pelos ensaios de fracionamento bio-guiado, identificamos os agentes apoptóticos. Avaliação morfológica após indução da apoptose das células cultivadas ea morfologia mitocondrial e nuclear por coloração com Mitotracker e 4,6-diamidino-2-fenilindol (DAPI), respectivamente, foram analisadas com microscopia fluorescente. A capacidade das células para migrar ou proliferar foi analisada hair ensaio Petit, seguido pelacoloração com azul de metileno. Una espectrometría de RMN y GC-MS para uso utilizado para una identificación de ácido palmítico y una escopoletina como fitoquímicos responsáveis ​​pelos efeitos observados.Fil: Musso, Florencia Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Pronsato, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Faraoni, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentin

High passage numbers induce resistance to apoptosis in C2C12 muscle cells

Cell lines with high passage numbers exhibit alterations in cell Morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.Fil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Ronda, Ana Carolina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Cátedra de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina

El 17β-Estradiol y la Testosterona protegen a las mitocondrias contra el estrés oxidativo en Células del Músculo Esquelético

En trabajos previos demostramos que la testosterona (T) y el 17β-estradiol (E2) protegen a las células musculares C2C12 de la apoptosis inducida por peróxido de hidrógeno (H2O2). Conjuntamente evidenciamos la existencia de receptores de estrógenos y andrógenos en las mitocondrias. El presente trabajo se ha centrado en caracterizar los efectos de ambos esteroides en esta organela, que conducen a la supervivencia celular. Específicamente, se evaluaron las acciones de T y E2 sobre el potencial de membrana mitocondrial con el colorante JC-1 y sobre el poro de permeabilidad transitoria mitocondrial (MPTP) mediante el método de calcein-acetoxymethylester/cobalt, utilizando microscopía de fluorescencia y citometría de flujo. Demostramos que T y E2 previenen la apertura del MPTP y la pérdida de potencial de membrana mitocondrial inducidas por H2O2. Además, observamos que el H2O2 aumenta los niveles de expresión proteica del canal aniónico dependiente de voltaje (VDAC) e induce la translocación de Bax a mitocondria. Sin embargo, en presencia de las hormonas la translocación de Bax fue inhibida lo cual sugiere que los miembros de la familia Bcl -2 pueden ser regulados por E2 y T. Los eventos moleculares desencadenados por E2 y T a nivel mitocondrial se reflejaron en la morfología de las organelas. El análisis microscópico de las células C2C12 y cultivos primarios de músculo esquelético de ratón, mediante tinciones con verde de Jano y Mitotracker reveló un efecto protector de los esteroides contra el daño por estrés oxidativo inhibiendo la redistribución y picnosis mitocondrial.We have previously shown that testosterone (T) and 17β-estradiol (E2) protect C2C12 muscle cells against apoptosis induced by hydrogen peroxide (H2O2). Since we also showed the presence of estrogen and androgen Receptors in mitochondria, this work was focused on the effects of both steroids on this organelle, which result in cellular survival. Specifically, we evaluated the actions of T and E2 on the mitochondrial membrane potential with JC-1 dye and on the mitochondrial permeability transition pore (MPTP) by the calceinacetoxymethylester (AM)/cobalt method, using fluorescence microscopy and flow cytometry. We demonstrated that T and E2 prevent MPTP opening and the loss of mitochondrial membrane potential induced by H2O2. In addition, it was observed that H2O2 increase voltage-dependent anion channel (VDAC) protein expression levels and induce translocation of Bax to mitochondria. However, in the presence of the steroids Bax translocation was abrogated suggesting that members of the Bcl-2 family may be regulated by E2 and T. The observed effects triggered by E2 and T were reflected on mitochondrial morphology. Microscopic analysis of C2C12 cells and primary cultures of mouse skeletal muscle, with Janus Green and Mitotracker staining revealed a protective effect of the steroids against oxidative stress damage which included mitochondrial redistribution and pyknosis of the organelle.Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Ronda, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto Argentino de Oceanografía (i); Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentin

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Subcellular Localization Of Estrogen Receptors

It is well established that estrogens elicit a variety of rapid effects in many tissues in addition to their actions on gene expression in the cell nucleus after Estrogen Receptors (ERs) dimerization and DNA-binding through their EREs. In this chapter we describe tissue distribution, subcellular localization, targets and roles of classical and non-classical ERs.Fil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Antiapoptotic effects of estrogens and androgens

As it was mentioned before, estrogens and androgens acts on different tissues, also estrogens and androgens receptors are ubiquitously expressed and have shown not only nuclear, but also non-classical intracellular sites like plasma membrane, mitochondria, golgi and endoplasmic reticulum, making increasing this properties a more complex function to the classical roles of estrogens and androgens (regulation of gene expression). E2 and T can trigger different pathways by a non-genomic mechanism through proteins that have the ability to interact with the steroid hormones (structural similar or different from known steroid receptors). So the hormones can regulate apoptotic events through those different signaling pathways. In mitochondria, a control point of apoptosis, it was demonstrated not only the presence of ER and AR but also an steroid protective action against different injuries that results in antiapoptotic effect. Here we summarize the molecular events, modulated by E2 and/or T in several tissues, during programmed cell death.Fil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Presence of estrogen receptor (ER)-like proteins and endogenous ligands for ER in solanaceae

The synthesis of steroid hormones in different plant species and the possibility that these molecules could regulate cell growth, tissue differentiation and germination has been reported. However, the mechanism of action of these endogenous steroids in plant cells is still poorly understood. In the present work, binding experiments with [3H]17β-estradiol in the presence and absence of an excess of unlabeled 17β-estradiol showed that Solanum glaucophyllum and Lycopersicon esculentum organs contain estrogen-binding sites. Scatchard analysis detected saturable [ 3H]17β-estradiol-binding sites (Kd∼6.6nM; B max∼1140fmol/mg protein) in S. glaucophyllum callus tissue. Estrogen-like compounds were detected in lipid extracts from S. glaucophyllum and L. esculentum. Moreover, the lipid fraction was able to compete with [ 3H]17β-estradiol for binding to estrogen receptor (ER) from breast cancer MCF-7 cells as well as with estrogen-binding sites present in both plant species. Western blot analysis with monoclonal antibodies against different domains of the ER α, detected a ∼67kDa band in various organs of both plant species. These proteins were able to bind estradiol in ligand blot assays using 17β-estradiol macromolecular derivatives as ligands. Western and ligand blot experiments of subcellular fractions from callus tissues of S. glaucophyllum showed that the ER-like protein of ∼67kDa was most concentrated in the nuclear fraction. Reactive bands of lower molecular weight were also detected. Altogether these results provide evidence about the existence of estrogen-binding proteins and endogenous ligands in Solanaceae.Fil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Boland, Ricardo Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentin

Presence of vitamin D3 receptor (VDR)-like proteins in solanum glaucophyllum

The detection of 1α,25(OH)2‐vitamin D3 [1α,25(OH)2D3] in Solanum glaucophyllum and other plant species has been reported. Little is known about the mode of action of 1α,25(OH)2D3 in plants. The steroid acts in animal systems through binding to a specific receptor (VDR). In the present work, competition assays allowed the detection of binding sites for [3H]1α,25(OH)2D3 in cultured S. glaucophyllum cells. The binding of [3H]1α,25(OH)2D3 to total protein from calli was a saturable process with respect to the ligand concentration (Kd= 3.49 ± 0.75 nM; Bmax= 255.7 ± 18.6 fmol/mg protein). Western blots employing antibodies directed against the avian VDR revealed three immunoreactive bands of approximately 72, 58 and 43 kDa in different plant organs, cells and callus cultures. The immunoreactive proteins were tyrosine phosphorylated, and the signal detected was increased using hypertonic buffers for protein extraction, suggesting that these proteins are associated with subcellular structures. When the callus cells were subjected to homogenization and differential centrifugation, a substantial proportion of the immunolabeling was localized in the nuclear and mitochondrial fractions. Ligand blot assays revealed the ability of the reactive bands to bind [3H]1α,25(OH)2D3. Immunocytochemistry detection using Oregon Green‐conjugated secondary antibodies showed immunofluorescent staining mainly in the nucleus, and in the nucleus, cytoplasmic corpuscles and outer cell membrane, for the monoclonal and polyclonal antibodies, respectively. [3H]Thymidine incorporation assays and immunoblot analysis of mitogen‐activated protein kinase (MAPK) indicated that 1α,25(OH)2D3 and cell lipid extracts stimulate DNA synthesis but do not activate MAPKs with TEY domains in S. glaucophyllum cells. These data demonstrate that S. glaucophyllum cells, which produce 1α,25(OH)2D3, also express a cognate binding protein(s) and are able to respond to 1α,25(OH)2D3. The exact functional role of the VDR‐like protein (s) remains to be established.Fil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentin