Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line

Toxic effects of ultraviolet (UV) radiation on skin include protein and lipid oxidation, and DNA damage. The latter is known to play a major role in photocarcinogenesis and photoaging. Many plant extracts and natural compounds are emerging as photoprotective agents. Castanea sativa leaf extract is able to scavenge several reactive species that have been associated to UV-induced oxidative stress. The aim of this work was to analyze the protective effect of C. sativa extract (ECS) at different concentrations (0.001, 0.01, 0.05 and 0.1 μg/mL) against the UV mediated-DNA damage in a human keratinocyte cell line (HaCaT). For this purpose, the cytokinesis-block micronucleus assay was used. Elucidation of the protective mechanism was undertaken regarding UV absorption, influence on 1O2 mediated effects or NRF2 activation. ECS presented a concentration-dependent protective effect against UV-mediated DNA damage in HaCaT cells. The maximum protection afforded (66.4%) was achieved with the concentration of 0.1 μg/mL. This effect was found to be related to a direct antioxidant effect (involving 1O2) rather than activation of the endogenous antioxidant response coordinated by NRF2. Electrochemical studies showed that the good antioxidant capacity of the ECS can be ascribed to the presence of a pool of different phenolic antioxidants. No genotoxic or phototoxic effects were observed after incubation of HaCaT cells with ECS (up to 0.1 μg/mL). Taken together these results reinforce the putative application of this plant extract in the prevention/minimization of UV deleterious effects on skin.

Effects of antihypertensive drugs in the differentiation and activation of human bone cells

O osso é um tecido metabolicamente ativo e a sua remodelação é importante para regular e manter a massa óssea. Esse processo envolve a reabsorção do material ósseo por ação dos osteoclastos e a síntese de novo material ósseo mediado pelos osteoblastos. Vários estudos têm sugerido que a pressão arterial elevada está associada a alterações no metabolismo do cálcio, o que leva ao aumento da perda de cálcio e da remoção de cálcio do osso. Embora as alterações no metabolismo ósseo sejam um efeito adverso associado a alguns fármacos antihipertensores, o conhecimento em relação a este efeito terapêutico ligado com os bloqueadores de canais de cálcio é ainda muito escasso. Uma vez que os possíveis efeitos no osso podem ser atribuídos à ação antihipertensiva dessas moléculas, ou através de um efeito direto nas atividades metabólicas ósseas, torna-se necessário esclarecer este assunto. Devido ao facto de que as alterações no metabolismo ósseo são um efeito adverso associado a alguns fármacos antihipertensores, o objetivo deste trabalho é avaliar o efeito que os bloqueadores dos canais de cálcio exercem sobre as células ósseas humanas, nomeadamente osteoclastos, osteoblastos e co-culturas de ambos os tipos celulares. Verificou-se que os efeitos dos fármacos antihipertensores variaram consoante o fármaco testado e o sistema de cultura usado. Alguns fármacos revelaram a capacidade de estimular a osteoclastogénese e a osteoblastogénese em concentrações baixas. Independentemente da identidade do fármaco, concentrações elevadas revelaram ser prejudiciais para a resposta das células ósseas. Os mecanismos intracelulares através dos quais os efeitos foram exercidos foram igualmente afetados de forma diferencial pelos diferentes fármacos. Em resumo, este trabalho demonstrou que os bloqueadores dos canais de cálcio utilizados possuem a capacidade de afetar direta- e indiretamente a resposta de células ósseas humanas, cultivadas isoladamente ou co-cultivadas. Este tipo de informação é crucial para compreender e prevenir os potenciais efeitos destes fármacos no tecido ósseo, e também para adequar e eventualmente melhorar a terapêutica antihipertensora de cada paciente.

New sensing materials of molecularly-imprinted polymers for the selective recognition of Chlortetracycline

As a result of the stressful conditions in aquaculture facilities there is a high risk of bacterial infections among cultured fish. Chlortetracycline (CTC) is one of the antimicrobials used to solve this problem. It is a broad spectrum antibacterial active against a wide range of Gram-positive and Gram-negative bacteria. Numerous analytical methods for screening, identifying, and quantifying CTC in animal products have been developed over the years. An alternative and advantageous method should rely on expeditious and efficient procedures providing highly specific and sensitive measurements in food samples. Ion-selective electrodes (ISEs) could meet these criteria. The only ISE reported in literature for this purpose used traditional electro-active materials. A selectivity enhancement could however be achieved after improving the analyte recognition by molecularly imprinted polymers (MIPs). Several MIP particles were synthesized and used as electro-active materials. ISEs based in methacrylic acid monomers showed the best analytical performance according to slope (62.5 and 68.6 mV/decade) and detection limit (4.1×10−5 and 5.5×10−5 mol L−1). The electrodes displayed good selectivity. The ISEs are not affected by pH changes ranging from 2.5 to 13. The sensors were successfully applied to the analysis of serum, urine and fish samples.

“Papel dos recetores purinérgicos no crescimento e temodelação do tecido cavernoso”

A adenosina é um nucleósido ubíquo envolvido na regulação de controlo do tónus vascular do tecido cavernoso, desempenhando um papel importante na fisiopatologia da Disfunção Erétil (DE) resistente aos fármacos relaxantes musculares clássicos. Apesar da importância comprovada dos recetores da adenosina na fisiopatologia da DE no homem, pouca informação é conhecida no que diz respeito à expressão e localização dos recetores purinérgicos no Tecido Cavernoso de Ratazana (TCR). Neste trabalho avaliou-se o fenótipo dos recetores purinérgicos responsáveis pela regulação do tónus do tecido erétil de ratazana por imunofluorescência indireta aplicada à microscopia confocal em co-culturas de células endoteliais e musculares lisas do TCR. Para além da caracterização imunofenotípica, desenvolveu-se uma técnica que permite diferenciar funcionalmente em tempo real (por microscopia confocal funcional) células musculares lisas e células endoteliais isoladas de TCR em co-cultura marcadas com a sonda fluorescente Fluo-4NW. Esta técnica permite distinguir cada um dos subtipos celulares mediante o padrão e a magnitude das oscilações dos níveis intracelulares de Ca2+ ([Ca2+]i) em resposta ao ATP (agonista P2) e à fenilefrina (PE, agonista α-adrenérgico). Nas células musculares lisas, observou-se uma resposta mais acentuada ao agonista α-adrenérgico, PE, e uma resposta menos significativa ao ATP. O contrário foi observado relativamente às células endoteliais. A incubação das células musculares lisas e endoteliais com ATP (300 μM) causou um aumento dos níveis de [Ca2+]i. O efeito do ATP (300 μM) parece envolver a ativação de recetores dos subtipos P2X1 e P2X3 sensíveis ao bloqueio com NF023 (3μM) e A317491 (100 nM), respetivamente. Já o aumento dos níveis [Ca2+]i produzido pelo ADP (300 μM) parece envolver a ativação de recetores P2Y1, P2Y12 e P2Y13 mediante o antagonismo produzido pelos antagonistas MRS 2179 (0,3μM), AR-C66096 (0,1 μM) e MRS 2211 (10μM), respetivamente. Os dois tipos celulares expressam imunorreatividade contra recetores A2A, A2B, P2X1, P2X3, P2Y1, P2Y12 e P2Y13.

Bisphenol A migration from plastic materials: direct insight of ecotoxicity in Daphnia magna

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) whose migration from food packaging is recognized worldwide. However, the real overall food contamination and related consequences are yet largely unknown. Among humans, children’s exposure to BPA has been emphasized because of the immaturity of their biological systems. The main aim of this study was to assess the reproductive impact of BPA leached from commercially available plastic containers used or related to child nutrition, performing ecotoxicological tests using the biomonitoring species Daphnia magna. Acute and chronic tests, as well as single and multigenerational tests were done. Migration of BPA from several baby bottles and other plastic containers evaluated by GC-MS indicated that a broader range of foodstuff may be contaminated when packed in plastics. Ecotoxicological test results performed using defined concentrations of BPA were in agreement with literature, although a precocious maturity of daphnids was detected at 3.0 mg/L. Curiously, an increased reproductive output (neonates per female) was observed when daphnids were bred in the polycarbonate (PC) containers (145.1±4.3 % to 264.7±3.8 %), both in single as in multigenerational tests, in comparison with the negative control group (100.3±1.6 %). A strong correlated dose-dependent ecotoxicological effect was observed, providing evidence that BPA leached from plastic food packaging materials act as functional estrogen in vivo at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non-PC bottles was slightly but not significantly enhanced (92.5±2.0 % to 118.8±1.8 %). Multigenerational tests also revealed magnification of the adverse effects, not only on fecundity but also on mortality, which represents a worrying trend for organisms that are chronically exposed to xenoestrogens for many generations. Two plausible explanations for the observed results could be given: a non-monotonic dose–response relationship or a mixture toxicity effect.

Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Obesidade e cancro da próstata: Avaliação do efeito dos adipócitos nas células RM1 de carcinoma da próstata

O cancro da próstata é o segundo cancro mais frequente e a sexta causa de morte mundial por cancro no sexo masculino. A obesidade tem sido associada ao aumento da incidência e mortalidade por cancro, com alguma controvérsia. As alterações nas expressões de adipocinas associadas à obesidade têm sido um dos diversos mecanismos propostos para explicar a associação entre a obesidade e o cancro da próstata, nomeadamente na promoção do desenvolvimento e progressão celular do tumor. O objetivo deste trabalho é avaliar o efeito dos fatores produzidos pelos pré-adipócitos e os adipócitos na proliferação, migração e invasão das células de carcinoma da próstata independentes dos androgénios. As células RM1 foram cultivadas na presença de diferentes concentrações de insulina e leptina, bem como em meio condicionado (MC) de pré-adipócitos e adipócitos e co-cultivadas em sistema de transwells, com as mesmas células. A proliferação celular das RM1 foi avaliada recorrendo a contagem celular em camara de Neubauer e em citometro de fluxo, e aos ensaios metabólicos alamar blue e XTT. Efetuou-se um ensaio de migração por dano nas células RM1 na presença dos meios condicionados. A invasão das células foi avaliada recorrendo a um sistema de transwells, com membrana de matrigel, quando cultivadas com pré-adipócitos e adipócitos. A insulina aumentou significativamente a proliferação celular, ao contrário da leptina que não teve efeito. O meio condicionado dos pré-adipócitos aumentou ligeiramente a proliferação, enquanto meio condicionado dos adipócitos de 1 e 2 dias aumentou significativamente a proliferação das células RM1 (p<0.01), quando avaliada por XTT. Na câmara de Neubauer não se verificaram diferenças significativas na proliferação celular. Relativamente à migração celular, observou-se um aumento significativo da migração das células RM1 cultivadas com meio condicionado de adipócitos (MCA) e pré-adipócitos (MCPA) em comparação com o controlo (p<0.01). Observou-se um aumento significativo da invasão de células RM1 cultivadas com adipócitos e pré-adipócitos (p <0.05). Os adipócitos aumentaram significativamente a proliferação das células RM1 em co-cultura (p<0.01). Em conclusão, as células RM1 parecem ser influenciadas por fatores secretados pelos adipócitos, capazes de aumentar a sua capacidade de proliferar, invadir e migrar.

Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma Cells

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

Picocyanobacteria From a Clade Of Marine Cyanobium Revealed Bioactive Potential Against Microalgae, Bacteria, And Marine

The production of bioactive compounds either toxic or with pharmacological applications by cyanobacteria is well established. However, picoplanktonic forms within this group of organisms have rarely been studied in this context. In this study, the toxicological potential of picocyanobacteria from a clade of marine Cyanobium strains isolated from the Portuguese coast was examined using different biological models. First, strains were identified by applying morphological and molecular approaches and cultured under lab conditions. A crude extract and three fractions reflecting a preliminary segregation of lipophilic metabolites were tested for toxicity with the marine microalga Nannochloropsis sp., the bacteria Pseudomonas sp., the brine shrimp Artemia salina, and fertilized eggs of the sea urchin Paracentrotus lividus. No significant apparent adverse effects were noted against Artemia salina. However, significant adverse effects were found in all other assays, with an inhibition of Nannochloropsis sp. and Pseudomonas sp. growth and marked reduction in Paracentrotus lividus larvae length. The results obtained indicated that Cyanobium genus may serve as a potential source of interesting bioactive compounds and emphasize the importance of also studying smaller picoplanktonic fractions of marine cyanobacteria.

New sensing materials of molecularly-imprinted polymers for the selective recognition of Chlortetracycline

As a result of the stressful conditions in aquaculture facilities there is a high risk of bacterial infections among cultured fish. Chlortetracycline (CTC) is one of the antimicrobials used to solve this problem. It is a broad spectrum antibacterial active against a wide range of Gram-positive and Gram-negative bacteria. Numerous analytical methods for screening, identifying, and quantifying CTC in animal products have been developed over the years. An alternative and advantageous method should rely on expeditious and efficient procedures providing highly specific and sensitive measurements in food samples. Ion-selective electrodes (ISEs) could meet these criteria. The only ISE reported in literature for this purpose used traditional electro-active materials. A selectivity enhancement could however be achieved after improving the analyte recognition by molecularly imprinted polymers (MIPs). Several MIP particles were synthesized and used as electro-active materials. ISEs based in methacrylic acid monomers showed the best analytical performance according to slope (62.5 and 68.6 mV/decade) and detection limit (4.1 × 10−5 and 5.5 × 10−5 mol L−1). The electrodes displayed good selectivity. The ISEs are not affected by pH changes ranging from 2.5 to 13. The sensors were successfully applied to the analysis of serum, urine and fish samples.

Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and -smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.