Copaiba Oil-Loaded Polymeric Nanocapsules: Production and In Vitro Biosafety Evaluation on Lung Cells as a Pre-Formulation Step to Produce Phytotherapeutic Medicine.

Copaiba oil has been largely used due to its therapeutic properties. Nanocapsules were revealed to be a great nanosystem to carry natural oils due to their ability to improve the bioaccessibility and the bioavailability of lipophilic compounds. The aim of this study was to produce and characterize copaiba oil nanocapsules (CopNc) and to evaluate their hemocompatibility, cytotoxicity, and genotoxicity. Copaiba oil was chemically characterized by GC-MS and FTIR. CopNc was produced using the nanoprecipitation method. The physicochemical stability, toxicity, and biocompatibility of the systems, in vitro, were then evaluated. Β-bisabolene, cis-α-bergamotene, caryophyllene, and caryophyllene oxide were identified as the major copaiba oil components. CopNc showed a particle size of 215 ± 10 nm, a polydispersity index of 0.15 ± 0.01, and a zeta potential of -18 ± 1. These parameters remained unchanged over 30 days at 25 ± 2 °C. The encapsulation efficiency of CopNc was 54 ± 2%. CopNc neither induced hemolysis in erythrocytes, nor cytotoxic and genotoxic in lung cells at the range of concentrations from 50 to 200 µg·mL-1. In conclusion, CopNc showed suitable stability and physicochemical properties. Moreover, this formulation presented a remarkable safety profile on lung cells. These results may pave the way to further use CopNc for the development of phytotherapeutic medicine intended for pulmonary delivery of copaiba oil.

Development and validation of a simple questionnaire for the identification of hereditary breast cancer in primary care.

BACKGROUND: Breast cancer is a significant public health problem worldwide and the development of tools to identify individuals at-risk for hereditary breast cancer syndromes, where specific interventions can be proposed to reduce risk, has become increasingly relevant. A previous study in Southern Brazil has shown that a family history suggestive of these syndromes may be prevalent at the primary care level. Development of a simple and sensitive instrument, easily applicable in primary care units, would be particularly helpful in underserved communities in which identification and referral of high-risk individuals is difficult. METHODS: A simple 7-question instrument about family history of breast, ovarian and colorectal cancer, FHS-7, was developed to screen for individuals with an increased risk for hereditary breast cancer syndromes. FHS-7 was applied to 9218 women during routine visits to primary care units in Southern Brazil. Two consecutive samples of 885 women and 910 women who answered positively to at least one question and negatively to all questions were included, respectively. The sensitivity, specificity and positive and negative predictive values were determined. RESULTS: Of the 885 women reporting a positive family history, 211 (23.8%; CI95%: 21.5-26.2) had a pedigree suggestive of a hereditary breast and/or breast and colorectal cancer syndrome. Using as cut point one positive answer, the sensitivity and specificity of the instrument were 87.6% and 56.4%, respectively. Concordance between answers in two different applications was given by a intra-class correlation (ICC) of 0.84 for at least one positive answer. Temporal stability of the instrument was adequate (ICC = 0.65). CONCLUSION: A simple instrument for the identification of the most common hereditary breast cancer syndrome phenotypes, showing good specificity and temporal stability was developed and could be used as a screening tool in primary care to refer at-risk individuals for genetic evaluations.

Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence.

BACKGROUND: Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an alpha-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface. RESULTS: The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. CONCLUSION: The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.

Biochemical responses of the marine mussel Mytilus galloprovincialis to petrochemical environmental contamination along the North-western coast of Portugal.

Following the development of urban and industrial centres petrochemical products have become a widespread class of contaminants. The aim of this study was to investigate the effects of petrochemical contamination in wild populations of mussels (Mytilus galloprovincialis) along the NW Atlantic coast of Portugal by applying antioxidant and energetic metabolism parameters as biomarkers. For that, mussels were collected at five sampling sites presenting different petrochemical contamination levels. To evaluate the mussels' antioxidant status, enzymatic activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione S-transferases, as well as glutathione redox status were evaluated in gills and digestive glands of mussels collected from the selected sites. Lipid peroxidation was determined in the same tissues to quantify cellular oxidative damage. Furthermore, to investigate how energetic processes may respond to these contaminants, the activity of NADP(+)-dependent isocitrate dehydrogenase was determined in mussels' digestive glands, and octopine dehydrogenase was determined in mussels' posterior adductor muscle. Furthermore, the concentrations of aliphatic hydrocarbons, unresolved complex mixture and polycyclic aromatic hydrocarbons (PAHs) were quantified in mussels' tissue, and abiotic parameters were quantified in water samples collected at each site. Several biomarkers showed statistically significant differences among sampling sites. The redundancy analysis (RDA) used to perform the integrated analysis of the data showed a clear separation of the sampling sites in three different assemblages, which are in agreement with the PAHs levels found in mussels tissues. In addition, the RDA indicated that some of the selected biomarkers may be influenced by abiotic parameters (e.g. salinity, pH, nitrates and ammonia). The approach selected for this study seems to be suitable for monitoring petrochemical contamination.

Effects of estuarine sediment contamination on feeding and on key physiological functions of the polychaete Hediste diversicolor: Laboratory and in situ assays.

This study aimed at integrating postexposure feeding and some biochemical parameters in the responses of the estuarine polychaeta, Hediste diversicolor, to controlled laboratory exposure conditions and to in situ exposures scenario of sediment contamination. Since H. diversicolor feeding may be considered as a major rate-limiting step in the processing of detritus in European estuaries, a reduction in feeding activity may have implications not only at the individual and population level of the species but also in detritus processing and in organic matter decomposition rates at the ecosystem level. The biochemical parameters were chosen as indicators of four key physiological functions: neurotransmission, metabolic condition, detoxification processes and antioxidant defences. The Mira and Sado estuaries, located in the Southwest coast of Portugal and classified as undisturbed and impacted, respectively, were selected as sites for this study. A significant depression in H. diversicolor postexposure feeding (from 30 to 70%) was consistently detected in all impacted sediments, supporting the sensitivity and responsiveness of feeding as a sublethal toxicity endpoint. Alongside with a reduced energy intake, an increased rate of organisms' anaerobic metabolism, as evidenced by an enhancement of lactate dehydrogenase activity (up to 1.5-fold), suggested a rapid need of additional energy to ameliorate chemical stress. Moreover, oxidative stress was shown to be an important mechanism of toxicity of the impacted sediments in H. diversicolor, as evidenced by a marked reduction in the glutathione redox status (up to 6.5-fold) and an increase in lipid peroxides levels (up to 2.3-fold) in organisms exposed to the most impacted sediments. Results of the in situ assay, conducted to assess the ecological relevance of sediment laboratory toxicity estimates and their application to make valid field extrapolations, revealed a lack of agreement in the response of catalase in organisms exposed to moderate impacted sediments. Our results support the utility of integrating responses at individual and sub-individual level to evaluate potential toxicant-induced changes in key physiological functions of H. diversicolor and to interpret their potential ecological consequences.

An in situ postexposure feeding assay with Carcinus maenas for estuarine sediment-overlying water toxicity evaluations.

This study developed and evaluated a short-term sublethal in situ toxicity assay for estuarine sediment-overlying waters, with the crab Carcinus maenas (L.) based on postexposure feeding. It consisted of a 48-h in situ exposure period followed by a short postexposure feeding period (30 min). A precise method for quantifying feeding, using the Polychaeta Hediste (Nereis) diversicolor Müller as food source, was first developed. The sensitivity of the postexposure feeding response was verified by comparing it to that of lethality, upon cadmium exposure. The influence of environmental conditions prevailing during exposure (salinity, temperature, substrate, light regime, and food availability) on postexposure feeding was also addressed. The potential of this in situ assay was then investigated by deploying organisms at ten sites, located in reference and contaminated Portuguese estuaries. Organism recovery ranged between 90% and 100% and a significant postexposure feeding depression (16.3-72.7%) was observed at all contaminated sites relatively to references.

Short-term sublethal (sediment and aquatic roots of floating macrophytes) assays with a tropical chironomid based on postexposure feeding and biomarkers.

This study proposes assays with a freshwater chironomid, Chironomus xanthus, distributed over South America, based on subindividual (acetylcholinesterase activity) and individual (survival and postexposure feeding) level endpoints. Sediment and aquatic-rooted floating macrophyte assays were developed, due to the importance of both substrates for toxicant exposure in subtropical/tropical environments. Assays were evaluated under realistic exposure scenarios by simulating a runoff over an agricultural field dosed with deltamethrin. In situ assays were performed within microcosms to discriminate the effects of deltamethrin from those of additional potential stress factors (organism handling and caging, microcosms, use of aquatic roots, and runoff per se). A laboratory sediment assay based on feeding was conducted with samples from the microcosms. In all assays, both sublethal endpoints were responsive to deltamethrin and more sensitive than survival. Survival and feeding were more sensitive in situ than in the laboratory. In the in situ sediment assays, both sublethal endpoints were within a similar range of sensitivity; they were significantly inhibited as of the lowest Decis dose, from approximately 20 to 70%. In situ feeding was more sensitive in the sediment than in the macrophyte assay, where it was inhibited significantly only at the two highest Decis doses (up to approximately 60%). Larval performance was not influenced significantly by any of the other potential stress factors.