7 results
Effects of exposure to oxide nanoparticles (Al2O3and ZnO) singly and mixtures onCarassius auratusgills
- M. Benavides, P. Coelho, C. Lodeiro, M.S. Diniz
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- Journal:
- Microscopy and Microanalysis / Volume 21 / Issue S6 / August 2015
- Published online by Cambridge University Press:
- 10 September 2015, pp. 18-19
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- August 2015
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Effects of ultra-sonication on the cyanobacteria Microcystis aeruginosa structure and growth
- H. Muelle, P. Barquinha, I. Ferreira, E. Fortunato, M. Conceição Santos, M.S. Diniz
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- Journal:
- Microscopy and Microanalysis / Volume 21 / Issue S6 / August 2015
- Published online by Cambridge University Press:
- 10 September 2015, pp. 50-51
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- August 2015
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Toxicity study of new metal nanoparticles functionalized with fluorescein derivatives as novel image systems
- A. Fernandéz-Lodeiro, J. Fernandéz-Lodeiro, C. Nuñez, E. Oliveira, H.M. Santos, C. Lodeiro, J.L. Capelo, M.S. Diniz
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 25-26
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- August 2013
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Nanoparticles in general (NPs) and/or nanomaterials offer remarkable opportunities in industrial production, daily consumables, medicine, biotechnology, electronics and numerous other important commercial and economical areas. Among all these areas, nanomedicine has opened novel treatments for problematic diseases such as viral, genetic, cancer, AIDS, etc. There is limited information available regarding translocation and distribution of NPs in the body and in the environment. Additionally, there is also need for more information on NPs toxicity. Recently has been demonstrated that physiological barriers such as pulmonary and gastro-intestinal tract are affected.
The main objective of this work is to use functionalized metal NPs, as emissive agent markers, assess their internalization in cells and evaluate toxicity to cells.
Using the emissive two probes synthesized in a one-pot reaction using fluoresceine as chromophore, several gold (Au), round shape, and silver (Ag) NPs (round and triangular shapes) were functionalized in organic media and water by Brust and Turkevish methodology, using tetraoctylammonium bromide (TOABr) as a common stabilizer and sodium borohydride as reducing agent. All has been characterized by UV-vis and emission spectroscopy, transmission electron microscopy (TEM) (Figure 1), and Light scattering. To study the route of internalization into the cell NP-complexes were injected intraperitoneally in fish (Carassius auratus). After 48 hour fish were sampled and sacrificed and liver and intestine processed for histology examination. Additional sub-samples were stored at – 80ºC for enzymatic analysis (glutathione-S-transferase and catalase). Blood was also collected from healthy non-injected fish, for leucocyte separation followed by incubation with the metal NPs and cell viability assays. The presence of emissive NPs in cells was examined by microscopy using a Leica microscope (ATC 2000) adapted for epifluorescence (EF).
The microscopy analysis showed that apparently both metal NPs were internalized by leucocytes and intestine cells (Figure 2a and 2b) but apparently not by hepatocytes. However, it is still to clarify if NPs internalization occurred in dead or dying cells only, with more permeable membranes, or also in living cells. Another possibility relates to the detection limits and resolution of the microscope used: the fraction of NPs entering is too low and not detectable with this type of equipment. No significant fluorescence was detected in controls. Viability assays showed higher mortality rates in leucocytes incubated with triangular Ag NPs suggesting that the type of metal and shape have influence in cell toxicity. In general, enzymatic assays indicate low oxidative stress for cells. However, GST results show significant (p > 0.05) differences in livers from fish injected with round Ag NPs. With respect to catalase, significant differences (p > 0.05) were detected in livers from fish injected with round Au NPs. Although the presented results are preliminary they suggest that functionalized NPs are able to penetrate cell membranes. On the other hand, the observed toxicity can be attributed to differences in shape and type of metal NPs.
The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/MAR/119068/2010 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.
Sea warming affects bream (Sparus aurata) tissues and stress proteins (HSP70)
- D. Madeira, C. Vinagre, R. Rosa, P.M. Costa, M.H. Costa, S. Caeiro, M. Galésio, H.M. Santos, C. Nuñez, E. Oliveira, L. Castro, I. Peres, C. Lodeiro, J.L. Capelo, M.S. Diniz
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 83-84
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- August 2013
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The increase in CO2 emissions from anthropogenic sources may not only result in temperature increase on a global scale but also in increased ocean acidification (OA), by lowering the ocean's capacity to absorb additional atmospheric CO2. As a consequence, changes in ocean chemistry are prone to occur through the imbalancing of sea-atmosphere gas exchange, thus affecting O2 absorption as well.
There are many studies on the potential effects of OA and increased ocean temperatures on the physiology of marine organisms but little is known about changes at histological level and there are still several gaps at molecular level which must be studied for a better understanding of all biological mechanisms involved. With regard to hypoxia, alterations to the stress response can provide information on the organisms’ physiological effects and coping strategies triggered by anoxia. For instance, it is known that organisms respond by reducing protein synthesis. Once temperature affects physiological, behavioral and ecological processes, there is a need to understand what mechanisms are behind the organisms’ response to stress, enhancing our predictive and environmental management capacities considering a climate change scenario. This is of great importance, in particular to countries with a sea-based economy.
The aim of the present study is to assess the stress response of a marine fish, sea bream (Sparus aurata) exposed to increasing water temperature and different water acidity (alone or in combination). Here we present preliminary data on temperature effects on S. aurata at a cellular and molecular level. In addition, tissue samples from muscle, livers, gills and intestine are examined to evaluate any alterations caused by altering this physical parameter.
Fish were distributed randomly in tanks (n=96) and allowed to acclimate at 18ºC (the same temperature of the hatchery) before the beginning of the bioassays. After assessment of the upper thermal limits (UPL), water temperature was increased at a rate of 1ºC per hour using a thermostatized bath with a constant rate of water-temperature until reaching the endpoint, following the dynamic method of Critical Thermal Maximum (CTM). Every 2ºC step, fish were euthanized by cervical transection and the selected organs removed and stored at -80ºC until further analysis. Sub-samples were taken and processed for histological examination following standard techniques. Frozen samples were analysed for heat stress proteins (HSP70) as described by Madeira et al. The histological observations were carried out using a Leica microscope (DMLB model). Preliminary results of the histological examination showed changes in the cellular structure, with visible damage at higher temperatures in liver and gills (Fig. 1). Regarding HSP70, significant changes were observed throughout the temperature assay. The results indicate that elevated water temperature can be a major stressor that will affect fish due to potential climate changes, thus compelling the need to perform these studies to enhance our predictive and environmental management capacities.
The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/MAR/119068/2010 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.
Histological and biochemical effects of exposure to TiO2 nanoparticles in livers of two freshwater fish species: Carassius auratus and Danio rerio
- M.S. Diniz, A.P. Alves de Matos, J. Lourenço, L. Castro, I. Peres, E. Mendonça, A. Picado
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 51-52
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- August 2013
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Nanoparticles (NPs), particles with at least one dimension less than 100 nm, are used in many industrial applications and to produce new types of materials with unique physicochemical properties. The aquatic environment is commonly the ultimate recipient for NPs and there is uncertainty of exposure as understanding and data regarding the potential detrimental effects of NPs on aquatic biota are missing. In this study, titanium dioxide (TiO2) was chosen for its potential use in technology and diverse industrial applications. The objective of this work is to evaluate the toxicity of TiO2 NPs on total liver glutathione-S-transferase (GST), lipid peroxidation and tissue structure of the livers of two freshwater fish species (Carassius auratus and Danio rerio).
Stock suspensions of TiO2 NPs, with an average size of 21 nm, were prepared using distilled water and then ultrasonicated (10 min, 35 KHz). The suspensions were added to 10L of tap water in exposure tanks, to obtain nominal concentrations (0.01; 0.1; 1, 10; 100 TiO2 mg/L). The test fish, C. auratus (N=144) and D. rerio (N=80), were randomly distributed by 6 exposure tanks and an additional tank with clean tap water was used as control. Fish were sampled after 7, 14, and 21 days. Six fish from both species were left for depuration in clean tap water during 14 days and then sacrificed. Immediately after sampling the fish were processed for enzymatic determination and histopathology. The GST activity was determined by following the procedure described by Habig et al. and lipid peroxidation was measured based on the Thiobarbituric Acid Reactive Species method. The tissues were processed essentially according to Martoja and Martoja for light microscopy (LM). For transmission electron microscopy (TEM) the samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite according to standard procedures. The histological and ultra-structural observations were carried out using a Leica microscope (Leica-ATC 2000) and a JEOL 100-SX electron microscope respectively.
The results showed increased activities of the GST in livers with increasing TiO2 NP concentrations after 7 days of exposure, however after 14 days a trend to decrease was observed for both species. The GST results suggest that the increase of activity of these detoxification enzymes can be a response to oxidative stress caused by the generation of reactive oxygen species by the NP. On the other hand, size, chemical composition, surface area, shape, solubility and aggregation may also contribute for NPs toxicity. The results from lipid peroxidation showed an increase according to tested concentrations suggesting that TiO2 NPs is able to cause cell damage and is in agreement with biochemical and histological findings. After 14 days of depuration, GST and lipid peroxidation levels were not significant different from controls suggesting that cells are able to recover in a certain degree. The results from LM (Fig. 1) showed that exposure to TiO2 NPs affected liver structure, with more pronounced changes detected in livers from fish exposed to higher concentrations. Observed changes include tissue degeneration, inflammation and pyknosis among others. The TEM analysis revealed also severe changes in liver cells compatible with oxidative stress. Hepatocytes of treated fish showed glycogen depletion, swollen mitochondria and increased lysosomes, compared to controls. After depuration, some cells recovered nearly normal morphology, but retained the lysosomes, while others underwent necrotic changes (Fig. 2). Differences among the two species studied were of a quantitative nature, and more pronounced in Danio rerio.
The results suggest that potential risk to fish health exist related to the TiO2 NPs release to the aquatic environment and may cause deleterious effects in aquatic organisms. It is evident that the effects of TiO2 NPs on environment is a matter of great concern and the precise mechanisms of toxicity of this and other types of NPs must be clarified.
The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/CTM/099446/2008 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.
Are squid statoliths hollow during embryogenesis?
- R. Rosa, G. Dionísio, A. Bensimon-Brito, A. Jacinto, M.S. Pimentel, K. Trübenbach, A. Moreno, P. Barquinha, M. Diniz
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 89-90
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- August 2013
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Squid statoliths are paired calcareous concretions essentially composed of calcium carbonate crystallised as aragoniteand found inside cranial fluid-filled cavities, the statocysts - the main organ of detection of gravity and movement. Statolith use as an ageing tool was validated in the 1980s, when the daily periodicity of the growth increments was confirmed by chemically marking these structures in animals maintained in aquaria. Although statolith analysis has brought major advances in the knowledge of squid population-dynamics, little is known about the processes involved in statolith’s calcification and increment deposition (e.g. during embryogenesis). According to Villanueva (2000), embryonic statolith development involves the formation of structures such as the focus, nucleus, postnuclear zone and natal ring.
The aim of this study was to investigatethe microstructure of the embryonic statolith, considering all main structures,in recently-hatched squids using optical (fluorescence) microscopy, scanning electron microscopy (SEM) and confocal microscopy.
Recently-spawned egg masses of the European squid, Loligo vulgaris, were collected in the west coast of Portugal. After hatching at 13-15ºC, statoliths were removed from the paralarvae andpreserved at -20ºC. Prior to confocal analysis, statoliths were submitted to Alizarin red S (ARS) staining (5% of ARS in 0.1% KOH) to verify the presence of calcium content. In addition, images from optical microscopy and scanning electron microscopy (SEM) were acquired.
The findings obtained by auto-fluorescence observation (Figure 1 A, B) and SEM (Figure 1 C), show that the embryonic statolith is hollow in some of the area between the nucleus and the natal ring. ARS stained samples analysed in confocal microscopy (Figure 2 A-G), confirmed the previous observation.
This surprising structural feature in the origin of CaCO3 sensorial structures has never been described before and suggests a production, with minimum of material and energy expenditure. This is fundamentally interesting not only in biological terms but also considering biotemplating orbiomimetics approaches (i.e. synthetic approaches), where hollow calcium carbonate structures have attracted considerable attention owing to their unique structural, optical, and surface properties that lead them to a wide range of applications, including as templates for functional architecture composite materials.
The Portuguese Foundation for Science and Technology (FCT) supported this study through project grant PTDC/BIA-BEC/103266/2008 to R. Rosa.
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TiO2 nanoparticles intake by fish gill cells following exposure
- M.S. Diniz, A.P. Alves de Matos, J. Lourenço, L. Castro, I. Peres, E. Mendonça, A. Picado
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- Journal:
- Microscopy and Microanalysis / Volume 19 / Issue S4 / August 2013
- Published online by Cambridge University Press:
- 06 August 2013, pp. 71-72
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- August 2013
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Engineered nanomaterials such as nanoparticles (NPs) are increasingly being used for commercial purposes in products within medicine, electronics, sporting goods, tires, textiles and cosmetics. They comprise diverse types of materials from metals, polymers, ceramic to biomaterials and have been defined as particles with at least one dimension in the order up to 100 nm1. The higher toxicological potential of NPs is mostly due to their small size, wide surface, increase of their chemical reactivity and biological activity and the capacity to generate free radicals. NPs also can have the ability to penetrate trough the biological barriers and to move easily through the biological systems. Therefore, is mandatory to assess the toxicity of these nanomaterials, since their industrial production and uses will also result in releases to the environment with unclear consequences.
The aim of the present work is to evaluate the toxicity of titanium dioxide (TiO2) NPs on gill gluthatione-S-transferase activity (GST), lipid peroxidation and on structure of the gills of two freshwater fish species (Carassius auratus and Danio rerio). Suspensions of TiO2 NPs, with an average size of 21 nm, were prepared using distillate water and then ultrasonicated (10 min, 35 KHz). The suspensions were added to 10L of tap water in exposure tanks, to obtain nominal concentrations (0.01; 0.1; 1, 10; 100 TiO2 mg/L). The test fish, C. auratus (N=144) and D. rerio (N=80), were randomly distributed by 6 exposure tanks and an additional tank with clean tap water was used as control. Fish were sampled after 7, 14, and 21 days. Six fish from both species were left for depuration in clean tap water during 14 days and then sacrificed. The GST activity was determined by following the procedure described by Habig et al. and lipid peroxidation was measured based on the Thiobarbituric Acid Reactive Species method. The tissues were processed essentially according to Martoja and Martoja for light microscopy (LM). For transmission electron microscopy (TEM) the samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite according to standard procedures. The histological and ultrastructural observations were performed using a Leica-ATC 2000 microscope and a JEOL 100-SX electron microscope respectively.
The results show a significant increase of GST in gills tissues for C.auratus exposed to 10 and 100 mg/L TiO2 NPs and a decrease following the depuration period. With respect to D. rerio a significant increase was observed in fish exposed to 1, 10 and 100 mg/L TiO2 NPs. Lipid peroxidation are in agreement with GST results but showing a significant increase for fish (C.auratus and D. rerio) exposed to concentrations of 0.1 TiO2 mg/L NPs and higher. Usually, the oxidative stress caused by exposure to TiO2 NPs is attributed to hydroxyl radicals (OH) generated by photochemical (UV/vis) processes but it may be also related to specific properties of TiO2 NPs such as size, surface area and solubility that can influence the degree of toxicity. The results from LM observations (Fig. 1) showed that exposure to TiO2 NPs affected gill tissues, with changes being detected in both fish species exposed to 0.1 TiO2 mg/L NPs and higher which is in accordance with biochemical results. Changes include different degrees of hyperplasia (from low to complete fusion of lamellae). The TEM analysis revealed that TiO2 NPs were internalized by gills epithelial cells accumulating in vacuoles inside these cells (Fig. 2). After the depuration period it was observed that the capability for gills to recover was not complete. The results show a strong response to oxidative stress caused by exposure to TiO2 NPs, possibly because they are in direct contact with the exposure medium and function as a first barrier against external aggression. However, the gills changes observed following exposure and a partial recover after depuration suggest that TiO2 NPs may cause deleterious effects in fish gills compromising fish homeostasis.
The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/CTM/099446/2008 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.