Histochemical technique for the detection of chloride cells in fish

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bioactivity of extracts from extremophile microalgae Coccomyxa sp. on chloride cells in operculum tissue of Fundulus heteroclitus

Microalgae have been studied because of their great potential as a source of new compounds with important value for biotechnology and to understand their strategies of survival in extreme environments. The microalgae Coccomyxa sp., studied in this thesis, is a poly-extremophile witch was isolated from the acid mine drainage of S. Domingos mine. This environment is characterized by low pH (<3) and high concentration of metals, such as copper and iron. The main purpose of the present work was to evaluate the potential bioactivity in an ex-vivo animal model (Fundulus heteroclitus), and expression on selected genes, of cellular extracts obtained from cultures of Coccomyxa sp. at pH 7 without or with exposure to copper (0.6mM Cu²+). The extracts of Coccomyxa sp. cultured at pH 7 exposed to copper show a great potential to be used as epithelial NKCC inhibitors, revealing their potential use as diuretics, but did not show significant effects on gene expression. Coccomyxa sp. could be a good source of cellular extracts with a great potential to be used in pharmaceutical and biotechnology industries.

Osmoregulatory actions of growth hormone in juvenile tilapia (Oreochromis niloticus)

Growth hormone (GH) effectively promotes seawater (SW) adaptation in salmonids, but little is known of its effect in tilapias. Experiments were performed to investigate the effects of recombinant eel GH (reGH) on osmoregulatory actions and ultrastructural features of gill chloride cells in juvenile tilapia, Oreochromis niloticus. Tilapia showed a markedly improved SW survival, when directly transferred from freshwater (FW) to 62.5% SW 24h after a single reGH injection (0.25 or 2.5 mu g g(-1)) or 3 reGH injections (0.25 mu g g(-1) every other day). Plasma Na+ and Mg2+ levels were significantly reduced by reGH (0.25 and 2.5 mu g g(-1)) compared with saline injections; Ca2+ concentrations were reduced significantly by high dose of reGH (2.5 mu g g(-1)) after SW transfer. However, fish failed to survive more than 24h when directly transferred to 70 % SW, although the fish treated with reGH could survive longer than the controls. When examined by electron microscopy, the chloride cells were identified as mitochondrion-rich and an extensive tubular system was induced by GH treatment. The results of the present study suggest that, similar to its effect on salmonids, GH also exerts acute osmoregulatory actions and enhances SW adaptation in juvenile tilapia. GH also stimulates the differentiation of chloride cells toward SW adaptation.

Histopathological biomarkers in pacu (Piaractus mesopotamicus) infected with aeromonas hydrophila and treated with antibiotics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Health variables and gill morphology in the tropical fish Astyanax fasciatus from a sewage-contaminated river

The relative condition factor (Kn), gonadosomatic index (GSI), selected hematological variables and gill morphology of the fish Astyanax fasciatus were analyzed in two sites (site I was unpolluted and site 2 was polluted with untreated domestic sewage) of a tropical river (Camanducaia river, São Paulo State, Brazil). The relationship between the body mass (M-B) and the standard length (L-S) of A. fasciatus from both sites was M-B = 0.00799 L-S(3.51843). Tyhe Kn values from both the males and females were significantly higher in site 2. The mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were higher in females from site 2. Gill tissue anomalies and gill parasites were rare in fish from both sites; however, the number of chloride cells was significantly higher in fish from site 1. A. fasciatus presents high capacity to live in ion-poor and soft water and is able to compensate for environmental changes caused by untreated domestic sewage discharges. (c) 2004 Elsevier B.V. All rights reserved.

Utilização de peixes nativos da Amazônia como biomarcadores na avaliação da qualidade da água de uma área industrial no rio Pará - PA - Brasil

A dissertação foi elaborada no formato de artigo, intitulado de “Immunohistochemical and structural biomarkers in two fish species exposed to the industrial area in Amazon Estuary”, submetido à revista Environmental Monitoring and Assessment, formatado segundo os padrões da revista, não contendo resumo em português.

Análise morfológica de brânquias de peixes Oreochromis niloticus (tilápia) expostos ao níquel

Water is an essential factor in maintaining the vital functions of living beings. We have observed a growing commitment of quality, are due to pollution from many sources and even entire watersheds, whether for industrial waste, sewage, or for substances used in farming such as pesticides, herbicides and fertilizers. Nickel is the 24th most abundant element on earth, is a heavy metal that, in the form of chloride, is a proven genotoxic and mutagenic. Due to its industrial use, there was considerable increase of its concentration in surface sediments. Fish combine characteristics that make them excellent experimental models for aquatic toxicology studies, which are particularly usable as warn about the potential danger of chemicals or the possibility of environmental pollution. Due to impaired water quality and the few published studies relating the nickel with the tissue change, this study aimed at assessing the consequences of the presence of nickel in the aquatic environment. For this analysis, we used individuals of Oreochromis niloticus, exposed for 96 hours at three different concentrations of nickel dissolved in water compared to a control group. After exposure, the gills were removed and these were analyzed by ultramorphological, histological and histochemical analysis. The results indicate that all concentrations used in the experiment altered the histophysiology of exposed individuals. We observed the following changes: rupture of paviment cells, thus resulting in bleeding, loss of microridges surface of these cells and epithelial loss in the gills of all animals in all treatments with nickel chloride, the histochemical analysis showed non-proliferation of chloride cells. However, there was a dose-dependent increase of mucus cells in all animals. Therefore, nickel has toxic potential to fish, from the smallest concentration used up to twice as permitted by law, indicating... (Complete abstract click electronic access below)

Freshwater to seawater acclimation of juvenile bull sharks ( Carcharhinus leucas): plasma osmolytes and Na +/K +-ATPase activity in gill, rectal gland, kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater ( SW). Juvenile C. leucas captured in FW ( 3 mOsm l(-1) kg(-1)) were acclimated to SW ( 980 - 1,000 mOsm l(-1) kg(-1)) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l(-1) kg(-1). In SW, bull sharks had significantly higher plasma osmolarities ( 940 mOsm l(-1) kg(-1)) than FW-acclimated animals and were slightly hypoosmotic to the environment. Plasma Na+, Cl-, K+, Mg2+, Ca2+, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na+/ K+-ATPase activity. Na+/ K+-ATPase activity in the gills and intestine was less than 1 mmol Pi mg(-1) protein h(-1) and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na+/ K+-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na+/ K+-ATPase activity was 5.6 +/- 0.8 and 9.2 +/- 0.6 mmol Pi mg(-1) protein h(-1), respectively. Na+/ K+-ATPase activity in the kidney of FW and SW acclimated animals was 8.4 +/- 1.1 and 3.3 +/- 1.1 Pi mg(-1) protein h(-1), respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.

Enhancing in vivo vascularized bone formation by cobalt chloride-treated bone marrow stromal cells in a tissue engineered periosteum model

The periosteum plays an indispensable role in both bone formation and bone defect healing. In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl(2))-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularization. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularization by micro-CT, histomorphometrical and immunohistochemical methods. The results showed that CoCl(2) pre-treated BMSCs induced higher degree of vascularization and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Ca2+ current and Ca(2+)-activated chloride current in isolated smooth muscle cells of the sheep urethra.

1. Isolated sheep urethral cells were studied using the perforated patch clamp technique (T = 37 degrees C). Depolarizing steps ranging from -40 to -10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of -80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 microM). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails. 2. Changing the chloride equilibrium potential (ECl) from 0 to +27 mV shifted the reversal potential of the tail currents from 1 +/- 1 to 27 +/- 1 mV (number of cells, n = 5). Chloride channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9AC, 1 mM), reduced the slow current and tails suggesting that these were Ca(2+)-activated Cl- currents, ICl(Ca). 4. Caffeine (10 mM) induced currents that reversed at ECl and were blocked by niflumic acid (10 microM). 5. In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials. 6. In conclusion, these cells have both L-type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current.

Effects of aluminium chloride and aluminium chlorohydrate on DNA repair in MCF10A immortalised non-transformed human breast epithelial cells

Use of underarm aluminium (Al)-based antiperspirant salts may be a contributory factor in breast cancer development. At the 10th Keele meeting, Al was reported to cause anchorage-independent growth and double strand DNA breaks in MCF10A immortalised non-transformed human breast epithelial cells. We now report that exposure of MCF10A cells to Al chloride or Al chlorohydrate also compromised DNA repair systems. Longterm (19–21 weeks) exposure to Al chloride or Al chlorohydrate at a 10−4 M concentration resulted in reduced levels of BRCA1 mRNA as determined by real-time RT-PCR and BRCA1 protein as determined by Western immunoblotting. Reduced levels of mRNA for other DNA repair genes (BRCA2, CHK1, CHK2, Rad51, ATR) were also observed using real-time RT-PCR. Loss of BRCA1 or BRCA2 gene function has long been associated with inherited susceptibility to breast cancer but these results suggest that exposure to aluminium-based antiperspirant salts may also reduce levels of these key components of DNA repair in breast epithelial cells. If Al can not only damage DNA but also compromise DNA repair systems, then there is the potential for Al to impact on breast carcinogenesis.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-_aprotein kinase C (_aPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and _aPKC (_aPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and _aPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar _aPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of _aKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.