Fernanda Malhão

Testing the effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens as found in the Douro River (Portugal) on the maturation of fish gonads—A stereological study using the zebrafish (Danio rerio) as model

In natural environments fish populations are exposed to many potential xenoestrogens, whereby understanding the impacts of mixtures continue to be of great interest. The main objective of this study was, therefore, to understand whether and how an environmentally relevant mixture of xenoestrogens found in the Douro River estuary can disrupt the normal gametogenesis in fish. For this purpose, adult zebrafish of both sexes were exposed for 21 days to an environmental mixture (MIX) of 11 xenoestrogens from diverse sources. A 100 ng/L ethinylestradiol (EE2) positive control was added. A quantitative (stereological) analysis with systematic sampling was made in the gonads, and using light microscopy both the relative and the absolute volumes of the gametogenic stages were estimated. Data point that the EE2 stimulus induced changes in structural compartments; with decreasing trends for the advanced maturation stages both in males and females. There was also a trend for a greater amount of interstitial tissue in males. Along with an interstitial fibrosis increase detected, the presence of a proteinaceous fluid was observed in both sexes and experimental groups (EE2 and MIX). Other histopathologic alterations were observed in the EE2 female group, such as the presence of foci of granulomatous inflammation and follicular mineralization in the germinal parenchyma and luminal areas. The most interesting finding of this study was that the exposure to the MIX caused a decrease of the relative volume of spermatozoa in zebrafish. This kind of estrogenic effect has not earlier been structurally quantified in such a fine detail with unbiased stereology in fish gonads. Despite the ultimate consequences of such disruptions being unknown, it could be logically argued that reduction or slowing-down of the appearance of the most mature cohorts and/or eventual interstitial fibrosis and other pathologic changes can adversely affect breeding. The findings add further explanatory bases for understanding the negative impacts of xenoestrogens.

Pancytopenia in a cat with visceral leishmaniasis

A 4-year-old, domestic shorthair, female spayed cat was presented for decreased appetite and depression. Severe pancytopenia with erythrocyte autoagglutination was found. The cat was seronegative for feline immunodeficiency and leukemia viruses. Immune-mediated hemolytic anemia was suspected but no response to treatment with a blood transfusion, enrofloxacin, and prednisone was observed. Blood and bone marrow smears obtained 11 days later contained Leishmania amastigotes in the cytoplasm of neutrophils and macrophages, respectively. Serologic and PCR testing of peripheral blood confirmed infection with Leishmania infantum. Despite treatment, the cat worsened clinically and was euthanized. At necropsy, visceral dissemination of the parasite was confirmed. The findings in this case indicate that visceral leishmaniasis should be considered as a differential diagnoses in cats with pancytopenia in areas endemic for Leishmania. In addition, amastigotes may be observed in peripheral blood neutrophils.

Cytological, immunocytochemical, ultrastructural and growth characterization of the rainbow trout liver cell line RTL-W1.

Despite its wide use in toxicology, a detailed characterization of RTL-W1 cell line lagged behind leaving ambiguities about its cell origin. We aimed to better characterize the line regarding cell phenotype and tumorigenic state. We studied RTL-W1 cells in monolayers and in (4-22-week-old) aggregates considering: (a) morphology (light and electron microscopy); (b) immunophenotype using AE1/AE3, vimentin, Cam5.2, CK7 and CK19 and e-cadherin antibodies and (c) growth behavior. RTL-W1 organelle content is constituted basically by mitochondria and abundant free ribosomes, with no (cytochemically) detectable peroxisomes and lysosomes. Immunocytochemistry showed a strong marking for AE1/AE3 and vimentin (in a cell subset). Since AE1/AE3 stained biliary epithelial ducts in trout liver, and considering the morphological characteristics and long term culture, RTL-W1 cells seem more similar to bile preductular epithelial cells (considered as stem cells in teleost liver). Also, we observed abnormal nuclear features described for both malignant cell lines and stem cells, so we could not conclude about tumorigenicity. Cell aggregates had signs of hepatocytic differentiation, such as the development of RER and microvillus-like projections into intercellular spaces. The morphological resemblance to the original tissue suggests that aggregates could have an added value in metabolic as well as in cell-to-cell interaction studies.

An unbiased stereological study on subpopulations of rat liver macrophages and on their numerical relation with the hepatocytes and stellate cells

Studies on liver macrophages have elucidated their key roles in immunological, fibrotic and regenerative responses, and shown that macrophages are not a homogeneous population. In the rat, two sets of liver macrophages coexist, identified by ED1 and ED2 antibodies. Those sets have different quantitative responses in liver injuries and may have different tasks throughout the injury and recovery phases. Nevertheless, the total number (N), number per gram (N g−1) and proportion of those macrophages in relation to other liver cells has never been quantified using design‐based stereology. Thus, we combined immunocytochemistry with those tools to produce an unbiased estimate of the N of ED1+ and of ED2+ cells. A smooth fractionator sampling scheme was applied to the liver of five male Wistar rats (3 months old), to obtain systematic uniform random sections (30 µm thick); these were immunostained with the monoclonal antibodies: ED1, a pan‐macrophagic marker; and ED2, which identifies the completely differentiated macrophages, i.e. Kupffer cells. The N of ED1+ cells was 340 × 106, estimated with a coefficient of error (CE) of 0.04, and that of ED2+ cells was 283 × 106, with a CE of 0.05. These figures correspond to 10.7% and 8.9%, respectively, of the total liver cells. The new data constitute reference values for correlative inferences. Also, the methodological strategy, by its accuracy and precision, is valuable for future investigations on the liver cell composition in various models of disease, and especially for studying the more subtle variations that occur during the injury and recovery phases.

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors

Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50μM; (2) an ER antagonist (ICI 182,780) at 10 and 50μM; and (3) mixtures of both (Mix I-10μM EE2 and 50μM ICI; Mix II-1μM EE2 and 10μM ICI and Mix III-1μM EE2 and 50μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17β-hydroxysteroid dehydrogenase 4-17β-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERβ-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic experiments.

Use of destained cytology slides for the application of routine special stains

BACKGROUND Special stains to demonstrate microorganisms or intra- and extracellular substances have not been evaluated in detail regarding their applicability and usefulness in destained cytologic specimens. OBJECTIVES The aim of this study is to compare the results of routine special stains on destained slides previously stained with Hemacolor and on fresh (unstained) specimens. METHODS Archival Hemacolor-stained fine needle aspirate specimens of inflammation with infectious agents (bacterial, mycobacterial, and fungal infections), neoplasia (melanoma, myxosarcoma, and mammary adenocarcinoma), and hemorrhage (pericardial effusion) from 14 dogs and 7 cats were selected. Cells in a minimum of 4 fields were photographed and 5 slides from each case were then destained by different methods (alcohol acid or microwave). Seven special stains were applied selectively to the destained slides, depending on the cytologic findings: periodic acid Schiff, Grocott-Gomori methenamine silver, Grams, Ziehl-Neelsen, Alcian blue, Fontana-Masson, and Prussian blue. The same fields were rephotographed and 2 observers evaluated the slides qualitatively, with comparison to fresh cytologic specimens from similar lesions. RESULTS Special stains applied to destained slides demonstrated the expected cellular and extracellular material or organisms independent of the destaining method. Staining intensity, nonspecific staining (background), cell morphology, and nuclear counterstaining results were similar to those of special stains applied to fresh unstained slides. CONCLUSIONS Destaining does not appear to affect the results of routine special staining for cytologic specimens. Destaining before special stains may be a valuable diagnostic strategy when few slides are present or only stained slides are available.

Stereological assessment of sexual dimorphism in the rat liver reveals differences in hepatocytes and Kupffer cells but not hepatic stellate cells

There is long‐standing evidence that male and female rat livers differ in enzyme activity. More recently, differences in gene expression profiling have also been found to exist; however, it is still unclear whether there is morphological expression of male/female differences in the normal liver. Such differences could help to explain features seen at the pathological level, such as the greater regenerative potential generally attributed to the female liver. In this paper, hepatocytes (HEP), Kupffer cells (KC) and hepatic stellate cells (HSC) of male and female rats were examined to investigate hypothesised differences in number, volume and spatial co‐localisation of these cell types. Immunohistochemistry and design‐based stereology were used to estimate total numbers, numbers per gram and mean cell volumes. The position of HSC within lobules (periportal vs. centrilobular) and their spatial proximity to KC was also assessed. In addition, flow cytometry was used to investigate the liver ploidy. In the case of HEP and KC, differences in the measured cell parameters were observed between male and female specimens; however, no such differences were detected for HSC. Female samples contained a higher number of HEP per gram, with more binucleate cells. The HEP nuclei were smaller in females, which was coincident with more abundant diploid particles in these animals. The female liver also had a greater number of KC per gram, with a lower percentage of KC in the vicinity of HSC compared with males. In this study, we document hitherto unknown morphological sexual dimorphism in the rat liver, namely in HEP and KC. These differences may account for the higher regenerative potential of the female liver and lend weight to the argument for considering the rat liver as a sexually dimorphic organ.

Marine-derived fungi extracts enhance the cytotoxic activity of doxorubicin in nonsmall cell lung cancer cells A459

Background: Drug resistance is a major concern in the current chemotherapeutic approaches and the combination with natural compounds may enhance the cytotoxic effects of the anticancer drugs. Therefore, this study evaluated the cytotoxicity of crude ethyl extracts of six marine-derived fungi – Neosartorya tsunodae KUFC 9213 (E1), Neosartorya laciniosa KUFC 7896 (E2), Neosartorya fischeri KUFC 6344 (E3), Aspergillus similanensis KUFA 0013 (E4), Neosartorya paulistensis KUFC 7894 (E5), and Talaromyces trachyspermum KUFC 0021 (E6) – when combined with doxorubicin (Dox), in seven human cancer cell lines. Materials and Methods: The antiproliferative activity was primarily assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: Two extracts, E1 and E2, demonstrated a significant enhancement of Doxs cytotoxicity in nonsmall cell lung cancer A549 cells. Accumulation of Dox in the nuclei increased when A549 cells were treated in combination with extracts E1 and E2, with induction of cell death observed by the nuclear condensation assay. The combination of E2 with Dox increased the DNA damage as detected by the comet assay. Ultrastructural observations by transmission electron microscopy suggest an autophagic cell death due to an increase of autophagic vesicles, namely with the combination of Dox with E1 and E2. Conclusion: These findings led to the conclusion that the fungal extracts E1 and E2 potentiate the anticancer action of Dox, through nuclear accumulation of Dox with induction of cell death mainly by cytotoxic autophagy.

Viability analysis of oocyte–follicle complexes and gonadal fragments of zebrafish as baseline for toxicity testing

Abstract To achieve more information about growth and development of oocytes in teleost fish or concerning toxicity testing, it is necessary to develop adequate in vitro oocyte culture conditions. Herein, initial stages of zebrafish oocytes (I, primary; II, cortical; III, vitellogenic) were analyzed under serum-free medium conditions as gonadal fragments or as separated oocyte–follicle complexes. Two vital dye staining methods (MTT, trypan blue) were applied to assess mitochondrial activity and membrane integrity of the oocytes during 4 days, and compared to morphological alterations studied by transmission electron microscopy. Vital dye staining indicated reduced viability at day 4 for all stages in both in vitro culture methods. Additionally, the viability decreased significantly in gonadal fragments at day 2 for stages III (MTT, TB) and II (TB only). Signs of degradation at the ultrastructural level (vacuoles, disintegration of endoplasmic reticulum and detachment of follicular cell layers) appeared in gonadal fragments at day 4 for stages II and III, and in separated oocyte–follicle complexes both at day 4 for stages I–III, and at day 2 for stage III. In conclusion, zebrafish oocytes at stages I and II seemed viable for 2 days as separated oocyte–follicle complexes considering their mitochondrial activity, membrane integrity and ultrastructural morphology. Cultured as gonadal fragments, the majority of analyses indicated similar results for stages I and II oocytes. In contrast, stage III oocytes seemed viable for not longer than 24 h. Results should be taken into consideration for the experimental design of in vitro assays using teleost fish oocytes.

Seasonal changes in hepatocytic lipid droplets, glycogen deposits, and rough endoplasmic reticulum along the natural breeding cycle of female ohrid trout (Salmo letnica Kar.)-A semiquantitative ultrastructural study.

This study on wild female Ohrid trout was primarily designed to provide a general overview of the breeding cycle influence upon selected aspects of hepatocytes. According with a semiquantitatively evaluation, some of these cells structural compartments change during the breeding cycle. Structural modifications were disclosed in the relative occurrence of lipid, glycogen, and RER content during breeding cycle. The relative amount of lipid deposits in the hepatocytes was much greater in previtellogenesis, and decreased postspawning. So, while the seasonal changes in RER were positively related with the ovary maturation status, those of the lipid droplets followed an opposite trend. The hepatocytic glycogen occurred rarely, mainly in late‐vitellogenesis and spawning, suggesting that in this species such kind of energy storage is comparatively unimportant. Lipid accumulation and later usage is, probably, the relevant biochemical pathway for Ohrid trout in the wild. While glycogen and RER contents were positively correlated with the gonadosomatic index, lipids were negatively correlated. Additionally, glycogen inclusions were positively correlated with the plasma estradiol levels. When comparing seasonal patterns from wild Ohrid trout with those from well‐studied rainbow and brown trout (specimens studied were from aquaculture), there are contradicting results as to lipid and glycogen reserves, and also as to RER loads. The differences among the mentioned trout can result from intrinsic interspecies differences or may be associated with natural feeding conditions versus feeding with commercially prepared diets, or other factors. This study offers new data useful as standard to access liver pathology in wild and aquacultured Ohrid trout. Microsc. Res. Tech. 79:700–706, 2016.