Frequency of micronuclei and of other nuclear abnormalities in erythrocytes of the grey mullet from the Mondego, Douro and Ave estuaries--Portugal.

Fish are bioindicators of water pollution, and an increased rate of their erythrocyte nuclear morphological abnormalities (ENMAs)-and particularly of erythrocyte micronuclei (EMN)-is used as a genotoxicity biomarker. Despite the potential value of ENMAs and MN, there is scarce information about fish captured in Iberian estuaries. This is the case of the Portuguese estuaries of the Mondego, Douro and Ave, suffering from different levels of environmental stress and where chemical surveys have been disclosing significant amounts of certain pollutants. So, the aim of this study was to evaluate genotoxicants impacts and infer about the exposure at those ecosystems, using the grey mullet (Mugil cephalus) as bioindicator and considering the type and frequency of nuclear abnormalities of erythrocytes as proxies of genotoxicity. Sampling of mullets was done throughout the year in the important Mondego, Douro and Ave River estuaries (centre and north-western Portugal). The fish (total n = 242) were caught in campaigns made in spring-summer and autumn-winter, using nets or fishing rods. The sampled mullets were comparable between locations in terms of the basic biometric parameters. Blood smears were stained with Diff-Quik to assess the frequencies of six types of ENMAs and MN (given per 1,000 erythrocytes). Some basic water physicochemical parameters were recorded to search for fluctuations matching the ENMAs. Overall, the most frequent nucleus abnormality was the polymorphic type, sequentially followed by the blebbed/lobed/notched, segmented, kidney shaped, vacuolated, MN and binucleated. The total average frequency of the ENMAs ranged from 73 ‰ in the Mondego to 108 ‰ in the Ave. The polymorphic type was typically ≥50 % of the total ENMAs, averaging about 51 ‰, when considering all three estuaries. The most serious lesion-the MN-in fish from Mondego and Douro had a similar frequency (≈0.38 ‰), which was significantly lower than that in the Ave (0.75 ‰). No significant seasonal differences existed as to the MN rates and seasonal differences existed almost only in the Douro, with the higher values in AW. In general, the pattern of ENMAs frequencies was unrelated with the water physicochemical parameters. Considering the data for both the total ENMAs and for each specific abnormality, and bearing in mind that values of MN in fish erythrocytes >0.3 ‰ usually reflect pollution by genotoxicants, it is suggested that mullets were likely being chronically exposed to such compounds, even in the allegedly less polluted ecosystem (Mondego). Moreover, data supported the following pollution exposure gradient: Mondego < Douro < Ave. The scenario and inferences nicely agree with the published data from chemical monitoring.

A stereological study of the volume-weighted volume and of the relative volume of the nucleus of normal and preneoplastic hepatocytes in a trout model of hepatocarcinogenesis.

The fish liver has been the main subject of the biomonitoring and laboratory studies dealing with environmental carcinogenesis. The foci of cellular alterations are accepted pre-neoplastic hepatic lesions, and histopathology is the primary tool for their characterization. Despite its potential, using stereology to study quantitatively nuclear features of those lesions has not been evaluated. Herein, we estimated the volume density and the volume-weighted volume of the nucleus of normal and preneoplastic hepatocytes, using stereology and the brown trout (Salmo trutta f. fario) as model. In the hepatocarcinogenesis protocol the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as initiator, and the 17-ß estradiol (E2) as promoter. Three groups of 30 animals were considered: negative controls (non-exposed), initiator exposed and initiator plus promoter exposed. Estimates of both stereological parameters were significantly higher in preneoplastic hepatocytes, also showing an excellent discriminatory power when used to differentiate those hepatocytes from the normal ones. Besides, in the normal parenchyma the two parameters also differed among the three tested groups. The exposure to MNNG and/or to E2 leaded to modifications in the hepatocyte nuclei that could be unbiasedly quantified with two stereological parameters. We showed that quantitative nuclear morphology represents a valuable auxiliary tool in assessing hepatocarcinogenesis in fishes.

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.

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal): evaluation of impacts on fish liver, by histopathology, stereology, vitellogenin and CYP1A immunohistochemistry, after sub-acute exposures of the zebrafish model.

Qualitative and quantitative approaches were tested to assess zebrafish liver effects after sub-acute exposures of certain pharmaceuticals. Carbamazepine, fenofibric acid, propranolol, sulfamethoxazole and trimethoprim were tested individually and in mixtures, including low environmental levels. Overall, data showed sex specific reactions in liver, with the major alterations being observed in males. Males treated with propranolol, fenofibric acid and with mixtures, showed an increase of vitellogenin immunostaining, compared with the control. Males also evidenced a tendency for an increased hepatic mass, after individual and mixture exposures. The volume-weighted nuclear volume of hepatocytes was high in males after exposures to either mixture, which together with the greater cytoplasmic eosinophilia and changes in cytochrome P450 1A immunoreactivity, point to an increase in metabolic/detoxification activity. These investigations revealed distinct impacts depending on the exposure type, and strengthened the importance of studying non-steroidal compounds in mixtures, including environmental levels and both sexes.

The use of design-based stereology to evaluate volumes and numbers in the liver: a review with practical guidelines.

Stereology offers a number of tools for the analysis of sections in microscopy (which usually provide only two-dimensional information) for the purpose of estimating geometric quantities, such as volume, surface area, length or number of particles (cells or other structures). The use of these tools enables recovery of the three-dimensional information that is inherent in biological tissues. This review uses the liver as a paradigm for summarizing the most commonly used state-of-the-art methods for quantitation in design-based stereology. Because it is often relevant to distinguish hyperplasia and hypertrophy in liver responses, we also focus on potential pitfalls in the sampling and processing of liver specimens for stereological purposes, and assess the existing methods for volume and number estimation. With respect to volume, we considered whole liver volume (V), volume density (V(V)) and so-called local volumes, including the number-weighted volume (V(N)) and the volume-weighted volume (V(V)). For number, we considered the total number (N) and the numerical density (N(V)). If correctly applied, current stereological methods guarantee that no bias is introduced in the estimates, which will be therefore accurate; additionally, methods can be tuned for obtaining precise quantitative estimates that can reveal subtle changes in the volume or number of selected hepatic cells. These methods have already detailed the effects of some substances and specific diets on the liver, and should be routinely included in the toolbox of liver research.

In vitro exposure of Nile tilapia (Oreochromis niloticus) testis to estrogenic endocrine disrupting chemicals: mRNA expression of genes encoding steroidogenic enzymes.

Many environmental pollutants can exert adverse effects on exposed organisms, including fish, leading to disruption of the endocrine system. Enzymes involved in the sex steroid biosynthesis are potential targets for the toxic action of pollutants. In this context, we investigated the hypothesis that selected estrogenic chemicals-the pharmaceutical estrogen ethinylestradiol (EE2), the phytoestrogen genistein (GEN), and the industrial compound bisphenol A (BPA)-may cause endocrine disruption by directly disturbing steps of fish steroidogenic pathways. We studied the mRNA expression of eight selected genes encoding steroidogenic enzymes (11ß-HSD2, 20ß-HSD, 3ß-HSD1, 17ß-HSD1, 17ß-HSD8, 17ß-HSD12, CYP19a, CYP19b) by quantitative real-time PCR. Testis slices from adult specimens of the model fish Nile tilapia were exposed in vitro for 3 and 8 h either to individual or to mixture solutions of EE2 (100 ng/L), GEN (200 ng/L), and BPA (10 µg/L); all at the peak concentrations observed in the Douro River estuary (Portugal). Our data revealed that only the mixture of the tested chemicals directly induced the expression of 11ß-HSD2, 17ß-HSD1, and 17ß-HSD12, after 8 h, whereas no effect was seen for chemicals tested individually. The gene expression pattern agrees with the concept of dose addition for environmental mixtures, and for the first time an interference of estrogenic EDCs is reported for 17ß-HSD1 and 17ß-HSD12.

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal): assessing impacts on gonadal maturation with a histopathological and stereological study of zebrafish ovary and testis after sub-acute exposures.

Concerns associated with pharmaceuticals in aquatic systems demand the establishment of links between xenobiotics and their respective concentrations and impacts on aquatic organisms. Herein, effects of non-steroidal pharmaceuticals in the gonadal maturation of zebrafish (Danio rerio) were evaluated by histopathological and stereological analyses after 21 days of exposure. Carbamazepine, fenofibric acid, propranolol, sulfamethoxazole and trimethoprim were selected, considering their detection in the Douro estuary (Portugal). Exposures were performed with single compounds and mixtures, the exposure concentrations including environmental levels. Overall, quantitative analyses showed a decreasing trend for late maturation stages in male and female gametogenesis with parallel increases in immature gametes. In females, and at the highest concentration mixture, a significant switch between the volume densities of late/mature oocytes versus primary oocytes was observed. On the verge of statistical significance, oocyte atresia was higher in both mixtures (5.75 ± 4.02% for MXA and 5.65 ± 5.27% for MXB) versus control (2.21 ± 1.88%), in accordance with the histological identification of large atretic areas in some fish. Unlike females, males showed significant effects with single exposures. Spermatozoa in controls totalled 53.25 ± 7.13% of the testis volume, decreasing with carbamazepine (47.19 ± 5.30%), fenofibric acid (46.36 ± 4.30%), propranolol (37.22 ± 2.38%) and sulfametoxazole (39.37 ± 5.15%). An increase in spermatocyte percentage was noted with propranolol (40.13 ± 7.36%) and sulfametoxazole (40.84 ± 1.66%) versus control (30.93 ± 6.53%). The changes in maturation dynamics did not impact the gonadosomatic index. The results show that pharmaceuticals from various therapeutic classes can disrupt the maturation dynamics of fish ovaries and testes. Further studies are justified to tackle the underlying mechanisms and to gauge the full extent of effects/risks.

Histological and stereological characterization of brown trout (Salmo trutta f. fario) trunk kidney.

The large variability in kidney morphology among fish makes it difficult to build a "universal" model on its function and structure. Therefore, a morphological study of brown trout trunk kidney was performed, considering potential seasonal and sex effects. Three-year-old specimens of both sexes were collected at four stages of their reproductive cycle. Kidney was processed for light and electron microscopy. The relative volumes of renal components, such as renal corpuscles and different nephron tubules, were estimated by stereological methods. Qualitatively, the general nephron structure of brown trout was similar to that described for other glomerular teleost species. Quantitatively, however, differences in the relative volume of some renal components were detected between sexes and among seasons. Particularly, highest values of vacuolized tubules and new growing tubules were observed after spawning, being more relevant in females. Despite seasonal changes, more linear correlations were found between those parameters and the reno-somatic index than the gonado-somatic index. Thus, we verified that some brown trout renal components undergo sex dependent seasonal variations, suggesting a morphological adaptation of the components to accomplish physiological needs. These findings constitute a baseline for launching studies to know which factors govern the morphological variations and their functional consequences.

The hepatocytes of the brown trout (Salmo trutta fario): a stereological study of some cytoplasmic components with the breeding cycle.

Sex differences exist in fish hepatocytes, but studies for characterizing their cytology throughout the breeding cycle are still scarce; suggesting changes, but most lacking quantitative data. To address this limitation, to complement baseline data generated from the brown trout model, and to prove that sex-specific seasonal changes exist, we made an unbiased stereological evaluation of the hepatocytic cytoplasm. Unprecedentedly for fish liver, the stereological design was exempt from model (biased) assumptions. Five (3 years old) animals per sex were studied in endogenous vitellogenesis, exogenous vitellogenesis, and spawning season end. Liver pieces for analysis were systematically sampled. Stereology was done in transmission electron microscopy (TEM) micrographs. Primary data generated relative volume estimates of the major cytoplasmic components. Such values were used for deriving absolute volumes (per cell and per liver). Lipid droplets did not show changes. As to other targets, trends at cell and liver levels were not always equal. If the hepatocyte was the reference space, the contents in mitochondria, dense bodies, glycogen, and cytosol changed seasonally, in both sexes. If taking the liver as the reference, changes attained the Golgi apparatus and rough endoplasmic reticulum (RER), besides dense bodies, glycogen (in females), and cytosol. The components volumes (namely per liver) were often positively (negatively for glycogen) correlated with the ovary weight, disclosing new associations and implications in fish. While also offering gold-standard data for backing morphofunctional correlations and pathology, we revealed a new process by which females increase the amount of RER and Golgi throughout vitellogenesis, breaking from the idea on how this event happens in fish.

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 microm 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 x 10(6), estimated with a coefficient of error (CE) of 0.04, and that of ED2(+) cells was 283 x 10(6), 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.

Spatial relationships of the intrahepatic vascular-biliary tracts and associated pancreatic acini of Nile tilapia, Oreochromis niloticus (Teleostei, Cichlidae): a serial section study by light microscopy.

Reports on teleost liver morphology reflect both controversial and confirmed interspecies variations. Choosing Nile tilapia as a model, we described the histology and 3D organization of all types of vascular-biliary tracts and their spatial relationships from the organ hilum toward the hepatic vein opening(s). The portal tracts entering the hilum, termed pancreatic-venous-biliary-arteriolar tracts (P-VBAT), are associated with pancreocytes and have an afferent axially located vein, plus biliary duct(s) and small artery(ies). The P-VBAT gradually disappears toward the anterior (efferent) end of the liver; those tracts ramify and originate new types of tracts, which may carry one type of element (vascular or biliary) or groups of two, in all possible combinations. Most tracts carrying afferent veins had pancreocytes, thus forming (pancreatic-venous tracts (P-VT), pancreatic-venous-biliary tracts (P-VBT), and pancreatic-venous-arteriolar tracts (P-VAT). There were terminal (and smaller) afferent isolated veins that had no associated pancreocytes. Also, the pancreatic sleeve of a vein could end abruptly or attenuate and disappear, reappearing in distal portions of the same vein. Thus, veins without pancreatic covering as seen in sections are not always efferent. Small arterioles can enter the liver retrogradely, via the adventitia of efferent hepatic veins, thus forming venous-arteriolar tracts (VAT). In comparison with the salmonid-liver type, there were no VBAT without associated pancreocytes and there was a smaller degree of ambiguity in identification of the afferent vs. efferent veins. Thus, the tilapine-liver type is proposed to be a more promising model for studying hepatic metabolic zonation in fish, defined not as in mammals, but eventually considering a gradient radiating from the hilum. Our data and differences from mammals supported the adequacy of the previously proposed nomenclature for the vascular-biliary tracts of fish livers, extending it to those that contain the exocrine pancreas.

Design-based stereological estimation of hepatocyte number, by combining the smooth optical fractionator and immunocytochemistry with anti-carcinoembryonic antigen polyclonal antibodies.

BACKGROUND/AIMS: Hepatocytes (HEP) have been the major target for structural quantification in the liver, but an estimation of their total number (N), their percentage in relation to the global number of liver cells and the evaluation of the percentage of binucleated hepatocytes (BnHEPs) have never been performed with modern design-based stereological techniques. The establishment of sound technical guidelines and baseline quantitative data in non-pathological conditions are relevant to properly evaluate HEP hyperplasia and BnHEP responses. METHODS: In this study, we combined immunocytochemistry with sound design-based stereology for estimating the N of HEP and the N of non-hepatocytic cells (NHCs). For obtaining systematic uniform random sections (30 microm thick), a smooth fractionator sampling scheme was applied to the liver of five male Wistar rats (3 month old). Those sections were immunostained with polyclonal antibodies against carcinoembryonic antigen. Because biliary canaliculi were then marked, an unequivocal counting of mononucleated hepatocytes (MnHEP) and BnHEP was allowed. RESULTS: The N of HEP was estimated to be 1.93 x 10(9), with a coefficient of error (CE) of 0.02, corresponding to 129 x 10(6) HEP/g of liver. BnHEP represented 26% of total HEP number. The N of NHC was estimated as 1.31 x 10(9) (CE=0.02). CONCLUSION: The strategy here presented provides a reliable method for accessing the N of HEP (distinguishing MnHEP from BnHEP) in situations in which these parameters are relevant, namely for evaluating the magnitude of an hyperplastic liver response from its very early onset.

Temperature and gender influences on the hepatic stroma (and associated pancreatic acini) of Nile tilapia, Oreochromis niloticus (Teleostei, Cichlidae): a stereological analysis by light microscopy.

The normal organ morphology and function in fishes varies according to several natural factors, and such variability is found in liver. Knowledge about the normal liver microanatomy is fundamental to pathological evaluation. Even though gender and temperature are important factors for modulating morphophysiological processes in fishes, their influences on liver stroma are virtually unknown. Because temperature- and gender-related changes exist in liver parenchyma, we predict both factors should also influence the normal stromal structure. Using Nile tilapia as a model, we undertook a study to: 1) establish baseline quantitative structural data on the hepatic stroma (and intimately associated pancreatic acini); 2) compare data with those available from other species, namely, salmonids that do not have a liver with pancreatic acini; and 3) test our hypothesis that, within normal healthy limits, the stroma and its structural components may vary significantly with temperature and gender. We used 1-year-old male and female specimens acclimated to 17 degrees C (breeding noncompatible) and 27 degrees C (breeding compatible) for 45 days. Basic morphometric fish parameters were recorded. After estimation of liver volume, the organ was sliced and pieces systematically sampled for light microscopy. Stereology allowed estimation of the relative volumes of organ components. The total volumes were computed by combining the relative volumes with the total liver volumes. Nile tilapia of both genders, held at 17 vs. 27 degrees C, showed structural quantitative differences in the relative volumes of stroma and most of its components, and in the total volumes of certain stromal elements. The total volume of the stroma and of associated pancreatic acini did not differ. We first established that, in fishes, the total amount (volume) of liver biliary ducts and of eosinophilic granule cells might significantly change (increase and decrease, respectively) with a higher acclimation temperature. Indeed, virtually all the stereological changes were, essentially, temperature- and not gender-related. At 27 degrees C, parallel changes in the parenchyma caused a decreased liver volume and hepatic-somatic index (HSI). The relative volumetric proportion of stroma vs. parenchyma in tilapia is higher than in salmonids. The differences found in this study could not be detected with a qualitative approach, thus stressing the importance of using stereology for analyzing histological patterns and for establishing reliable baseline values in healthy conditions. It was also anticipated that in experimental settings with fish the baseline liver stromal architecture may be different according to temperature and breeding status; in consequence, the effects of the tested variable may also diverge. Our data do not fully explain the lower liver volume and HSI at 27 degrees C, thus justifying studies on the parenchyma, particularly on cell size and number.

A new approach to an unbiased estimate of the hepatic stellate cell index in the rat liver: an example in healthy conditions.

Evaluation of activation and proliferation of hepatic stellate cells (HSCs) must be grounded on solid quantitative data under normal conditions. The HSC index (HSCI), number of HSCs per 1000 hepatocytes (HEP), is often used in hepatology but has been never determined using stereology. Systematically sampled sections were immunostained against glial fibrillary acidic protein and carcinoembryonic antigen, allowing unequivocal distinction of HSC and mononuclear/binuclear HEP. With the optical disector the HSCI was estimated as 109 (coefficient of error = 0.04). This work provides a sound technical basis for experiments in which the estimation of HSCI and/or simultaneous quantification of HSC and HEP are relevant.

Microanatomical organization of hepatic stroma of the brown trout, Salmo trutta fario (Teleostei, Salmonidae): A qualitative and quantitative approach.

Despite the absence of lobulation, light microscopy of serial sections of the liver of brown trout, Salmo trutta fario, reveals that the stromal elements are spatially organized as venous-biliary-arteriolar tracts (VBAT), venous-arteriolar tracts (VAT), biliary-arteriolar tracts (BAT), venous-biliary tracts (VBT), biliary tracts (BT), arteriolar tracts (AT), and isolated veins. These components are not two- but three-dimensional entities, and the anatomical interrelationships among all entities are displayed. The VBAT, VAT, and VBT are considered portal tracts; the adjacent parenchymal zones are viewed as periportal areas. The veins emerging from those tracts are regarded as afferent, and related with periportal zones. The veins that do not communicate with the VBAT, VAT, or VBT are viewed as efferent. Only serial sectioning allows a definite recognition of afferent from efferent isolated veins. The morphometric study discloses that isolated veins occupy around 60% of the stromal areas. Nevertheless, the VBAT, VAT, and BT are also considerably important, occupying variable proportions of the stromal areas (8-12%). The VBT and BAT are less important in quantitative terms. No sexual diffences appear in either qualitative or quantitative terms. There is no structural support for an eventual macroorganization of hepatic tissues. It is suggested that the quantitative data can be useful, as standards for the normal hepatic architecture of brown trout. The paper emphasizes the importance of a general structural model for the fish liver and of the use of an internationally acceptable nomenclature. © 1995 Wiley-Liss, Inc.