A Gill Histopathology Study in two Native Fish Species from the Hydrographic Douro Basin.

Gill histopathology is a valuable tool to evaluate ecosystems health, because the gill is a key site of waterborne pollutant uptake and the first target organ to their toxicity. Consequently, this makes it important in biomonitoring programs. This study aims to evaluate gill histopathological differences in Douro basin native fish species and determine possible associations with water quality and the ecological status classifications. Two native fish species (Pseudochondrostoma duriense and Luciobarbus bocagei) were sampled in four points of the Douro basin: two reference points, Ameixiosa (Paiva River) and Covelas (Bestança River), both classified with an excellent ecological status; and two disturbed points, Castro Daire (Paiva River) and Alvações do Corgo (Corgo River), categorized with a good and a moderate ecological status, respectively. Gill histopathological differences were qualitative and quantitatively analyzed. The histological analysis showed that, in all sampling locations, both species presented some degree of gill differences, such as epithelial lifting, lamellar fusion, and/or necrosis. The histopathological differences evaluation emphasized some variances in the responses between the two species. In nase, the filament and lamellar epithelium proliferation were the histopathological differences that better reflected the river ecological status classification, proving their usefulness in biomonitoring programs.

Screening and identification of potential sex-associated sequences in Danio rerio.

Current knowledge on zebrafish (Danio rerio) suggests that sex determination has a polygenic genetic basis in this species, although environmental factors may also be involved. This study aimed to identify sex-associated genomic regions using two different marker systems: inter-simple sequence repeats (ISSRs) and random-amplified polymorphic DNA (RAPDs). Two bulks were constructed: one with DNA from zebrafish females and the other from males; then, a total of 100 ISSR and 280 RAPD primers were tested. Three DNA fragments presenting sexual dimorphism (female-linked: OPA17436 and OPQ191027 ; male-linked: OPQ19951 ) were determined from sequential analysis of the bulks followed by assessment in individuals. These fragments were cloned and convert into the following sequenced characterized amplified regions (SCAR): DrSM_F1, DrSM_F2, and DrSM_M, which share identities with sequences located in chromosomes 2, 3, and 11 (Zv9), respectively. Using these potential markers in zebrafish samples it was possible to correctly identify 80% of the males (DrSM_M) and 100% of the females (DrSM_F1 + DrSM_F2) in the analyzed population.

Neuroendocrine and Eosinophilic Granule Cells in the Gills of Tilapia, Oreochromis niloticus: Effects of Waterborne Copper Exposure.

The contamination of aquatic ecosystems with copper (Cu) poses a serious threat to aquatic organisms. Although the histopathological changes caused by Cu in fish gills are well documented, knowledge about the impact of this metal in gill specific cell types, such as neuroendocrine cells (NECs) and eosinophilic granule cells (EGCs), is still limited. In the present work, Nile tilapia (Oreochromis niloticus) were exposed for 21 days to nominal concentrations of Cu (40 and 400 µg L(-1)). Stereological methods were used to estimate the volumetric density of both NECs and EGCs in fish gill filament after 3, 7, 14, and 21 days of exposure. The results showed that Cu significantly increased the relative volume of NECs, whereas the relative volume of EGCs decreased. NECs were more affected by Cu in the first 7 days of exposure, during which a greater increase in their relative volume was observed. The Cu exposure induced a progressive decrease in the relative volume of EGCs, which reached statistical significance after 14 days of exposure. An exception was observed in subepithelial EGCs with a slight increase in their relative volume after 3 days of exposure. Our findings confirm that Cu can modulate both neuroendocrine and immune systems and becomes immunotoxic after a prolonged exposure.

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.

Mullet and gudgeon liver histopathology and macroinvertebrate indexes and metrics upstream and downstream from a wastewater treatment plant (Febros River--Portugal).

The increased pollution in ecosystems reinforces the importance of both chemical monitoring and biological monitoring of streams and rivers, as an effective water quality-based approach to assess aquatic ecosystem health. In this study, gudgeon (Gobio gobio) and mullet (Mugil cephalus) liver histopathology (biomarker) and some macroinvertebrate community indexes and metrics (bioindicator) were used to evaluate the effect of the wastewater treatment plant (WWTP) of Febros (Avintes) in Febros River water quality and ecosystem health. Regarding macroinvertebrate communities, the Belgian Biotic Index (BBI) and Iberian Biological Monitoring Working Party (IBMWP) indexes suggested that Febros water was slightly polluted, even though the worst situation was found downstream the WWTP discharge. Concerning community metrics, upstream percent of individuals in five numerically dominant taxa (80%) was slightly more superior than the downstream (78%). The presence of intolerant or sensible individuals, determined by percent of Ephemeroptera, Plecoptera, and Trichoptera individuals and number of Ephemeroptera, Plecoptera, and Trichoptera families metrics, was higher upstream WWTP, reflecting a better water quality. The histopathology shows the presence of hepatic lesions in gudgeon and mullet. The statistical analysis of the lesion gradation showed that only necrosis was significantly higher in gudgeon captured downstream the WWTP, while differences were not observed for mullet. The multivariate analysis of data confirmed the existence of differences in hepatic lesions between gudgeon and mullet and between sampling sites. Regarding macroinvertebrate community, this analysis showed that the organic contamination reflected by the BBI and IBMWP indexes values was a determinant factor in the spatial distribution of macroinvertebrates. This work showed that the study of different biological organization levels can be used for a better assessment of ecosystem ecological integrity and can be used as a tool to reveal anthropogenic activity effects in macroinvertebrate diversity and in fish liver pathology from Febros River.

Uncommon hepatic macrophagic foamy-cell nodules in Iberian barbel (Luciobarbus bocagei Steindachner, 1864) from the Vizela River (Portugal).

The main intent of this work (after the by chance finding, in archived histological slides) is to characterize one previously non-described liver lesion of the Iberian barbel from the Vizela River (Portugal). This ran through a textile and dyeing industrial region. The lesion type was made of groups of foamy cells (presumptive macrophages), which appear either as a "smaller non-nodular form," without a connective tissue capsule and displaying an irregular profile, or as a "bigger nodular form," presenting a thin capsule and a circular profile. The nodular forms could appear multi-layered, resembling "cross-sectioned onions". The lesions number, dimension, and structural complexity were greater in bigger fish, appearing only after a history of poor water quality. In extreme cases, the lesions slightly protruded the liver surface. Special histological staining proved the connective tissue nature of the capsule (and its eventual septa), the presence of proteins, glycoproteins, lipofuscin, melanin, iron (putative hemosiderin), and copper, in a variable number of foamy cells within the lesions. At times, degenerating hepatocytes appeared at the border of the lesions. It is proposed that this lesion type incorporates both macrophages and degenerating hepatocytes, looking as one (unpublished) form of a macrophage aggregate. The term "foamy-cell nodules" was advanced for this abnormality. The lesion could have been induced by pollution, because: no parasites were ever associated with the lesion; there was co-existence of the lesion with a water quality status scored as "bad"; such lesion neither existed in fish sampled after mitigation and remediation measures nor in reference fish.

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.

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.

Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole).

Exposure of wildlife to endocrine disrupting chemicals (EDCs) is not necessarily continuous. Due to seasonal changes and variable industrial and agricultural activities it often occurs intermittently. Thus, it is possible that aquatic organisms may be more affected by periodic peak exposure than by chronic exposure. Therefore, an experimental scenario including an exposure from 2h to 90 days post-fertilization (dpf) and a subsequent recovery period until 150 dpf was chosen to assess the potential reversibility of the effects of sex steroids on sexual and gonad development of zebrafish (Danio rerio). The aim of this study was to investigate the persistence of the endocrine effects of an estrogen (EE2-17α-ethinylestradiol, 4ng/L), an inhibitor of estrogen synthesis (Fad-fadrozole, 50µg/L) or their binary mixture (Mix-EE2+ Fad, 4ng/L+50µg/L). Afterwards, a semi-quantitative histological assessment was used to investigate histopathological changes on gonad differentiation and development. The data showed that fadrozole, alone or in combination with EE2, permanently disrupts the sexual development, inducing masculinization and causing severe pathological alterations in testis, such as intersex associated to the enlargement of sperm ducts, interstitial changes, asynchronous development and detachment of basal membrane. After exposures to both EDCs and their mixture, the gonad histopathology revealed interstitial proteinaceous fluid deposits and, in ovaries, there were atretic oocytes, and presumably degenerative mineralization. On the other hand, the gonadal changes induced by EE2 alone seem to be partially reversible when the exposure regime changed to a recovery period. In addition, EE2 enhanced zebrafish growth in both genders, with male fish presenting signs of early obesity such as the presence of adipocytes in testis. Moreover, sex ratio was slightly skewed toward females, at 90 and 105 dpf, in zebrafish exposed to EE2. The data further indicate that long-term studies on impacts of single EDCs and their mixtures with recovery periods are crucial to reveal the possibility of sex reversal and pathological changes of gonads that can adversely affect breeding.

Effects of 17α-ethinylestradiol at different water temperatures on zebrafish sex differentiation and gonad development.

In the current climate change scenario, studies combining effects of water contaminants with environmental parameters, such as temperature, are essential to predict potentially harmful impacts on aquatic organisms. In zebrafish (Danio rerio), sex determination seems to have a polygenic genetic basis, which can be secondarily influenced by environmental factors, such as temperature and endocrine disrupting chemicals (EDCs). The present study aimed to evaluate the effects of the EDC 17α-ethinylestradiol (EE2), a potent synthetic estrogen, on zebrafish sex differentiation and gonad development at different water temperatures. Therefore, zebrafish raised at three distinct water temperatures (23, 28 or 33±0.5°C), were exposed to 4ng/L of EE2, from 2hours to 60days post-fertilization (dpf). Subsequently, a quantitative (stereological) assessment of zebrafish gonads was performed, at 35 and 60dpf, to identify alterations on gonadal development and differentiation. The results show that low temperature delayed general growth of zebrafish, as well as gonad differentiation and maturation, while high temperature induced an opposite effect. Moreover, sex ratio was skewed toward males when zebrafish were exposed to the high temperature. In general, EE2 exposure promoted gonad maturation in both genders, independently of the temperature. However, at the high temperature condition, exposure to EE2 induced a delay in the male gonad development, with some individuals still showing differentiating gonads at 60dpf. The findings of this study support the notion that zebrafish has a genetic sex determination mechanism highly sensitive to environmental factors and show that it is essential to study the effects of water contaminants at different climate scenarios in order to understand potential future impacts on organisms.

Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures.

Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE2, 4ng/L) and fadrozole (Fad, 50µg/L) from 2h to 35days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and -6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the "juvenile ovary" development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved in zebrafish spermatogenesis. Both EDCs, EE2 and Fad, increased the apoptosis stimulus in zebrafish gonad. It was noticed that the few females that were resistant to Fad-induced sex reversal had increased anti-apoptotic factor levels, while males exposed to EE2 showed increased pro-apoptotic genes/proteins and were more advanced in gonad differentiation. Overall, our findings show that apoptosis pathways are involved in zebrafish gonad differentiation and that EDCs can disrupt this process.

Effects of Cd injection on osmoregulation and stress indicators in freshwater Nile tilapia.

Freshwater tilapia (Oreochromis niloticus) were intraperitoneally injected with sublethal doses of cadmium (1.25 or 2.5 mg Cd kg(-1) body mass) and sampled after 1, 4 and 7 days in order to evaluate the mechanisms of Cd toxicity at physiological and biochemical levels. Cd levels were significantly elevated in the gill and kidney following injection however levels in the kidney continued to accumulate while levels in the gill either did not change or decreased with time. Cd caused a generalized stress condition as indicated by an increase in blood glucose, lactate and cortisol levels as well as an oxidative stress indicated by increases in lipid peroxidation and protein carbonyl content. Furthermore, tilapia exhibited impairment in their osmoregulatory status based on the fall in plasma sodium levels. Concerning ion regulatory disruption, the kidney was the most affected organ since there was a generalized increase in renal Na(+)/K(+)-ATPase activity after 1 day of exposure to Cd followed by a significant decrease in day 7. This study provides some insights into the mechanisms of Cd toxicity at physiological and biochemical levels and complements previously reported findings on O. niloticus. The disruption of ion homeostasis, alterations in Na(+)/K(+)-ATPase activity and oxidative damage are the effects of Cd exposure that can be integrated in a comprehensive model for Cd impacts.

Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study.

Current knowledge on zebrafish (Danio rerio) sex determination suggests that this trait has a polygenic genetic basis, although environmental factors, such as endocrine disrupting chemicals (EDC), may also be involved in modeling or disturbing the species sex differentiation and development. This study aimed to assess how sex steroids imbalance triggers impact on sex differentiation and gonad development in zebrafish. Fish where exposed to an estrogen (EE2, i.e. 17α-ethinylestradiol, 4ng/L), to an inhibitor of estrogen synthesis (Fad, i.e. fadrozole, 50µg/L) or to their binary mixture (Mix-EE2+ Fad, 4ng/L+50µg/L), from 2h to 60 days post-fertilization (dpf). Afterwards, a quantitative (stereological) analysis using light microscopy, based on systematic sampling, was made at 35 and 60dpf, to identify alterations on gonad differentiation and development. During the sex differentiation period, our histological data showed that not all zebrafish males develop a "juvenile ovary", contrarily to what is currently taken for granted. Furthermore, the stereological analysis suggests that EE2 alone enhanced both zebrafish growth and gonad development. On the other hand, exposure to Fad affected the sexual development in zebrafish, inducing masculinization of the specimens, with some degree of intersex observed in males. In addition, the binary mixture allowed identifying sex-dependent roles of steroid hormones in the general growth and gonad development of zebrafish, with estrogens acting as growth promoters in females and being essential for ovary development. Data further support that sex-specific and single EDC impact studies are important, but clearly not sufficient to understand what may occur in the environment.

Frequency of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from the Douro River estuary, Portugal.

Liver lesions in wild fish have been associated with xenobiotic exposure. Facing reports of pollution in the Douro River estuary (north of Portugal), we have been making field surveys using fishes and targeting histopathological biomarkers of exposure and effect. Herein, we intended to better characterize and report the rate of one poorly understood lesion-hepatocellular fibrillar inclusions (HFI)-found in European flounder (Platichthys flesus). With this report, we aimed to establish sound baseline data that could be viewed as a starting point for future biomonitoring, while offering the world's second only pool of field data on such a liver toxicopathic lesion, which could be compared with data available from the UK estuaries. Sampling was done in the Douro River estuary over 1 year. A total of 72 animals were fished with nets, in spring-summer (SS) and autumn-winter (AW) campaigns. Livers were processed for histopathology and both routine and special staining procedures (alcian blue, periodic acid Schiff (PAS), tetrazonium coupling reaction). Immunohistochemistry targeted AE1/AE3 (pan cytokeratins). The severity of the HFI extent was graded using a system with four levels, varying from 0 (absence of HFI) to 3 (high relative density of cells with HFI). Cells (isolated/groups) with HFI appeared in 35 % or more of the fish, in the total samples of each season, and over 40 % in more homogeneous sub-samples. There were no significant differences when comparing samples versus sub-samples or SS versus AW. When merging the data sets from the two seasons, the frequency of fish with HFI was ≈36 % for the total sample and ≈49 % for the sub-sample. The extreme group (biggest and smallest fish) revealed a HFI frequency of only 16 %, which differed significantly from the total and sub-sampled groups. Immunostaining and PAS were negative for the HFI, and alcian blue could, at times, faintly stain the inclusions. These were positive with the tetrazonium reaction. We showed the presence of HFI in European flounder from the Douro River estuary, proving that they are essentially protein in nature, that no seasonal changes existed in the HFI frequency, and that it was rarer in the smallest and biggest fish groups. Within the ranges of weight/size of our total sample, we estimate that the frequency of HFI in the local flounder is ≈35 %. That rate stands as a baseline value for future assessments, namely for biomonitoring purposes targeting correlations with the estuary pollution status.

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.

Copper induced upregulation of apoptosis related genes in zebrafish (Danio rerio) gill.

Copper (Cu) is an essential micronutrient that, when present in high concentrations, becomes toxic to aquatic organisms. It is known that Cu toxicity may induce apoptotic cell death. However, the precise mechanism and the pathways that are activated, in fish, are still unclear. Thus, this study aimed to assess which apoptotic pathways are triggered by Cu, in zebrafish (Danio rerio) gill, the main target of waterborne pollutants. Fish where exposed to 12.5 and 100 µg/L of Cu during 6, 12, 24 and 48 h. Fish gills were collected to TUNEL assay and mRNA expression analysis of selected genes by real time PCR. An approach to different apoptosis pathways was done selecting p53, caspase-8, caspase-9 and apoptosis inducing factor (AIF) genes. The higher incidence of TUNEL-positive cells, in gill epithelia of the exposed fish, proved that Cu induced apoptosis. The results suggest that different apoptosis pathways are triggered by Cu at different time points of the exposure period, as the increase in transcripts was sequential, instead of simultaneous. Apoptosis seems to be initiated via intrinsic pathway (caspase-9), through p53 activation; then followed by the extrinsic pathway (caspase-8) and finally by the caspase-independent pathway (AIF). A possible model for Cu-induce apoptosis pathways is proposed.

An immunohistochemical study of gill epithelium cells in the Nile tilapia, Oreochromis niloticus.

This study reports the first complete mapping of the gill epithelium in a tilapia species. Different gill epithelial cell types of the Nile tilapia, Oreochromis niloticus L. have been identified and located using different antisera against mammalian proteins and various histochemical techniques: Periodic Acid Schiff (PAS), Alcian Blue pH 1.0, 2.5, 3.5, Giemsa and Grimelius. The results show that the stratified filament epithelium of O. niloticus gill can be divided into two distinct regions, a superficial layer, where pavement, mucous and mitochondria rich cells can be found, and a deep layer, constituted by undifferentiated, myoepithelial-like, granular and neuroendocrine cells. V-ATPase and Na+/K+-ATPase presence allowed the identification of pavement and mitochondria-rich cells, respectively, suggesting that, in O. niloticus, pavement cells are implicated in Na+ uptake, whereas mitochondria-rich cells have a role in Cl- uptake. The use of PAS and Alcian Blue allowed the recognition of different sub-populations of mucous cells that differentiate from a common deeper precursor. Neuroendocrine markers were detected in different cell populations, stating evidence for a neuroendocrine role of mitochondria- rich cells, and suggesting the existence of distinct neural pathways, a putative O2-chemosensory and an ion regulatory pathway. A defence role was attributed to the deep filament epithelium, suggested by the presence of resident giemsa positive- eosinophil granular cells. The antibody raised against proliferating cell nuclear antigen identified two different cell types, the undifferentiated cells and myoepithelial-like cells. In the superficial layer, it is here stated for the first time the existence of vimentin positive support cells.

Fine structure of the branchial epithelium in the teleost Oreochromis niloticus.

We have studied the gill epithelium of Oreochromis niloticus using transmission electron microscopy with the particular interested relationship between cell morphology and osmotic, immunoregulatory, or other non-regulatory functions of the gill. Pavement cells covered the filament epithelium and lamellae of gills, with filament pavement cells showing distinct features from lamellar pavement cells. The superficial layer of the filament epithelium was formed by osmoregulatory elements, the columnar mitochondria-rich, mucous and support cells, as well as by their precursors. Light mitochondria-rich cells were located next to lamellae. They exhibited an apical crypt with microvilli and horizontal small dense rod-like vesicles, sealed by tight junctions to pavement cells. Dark mitochondria-rich cells had long dense rod-like vesicles and a small apical opening sealed by tight junctions to pavement cells. The deep layer of the filament epithelium was formed by a network of undifferentiated cells, containing neuroepithelial and myoepithelial cells, macrophage and eosinophil-like cells and their precursors, as well as precursors of mucous cells. The lateral-basal surface was coated by myoepithelial cells and a basal lamina. The lamellar blood lacunae was lined by pillar cells and surrounded by a basal lamina and pericytes. The data presented here support the existence of two distinct types of pavement cells, mitochondria-rich cells, and mitochondria-rich cells precursors, a structural role for support cells, a common origin for pavement cells and support cells, a paracrine function for neuroepithelial cells in the superficial layer, and the control of the lamellar capillary base by endocrine and contractile cells. Data further suggest that the filament superficial layer is involved in gill osmoregulation, that may interact, through pale mitochondria-rich cells, with the deep layer and lamellae, whereas the deep layer, through immune and neuroendocrine systems, acts in the regeneration and defense of the tissue.

Copper toxicity in gills of the teleost fish, Oreochromis niloticus: effects in apoptosis induction and cell proliferation.

Recent in vitro studies have demonstrated that copper may induce apoptosis triggering the activation of caspase-3, a central effector of apoptotic cell death. However, the precise mechanism of copper-induced apoptosis is still unclear, even less so in Oreochromis niloticus where no caspase genes have been reported so far. This study aimed to assess the in vivo role of copper in apoptosis induction on O. niloticus gill, simultaneously contributing to elucidate the mechanism of copper-induced apoptosis. Caspase-3 gene was partially sequenced and, after in vivo exposures to 40 and 400 microgL(-1) of copper, its mRNA expression was evaluated by real-time PCR. Apoptosis was also evaluated by TUNEL assay and cell proliferation identified using an antibody against proliferating cell nuclear antigen (PCNA). The copper concentrations used did not induce the upregulation of caspase-3 gene in O. niloticus gill. In addition, in the gills of fish exposed to copper there was no increase in the estimated relative volume of apoptotic cells, indicating that neither the caspase-3-dependent or caspase-independent apoptotic pathways were induced. On the other hand, the increase in the volumetric density of epithelial proliferating cells suggests a concentration-dependent repair response.

Histopathological gill changes in wild leaping grey mullet (Liza saliens) from the Esmoriz-Paramos coastal lagoon, Portugal.

The histopathological changes are among the most recognized responses to environmental stressors, namely heavy metals. Liza saliens were sampled, in the Portuguese Esmoriz-Paramos coastal lagoon to assess their gill histopathological response to Cu and Zn contaminated sediments. A lesion prevalence index, severity, and extension scores of each lesion, as well as an assessment value (severity x extension), were determined to evaluate the effect of environmental heavy metal exposure. The main histopathological changes observed were aneurisms, hyperplasia, lifting, and vasodilation. A high prevalence for each lesion (65-85%) was found, in addition to a high number of simultaneous lesions. Vasodilation and hyperplasia were the lesions that showed higher prevalence indexes. Lifting, followed by hyperplasia, were the lesions with highest assessment value. The prevalence of the number of the lesions found in L. saliens collected from the Esmoriz-Paramos lagoon was high when compared with mullet caught in the sea. A previous work revealed that fish collected in the lagoon showed elevated Cu and Zn levels in their gills, in a positive correlation with age. However, no positive correlation was observed between assessment value and gill metal levels.