Sandra M. Monteiro

Histopathological changes in liver and gill epithelium of Nile tilapia, Oreochromis niloticus, exposed to waterborne copper

Nile tilapia, Oreochromis niloticus, of both sexes were reared in freshwater and exposed to 0.5, 1.0 and 2.5mg L-1 of waterborne copper for a period of 21 days. Liver and gill samples were collected after 21 days of exposure to copper and lesions were analyzed by light microscopy. The main histopathological changes observed in gills exposed to the highest concentration were edema, lifting of lamellar epithelia and an intense vasodilatation of the lamellar vascular axis. Although less frequent, lamellar fusion caused by the filamentar epithelium proliferation and some lamellar aneurisms were also found. The liver of control group exhibited a quite normal architecture, while the fish exposed to copper showed vacuolation and necrosis. These hepatic alterations were more evident in fish exposed to 1.0 and 2.5mg L-1 copper concentrations. The number of hepatocytes nucleus per mm2 of hepatic tissue decreased with the increase of copper concentration. In contrast, the hepatic somatic index was high in fish exposed at 2.5mg L-1 of copper. In short, this work advance new knowledge as influence of copper in the gill and liver histology of O. niloticus and demonstrated that their effects could be observed at different concentrations.

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.

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.

A stereological study of copper toxicity in gills of Oreochromis niloticus

Stereological methods were used to estimate the volumetric density (V(V)) of the filamentar epithelium (FE, 39%), lamellae (L, 28%), central venous sinus (CVS, 14%), central axis (16%), mucous cells (MC, 2%) and chloride cells (CC, 1%) in the gill filament of control Nile Tilapia. The relative volumes of FE and L, and the relative volumes of CVS and central axis, varied inversely under exposure to copper, with high copper toxic levels declanching a chronic defence mechanism that was, nevertheless, overcome, and low copper toxic levels causing adaptation within a moderate acute phase type of response. Copper also induced a decrease of the V(V) (MC, gill filament) due to reduction of surface MC, despite the marked increase of stem MC at chronic exposure to high copper toxic levels. Diminution of the numerical density of filamentar CC was responsible for the decreased V(V) (CC, gill filament), although lamellar CC significantly increased at chronic exposure to low copper toxic levels. The present results demonstrate that cell relative volumes, mean volumes and numerical densities are dependent on the variations of the FE and L, which without a quantitative approach may be misinterpreted, thus stressing the importance of using stereological tools for analyzing histopathological patterns.

Gill histopathological and oxidative stress evaluation in native fish captured in Portuguese northwestern rivers.

The Northwestern Portuguese region is densely populated and highly industrialized, suffering from high anthropogenic pressure. To assess the biological effect of the several pollutants that are constantly released to the water, a biomarker-based biomonitoring is a promising approach that may provide early-warning signals of pollutants exposure. Fish gill is the first target of pollutants action, thus histopathological and biochemical changes may constitute potential biomarkers. To evaluate this hypothesis, three native fish species (barbel-Luciobarbus bocagei, chub-Squalius carolitertii and nase-Pseudochondrostoma sp.) were sampled in Northwestern Portuguese rivers, the gill histopathological changes were qualitative and quantitatively analyzed and the lipid peroxidation and glutathione-S-transferase activity were determined. A multivariate statistical analysis was performed to establish correlations between these biological responses, environmental variables and ecological status. The quantitative evaluation of the main histopathological changes and oxidative stress responses emphasize the differences, among species, in the responses to the presence of contaminants in water. Discriminant canonical analysis showed that filament epithelium proliferation, necrosis and GST activity were the main contributors to discriminate the ecological status classification. In addition, the results showed that a wide range of environmental factors are influencing fish physiology. In conclusion, the gill biological responses, although not reflecting specific contaminants, can be used as biomarkers of ecosystems perturbation.

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. J. Morphol. 2010.

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.

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.

Gill histopathological alterations in Nile tilapia, Oreochromis niloticus exposed to treated sewage Water

Adult Nile tilapia, Oreochromis niloticus, of both sexes were exposed in wastewater from a sewage treatment plant for a period of 4 days. Gill samples were collected after 24, 48, 72 and 96 h and histopathological changes were analyzed by light and scanning electronic microscopy. Gill epithelium of control O. niloticus (freshwater group) was similar to that of other teleosts, while histopathological lesions were observed in exposed fishes. The main histopathological changes were edema, lifting of lamellar and filamentar epithelia and lamellar fusion. Cell proliferation with consequent thickening of the filament epithelium was also found in fishes exposed to the treated sewage water. The severity of the lesions increased with the time of exposure, namely the hyperplasia of the epithelial cells with proliferation of filamentar epithelium and fusion of lamellae observed at 96 h. Additionally, several histopathological results obtained by light microscopy were confirmed through scanning microscopy.

Biochemical and histological changes in the liver and gills of Nile tilapia Oreochromis niloticus exposed to Red 195 dye

The present study investigates the biochemical and morphological responses induced in the liver and gills of Nile tilapia Oreochromis niloticus by exposure to various Red 195 dye concentrations (0.05, 0.1 and 0.2 mg L−1) for various durations (7, 14 and 21 days). The histology and antioxidant activities of catalase (CAT), glutathione reductase (GR) and glutathione S-transferase (GST) were monitored and evaluated during exposure and after recovery in clean water. The results revealed that CAT activity decreased after 7 and 21 days of exposure to 0.2 mg L−1 concentrations and increased only after 7 days of exposure to high concentrations compared to the liver and gill control samples, respectively. Both organs were, however, noted to undergo a decrease in GST activity after 7 days of exposure to low Red 195 dye concentrations. Compared to the control, the gills of the tilapia exposed to 0.05 and 0.1 mg L−1 concentrations for longer periods underwent an increase in GST activity. Similarly, GR activity was higher in the liver of tilapia exposed to this dye at the sampling days, except for the highest concentration (0.2 mg L−1) after 21 days of exposure. The GR activity in the gills decreased significantly after 7 and 21 days of exposure to 0.05 and 0.1 mg L−1 concentrations, respectively. The results of the recovery group revealed that the liver and gills displayed insignificant differences in antioxidant enzyme activities. The liver and gill tissues of the fish exposed to Red 195 showed several histopathological changes. Liver damages included an increase in cytoplasmic vacuolization, disruption of endothelial lining, metabolic zonation, necrotic cells, rupture of hepatocyte membrane, and decline of higher eosinophilia. The gills also exhibited some necrotic cells, edema, lifting of filaments and lamellar epithelium, and vascular disorders, such as extreme vasodilatation and proliferation of filament epithelium. A correlation between the biochemical and histological changes of the liver and gill tissues was established, attributing the tissue and cell damages to the accumulation of hydrogen peroxide or production of other radicals via a Fenton reaction.