Ana M. Coimbra

Disruption of zebrafish (Danio rerio) embryonic development after full life-cycle parental exposure to low levels of ethinylestradiol.

Exposure of fish to the synthetic estrogen ethinylestradiol (EE2) has been shown to induce a large set of deleterious effects. In addition to the negative impact of EE2 in reproductive endpoints, concern has recently increased on the potential effects of EE2 in fish embryonic development. Therefore, the present study aimed at examining the effects of EE2 on the full embryonic development of zebrafish in order to identify the actual phases where EE2 disrupts this process. Hence, zebrafish were exposed to environmentally relevant low levels of EE2, 0.5, 1 and 2ng/L (actual concentrations of 0.19, 0.24 and 1ng/L, respectively) from egg up to eight months of age (F(1)), and the survival as well as the occurrence of abnormalities in their offsprings (F(2)), per stage of embryonic development, was investigated. A thorough evaluation of reproductive endpoints and transcription of vtg1 gene in the parental generation (F(1)) at adulthood, was performed. No significant differences could be observed for the two lowest EE2 treatments, in comparison with controls, whereas vtg1 transcripts were significantly elevated (40-fold) in the 2ng/L EE2 treatment. In contrast to the findings in the F(1) generation,a significant concentration-dependent increase in egg mortality between 8 and 24hours post-fertilization (hpf) was observed for all EE2 treatments, when compared with controls. The screening of egg and embryo development showed a significant increase in the percentage of abnormalities at 8 hpf for the highest EE2 concentration, a fact that might explain the increased embryo mortality at the 24 hpf time-point observation. Taken together, these findings indicate that the two lowest tested EE2 concentations impact late gastrulation and/or early organogenesis, whereas exposure to 2ng/L EE2 also disrupts development in the blastula phase. After early organogenesis has been completed (24 hpf), no further mortality was observed. These results show that increased embryo mortality occurs at EE2 levels below those inducing reproductive impairment and vtg1 gene induction in the male parental generation, thus suggesting that EE2 may impact some fish populations at levels below those inducing an increase in vtg1 transcripts. Hence, these findings have important implications for environmental risk assessment, strongly supporting the inclusion of embryonic development studies in the screening of endocrine disruption in wild fish populations.

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.

Ketamine NMDA receptor-independent toxicity during zebrafish (Danio rerio) embryonic development

Concerns have been raised that the effect of anaesthetic drugs on the central nervous system may result in long-term impairment, namely when ketamine is used during embryogenesis. In addition, the cell and molecular basis of anaesthetics teratology and toxicity are still uncertain and its implications in the development remain to be clarified. More recently, the potential risks for human, and animal, exposure through environmental contamination also became an important question. In this study, the effects of sub- and over anaesthetic doses of ketamine were investigated during zebrafish (Danio rerio) embryonic development by exposing zebrafish embryos to ketamine concentrations (0.2, 0.4 and 0.8 mg mL(-1)) for a period of 20 min during the blastula stage. Ethanol 2% was used as a positive control. Morphological parameters, the overall pattern of cell death using acridine orange and overall degree of oxidative stress levels by 2,7-dichlorodihydrofluorescein-diacetate were determined. Lethality and/or developmental anomalies were measured based on specific time endpoints until 144 h post fertilisation. Results showed a concentration-dependent increase in anomalies and mortality. Cephalic disorders, enlarged organs and tail/spine anomalies were the most prominent deformities observed at 144 hpf. Acridine orange images revealed no differences in cellular death pattern in exposed embryos at 24 hpf. At the same time point, the cellular redox processes were found to be similar among groups. In summary, this study shows that ketamine is teratogen and toxic, interfering with the normal developmental pathways of embryogenesis, suggesting that ketamine exerts an independent NMDA receptor action during the zebrafish blastula stage.

Developmental toxicity of endocrine disruptors in early life stages of zebrafish, a genetic and embryogenesis study.

Endocrine disrupting compounds (EDCs) are capable of interfering with the endocrine system and are increasingly widespread in the aquatic environments. In the present study, zebrafish (Danio rerio) embryos and larvae were used to assess how EDCs may interfere with embryogenesis. Therefore, zebrafish embryos were exposed to 17α-ethinylestradiol (EE2: 0.4, 2, 4 and 20 ng/L), genistein (Gen: 2, 20, 200 and 2000 ng/L) and fadrozole (Fad: 2, 10, 50 and 250 μg/L), between 2 and 144 h post-fertilization (hpf). Somite development, heartbeat, malformations, mortality and hatching rates were evaluated. In parallel, the expression patterns of hormone receptors (esr1, esr2a, esr2b and ar) and apoptotic pathways related genes (p53 and c-jun) were determined using quantitative real-time PCR. Results showed that EE2, Gen and Fad caused a higher mortality and also malformations in larvae compared with control. A significant toxic effect was observed in the heartbeat rate, at 144 hpf, in larvae exposed to EE2 and Fad. QPCR revealed alterations in the expression levels of all the evaluated genes, at different time points. esr1 and c-jun genes were upregulated by EE2 and Gen exposure while the expression of esr2a, esr2b and ar genes was downregulated. Fad exposure decreased esr1, p53 and c-jun expression levels. This study shows a toxic effect of EE2, Gen and Fad to vertebrate embryogenesis and a relation between hormones action and apoptosis pathways.

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.

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.

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect.

Embryonic Stage-Dependent Teratogenicity of Ketamine in Zebrafish (Danio rerio)

Ketamine, a widely used anesthetic, has been shown to have NMDA receptor dependent and independent actions during zebrafish (Danio rerio) embryogenesis. Notwithstanding, the effects of developmental toxicity and the mechanisms of ketamine action on fish embryos are still not well understood, and its implications for early vertebrate development remains to be clarified. In this work, zebrafish embryos were exposed to ketamine (0.2, 0.4, and 0.8 mg mL(-1)) in order to study the stage-developmental toxicity of this pharmaceutical. During 256-cell (2.5 h post-fertilization, hpf), 50% epiboly (5.5 hpf) and 1-4 somites (10.5 hpf), embryos were exposed to the referred ketamine concentrations for a period of 20 min and were allowed to grow until 144 hpf. Both lethal and nonlethal parameters were evaluated. Skeletal development was assessed by alcian blue and calcein staining. Additionally, the expression of the developmental genes sonic hedgehog a (shh a) and noggin 3 (nog3) was evaluated. Similar to our previous work, bone and cartilage malformations were observed after 256-cell exposure. During 50% epiboly, ketamine exposure induced concentration-dependent mortality and malformations, such as lordosis and/or kyphosis and microcephaly, namely, at higher concentrations. Conversely, exposure during 1-4 somites showed the induction of nonspecific effects with no rise in mortality. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed differences in shh a and nog3 expressions comparatively to the control group. Overall, this study shows that the ketamine toxic profile is developmental phase-dependent with 256-cell being the most susceptible phase. The effects observed may result from ketamine interaction with cellular signaling pathways that merits further investigation.

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 (Mugilcephalus) 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.