Inhibitory effects of estrogenic endocrine disrupting chemicals on fin regeneration in zebrafish are dependent on estrogen receptors.

For fish and other aquatic organisms, disrupting their capacity for repair and regeneration will reduce their quality of life and survivorship in the wild. Studies have shown that 17α-ethinylestradiol (EE2), a synthetic estrogenic endocrine disrupting chemical (EEDC), can inhibit caudal fin regeneration in larval zebrafish following fin amputation. However, whether the inhibitory effects of EE2 are dependent on estrogen receptor (ER) remains unknown. Therefore, in this study, amputated zebrafish larvae were exposed to the ER agonist EE2 alone and in combination with the ER antagonist ICI 182,780 (ICI), and the change in regenerative capacity was determined. The inhibition of fin regeneration caused by EE2 alone (100 ng/L) was ameliorated after combination with ICI (30-300 µg/L), and these changes in regeneration-related signaling and the immune system corresponded with morphological observations, implying that the effects of EE2 on regeneration were possibly initiated by the activation of ER. Furthermore, the role of ER was confirmed with a natural ligand of ER, namely, 17ß-estradiol (E2), and as expected, the effects of E2 (10, 100 and 1000 ng/L) paralleled those of EE2. In conclusion, EEDCs can disrupt the regenerative capacity in zebrafish, possibly due to the binding and activation of ERs and the consequent alteration of signaling pathways that regulate fin regeneration and immune competence. Given that EEDCs appear to be ubiquitous in the aquatic environment, the risk of these chemicals might be readdressed regarding their potential effects on tissue repair and regeneration.

Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish.

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The bioaccumulation of microplastics plays a key role in their toxic effects; however, as a particulate, their bioaccumulations are different from many other pollutants. Described here is a feasible method to visually determine the accumulation and distribution of microplastics in zebrafish embryos or larvae using fluorescent microplastics. Embryos are exposed to different concentrations (0.1, 1, and 10 mg/L) of fluorescent microplastics with a diameter of 500 nm for 120 h. It is shown in the results that microplastics can bioaccumulate in zebrafish embryos/larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion; while in zebrafish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics. The results demonstrate the uptake and internalization of microplastics in zebrafish at early life stages, which will provide basis for better understanding the impact of microplastics on aquatic animals.

Development and validation of an activated immune model with zebrafish eleutheroembryo based on caudal fin acupuncture.

Environmental pollutants are ubiquitous in global aquatic ecosystems and may cause immunotoxicity in aquatic organisms. However, disadvantages remain in the existing in vivo immunotoxicological methods, which make it difficult to meet the increasing demands for screening and for discriminating the immunotoxicity of environmental pollutants. In this study, the immune response in zebrafish eleutheroembryo was activated by acupuncture of the caudal fin at 72 hours post fertilization (hpf), and this immune model was further validated with a well-defined immunosuppressor, beclomethasone dipropionate (BDP). It was shown that acupuncture resulted in no increase in mortality in zebrafish eleutheroembryos. The transcription and protein levels of most immune genes were significantly increased after acupuncture, which indicated that acupuncture can effectively activate the immune response in zebrafish eleutheroembryos. Following exposure to BDP (0.01-1 µmol/L), the suppressive effects on the immune system were more significant in zebrafish that received acupuncture than in zebrafish that did not receive acupuncture. Considering these advantages, including its sensitivity, safety, and simple operation, over existing methods, the established immune model of zebrafish is promising for assessing the immunotoxicity of environmental pollutants.

Inhibitory effects of polystyrene microplastics on caudal fin regeneration in zebrafish larvae.

Microplastic pollution is pervasive in aquatic environments, but the potential effects of microplastics on aquatic organisms are still under debate. Given that tissue damage is unavoidable in fish and the available data mostly concentrate on healthy fish, there is a large chance that the ecotoxicological risk of microplastic pollution is underrated. Therefore, in this study, the effects of microplastics on the regenerative capacity of injured fish were investigated using a zebrafish caudal fin regeneration model. After fin amputation at 72 h post fertilization, the larvae were exposed to polystyrene microplastics (0.1-10 mg/L) with diameters of 50 or 500 nm. Microplastic exposure significantly inhibited fin regeneration, both morphologically and functionally. Furthermore, the signaling networks that regulate fin regeneration, as well as reactive oxygen species signaling and the immune response, both of which are essential for tissue repair and regeneration, were altered. Transcriptomic analyses of the regenerating fin confirmed that genes related to fin regeneration were transcriptionally modulated in response to microplastic exposure and that metabolic pathways were also extensively involved. In conclusion, this study demonstrated for the first time that microplastic exposure could disrupt the regenerative capacity of fish and might eventually impair their fitness in the wild.

Effects of 17α­ethinylestradiol on caudal fin regeneration in zebrafish larvae.

The ability to restore tissue function and morphology after injury is a key advantage of many fish for a greater chance of survival. The tissue regeneration process is regulated by multiple pathways, and it can therefore be hypothesized that environmental contaminants targeting components of these signaling pathways, may disrupt the fish's capability to repair or regenerate. This could lead to higher mortality and eventually even to a decline in populations. In this study, the effects of 17α­ethinylestradiol (EE2), a synthetic estrogen, were assessed on the regenerative capacity of larval zebrafish. Zebrafish aged 2 hour post fertilization (hpf) were exposed to 1, 10, or 100 ng/L EE2, and the caudal fins were amputated at 72 hpf. It was found that EE2 exposure significantly inhibited fin regeneration and changed locomotor behavior. The transcription levels for most of the genes involved in the signaling networks regulating the fin regeneration, such as axin2, fgfr1, bmp2b and igf2b, were down-regulated in the amputated fish in response to EE2 exposure, which was in contrast to their increased patterns in the vehicle-exposed control fish. Additionally, the mRNA levels of several immune-related genes, such as il-1ß, il-6, il-10 and nf-κb2, were significantly decreased after EE2 exposure, accompanied by a lower density of neutrophils migrated into the wound site. In conclusion, the present study indicated for the first time that estrogenic endocrine disrupting chemicals (EEDCs) could inhibit the regenerative capacity of zebrafish, and this effect was speculated to be mediated through the alteration in regeneration-related signaling pathways and immune competence. This work expands our knowledge of the potential effects of EEDCs on injured aquatic organisms, and highlights the ecotoxicological significance of relationships between regenerative process and endocrine system. This study also implies the potential application of fin regeneration assay for assessing immunotoxicity in ecotoxicological risk assessment.