Detection of microplastics in zebrafish housing systems: Can microplastic background contamination affect the final results of microplastic-related toxicological tests?

Concentrations of microplastics (MPs) were determined in three commonly used zebrafish housing systems to see if their levels could affect the final results of laboratory microplastic-related toxicology tests. MPs have received notable attention in the last few years, and their toxicology tests have also come to the fore. Zebrafish (Danio rerio), kept in fish housing systems, are widely used as models for MPs studies. Most of these systems contain a significant number of parts made of different polymers. As usage and amortization can erode these parts, MPs might appear in the keeping water or the fish body, which may represent a background load and possibly influence the results of microplastic-related toxicological tests. To take representative water samples from systems, two in-situ filtration techniques, a newly developed peristaltic pump-, and a jet pump-driven method were applied. The collected MP particles were analyzed with a Fourier-transform infrared microscope (detection limit 50 µm), and their possible origin was also investigated. The newly developed technique was more sufficient for sampling as it had a higher MPs recovery, especially in the smaller size range. Polyester, polyethylene and polypropylene were the most frequently detected polymers in the examined fish housing systems, the highest detected concentration was 0.31±0.12 particles/liter (0.22±0.16 µg/liter). These values are negligible compared to the literature data reporting enormously high applied MPs concentrations (104 - 2.21 × 108 particles/liter) during toxicology tests. The results also show that some detected MPs did not originate from the systems, their origin was presumed to be external.

In vivo estrogenicity of glyphosate, its formulations, and AMPA on transgenic zebrafish (Danio rerio) embryos.

In this study, the disrupting effects of glyphosate (GLY), aminomethylphosphonic acid (AMPA), and three glyphosate-based herbicides (GBHs) on vitellogenesis in a non-concentration-dependent manner are reported for the first time in 120 h of acute exposure of zebrafish at environmentally relevant concentrations. GBHs are commonly used worldwide in weed control management. Due to their extensive application, they frequently occur in aquatic ecosystems and may affect various organisms. The active substance GLY and its major by-product, AMPA, are the most thoroughly studied chemicals; however, the adverse effects of the complex formulas of GBHs with diverse and unknown content of co-formulants are still not sufficiently researched. This study focused on the embryotoxicity, sublethal malformations, and estrogenic potency of GLY, AMPA, and four commonly used GBHs on zebrafish embryos using a wild type and an estrogen-sensitive, transgenic zebrafish line (Tg(vtg1:mCherry)). After 120 h of exposition, AMPA did not cause acute toxicity, while the LC50 of GLY was 160 mg/L. The GBHs were more toxic with LC50 values ranging from 31 to 111 GLY active equivalent (a.e.) mg/L. Exposure to 0.35-2.8 mg/L GBHs led to sublethal abnormalities: typical symptoms were structural deformation of the lower jaw and anomalies in the olfactory region. Deformity rates were 10-30% in the treated groups. In vivo, fluorescently expressed vtg1 mCherry protein in embryonic liver was detected by a non-invasive microscopic method indicating estrogenic action through vitellogenin production by GLY, AMPA, and GBHs. To confirm the in vivo findings, RT-qPCR method was performed to determine the levels of the estrogenicity-related vtg1 mRNA. After 120 h of exposure to GLY, AMPA, and three GBHs at a concentration of 0.35 mg/L, the expression of vtg1 gene was significantly up-regulated. Our results highlight the risk that short-term GLY and GBH exposure can cause developmental malformations and disrupt the hormonal balance in zebrafish embryos.

Interaction of Fumonisin B1, N-Palmitoyl-Fumonisin B1, 5-O-Palmitoyl-Fumonisin B1, and Fumonisin B4 Mycotoxins with Human Serum Albumin and Their Toxic Impacts on Zebrafish Embryos.

Fumonisins are frequent food contaminants. The high exposure to fumonisins can cause harmful effects in humans and animals. Fumonisin B1 (FB1) is the most typical member of this group; however, the occurrence of several other derivatives has been reported. Acylated metabolites of FB1 have also been described as possible food contaminants, and the very limited data available suggest their significantly higher toxicity compared to FB1. Furthermore, the physicochemical and toxicokinetic properties (e.g., albumin binding) of acyl-FB1 derivatives may show large differences compared to the parent mycotoxin. Therefore, we tested the interactions of FB1, N-palmitoyl-FB1 (N-pal-FB1), 5-O-palmitoyl-FB1 (5-O-pal-FB1), and fumonisin B4 (FB4) with human serum albumin as well as the toxic effects of these mycotoxins on zebrafish embryos were examined. Based on our results, the most important observations and conclusions are the following: (1) FB1 and FB4 bind to albumin with low affinity, while palmitoyl-FB1 derivatives form highly stable complexes with the protein. (2) N-pal-FB1 and 5-O-pal-FB1 likely occupy more high-affinity binding sites on albumin. (3) Among the mycotoxins tested, N-pal-FB1 showed the most toxic effects on zebrafish, followed by 5-O-pal-FB1, FB4, and FB1. (4) Our study provides the first in vivo toxicity data regarding N-pal-FB1, 5-O-pal-FB1, and FB4.

Chronic Effects of Carbamazepine, Progesterone and Their Mixtures at Environmentally Relevant Concentrations on Biochemical Markers of Zebrafish (Danio rerio).

The impact of pharmaceuticals on non-target organisms in the environment is of increasing concern and study. Pharmaceuticals and other pollutants are often present as mixtures in an environmental compartment. Studies on the toxicological implications of these drugs on fish, particularly as mixtures at environmentally relevant concentrations, are very limited. Thus, this study aimed to evaluate the chronic effects of the anticonvulsant drug carbamazepine (CBZ) and progesterone (P4) at environmentally relevant concentrations, individually and in binary mixtures, applying a suite of biomarkers at the molecular level in zebrafish (Danio rerio). The effects on biotransformation enzymes 7-ethoxyresorufin O-deethylase (EROD) and glutathione-S-transferase (GST), antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidases (GPxSe and GPxTOT), and glutathione reductase (GR), and markers of damage, such as DNA strand breaks (DNAsb), lactate dehydrogenase (LDH), lipid peroxidation (LPO), and vitellogenin-like proteins (VTG), were evaluated. Analyses of the biochemical markers indicated that a synergistic dose-ratio-dependent effect of CBZ and P4 in zebrafish occurs after chronic exposure regarding VTG, biotransformation enzymes (EROD, GST), and oxidative stress marker (DNAsb). The results suggest a synergistic effect regarding VTG, thus indicating a high risk to the reproductive success of fish if these pharmaceuticals co-occur.

Testing the protective effects of cyclodextrins vs. alternariol-induced acute toxicity in HeLa cells and in zebrafish embryos.

Alternariol (AOH) is a mycotoxin produced by Alternaria fungi, it appears as a contaminant in tomatoes, grains, and grapes. The chronic exposure to AOH may cause carcinogenic and xenoestrogenic effects. Cyclodextrins (CDs) are cyclic oligosaccharides, they form host-guest complexes with apolar molecules. In this study, the interactions of AOH with CD monomers and polymers were examined employing fluorescence spectroscopy. Thereafter, the protective effects of certain CDs vs. AOH-induced toxicity were investigated on HeLa cells and on zebrafish embryos. Our major observations are the following: (1) Sugammadex forms highly stable complex with AOH (K = 4.8 ×104 L/mol). (2) Sugammadex abolished the AOH-induced toxicity in HeLa cells, while native ß-CD did not show relevant protective effect. (3) Each CD tested decreased the AOH-induced mortality and sublethal adverse effects in zebrafish embryos: Interestingly, native ß-CD showed the strongest protective impact in this model. (4) CD technology may be suitable to relieve AOH-induced toxicity.

Biochemical Marker Assessment of Chronic Carbamazepine Exposure at Environmentally Relevant Concentrations in Juvenile Common Carp (Cyprinus carpio).

Worldwide, the anticonvulsant drug carbamazepine (CBZ) is the most frequently identified pharmaceutical residue detected in rivers. Reported chronic effects of CBZ in non-target freshwater organisms, particularly fish, include oxidative stress and damage to liver tissues. Studies on CBZ effects in fish are mostly limited to zebrafish and rainbow trout studies. Furthermore, there are only a few chronic CBZ studies using near environmental concentrations. In this study, we provide data on subacute effects of CBZ exposure (28 days) to common carp (Cyprinus carpio), employing a set of biochemical markers of damage and exposure. CBZ was found to induce a significant change in the hepatic antioxidant status of fish subjected to 5 µg/L. Moreover, with increasing concentrations, enzymatic and non-enzymatic biomarkers of oxidative defence (catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), DNA strand breaks)), toxicant biotransformation (ethoxyresorufin-o-demethylase (EROD), glutathione-S-transferase (GST)), and organ and tissue damage (lactate dehydrogenase (LDH), cetylcholinesterase (AChE)) were altered. The AChE, LDH, and lipid peroxidation (LPO) results indicate the occurrence of apoptotic process activation and tissue damage after 28 days of exposure to CBZ. These findings suggest significant adverse effects of CBZ exposure to common carp at concentrations often found in surface waters.

Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method.

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.

The individual and combined effects of ochratoxin A with citrinin and their metabolites (ochratoxin B, ochratoxin C, and dihydrocitrinone) on 2D/3D cell cultures, and zebrafish embryo models.

Ochratoxin A and citrinin are nephrotoxic mycotoxins produced by Aspergillus, Penicillium, and/or Monascus species. The combined effects of ochratoxin A and citrinin have been examined in more studies; however, only limited data are available regarding the co-exposure to their metabolites. In this investigation, the individual toxic effects of ochratoxin A, ochratoxin B, ochratoxin C, citrinin, and dihydrocitrinone were tested as well as the combinations of ochratoxin A with the latter mycotoxins were examined on 2D and 3D cell cultures, and on zebrafish embryos. Our results demonstrate that even subtoxic concentrations of certain mycotoxins can increase the toxic impact of ochratoxin A. In addition, typically additive effects or synergism were observed as the combined effects of mycotoxins tested. These observations highlight that different cell lines (e.g. MDBK vs. MDCK), cell cultures (e.g. 2D vs. 3D), and models (e.g. in vitro vs. in vivo) can show different (sometimes opposite) impacts. Mycotoxin combinations considerably increased miR-731 levels in zebrafish embryos, which is an early marker of the toxicity on kidney development. These results underline that the co-exposure to mycotoxins (and/or mycotoxin metabolites) should be seriously considered, since even the barely toxic mycotoxins (or metabolites) in combinations can cause significant toxicity.

Ovarian inseminated sperm impacts spawning success in zebrafish, Danio rerio (Hamilton, 1822) even in the absence of a male stimulus.

Reproductive obstacles have led scientists to develop novel techniques/technologies for artificial reproduction. We aimed to investigate the possibility of propagating zebrafish females using sperm ovarian lavage with and without presence of male stimulus. This experiment consisted of several treatments: traditional spawning approaches with females and wild-type males (AB♀ × AB♂); no males present with non-manipulated females (AB♀); no males present with females inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)]; no males present with females inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)]; non-manipulated females kept separately from wild-type males (AB♀|AB♂); females kept separately from wild-type males and inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)|AB♂]; and females kept separately from wild-type males and inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)|AB♂]. There were no released eggs in both negative control treatments (AB♀ and AB♀|AB♂). Egg production increased (ranged from 0 to 28.5 eggs/female) when females were injected in the presence [AB♀ (inj.NaCl) |AB♂] or absence of male stimulus [AB♀ (inj.NaCl) and (AB♀(inj.Tg♂)]. A further increase in egg production [relative to AB♀, AB♀ (inj.NaCl), and AB♀|AB♂] was detected when females were inseminated with pooled sperm from transgenic males in the presence of male stimulus [AB♀(inj.Tg♂)|AB♂; ranged from 2.5 to 55 eggs/female] or when using traditional spawning approaches (AB♀ × AB♂; ranged from 25 to 131 eggs/female). Females inseminated with sperm produced embryos, although no differences were detected when females were inseminated with pooled sperm from transgenic males in presence (11.8 ± 16.3%) or absence (average = 12.6 ± 9.2%) of male stimulus. Traditional spawning approaches produced the most eggs (81.2 ± 42.3 per female) and highest fertilization rate (81.3 ± 10.4).

Evaluation of the Multimycotoxin-Degrading Efficiency of Rhodococcus erythropolis NI1 Strain with the Three-Step Zebrafish Microinjection Method.

The multimycotoxin-degrading efficiency of the Rhodococcus erythropolis NI1 strain was investigated with a previously developed three-step method. NI1 bacterial metabolites, single and combined mycotoxins and their NI1 degradation products, were injected into one cell stage zebrafish embryos in the same doses. Toxic and interaction effects were supplemented with UHPLC-MS/MS measurement of toxin concentrations. Results showed that the NI1 strain was able to degrade mycotoxins and their mixtures in different proportions, where a higher ratio of mycotoxins were reduced in combination than single ones. The NI1 strain reduced the toxic effects of mycotoxins and mixtures, except for the AFB1+T-2 mixture. Degradation products of the AFB1+T-2 mixture by the NI1 strain were more toxic than the initial AFB1+T-2 mixture, while the analytical results showed very high degradation, which means that the NI1 strain degraded this mixture to toxic degradation products. The NI1 strain was able to detoxify the AFB1, ZEN, T-2 toxins and mixtures (except for AFB1+T-2 mixture) during the degradation experiments, which means that the NI1 strain degraded these to non-toxic degradation products. The results demonstrate that single exposures of mycotoxins were very toxic. The combined exposure of mycotoxins had synergistic effects, except for ZEN+T-2 and AFB1+ZEN +T-2, whose mixtures had very strong antagonistic effects.

Deregulation of whole-transcriptome gene expression in zebrafish (Danio rerio) after chronic exposure to low doses of imatinib mesylate in a complete life cycle study.

Imatinib mesylate (IM) is an anticancer drug that belongs to tyrosine kinase inhibitors. We report the results of the first investigation of the chronic exposure of zebrafish (Danio rerio) to IM. The exposure to IM (0.01, 1 and 100 µg/L) was initiated in adult fish and continued through hatching and the offspring generation for seven months. In addition to standard toxicological endpoints, induction of genotoxic effects and whole-genome transcriptome of liver samples of offspring generation of zebrafish were analysed. Exposure to IM did not affect the survival and growth of zebrafish, did not cause any histopathological changes, but it induced a marginal increase in the chromosomal damage in blood cells. The whole-genome transcriptome analyses demonstrated dose-dependent increase in the number of differentially expressed genes with a significantly higher number of deregulated genes in female fish compared to male. Differentially expressed genes included genes involved in response to DNA damage, cell cycle control and regulation of circadian rhythm. Based on the low genotoxic activity and the pattern of the changes in DNA damage responsive genes we consider that at current environmental exposure levels, IM represents low risk for genotoxic effects in aquatic organisms. Exposure to IM also induced deregulation of the expression of genes associated with steroidogenesis and hormone metabolism and function, which indicates hormone-disrupting activity of IM that has not been studied so far. The study provide new information on the potential consequences of chronic exposure to the residues of tyrosine kinase inhibitors, which remain to be further explored.

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds.

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, α- and ß-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

Qualifying the T-2 Toxin-Degrading Properties of Seven Microbes with Zebrafish Embryo Microinjection Method.

T-2 mycotoxin degradation and detoxification efficiency of seven bacterial strains were investigated with zebrafish microinjection method in three steps ((1) determination of mycotoxin toxicity baseline, (2) examination of bacterial metabolites toxicity, (3) identification of degradation products toxicity). Toxicity of T-2 was used as a baseline of toxic effects, bacterial metabolites of strains as control of bacterial toxicity and degradation products of toxin as control of biodegradation were injected into one-cell stage embryos in the same experiment. The results of in vivo tests were checked and supplemented with UHPLC-MS/MS measurement of T-2 concentration of samples. Results showed that the Rhodococcus erythropolis NI1 strain was the only one of the seven tested (R. gordoniae AK38, R. ruber N361, R. coprophilus N774, R. rhodochrous NI2, R. globerulus N58, Gordonia paraffinivorans NZS14), which was appropriated to criteria all aspects (bacterial and degradation metabolites of strains caused lower toxicity effects than T-2, and strains were able to degrade T-2 mycotoxin). Bacterial and degradation metabolites of the NI1 strain caused slight lethal and sublethal effects on zebrafish embryos at 72- and 120-h postinjection. Results demonstrated that the three-step zebrafish microinjection method is well-suited to the determination and classification of different bacterial strains by their mycotoxin degradation and detoxification efficiency.

Protective effects of beta-cyclodextrins vs. zearalenone-induced toxicity in HeLa cells and Tg(vtg1:mCherry) zebrafish embryos.

Zearalenone is a xenoestrogenic mycotoxin produced by Fusarium species. High exposure with zearalenone induces reproductive disorders worldwide. Cyclodextrins are ring-shaped host molecules built up from glucose units. The apolar cavity of cyclodextrins can entrap so-called guest molecules. The formation of highly stable host-guest type complexes with cyclodextrins can decrease the biological effect of the guest molecule. Therefore, cyclodextrins may be suitable to decrease the toxicity of some xenobiotics even after the exposure. In this study, the protective effect of beta-cyclodextrins against zearalenone-induced toxicity was investigated in HeLa cells and zebrafish embryos. Fluorescence spectroscopic studies demonstrated the formation of stable complexes of zearalenone with sulfobutyl-, methyl-, and succinyl-methyl-substituted beta-cyclodextrins at pH 7.4 (K = 1.4-4.7 × 104 L/mol). These chemically modified cyclodextrins considerably decreased or even abolished the zearalenone-induced loss of cell viability in HeLa cells and mortality in zebrafish embryos. Furthermore, the sublethal effects of zearalenone were also significantly alleviated by the co-treatment with beta-cyclodextrins. To test the estrogenic effect of the mycotoxin, a transgenic bioindicator zebrafish model (Tg(vtg1:mCherry)) was also applied. Our results suggest that the zearalenone-induced vitellogenin production is partly suppressed by the hepatotoxicity of zearalenone in zebrafish. This study demonstrates that the formation of stable zearalenone-cyclodextrin complexes can strongly decrease or even abolish the zearalenone-induced toxicity, both in vitro and in vivo. Therefore, cyclodextrins appear as promising new mycotoxin binders.

Preservation of zebrafish genetic resources through testis cryopreservation and spermatogonia transplantation.

Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

Biological evaluation of microbial toxin degradation by microinjected zebrafish (Danio rerio) embryos.

The use of microinjection of newly fertilized zebrafish eggs as an appropriate tool for qualifying the biodetoxification properties of toxin-degrading microbes was investigated. Ochratoxin A (OTA), bacterial degradation products of OTA and bacterial metabolites of the Cupriavidus basilensis OR16 strain were microinjected. Results showed that variations in the injected droplet size, and thus treatment concentrations, stayed within ±20%, moreover embryo mortality did not exceed 10% in controls, that is in accordance with the recommendations of the OECD 236 guideline. The highest lethality was caused by OTA with a significantly higher toxicity than that of bacterial metabolites or OTA degradation products. However, toxicity of the latter two did not differ statistically from each other showing that the observed mortality was due to the intrinsic toxicity of bacterial metabolites (and not OTA degradation products), thus, the strain effectively degrades OTA to nontoxic products. Sublethal symptoms also confirmed this finding. RESULTS: confirmed that microinjection of zebrafish embryos could be a reliable tool for testing the toxin-degrading properties of microbes. The method also allows comparisons among microbial strains able to degrade the same toxin, helping the selection of effective and environmentally safe microbial strains for the biodetoxification of mycotoxins in large scale.

A Method for Zebrafish Follicle Transplantation into Recipient Mothers for the Generation of Fertilizable Eggs and Viable Offspring.

In zebrafish and other externally developing animals, maternal effect gene products significantly contribute to embryonic and larval development. Several methods exist for both forward and reverse genetic manipulation of maternal gene products, yet it remains technically difficult to interfere with maternal gene products. Therefore, alternative methods to manipulate maternal factors in oocytes remain of interest. Here we describe a method which allows manipulation and subsequent transplantation of stage I-II follicles of zebrafish into recipient mothers where donor oocytes can develop into mature eggs and produce viable offspring. Additionally, we describe a simple microinjection protocol for injecting reporter constructs into follicles as a proxy for manipulating maternal effect genes.

Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples.

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations. The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites. A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos. Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

l-Alpha Glycerylphosphorylcholine as a Potential Radioprotective Agent in Zebrafish Embryo Model.

This work establishes the zebrafish embryo model for ionizing radiation (IR) modifier research and also evaluates the protective effect of l-alpha glycerylphosphorylcholine (GPC). Embryos were exposed to a single-fraction whole-body gamma irradiation (5, 10, 15, and 20 Gy) at different postfertilization time points and were serially assessed for viability and macro- and micromorphologic abnormalities. After toxicity evaluation, 194 µM of GPC was added for certain groups with 3-h incubation before the radiation. Nuclear factor kappa B (NF-κB) and interleukin-1ß (IL-1ß) expression changes were measured using quantitative real-time polymerase chain reaction. A higher sensitivity could be observed at earlier stages of the embryogenesis. The lethal dose (LD50) for 6 hours postfertilization (hpf) embryos was 15 Gy and for 24 hpf was 20 Gy on day 7, respectively. GPC administration resulted in a significant improvement in both the distortion rate and survival of the 24 hpf embryos. Qualitative evaluation of the histological changes confirmed the protective effect of GPC. IL-1ß and NF-κB overexpression due to 10 Gy irradiation was also reduced by GPC. GPC exhibited promising radioprotective effects in our zebrafish embryo model, decreasing the irradiation-induced morphological damage and lethality with significant reduction of IR-caused pro-inflammatory activation.