Background:
In multiple sclerosis, development of screening tools for remyelination-promoting molecules is timely.
Objective:
A Xenopus transgenic line allowing conditional ablation of myelinating ...oligodendrocytes has been adapted for in vivo screening of remyelination-favoring molecules.
Methods:
In this transgenic, the green fluorescent protein reporter is fused to E. coli nitroreductase and expressed specifically in myelinating oligodendrocytes. Nitroreductase converts the innocuous pro-drug metronidazole to a cytotoxin. Spontaneous remyelination occurs after metronidazole-induced demyelinating responses. As tadpoles are transparent, these events can be monitored in vivo and quantified. At the end of metronidazole-induced demyelination, tadpoles were screened in water containing the compounds tested. After 72 h, remyelination was assayed by counting numbers of oligodendrocytes per optic nerve.
Results:
Among a battery of molecules tested, siponimod, a dual agonist of sphingosine-1-phosphate receptor 1 and 5, was among the most efficient favoring remyelination. Crispr/cas9 gene editing showed that the promyelinating effect of siponimod involves the sphingosine-1-phosphate receptor 5.
Conclusion:
This Xenopus transgenic line constitutes a simple in vivo screening platform for myelin repair therapeutics. We validated several known promyelinating compounds and demonstrated that the strong remyelinating efficacy of siponimod implicates the sphingosine-1-phosphate receptor 5.
The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, ...and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.
Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase ...extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.
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•Effect-based triggers (EBTs) for bioassays discriminate good from poor water quality.•EBTs can be derived by read across from existing water quality guideline values.•Mixture factor warranted for bioassays responding to many different chemicals.•EBT derivation method applicable to every bioassay subject to data availability•Here we derived preliminary EBTs for 32 bioassays and discuss many more.
Ovulated eggs possess maternal apoptotic execution machinery that is inhibited for a limited time. The fertilized eggs switch off this time bomb whereas aged unfertilized eggs and parthenogenetically ...activated eggs fail to stop the timer and die. To investigate the nature of the molecular clock that triggers the egg decision of committing suicide, we introduce here Xenopus eggs as an in vivo system for studying the death of unfertilized eggs. We report that after ovulation, a number of eggs remains in the female body where they die by apoptosis. Similarly, ovulated unfertilized eggs recovered in the external medium die within 72 h. We showed that the death process depends on both cytochrome c release and caspase activation. The apoptotic machinery is turned on during meiotic maturation, before fertilization. The death pathway is independent of ERK but relies on activating Bad phosphorylation through the control of both kinases Cdk1 and JNK. In conclusion, the default fate of an unfertilized Xenopus egg is to die by a mitochondrial dependent apoptosis activated during meiotic maturation.
Over the previous decade, numerous new approach methodologies (NAMs) have been developed and validated for the detection of endocrine activity of individual chemicals or environmental samples. These ...NAMs can be largely separated into three categories, in silico tools, in vitro assays, and in vivo assays using organisms or life stages not considered as laboratory animals, each with their own advantages and disadvantages. While in vitro assays provide more mechanistic information, the use of whole organisms such as fish or amphibian embryos provides a more holistic view of the net effects of an environmental sample on hormonal activity. A panel of bioassays was used to test the endocrine activity of several samples from the Danube River at Novi Sad, Serbia. The results of the in vitro assays have been published previously. Here, we present the results of the in vivo assays that were performed at the same time on the same samples. These whole organism assays are based on the use of transgenic fish and amphibian eleuthero‐embryos and included the Xenopus Eleuthero‐embryo Thyroid Assay (XETA), the Rapid Estrogen ACTivity In Vivo assay (REACTIV), and the Rapid Androgen Disruption Activity Reporter (RADAR) assay. Discrepancies between the different in vitro assays have previously been reported. The results of the in vivo studies also indicate discrepancies between the in vivo and in vitro data with an underestimation of the endocrine activity by the in vitro tests. Therefore, a battery of tests is advised with the initial diagnostic performed with in vivo tests to cover a wider range of modes of action and to allow the appropriate in vitro assay(s) to be selected to confirm the mode of action.
Practitioner Points
Endocrine activity was quantified in surface water using in vitro and in vivo models.
The in vivo results fit with previously reported in vitro results.
Higher activity was observed in water samples with in vivo models, which cover a wider range of modes of action.
Endocrine activity of surface water samples may be underestimated when measured with in vitro models.
Endocrine activity was quantified in surface water using in vitro and in vivo models and direct comparisons made between the results. The in vivo models, which cover a wider range modes of action, show higher endocrine activity than that measured in vitro.
Wastewaters contain complex mixtures of chemicals, which can cause adverse toxic effects in the receiving environment. In the present study, the toxicity removal during wastewater treatment at seven ...municipal wastewater treatment plants (WWTPs) was investigated using an effect-based approach. A battery of eight bioassays was applied comprising of cytotoxicity, genotoxicity, endocrine disruption and fish embryo toxicity assays. Human cell-based CALUX assays, transgenic larval models and the fish embryo toxicity test were particularly sensitive to WWTP effluents. The results indicate that most effects were significantly reduced or completely removed during wastewater treatment (76–100%), while embryo toxicity, estrogenic activity and thyroid disruption were still detectable in the effluents suggesting that some harmful substances remain after treatment. The responsiveness of the bioassays was compared and the human cell-based CALUX assays showed highest responsiveness in the samples. Additionally, the fish embryo toxicity test and the transgenic larval models for endocrine disrupting effects showed high responsiveness at low sample concentrations in nearly all of the effluent samples. The results showed a similar effect pattern among all WWTPs investigated, indicating that the wastewater composition could be rather similar at different locations. There were no considerable differences in the toxicity removal efficiencies of the treatment plants and no correlation was observed with WWTP characteristics, such as process configuration or sludge age. This study demonstrated that a biotest battery comprising of multiple endpoints can serve as a powerful tool when assessing water quality or water treatment efficiency in a holistic manner. Rather than analyzing the concentrations of a few selected chemicals, bioassays can be used to complement traditional methods of monitoring in the future by assessing sum-parameter based effects, such as mixture effects, and tackling chemicals that are present at concentrations below chemical analytical detection limits.
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•Toxicity removal during wastewater treatment at 7 municipal WWTPs was investigated.•Removal efficiency was assessed by an effect-based approach comprising of multiple endpoints.•Large volumes of influent and effluent samples were extracted with a novel device.•Embryo toxicity, estrogenic activity and thyroid disruption were detected in effluent samples.•The results showed a similar effect pattern among all the WWTPs.
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•eAOP efficiency evaluated by UHPLC-QTOF-MS and in vivo estrogenic bioassay.•Over 99.9% EE2 removal obtained at optimal conditions.•Estrogenic activity decreases following the ...degradation of EE2.•Electro-generated radicals readily attack phenolic moiety of EE2.•79 transformation products identified and monitored.
Conventional water treatment methods are not efficient in eliminating endocrine disrupting compounds (EDCs) in wastewater. Electrochemical Advanced Oxidation Processes (eAOPs) offer a promising alternative, as they electro-generate highly reactive species that oxidize EDCs. However, these processes produce a wide spectrum of transformation products (TPs) with unknown chemical and biological properties. Therefore, a comprehensive chemical and biological evaluation of these remediation technologies is necessary before they can be safely applied in real-life situations. In this study, 17α-ethinylestradiol (EE2), a persistent estrogen, was electrochemically degraded using a boron doped diamond anode with sodium sulfate (Na2SO4) and sodium chloride (NaCl) as supporting electrolytes. Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was used for the quantification of EE2 and the identification of TPs. Estrogenic activity was assessed using a transgenic medaka fish line. At optimal operating conditions, EE2 removal reached over 99.9% after 120 min and 2 min, using Na2SO4 and NaCl, respectively. The combined EE2 quantification and in vivo estrogenic assessment demonstrated the overall estrogenic activity was consistently reduced with the degradation of EE2, but not completely eradicated. The identification and time monitoring of TPs showed that the radical agents readily oxidized the phenolic A-ring of EE2, leading to the generation of hydroxylated and/or halogenated TPs and ring-opening products. eAOP revealed to be a promising technique for the removal of EE2 from water. However, caution should be exercised with respect to the generation of potentially toxic TPs.
Widespread environmental antiandrogen contamination has been associated with negative impacts on biodiversity and human health. In particular, many pesticides are antiandrogenic, creating a need for ...robust and sensitive environmental monitoring. Our aim was to develop a sensitive and specific transgenic medaka (Oryzias latipes) model bearing an androgen responsive fluorescent reporter construct for whole organism-based environmental screening of pro- and antiandrogens. We analyzed the 5′ regions of the androgen responsive three-spined stickleback (Gasterosteus aculeatus) spiggin genes in silico, revealing conserved blocks of sequence harboring androgen response elements. Identified putative promoters were cloned upstream of GFP. Germinal transgenesis with spg1-gfp led to stable medaka lines. GFP induction was exclusive to the kidney, the site of spiggin protein production in sticklebacks. Significant GFP expression was induced by three or four-day androgen treatment of newly hatched fry, but not by estrogens, mineralocorticoids, glucocorticoids or progestogens. The model responded dose-dependently to androgens, with highest sensitivity to 17MT (1.5 μg/L). In addition to flutamide, the biocides fenitrothion, vinclozolin and linuron significantly inhibited 17MT-induced GFP induction, validating the model for detection of antiandrogens. The spg1-gfp medaka model provides a sensitive, specific, and physiologically pertinent biosensor system for analyzing environmental androgen activity.
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