Periodic Reporting for period 1 - NRgCod (Developing an Adverse Outcome Pathway for metabolic Nuclear Receptor-binding compounds in Atlantic cod)
Período documentado: 2021-10-01 hasta 2023-09-30
Human-made chemicals like pesticides, persistent organic pollutants (POPs), plastics, and plasticizers are accumulating in the environment. Recent research suggests that these contaminants might subtly impact the behavior of aquatic animals. Even at low exposure levels, changes in feeding, mating, and daily activity can profoundly affect population structures and ecosystems, as seen in fish. However, the biological mechanisms behind these behavioral shifts remain mostly unknown. It is important to understand these biological mechanisms to develop fast and cost-effective screening tools to assess the environmental risks of ongoing and future ocean pollution.
The NRgCod project aimed to uncover how disruptions in energy metabolism, especially lipid and glucose metabolism, might drive these behavioral changes. Focusing on the commercially valuable Atlantic cod, the project had three main goals:
1. Identify environmentally relevant chemicals that activate a metabolic nuclear receptor and disrupt glucose metabolism in Atlantic cod.
2. Develop a luciferase reporter assay to test the activity of the chosen receptor and quickly screen numerous environmental contaminants.
3. Investigate the link between receptor activity, changes in glucose metabolism, and behavior in Atlantic cod.
The NRgCod project aimed to uncover how disruptions in energy metabolism, especially lipid and glucose metabolism, might drive these behavioral changes. Focusing on the commercially valuable Atlantic cod, the project had three main goals:
1. Identify environmentally relevant chemicals that activate a metabolic nuclear receptor and disrupt glucose metabolism in Atlantic cod.
2. Develop a luciferase reporter assay to test the activity of the chosen receptor and quickly screen numerous environmental contaminants.
3. Investigate the link between receptor activity, changes in glucose metabolism, and behavior in Atlantic cod.
The NRgCod project focused on the glucocorticoid receptor (GR), which can be activated by environmental pharmaceuticals like dexamethasone. Literature review showed that several contaminants could bind to the human GR, but their effects on the Atlantic cod GR were unknown. A comparison of GR sequences from Atlantic cod, zebrafish, and humans revealed that, while overall sequences differ, the active site residues are conserved, suggesting similar ligand binding across species.
Next, the team developed a luciferase reporter assay to test GRs from Atlantic cod, zebrafish, and humans. The environmental contaminants from the literature review were screened for their ability to activate the GR of all three species and additionally two wastewater samples from a treatment plant in Stavanger were included (in collaboration with Daniela Pampanin from the University of Stavanger). Results showed that only dexamethasone and prednisolone activated the GR in all three species, while most other contaminants did not. The contaminants were also tested for their potential to inhibit an agonist-stimulated response (antagonism). While the analysis is still ongoing, the data indicates that some substances, like bisphenol B and triphenyl phosphate, displayed antagonistic effects on the GR in Atlantic cod, with similar results for zebrafish GR. Human GR antagonism tests are still pending. Thus, most contaminants previously thought to interact with the GR could not be confirmed as GR activators or inhibitors.
To address objective 3, two in vivo experiments with juvenile Atlantic cod were designed and performed. Dexamethasone was chosen as a compound due to its clear agonistic effect on the Atlantic cod GR in the in vitro assay and its environmental presence. In the first experiment, cod were exposed to environmentally relevant dexamethasone concentrations for two weeks, while in the second, they were exposed for six hours to much higher doses. Cod behavior was recorded to assess movement and feeding. Subsequently, the liver was sampled to measure changes in gene expression of genes relevant to glucose metabolism in exposed fish in comparison to the control. Transcriptomic changes in liver samples from the highest dose were also evaluated using RNA sequencing (RNA-seq). Blood samples were analyzed for glucose, cholesterol, triglycerides, and cortisol levels, and water samples verified the exposure concentrations. Both exposure scenarios did not significantly affect any of the measured parameters. Except for the RNA-seq analysis, which highlighted the steroid biosynthesis, glycolysis/gluconeogenesis, and metabolic pathways as significantly altered pathways in the highest dose group. These two in vivo experiments thus suggest that while GR ligands can induce changes in metabolism, including gluconeogenesis, in Atlantic cod, such alterations are not cascading into measurable changes in locomotion and feeding.
In summary, the NRgCod project examined the effects of environmental contaminants and dexamethasone on the GR in Atlantic cod. Only dexamethasone and prednisolone activated the GR, while some contaminants showed antagonistic effects on cod and zebrafish GRs. In vivo tests with dexamethasone revealed no measurable changes in cod behavior or feeding, though high-dose exposure influenced certain metabolic pathways in the liver. These results suggest that while GR ligands may alter metabolism in cod, they do not necessarily translate to physiological parameters or behaviors.
The results of the NRgCod project are disseminated in two forthcoming publications on the activation/inhibition of GR in three species by various environmental contaminants and on the in vivo experiment investigating the response of Atlantic cod to a GR ligand. The results were also shared at two national conferences (the Norwegian Environmental Chemistry Symposium 2022, 2023) and an international conference (PRIMO in Gothenburg, 2022). The laboratory protocols are shared on protocols.io. The knowledge gained in the NrgCod project was transferred to two MSc students who did their thesis work on the project. On the university's project webpage, an overview of the project and initial results are communicated.
Next, the team developed a luciferase reporter assay to test GRs from Atlantic cod, zebrafish, and humans. The environmental contaminants from the literature review were screened for their ability to activate the GR of all three species and additionally two wastewater samples from a treatment plant in Stavanger were included (in collaboration with Daniela Pampanin from the University of Stavanger). Results showed that only dexamethasone and prednisolone activated the GR in all three species, while most other contaminants did not. The contaminants were also tested for their potential to inhibit an agonist-stimulated response (antagonism). While the analysis is still ongoing, the data indicates that some substances, like bisphenol B and triphenyl phosphate, displayed antagonistic effects on the GR in Atlantic cod, with similar results for zebrafish GR. Human GR antagonism tests are still pending. Thus, most contaminants previously thought to interact with the GR could not be confirmed as GR activators or inhibitors.
To address objective 3, two in vivo experiments with juvenile Atlantic cod were designed and performed. Dexamethasone was chosen as a compound due to its clear agonistic effect on the Atlantic cod GR in the in vitro assay and its environmental presence. In the first experiment, cod were exposed to environmentally relevant dexamethasone concentrations for two weeks, while in the second, they were exposed for six hours to much higher doses. Cod behavior was recorded to assess movement and feeding. Subsequently, the liver was sampled to measure changes in gene expression of genes relevant to glucose metabolism in exposed fish in comparison to the control. Transcriptomic changes in liver samples from the highest dose were also evaluated using RNA sequencing (RNA-seq). Blood samples were analyzed for glucose, cholesterol, triglycerides, and cortisol levels, and water samples verified the exposure concentrations. Both exposure scenarios did not significantly affect any of the measured parameters. Except for the RNA-seq analysis, which highlighted the steroid biosynthesis, glycolysis/gluconeogenesis, and metabolic pathways as significantly altered pathways in the highest dose group. These two in vivo experiments thus suggest that while GR ligands can induce changes in metabolism, including gluconeogenesis, in Atlantic cod, such alterations are not cascading into measurable changes in locomotion and feeding.
In summary, the NRgCod project examined the effects of environmental contaminants and dexamethasone on the GR in Atlantic cod. Only dexamethasone and prednisolone activated the GR, while some contaminants showed antagonistic effects on cod and zebrafish GRs. In vivo tests with dexamethasone revealed no measurable changes in cod behavior or feeding, though high-dose exposure influenced certain metabolic pathways in the liver. These results suggest that while GR ligands may alter metabolism in cod, they do not necessarily translate to physiological parameters or behaviors.
The results of the NRgCod project are disseminated in two forthcoming publications on the activation/inhibition of GR in three species by various environmental contaminants and on the in vivo experiment investigating the response of Atlantic cod to a GR ligand. The results were also shared at two national conferences (the Norwegian Environmental Chemistry Symposium 2022, 2023) and an international conference (PRIMO in Gothenburg, 2022). The laboratory protocols are shared on protocols.io. The knowledge gained in the NrgCod project was transferred to two MSc students who did their thesis work on the project. On the university's project webpage, an overview of the project and initial results are communicated.
The NRgCod project has developed a GR receptor-ligand-based luciferase reporter assay for three species, including the Atlantic cod. Such a screening tool for ecologically relevant benthopelagic species expands and complements the screening assays based on zebrafish currently used by the European REACH regulation. The environmental contaminants screened in this project showed little interaction with the GR which will help to guide future research efforts towards other nuclear receptors. Furthermore, the project delivered one of the first studies on Atlantic cod movement and feeding behavior. The behavioral setup developed in this project can guide other behavioral studies with this species and thereby advance the assessment of a sensitive yet underappreciated endpoint that currently is under discussion to be included in risk assessments.