Periodic Reporting for period 1 - SPHEROTOX (Warming Impacts on Estrogenic and Progestagenic Endocrine Disruptors Effects in Fish Liver - A Proof of Concept Study Using Single- and Novel Multi-Cell 3D Cultures of the European Native Brown Trout)
Reporting period: 2021-11-01 to 2022-10-31
Contamination of freshwater ecosystems originates from many sources, but the majority of pollutants that end up in water bodies are of anthropogenic origin (man-made). Several classes of pollutants are contributing to water contamination and have been studied for decades: organic waste, plant nutrients, inorganic chemicals and minerals. However, another class of contaminants is not extensively studied and is termed “emerging pollutants”. These substances are not yet regulated but may be of environmental or human health concern, and they include antibiotics, drugs, industrial additives, microbeads, microplastics and hormones. The focus of this research project was to tackle the pollution of aquatic ecosystems with one specific class of hormones - progestins. Progestins (also called gestagens, progestogens) are frequently used in birth control pills, cancer therapy and animal husbandry. After use, progestins are excreted from the human or animal body, and they end up in the water since wastewater treatment plants cannot completely degrade them. If concentrations increase in the water, progestins can cause deleterious effects on aquatic fauna and in the first place, they negatively affect the endocrine system of animals. This phenomenon is called “endocrine disruption” since progestins modulate and ultimately disturb the normal hormonal status of the animal. They may cause changes in many organs of fish, but the effects are most severe in endocrine glands, gonads and liver.
Apart from fish, all other aquatic animals are at risk, as well as complete ecosystems (not only freshwater, but also marine ecosystems). In previous experiments, entire fish populations collapsed if they were exposed to extremely low concentrations of hormones (6 ng/L) for three years. Those concentrations are usually found in rivers across the world, including Europe. Furthermore, the presence of hormones in drinking water poses a significant risk to the human population.
The main objective of the SPHEROTOX project was to study effects of two progestins (levonorgestrel and megestrol-acetate), as well as 17α-ethynylestradiol (estrogen) and their binary mixture on the liver of brown trout, an indigenous species for the European continent. Moreover, we also wanted to assess the impact of one key element of climate change on brown trout by exposing them to hormones at two different temperatures: 18 and 21°C. Fish are poikilothermic animals, and their body temperature depends on the water temperature in which they live. We hypothesised that at a higher temperature, the toxicity of endocrine disruptors would have a different impact compared to a lower temperature. Instead of conducting toxicological assays in vivo, we used three-dimensional cellular agglomerations called “spheroids” in vitro. The advantage of using spheroids compared to routine 2D cell cultures is their ability to more closely mimic conditions in real organs. Spheroids have specific microenvironments, cell-to-cell interactions, and metabolism more similar to in vivo. Using this technique, we managed to culture spheroids and exposed them to hormones, either as a single chemical or in mixtures. These are more relevant to environmental pollution as pollutants in the water are found in complex mixtures, which have additional adverse effects on aquatic animals.
Apart from fish, all other aquatic animals are at risk, as well as complete ecosystems (not only freshwater, but also marine ecosystems). In previous experiments, entire fish populations collapsed if they were exposed to extremely low concentrations of hormones (6 ng/L) for three years. Those concentrations are usually found in rivers across the world, including Europe. Furthermore, the presence of hormones in drinking water poses a significant risk to the human population.
The main objective of the SPHEROTOX project was to study effects of two progestins (levonorgestrel and megestrol-acetate), as well as 17α-ethynylestradiol (estrogen) and their binary mixture on the liver of brown trout, an indigenous species for the European continent. Moreover, we also wanted to assess the impact of one key element of climate change on brown trout by exposing them to hormones at two different temperatures: 18 and 21°C. Fish are poikilothermic animals, and their body temperature depends on the water temperature in which they live. We hypothesised that at a higher temperature, the toxicity of endocrine disruptors would have a different impact compared to a lower temperature. Instead of conducting toxicological assays in vivo, we used three-dimensional cellular agglomerations called “spheroids” in vitro. The advantage of using spheroids compared to routine 2D cell cultures is their ability to more closely mimic conditions in real organs. Spheroids have specific microenvironments, cell-to-cell interactions, and metabolism more similar to in vivo. Using this technique, we managed to culture spheroids and exposed them to hormones, either as a single chemical or in mixtures. These are more relevant to environmental pollution as pollutants in the water are found in complex mixtures, which have additional adverse effects on aquatic animals.
As already pointed out, the spheroids were successfully cultured in the Laboratory for Histology and Embryology at ICBAS - School of Medicine and Biomedical Sciences, University of Porto. They need around 8 days to mature and become organized like cells in the liver. However, we chose to cultivate them for 12 days before exposing them to the aforementioned hormones for 6 days. Hormones were applied in either single concentrations (0.3 or 0.6 µM and in the binary mixtures of 0.3 µM), while we also had control and solvent control groups. At the end of the exposure period, spheroids were collected for several laboratory assays to check viability and effects of hormones: (1) gene expression assays; (2) biochemical analysis of viability; (3) light microscopy (morphology/immunohistochemistry); (4) Raman spectroscopy and (5) biometry. Results showed that in terms of metabolism (measured by two biochemical assays – LDH and resazurin), biometry (measuring of diameter, surface area and sphericity) and morphology of cells – there were no observable or statistically significant differences among groups. In other words, hormones did not cause changes to the viability and survival of spheroids/cells or their size. However, we demonstrated the difference between the two culture temperatures in terms of biochemical assays, meaning that global warming negatively impacted the metabolism of spheroids. Using a specific antibody (vitellogenin A) and gene expression assays, we also showed that hormones negatively impacted genes related to hepatic lipid metabolism, production of yolk and eggshell proteins, and expression of estrogen receptors in exposed spheroids.
Using in-vitro methods, such are spheroids in the SPHEROTOX project, spared many lives of experimental animals. As described in the experimental setup, the exposure of spheroids to three hormones at two temperatures (eight combinations in total) allowed us to sacrifice only 8 brown trouts to achieve the research aim. If the same experimental design were conducted in vivo (using live animals), it would be necessary to sacrifice at least 300 fish. Therefore, our strategy allowed an enormous reduction in experimental animals (more than 97%). Annually, approximately 10.4 million experimental animals were used in the EU for various testing purposes, and 24.6% of that number accounted for fish (approx. 2.5 million fish), which means there is a way of reducing these huge numbers. The introduction of in vitro testing covering subacute and chronic toxicity has considerable ethical and economic benefits for society besides allowing new research opportunities and insights. Currently, traditional cell cultures are routinely run only in acute toxicity testing since they typically last 72 hours. Still, spheroids can survive, be exposed to much longer periods, and last for more than 30 days in the laboratory. This allows scientists to test the effects of various harmful chemicals, such as endocrine disruptors in the current project, for a longer period and assess their potential chronic toxicity. In this project, we showed that liver spheroids might react to hormones in similar ways as in vivo and that the methodology is robust since a large number of experimental groups is possible simultaneously, without the need for adding expensive equipment to already running cell culture laboratories.