TY  - CONF
AU  - Madureira, Tania Vieria
AU  - Alves, Rodrigo
AU  - Lopes, Celia
AU  - Raskovic, Bozidar
AU  - Rocha, Eduardo
PY  - 2023
UR  - http://aspace.agrif.bg.ac.rs/handle/123456789/6937
AB  - Three-dimensional (3D) culture models of mammalian hepatocytes have been shown to replicate in vivo liver features accurately. The use of these 3D models avoids the constraints of an in vivo assay while sustaining the morphology and functionality of the hepatocytes throughout several days in culture. The feasibility of generating hepatic spheroids from other species, including fish, seems promising but has received less attention than in mammal models. Primary brown trout hepatocyte spheroids were first obtained by our research group from juvenile fish and cultured for over 30 days under orbital shaking. Optimization assays showed that these spheroids achieved biometric, morphological and basal gene expression stability from day 12 to day 20. This multiparametric characterization allowed the selection of a temporal window of culture days, which is being explored in several assays. The 3D primary hepatocyte spheroids were exposed to estrogens (e.g., 17α-ethinylestradiol) at different concentrations. The effect of increasing temperature was also evaluated in this model by maintaining cultures at 18°C and 21°C. Data collected include microscopy analysis, target gene expression of estrogenic and lipid pathways, and cellular biochemistry. The outputs indicate that hepatic spheroids are metabolically active and respond to different hormonal and temperature stimuli. The 3D model developed from primary brown trout hepatocytes seems to be a viable alternative for studying hepatic disruptions caused by (at least) estrogenic compounds and varied temperatures.
PB  - Iberian Association of Comparative Endocrinology
C3  - XIV Congress of the Iberian Association of Comparative Endocrinology
T1  - Brown trout primary hepatocyte spheroids – characterization and applications in endocrinology and beyond
EP  - 87
SP  - 87
UR  - https://hdl.handle.net/21.15107/rcub_agrospace_6937
ER  - 

@conference{
author = "Madureira, Tania Vieria and Alves, Rodrigo and Lopes, Celia and Raskovic, Bozidar and Rocha, Eduardo",
year = "2023",
abstract = "Three-dimensional (3D) culture models of mammalian hepatocytes have been shown to replicate in vivo liver features accurately. The use of these 3D models avoids the constraints of an in vivo assay while sustaining the morphology and functionality of the hepatocytes throughout several days in culture. The feasibility of generating hepatic spheroids from other species, including fish, seems promising but has received less attention than in mammal models. Primary brown trout hepatocyte spheroids were first obtained by our research group from juvenile fish and cultured for over 30 days under orbital shaking. Optimization assays showed that these spheroids achieved biometric, morphological and basal gene expression stability from day 12 to day 20. This multiparametric characterization allowed the selection of a temporal window of culture days, which is being explored in several assays. The 3D primary hepatocyte spheroids were exposed to estrogens (e.g., 17α-ethinylestradiol) at different concentrations. The effect of increasing temperature was also evaluated in this model by maintaining cultures at 18°C and 21°C. Data collected include microscopy analysis, target gene expression of estrogenic and lipid pathways, and cellular biochemistry. The outputs indicate that hepatic spheroids are metabolically active and respond to different hormonal and temperature stimuli. The 3D model developed from primary brown trout hepatocytes seems to be a viable alternative for studying hepatic disruptions caused by (at least) estrogenic compounds and varied temperatures.",
publisher = "Iberian Association of Comparative Endocrinology",
journal = "XIV Congress of the Iberian Association of Comparative Endocrinology",
title = "Brown trout primary hepatocyte spheroids – characterization and applications in endocrinology and beyond",
pages = "87-87",
url = "https://hdl.handle.net/21.15107/rcub_agrospace_6937"
}

Madureira, T. V., Alves, R., Lopes, C., Raskovic, B.,& Rocha, E.. (2023). Brown trout primary hepatocyte spheroids – characterization and applications in endocrinology and beyond. in XIV Congress of the Iberian Association of Comparative Endocrinology
Iberian Association of Comparative Endocrinology., 87-87.
https://hdl.handle.net/21.15107/rcub_agrospace_6937

Madureira TV, Alves R, Lopes C, Raskovic B, Rocha E. Brown trout primary hepatocyte spheroids – characterization and applications in endocrinology and beyond. in XIV Congress of the Iberian Association of Comparative Endocrinology. 2023;:87-87.
https://hdl.handle.net/21.15107/rcub_agrospace_6937 .

Madureira, Tania Vieria, Alves, Rodrigo, Lopes, Celia, Raskovic, Bozidar, Rocha, Eduardo, "Brown trout primary hepatocyte spheroids – characterization and applications in endocrinology and beyond" in XIV Congress of the Iberian Association of Comparative Endocrinology (2023):87-87,
https://hdl.handle.net/21.15107/rcub_agrospace_6937 .

TY  - GEN
AU  - Lopes, Celia
AU  - Teixeira, Ingrid
AU  - Raskovic, Bozidar
AU  - Rocha, Eduardo
PY  - 2022
UR  - http://aspace.agrif.bg.ac.rs/handle/123456789/6939
AB  - Global warming is an undeniable reality, and one consequence is a continuous increase in the temperature of different water bodies. This phenomenon affects numerous processes in the aquatic ecosystems, including the impacts of the widely present endocrine-disrupting chemicals (EDCs). Within EDCs, ethinylestradiol (EE2) and progestins, such as levonorgestrel (LEV), are commonly detected in environmental waters. It is thus imperative to study the effects of temperature changes in the toxicology of EDCs to aquatic animals, including fish. Endocrine disruption research often relies on in vitro models, complying with the 3Rs principles. Primary hepatocytes are commonly used but also are specific and well-characterized cell lines, such as the rainbow trout liver-derived RTL-W1. This cell line presents several functional features of differentiated hepatocytes. To test the cell line potential to study the way temperature models the effects of EDCs, we exposed RTL-W1 cells (72h) to EE2, LEV or a mixture of both synthetic hormones (at 10 μM). Exposures were carried out at 18 ºC and 21 ºC, in 24-well plates, 80.000 cells per well. The cells grew at either temperature. At the end of exposures, cells were trypsinized, and the viability measured using the trypan blue exclusion assay. Cell suspensions were then centrifuged at 200 rcf, for 5 min, and pellets were frozen at -80 ºC for gene expression analysis. No differences were found in cell viability among experimental groups. The expression of two CYP P450 enzymes enrolled in xenobiotic metabolism was analyzed by qRT-PCR. The Two-way ANOVA results revealed that CYP1A was affected by temperature as its expression was lower at 18ºC than at 21ºC. As to CYP3A27 expression, it was independent of temperature but was significantly higher in the EE2 + LEV mixture compared with control and solvent control conditions. Given these results, the RTL-W1 cell line seems like a promising model to study the interactions of temperature and exposure effects of EDCs. Studies are undergoing to include genes from other relevant signalling pathways such as estrogenic, progestogenic and lipid metabolism.
PB  - European Society for Comparative Endocrinology and International Society for Fish Endocrinology
T2  - 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology
T1  - Testing the trout liver RTL-W1 cell line potential to study the influences of temperature on the effects of endocrine disruptors
EP  - 202
SP  - 202
UR  - https://hdl.handle.net/21.15107/rcub_agrospace_6939
ER  - 

@misc{
author = "Lopes, Celia and Teixeira, Ingrid and Raskovic, Bozidar and Rocha, Eduardo",
year = "2022",
abstract = "Global warming is an undeniable reality, and one consequence is a continuous increase in the temperature of different water bodies. This phenomenon affects numerous processes in the aquatic ecosystems, including the impacts of the widely present endocrine-disrupting chemicals (EDCs). Within EDCs, ethinylestradiol (EE2) and progestins, such as levonorgestrel (LEV), are commonly detected in environmental waters. It is thus imperative to study the effects of temperature changes in the toxicology of EDCs to aquatic animals, including fish. Endocrine disruption research often relies on in vitro models, complying with the 3Rs principles. Primary hepatocytes are commonly used but also are specific and well-characterized cell lines, such as the rainbow trout liver-derived RTL-W1. This cell line presents several functional features of differentiated hepatocytes. To test the cell line potential to study the way temperature models the effects of EDCs, we exposed RTL-W1 cells (72h) to EE2, LEV or a mixture of both synthetic hormones (at 10 μM). Exposures were carried out at 18 ºC and 21 ºC, in 24-well plates, 80.000 cells per well. The cells grew at either temperature. At the end of exposures, cells were trypsinized, and the viability measured using the trypan blue exclusion assay. Cell suspensions were then centrifuged at 200 rcf, for 5 min, and pellets were frozen at -80 ºC for gene expression analysis. No differences were found in cell viability among experimental groups. The expression of two CYP P450 enzymes enrolled in xenobiotic metabolism was analyzed by qRT-PCR. The Two-way ANOVA results revealed that CYP1A was affected by temperature as its expression was lower at 18ºC than at 21ºC. As to CYP3A27 expression, it was independent of temperature but was significantly higher in the EE2 + LEV mixture compared with control and solvent control conditions. Given these results, the RTL-W1 cell line seems like a promising model to study the interactions of temperature and exposure effects of EDCs. Studies are undergoing to include genes from other relevant signalling pathways such as estrogenic, progestogenic and lipid metabolism.",
publisher = "European Society for Comparative Endocrinology and International Society for Fish Endocrinology",
journal = "30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology",
title = "Testing the trout liver RTL-W1 cell line potential to study the influences of temperature on the effects of endocrine disruptors",
pages = "202-202",
url = "https://hdl.handle.net/21.15107/rcub_agrospace_6939"
}

Lopes, C., Teixeira, I., Raskovic, B.,& Rocha, E.. (2022). Testing the trout liver RTL-W1 cell line potential to study the influences of temperature on the effects of endocrine disruptors. in 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology
European Society for Comparative Endocrinology and International Society for Fish Endocrinology., 202-202.
https://hdl.handle.net/21.15107/rcub_agrospace_6939

Lopes C, Teixeira I, Raskovic B, Rocha E. Testing the trout liver RTL-W1 cell line potential to study the influences of temperature on the effects of endocrine disruptors. in 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology. 2022;:202-202.
https://hdl.handle.net/21.15107/rcub_agrospace_6939 .

Lopes, Celia, Teixeira, Ingrid, Raskovic, Bozidar, Rocha, Eduardo, "Testing the trout liver RTL-W1 cell line potential to study the influences of temperature on the effects of endocrine disruptors" in 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology (2022):202-202,
https://hdl.handle.net/21.15107/rcub_agrospace_6939 .

TY  - GEN
AU  - Raskovic, Bozidar
AU  - Madureira, Tania Vieria
AU  - Lopes, Celia
AU  - Rocha, Eduardo
PY  - 2022
UR  - http://aspace.agrif.bg.ac.rs/handle/123456789/6940
AB  - Progestins are classified as emerging micropollutants in freshwater ecosystems. The concentrations of this class of contaminants in water have been increasing, since they are of anthropogenic origin and their use in human and veterinary medicine is in expansion. Apart from well-studied effects of progestins on fish, such as alterations of behavioral processes, impaired reproduction and involvement in intersex, the effects on structure and metabolism of liver are understudied; namely when progestins and estrogens are concurrent. Thus, this study is exploring sub-chronic effects of in vitro exposure hepatocyte spheroids to progestins and 17α-ethinylestradiol. Primary hepatocytes were isolated from brown trout (n = 3) and cultured for 12 days in non-adhesive plates which were constantly agitated with frequency of 60 times per minute at 18 °C. Shortly after plating, hepatocytes started to spontaneously form spheroids (3D cellular aggregates), which increased their size over time. At day 12, spheroids were exposed to either 17α-ethinylestradiol (0.3 μM), levonorgestrel and megestrol acetate in two concentrations (0.3 and 0.6 μM) or binary mixtures of these chemicals in 0.1% ethanol as a solvent. After a 6 days exposure, spheroids were measured and sampled for biochemical (lactate dehydrogenase and resazurin assays), biometrical, immunohistochemical and gene expression analyses. Both biochemical assays did not show differences between exposed groups or between exposed and control and solvent control groups, which supported that viability of exposed spheroids was not compromised. For biometry, no significant differences in sphericity, area and diameter of spheroids were found between groups. However, when anti-vitellogenin antibody was applied on histological sections, spheroids from all exposed groups had increased expression comparing with controls, proving all three chemicals could modify the hepatocyte function. Besides estrogenic targets, genes related with lipid-metabolism pathways are under study. As a perspective, analysis of the modeling effect of temperature in the same targets is being considered.
PB  - European Society for Comparative Endocrinology and International Society for Fish Endocrinology
T2  - 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology
T1  - The effects of two progestins and 17α-ethinylestradiol on cultured hepatocyte spheroids of brown trout (salmo trutta)
EP  - 203
SP  - 203
UR  - https://hdl.handle.net/21.15107/rcub_agrospace_6940
ER  - 

@misc{
author = "Raskovic, Bozidar and Madureira, Tania Vieria and Lopes, Celia and Rocha, Eduardo",
year = "2022",
abstract = "Progestins are classified as emerging micropollutants in freshwater ecosystems. The concentrations of this class of contaminants in water have been increasing, since they are of anthropogenic origin and their use in human and veterinary medicine is in expansion. Apart from well-studied effects of progestins on fish, such as alterations of behavioral processes, impaired reproduction and involvement in intersex, the effects on structure and metabolism of liver are understudied; namely when progestins and estrogens are concurrent. Thus, this study is exploring sub-chronic effects of in vitro exposure hepatocyte spheroids to progestins and 17α-ethinylestradiol. Primary hepatocytes were isolated from brown trout (n = 3) and cultured for 12 days in non-adhesive plates which were constantly agitated with frequency of 60 times per minute at 18 °C. Shortly after plating, hepatocytes started to spontaneously form spheroids (3D cellular aggregates), which increased their size over time. At day 12, spheroids were exposed to either 17α-ethinylestradiol (0.3 μM), levonorgestrel and megestrol acetate in two concentrations (0.3 and 0.6 μM) or binary mixtures of these chemicals in 0.1% ethanol as a solvent. After a 6 days exposure, spheroids were measured and sampled for biochemical (lactate dehydrogenase and resazurin assays), biometrical, immunohistochemical and gene expression analyses. Both biochemical assays did not show differences between exposed groups or between exposed and control and solvent control groups, which supported that viability of exposed spheroids was not compromised. For biometry, no significant differences in sphericity, area and diameter of spheroids were found between groups. However, when anti-vitellogenin antibody was applied on histological sections, spheroids from all exposed groups had increased expression comparing with controls, proving all three chemicals could modify the hepatocyte function. Besides estrogenic targets, genes related with lipid-metabolism pathways are under study. As a perspective, analysis of the modeling effect of temperature in the same targets is being considered.",
publisher = "European Society for Comparative Endocrinology and International Society for Fish Endocrinology",
journal = "30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology",
title = "The effects of two progestins and 17α-ethinylestradiol on cultured hepatocyte spheroids of brown trout (salmo trutta)",
pages = "203-203",
url = "https://hdl.handle.net/21.15107/rcub_agrospace_6940"
}

Raskovic, B., Madureira, T. V., Lopes, C.,& Rocha, E.. (2022). The effects of two progestins and 17α-ethinylestradiol on cultured hepatocyte spheroids of brown trout (salmo trutta). in 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology
European Society for Comparative Endocrinology and International Society for Fish Endocrinology., 203-203.
https://hdl.handle.net/21.15107/rcub_agrospace_6940

Raskovic B, Madureira TV, Lopes C, Rocha E. The effects of two progestins and 17α-ethinylestradiol on cultured hepatocyte spheroids of brown trout (salmo trutta). in 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology. 2022;:203-203.
https://hdl.handle.net/21.15107/rcub_agrospace_6940 .

Raskovic, Bozidar, Madureira, Tania Vieria, Lopes, Celia, Rocha, Eduardo, "The effects of two progestins and 17α-ethinylestradiol on cultured hepatocyte spheroids of brown trout (salmo trutta)" in 30th CECE & 9th ISFE Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology (2022):203-203,
https://hdl.handle.net/21.15107/rcub_agrospace_6940 .

TY  - GEN
AU  - Raskovic, Bozidar
AU  - Madureira, Tania Vieria
AU  - Lopes, Celia
AU  - Rocha, Eduardo
PY  - 2022
UR  - http://aspace.agrif.bg.ac.rs/handle/123456789/6941
AB  - In 2018, approximately 10.5 million experimental animals were used in the EU for various testing purposes, and 26% of that number accounted for fish. The scientific community, regulatory agencies, and national and EU policymakers endorse the refinement, reduction, and replacement (3Rs) of assays to reduce the number of sacrificed experimental animals. Despite the guidelines in fish exposure assays being well established (such as OECD and US EPA) and the number of test animals kept as low as feasible, each exposure study still requires at least 105 fish. For this reason, many in vitro methods were developed or improved for use in fish assays, which provided a good platform for testing the effects of chemicals, complementing, or replacing in vivo studies. Methods from mammalian in vitro testing were used to develop primary piscine cultures and cell lines, typically cultured in monolayers, viz. two-dimensional (2D). The advantages of 2D cultures are rapid exposure tests, being easy for handling and requiring low-cost maintenance. However, the uses of 2D cultures also have downsides: cells cannot last for a long time and cannot mimic the organization of tissues since they are cultured in the bottom of flasks and plates. To solve these problems, researchers created three-dimensional (3D) cultures. The microarchitecture and physiology of cells in 3D cultures are closer to in vivo systems. 3D primary cultures in fish have been seldom used, but they may provide an excellent research tool for the toxicological assessment of chemicals. To further use fish spheroids in toxicology, we are expanding a protocol for routine isolation and culture/co-culture of cells from brown trout (Salmo trutta) liver. The protocol is compatible with exploring differential centrifugation of isolated cells to change the composition of spheroids in terms of the ratio of hepatocytes and biliary epithelial cells. After plating in non-adhesive plates, cells were cultured for 12 days until maturation, using orbital shakers, in which they slowly aggregated and formed spheroids in the incubator (at 18 °C). DMEM enabled the formation of spheroids with sufficient size, with an absence of significant necrotic centres, which usually occur in spheroids due to hypoxia. From day 12 to 18, liver spheroids were exposed to single or mixtures of endocrine disruptors (17α-ethinylestradiol and the progestins levonorgestrel and megestrol acetate) to assess their advert effects on cells. Spheroids were evaluated using light and electron microscopy (routine staining and immunohistochemistry), gene expression (a set of genes related to lipid metabolism, yolk proteins and vitellogenin), and biochemical assays (lactate dehydrogenase and resazurin assays). The spheroids were able to respond to the stimuli.
PB  - Czechoslovak Microscopy Society
T2  - 16th Multinational Congress on Microscopy
T1  - Spheroids: in vitro 3D cell cultures of brown trout liver as a model for ecotoxicology research
EP  - 235
SP  - 235
UR  - https://hdl.handle.net/21.15107/rcub_agrospace_6941
ER  - 

@misc{
author = "Raskovic, Bozidar and Madureira, Tania Vieria and Lopes, Celia and Rocha, Eduardo",
year = "2022",
abstract = "In 2018, approximately 10.5 million experimental animals were used in the EU for various testing purposes, and 26% of that number accounted for fish. The scientific community, regulatory agencies, and national and EU policymakers endorse the refinement, reduction, and replacement (3Rs) of assays to reduce the number of sacrificed experimental animals. Despite the guidelines in fish exposure assays being well established (such as OECD and US EPA) and the number of test animals kept as low as feasible, each exposure study still requires at least 105 fish. For this reason, many in vitro methods were developed or improved for use in fish assays, which provided a good platform for testing the effects of chemicals, complementing, or replacing in vivo studies. Methods from mammalian in vitro testing were used to develop primary piscine cultures and cell lines, typically cultured in monolayers, viz. two-dimensional (2D). The advantages of 2D cultures are rapid exposure tests, being easy for handling and requiring low-cost maintenance. However, the uses of 2D cultures also have downsides: cells cannot last for a long time and cannot mimic the organization of tissues since they are cultured in the bottom of flasks and plates. To solve these problems, researchers created three-dimensional (3D) cultures. The microarchitecture and physiology of cells in 3D cultures are closer to in vivo systems. 3D primary cultures in fish have been seldom used, but they may provide an excellent research tool for the toxicological assessment of chemicals. To further use fish spheroids in toxicology, we are expanding a protocol for routine isolation and culture/co-culture of cells from brown trout (Salmo trutta) liver. The protocol is compatible with exploring differential centrifugation of isolated cells to change the composition of spheroids in terms of the ratio of hepatocytes and biliary epithelial cells. After plating in non-adhesive plates, cells were cultured for 12 days until maturation, using orbital shakers, in which they slowly aggregated and formed spheroids in the incubator (at 18 °C). DMEM enabled the formation of spheroids with sufficient size, with an absence of significant necrotic centres, which usually occur in spheroids due to hypoxia. From day 12 to 18, liver spheroids were exposed to single or mixtures of endocrine disruptors (17α-ethinylestradiol and the progestins levonorgestrel and megestrol acetate) to assess their advert effects on cells. Spheroids were evaluated using light and electron microscopy (routine staining and immunohistochemistry), gene expression (a set of genes related to lipid metabolism, yolk proteins and vitellogenin), and biochemical assays (lactate dehydrogenase and resazurin assays). The spheroids were able to respond to the stimuli.",
publisher = "Czechoslovak Microscopy Society",
journal = "16th Multinational Congress on Microscopy",
title = "Spheroids: in vitro 3D cell cultures of brown trout liver as a model for ecotoxicology research",
pages = "235-235",
url = "https://hdl.handle.net/21.15107/rcub_agrospace_6941"
}

Raskovic, B., Madureira, T. V., Lopes, C.,& Rocha, E.. (2022). Spheroids: in vitro 3D cell cultures of brown trout liver as a model for ecotoxicology research. in 16th Multinational Congress on Microscopy
Czechoslovak Microscopy Society., 235-235.
https://hdl.handle.net/21.15107/rcub_agrospace_6941

Raskovic B, Madureira TV, Lopes C, Rocha E. Spheroids: in vitro 3D cell cultures of brown trout liver as a model for ecotoxicology research. in 16th Multinational Congress on Microscopy. 2022;:235-235.
https://hdl.handle.net/21.15107/rcub_agrospace_6941 .

Raskovic, Bozidar, Madureira, Tania Vieria, Lopes, Celia, Rocha, Eduardo, "Spheroids: in vitro 3D cell cultures of brown trout liver as a model for ecotoxicology research" in 16th Multinational Congress on Microscopy (2022):235-235,
https://hdl.handle.net/21.15107/rcub_agrospace_6941 .