Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis

Autores
Miszczuk, Gisel Sabrina; Barosso, Ismael Ricardo; Zucchetti, Andrés Ernesto; Boaglio, Andrea Carolina; Pellegrino, Jose Manuel; Sanchez Pozzi, Enrique Juan; Roma, Marcelo Gabriel; Crocenzi, Fernando Ariel
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17β-dglucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 μM) or TLC (2.5 μM) for 30 min, with or without pre-incubation with DBcAMP (10 μM) for 15 min. Then, he increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.
Fil: Miszczuk, Gisel Sabrina. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Barosso, Ismael Ricardo. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Zucchetti, Andrés Ernesto. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Boaglio, Andrea Carolina. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Pellegrino, Jose Manuel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Sanchez Pozzi, Enrique Juan. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Crocenzi, Fernando Ariel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Materia
Sandwich-Cultured Rat Hepatocytes
Mrp2 Transport Activity
Canalicular Volume And Width
Choleresis
Cholestasis
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/6093
Acceder
id CONICETDig_a01beaa871f5d158bdbc3e329216eed3
oai_identifier_str oai:ri.conicet.gov.ar:11336/6093
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasisMiszczuk, Gisel SabrinaBarosso, Ismael RicardoZucchetti, Andrés ErnestoBoaglio, Andrea CarolinaPellegrino, Jose ManuelSanchez Pozzi, Enrique JuanRoma, Marcelo GabrielCrocenzi, Fernando ArielSandwich-Cultured Rat HepatocytesMrp2 Transport ActivityCanalicular Volume And WidthCholeresisCholestasishttps://purl.org/becyt/ford/3.1https://purl.org/becyt/ford/3At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17β-dglucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 μM) or TLC (2.5 μM) for 30 min, with or without pre-incubation with DBcAMP (10 μM) for 15 min. Then, he increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.Fil: Miszczuk, Gisel Sabrina. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaFil: Barosso, Ismael Ricardo. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaFil: Zucchetti, Andrés Ernesto. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaFil: Boaglio, Andrea Carolina. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaFil: Pellegrino, Jose Manuel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaFil: Sanchez Pozzi, Enrique Juan. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaFil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaFil: Crocenzi, Fernando Ariel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); ArgentinaSpringer2014-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/6093Miszczuk, Gisel Sabrina; Barosso, Ismael Ricardo; Zucchetti, Andrés Ernesto; Boaglio, Andrea Carolina; Pellegrino, Jose Manuel; et al.; Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis; Springer; Archives of Toxicology; 89; 6; 6-2014; 979-9900340-5761enginfo:eu-repo/semantics/altIdentifier/url/http://link.springer.com/article/10.1007%2Fs00204-014-1283-xinfo:eu-repo/semantics/altIdentifier/doi/10.1007/s00204-014-1283-xinfo:eu-repo/semantics/altIdentifier/doi/info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:16:39Zoai:ri.conicet.gov.ar:11336/6093instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-15 15:16:39.977CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
title Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
spellingShingle Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
Miszczuk, Gisel Sabrina
Sandwich-Cultured Rat Hepatocytes
Mrp2 Transport Activity
Canalicular Volume And Width
Choleresis
Cholestasis
title_short Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
title_full Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
title_fullStr Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
title_full_unstemmed Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
title_sort Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis
dc.creator.none.fl_str_mv Miszczuk, Gisel Sabrina
Barosso, Ismael Ricardo
Zucchetti, Andrés Ernesto
Boaglio, Andrea Carolina
Pellegrino, Jose Manuel
Sanchez Pozzi, Enrique Juan
Roma, Marcelo Gabriel
Crocenzi, Fernando Ariel
author Miszczuk, Gisel Sabrina
author_facet Miszczuk, Gisel Sabrina
Barosso, Ismael Ricardo
Zucchetti, Andrés Ernesto
Boaglio, Andrea Carolina
Pellegrino, Jose Manuel
Sanchez Pozzi, Enrique Juan
Roma, Marcelo Gabriel
Crocenzi, Fernando Ariel
author_role author
author2 Barosso, Ismael Ricardo
Zucchetti, Andrés Ernesto
Boaglio, Andrea Carolina
Pellegrino, Jose Manuel
Sanchez Pozzi, Enrique Juan
Roma, Marcelo Gabriel
Crocenzi, Fernando Ariel
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Sandwich-Cultured Rat Hepatocytes
Mrp2 Transport Activity
Canalicular Volume And Width
Choleresis
Cholestasis
topic Sandwich-Cultured Rat Hepatocytes
Mrp2 Transport Activity
Canalicular Volume And Width
Choleresis
Cholestasis
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.1
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17β-dglucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 μM) or TLC (2.5 μM) for 30 min, with or without pre-incubation with DBcAMP (10 μM) for 15 min. Then, he increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.
Fil: Miszczuk, Gisel Sabrina. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Barosso, Ismael Ricardo. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Zucchetti, Andrés Ernesto. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Boaglio, Andrea Carolina. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Pellegrino, Jose Manuel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Sanchez Pozzi, Enrique Juan. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
Fil: Crocenzi, Fernando Ariel. Consejo Nacional de Investigaciones Cient__itilde__ficas y T__etilde__cnicas. Centro Cient__itilde__fico Tecnol__otilde__gico Rosario. Instituto de Fisiolog__itilde__a Experimental (i); Argentina
description At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17β-dglucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 μM) or TLC (2.5 μM) for 30 min, with or without pre-incubation with DBcAMP (10 μM) for 15 min. Then, he increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.
publishDate 2014
dc.date.none.fl_str_mv 2014-06
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/6093
Miszczuk, Gisel Sabrina; Barosso, Ismael Ricardo; Zucchetti, Andrés Ernesto; Boaglio, Andrea Carolina; Pellegrino, Jose Manuel; et al.; Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis; Springer; Archives of Toxicology; 89; 6; 6-2014; 979-990
0340-5761
url http://hdl.handle.net/11336/6093
identifier_str_mv Miszczuk, Gisel Sabrina; Barosso, Ismael Ricardo; Zucchetti, Andrés Ernesto; Boaglio, Andrea Carolina; Pellegrino, Jose Manuel; et al.; Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis; Springer; Archives of Toxicology; 89; 6; 6-2014; 979-990
0340-5761
dc.language.none.fl_str_mv eng
language eng
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info:eu-repo/semantics/altIdentifier/doi/10.1007/s00204-014-1283-x
info:eu-repo/semantics/altIdentifier/doi/
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
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dc.publisher.none.fl_str_mv Springer
publisher.none.fl_str_mv Springer
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.22299

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