Characterization of Sandwich-Cultured Hepatocytes As an in Vitro Model to Assess the Hepatobiliary Disposition of Copper

Abstract

ABSTRACT:Sandwich-cultured hepatocytes (SCH) from rats (SCRH), dogs (SCDH), and humans (SCHH) were used as an in vitro model to assess the hepatobiliary disposition of copper (Cu). The expression of Cu transporters, ceruloplasmin synthesis, Cu uptake, and biliary excretion and species differences in drug-induced alterations in Cu disposition were determined in SCH from all species. Western blot analysis verified basolateral Cu uptake transporter 1 (CTR1) and canalicular Cu efflux transporter (ATP7B) expression: enzymelinked immunosorbent assay verified synthesis/secretion of ceruloplasmin (major Cu binding protein found in blood). Endogenous Cu in SCRH, SCDH, and SCHH were 17.2 ؎ 7.00, 490 ؎ 44.8, and 43.5 ؎ 15.8 ng/well, respectively. The hepatobiliary disposition of Cu as measured by uptake (increase in intracellular Cu in comparison to endogenous levels) and biliary excretion (increase in Cu in wash solutions obtained from hepatocytes exposed to calciumfree versus standard buffer) was determined as a function of Cu concentration and incubation time. In general, an increase in Cu concentration or incubation time resulted in an increase in Cu uptake and/or biliary excretion; however, the extent to which they affected Cu disposition was species dependent. 5-(1,1-Dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (L-000870810) (an anti-HIV compound, the development of which was halted due to an observed Cu-specific toxicity in the liver and kidneys of dogs after long-term exposure) showed no effect on Cu disposition in SCRH; however, it increased the biliary excretion of Cu in SCDH and SCHH. This is the first report to demonstrate the utility of SCH as a model to assess hepatobiliary disposition of Cu in an in vitro system.

Transcriptional regulation of copper metabolism genes in the liver of fetal and neonatal control and iron-deficient rats

“…From then, levels remained high until about the second week of life, thereafter decreasing to adult levels by six weeks of age (Terao and Owen 1977 ). In mammalian cells, copper import is primarily mediated by CTR1 protein (Ansede et al 2009 ; Kim et al 2009 ; Lee et al 2002 ; Nose et al 2006 ; Van den Berghe and Klomp 2010 ). Kuo et al ( 2006 ) showed that in mice and rats CTR1 expression is regulated by copper status (Bauerly et al 2005 ; Kuo et al 2006 ).…”

Sandwich-cultured hepatocytes: anin vitromodel to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity

“…For example, cyclosporine A and glyburide decreased total accumulation (cells+bile), increased cellular accumulation, and decreased BEI and Cl biliary of deuterium-labeled taurocholate (d 8 -TC), suggesting that bile acid efflux was affected primarily (Ansede et al, 2009 in press). In contrast, erythromycin-estolate, troglitazone and bosentan decreased d 8 -TC accumulation (both cells+bile and cells), BEI and Cl biliary of d 8 -TC, suggesting that bile acid uptake was the predominant pathway affected.…”

“…SCH recently were evaluated for the potential to assess hepatobiliary disposition of copper (Ansede et al, 2009). Copper (Cu) is an essential nutrient necessary for the activity of various enzymes, but also can accumulate to toxic levels when Cu elimination is disrupted by drugs or genetic disorders, such as in Wilson's Disease, which is characterized by massive Cu accumulation in lysosomes and lack of excretion in bile, the major route (85%) of Cu elimination.…”