A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

Abstract

Hepatic cell lines serve as economical and reproducible alternatives for primary human hepatocytes. However, the utility of hepatic cell lines to examine bile acid homeostasis and cholestatic toxicity is limited due to abnormal expression and function of bile acid-metabolizing enzymes, transporters, and the absence of canalicular formation. We discovered that culturing HuH-7 human hepatoma cells with dexamethasone (DEX) and 0.5% dimethyl sulfoxide (DMSO) for two weeks, with Matrigel overlay after one week, resulted in a shorter and improved differentiation process. These culture conditions increased the expression and function of the major bile acid uptake and efflux transporters, sodium taurocholate co-transporting polypeptide (NTCP) and the bile salt export pump (BSEP), respectively, in two-week cultures of HuH-7 cells. This in vitro model was further characterized for expression and function of bile acid-metabolizing enzymes, transporters, and cellular bile acids. Differentiated HuH-7 cells displayed a marked shift in bile acid composition and induction of cytochrome P450 (CYP) 7A1, CYP8B1, CYP3A4, and bile acid-CoA: amino acid N-acyltransferase (BAAT) mRNAs compared to control. Inhibition of taurocholate uptake and excretion after a 24-h treatment with prototypical cholestatic drugs suggests that differentiated HuH-7 cells are a suitable model to examine cholestatic hepatotoxicity.

Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro

“…Concentration- and time-dependent inhibition of everolimus was examined without pre-treatment to differentiate between phosphorylation-dependent and direct NTCP inhibition. Differentiated HuH-7 cells cultured for two weeks expressed functional NTCP after addition of supplements (1 µM DEX, 0.5% DMSO and overlay) ( Saran et al, 2022a ). Undifferentiated HuH-7 cells (HuH-7 Ctrl) showed no difference in TCA accumulation between Na + and Chol + ECF buffers, indicating that functional NTCP was absent.…”

“…In contrast to HuH-7 cells, HEK293-derived cells show very low endogenous transporter expression ( Ahlin et al, 2009 ). In differentiated HuH-7 cells ( Figure 2A ), TCA accumulation in control cells was lower than Mock-GFP transfected HuH-7 cells ( Figure 1A ) due to decreased OATP1B1 and OATP1B3 protein expression after differentiation ( Saran et al, 2022a ).…”

“…Differentiated and undifferentiated HuH-7 were cultured as described previously ( Saran et al, 2022a ). Briefly, HuH-7 cells were seeded at 0.3–0.4 million cells per well in 24-well plates and the maintenance medium was supplemented with 1 μM dexamethasone (DEX) and 0.5% DMSO after 2 days to induce NTCP expression.…”

“…This is because of minimal NTCP expression in human hepatoma cell lines and downregulation of NTCP in primary human hepatocytes after isolation and depolarization ( Yan et al, 2019 ; Appelman et al, 2021 ). Therefore, overexpressing cell lines with human NTCP expression or differentiated HuH-7 cells were employed to explore kinase-mediated NTCP regulation ( Appelman et al, 2021 ; Saran et al, 2022a ). Pretreatment with rapamycin reduced preS1 attachment by NTCP inhibition through a direct interaction with NTCP ( Saso et al, 2018 ).…”

<i>In Vitro</i> Assessment of Drug-Induced Liver Injury Using Cell-Based Models: A Review

“…К общим недостаткам бессмертных клеточных линий относится их ограниченная метаболическая и транспортная активность [14]. Есть сообщения, что при длительном выращивании у клеток линии HuH7 проявляется активность некоторых изоферментов Р430 и транспортеров, участвующих в переносе желчных кислот [15], но широкого распространения эта линия для исследования лекарственного транспорта и метаболизма не получила.…”