scholarly journals mRNA Expression and Activity of Nucleoside Transporters in Human Hepatoma HepaRG Cells

Pharmaceutics ◽  
2018 ◽  
Vol 10 (4) ◽  
pp. 246
Author(s):  
Abdullah Mayati ◽  
Amélie Moreau ◽  
Elodie Jouan ◽  
Marie Febvre-James ◽  
Claire Denizot ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Human Hepatocytes ◽  
Nucleoside Transporters ◽  
Nucleoside Transporter ◽  
Human Hepatoma ◽  
Heparg Cell Line ◽  
Heparg Cells ◽  
Pkc Activation ◽  
Expression And Activity

The HepaRG cell line is a highly differentiated human hepatoma cell line, displaying the expression of various drug transporters. However, functional expression of nucleoside transporters remains poorly characterized in HepaRG cells, although these transporters play a key role in hepatic uptake of antiviral and anticancer drugs. The present study was, therefore, designed to characterize the expression, activity and regulation of equilibrative (ENT) and concentrative (CNT) nucleoside transporter isoforms in differentiated HepaRG cells. These cells were found to exhibit a profile of nucleoside transporter mRNAs similar to that found in human hepatocytes, i.e., notable expression of ENT1, ENT2 and CNT1, with very low or no expression of CNT2 and CNT3. ENT1 activity was, next, demonstrated to be the main uridine transport activity present in HepaRG cells, like in cultured human hepatocytes. Various physiological factors, such as protein kinase C (PKC) activation or treatment by inflammatory cytokines or hepatocyte growth factor (HGF), were additionally found to regulate expression of ENT1, ENT2 and CNT1; PKC activation and HGF notably concomitantly induced mRNA expression and activity of ENT1 in HepaRG cells. Overall, these data suggest that HepaRG cells may be useful for analyzing cellular pharmacokinetics of nucleoside-like drugs in human hepatic cells, especially of those handled by ENT1.

Bis(hydroxyphenyl)methane—bisphenol F—metabolism by the HepG2 human hepatoma cell line and cryopreserved human hepatocytes

2011 ◽  
Vol 34 (4) ◽  
pp. 445-453 ◽  
Author(s):  
Coralie Dumont ◽  
Elisabeth Perdu ◽  
Georges de Sousa ◽  
Laurent Debrauwer ◽  
Roger Rahmani ◽  
...  
Keyword(s):  
Cell Line ◽  
Hepatoma Cell ◽  
Human Hepatocytes ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Human Hepatoma Cell Line ◽  
Bisphenol F

Ethanol inhibits asialoglycoprotein receptor synthesis but augments its mRNA expression in a human hepatoma cell line

1994 ◽  
Vol 29 (5) ◽  
pp. 598-604 ◽  
Author(s):  
Yutaka Kohgo ◽  
Yoshihiro Mogi ◽  
Junji Kato ◽  
Reiji Nakaya ◽  
Masahiro Nakajima ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Hepatoma Cell ◽  
Asialoglycoprotein Receptor ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Human Hepatoma Cell Line

scholarly journals Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor

Hepatology ◽  
2007 ◽  
Vol 45 (4) ◽  
pp. 957-967 ◽  
Author(s):  
Virginie Cerec ◽  
Denise Glaise ◽  
Delphine Garnier ◽  
Serban Morosan ◽  
Bruno Turlin ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Hepatoma ◽  
Heparg Cell ◽  
Heparg Cell Line

scholarly journals Expression and hepatobiliary transport characteristics of the concentrative and equilibrative nucleoside transporters in sandwich-cultured human hepatocytes

2008 ◽  
Vol 295 (3) ◽  
pp. G570-G580 ◽  
Author(s):  
Rajgopal Govindarajan ◽  
Christopher J. Endres ◽  
Dale Whittington ◽  
Edward LeCluyse ◽  
Marçal Pastor-Anglada ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Rank Order ◽  
Human Hepatocytes ◽  
Nucleoside Transporters ◽  
Hepatic Tissue ◽  
Cultured Hepatocytes ◽  
Hepatobiliary Transport ◽  
Hepatic Diseases ◽  
Nucleoside Drugs

We previously reported that both the concentrative (hCNT) and equilibrative (hENT) nucleoside transporters are expressed in the human liver ( 21 ). Here we report a study that investigated the expression of these transporters (transcripts and proteins) and their role in the hepatobiliary transport of nucleosides/nucleoside drugs using sandwich-cultured human hepatocytes. In the hepatic tissue, the rank order of the mRNA expression of the transporters was hCNT1 ≈ hENT1 > hENT2 ≈ hCNT2 > hCNT3. In sandwich-cultured hepatocytes, the mRNA expression of hCNT2 and hENT2 was comparable to that in hepatic tissue, whereas the expression of corresponding transporters in the two-dimensional hepatocyte cultures was lower. Colocalization studies demonstrated predominant localization of these transporters at the sinusoidal membrane and of hENT1, hCNT1, and hCNT2 at the canalicular membrane. In the sandwich-cultured hepatocytes, ENTs were the major contributors to the transport of thymidine (hENT1, 63%; hENT2, 23%) or guanosine (hENT1, 53%; hENT2, 24%) into the hepatocytes followed by hCNT1 (10%) for thymidine or hCNT2 (23%) for guanosine. Although ribavirin was predominately transported (89%) into the hepatocytes by hENT1, fialuridine (FIAU) was transported by both hENT1 (30%) and hCNTs (61%). The extensively metabolized natural nucleosides were not effluxed into the bile, whereas significant biliary-efflux was observed of FIAU (19%), ribavirin (30%), and formycin B (35%). We conclude that the hepatic activity of hENT1 and hCNT1/2 transporters will determine the in vivo hepatic distribution and therefore the efficacy and/or toxicity of nucleoside drugs used to treat hepatic diseases.

scholarly journals Interactions between hepatitis B virus and aflatoxin B1: effects on p53 induction in HepaRG cells

2012 ◽  
Vol 93 (3) ◽  
pp. 640-650 ◽  
Author(s):  
Myriam Lereau ◽  
Doriane Gouas ◽  
Stéphanie Villar ◽  
Ahmad Besaratinia ◽  
Agnès Hautefeuille ◽  
...  
Keyword(s):  
Risk Factors ◽  
Dna Damage ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Line ◽  
Hbv Infection ◽  
Hbv Replication ◽  
Heparg Cell Line ◽  
Heparg Cells ◽  
B Virus

Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B1 (AFB1) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB1 activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB1 on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte-like morphology that metabolizes AFB1 and supports HBV infection. Exposure to AFB1 up to 5 µM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB1-DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB1 was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB1 exposure decreases HBV replication, whereas DNA damage by AFB1 and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB1 and HBV do not cooperate to increase DNA damage by AFB1. Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects.

scholarly journals Assessing hepatoprotective effects of antioxidants on amiodarone-induced cytotoxicity in human hepatoma HepaRG cell line

2021 ◽  
Author(s):  
KS Filimonova ◽  
NYu Rogovskaya ◽  
PP Beltyukov ◽  
VN Babakov
Keyword(s):  
Cell Line ◽  
P53 Protein ◽  
Toxic Effects ◽  
Human Hepatoma ◽  
Cell Cycle Regulators ◽  
Human Hepatoma Cells ◽  
Heparg Cell ◽  
Heparg Cell Line ◽  
Ic50 Value ◽  
Hepatoprotective Effects

Effective therapy of amiodarone-induced hepatotoxicity requires studying the mechanisms of the toxic effects of amiodarone on hepatocytes and assessing the potential impact of hepotoprotective agents. The study was aimed to assess hepatoprotective effects of antioxidants on the amiodarone-induced hepatotoxicity with the use of immortalized human hepatoma cells of the HepaRG cell line. Cell viability was evaluated upon exposure to amiodarone and in the mixture with vitamin Е, N-acetylcysteine and S-adenosylmethionine by impedance measurement; the levels of some hepatotoxicity biomarkers were defined using the Luminex xMAP technology. As a result of the research, the dose-dependent toxic effects of amiodarone were established. The IC50 value of amiodarone in the HepaRG cell line was 3.5 μМ. It is shown that cytotoxic effects decrease and the IC50 value increases in the presence of vitamin Е, N-acetylcysteine and S-adenosylmethionine. Amiodarone reduces the activity of cell cycle regulators: AKT, JNK kinases, and p53 protein. Exposure to amiodarone results in reduced intracellular ATP levels and the release of intracellular enzymes (malate dehydrogenase 1, glutathione S-transferase, sorbitol dehydrogenase, 5'-nucleotidase) into conditioned medium, indicating the necrotic cell death. Thus, vitamin Е, S-adenosylmethionine and N-acetylcysteine reduce amiodarone cytotoxicity in the model of amiodarone-induced damage to hepatocytes and can be considered as hepatoprotective agents in case of the need to protect liver against the hepatotoxic effects of amiodarone.

scholarly journals Effect of different growth hormone (GH) mutants on the regulation of GH-receptor gene transcription in a human hepatoma cell line

2002 ◽  
pp. 573-581 ◽  
Author(s):  
J Deladoey ◽  
G Gex ◽  
JM Vuissoz ◽  
CJ Strasburger ◽  
MP Wajnrajch ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Mrna Expression ◽  
Cell Line ◽  
Autosomal Dominant ◽  
Hepatoma Cell ◽  
Receptor Gene ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Gh Receptor

OBJECTIVE: G to A transition at position 6664 of the growth hormone (GH-1) gene results in the substitution of Arg183 by His (R183H) in the GH protein and causes a new form of autosomal dominant isolated GH deficiency (IGHD type II). The aim of this study was to assess the bioactivity of this R183H mutant GH in comparison with both other GH variants and the 22-kDa GH in terms of GH-receptor gene regulation. DESIGN AND METHODS: The regulation of the GH-receptor gene (GH-receptor/GH binding protein, GHR/GHBP) transcription following the addition of variable concentrations (0, 12.5, 25, 50 and 500 ng/ml) of R183H mutant GH was studied in a human hepatoma cell line (HuH7) cultured in a serum-free hormonally defined medium. In addition, identical experiments were performed using either recombinant human GH (22-kDa GH) as a positive control or two GH-receptor antagonists (R77C mutant GH and pegvisomant (B-2036-PEG)) as negative controls. GHR/GHBP mRNA expression was quantitatively assessed by RT-PCR amplification after 0, 1, 3 and 6 h incubation. RESULTS: Following the addition of R183H mutant GH, GHR/GHBP mRNA changed at a similar rate to that seen in experiments where 22-kDa GH was added, indicating equal bioactivity. At all times and concentrations studied, the addition of R77C mutant GH, however, resulted in a significantly lower increase (P<0.001) of GHR/GHBP mRNA concentration compared with that caused by the addition of either 22-kDa GH or R183H mutant GH. Furthermore, in additional experiments, pegvisomant resulted in an absolute block of GHR/GHBP mRNA expression identical to that seen in control experiments where no 22-kDa GH was added at all. CONCLUSIONS: These data indicate that the R183H mutant GH, although causing an autosomal dominant form of IGHD has an identical effect on GHR/GHBP transcription as its wild-type, the 22-kDa GH. This implies that the IGHD caused by the R183H heterozygous mutation of the GH-1 gene is mainly due to a block of its regulated GH secretion. In addition, the R77C-GH variant and pegvisomant have an antagonistic effect at the level of GHR/GHBP transcription. All these data were confirmed by run-on experiments. In addition, these data highlight, as far as the GH variants are concerned, that a mutational alteration within the GH-1 gene might cause short stature also on the basis of an altered secretory pathway. This fact has to be taken into consideration when growth retardation is clinically diagnosed and studied at the molecular level. Secretory pathways and, therefore, cell-biological mechanisms are of importance and have to be considered in future not only at the scientific but also at the clinical level.

scholarly journals Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies

2014 ◽  
Vol 60 (3) ◽  
pp. 451-454 ◽  
Author(s):  
Liesbeth Ceelen ◽  
Jurgen De Craene ◽  
Ward De Spiegelaere
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Heparg Cell ◽  
Expression Studies ◽  
Heparg Cell Line ◽  
Heparg Cells ◽  
Gene Expression Studies

Abstract BACKGROUND The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. CONTENT When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored. SUMMARY In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.

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