Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

Liu, Yitong; Flynn, Tom; Xia, Menghang; Wiesenfeld, Paddy L.; Ferguson, Martine

doi:10.1007/s10565-015-9306-9

Cited by 19 publications

(10 citation statements)

References 30 publications

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“…CYP3A4 subfamily enzymes play a major role in the metabolism of~30% of clinically used drugs from almost all therapeutic categories [30][31][32]. As previously reported [33], this major P450 enzyme is involved in CPZ oxidative metabolism, which is active in CPZ-treated in HepaRG cells.…”

Section: Discussionmentioning

confidence: 63%

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Brzeszczyńska

Brzeszczyński

Samuel

et al. 2020

Cells

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Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 'housekeeping' genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of 'classical' reference genes such as GAPDH (glyceralde-hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

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Section: Discussionmentioning

confidence: 63%

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Brzeszczyńska

Brzeszczyński

Samuel

et al. 2020

Cells

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“…Hepatocellular carcinoma (HCC) is one of the most malignant cancers all over the world, with the third cause of cancer mortality (Poon 2011 ; Liu et al 2015 ). A number of risk factors have been shown to drive this process, including hepatitis virus infections, non-alcoholic fatty liver diseases, and alcoholic liver diseases (Cavazza et al 2009 ).…”

Section: Introductionmentioning

confidence: 99%

Silencing GTSE-1 expression inhibits proliferation and invasion of hepatocellular carcinoma cells

Guo

Zhang

et al. 2016

Cell Biol Toxicol

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G2 and S phase-expressed-1 (GTSE1) was recently reported to upregulate in several types of human cancer, based on negatively regulate p53 expression. However, its expression and functional roles in hepatocellular carcinoma (HCC) remain unknown. In this study, GTSE1 was observed to be highly expressed in HCC specimens and cell lines both at messenger RNA (mRNA) and protein levels. Furthermore, high GTSE1 expression was positively associated with tumor size, venous invasion, advanced tumor stage, and short overall survival. Moreover, we generated stable GTSE1 knockdown HCC cell lines to explore the effects of GTSE1 silencing on the growth and invasion of HCC in vitro. In determining the pathway through which GTSE1 regulated cell proliferation and invasion, GTSE1 silencing was found to inhibit AKT phosphorylation and downregulated cell cycle-related protein. In addition, GTSE1 downregulation decreased the growth of xenografts. In conclusion, these results indicated for the first time that overexpression of GTSE1 was involved in the progress of HCC, enhancing proliferation and promoting cell invasion in HCC cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s10565-016-9327-z) contains supplementary material, which is available to authorized users.

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“…However, HepG2 cells show poor metabolic enzymes such as CYP enzymes. 33,34 Researchers have attempted to create a stable cell line that expresses CYP enzymes to screen for hepatotoxicity, [35][36][37] as CYP expression is an important parameter for evaluating hepatotoxicity and drug metabolism under similar conditions as those in vivo. In this study, we confirmed that differentiated HepaRG cells express major drug-metabolizing enzymes.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Evaluation of Cytochrome P450 3A4 Inhibition and Hepatotoxicity in HepaRG 3-D Spheroids

et al. 2018

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Predicting drug-drug interactions (DDIs) is an important step during drug development to avoid unexpected side effects. Cytochrome P450 (CYP) 3A4 is the most abundant human hepatic phase I enzyme, which metabolizes >50% of therapeutic drugs. Therefore, it is essential to test the potential of a drug candidate to induce CYP3A4 expression or inhibit its activity. Recently, 3-dimensional (3-D) mammalian cell culture models have been adopted in drug discovery research to assess toxicity, DDIs, and pharmacokinetics. In this study, we applied a human 3-D spheroid culture protocol using HepaRG cells combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess its ability to predict CYP3A4 inhibition. Levels of midazolam, a specific substrate of CYP3A4, were used to determine the long-term metabolic capacity of CYP3A4. Midazolam was decreased in the 3-D HepaRG culture system by ∼80% over 7 days, whereas its primary metabolite, 1-hydroxymidazolam, increased by ∼40%. Next, we assessed hepatotoxicity by determining the cytotoxicity of known hepatotoxicants in HepaRG spheroids, HepG2 cells, and primary human hepatocytes. Significant differences in cytotoxicity were detected in the system using 3-D HepaRG spheroids. These results suggest that 3-D HepaRG spheroids are a good model for prediction of CYP inhibition and hepatotoxicity in screening of early drug candidates.

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Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

Cited by 19 publications

References 30 publications

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Silencing GTSE-1 expression inhibits proliferation and invasion of hepatocellular carcinoma cells

Quantitative Evaluation of Cytochrome P450 3A4 Inhibition and Hepatotoxicity in HepaRG 3-D Spheroids

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