Human hepatocyte systems for in vitro toxicology analysis

Kammerer, Sarah; Küpper, Jan-Heiner

doi:10.3233/jcb-179012

Cited by 38 publications

(26 citation statements)

References 74 publications

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“…No differences in cell viability were observed over the 14 days. Based on this metabolic data, it would appear that the HepaRG model exhibits a slight advantage over HepG2, which agrees with observations by others, although they are largely comparable ( 12 ).…”

Section: Discussionsupporting

confidence: 91%

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Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations

et al. 2020

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Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0–2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

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Section: Discussionsupporting

confidence: 91%

“…Whilst 3D primary hepatocytes are relevant for longer-term toxicity testing, they are not suitable for use with the regulatory accepted micronucleus (MN) assay. The success of this assay relies on cellular proliferation; however, many primary or differentiated 3D hepatic models are static ( 12 ).…”

Section: Introductionmentioning

confidence: 99%

Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations

et al. 2020

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“…Seeing that poor PTEN expression is common in toxicity, we further determined the effects of FB 1 on the epigenetic regulation of PTEN via miR-30c and H3K4me3 in human hepatoma G2 (HepG2) cells. The liver is one of the primary organs in which FB 1 is thought to accumulate, and is usually the initial site for the metabolism and detoxification of food and food contaminants [ 47 , 48 ]. Due to the limitations of primary hepatocytes such as poor availability, short life span, inter-donor variability, loss of hepatic function, and early phenotypic changes, we opted to use the HepG2 cell line for this study [ 49 , 50 ].…”

Section: Discussionmentioning

confidence: 99%

Fumonisin B1 Epigenetically Regulates PTEN Expression and Modulates DNA Damage Checkpoint Regulation in HepG2 Liver Cells

Arumugam

Ghazi

Chuturgoon

2020

Toxins

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Fumonisin B1 (FB1), a Fusarium-produced mycotoxin, is found in various foods and feeds. It is a well-known liver carcinogen in experimental animals; however, its role in genotoxicity is controversial. The current study investigated FB1-triggered changes in the epigenetic regulation of PTEN and determined its effect on DNA damage checkpoint regulation in human liver hepatoma G2 (HepG2) cells. Following treatment with FB1 (IC50: 200 µM; 24 h), the expression of miR-30c, KDM5B, PTEN, H3K4me3, PI3K, AKT, p-ser473-AKT, CHK1, and p-ser280-CHK1 was measured using qPCR and/or Western blot. H3K4me3 enrichment at the PTEN promoter region was assayed via a ChIP assay and DNA damage was determined using an ELISA. FB1 induced oxidative DNA damage. Total KDM5B expression was reduced, which subsequently increased the total H3K4me3 and the enrichment of H3K4me3 at PTEN promoters. Increased H3K4me3 induced an increase in PTEN transcript levels. However, miR-30c inhibited PTEN translation. Thus, PI3K/AKT signaling was activated, inhibiting CHK1 activity via phosphorylation of its serine 280 residue preventing the repair of damaged DNA. In conclusion, FB1 epigenetically modulates the PTEN/PI3K/AKT signaling cascade, preventing DNA damage checkpoint regulation, and induces significant DNA damage.

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“…Widely used HepG2 cells, a human hepatoblastoma cell line, exhibit only low expressions and activities of nearly all phase I CYP enzymes and therefore have limited value for prediction of hepatic biotransformation. However, several approaches to increase HepG2 liver function were described in the literature [25,26]. These include genetic engineering of the cell line to enhance CYP expression.…”

Section: Introductionmentioning

confidence: 99%

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

Schulz

Kammerer

Küpper

2019

Self Cite

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BACKGROUND: Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte ® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte ® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes. OBJECTIVE: For Upcyte ® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte ® hepatocytes representing proliferating primary-like hepatocytes. METHODS: POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method. RESULTS: The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3

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Human hepatocyte systems for in vitro toxicology analysis

Cited by 38 publications

References 74 publications

Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations

Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations

Fumonisin B1 Epigenetically Regulates PTEN Expression and Modulates DNA Damage Checkpoint Regulation in HepG2 Liver Cells

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

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