High content analysis assay for prediction of human hepatotoxicity in HepaRG and HepG2 cells

Saito, Junichiro; Okamura, Ai; Takeuchi, Kenichiro; Hanioka, Kenichi; Okada, Akinobu; Ohata, Takeji

doi:10.1016/j.tiv.2016.02.019

Cited by 47 publications

(35 citation statements)

References 39 publications

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“…Exponentially growing cells (2 × 10 3 cells/well) were seeded in 96-well plates for MTT cell viability assay [ 113 ]. The cells were grown for 24 h, then treated with serial concentrations of Suaeda vermiculata aq.-ethanolic extract (up 500 µg/mL) and DOX (100 µg/mL) for 24 h and with MTT solution (0.5 mg/mL) for 4 h. The DMSO dissolved the reaction product (crystals).…”

Section: Methodsmentioning

confidence: 99%

Suaeda vermiculata Aqueous-Ethanolic Extract-Based Mitigation of CCl4-Induced Hepatotoxicity in Rats, and HepG-2 and HepG-2/ADR Cell-Lines-Based Cytotoxicity Evaluations

Mohammed

Khan

El-Readi

et al. 2020

Plants

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Suaeda vermiculata, an edible halophytic plant, used by desert nomads to treat jaundice, was investigated for its hepatoprotective bioactivity and safety profile on its mother liquor aqueous-ethanolic extract. Upon LC-MS (Liquid Chromatography-Mass Spectrometry) analysis, the presence of several constituents including three major flavonoids, namely quercetin, quercetin-3-O-rutinoside, and kaempferol-O-(acetyl)-hexoside-pentoside were confirmed. The aqueous-ethanolic extract, rich in antioxidants, quenched the DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals, and also showed noticeable levels of radical scavenging capacity in ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) assay. For the hepatoprotective activity confirmation, the male rat groups were fed daily, for 7 days (n = 8/group, p.o.), either carboxyl methylcellulose (CMC) 0.5%, silymarin 200 mg/kg, the aqueous-ethanolic extract of the plant Suaeda vermiculata (100, 250, and 500 mg/kg extract), or quercetin (100 mg/kg) alone, and on day 7 of the administrations, all the animal groups, excluding a naïve (250 mg/kg aqueous-ethanolic extract-fed), and an intact animal group were induced hepatotoxicity by intraperitoneally administering carbon tetrachloride (CCl4). All the animals were sacrificed after 24 h, and aspartate transaminase and alanine transaminase serum levels were observed, which were noted to be significantly decreased for the aqueous-ethanolic extract, silymarin, and quercetin-fed groups in comparison to the CMC-fed group (p < 0.0001). No noticeable adverse effects were observed on the liver, kidney, or heart’s functions of the naïve (250 mg/kg) group. The aqueous-ethanolic extract was found to be safe in the acute toxicity (5 g/kg) test and showed hepatoprotection and safety at higher doses. Further upon, the cytotoxicity testings in HepG-2 and HepG-2/ADR (Adriamycin resistant) cell-lines were also investigated, and the IC50 values were recorded at 56.19±2.55 µg/mL, and 78.40±0.32 µg/mL (p < 0.001, Relative Resistance RR 1.39), respectively, while the doxorubicin (Adriamycin) IC50 values were found to be 1.3±0.064, and 4.77±1.05 µg/mL (p < 0.001, RR 3.67), respectively. The HepG-2/ADR cell-lines when tested in a combination of the aqueous-ethanolic extract with doxorubicin, a significant reversal in the doxorubicin’s IC50 value by 2.77 folds (p < 0.001, CI = 0.56) was noted as compared to the cytotoxicity test where the extract was absent. The mode of action for the reversal was determined to be synergistic in nature indicating the role of the aqueous-ethanolic extract.

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Section: Methodsmentioning

confidence: 99%

Suaeda vermiculata Aqueous-Ethanolic Extract-Based Mitigation of CCl4-Induced Hepatotoxicity in Rats, and HepG-2 and HepG-2/ADR Cell-Lines-Based Cytotoxicity Evaluations

Mohammed

Khan

El-Readi

et al. 2020

Plants

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“…Immortalized human liver cell lines, such as the HepG2 or HepaRG cell lines, are often employed to obtain an initial indication of the crude DILI risk of drug candidates using supraphysiological drug concentrations (194)(195)(196)(197). Though these cell lines are wellestablished, cheap, easy to handle, and allow generation of reproducible data in a highthroughput setting, they are limited by their immature phenotypes.…”

Section: Conventional In Vitro Modelsmentioning

confidence: 99%

Inter-individual differences in the susceptibility of primary human hepatocytes towards drug-induced cholestasis are compound and time dependent

Parmentier¹,

Hendriks

Heyd

et al. 2018

Toxicology Letters

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“…These issues highlight the need for alternative human liver derived cell lines that can be used for general pharmacokinetics and hepatotoxicity evaluation studies [ 5 ]. Currently available alternative in vitro models utilizing a human liver cancer cell line HepG2 is used to provide supplementary data to support findings obtained from human hepatocytes [ 6 , 7 ]. However, the expression levels of drug-metabolizing enzymes are significantly lower in the cell line than in primary human hepatocytes in vitro [ 8 – 13 ].…”

Section: Introductionmentioning

confidence: 99%

Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

Satoh¹,

Iwado²,

Abe³

et al. 2017

PLoS ONE

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The utility of HepG2 cells to assess drug metabolism and toxicity induced by chemical compounds is hampered by their low cytochrome P450 (CYP) activities. To overcome this limitation, we established HepG2 cell lines expressing major CYP enzymes involved in drug metabolism (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and CYP oxidoreductase (POR) using the mammalian-derived artificial chromosome vector. Transchromosomic HepG2 (TC-HepG2) cells expressing four CYPs and POR were used to determine time- and concentration-dependent inhibition and toxicity of several compounds by luminescence detection of CYP-specific substrates and cell viability assays. Gene expression levels of all four CYPs and POR, as well as the CYP activities, were higher in TC-HepG2 clones than in parental HepG2 cells. Additionally, the activity levels of all CYPs were reduced in a concentration-dependent manner by specific CYP inhibitors. Furthermore, preincubation of TC-HepG2 cells with CYP inhibitors known as time-dependent inhibitors (TDI) prior to the addition of CYP-specific substrates determined that CYP inhibition was enhanced in the TDI group than in the non-TDI group. Finally, the IC50 of bioactivable compound aflatoxin B1 was lower in TC-HepG2 cells than in HepG2 cells. In conclusion, the TC-HepG2 cells characterized in the current study are a highly versatile model to evaluate drug-drug interactions and hepatotoxicity in initial screening of candidate drug compounds, which require a high degree of processing capacity and reliability.

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High content analysis assay for prediction of human hepatotoxicity in HepaRG and HepG2 cells

Cited by 47 publications

References 39 publications

Suaeda vermiculata Aqueous-Ethanolic Extract-Based Mitigation of CCl4-Induced Hepatotoxicity in Rats, and HepG-2 and HepG-2/ADR Cell-Lines-Based Cytotoxicity Evaluations

Suaeda vermiculata Aqueous-Ethanolic Extract-Based Mitigation of CCl4-Induced Hepatotoxicity in Rats, and HepG-2 and HepG-2/ADR Cell-Lines-Based Cytotoxicity Evaluations

Inter-individual differences in the susceptibility of primary human hepatocytes towards drug-induced cholestasis are compound and time dependent

Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

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