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Evaluation of HepaRG cells for the assessment of indirect drug-induced hepatotoxicity using INH as a model substance

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Abstract

HepaRG cells are widely used as an in vitro model to assess drug-induced hepatotoxicity. However, only few studies exist so far regarding their suitability to detect the effects of drugs requiring a preceding activation via the cytochrome P450 (CYP) system. A prototypic substance is the anti-tuberculosis agent INH, which is metabolized into N-acetylhydrazine, which then triggers hepatotoxicity. Therefore, the aim of the present study was to test if this effect can also be detected in HepaRG cells and if it can be counteracted by the known hepatoprotectant silibinin. For this purpose, differentiated HepaRG cells were treated with increasing concentrations of INH (0.1–100 mM) or 10 mM INH plus escalating concentrations of silibinin (1–100 µM). After 48 h of treatment, cell morphology and parameters indicating cell vitality, oxidative stress, and liver cell function were assessed. High concentrations of INH led to severe histopathological changes, reduced cell vitality and glutathione content, increased LDH and ASAT release into the medium, enhanced lipid peroxidation, and elevated cleaved caspase-3 expression. Additionally, glycogen depletion and reduced biotransformation capacity were seen at high INH concentrations, whereas at low concentrations an induction of biotransformation enzymes was noticed. Silibinin caused clear-cut protective effects, but with few parameters INH toxicity was even aggravated, most probably due to increased metabolization of INH into its toxic metabolite. In conclusion, HepaRG cells are excellently suited to evaluate the effects of substances requiring prior toxification via the CYP system, such as INH. They additionally enable the identification of complex substance interactions.

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Abbreviations

ASAT:

Aspartate transaminase

CDNB:

1-Chloro-2,4-dinitrobenzene

CYP:

Cytochrome P450

DMSO:

Dimethylsulfoxide

ECOD:

Ethoxycoumarin-O-deethylation

EROD:

Ethoxyresorufin-O-deethylation

GSH:

(Reduced) glutathione

GST:

Glutathione-S-transferase

INH:

Isoniazid

LDH:

Lactate dehydrogenase

LPO:

Lipid peroxidation products

NAT:

N-Acetyltransferase

PHH:

Primary human hepatocytes

PNPH:

p-Nitrophenol hydroxylase

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Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Drackendorfer Str. 1, 07747, Jena, Germany

    Anika Mann, Thomas Pelz & Amelie Lupp

  2. Institute of Biochemistry II, Jena University Hospital, Jena, Germany

    Knut Rennert & Alexander Mosig

  3. Institute for Pharmacy and Food Chemistry, University of Würzburg, Würzburg, Germany

    Michael Decker

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  1. Anika Mann
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13577_2017_175_MOESM1_ESM.tif

Supplementary material 1 (TIFF 2139 kb) Supplemental Figure S1 INH induced cytotoxicity in undifferentaited and in differentiated HepaRG cells. Undifferentiated and differentiated HepaRG cells were either left untreated (control) or were treated for 48 h with 10 mM or 100 mM INH after which phase contrast microscopy was performed. Original magnification: × 100. Representative photomicrographs from 3 independent experiments with three independent batches of cells are shown. Arrows: hepatocyte-like cells; asterisks: cholangiocyte-like cells

13577_2017_175_MOESM2_ESM.tif

Supplementary material 2 (TIFF 2414 kb) Supplemental Figure S2 Induction of CYP enzyme activities by prototypical inducers in HepaRG cells. HepaRG cells were either left untreated or were treated for 24 h with 25 µM β-naphthoflavone (BNF) or with 100 µM phenobarbital (PB). The cells were then harvested and (A) ethoxyresorufin-O-deetylation (EROD) activity or (B) ethoxycoumarin-O-deethylation (ECOD) activity was measured. Data are given as mean ± standard error of the mean (SEM), n = 3 independent batches of cells. *: p ≤ 0.05 versus untreated cells (control) (Dunnett’s post hoc test)

13577_2017_175_MOESM3_ESM.tif

Supplementary material 3 (TIFF 5997 kb) Supplemental Figure S3 CYP enzyme activities in HepG2 cells, in HepaRG cells, and in human liver 9000 g supernatants. (A) Ethoxyresorufin-O-deetylation (EROD) activity, (B) ethoxycoumarin-O-deethylation (ECOD) acitivity, (C) glutathione-S-transferase activity using 1-chloro-2,4-dinitrobenzene as a substrate, and (F) p-nitrophenol hydroxylase (PNPH) activity. For preparation of human liver 9000 g supernatants, human liver samples were homogenized in 0.1 M sodium phosphate buffer pH 7.4 (1:3 w/v) and centrifuged at 9000 × g for 30 min. Data are given as mean ± standard error of the mean (SEM), n = 3 independent batches of cells or 3 different human liver 9000 g supernatants

13577_2017_175_MOESM4_ESM.tif

Supplementary material 4 (TIFF 8909 kb) Supplemental Figure S4 Influence of different concentrations of silibinin on different parameters for cytotoxicity in HepaRG cells. Differentiated HepaRG cells were incubated for 48 h with increasing concentrations (0 µM-100 µM) of silibinin after which (A) cell viability was quantified by means of the CCK-8 assay, (B) LDH leakage, (C) ASAT release into the medium was measured. Data are given as mean ± standard error of the mean (SEM), n = 8 (A) or n = 3 (B, C) independent batches of cells. *: p ≤ 0.05 versus untreated cells (control) (Dunnett’s post hoc test). (D, E) Silibinin-treated cells were embedded in paraffin and 4-µm-sections were prepared from the paraffin blocks. (D) Hematoxylin-eosin staining; original magnification: x630. (E) PAS staining; original magnification: x630. Representative photomicrographs from 3 independent experiments with three independent batches of cells are shown

13577_2017_175_MOESM5_ESM.tif

Supplementary material 5 (TIFF 5928 kb) Supplemental Figure S5 Influence of silibinin on oxidative state and apoptosis rate in HepaRG cells. Differentiated HepaRG cells were incubated for 48 h with increasing concentrations (0 µM-100 µM) of silibinin after which (A) protein content of the cell pellet, (B) the cellular content of reduced glutathione, and (C) the concentration of lipid peroxidation products were measured. Data are given as mean ± standard error of the mean (SEM), n = 6 (A) or n = 3 (B, C) independent batches of cells. *: p ≤ 0.05 versus untreated cells (control) (Dunnett’s post hoc test). (D) Silibinin-treated cells were embedded in paraffin and 4-µm-sections were prepared from the paraffin blocks and stained for cleaved caspase-3 expression. Immunohistochemistry, counterstaining with hematoxylin; original magnification: x630. Representative photomicrographs from 3 independent experiments with three independent batches of cells are shown

13577_2017_175_MOESM6_ESM.tif

Supplementary material 6 (TIFF 11069 kb) Supplemental Figure S6 Influence of silibinin on biotransformation capacity in HepaRG cells. Differentiated HepaRG cells were incubated for 48 h with increasing concentrations (0 µM-100 µM) of silibinin after which (A) ethoxyresorufin-O-deetylation (EROD) activity, (B) ethoxycoumarin-O-deethylation (ECOD) activity, (C) glutathione-S-transferase activity using 1-chloro-2,4-dinitrobenzene as a substrate, and (F) p-nitrophenol hydroxylase (PNPH) activity were measured. Data are given as mean ± standard error of the mean (SEM), n = 3 independent batches of cells. *: p ≤ 0.05 versus untreated cells (control) (Dunnett’s post hoc test). (D, E) Silibinin-treated cells were embedded in paraffin and 4-µm-sections were prepared from the paraffin blocks and stained for (D) CYP1A2, (E) CYP2E1 expression. Immunohistochemistry, counterstaining with hematoxylin; original magnification: x630. Representative photomicrographs from 3 independent experiments with three independent batches of cells are shown

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Mann, A., Pelz, T., Rennert, K. et al. Evaluation of HepaRG cells for the assessment of indirect drug-induced hepatotoxicity using INH as a model substance. Human Cell 30, 267–278 (2017). https://doi.org/10.1007/s13577-017-0175-9

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