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

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.

An Evaluation of the In Vitro Roles and Mechanisms of Silibinin in Reducing Pyrazinamide- and Isoniazid-Induced Hepatocellular Damage

“…the most efficacious within a specific window of INH concentrations that are neither too severe nor too mild. This observation is consistent with other in vitro findings, which involve the characterisation of silibinin's hepatoprotective effects at toxicant concentrations around the IC 50 values of their respective assays (Mann et al, 2017;Singh et al, 2012). The existence of INH's "Goldilocks zone" has two clinical implications in discussing silibinin's role as a rescue adjuvant.…”

“…The optimal silibinin concentration from our viability experiments was determined to be 25 μ M when DILI was induced with 80 mM INH ( Fig. 2A), which may be explained by silibinin's pro-oxidative and pro-apoptotic effects at higher concentrations, an observation that corroborates experiments conducted by other research groups in rats (Malekinejad et al, 2012) and other in vitro cell lines (Mann et al, 2017;Procházková et al, 2011;Surai, 2015).…”

“…Notably, HepaRG's poor expression of CYP2E1 has cast doubt on its relevance in INH-induced hepatotoxicity models (Du et al, 2016). Concurrently, our in vitro experiments using human cell lines serve as useful cross-references for other in vitro (Mann et al, 2017;Singh et al, 2012), in vivo animal (Srivastava et al, 2008), and human (Luangchosiri et al, 2015) studies on silibinin's protection against PIER-induced hepatotoxicity.…”

“…Unfortunately, despite the extensive and rigorous research on the basis for silibinin's hepatoprotective effect in recent years (Ramappa and Aithal, 2013;Roubalová et al, 2017;Surai, 2015), the in vivo and in vitro biological markers which silibinin modulates have not been conclusively linked to the reduction of DILI (Mann et al, 2017). Furthermore, the exact biochemical mediators behind silibinin's hepatoprotective effect have also not been identified (Raghu and Karthikeyan, 2016).…”

A lung/liver-on-a-chip platform for acute and chronic toxicity studies

“…21 Our 3D liver spheroid model was built using human HepaRG™, which is considered a good hepatic model, as it exhibits high and stable secretion of albumin and metabolic capacity. [22][23][24][25] Multiple studies have shown that the toxicity of the anti-tuberculosis agent isoniazid, 26 acetaminophen, 27 or aflatoxin B1 (AFB1) 28 could be predicted accurately using 3D liver spheroid models. For the lung/liver-on-a-chip system, the 3D organotypic bronchial culture was prepared using normal human bronchial epithelial (NHBE) cells cultured at the ALI.…”