The Performance of HepG2 and HepaRG Systems through the Glass of Acetaminophen-Induced Toxicity

Abstract

Investigation of drug-induced liver injuries requires appropriate in vivo and in vitro toxicological model systems. In our study, an attempt was made to compare the hepatocarcinoma HepG2 and the stem cell-derived HepaRG cell lines both in two- and three-dimensional culture conditions to find the most suitable model. Comparison of the liver-specific characteristics of these models was performed via the extent and mechanism of acetaminophen (APAP)-induced hepatotoxicity. Investigating the detailed mechanism of APAP-induced hepatotoxicity, different specific cell death inhibitors were used: the pan-caspase inhibitor zVAD-fmk and dabrafenib significantly protected both cell lines from APAP-induced cell death. However, the known specific inhibitors of necroptosis (necrostatin-1 and MDIVI) were only effective in differentiated HepaRG, which suggest a differential execution of activated pathways in the two models. By applying 3D culture methods, CYP2E1 mRNA levels could be elevated, but we failed to achieve a significant increase in hepatocyte function; hence, the 3D cultivation especially in APAP toxicity studies is not necessarily worth the complicated maintenance. Based on our findings, the hepatocyte functions of HepaRG may stand between the properties of HepG2 cells and primary hepatocytes (PHHs). However, it should be noted that in contrast to PHHs having many limitations, HepaRG cells are relatively immortal, having a stable phenotype and CYP450 expression.

Investigation of TGF‐α‐overexpressing mouse hepatocytes (TAMH) cultured as spheroids for use in hepatotoxicity screening of drug candidates

“…It is reported in the literature that IC 50 values of APAP-treated 2D mouse hepatocytes ranged from 3.8 to 20 mM (Fey & Wrzesinski, 2012), consistent with our findings for the TAMH cell line. HepG2 cells in this study appeared to be more sensitive to APAP-treatment (IC 50 < 10 mM) than reported in literature, where values of $10-30 mM for 2D cultured HepG2 cells were determined (Aritomi et al, 2014;L} orincz et al, 2021). This discrepancy could be attributed maximal plasma concentrations (c max ) were 2.8 (Siegmund et al, 2015) and 1.4 μM (Ferron et al, 2002), respectively.…”

Investigation of the effect of 4-hydroxyphenylboronic acid on acetaminophen-induced liver cell injury in HEPG2 cell line.

“…Hücrelerin yaklaşık olarak %90 oranında yüzeyi kaplaması beklendikten sonra adhezyon için 24 saat inkübe edilmiştir. Sonra, hücreler 20mM APAP ile 4 saat muamele edilmiştir [30,31]. APAP verilişinden sonra 4-OHFBA'nın 31,25µM, 62,5µM, 125µM ve 250µM dozları kuyucuklara eklenerek 24. ve 48. saatlerde hücre canlılığı değerlendirilmiştir [30].…”

“…Sonra, hücreler 20mM APAP ile 4 saat muamele edilmiştir [30,31]. APAP verilişinden sonra 4-OHFBA'nın 31,25µM, 62,5µM, 125µM ve 250µM dozları kuyucuklara eklenerek 24. ve 48. saatlerde hücre canlılığı değerlendirilmiştir [30]. Tedavi grupları, kontrol grupları ile kıyaslanmıştır.…”

Mera: A scalable high throughput automated micro-physiological system

“…Approximately 5-10% of APAP is oxidised by CYP450s into N-acetyl-p-benzoquinone imine (NAPQI) which is detoxified with glutathione (GSH) upon conjugation. It is thought that the toxicity of APAP is directly linked to the binding of NAPQI to mitochondrial proteins (Lőrincz et al, 2021). To demonstrate the improved function of MTs, HepG2 cells grown in a 2D monolayer were compared to 3D MTs before and after treatment with 30mM APAP where APAP was used to induce cell death.…”