An adaptation of the human HepaRG cells to the in vitro micronucleus assay

Abstract

The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of chemicals because of its simplicity of scoring and wide applicability in different cell types. However, most of the cells currently in use are devoid of the enzyme equipment required for activation of promutagens in the genotoxic metabolites. We postulated that the human HepaRG cell line, which can express xenobiotic metabolising enzymes at levels close to those found in primary human hepatocytes and has retained the indefinite growth capacity of transformed cells, could represent a more suitable model for genotoxicity testing of chemicals requiring metabolic activation. Based on the recommendations of the Organisation for Economic Co-operation and Development test guideline TG 487 for testing of chemicals, HepaRG cell cultures containing >80% mature hepatocytes were treated in situ with various chemicals for 24 h followed by a 3-day mitogenic stimulation with epidermal growth factor without cytokinesis block. In such culture conditions, HepaRG cells underwent >1.5 cell cycle per cell during the mitogenic stimulation. While non-genotoxic compounds (mannitol and staurosporine) did not increase the rate of micronucleated mononucleated cells, all aneugens (colchicine, nocodazole and dichlorodiphenyldichloroethylene) as well as the direct acting clastogen methyl methanesulfonate and clastogens requiring metabolic activation (aflatoxin B1, benzo(a)pyrene and 2-nitrofluorene) induced a statistically significant concentration-related increase in the number of mono-micronucleated cells. The micronucleus test was also performed after 7-day repeat exposure of HepaRG cells to the chemicals. Noticeably, a time-dependent effect was obtained with the three clastogens requiring metabolic activation. In conclusion, our results obtained with HepaRG hepatocytes exposed to various genotoxic compounds requiring or not bioactivation, compared favorably with those reported in various other cell types. They support the view that metabolically competent HepaRG cells have unique potential benefits for testing genotoxic compounds using the in vitro micronucleus assay.

Impact of isomalathion on malathion cytotoxicity and genotoxicity in human HepaRG cells

“…In agreement, we observed that malathion and isomalathion did not cause any cell loss in HepG2 cells when using the same range of concentrationsas those used for HepaRG cells. These results support our previous data showing that HepaRG cells are much more sensitive to compounds requiring liver metabolism to exert their toxic effects than HepG2 cells that did not express most of the major cytochromes P450 [21,26].…”

“…Genotoxic effects of malathion and isomalathion either individually or combined, were evaluated by the micronucleus assay as previously described [21]. Briefly, after 24 h of treatment with pesticides, followed by a 72h epidermal growth factor (EGF) stimulation corresponding to the recovery time, HepaRG cells were fixed for 10 min in …”

“…In order to evaluate malathion and isomalathion genotoxic effects, the micronucleus assay was performed in HepaRG cells as previously described [21]. Two positive controls were used: MMS, a classical DNA damaging agent and AFB1, a metabolism-requiring genotoxic compound (Table 2) Figure 5D).…”

Long‐term maintenance of HepaRG cells in serum‐free conditions and application in a repeated dose study

“…Green to blue colour indicates increasing consumption. until now, were exclusively performed in serum-supplemented medium and usually these studies did not exceed 2 weeks of cultivation after the differentiation of the HepaRG cells (Antherieu et al, 2011;Josse et al, 2012). Since HepaRG cells show stable CYP activity, they may be reliably applied in long-term repeated dose toxicity studies especially in the assessment of metabolism mediated adverse effects.…”

“…Dimethylsulfoxide (DMSO) is essential for the differentiation of HepaRG cells and induction of CYP expression (Gripon et al, 2002;Hoekstra et al, 2011). Studies that involve HepaRG cells are routinely carried out in medium containing high DMSO concentrations (≈2%) and in the presence of fetal bovine serum (FBS) (Josse et al, 2012;Leite et al, 2012). As a result of undefined composition and batch-to-batch variations, the use of FBS poses a problem in reproducibility and standardization (Hahne and Reichl, 2011).…”