Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins: a new hope for laboratory animals?

Abstract

Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB₁ (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.

Keywords: Comet assay; Genotoxicity; Hepatic cell lines; p53.

Fig. 1

Induction of DNA damage in different human-derived liver cell lines by H₂O₂. Bars indicate mean ± SD of medians of four measurements (per experimental point in total 200 cells), asterisks indicate statistical significance (Dunnett’s Multiple Comparison Test, P ≤ 0.05)

Fig. 2

Reproducibility of SCGE experiments with different model mutagens in hepatoma cell lines. The cells were treated with different concentrations of the model compounds either for 24 h (AFB₁, B(a)P) or for 48 h (IQ and PhIP), and H₂O_2. IF values represent the ratio of chemically induced comet formation (% DNA in tail) vs. DNA migration in corresponding controls. Bars show the results of two separate experiments; for each experimental point, at least two different cultures were set up in parallel, one slide with two gels was made per culture and 100 cells were counted per slide. Bars indicate mean ± SD of medians of four measurements; asterisks indicate statistical significance (Student T Test, p ≤ 0.05)