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.
Highlights
One of the fundamental problems of in vitro tests is the inadequate representation of drug metabolizing enzymes in cell lines which are currently used in routine screening of chemicals
We studied characteristics of the different cell lines which are relevant in regard to their potential use in genotoxicity tests, namely, (1) the morphology of the cells which provides information about their origin and similarity to primary liver cells, (2) their karyotype allowing to draw conclusions concerning their chromosomal stability, (3) measurements of the mitotic activities providing information about the duration of the repair phase which is needed for the design of tests which require cell division and (4) the p53 status of the cells as it was found that it plays an important role in regard to the reliability of results obtained with mammalian cell lines in mutagenicity experiments (Kirkland et al 2007; Pfuhler et al 2011)
The features of HuH6 cells differ from those of the other cell types, i.e., they are relatively undifferentiated and we found in agreement with previous studies that they contain numerous glycogen granules, which are only rarely seen in other liver-derived cells (Figure S1E) (Doi 1976)
Summary
One of the fundamental problems of in vitro tests is the inadequate representation of drug metabolizing enzymes in cell lines which are currently used in routine screening of chemicals. The homogenates contain phase I enzymes that convert chemicals to genotoxic metabolites and are added in experiments with bacteria and mammalian cells to mimic the biotransformation of chemicals in humans (Brandon et al 2003). These experimental models do not reflect the situation in vivo, for example, the detoxification of electrophilic DNA reactive intermediates by phase II enzymes. Often false results are obtained and animal experiments with rodents are performed which could be avoided with more reliable in vitro models (Kirkland et al 2007). Epithelial, grow in multilayered islands, often piled up, peripheral cells surrounding the island appeared to be flattened JHH6
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