Cytome micronucleus assays with a metabolically competent human derived liver cell line (Huh6): A promising approach for routine testing of chemicals?

Abstract

One of the main problems of in vitro genotoxicity tests is the inadequate representation of drug metabolizing enzymes in most indicator cell lines which are currently used. We identified recently a human derived liver cell line (Huh6) which detected induction of DNA damage by representatives of different groups of promutagens without enzyme mix and showed that these cells are more suitable in terms of reproducibility and sensitivity as other currently used liver derived lines. We developed a protocol for micronucleus (MN) cytome assays with these cells and validated the procedure in experiments with representatives of different groups of directly and indirectly acting genotoxic carcinogens (MMS, cisplatin, PhIP, IQ, NDMA, B(a)P, AFB1, etoposide, and H 2 O 2 ). The optimal cytochalasin B concentration in combination with 48 hr treatment was found to be 1.5 μg/mL and leads to a cytokinesis block proliferation index in the range between 1.7 and 2.0. The morphological characteristics of different nuclear anomalies which reflect DNA damage (MN, nuclear bridges, and buds) and their baseline frequencies in untreated cells were characterized, and the rates which are required to cause significant effects were calculated. All compounds caused dose dependent induction of MN when the cells were treated for 24 hr, longer and shorter exposure times were less effective. Experiments with different serum levels (fetal bovine serum [FBS]) showed that 10% FBS in the medium (instead of 4%) causes a substantial increase of the sensitivity of the cells. Our results indicate that the new protocol is a promising approach for routine testing of chemicals. Environ. Mol. Mutagen. 60: 134–144, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Development of a micronucleus test using the EpiAirway™ organotypic human airway model

“…We first optimized the CB concentration and incubation period to obtain as many BNCs as possible in our organotypic human airway model. Figure 1b and d show that a plateau in %BNCs was reached at approximately 3 μg/mL CB at 72-h incubation with medium exchanged every 24 h, based on the present optimization process using a range of 1-10 μg/mL CB and three incubation periods of 48, 72, and 120 h. This suggests that a higher concentration of CB over a longer incubation period may not linearly increase the %BNCs in EpiAirway ™ , consistent with previous studies in human peripheral blood lymphocytes [32] and Huh6, a human-derived liver cell line [33], which found that a higher concentration of stimulant over a longer incubation period did not produce a linear increase in cell proliferation.…”

Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay

“…Nevertheless, as a positive result was obtained with 2-AAF, we expect that the 3D HepaRG model possesses at least some NAT activities. Importantly, in the comet assay, positive results with 2-AAF were observed on 3 hepatic cell lines (HepG2, HCC1.2, and Huh6), whereas IQ was negative in the same cell lines as well as negative in the micronucleus test on HepG2 cells 2,31 , but positive in Huh6 cells 32 . The genotoxic mechanism of action of IQ in vitro remains unclear: the link between the positive response in the comet assay and the formation of DNA-reactive nitrenium ions is hypothetic 31 .…”

In vitro determinants of e-cig aerosol condensate bioavailability and toxicity: Influence of cell culture parameters and utility of a normalized dose metric in e-cigarette studies

“…The presence of serum in cell culture medium could be a complicating factor in cytotoxicity and genotoxicity assays because of the potential for interactions between serum proteins and the test substances [39–41]. It remains unclear whether serum proteins could influence the cytotoxic potential of ECAC.…”