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 H₂ O₂ ). 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.

Keywords: Huh6; micronuclei; nucleoplasmatic bridges; serum.

Figure 1A,B.

Impact of Cyt B treatment on the proliferation of Huh6 cells. A: Impact of different treatment periods in combination with 3.0 μg/mL Cyt B on CBPI values. B: Impact of different Cyt B concentrations on the CBPI values (exposure time 48 hr). Data show means ± SD of results obtained with two cultures per experimental point. From each culture at least 500 cells were evaluated.

Figure 2

Photographic images of the cells and different nuclear anomalies (NBs – nucleoplasmic briges; NBuds – nucleoplasmic buds), stain Diff Quick, magnification 400×, (A) mononucleated epithelioid cell with some cytoplasmic vacuoles and few desmosomes; (B) binucleated epithelioid cell; (C) multinucleated epithelioid cell with desmosomes; (D) binucleated epithelioid cells with some cytoplasmic vacuoles and one or two nucleoplasmic bridges; (E) binucleated epithelioid cells with a micronucleus in different position; (F) binucleated epithelioid cells with nuclear buds; (G) mononucleated apoptotic cells; (H) mononucleated necrotic cells.

Figure 3

Induction of MN after treatment of Huh6 cells with different direct (A–D) and indirect (E–I) acting mutagens for different time periods. CDDP, H₂O₂, MMS, and NDMA were dissolved in medium, Etop, AFB1, B(a)P, IQ, and PhiP were dissolved in DMSO. Bars show means ± SD of results obtained in a representative experiment. Two cultures were treated per experimental point and from each, at least 1,000 BNC cells were evaluated. Data were evaluated by generalized linear model with Poisson counts. Chi‐square tests for overdispersion were applied. Stars indicate statistical significance (P ≤ 0.05).

Figure 4

Impact of different serum conditions (4% and 10%) on MN induction by various model mutagens. The cells were tested with the chemicals for 24 hr and Cyt B (1.5 μg/mL) was added for 48 hr. Bars show means ± SD of results obtained in a representative experiment; two cultures were treated per experimental point and from each at least 1,000 BNC cells were evaluated. Data were evaluated by generalized linear model with Poisson counts. Chi‐square tests for overdispersion were applied. Stars indicate statistical significance (P ≤ 0.05).