Please cite this article in press as: W.M.A. Westerink, et al., Development and validation of a high-volume screening in vitro micronucleus assay in CHO-k1 and HepG2 cells, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2011), doi:10.1016/j.mrgentox.2011.05.007 ARTICLE IN PRESS G Model MUTGEN 402007 1–15 Mutation Research xxx (2011) xxx–xxx 1 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis jou rnal h om epage: www.elsevier.com/locate/gentox C om mun i ty a ddress: www.elsevier.com/locate/mutres Development and validation of a high-volume screening in vitro micronucleus assay in CHO-k1 and HepG2 cells 1 2 Walter M.A. Westerink ∗,1 , Tom J.J. Schirris 1 , G. Jean Horbach, Willem G.E.J. Schoonen 3 Toxicology and Drug Disposition, MSD, PO Box 20, 5342 CC Oss, The Netherlands Q1 4 5 a r t i c l e i n f o 6 7 Article history: 8 Received 17 January 2011 9 Received in revised form 22 March 2011 10 Accepted 24 April 2011 11 Available online xxx a b s t r a c t In the present study an automated image analysis assisted in vitro micronucleus assay was developed Q2 with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these cells of a functionally active p53 protein, a functionally competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system. These properties may result in an assay with a high predictivity for in vivo genotoxicity. The assays with CHO-k1 and HepG2 cells were both evaluated by testing a set of compounds rec- ommended by the European Centre for the Validation of Alternative Methods (ECVAM), among which are in vivo genotoxins and non-genotoxins. The CHO-k1 cell line showed a high sensitivity (percentage of genotoxic compounds that gave a positive result: 80%; 16/20) and specificity (percentage of non- genotoxic compounds that came out negative: 88%; 37/42). Although the sensitivity of the HepG2 cell line was lower (60%; 12/20), the specificity was high (88%; 37/42). These results were confirmed by test- ing an additional series of 16 genotoxic compounds. For both the CHO-k1 and the HepG2 cell line it was possible to size-classify micronuclei, enabling distinguishing aneugens from clastogens. It is concluded that two high-throughput micronucleus assays were developed that can detect geno- toxic potential and allow differentiation between clastogens and aneugens. The performance scores of the CHO-k1 and HepG2 cell lines for in vivo genotoxicity were high. Application of these assays in the early discovery phase of drug development may prove to be a useful strategy to assess genotoxic potential at an early stage. © 2011 Published by Elsevier B.V. 1. Introduction 12 In drug development, toxicity is an important factor for drug Q3 13 attrition. Genotoxicity is one of the causes of toxicity. The current 14 regulatory in vitro genotoxicity assays used to determine genotoxic 15 potential have a low throughput and need a relatively large amount 16 of test compound. Therefore, these assays are in their present for- 17 mat less applicable in the early discovery phase. Toxicity screening 18 in the early phase requires assays that have a high throughput and 19 require only a small amount of compound. High-throughput assays 20 based on the use of bacteria, yeast, and rodent/human cell lines are 21 proposed as useful in vitro models. 22 In the present study a high-volume screening (HVS) technique 23 was used to develop a high-throughput in vitro micronucleus assay 24 in the rodent Chinese Hamster Ovarian k1 (CHO-k1) cell line and 25 in the human HepG2 cell line. In the HVS technique (fluorescence) 26 ∗ Corresponding author. E-mail address: westeriw@gmail.com (W.M.A. Westerink). 1 These authors contributed equally to the work. microscopy is used to analyze cells in 96-well plates (or even 384- 27 well plates). Aspects of importance for development of a fast HVS- 28 micronucleus assay are automatic scoring, the choice of the cell line, 29 the validation with proper reference compounds, and the ability of 30 the test system to discriminate between clastogens and aneugens. 31 Scoring of micronuclei in the regulatory in vitro micronucleus 32 (IVMN) assay is a labor-intensive method that is performed manu- 33 ally on microscope slides by trained operators. This manual scoring 34 of micronuclei results in inter-observer and intra-observer vari- 35 ability, with a scorer-specific coefficient of variation between 5.5 36 and 9.5% [1]. One approach to improve the efficiency is the auto- 37 mated counting of micronuclei by use of image-analysis systems 38 [1]. With this aim, Diaz et al. [2] developed a HVS IVMN assay in 39 CHO-k1 cells. Validation with reference compounds showed that 40 this CHO-k1 HVS IVMN assay was a high throughput alternative 41 to the manual scoring of micronuclei on microscope slides. In the 42 present study this method was further optimized with respect to 43 speed and stability of the stains. 44 To assess the use of HVS in the development of an IVMN assay 45 two cell lines were compared in the present study, viz CHO-k1 and 46 HepG2. The CHO-k1 cell line is frequently used in the regulatory 47 1383-5718/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.mrgentox.2011.05.007