6th International Conference on Environmental Mutagens in Human Populations, Doha, Qatar,
March 26-29, 2012
Automated image analysis of the micronucleus cytome assay and comet assay for high throughput human biomonitoring and in vitro/in vivo genotoxicity testing in industry studies: IMSTAR system
F. Soussaline (1), A. Papine (1), E. Tatarinova (1), I. Decordier (2), J. Dumont (3), A. Azqueta-Oscoz (4), A.K. Sharma (5), M-L. Binderup (5), A. Collins (4), E. Lorge (3), M. Kirsch-Volders (2),
(1) IMSTAR S.A. 60 rue Notre-Dame-des-Champs, 75006 Paris, France; (2) Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussels, Belgium; (3) Biologie Servier Group, route de Servan 45520 Gidy, France ; (4) Department of Nutrition, University of Oslo, Blindern, 0316 Oslo, Norway; (5) Technical University of Denmark, National Food Institute, Mørkhøj Bygade 2860 Søborg, Denmark
Genetic toxicology tests such as micronuclei and comet assays are providing crucial information about DNA damages in environmental risk assessment, biomonitoring and compound testing. They require both substantial time and expertise when performed by conventional screening.
Analysis of micronuclei (MN) is the most widely used for biomonitoring or in vitro/in vivo genotoxicity testing to assess genetic damage . It has been shown that scoring MN frequencies in binucleated and mononucleated cells in cytokinesis-blocked human lymphocytes is a predictive biomarker for cancer risk and a reliable, sensitive method for biomonitoring of early genetic effects . However, automation of MN analysis is needed for more reliable, faster detection, and improved scoring accuracy. It also allows large scale analysis for multi-centres cohort studies. Within the framework of the EU project NewGeneris [3|, an automated image analysis system for scoring MN frequencies in human lymphocytes was developed in collaboration between VUB Cellulaire Genetica Lab and IMSTAR [4, 5]. The IMSTAR PathfinderTM automated imaging system is based on solid and specific algorithms for image analysis, starting from the detection of each individual cell in the whole sample; then, within every cell, nuclei and micronuclei are accurately detected, and the scoring in mono, bi and polynucleated cells is proposed and validated by an expert technician.
In parallel, in order to increase the scoring throughput and robustness, and to decrease operator-dependency of MN scoring for industrial compound screening of several cell lines, e.g. L5178Y, V79, CHO, TK6, and HepG2, we implemented an automated imaging module to be used in further regulatory in vitro micronucleus studies compliant with Good Laboratory Practice (OECD Principles of GPL [C(97)186/Final]). Mouse lymphoma L5178Y cells were exposed to known genotoxic compounds at various concentrations according to different treatment conditions. The scores of micronucleated cells were compared between visual and automated scoring in terms of time-saving, repeatability, reproducibility and accuracy. The results showed that the PathfinderTM imaging system was able to detect micronucleated cells as accurately as visual scoring, while saving scoring time by 10 to 15 fold. Moreover, in this study, a better homogeneity was found between two cultures of a same treated group with the automated method.
The comet assay or single-cell gel electrophoresis is a well-established genotoxicity test and a simple to use method to detect a broad spectrum of DNA damage with high sensitivity. It is widely used in regulatory, mechanistic and biomonitoring studies . Moreover, it has been recently recommended that the in vivo comet assay could be preferred to the OECD guideline rat liver UDS assay for pharmaceuticals intended for human use testing, as a second in vivo assay when positive results are obtained in vitro but the in vivo micronucleus test is negative .
Within the COMICS European project , we validated the Pathfinder COMET system  and demonstrated the added-value of high-content screening methods using the IMSTAR automated scoring system in terms of time saving and, more important, providing new information for empowered sensitivity and versatility of the test, as a genuine alternative to animal testing.
More specifically, automated image analysis and scoring algorithms were developed by IMSTAR in collaboration with the National Food Institute of Denmark, to fit a wide range of cell lines for in vitro comet assay: HEK, V79, keratinocytes, fibroblasts, Hela and Caco2. As for in vivo comet assay, dedicated modules for preparations from rat’s bone marrow, liver, lung and stomach cells are now available. In studies where fully automated was compared to conventional procedure, the scoring was faster, sensitivity higher, variability lower, while the several thousands of comets scored per sample enables to work with parameter distributions.
 Kirsch-Volders et al., Arch Toxicol. 2011, 85: 873-899:  Kirsch-Volders and Fenech, Mutagenesis 2001, 16: 51-58;  EU Integrated Project “NewGeneris (Contract n° FOOD-CT-2005-016320);  Decordier et al., Mutagenesis, 2009, 24: 85-93;  Decordier et al., Mutagenesis, 2011, 26: 163-168;  Wiklund SJ, et al. Mutagenesis 2003; 18:167-175;  EU STREP COMICS project, (contract n° LSHB-CT-2006-037575);  Azqueta A. et al., Mutagenesis 2011; 26:393-399;  OCDE 486 Guide line.