Supplementary Material A novel toxicogenomics-based approach to categorize (non-)genotoxic carcinogens Mirjam M Schaap, Paul FK Wackers, Edwin P Zwart, Ilse Huijskens, Martijs J Jonker, Giel Hendriks, Timo M Breit, Harry van Steeg *, Bob van de Water, Mirjam Luijten * Corresponding author: Dr. Harry van Steeg, Center for Health Protection, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, the Netherlands, Tel: +31 30 274 2102, Email: harry.van.steeg@rivm.nl Table of contents: Figure S1, Cytotoxicity plots primary mouse hepatocytes 2 Table S1, Overview concentration selection procedure mouse embryonic 7 stem cells Figure S2, Apoptosis plots embryonic stem cells 8 Figure S3, Cytotoxicity plots embryonic stem cells, 24 hours 9 Figure S4, Cytotoxicity plots embryonic stem cells, 72 hours 10 Table S2, Gene lists comparison approach primary mouse hepatocytes * Table S3, Gene lists comparison approach embryonic stem cells * * These tables are provided in separate excel files. 1
Supplementary Figure S1. Cell viability of the 26 substances in primary mouse hepatocytes. The cells were exposed to one of the substances for 24 hours, followed by an 48- hour incubation time in normal culture medium. Thereafter, cell viability was measured using a MTT test. Control cells were vehicle-treated (DMSO or PBS, 0.5%) and represent 100% cell viability. 2
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Supplementary Table S1. Concentration selection for mouse embryonic stem cells. Apoptotic response Cell viability after 24 hours Cell viability after 72 hours Chemicals Range Result Range Result Range Result Conc. used for GEP NGTXC TCE 0.1-20 mm no observed increase 0.1-20 mm no observed decrease 0.1-20 mm no observed decrease 3 mm TCPOBOP 1-25 µm no observed increase 1-25 µm 20% decrease at 5 um 1-25 µm no observed decrease 5 µm TCDD 0.01-155 nm no observed increase 0.01-155 nm no observed decrease 0.01-155 nm Figure S4 A 100 nm ARO 5-250 µm Figure S2 A 1-250 µm Figure S3 A 1-250 µm no observed decrease 25 µm CA 0.05-2.5 nm no observed increase 0.05-2.5 nm Figure S3 B 0.05-2.5 nm Figure S4 B 0.5 nm CT 0.1-20 mm no observed increase 2.5-20 mm Figure S3 C 2.5-20 mm Figure S4 C 3 mm CF 0.02-200 µm no observed increase 0.02-200 µm Figure S3 D 0.02-200 µm no observed decrease 100 µm HCE 5-60 µm Figure S2 B Figure S3 E Figure S4 D 5 µm LAC 0.01-400 µm no observed increase 0.01-100 µm Figure S3 F 0.01-100 µm Figure S4 E 10 µm OA 0.01-200 nm Figure S2 C 0.01-200 nm NA 0.01-200 nm Figure S4 F 4 nm PB 1.5-4 mm Figure S2 D NA NA 1.5-4 mm Figure S4 G 3 mm SAR 0.5-2.5 µm Figure S2 E 0.5-2.5 µm Figure S3 G 0.5-2.5 µm Figure S4 H 2 µm FK506 3-50 µm no observed increase 3-50 µm no observed decrease 3-50 µm Figure S4 I 10 µm HCH 1-1000 µm no observed increase 1-50 µm no observed decrease 1-50 µm Figure S4 J 50 µm NC BPA 0.1-1000 µm Figure S2 F 0.1-1000 µm Figure S3 H 0.1-1000 µm Figure S4 K 80 µm DIDP 0.1-4 mm no observed increase 0.1-4 mm no observed decrease 0.1-4 mm no observed decrease 2 mm D-M 0.1-4.5 mm no observed increase 0.1-4.5 mm no observed decrease 0.1-4.5 mm no observed decrease 2 mm TBTO 50-1500 nm Figure S2 G 50-1500 nm Figure S3 I 50-1500 nm Figure S4 L 250 nm The concentration used for gene expression profiling was based on the following criteria: a) an increase in apoptosis (max. 30%); b) a decrease in cell viability after 24/72 hours (max. 20%). If no increase in apoptosis and no decrease in cell viability was observed a maximal concentration of 3 mm (NGTXC) or 2 mm (NC) was used. The concentration used for the non-genotoxic carcinogens CSA and WY and the additional tested chemicals CSPT, DEM, ETP, MEN, MMC and MNU and was based on previously performed experiments (1). 1. Hendriks, G., et al. (2011) Sensitive DsRed fluorescence-based reporter cell systems for genotoxicity and oxidative stress assessment. Mutat Res, 709-710, 49-59. 7
Supplementary Figure S2. Apoptotic response in embryonic stem cells upon exposure to one of the 26 substances. An increase in apoptosis was measured by FACS analysis. Only chemicals that induced apoptosis by at least 30% are shown. 8
Supplementary Figure S3. Cell viability of embryonic stem cells after 24 hours of exposure. Cell viability was measured using an MTT test. Control cells were vehicle-treated (DMSO or PBS, 0.5%) and represent 100% cell viability. A graph is only depicted if a clear decrease of the cell viability was observed. 9
Supplementary Figure S4. Cell viability of embryonic stem cells after 72 hours of exposure. Cell viability was measured using an MTT test. Control cells were vehicle-treated (DMSO or PBS, 0.5%) and represent 100% cell viability. A graph is only depicted if a clear decrease of the cell viability was observed. 10
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