Transcriptomic-based biomarker for detection of genotoxicants

The GENOMARK tool in metabolically competent human HepaRG cells is based on the transcriptomic results obtained with 12 genotoxic (in vivo positive results) and 12 non-genotoxic (in vivo negative results) reference compounds. Genotoxic compounds were selected to cover different mechanisms of action including bulky adduct formation, DNA alkylation, cross-linking, radical generation causing DNA strand breaks, inhibition of tubulin polymerization and base analogues. The GENOMARK biomarker includes 84 genes (+ 5 housekeeping genes) of which the expression can be assessed in an easy-to-handle qPCR array. Briefly, after cultivation for 7 days, cells are exposed for 72 hours to the test compound at the IC10 concentration as determined by an MTT test (cytotoxicity formazan-forming test). Next, RNA is isolated and transcribed to cDNA which is applied on the qPCR pre-spotted plate to investigate the expression of the 84 genes included in the GENOMARK tool. Gene expression results are analyzed in R (software environment for statistical computing and graphics). All experiments are performed in triplicate with different batches of HepaRGTM cells to ensure reproducibility.