Application of the TGx-28.65 toxicogenomics biomarker to classify genotoxic and non-genotoxic chemicals in human TK6 cells in the presence of rat liver S9

In vitro toxicogenomics signatures to predict mode of action can facilitate chemical screening. We recently demonstrated the use of the TGx-28.65 genomic biomarker (representing: toxicogenomics (TGx), developed using a 28 chemical training set, and comprising 65 genes) in classifying agents as genotoxic (DNA damaging) and non-genotoxic in human lymphoblastoid TK6 cells. Because TK6 cells do not possess cytochrome P450 activity, we confirmed accurate classification in cells co-exposed to 1% 5,6 benzoflavone/phenobarbital-induced rat liver S9 for metabolic activation. However, chemicals may require different types of S9 for activation. Here we conducted experiments in TK6 cells exposed to chemicals in the presence of 2% or 10% Aroclor-induced, or 5% ethanol-induced rat liver S9 to expand TGx-28.65 biomarker application. Gene expression profiles were produced 3-4 hr following a 4 hr co-exposure of TK cells to test chemicals (seven genotoxic and two non-genotoxic, three concentrations, and concurrent solvent controls) and S9. Relative survival and micronucleus frequency was assessed by flow cytometry in cells 20 hr after the exposure. We found that genotoxicity/non-genotoxicity could be accurately predicted for the test compounds using the different S9s. One technical replicate of cells co-treated with dexamethasone and 10% Aroclor-induced S9 was falsely predicted as genotoxic, suggesting caution in using high concentrations of S9. TGx-28.65 correctly classified low concentrations of genotoxic chemicals (those not causing cytotoxicity or micronuclei) as genotoxic, demonstrating that it is a sensitive biomarker of genotoxicity. Overall, the work confirms that different S9s can be used in TK6 cells without impairing predictivity using the TGx-28.65 biomarker. This experiment examined the whole genome transcriptional response of TK6 cells exposed to 9 test chemicals, including acetaminophen, 2-aminoanthracene, cyclophosphamide, dibenz[a,h]anthracene, dimethylnitrosamine, furan, 2-nitrofluorene, phenanthrene, sucrose, and for 4 hours followed by a recovery time of 3-4h in fresh media at 3 different concentrations, including a low, a medium, and a high concentration, in addition to vehicle controls (+S9). Each concentration and time point had 3 biological replicates, which were pooled for gene expression analysis. There were a total 31 samples (9 arrays) included in the final analysis using a two-colour dye swap design.