Assessment of the Performance of the TGx-DDI Biomarker to Detect DNA Damage-Inducing Agents Using Quantitative RT- PCR in TK6 Cells

Gene expression biomarkers are now available for application in the identification of genotoxic hazards. The TGx-DDI transcriptomic biomarker can accurately distinguish DDI from non-DDI exposures based on changes in the expression of 64 biomarker genes. The bioamarker was originally derived from DNA microarray gene expression profiles of TK6 human lymphoblastoid cells post 4-hour exposure to 28 reference DDI and non-DDI agents [Li et al. 2015]. To broaden the applicability of TGx-DDI, the biomarker was tested using quantitative RT-PCR (qPCR), which is accessible to most molecular biology laboratories. To assess the classification capability of the biomarker using qPCR, a custom 96-well TaqMan qPCR array (TGx-DDI qPCR array) was constructed using the 64 biomarker genes. TK6 cells were exposed to each of the 28 reference agents and their vehicle controls for 4 hours and the expression level of the TGx-DDI genes were profiled using the TaqMan arrays. This study provides reference qPCR expression profiles of the TGx-DDI biomarker for DDI chemical classification using qPCR. TK6 human lymphoblastoid cells were exposed to 28 reference DDI and non-DDI agents (29 treatment samples in total; two concentrations of EtOH were included) and vehicle controls (9 control samples) for 4 hours. Two biological replicates for each treatment were generated (76 samples in total). Following RNA extraction and cDNA synthesis, each sample was loaded on one TGx-DDI qPCR array for gene expression profiling. Each 96-well TaqMan qPCR array contained 64 TGx-DDI biomarker genes and housekeeping genes (HPRT and GUSB; the average Ct value of the two genes were used) for normalization. The treatment concentration of each compound was determined in the original TGx-DDI publication by Li et al. [2015]. Briefly, TK6 cells were exposed to increasing concentrations of each agent for 4 hours. Cell viability was measured using MTT assay after 20-hour recovery (at 24h) and the expression of three genes, ATF3, GADD45A, and CDKN1A, were measured at 4h. The selected concentration maximally induced the three indicator genes, while maintaining cell viability above 50% at 24h [Li et al., 2015]. Following normalization of each gene using the housekeeping genes, gene expression fold changes due to chemical exposure were determined by normalizing each chemical treatment to its vehicle control. The current dataset contains 61 genes; Ct values for three of the 64 TGx-DDI genes were above 40 in majority of the samples and, thus, were excluded.