Citation:

Cho, E., J. Buick, A. Williams, R. Chen, H. Li, C. Corton, A. Fornace, J. Aubrecht, AND C. Yauk. Assessment of the performance of the TGx-DDI biomarker to detect DNA damage-inducing agents using quantitative RT- PCR in TK6 cells. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS. John Wiley & Sons, Inc, Hoboken, NJ, 60(2):122-133, (2019). https://doi.org/10.1002/em.22257

Impact/Purpose:

Gene expression signatures of DNA damage-inducing (DDI) agents can be used as mechanistic markers of genotoxicity for in vitro hazard identification. Indeed, the TGx-DDI transcriptomic biomarker can accurately distinguish DDI from non-DDI exposures based on changes in the expression of 64 biomarker genes. The 64 genes were previously derived from whole transcriptome DNA microarray profiles of 28 reference agents (13 DDI and 15 non-DDI) after 4-hour treatments of TK6 human lymphoblastoid cells. To broaden the applicability of TGx-DDI, we tested the biomarker using quantitative RT-PCR (qPCR), which is accessible to most molecular biology laboratories. Information from this product should be useful to OCSPP and other program offices as part of ongoing efforts to evaluate the health risks of chemicals which activate AOPs leading to cancer. Additionally, our results provide further evidence that gene expression biomarkers can be effectively used to predict molecular initiating events or key events in AOPs that will help to interpret gene expression profiling data in Tier 0 screening. The work presented in this paper will contribute to the FY18 RAP product “Genomic biomarkers for molecular initiating events and early key events in AOPs.” in the CSS projects 16.01.01 (Task Title: High throughput global transcriptomic analysis) and 17.01.01 (Task Title: Adverse Outcome Pathway Discovery and Development).

Description:

Gene expression signatures of DNA damage-inducing (DDI) agents can be used as mechanistic markers of genotoxicity for in vitro hazard identification. Indeed, the TGx-DDI transcriptomic biomarker can accurately distinguish DDI from non-DDI exposures based on changes in the expression of 64 biomarker genes. The 64 genes were previously derived from whole transcriptome DNA microarray profiles of 28 reference agents (13 DDI and 15 non-DDI) after 4-hour treatments of TK6 human lymphoblastoid cells. To broaden the applicability of TGx-DDI, we tested the biomarker using quantitative RT-PCR (qPCR), which is accessible to most molecular biology laboratories. First, we selectively profiled the expression of the 64 biomarker genes using TaqMan qPCR assays in 96-well arrays after exposing TK6 cells to the 28 reference agents for 4 hours. To evaluate the classification capability of the qPCR profiles, we used the reference qPCR signature to classify 24 external validation chemicals using two different methods – a combination of three statistical analyses and an alternative, the Running Fisher test. The qPCR results for the reference set were comparable to the original microarray biomarker; 26 of the 28 reference agents (93%) were accurately classified. Moreover, the two classification approaches supported the conservation of TGx-DDI classification capability using qPCR; the combination of the two approaches accurately classified 20 of the 24 external validation chemicals, demonstrating 88% sensitivity, 81% specificity, and 84% balanced accuracy. This study demonstrates that qPCR can be used when applying the TGx-DDI biomarker and will improve the accessibility of TGx-DDI for genotoxicity screening.

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