Assessment of the performance of the TGx-DDI biomarker to detect DNA damage-inducing agents using quantitative RT-PCR in TK6 cells
- DOI
- Language of the publication
- English
- Date
- 2018-11-29
- Type
- Article
- Author(s)
- Cho, Eunnara
- Buick, Julie K.
- Williams, Andrew
- Chen, Renxiang
- Li, Heng-Hong
- Corton, J. Christopher
- Fornace Jr., Albert J.
- Aubrecht, Jiri
- Yauk, Carole L.
- Publisher
- Wiley
Abstract
Gene expression biomarkers are now available for application in the identification of genotoxic hazards. The TGx-DDI transcriptomic biomarker can accurately distinguish DNA damage-inducing (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 (14 DDI and 14 non-DDI) after 4 h 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 h. 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; 27 of the 28 reference agents (96%) 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 21 of the 24 external validation chemicals, demonstrating 100% sensitivity, 81% specificity, and 91% 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.
Plain language summary
Health Canada is responsible for assessing and managing health risks posed by chemicals to the Canadian population. Chemicals that are genotoxic can damage DNA and cause adverse effects such as cancer and inherited genetic diseases. Thus, genotoxicity testing is a critical component of human health risk assessment. Health Canada previously collaborated with members of a Health and Environmental Sciences Institute (HESI) consortium to develop a test method in human cells in culture that measures changes in gene expression as an indicator (biomarker) of a chemical’s potential to cause DNA damage (Li et al and Buick et al, 2015; Yauk et al, 2016; Buick et al and Li et al, 2017). Prior to the present study, this biomarker could only be analyzed using DNA microarrays, a platform that measures the expression of thousands of genes simultaneously. The equipment is costly and not available in most molecular biology laboratories. Thus, the research group tested the biomarker using a more widely accessible gene expression platform to determine whether the ability to predict genotoxicity using the biomarker is reproducible on different technologies. Real Time Quantitative Polymerase Chain Reaction (qPCR) is a platform that is commonly used to measure the expression of user-selected genes and is available in most molecular biology laboratories. The scientists found that the changes in gene expression measured using qPCR also accurately predict whether a chemical is genotoxic or non-genotoxic. These data indicate that the qPCR platform can be used to measure the biomarker for genotoxicity testing. The method will improve the accessibility of the biomarker to laboratories around the world to identify potentially genotoxic agents in human cells in culture for chemical assessments. Health Canada is currently using the biomarker to assess the potential genotoxicity of chemicals with little conventional data under the Chemicals Management Plan.
Subject
- Health,
- Health and safety