Abstract
The increasing number of compounds under development and chemicals in commerce that require safety assessments pose a serious challenge for regulatory agencies worldwide. In vitro screening using toxicogenomic biomarkers has been proposed as a first-tier screen in chemical assessment and has been endorsed internationally. We previously developed, evaluated, and validated an in vitro transcriptomic biomarker responsive to DNA damage-inducing (DDI) agents, namely TGx-DDI, for genotoxicity testing in human cells and demonstrated the feasibility of using TGx-DDI in a medium-throughput, cell-based genotoxicity testing system by implementing this biomarker with the Nanostring nCounter system. In this current study, we took advantage of Nanostring nCounter Plexset technology to develop a highly automated, multiplexed, and high-throughput genotoxicity testing assay, designated the TGx-DDI Plexset assay, which can increase the screening efficiency eight-fold compared to standard nCounter technology while decreasing the hands-on time. We demonstrate the high-throughput capability of this assay by eliminating concentration determination and RNA extraction steps without compromising the specificity and sensitivity of TGx-DDI. Thus, we propose that this simple, highly automated, multiplexed high-throughput pipeline can be widely used in chemical screening and assessment.
Highlights
Chemicals that are capable of inducing genetic changes, such as mutations and chromosome damage, are defined as genotoxic (GTX) compounds
We focused on developing a new highly-automated and high-throughput genotoxicity testing assay by integrating TGx-DDI with Nanostring nCounter PlexsetTM technology, designated as TGx-DDI Plexset assay, which can increase the screening efficiency eight-fold compared to standard nCounter Codeset technology, which is referred as the Codeset method hereafter, while decreasing the hands-on time
Each set is comprised of the identical genes, i.e., the 64 genes in TGx-DDI and eight housekeeping genes that were selected based on stability and expression level (Li et al, 2017)
Summary
Chemicals that are capable of inducing genetic changes, such as mutations and chromosome damage, are defined as genotoxic (GTX) compounds. Genotoxicity testing is an essential part of the safety assessment for predicting the carcinogenic potential of all drugs and chemicals. There is not sufficient toxicity information available for risk assessment (Judson et al, 2010). The inventory of the TSCA (Toxic Substances Control Act) has continued to grow since the initial reporting period and lists more than 86,000 chemicals. The increasing number of compounds under development and chemicals in commerce that require safety assessments pose a serious challenge for regulatory agencies. There are compelling needs for developing high-throughput screening (HTS) assays to assist regulatory agencies in making informed decisions and policies regarding chemical safety
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