Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform

Abstract

DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 (-/-) /RAD54 (-/-) and REV3 (-/-) ) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity-2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether-were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA.

Tox21Challenge to Build Predictive Models of Nuclear Receptor and Stress Response Pathways as Mediated by Exposure to Environmental Chemicals and Drugs

“…The Tox21 consortium leverages its partners' resources and expertise to predict more effectively how a collection of ∼10,000 compounds (referred to as Tox21 10K library) composed of environmental chemicals and approved drugs will affect human health and the environment. The Tox21 10K library has been tested in a quantitative high-throughput screening (qHTS) format against a panel of nuclear receptor (NR) (Huang et al, 2011(Huang et al, , 2014Hsu et al, 2014;Chen et al, 2015) and stress response (SR) pathway assays (Attene-Ramos et al, 2015;Nishihara et al, 2016), producing over 50 million data points to date (PubChem, 2013b). These data can serve as a knowledge-base to correlate chemical structures to their biological activities to develop QSAR models.…”

The Next Generation of Risk Assessment Multi-Year Study—Highlights of Findings, Applications to Risk Assessment, and Future Directions

“…QSAR, in vitro, and in silico methods are proving useful for screening and ranking large numbers of chemicals for further assessment and urgent-response situations where traditional data are lacking (Adeleye et al 2015; ECHA 2016a, 2016b; Eduati et al 2015; Judson et al 2015; Knudsen et al 2015; NAFTA Technical Working Group on Pesticides 2012; OECD 2016a, 2016b; Ryan et al 2016; Nishihara et al 2016). Current estimates of human disease risks based exclusively on QSAR and in vitro HT screening generally are too uncertain for many applications (Casey et al 2015; Cox et al 2014; EC and JRC 2015; U.S.…”

Advances in high-throughput screening technology for toxicology

“…The utility of this cell line for HTS in a 1,536-well format is still under investigation. Cell viability assays have also been used to evaluate the effect of DNA-damage inducing compounds on a group of isogenic DNA repair-deficient chicken DT40 cell lines (Yamamoto et al, 2011;Nishihara et al, 2016). Differential cytotoxicity patterns were observed in these cell lines depending on the type of DNA-damage inducing compounds, making them good research tools for identifying genotoxic compounds and associated mechanisms.…”