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Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high
Kana Nishihara1, Ruili Huang2, Jinghua Zhao2
1Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo, Kyoto 606-8501, Japan, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC: 3375 Bethesda, MD 20892, USA and.
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.
Insights
This study used DNA repair-deficient DT40 cells to screen chemicals for genotoxicity. Three new genotoxic compounds were identified, highlighting the approach
Area of Science:
- Molecular Biology
- Toxicology
- Genetics
Background:
- DNA repair pathways are crucial for maintaining genomic stability and preventing cellular dysfunction.
- DNA damage can result from endogenous processes and exposure to environmental chemicals (xenobiotics).
- Genomic instability, stemming from DNA damage, is linked to disease development, including cancer.
Purpose of the Study:
- To identify genotoxic compounds using a high-throughput screening platform with isogenic DNA repair-deficient DT40 cell lines.
- To characterize the genotoxic potential of compounds within the Tox21 10K compound library.
- To validate the utility of DT40 cell lines for detecting and prioritizing DNA-damaging agents.
Main Methods:
- Screened the Tox21 10K compound library against wild-type and DNA repair-deficient (KU70(-/-)/RAD54(-/-) and REV3(-/-)) DT40 cell lines.
- Utilized a cell viability assay measuring intracellular adenosine triphosphate (ATP) levels for primary screening.
- Further evaluated a subset of compounds for their ability to induce micronuclei and phosphorylated H2AX (γH2AX).
Main Results:
- The screening identified known genotoxicants and several compounds with previously unevaluated genotoxicity.
- Three compounds—2-oxiranemethanamine, AD-67, and tetraphenylolethane glycidyl ether—were confirmed as genotoxic.
- The study successfully identified and prioritized compounds with potential DNA-damaging properties.
Conclusions:
- Isogenic DNA repair-deficient DT40 cell lines are effective tools for high-throughput screening of genotoxic compounds.
- This approach aids in identifying novel genotoxic agents and understanding DNA damage mechanisms.
- The findings support the use of this methodology for prioritizing chemical safety assessments.
