Development and Validation of an Assay for the Characterization of the DNA Damage Response to Damage Caused by Superfund Chemicals

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  • March 20, 2019
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  • Ridpath, John Robert
    • Affiliation: Gillings School of Global Public Health, Department of Environmental Sciences and Engineering
Abstract
  • Determination of intracellular mode of action regarding how chemicals induce toxicity plays an important role for establishing risk assessment. Although several methods exist to ascertain how toxic substances act intracellularly, the amount of information obtained from these assays usually fails to fully clarify the route to toxicity. This projects goal was to establish and validate a new assay to provide more information with regard to the DNA damage response of cells to toxic agents. This assay uses a reverse-genetic approach similar to that used with yeast mutants. We used a cell line known as DT40 from a vertebrate source whose mutants have been derived from a single parent making the entire cell line isogenic. The DT40 cell line includes more than 50 mutants for proteins involved in DNA repair, DNA damage bypass and cell cycle checkpoints. This line is used to study several cellular metabolic processes, but we believe we are the first to exploit this exceptional tool to aid in the determination of toxicological end-points. First we developed the assay into a relatively rapid and cost-efficient method that may be used to screen chemicals, but with accuracy, reliability and sensitivity. For this, we measured the sensitivity of DT40 mutants to chemicals using XTT, a dye reduced only in respirating cells, providing a direct measurement of cell proliferation after treatment. We found this method both reliable and sensitive as evidenced by validations with cisplatin, methyl methanesulfonate and acrolein. We next applied our method to study the DNA damage response to formaldehyde, an agent shown to induce DNA-protein crosslinks. Little was known as to how cells dealt with these crosslinks. Our assay was able to demonstrate the FANC/BRCA damage response pathway is important for dealing with formaldehyde-induced DNA damage. Finally, we turned our attention to help clarify the type of DNA damage induced by hexavalent chromium whose inorganic salts are known cancer inducers but for which the mode of action is controversial. We were able to provide valuable information concerning the cellular response to chromate by showing that chromate probably induces mutagenicity by interfering with replication but not through oxidative stress.
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  • In Copyright
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Environmental Sciences and Engineering."
Advisor
  • Nakamura, Jun
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  • Chapel Hill, NC
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  • Open access
Date uploaded
  • March 18, 2013

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