Mechanisms of cytotoxicity for bifunctional epoxide alkylating agents

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The discovery that bifunctional alkylating agents have antitumor activity led to the development of cancer chemotherapy over 50 years ago, yet these compounds also lead to an increased risk of secondary cancers. Bifunctional alkylating agents form a variety of DNA adducts, including monoadducts, intrastrand cross‐links, and interstrand cross‐links. Interstrand cross‐links are believed to be the most lethal, impeding both replication and expression of the genetic material. We are characterizing the mechanisms by which diepoxybutane (DEB) and epichlorohydrin (ECH) exert their cytotoxic effects in cultured cells. Our first goal is to determine the relationship between interstrand cross‐linking and cytotoxicity in human acute myeloid leukemia (HL60) cells. HL60 cells are treated with varying concentrations of the compound of interest and assayed to determine the fraction of cells that remain viable, allowing determination of LD50 values. Cross‐linking ability is assessed using an ethidium bromide assay to determine the amount of duplex DNA following alkaline denaturation. Only cross‐linked DNA reanneals rapidly and interacts with the dye, which is highly fluorescent when bound to duplex DNA. Preliminary results suggest a correlation between cross‐linking and LD50 values for these compounds, with DEB a more efficient cross‐linker than ECH.Our second goal is to identify the structures of ECH‐cross‐linked lesions via ESI‐TOF‐MS, which may provide information about the mechanism of this agent. This research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.