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44839-B4
Epihalohydrin Cross-Linking of DNA

Julie Tamsen Millard, Colby College

The goal of this project is to characterize DNA damage by the epihalohydrins. In the past year, we have made progress in the following areas:

1. We have continued to assess epibromohydrin (EBH) interstrand cross-links for bending.
2. We are exploring the structure-function relationship of cross-linking by comparing epichlorohydrin (ECH) cross-linking to that of (1-chloroethenyl) oxirane.
3. We are characterizing the mechanism of cell death induced by ECH in 6C2 chicken erythroid cells.

Goal 1 Accomplishments

We are using native gel electrophoresis to examine potential DNA bending by cross-linkers such as the epihalohydrins. Cross-linked duplexes and control native duplexes are ligated to form a population of multimers that appear as a ladder of bands on a native gel. If the parent cross-linked duplex is bent, then this ladder of bands will appear retarded relative to a control lane of size standards of equal lengths. The magnitude of the anomalous mobility can be defined in terms of the ratio of the apparent length to the actual length in base pairs and can be used to calculate the angle of absolute curvature. We are comparing EBH cross-links to diepoxybutane (DEB) cross-links to assess the effect of chain length on bending. In a panel of duplexes 30-33 base pairs in length, bending is maximal in the 30- and 31-mers. DEB induces a bend of circa 19? per helical turn, whereas epibromohydrin induces a bend of circa 17?. We are currently assessing the direction of bending in A-tract-containing duplexes by determining whether cross-linking is additive or subtractive to the bending induced by the A tract.

Goal 2 Accomplishments

We are investigating the structure-function relationship for cross-linking. ?-Chloroprene is an important industrial chemical used in manufacture of polychloroprene, a solvent-resistant elastomer. Correlation between occupational exposure to chloroprene and lung cancer has been reported. The active compound is believed to be (1-chloroethenyl) oxirane, a bifunctional metabolite structurally related to ECH. We have synthesized this compound and found evidence for DNA interstrand cross-linking. The optimal pH for cross-linking varies, with more cross-linking at pH 5.0 than at pH 7.0. This pH dependence for cross-linking, which is similar to that of ECH, suggests initial reaction at the epoxide. However, (1-chloroethenyl) oxirane appears to be a more efficient cross-linker than ECH.

Goal 3 Accomplishments

We are exploring the role of apoptosis (programmed cell death) versus necrosis (direct damage) in the cytotoxicity of ECH and related compounds. Bifunctional alkylating agents react with many biological molecules in vivo. Reaction with DNA halts the cell cycle for DNA repair, which can trigger apoptosis. However, many cancer cells have defects in apoptotic control, in which case necrosis can be the mechanism of cytotoxicity.

We are establishing protocols to assay for apoptosis within a 6C2 chicken erythroid cell line. Most successful has been monitoring for activity of the apoptosis-signaling proteins caspase-3/7 via cleavage of a pro-luminescent substrate. Although the positive control (1 mM camptothecin, 4 h treatment) displayed high luminescence, 6C2 cells treated with 250 mM DEB or ECH for up to 24 h did not. Cells treated with 50 ?M DEB did show luminescence after 21 h, but those treated with 50 ?M ECH did not. These results suggest different modes of cell death for DEB and ECH: apoptosis and necrosis, respectively. Future work will include a systematic evaluation of the effects of concentration and incubation time.

Impact on Students

The Department of Chemistry at Colby College is firmly committed to providing significant independent research opportunities for our students, and we therefore require independent research of all our majors. Independent research reinforces classroom instruction and requires students to develop time-management skills, perseverance, and the ability to work in a group setting. Several students have been involved in this work in the Millard laboratory over the past year. Megan Watts '08 is entering medical school at the University of Vermont, Erin McGowan '08 is entering veterinary school at the University of Pennsylvania, Mathew Stein '08 is working as a research assistant at Boston Children's Hospital, and Brian Wadugu '09 and Sharonda Bradley '10 are entering their senior and junior year at Colby College, respectively. Ms. Watts, Mr. Wadugu, and Ms. Bradley all presented their work both at the Northeast Regional Meeting of the ACS and at the Colby Undergraduate Summer Research Retreat this summer.

Millard PRF Progress Report, September 2008 2

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