Mapping of Diepoxybutane Damage in the Cytochrome b Domain of Mitochondrial DNA and the β-globin Domain of Nuclear Chicken DNA Using Quantitative PCR

Diepoxybutane (DEB), a known industrial carcinogen, reacts with DNA primarily at the N7 position of deoxyguanosine residues and creates interstrand cross-links at the sequence 5'-GNC. Since N7-N7 cross-links cause DNA to fragment upon heating, quantative polymerase chain reaction (QPCR) is being used in this experiment to measure the amount of DEB damage (lesion frequency) with three different targets-mitochondrial (unpackaged), open chromatin region, and closed chromatin region. Initial measurements of DEB damage within these three targets were not consistent because the template DNA was not the limiting reagent in the PCR. Follow-up PCR trials using a limiting amount of DNA are still in progress although initial experimentation looks promising. Sequencing of these three targets to confirm the primer targets has only been successfully performed for the closed chromatin target and does not match the sequence from NIH used to design that primer pair. Further sequencing trials need to be conducted on all three targets to assure that a mitochondrial, open chromatin, and closed chromatin region are actually being amplified in this experimental series.

Cytotoxicity of Diepoxybutane and Epichlorohydrin in Relation to Stages of the Cell Cycle

Work conducted in the Millard Biochemistry Research Laboratory examines the dual nature of molecules as carcinogens and anti-tumor agents through the molecular mechanisms of duplex DNA damage by bifunctional alkylating agents. Diepoxybutane (DEB) and epichlorohydrin (ECH) are polar molecules that form covalent DNA interstrand lesions by cross-linking the N7 position of deoxyguanosine residues. A recent experiment indicated that ECH preferentially targets nuclear DNA over mitochondrial DNA, whereas DEB shows similar rates of lesion formation for both loci. It was concluded that preferential targeting of nuclear DNA results from relatively poor uptake of ECH across the mitochondrial membrane. The objective of my honors research was to determine if the cytotoxicities of DEB and ECH vary according to the presence of the nuclear envelope in 6C2 chicken erythro-progenitor cells. The cytotoxicity of DEB and ECH was compared between cells randomly distributed throughout the cell cycle (Go/G, and S » G2/M) and cells enriched in G2/M stages. Results indicated that ECH is more cytotoxic than DEB in both unsynchronized control 6C2 cells and synchronized 6C2 cells enriched in G2/M stages of the cell cycle. Treatment with either bifunctional alkylating agent induced greater cytotoxicity in 6C2 cells enriched in G2/M stages than in unsynchronized control 6C2 cells, suggesting that the presence of the nuclear envelope-or any plasma membrane-may inhibit the reactivity of DEB and ECH.

Changes in Apoptotic Gene Expression Induced by DNA Cross-Linkers

The Millard Research Laboratory is interested in the cytotoxic mechanisms of the bifunctional alkylators diepoxybutane (DEB), epichlorohydrin (ECH), and (1-chloroethenyl) oxirane (COX). Studies performed in the laboratory examine the dual nature of these DNA cross-linking compounds that can act as carcinogens or anti-cancer agents. The mechanisms through which these compounds induce cell death are explored in this study. Cells either undergo cell death due to necrosis or apoptosis. HL-60 cells were treated with varying concentrations of DEB, ECH, or COX. A caspase 3/7 assay was used to test for induction of apoptosis in the treated cells at varying incubation times. It was concluded that DEB induces apoptosis in HL-60 cells treated with 100 μM for 24 hours. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was then used to explore the changes in gene expression of various genes involved in apoptosis signaling. The results were inconclusive as to specific genes involved in DEB induced apoptosis, but the data does suggest that apoptosis is induced by a mitochondrial-mediated apoptosis signaling pathway.