Tenzin PassangFollow

Date of Award

2019

Document Type

Honors Thesis (Colby Access Only)

Department

Colby College. Chemistry Dept.

Advisor(s)

Julie T. Millard

Abstract

Over 1.5 million tons of epichlorohydrin (ECH) are used annually in the manufacturing of epoxy resins and polymers. A probable human carcinogen, ECH shares structural homology to nitrogen mustards, the oldest class of anti-tumor drugs. Such bifunctional alkylating agents can react with DNA and proteins, leading to genotoxic effects. Previous work in our lab has shown that treating human leukemia (HL-60) cells with toxic doses of ECH leads to the formation of cross-links and triggers apoptosis. In these studies, cross-linking was quantified via single cell gel electrophoresis (the comet assay), which does not distinguish between DNA-DNA cross-links and DNA-protein cross-links (DPCs). The goal of this work is to further characterize ECH-induced DPCs. Nuclear proteins that bind to biotinylated DNA after treating with ECH under different conditions were captured via streptavidin beads. Cross-linked proteins were then characterized through gel electrophoresis. A proteinase K-modified alkaline comet assay, specific for DPCs was further used to study the phenomenon ex-vivo in HL-60 cells. The tail length and the olive tail moment of the comets visualized via fluorescent microscopy were determined. Our data strongly indicates that ECH does induce DPC formation both in-vitro and ex-vivo, with greater cross-linking efficiency at higher concentrations of ECH. Our findings could have significance for understanding the occupational risks of ECH exposure among industrial workers.

Keywords

Epichlorohydrin, Crosslinks, DNA-Protein Crosslinks, Affinity Capture, Comet Assay, Human Leukemia Cells

Download

Share

COinS