Penny J. Beuning, Geoffrey Davies
Date of Award
Master of Science
Department or Academic Unit
College of Science. Department of Chemistry and Chemical Biology.
chemistry, 4-ABP, DNA adduct, liquid chromatography, mass spectrometry, urothelial
Liquid chromatography, Carcinogens
4-aminobiphenyl (4-ABP) is a known tobacco smoke carcinogen that has been shown to form DNA adducts in the bladder epithelium, which may lead to cancer development. Our laboratory has developed and implemented a highly sensitive and quantitative nano-flow liquid chromatography electrospray ionization mass spectrometry (nano-LC/ESI-MS/MS) method for the analysis of 4-ABP DNA adducts in vitro from liver and bladder tissue. In collaboration with Roswell Park Cancer Institute we sought to apply our method for the analysis of 4-ABP DNA adducts from bladder epithelial cells excreted in urine during the course of a smoking cessation study. However, significant challenges were presented when applying this method to exfoliated human urinary epithelial cells. One such challenge is that DNA yields from urine samples are often low and extremely variable, due to factors such as gender of the donor, sample storage conditions, extent of bacterial contamination, and release of nucleases from hydrolyzed cells. The major impediment in this study was the sediment that forms in the samples as a result of storage at -80 °C. Most of this sediment is due to the formation of urinary crystals, such as calcium oxalate, magnesium ammonium phosphate, and uric acid. Treatment of the urine samples with a 5 mM solution of EDTA (ethylenediaminetetraacetic acid) has proven to significantly reduce these crystalline precipitates, and has allowed for successful isolation of urinary epithelial cell DNA from frozen urine samples. Our current nano-LC/ESI-MS/MS method is ideal for urinary epithelial cell DNA isolation, in which DNA yields are often limited, for it requires only 2 μg of DNA, which is digested into mononucleosides for analysis. 4-ABP adducts are detectable at attomole levels (LOD 140 amol, LOQ 560 amol). Preliminary results from a small blind subset of patient samples show no detection of 4-ABP DNA adducts. Analysis of remaining patient samples is in progress.
Samantha J. Sokup
Sokup, Samantha J., "Isolation of urinary epithelial cell DNA for the analysis of 4-Aminobiphenyl DNA adducts by nano-LC/ESI-MS/MS" (2011). Chemistry Master's Theses. Paper 21. http://hdl.handle.net/2047/d20002309
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