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|Title:||Development and application of a fluorescent postlabelling assay for the detection of N7-alkylguanines|
|Presented at:||University of Leicester|
|Abstract:||DNA reacts with many alkylating carcinogens to give N-alkylated bases as major products, which can be used as biomarkers of human exposure to carcinogens. However, quantitation of this DNA damage is found to be difficult, largely due to the instability of the modified bases and hazardous nature of some detection methods (32P-postalabelling). This instability is utilised in an approach that uses a non-radioactive postlabelling technique to detect and quantify N7-alkylguanine bases. The technique is based on the reaction of phenylmalondialdehyde with N7-alkylguanines to give fluorescent pyrimidopurines, i.e. 7-phenyl-10-oxo-1-alkyl,9,10-dihydropyrimido-[1,2,a]-purines. N2-Carboxymethyl-N7-ethygluanine was coupled with methylated bovine serum albumin, and used to immunise mice, to successfully produce monoclonal antibodies specific for N7-ethylguanine. The monoclonal antibodies were subsequently used to manufacture immunoaffinity columns, which were incorporated into the fluorescent postlabelling assay.;The sensitivity and application of the approach is exemplified by the quantitation of N7-methylguanine and N7-ethylguanine in DNA. Calf thymus DNA treated in vitro with synthesised 2-diazopropanoic acid (a possible precursor to an ethylating agent, formed from alanine in tobacco after undergoing nitrosatin and decarboxylation on burning), dimethylsulphate, diethylsulphate and exposed to tobacco smoke, was analysed by HPLC fluorescence. The assay is shown to be very sensitive with a limit of detection being approximately 0.8 pmol of adduct for a given sample of DNA. This has enabled the detection of one N7-methylguanine addcut/106 nucleotides from 1 mg of DNA. Unfortunately, the assay was unsuccessful in detecting significant levels of N7-ethylguanine from DNA exposed to tobacco smoke and 2-diazopropanoic acid.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, MRC Toxicology Unit|
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