Analysis of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine in rat and human tissues by alkaline hydrolysis and gas chromatography/electron capture mass spectrometry: validation by comparison with 32P-postlabeling.

Abstract

A sensitive and specific method has been developed to measure levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) adducted to DNA in tissues. The method is based on alkaline hydrolysis of PhIP from DNA, followed by organic solvent extraction, derivatization to form the electron-capturing bis(pentafluorobenzyl) derivative, and analysis by gas chromatography/electron capture mass spectrometry (GC/MS) using a deuterium-labeled internal standard. The method can detect PhIP-DNA adducts at levels down to 0.03 fmol of PhIP/micrograms of DNA (1 PhIP adduct/10(8) normal nucleotides) for a 100 micrograms sample of DNA. The method is reproducible for sample sizes ranging up to at least 1000 micrograms of DNA. A series of 20 DNA samples from 5 tissues of rats treated with a single oral dose of PhIP were analyzed both by alkaline hydrolysis-GC/MS and by 32P-postlabeling. Results from the two methods were highly correlated (r2 = 0.83), with adduct levels determined by alkaline hydrolysis-GC/MS averaging about 60% of the levels determined by 32P-postlabeling. A pilot survey of 24 individual human tissue DNA samples, including pancreas (n = 12), colon mucosa (n = 6), and urinary bladder epithelium (n = 6), was carried out by alkaline hydrolysis-GC/MS and 32P-postlabeling. Both methods provided evidence for PhIP-DNA adducts in two of the colon samples, but not in the samples from human pancreas or urinary bladder.