TY - JOUR
T1 - 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
T2 - Validation by Comparison with 32P-Postlabeling
AU - Friesen, Marlin D.
AU - Kaderlik, Keith
AU - Lin, Dongxin
AU - Garren, Liliane
AU - Bartsch, Helmut
AU - Lang, Nicholas P.
AU - Kadlubar, Fred F.
PY - 1994/11/1
Y1 - 1994/11/1
N2 - 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//μg of DNA (1 PhIP adduct/108 normal nucleotides) for a 100 μg sample of DNA. The method is reproducible for sample sizes ranging up to at least 1000 μg 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.
AB - 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//μg of DNA (1 PhIP adduct/108 normal nucleotides) for a 100 μg sample of DNA. The method is reproducible for sample sizes ranging up to at least 1000 μg 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.
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U2 - 10.1021/tx00042a004
DO - 10.1021/tx00042a004
M3 - Article
C2 - 7696526
AN - SCOPUS:0028110295
SN - 0893-228X
VL - 7
SP - 733
EP - 739
JO - Chemical research in toxicology
JF - Chemical research in toxicology
IS - 6
ER -