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

Marlin D. Friesen, Keith Kaderlik, Dongxin Lin, Liliane Garren, Helmut Bartsch, Nicholas P. Lang, Fred F. Kadlubar

Research output: Contribution to journalArticle

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/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.

Original languageEnglish (US)
Pages (from-to)733-739
Number of pages7
JournalChemical Research in Toxicology
Volume7
Issue number6
StatePublished - 1994
Externally publishedYes

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DNA Adducts
Gas chromatography
Gas Chromatography
Mass spectrometry
Rats
Hydrolysis
Mass Spectrometry
Electrons
Tissue
DNA
Pancreas
Colon
Urinary Bladder
Deuterium
Sample Size
Solvent extraction
2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine
Mucous Membrane
Organic solvents
Epithelium

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

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. / Friesen, Marlin D.; Kaderlik, Keith; Lin, Dongxin; Garren, Liliane; Bartsch, Helmut; Lang, Nicholas P.; Kadlubar, Fred F.

In: Chemical Research in Toxicology, Vol. 7, No. 6, 1994, p. 733-739.

Research output: Contribution to journalArticle

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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/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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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

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AU - Friesen, Marlin D.

AU - Kaderlik, Keith

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AU - Lang, Nicholas P.

AU - Kadlubar, Fred F.

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