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.
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.
Friesen
M D
MD
International Agency for Research on Cancer, Lyon, France.
Kaderlik
K
K
Lin
D
D
Garren
L
L
Bartsch
H
H
Lang
N P
NP
Kadlubar
F F
FF
D000818:Animals; D002849:Chromatography, Gas; D003106:Colon / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D007093:Imidazoles / Q000032:analysis*; D008099:Liver / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D009153:Mutagens / Q000032:analysis*; D010179:Pancreas / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D051381:Rats; D011916:Rats, Inbred F344
D018736:DNA Adducts; D007093:Imidazoles; D009153:Mutagens; D010761:Phosphorus Radioisotopes; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine