Carcinogenesis Advance Access published online on January 23, 2004
Carcinogenesis, doi:10.1093/carcin/bgh084
© 2004 by Oxford University Press
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CARCINOGENESIS
1 Section of Experimental Medicine and Toxicology, Division of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK
* Corresponding author. E-mail: s.murray{at}imperial.ac.uk.
Received 7 October 2003
; revised 9 January 2004
; accepted 10 January 2004
The food-derived, heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is genotoxic and is carcinogenic in experimental animals. Studies on the role of PhIP in human diet-related cancer would be aided considerably by the availability of a readily applicable biomarker of the internal dose of the ultimate genotoxic species. PhIP has been shown to adduct primarily at C-8 of deoxyguanosine in DNA and so the DNA repair product N2-(2'-deoxyguanosin-8-yl)PhIP is a potential biomarker of DNA adduction and repair after exposure to PhIP. An assay for N2-(2'-deoxyguanosin-8-yl)PhIP in urine has been developed based on liquid chromatography mass spectrometry, using a deuterated analogue of the nucleoside as an internal standard and an antibody mediated extraction procedure. Polyclonal antibodies were raised against the PhIP-nucleotide, PhIP-nucleoside and PhIP-guanine base adducts conjugated to keyhole limpet haemocyanin. Following their evaluation, the immobilised PhIP nucleotide antibody was used for the extraction of N2-(2'-deoxyguanosin-8-yl)PhIP from urine. The limit of detection of the assay was 125 pg and the limit of quantification 200 pg for a 50 mL human urine sample. Following oral administration of PhIP (20 mg/kg body wt / day) to rats for six days, N2-(2'-deoxyguanosin-8-yl)PhIP was readily detected in the urine, reaching steady state over 3 days. This is the first direct demonstration of the urinary elimination of this adduct following exposure to parent amine. The half-life of the adduct with DNA was estimated to be approximately 20 h. The total amount of PhIP recovered in the urine as adduct was <0.5 x 10-3% of the dose administered. Levels of the PhIP adduct in urine collections from human subjects ingesting the amine (4.9 µg) in cooked meat were below the limits of detection, indicating that humans are exposed to a bioactive dose of <3 x 10-4 of that associated with a non-carcinogenic level in rats.
Urinary N2-(2'-deoxyguanosin-8-yl)PhIP as a biomarker for PhIP exposure
2 Cancer Research UK, Lincoln's Inn Fields, London WC2A 3PX, UK
3 Section on Proteomics, Division of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK
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