Journal Article

Urinary <i>N</i><sup>2</sup>-(2′-deoxyguanosin-8-yl)PhIP as a biomarker for PhIP exposure

Min Fang, Robert J. Edwards, Michael Bartlet-Jones, Graham W. Taylor, Stephen Murray and Alan R. Boobis

in Carcinogenesis

Volume 25, issue 6, pages 1053-1062
Published in print June 2004 | ISSN: 0143-3334
Published online June 2004 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgh084
Urinary N2-(2′-deoxyguanosin-8-yl)PhIP as a biomarker for PhIP exposure

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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 immobilized 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 6 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 ∼20 h. The total amount of PhIP recovered in the urine as adduct was <0.5 × 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 × 10−4 of that associated with a non-carcinogenic level in rats.

Keywords: ELISA, enzyme-linked immunosorbent assay; HAA, heterocyclic aromatic amine; MRM, multiple reaction monitoring; OVA, ovalbumin; PBS, phosphate-buffered saline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Journal Article.  7986 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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