title: Urinary N2-(2'-deoxyguanosin-8-yl)PhIP as a biomarker for PhIP exposure.
abstract: 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 N(2)-(2'-deoxyguanosin-8-yl)PhIP is a potential biomarker of DNA adduction and repair after exposure to PhIP. An assay for N(2)-(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 N(2)-(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, N(2)-(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 micro g) 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.
authors.affiliation: Section of Experimental Medicine and Toxicolog, Division of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK.
authors.forename: Min
authors.initials: M
authors.lastname: Fang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Edwards
authors.forename: Michael
authors.initials: M
authors.lastname: Bartlet-Jones
authors.forename: Graham W
authors.initials: GW
authors.lastname: Taylor
authors.forename: Stephen
authors.initials: S
authors.lastname: Murray
authors.forename: Alan R
authors.initials: AR
authors.lastname: Boobis
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000652:urine*; D002273:Carcinogens / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003849:Deoxyguanosine / Q000652:urine*; D007093:Imidazoles / Q000652:urine*; D008297:Male; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D002273:Carcinogens; D007093:Imidazoles; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D003849:Deoxyguanosine