Validation in rats of two biomarkers of exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): PhIP-DNA adducts and urinary PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts in white blood cells and tissues and unmetabolized PhIP in urine were validated as biomarkers of exposure in male Fischer-344 rats treated with daily PhIP doses ranging from 1 to 0.0001 mg/kg. At the end of the 23 day treatment period all rats were killed and their blood and 10 tissues were collected for isolation of DNA and analysis of PhIP-DNA adducts by 32P-postlabeling and alkaline hydrolysis with GC/MS. PhIP-DNA adducts could be detected only in animals receiving 1 or 0.1 mg/kg/day, with highest adduct levels in the pancreas, heart and kidneys. There was a good correlation (r = 0.77, P < 0.005) between the two methods of analysis, with average adduct levels determined by 32P-postlabeling approximately 1.4 times higher than those determined by alkaline hydrolysis with GC/MS. PhIP-DNA adducts accumulated in most tissues, especially in the liver, kidneys, heart and pancreas, with lower levels in the white blood cells, small intestine, stomach, colon and cecum. Using GC/MS levels of unmetabolized PhIP were measurable in four weekly 24 h urine samples even at 0.0001 mg/kg/day, a dose comparable with reported human dietary exposure. A linear dose-response was obtained for excretion of unmetabolized PhIP across the range of doses, with approximately 1.8% of the dose excreted daily, largely independent of the number of doses. No PhIP was detected in the urine of untreated rats. If it can be shown that a constant percentage of PhIP is excreted unchanged in human urine, irrespective of dose, as has been found with the rat, measurement of urinary PhIP could be used as an accurate measure of dietary exposure to this amine in man.