METABOLIC-ACTIVATION PATHWAY FOR THE FORMATION OF DNA-ADDUCTS OF THE CARCINOGEN 2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE (PHIP) IN RAT EXTRAHEPATIC TISSUES

The food-borne mutagen 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) induces tumors in colon of male rats and has been implicated in the etiology of human cancers, particularly colorectal cancer. This s tudy was conducted to examine: (1) the biliary and/or circulatory tran sport of N-hydroxy-PhIP and its N-glucuronides, N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximate and ultimate carcinogenic metabolites of PhIP; (3) the potential role of glutathione in modulat ing PhIP-DNA adduct formation. PhIP-DNA adducts, measured by the P-32- postlabeling method, were highest in the pancreas (361 adducts/10(8) n ucleotides or 100%), followed by colon (56%), lung (28%), heart (27%) and liver (2%), at 24 h after a single oral dose of PhIP (220 mu mol/k g) to male rats. In each tissue examined, we observed two major adduct s, each of which accounted for 35-45% of the total, and one minor addu ct, which represented about 10-20% of the total. One of the major addu cts was identified as osin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5- b] pyridine by chromatographic comparisons with an authentic standard. The major urinary metabolites of PhIP in these rats were 4'-hydroxy-P hIP and its glucuronide and sulfate conjugates, followed by N-hydroxy- PhIP N3-glucuronide, N-hydroxy-PhIP N-2-glucuronide and unchanged PhIP . In bile duct-ligated rats, the urinary excretion of the N-OH-PhIP N3 -glucuronide was increased two-fold, but there was no effect on PhIP-D NA adduct formation in the colon, heart, lung, pancreas or liver. 2,6- Dichloro-4-nitrophenol, which strongly inhibits arylsulfotransferase-m ediated DNA binding in vivo, had no effect on PhIP-DNA adduct levels i n liver or in extrahepatic tissues. Pretreatment of rats with buthioni ne sulfoximine, which results in hepatic glutathione depletion, caused a five-fold increase in adduct formation in the liver. Intravenous ad ministration (10 mu mol/kg) of N-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels of PhIP-DNA adducts in each of the extrahepatic tis sues examined. Adduct levels ranged from two- to six-fold higher (for N-hydroxy-PhIP and four- to 28-fold higher (for N-acetoxy-PhIP) as com pared to that after an i.v. dose of the parent compound, indicating th at these two bioactivated derivatives of PhIP are sufficiently stable to be transported through the circulation to extrahepatic tissues. Ana lyses of whole blood obtained at 2-8 h after oral administration of [H -3]PNP failed to detect N-hydroxy-PhIP (<0.1% of the radioactivity), h owever, a decomposition product of N-acetoxy-PhIP was found to account for about 80% of the total radioactivity in the blood. These results suggest that transport of N-acetoxy-PhIP, and perhaps N-hydroxy-PhIP, via the bloodstream and not biliary transport and deconjugation of N-h ydroxy-PhIP N-glucuronides is primarily responsible for PhIP-DNA adduc t formation in rat colon and other extrahepatic tissues.