Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-l-methyl-6-phenyUmidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues

The food-borne mutagen 2-amino-l-methyl-6-phenylimidazo 4,5-b]pyridine(PhIP) induces tumors in colon of male rats and has been implicatedin the etiology of human cancers, particularly colorectal cancer.This study was conducted to examine: (1) the biliary and/orcirculatory transport of N-hydroxy- PhIP and its N-glucuronides,N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximateand ultimate carcinogenic metabolites of PhIP; (3) the potentialrole of glutathione in modulating PhIP-DNA adduct formation.PhIP-DNA adducts, measured by the ³²P-postlabeling method, werehighest in the pancreas (361 adducts/10⁸ nucleotides or 100%),followed by colon (56%), lung (28%), heart (27%) and liver (2%),at 24 h after a single oral dose of PhIP (220 µmol/kg)to male rats. In each tissue examined, we observed two majoradducts, each of which accounted for 35–45% of the total,and one minor adduct, which represented about 10–20% ofthe total. One of the major adducts was identified as N-(deoxyguanosin-8-yl)-2-amino-l-methyl-6-phenylimidazo4,5-b]pyridine by chromatographic comparisonswith an authentic standard. The major urinary metabolites ofPhIP in these rats were 4'-hydroxy-PhIP and its glucuronideand sulfate conjugates, followed by N-hydroxy-PhIP N3-glucuronide,N-hydroxy-PhIP N-glucuronide and unchanged PhIP. In bile duct-ligatedrats, the urinary excretion of the N-OH-PhIP N3-glucuronidewas increased two-fold, but there was no effect on PhIP-DNAadduct formation in the colon, heart, lung, pancreas or liver.2,6-Dichloro-4-nitrophenol, which strongly inhibits arylsulfo-transferase-mediatedDNA binding in vivo, had no effect on PhIP-DNA adduct levelsin liver or in extrahepatic tissues. Pretreatment of rats withbuthionine sulfoximine, which results in hepatic glutathionedepletion, caused a five-fold increase in adduct formation inthe liver. Intravenous administration (10 µmol/kg) ofN-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels ofPhIP-DNA adducts in each of the extrahepatic tissues 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 comparedto that after an i.v. dose of the parent compound, indicatingthat these two bioactivated derivatives of PhIP are sufficientlystable to be transported through the circulation to extrahepatictissues. Analyses of whole blood obtained at 2—8 h afteroral administration of ³H]PhIP failed to detect N-hydroxy-PhIP(<0.1% of the radioactivity), however, a decomposition productof N-acetoxy-PhIP was found to account for about 80% of thetotal radioactivity in the blood. These results suggest thattransport of N-acetoxy-PhIP, and perhaps N-hydroxy-PhIP, viathe bloodstream and not biliary transport and deconjugationof N-hydroxy-PhIP N-glucuronides is primarily responsible forPhIP-DNA adduct formation in rat colon and other extrahepatictissues.