A major target tissue for carcinogenesis from the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rodents is the colon, yet the role of colon metabolism on the carcinogenicity of PhIP is not clearly understood. The mutagenic potency of PhIP is highly dependent upon cytochrome P450 N-hydroxylation. In the present study, the ability of rat colon tissue to activate PhIP to a mutagen was investigated in Salmonella typhimurium (strains TA98 and YG1024) and rat colon tissue slices. In the Ames/Salmonella assay, using rat colon S9 as the activating system, no mutations were evident from bacteria exposed to PhIP at any concentration tested. However, mutations were observed when bacteria were exposed to 2-aminoanthracene (2AA) and colon S9, indicating sufficient P450 activity in the S9 to activate 2AA but not PhIP. In rat colon slice preparations, the sulfotransferase and acetyltransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate DNA adduct and metabolite formation. Incubations of 3-methylcholanthrene-induced colon slices dosed with 50 µm [3H]PhIP produced no detectable metabolites. However, incubations of uninduced slices exposed to 10 µm of the reactive intermediate, [3H]2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP), produced a single detectable metabolite, a glucuronide conjugate of N-hydroxy-PhIP. This metabolite decreased when PCP or DCNP was added to the incubation medium. DNA adducts were detected in colon slices exposed to N-hydroxy-PhIP at approximately 33 adducts/107 nucleotides. Interestingly, when PCP was added to the incubation mixture, an increase in DNA adduct levels was detected, whereas DCNP produced a decrease in adducts. Because these inhibitors are thought to have similar mechanisms with regard to sulfotransferase inhibition, the inverse relationship in DNA adduct levels due to PCP or DCNP treatment is at present unexplainable. The formation of DNA adducts and metabolites from colon slices exposed to N-hydroxy-PhIP but not PhIP implies that there is insufficient P450 activity in the rat colon to activate PhIP to hydroxylated metabolites, suggesting that the rat colon is a site of Phase II metabolism for PhIP and that the liver is the primary source for hydroxylation.


This work was performed under the auspices of the United States Department of Energy by Lawrence Livermore National Laboratory under contract W-7405-Eng-48 and supported by National Cancer Institute Grant CA55861 and USAMRDC MM4559FLB.

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