The capability of rat colon tissue slices to metabolize the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

A major target tissue for carcinogenesis from the cooked-food carcinogen 2-amino-l-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 YGI024) 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 microMolar [(3)H]PhIP produced no detectable metabolites. However, incubations of uninduced slices exposed to 10 microMolar of the reactive intermediate, [(3)H]2-(hydroxyamino)-l-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/10(7) 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.

Biotransformation of a somatostatin analogue in precision-cut liver and kidney slices from rat, dog and man.

1. Cleavage of the glucopyranosyl moiety of the somatostatin analogue SDZ CO 611 results in the formation of the major metabolite, SDZ CO 610, in liver and kidney slices of rat, dog and man, as well as in liver S9 and cytosol of rat and man. 2. The rates of SDZ CO 610 formation (nmol/h/mg slice protein) for all three species were determined in liver slices for 24 h and the relative order was: rat (0.12) > dog (0.096) = man (0.095). The rates of SDZ CO 610 formation (nmol/h/mg slice protein) for all three species in kidney were determined, and the relative order was: rat (0.29) > dog (0.16) > man (0.10). 3. SDZ CO 610 was rapidly formed by rat gut contents in the absence of NADPH, possibly by disaccharide-splitting enzymes. 4. Biotransformation of SDZ CO 611 to SDZ CO 610 in human and rat liver S9 and cytosol was similar to that found in liver slices cultures indicating that cleavage of the glucopyranosyl moiety of SDZ CO 611 could occur in the presence and in the absence of cytochrome P450, possibly by glucosidases in liver cytosol. 5. Rat intestinal homogenate also formed SDZ CO 610 but metabolism was dependent upon NADPH, suggestive of a cytochrome P450-dependent reaction.

Biotransformation of the antiemetic 5-HT3 antagonist tropisetron in liver and kidney slices of human, rat and dog with a comparison to in vivo.

Species differences in the biotransformation of the antiemetic tropisetron, a potent 5-hydroxytryptamine type 3 (5-HT3) receptor antagonist, were evident in liver slice incubates of human, rat and dog, and reflected the species differences observed in vivo with respect to the relative importance of individual pathways. The dominant biotransformation pathway of tropisetron (10 microM) in human liver slices was formation of 6-hydroxy-tropisetron, whereas in rat liver slices it was 5-hydroxy-tropisetron, and in dog liver slices N-oxide formation. Initial rates of tropisetron metabolite formation in the liver slices (8 mm in diameter, 200 +/- 25 microns thickness) of human (83 +/- 61 pmol/h/mg slice protein), rat (413 +/- 98 pmol/h/mg slice protein) and dog (426 +/- 38 pmol/h/mg slice protein) would predict less of a first-pass effect in humans compared to the rat or the dog. For human and rat, the prediction matched well with the species ranking of tropisetron bioavailability; however, for dog the in vitro data overestimated the apparent first-pass effect. The jejunum is not expected to contribute to the first-pass effect in humans, since human jejunum microsomes did not metabolize tropisetron. The major organ of excretion for tropisetron and its metabolites is the kidney, but the contribution of the kidney to the overall metabolism of tropisetron would be small. Species independent N-oxide formation (2-12 pmol/h/mg slice protein) was the major pathway in human, rat and dog kidney slices, and was comparable to N-oxide formation in the rat and human liver slices but was 1/10 the rate in dog liver slices. This study has demonstrated that the liver is the primary site of tropisetron biotransformation, and the usefulness of organ slices to characterize cross species differences in the dominant biotransformation pathways.

The metabolism and DNA binding of the cooked-food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in precision-cut rat liver slices.

Precision-cut liver slices prepared from Aroclor 1254 pretreated male rats were used to investigate the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The acetyltransferase and sulfotransferase inhibitors, pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), and the cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), were used to modulate PhIP metabolism and DNA and protein adduct formation. PCP and DCNP had similar effects on the formation of some PhIP metabolites. PCP and DCNP decreased the formation of 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP)-glucuronide to 10% and 55% of controls, respectively. 2-Amino-1-methyl-4'-hydroxy-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) was increased by 50% relative to control levels due to PCP and DCNP treatment. PCP and DCNP had different effects on the formation of other PhIP metabolites. Metabolite formation as percent of control for the uncharacterized metabolite, 'Peak A', was 50% and 100% in incubations with PCP and DCNP, respectively. Formation of 4'-hydroxy-PhIP-glucuronide was decreased to 10% of controls with PCP and increased to 147% of controls with DCNP. PCP and DCNP had no effect on the formation of an unidentified metabolite, 'Peak B'. ANF decreased metabolite formation by 60-95%. None of the enzyme inhibitors had a statistically significant effect on PhIP-DNA binding. Covalent binding of PhIP to protein was slightly decreased in incubations containing DCNP or PCP. The lack of significant changes in covalent binding to either DNA or protein suggests that additional pathways may be important in PhIP bioactivation in rat liver slices. With ANF, there was a significant decrease (35%) in protein binding. These observations on the effects of PCP, DCNP and ANF on PhIP metabolism as well as on covalent binding of PhIP to tissue macromolecules are in close agreement with what was reported earlier in hepatocytes. This indicates that tissue slices from various target tissues for tumorigenesis will be a useful in vitro tool for future studies on heterocyclic amine metabolism. This study provides another important example of the utility of precision-cut tissue slices to investigate xenobiotic metabolism and toxicity.

Conjugation of polychlorinated agrochemical sulphoxides and sulphones by glutathione.

1. Pentachlorophenyl methyl sulphoxide and pentachlorophenyl methyl sulphone were found to be substrates for microsomal and cytosolic glutathione-S-transferase of rabbit, monkey, chicken and human liver, covalently immobilized on beaded sepharose. 2. Protein was immobilized with greater than 95% transferase activity, measured by dinitrochlorobenzene. Immobilized rabbit liver microsomal transferase activity was more stable than immobilized cytosolic activity. 3. The sulphoxide moiety was displayed by glutathione in the presence of chicken liver microsomal protein. The sulphone moiety was displayed by glutathione in the formation of a diglutathione under catalysis by rhesus monkey liver cytosolic and microsomal protein. 4. Chlorine was displaced by transferases from all species to form regioisomeric monoglutathiones. 5. Qualitative and quantitative differences were observed in product distributions between species and between microsomal and cytosolic protein.