Binding of the food mutagen PhIP in pigmented tissues of mice.

The distribution of the 14C-labelled food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the tissues of C57B1/6 and NMRI mice was studied. The results showed a high and selective binding of radioactivity in the pigment epithelium of the eye and in the fur following a single dose (0.3-4 mg/kg) of [14C]PhIP in the pigmented C57B1/6 mice whereas no such localization of radioactivity was present in the albino NMRI mice. A low but selective covalent binding of radioactivity was observed in the liver, inner cortex of the kidney and in the tracheal mucosa of [14C]PhIP-injected mice. PhIP was firmly bound to synthetic melanin pigment in vitro; only 3% was released by extraction with a phosphate buffer (pH 7) whereas 72% was released by extraction by methanol:conc. NH3 (15:1). Three hours to 7 days following a single injection of [14C]PhIP in C57B1/6 mice the radioactivity in the eye was 3- to 6-fold higher than that in the liver or kidney. Almost 60% of the radioactive material present in the pigmented epithelium of the eye 3 and 24 h following injection could be extracted by basic methanol and identified as unchanged PhIP. The residual radioactivity in the pigmented epithelium of the eyes may represent a covalent binding of [14C]PhIP metabolites to cellular constituents or to a basic methanol-resistant binding of [14C]PhIP to melanin. The results indicate that pigmented tissues may be potential target tissues for the toxic effects of PhIP and suggest that the use of hair for biological monitoring of PhIP should be examined.     

The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.     

Fate and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats

Fischer 344 rats were given a single dose of 0.60 mg/animal of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by gavage; and radioactivity contained in feces, urine, blood, serum proteins, hemoglobin, and tissues was determined at 12, 24, 48 and 96 h after dosing. One major and four minor radioactivity-containing fractions were found in the urine and one major and two minor radioactivity-containing fractions were found in the feces. The feces was the major route of excretion, representing 78% of dose during the first 24 h, and unchanged PhIP in the feces accounted for 51% of the dose. Unmetabolized PhIP was also shown to be the major radioactive fraction in bile and feces from animals given a single dose by i.p. injection. Blood contained a small fraction of the dose and the major, persistently-bound form of PhIP in the blood was to hemoglobin. At 12 h after administration of the dose the colon and cecum contained the highest concentration of radioactivity, while at later times the kidney and liver showed the highest concentration. Of the tissue-contained radioactivity 80-90% was ethanol insoluble at time points later than 24 h, suggesting that it was covalently bound to macromolecules.     

Fate and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in mice at a human dietary equivalent dose.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine rodent carcinogen that is found at the ppb level in cooked meat. Most laboratory studies are at 10(4)-10(7)-fold greater concentrations than actual ingested human doses. We report the first study of the bioavailability and fate of this heterocyclic amine at a human dietary equivalent dose using the high sensitivity offered by accelerator mass spectrometry. [2-14C]PhIP was administered to C57BL/6 male mice (41 ng/kg) by gavage. Tissues and excreta were collected over the subsequent 96 h. One hundred % of the administered dose was excreted in urine (90%) and feces (10%) over the length of the study. Absorption of the radiocarbon-tagged PhIP from the gastrointestinal tract was rapid, with radiocarbon levels peaking in the whole blood and urine within 1 h of exposure. Fecal 14C levels peaked at 12 h. Tissue levels peaked by 3 h with the highest concentrations of radiolabel in the intestine, stomach, and liver, followed by the kidney, pancreas, lung, and spleen. Low levels of 14C from PhIP (0.01-0.04% of the administered dose) could be detected in the tissues 48-96 h after exposure, possibly due to covalent binding to protein or DNA. The calculated half-life of PhIP at this dose was 1.14 h. This study is the first example of how accelerator mass spectrometry can be used to gather biological information about carcinogenic compounds at environmental levels of exposure.     

Distribution and metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) in female rats and their pups at dietary doses

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary carcinogen in female rats and is present in a wide variety of cooked meats. We address here the excretion of PhIP and its metabolites into the breast-milk of lactating rats and the ability of chlorophyllin, a food product derivative with chemopreventive properties, to affect these levels at low PhIP doses. Lactating female F344 rats with suckling pups were orally administered 50, 500 and 1000 ng [14C]PhIP/kg body weight. The excretion of the [14C]PhIP into milk and its distribution among the mammary tissue, liver and blood of the dam, as well as among stomach contents and liver of their suckling pups was measured using accelerator mass spectrometry (AMS). PhIP, PhIP-4'- sulfate, 4'-hydroxy-PhIP, and N2-hydroxy-PhIP-N3-glucuronide were found in the milk at all doses. The chlorophyllin (500 microg/kg) co- administration with PhIP (500 ng/kg) caused increased levels of [14C]PhIP in the milk (32%) and stomach contents (35%) of the pups relative to the animals not receiving chlorophyllin at these low PhIP doses. In contrast, lower [14C]PhIP levels in the chlorophyllin treated animals were observed in the blood (47%) and mammary tissue (68%) of the dam, as well as the pup's liver tissue (37%) compared to the animals receiving only PhIP. Chlorophyllin co-administration resulted in an increased amount of N2-hydroxy-PhIP-N3-glucuronide (42%), increased PhIP (79%) and decreased levels of PhIP-4'-sulphate (77%) relative to the animals not receiving chlorophyllin. These results suggest that PhIP and PhIP metabolites are present in the breast-milk of lactating rats at human dietary PhIP exposures and that PhIP is absorbed by the newborn. Furthermore, these results suggest that other dietary components can affect the dosimetry of PhIP in breast-feeding offspring.      

Differential metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rat and human hepatocytes.

Metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been compared in human and rat hepatocytes. The identities of seven metabolites were confirmed by UV and mass spectroscopy and by co-elution with reference standards using HPLC. In human hepatocytes, the major biotransformation pathway of PhIP was cytochrome P4501A2 (CYP1A2)-mediated N-oxidation to form the genotoxic metabolite 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which underwent glucuronidation at the N(2) and N3 positions of PhIP to form stable conjugates. These products combined accounted for as much as 60% of the added PhIP. Direct glucuronidation of PhIP at the N(2) and N3 positions also occurred, accounting for up to 20% of the amount added. Glucuronide and sulfate conjugates of 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-HO-PhIP) were also detected, comprising 5 and 12% of the products, respectively. The CYP1A2 inhibitor furafylline diminished the formation of both HONH-PhIP glucuronide conjugates in a concentration-dependent manner, however, levels of 4'-HO-PhIP were unchanged, indicating that CYP1A2 does not significantly contribute to 4'-hydroxylation of PhIP. Hepatocytes of male rats, both untreated and pretreated with the CYP1A2 inducer 3-methylcholanthrene (3-MC) transformed PhIP into 4'-HO-PhIP as the prominent product. Unconjugated and conjugated 4'-HO-PhIP metabolites combined accounted for 18 and 46% of the PhIP products in untreated and in 3-MC-pretreated rat hepatocytes, respectively. The isomeric glucuronide conjugates of HONH-PhIP combined accounted for 11 and 26% of the PhIP, respectively, in untreated and 3-MC-pretreated hepatocytes. The regioselectivity of glucuronidation of PhIP was different in human and rat hepatocytes. Human liver UDP-glucuronosyltransferases favored conjugation to the N(2) positions of PhIP and HONH-PhIP, while the N3 atom was the preferred site of conjugation for the rat enzymes. Thus, important differences exist between human and rat enzymes in catalytic activity and regioselectivity of PhIP metabolism. Some human hepatocyte populations are more active at transforming PhIP to a genotoxic species than rat hepatocytes pretreated with the potent CYP1A2 inducer 3-MC.     

Metabolism of the food-derived mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) in nonhuman primates

Metabolism of the food-derived heterocyclic amine mutagen/carcinogen2-amino-1-methy1–6-phenylimidazo[4, 5-b]pyridine (PhIP)was examined in cynomolgus monkeys. [3H]PhiP (50µmol/kg,p.o.) was extensively metabolized, with only 1% of the doseexcreted into the urine as parent compound. Four metaboliteswere isolated by HPLC and identified: PhIP-4–0-glucuronide,PhIP-4‘-sulfate, 4’-hydroxy-PhIP and a glucur-onideconjugate of N-hydroxy-PhIP. All four metabolites were detectedin urine, bile and plasma of monkeys. 4‘-Hydroxy-PhIPand PhIP were found in feces. The major PhIP metabolite in urine,bile and plasma was PhIP-4’-sulfate. Inurine this metaboliteconstituted -64–72% of the radioactivity excreted. Theclearance of PhIP and PhIP metabolites from plasma was rapid,with the largest elimination occurring within 8 h. Administrationof nine consecutive daily doses of unlabeled PhIP (50 µmol/kg,p.o.) prior to administration of [3H]PhIP (50 µmol/kg,p.o.) did not alter the plasma clearance of radiolabeled PhIPor PhIP metabolites, suggesting that this multiple-dose regimendid not induce or alter PhIP metabolism. PhIP formed DNA adductsin white blood cells, as determined by the 32P-postlabelingmethod. The levels of PhIP-DNA adducts in blood appeared topeak 3 h after administering a single dose of PhIP (50µmol/kg,p.o.) and were still detected 1 week after dosing. The presenceof the glucuronide conjugate of N-hydroxy-PhIP in urine, bileand plasma, and the presence of PhIP-DNA adducts in white bloodcells indicate that PhIP undergoes metabolic activation viaN-hydroxylation in cynomolgus monkeys. The results suggest thatPhIP is activated in vivo to genotoxic metabolites in nonhumanprimates and thus is a potential carcinogen in this species.     

Interaction of aflatoxin B1 metabolites with cellular macromolecules in neonatal rats receiving carcinogen through mother's milk

A single dose of 2 microCi [3H] aflatoxin B1 (AFB1) containing 20 micrograms AFB1 was administered to the lactating rat. This resulted in the excretion of AFB1-metabolites in the milk, which were transferred and distributed in many tissues of the offspring consuming milk for 48 h. An equal distribution of radioactivity in liver and lung of suckling rats as well as in their sub-cellular fractions was observed. No AFB1 metabolite binding was apparent with tissue DNA. However, considerable binding of aflatoxin residues was observed with protein followed by RNA. Three metabolites have been detected in the milk sample derived from the stomach of the suckling rats on thin layer chromatogram with Rf values of 0.2, 0.4 and 0.8. Pretreatment of lactating rats with phenobarbitone (PB: 80 mg/kg/3 days) prior to the administration of [3H]AFB1 caused an overall decrease in the radioactivity in the neonatal tissues, particularly lung and intestine (P less than 0.05). The fluorescent bands with Rf values of 0.2 and 0.4 were not visualized in the milk sample from the PB group. Subsequently, the binding of AFB1 residues to cellular macromolecules of neonatal tissues was decreased. PB treatment further resulted in lowering the glucuronide/sulphate conjugates of AFB1 in the milk consumed by suckling rats; AFB1-glutathione (GSH) conjugates were slightly increased. These results point out the possibility that aflatoxin metabolites bind to the tissues of offspring fed by mothers consuming aflatoxin contaminated diets thus leading to chronic effects.     

Immunochemical detection of rodent hepatic and urinary metabolites of cooking-induced food mutagens

Monoclonal antibodies, previously selected to bind either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were evaluated to determine their binding properties with several known metabolites of these compounds purified from rat hepatocyte cultures. Both 2-N- and 5-substituted MeIQx metabolites were recognized by antibodies AIA-2 and AIA-11, while antibodies AIA-1 and AIA-12 bound N-substituted metabolites only. Anti-PhIP antibodies bound N-hydroxy-PhIP, N-sulfinamide-glutathione-PhIP and N-hydroxy-glucuronide-PhIP. Immunoaffinity columns incorporating these antibodies were used to concentrate and purify both the unmetabolized parent compounds and several relatively non-polar metabolites from the urine of rats exposed either to MeIQx or PhIP. Several of these metabolites corresponded with available standards of previously identified polar conjugate metabolites, e.g. N-sulfamate-MeIQx and N(OH)-gluc-PhIP, while others are as yet unidentified. Immunoaffinity chromatography is demonstrated as a promising means of selectively concentrating metabolites of PhIP and MeIQx from urine.     

Regiospecificity in the metabolism of the homologous cyclic nitrosamines, N' -nitrosonornicotine and N' -nitrosoanabasine

We compared the metabolism in the F-344 rat of the moderatelypotent esophageal carcinogen N' -nitrosonornicotine (NNN, 2'-(3-pyridyl)-N-nitrosopyrrolidine) and its weakly active homologueN'-nitrosoanabasine (NAB, 2' -(3-pyridyl)-N-nitrosopiperldine).Urine was the major pathway of excretion for both nitrosamines.The major urinary metabolites of dl-NNN resulted from 2' -hydroxylation(8.1% of the dose), 5' -hydroxylation (37.6%), and pyridineN-oxidation (10.8%). The percentages of the dose of the correspondingmetabolites of dl-NAB were: 2'-hydroxylation (not detected),6'-hydroxylation (9.8%), pyridine-N-oxidation (30.0%). Similarresults were obtained when the urinary metabolites of l-NNNand l-NAB were compared. In 48 h cultures of rat esophagus,the major metabolites of [2' -¹⁴C]dl-NNN resulted from 2' -hydroxylation(47%) and to a lesser extent from 5'-hydroxylation (15%). Incontrast the major metabolite of [2'-¹⁴C]dl-NAB resulted from6'-hydroxylation (35%) with lesser amounts from 2'-hydroxylation(8%). 6'-Hydroxylation of [2'-¹⁴C]dl-NAB also exceeded 2' -hydroxylationin cultures of 3,6,12 or 24 h duration. Pyridine-N-oxidationwas not observed in the esophagus for either nitrosamine. Theseresults demonstrate a high degree of regiospecificity in themetabolism of these structurally related nitrosamines. Amongthe identified urinary metabolites the ratio of -hydroxylationto N-oxidation was 4.2 for NNN and 0.3 for NAB. Among the 48h esophageal metabolites the ratio of 2'-hydroxylation to 5'- or 6' -hydroxylation was 3.1 for NNN and 0.2 for NAB. Theresults also suggest a basis for the weak carcinogenicity ofNAB: facile excretion as its pyridine-N-oxide and detoxificationin the esophagus by 6' -hydroxylation.     

In vivo human metabolism of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

To better understand the interactions of the pathways of activation and detoxification on the metabolism of the putative carcinogen, PhIP, we administered a dose of 70-84 microg [2-14C] PhIP (17.5 [microCi 14C) 48-72 h before scheduled colon surgery. Blood and urine collected for the next 48-72 h was evaluated by linear accelerator mass spectroscopy (AMS) and scintillation counting LC-MS to identify specific PhIP metabolites. The thermostable phenol sulfotransferase (SULT1A1) phenotype was correlated with the 4'-PhIP-SO4 levels in the urine at 0-4 h (R = 0.86, P = 0.059). The CYP1A2 activity had a negative correlation with PhIP serum levels at 1 h (R = 0.94, P = 0.06) and a positive correlation with urine N-OH-PhIP levels at 0-4 h (R = 0.85, P = 0.15). This low level radioisotope method of determining the influence of phenotype on metabolism will significantly improve our understanding of the interrelationships of these pathways and provide a critical foundation for the development of individual risk assessment.     

Disposition and metabolism of the food mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rats

The disposition and metabolism of a common food mutagen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), was studied in rats. Five rats of both sexes were given a single oral dose of 14C-labeled MeIQx (3-4 mg/kg body wt). The male rats excreted 36% of the radioactivity and 15% of the mutagenic activity of the dose given in the urine collected during the first 24 h. In the females the corresponding urine contained 41% of the radioactivity and 12% of the mutagenicity. During the next 48 h only 1-3% of the radioactive dose was excreted in urine. The remaining dose was excreted in the feces except of less than 1% that was retained by the tissues after 72 h. The liver and kidney retained more radioactivity than other organs. In a separate study the metabolites of bile, urine and feces of both sexes were investigated. After a single oral dose of 20 mg 14C-labeled MeIQx/kg body wt, three major non-mutagenic metabolites were identified. These were 2-amino-4(or 5)-(beta-D-glucuronopyranosyloxy)-3,8-dimethylimidazo[4,5-f] quinoxaline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxalin-4(or 5)-yl sulfate and N-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl) sulfamate. Another two metabolites present in bile, urine and feces were 2-(beta-D-glucuronopyranosylamino)-3,8-dimethylimidazo[4,5-f ] quinoxaline and 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxalin-4 (or 5)yl sulfate. All metabolites were essentially non-mutagenic. Most of the mutagenicity still present in bile, urine and feces could be explained by unchanged MeIQx. Unchanged MeIQx was the most abundant form excreted in urine.     

Identification of urine metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine following consumption of a single cooked chicken meal in humans

Many studies suggest that mutagenic/carcinogenic chemicals in the diet, like 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), may play a role in human cancer initiation. We have developed a method to quantify PhIP metabolites in human urine and have applied it to samples from female volunteers who had eaten a meal of cooked chicken. For this analysis, urine samples (5 ml) were spiked with a deuterium-labeled internal standard, adsorbed to a macroporous polymeric column and then eluted with methanol. After a solvent exchange to 0.01 M HCl, the urine extracts were passed through a filter, applied to a benzenesulfonic acid column, washed with methanol/acid and eluted with ammonium acetate and concentrated on a C(18) column. The metabolites were eluted from the C(18) column and quantified by LC/MS/MS. In our studies of human PhIP metabolism, eight volunteers were fed 200 g of cooked chicken containing a total of 27 microg PhIP. Urine samples were collected for 24 h after the meal, in 6 h aliquots. Although no metabolites could be found in urine collected from volunteers before eating the chicken, four major human PhIP metabolites, N:(2)-OH-PhIP-N:(2)-glucuronide, PhIP-N:(2)-glucuronide, 4'-PhIP-sulfate and N:(2)-OH-PhIP-N:3-glucuronide, were found in the urine after the chicken meal. The volunteers in the study excreted 4-53% of the ingested PhIP dose in the urine. The rate of metabolite excretion varied among the subjects, however, in all of the subjects the majority of the metabolites were excreted in the first 12 h. Very little metabolite was detected in the urine after 18 h. In humans, N:(2)-OH-PhIP-N:(2) glucuronide is the most abundant urinary metabolite, followed by PhIP-N:(2)-glucuronide. The variation seen in the total amount, excretion time and metabolite ratios with our method suggests that individual digestion, metabolism and/or other components of the diet may influence the absorption and amounts of metabolic products produced from PhIP.     

Characterization of benzopyrene metabolism in rat pancreas

The present studies characterized the pancreatic metabolic system which biodegrades benzo[a]pyrene (BP), in male Sprague-Dawley rats pretreated with 20 mg/kg 3-methylcholanthrene (3MC), 75 mg/kg phenobarbital (PB) or solvent as control. Type of cytochrome involved was examined by measuring the reaction in the presence of 7,8-benzoflavone (BF), an inhibitor of the cytochrome P-448-related enzyme activity. The data indicated that the enzyme activity was induced by pretreatment with PB but not with 3MC. These pretreatment protocols resulted in changes both in Km and Vmax. Studies with BF suggested that pancreatic tissue may not contain more than one class of hemoprotein. These studies pointed out species differences between the rat and guinea pig, and confirmed the pancreatic capability to metabolize procarcinogens and to further degrade carcinogenic metabolites.     

Disposition and metabolism of aniline in Fischer 344 rats and C57BL/6 X C3H F1 mice

We examined the metabolism and disposition of aniline, which induces spleen hemangiosarcomas in rats but no tumors in mice, in normal and predosed Fischer 344 rats, and C57BL/6 X C3H F1 mice administered low (50 and 100 mg/kg, respectively) or high (250 and 500 mg/kg, respectively) doses. Of 11 tissues examined, the highest levels of binding of [14C]aniline to DNA were in the kidney, large intestine, and spleen of high-dose rats that had received prior dosing; these tissues had covalent binding indices of 14.2, 4.3, and 3.7 mumol/mol nucleotides/dose, respectively. Protein and RNA were the major macromolecular targets for binding of radioactivity from [14C]aniline. Relative to controls, most tissues from predosed mice (low dose and high dose) showed less binding to protein and RNA; but for most tissues from predosed rats administered 50-mg/kg doses of [14C]aniline, there was more extensive binding. Also relative to controls, binding of radioactivity in the spleen of predosed rats given [14C]aniline (50 mg/kg) was 148% greater for protein and 302% greater for RNA. For rats administered 250 mg of [14C]aniline per kg, however, there were no outstanding differences in binding to RNA and protein between normal and predosed animals. The profiles of urinary metabolites produced by rats and mice were not appreciably different in animals predosed with aniline. For rats, however, the profiles were different for the low and high doses, suggesting that the main metabolic pathway was saturated at the higher dose. p-Acetamidophenyl sulfate represented over 70% of the total radioactivity recovered from the urine of rats dosed with 50 mg of aniline per kg but only 30% in the urine of those dosed with 250 mg/kg. The urine of the high-dose rats contained greater percentages of p-aminophenyl sulfate, p-acetamidophenyl glucuronide, and unconjugated metabolites. In mouse urine, p-acetamidophenyl glucuronide, representing 29 to 32% of the total radioactivity, was the major metabolite. Nevertheless, mice produced more ortho derivatives than did rats, for in acid-treated urine, the ratio of p- to o-aminophenol was 8.1 for rats and 1.6 for mice. Predosing of rats and mice did not change the kinetic values for liver aniline p-hydroxylase or N-hydroxylase but increased the amount of mouse liver cytochrome P-450 from 0.231 to 0.491 nmol/mg protein. For p-hydroxylase of rat liver, the apparent Km value was higher, and the apparent Vmax value lower than in mouse liver. Kinetic values for rat and mouse N-hydroxylase were similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

4-(2-amino-1-methylimidazo pyrid-6-yl)[4,5-b]phenyl sulfate--a major metabolite of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b] (PhIP) in the rat

Isolated rat hepatocytes (4 ? 10⁶ cells/ml metabolized the foodmutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)(100 µM) to at least eight different metabolites in a4 h duration. The major metaboilte formed was 4-(2-amino-1-methylimldazo[4,5-b]pyrid-6-yl)sulfate (4'-PhIP sulfate). Its rate of formation was increasedin hepatocytes from PCB pretreated animals in comparison tohepatocytes from untreated animals. One of the other metaboliteswas the unconjugated derivative of the sulfate (4'-OH-PhIP).This metabolite was also found after in-vitro incubations ofrat liver microsomes from PCB or ß-naphtho flavonepretreated animals. The evidence for the proposed structureof the major metabolite is based on [¹H]NMR and UV spectroscopy,incorporation of radiolabelled sulfate and arylsulfatase-labilhty.The formation of 4'-PhIP sulfate was inhibited by the P-450inhibitors -naphthoflavone and metyrapone and when incubatedin sulfate-free medium added the sulfotransferase Inhibitorpentachlorophenol. 4'-PhIP sulfate was also the major metaboliteof PhIP in the urine of exposed rat.     

Synthesis and excretion profile of 1,4- [14C]phenylenebis(methylene)selenocyanate in the rat

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits chemically induced tumors in several laboratory animal models. To understand its mode of action, we synthesized p-[14C]XSC, examined its excretion pattern in female CD rats and also the nature of its metabolites. p- [14C]XSC was synthesized from alpha,alpha-dibromo-p-[ring-14C]xylene in 80% yield. The excretion profile of p-[14C]XSC (15.8 mg/kg body wt, 200 microCi/rat, oral administration, in 1 ml corn oil) in vivo was monitored by measuring radioactivity and selenium content. On the basis of radioactivity, approximately 20% of the dose was excreted in the urine and 68% in the feces over 3 days. The cumulative percentages of the dose excreted over 7 days were 24% in urine and 75% in feces, similar to excretion rates of selenium. According to selenium measurement, <1% of the dose was detected in exhaled air; radioactivity was not detected. Only 15% of the dose was extractable from the feces with EtOAc and was identified as tetraselenocyclophane (TSC). Most of the radioactivity remained tightly bound to the feces. Approximately 10% of this bound material converted to TSC on reduction with NaBH4. Organic soluble metabolites in urine did not exceed 2% of the dose; sulfate (9 % of urinary metabolites) and glucuronic acid (19.5% of urinary metabolites) conjugates were observed but their structural identification is still underway. Co-chromatography with a synthetic standard led to the detection of terephthalic acid (1,4- benzenedicarboxylic acid) as a minor metabolite. The major urinary conjugates contained selenium. Despite the low levels of selenium in the exhaled air, the reductive metabolism of p-XSC to H2Se cannot be ruled out. Identification of TSC in vivo indicates that a selenol may be a key intermediate responsible for the chemopreventive action of p- XSC.      

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice.

The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), a heterocyclic amine carcinogen detected in cooked meats, was investigated in mice. In 3-methylcholanthrene-induced mice administered 0.1, 1.0 and 10 mg/kg [14C]PhIP (i.p.), urinary and fecal excretion over 24 h accounted for 16% and 42-56% of the dose respectively. Urinary excretion of unchanged parent compound accounted for only 0.5-0.8% of the administered dose. At all doses, the major urinary metabolite was identified as 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate and this metabolite comprised approximately 5% of the dose. Uninduced mice excreted greater than 13% of a 10 mg/kg dose as the sulfate conjugate. Urinary excretion of both 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) and a glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (N-hydroxy-PhIP) was also higher (4-fold) in uninduced versus induced mice. The decreased urinary excretion of P450-derived metabolites via induction contrasted with increased metabolite formation by hepatic microsomal preparations. 4'-Hydroxy-PhIP and N-hydroxy-PhIP were produced in amounts nearly 7- and 3-fold higher respectively by induced versus uninduced microsomal incubations at 50 microM [3H]PhIP. At concentrations less than 10 microM, PhIP was almost exclusively converted by the induced preparations to an unidentified metabolite that was not retained by the C18 column. This metabolite, which also was formed in incubations with either 4'-hydroxy-PhIP or N-hydroxy-PhIP, was produced by microsomes from uninduced animals at a much slower rate. Covalent binding to microsomal protein in incubations with [3H]PhIP was concentration-dependent and 2- to 4-fold higher in induced than uninduced preparations. Covalent binding in liver and kidney of induced mice administered [14C]PhIP was dose dependent. At 10 mg/kg PhIP, adducts were produced at 1.7-fold higher levels in livers of induced versus uninduced mice, but renal binding was higher in uninduced animals. These studies indicate the importance of cytochrome P450 and other xenobiotic enzymes in the metabolism, disposition and activation of PhIP.     

Excretion of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, PhIP- dG, PhIP-DNA and DiMeIQx-DNA from the rat

The heterocyclic aromatic amines, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) are formed during frying of meat. PhIP and 4,8-DiMeIQx have, after metabolic activation, been shown to form adducts with DNA at the C8 of guanine both in vitro and in vivo. In order to investigate possible urinary biomarkers for estimation of the genotoxic dose of PhIP and 4,8-DiMeIQx, [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx- DNA were injected i.p. to rats and the excretion of radioactivity in urine and faeces were measured. For all three [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA 15-20% of the dose were excreted in the urine and 80-85% of the dose were excreted in the faeces. Urinary excretion showed maximum to 24 h (90%) with a rapid decline, 10% to 48 h and 0% to 72 h. Faecal excretion also showed maximum to 24 h (60%) with a slower decline, 30% to 48 h and 10% to 72 h. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-dG, showed that approximately 90% of the radioactivity co-eluted with PhIP- dG, indicating that PhIP-dG is excreted unmetabolized. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-DNA, showed that approximately 85% of the radioactivity co- eluted with PhIP-dG, indicating that PhIP-DNA adducts is mainly excreted as nucleoside adducts. Approximately 5% of the radioactivity excreted in the urine co-eluted with PhIP-G, indicating loss of deoxyribose. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [14C]4,8-DiMeIQx-DNA, showed that approximately 90% of the radioactivity co-eluted with 4,8-DiMeIQx-dG, indicating that 4,8-DiMeIQx-DNA adducts is mainly excreted as nucleoside adducts. Man is able to eliminate compounds of a higher mol. wt in the urine than the rat, the percentage of PhIP-dG and 4,8-DiMeIQx eliminated in the urine of man would therefore be expected to be higher than in the rat. Measurement of urinary nucleoside adducts of PhIP and 4,8-DiMeIQx could therefore provide a basis for the development of a biomonitoring strategy for the genotoxic dose of these food derived HAA.      

Metabolism and macromolecular covalent binding of [14C]-1-nitropyrene in isolated perfused and ventilated rat lungs

1-Nitropyrene (1-NP) originating from such sources as diesel exhaust emissions and coal combustion fly ash has been detected in the environment. 1-NP, in addition to being both a bacterial and mammalian mutagen, is carcinogenic in rats. The purpose of this study was to quantitate 1-NP metabolism and macromolecular covalent binding in the isolated perfused rat lung. Rat lungs were perfused with 2, 5, or 24 microM [14C]-1-NP for 90 min. Tidal volume and dynamic lung compliance were monitored continually throughout the perfusion to document the ventilatory pattern and the decay of tissue elasticity. Perfusate was sampled periodically throughout the duration of the experiment and analyzed for 1-NP metabolites with high-performance liquid chromatography. In all experiments, both dynamic lung compliance and tidal volume declined in a nearly linear manner and were approximately 60% of the initial value at the end of 90 min of perfusion. At all concentrations of [14C]-1-NP tested, less than 5 to 6% of the total amount of [14C]-1-NP added was metabolized in lungs from control and phenobarbital (PB)-treated rats. Lungs from control and PB- and 3-methylcholanthrene (3-MC)-treated rats metabolized [14C]-1-NP to oxidized, reduced, and conjugated metabolites. The major metabolites were 3-, 6-, and 8-hydroxynitropyrene. Small quantities of 10-hydroxynitropyrene, aminopyrene, and acetylaminopyrene were also detected in perfusates (less than 10% of the total metabolites). Treatment of rats with PB resulted in a 60% increase in the total metabolism of [14C]-1-NP, whereas treatment of rats with 3-MC resulted in a 10-fold increase in the rate of metabolism of [14C]-1-NP when compared to controls. Conjugate hydrolysis studies indicated that the water-soluble metabolites from lungs of control and PB- and 3-MC-treated rats consisted of hydroxynitropyrene glucuronides and hydroxynitropyrene sulfate conjugates. Quantities of 14C covalently bound to lung macromolecules after 90 min of perfusion from lungs of control and PB-treated rats were 0.06 to 0.21 nmol equivalents/g lung. However, in lungs from 3-MC-treated rats, there was a 20-fold increase in quantities of 14C covalently bound when compared to lungs from either control or PB-treated rats. The results from these studies point to the potential importance of lung metabolism in contributing to the metabolic fate of inhaled 1-NP.