Carcinogenesis vol.11 no 6 pp.941-946. 1990
Metabolism of 2-ainnLmo-l-metlhyl-6-pIhe]iiylliimdazo[4,S-Z>] pyridime (PMP) by liver microsomes and isolated rabbit cytochrome P4S© isozymes
K.W.Turteltaub, M.G.Knize, M.H.Buonarati, M.E.McManus', M.E.Veronese1, J.A.Mazrimas and J.S.Felton Biomedical Sciences Division, Lawrence Livermore National Laboratory, PO Box 5507, Livermore, CA 94550, USA and 'Department of Clinical Pharmacology, School of Medicine, Flinders University of South Australia, Bedford Park 5042, Australia
Introduction 2-Amino-I-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP*), a potent Salmonella and mammalian cell mutagen, was originally identified in fried beef and has since been reported in cooked pork, fish and sausage products (1 —4). PhlP is believed to be formed via the condensation of creatine and phenylalanine when meat is cooked at temperatures ranging from 150 to 25O°C (5,6). These temperatures approximate cooking conditions employed in the normal Western household, which suggests PhlP may be a potential risk factor in human carcinogenesis. To understand the cancer risk posed by exposure to PhlP, it is necessary to elucidate its activation and detoxification pathways in the animal models used to define the carcinogenicity of PhlP. The work presented here is an initial step towards that end. PhlP belongs to the amino-imidazoazaarene (AIA) family of compounds (7). Several members of this family, including PhlP, have been found to produce tumors in multiple species and at multiple sites (8—13). Thus the AIA mutagens may constitute •Abbreviations: PhJP, 2-amino-l-rnethyl-6-phenylimidazo{4,5-t]pyridine; ALA, amino-imidazoazaarene; TCDD, 2,3,7,8-tetrachlorodibenzo-/7-dioxin; 4'-hydroxyPWP, 2-amino-l-rnethyl-4'-hydroxy-6-phenylirrudazo(4,5-ft]pyridine; rutro-PhIP, 2-nitro-l-methyl-6-phenylimidaio[4,5-^]pyrkline; 3-MC, 3-methylcholanthrene; FAB, fast atom bombardment.
Materials and methods Chemicals [l4C]PhIP and PhlP were synthesized in our laboratory as previously described (27,28). Radiochemical punty was shown to be >98% by HPLC. 2-Nitro-lmethyl-6-phenylimidazo[4,5-6]pyridine (nitro-PhIP) standard was the kind gift of Dr S.Grivas, Swedish University of Agricultural Sciences. All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO). The Ames/'Salmonella tester strain TA98 was thelcind gift of Dr Bruce Ames, (University of California, Berkeley, CA).
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The cytochrome P450-dependent metabolism of the heterocyclic amine mutagen 2-amino-l-methyl-6-phenylimidazo [4,5-A]pyridine (PhlP) has been determined. We investigated the in vitro metabolism of PhlP by polycyclic hydrocarboninduced mouse and rabbit liver microsomes, and by purified rabbit liver P450 isozymes. Following a 60 min incubation, 3-methylcbolanthrene-induced mouse microsomes converted 36% of the PhlP to two major metabolites, N-hydroxy-PhIP and 4-hydroxy-PhIP, with 43% total metabolism. Rabbit P450 form 6 and form 4 produced the same two major metabolites (20 and 5% total metabolism respectively). Additional metabolites were produced in low yields and amounts varied depending on the isozyme used (1-5%). Metabolites were not detected in incubations of PhlP with P450 forms 2 and 3C. N-Hydroxy-PhIP was found to be directly mutagenic to Salmonella TA98, while the 4'-hydroxyPhlP was not mutagenic either with or without additional metabolic activation. These data suggest that the cytochrome P450IA isozymes are involved in the metabolism of PhlP by rabbit liver and that formation of N-hydroxy-PhIP is involved in the mutagenicity of PhlP.
a significant dietary risk for humans, particularly for cancers of the colon and gastrointestinal tract, since these tissues would have direct contact with these compounds. PhlP may be particularly important in this regard, since it has been reported to account for - 7 5 % , by mass, of the AIAs found in well-done cooked beef and 91% in fried fish (4,14). PhlP contains a phenylpyridine structure which differentiates it from its quinoline and quinoxaline AIA counterparts. PhlP is the least potent heterocyclic amine mutagen isolated from meat in Salmonella mutation assays, yet is the most potent clastogen in Chinese hamster ovary cells grown in culture (14,15). PhlP has recently been shown to cause DNA strand breaks in suspensions of rat hepatocytes, and is —4 times more potent than 2-amino-3,8-dimethylimidazo[4,5-/|quinoxaline in the induction of sister chromatid exchange in V79 cells (16). PhlP also has been shown to cause sister chromatid exchange in mouse bone marrow and in peripheral blood (17). These data suggest that a number of tissues may be susceptible to the genotoxic effects of PhlP. The genotoxic activity of PhlP, like other heterocyclic amine mutagens, presumably depends on metabolism of the compound to electrophilic intermediates. None of the AIAs are mutagenic in vitro without activation. Activation of 2-amino-3-methylimidazo[4,5-/|quinoline and 2-amino-3,8-dimethylimidazo[4,5-/] quinoxaline has been shown to require oxidation of the exocyclic amino group to the corresponding hydroxyamino derivative by cytochrome P450IA1 and P450IA2 (18-22). This is believed to be followed by esterification to N-acetoxy metabolites (23 -25). In an earlier report (26), PhlP was shown to be mutagenic to Salmonella in the presence of 2,3,7,8-tetrachlorodibenzo-/?-dioxin (TCDD)-induced rabbit liver and lung microsomes whereas control, acetone-induced, phenobarbital-induced and rifampicininduced rabbit liver homogenates were less effective. These data suggested the involvement of the P450IA family of monooxygenases in the activation of PhlP. Purified rabbit P450 forms 4 (P450IA2) and 6 (P450IA1) also were shown to be efficient in converting PhlP to a Salmonella mutagen, while forms 5 (P450IV1), 2 (P450ITB1), 3b (P450HC3) and 3c (P450IIIA6) were inactive (26). In the present paper we have isolated and identified the metabolic products of [ C]PhTP formed by mouse and rabbit cytochromes P450. The major metabolites produced were 2-amino-l-methyl-4'-hydroxy-6-phenylimidazo[4,5-£] pyridine (4'-hydroxy-PhIP) and 2-hydroxyamino-l-methyl-6phenylimidazo[4,5-fc]pyridine (N-hydroxy-PhIP), with the latter metabolite being the mutagenic species.
K.W.Turtdtaub et al. Animals C57BL/6N male mice were obtained from Simonsen Farms (Gilroy, CA). Animals were housed individually on hard-wood bedding in polystyrene cages with free access to food and water. Animals were induced with a single i.p. injection of 3-methylcholanthrene (3-MC; 80 mg/kg in corn oil) and killed 2 days later. Livers were harvested and immediately used to prepare microsomes (29). Preparation of rabbit microsomes and isolation of P450 isozymes were carried out as previously reported (26). Microsomes and purified P45Os were stored at —80°C until use.
In vitro metabolism PhIP was incubated with 3-MC-induced mouse hepatic microsomes, TCDDrnduced rabbit hepatic microsomes and highly purified rabbit cytochrome P45O forms 2, 3c, 4 and 6. Assays with mouse and rabbit microsomes were conducted in a buffer consisting of 8 mM MgCI2, 33 mM KC1 and 200 mM phosphate, pH 7.4. The protein concentration used for the mouse and rabbit TCDD-induced microsomes was 2 mg/ml. Reactions were initiated after a 3 min preincubation at 37°C by the addition of cofactor (5 mM glucose-6-phosphate, 5 mM NADPH, and 4 mM NADP final concentration). The total volume of each reaction mixture was 0 5 ml. Reactions were terminated with the addition of an equal volume of methanol Incubations using the isolated P450 forms were conducted in a similar fashion except that 0.5 unit of NADPH-cytochrome P450 reductase and 75 ng of dilauroylL-a-lecithin were added. P45O protein concentrations corresponded to 0.4
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Results Figure 1 shows representative PhIP metabolite profiles from mouse microsomes and purified rabbit P450 isozymes. Radioactivity for PhIP incubated with 3-MC-induced microsomes and no cofactors is shown (Figure 1 A) to be associated only with
40 44 4S
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Mutageniaty testing Fractions taken directly from the HPLC were tested for mutagenicity using Salmonella TA98. Fractions (100 /J) were tested both with and without rat liver Aroclor-induced S9 (2 mg protein/plate). Ames assays were conducted as previously described (33).
4
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Fraction (min) Fig. I. HPLC radiochromatograms of PhIP (A) and PhIP metabolites after I h of incubation with mouse 3-MC-induced hepatic microsomes (B). purified rabbit hepatic P450 forms 2 (C), 3c (D) 4 (E) and 6 (F). Incubations were conducted at 37°C for 1 h using 0.4 nmol/ml P450 protein or 2 mg/ml microsomes with 0.18 /iM PhIP. Peaks 1 and 2 on (B) were identified as 4'-hydroxy-PhIP and N-hydroxy-PhIP respectively.
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Synthesis of N-hydroxy-PhIP N-Hydroxy-PhIP was synthesized using a two-step procedure based upon modifications of previously descnbed methods for the synthesis of nitro-imidazoquinoxalines (30) and arylhydroxylamines (31). Briefly, 19 mg PhIP, dissolved in 1.5 ml of 5096 acetic acid in water, was added dropwise to a solution of 0.3 g NaNO^ in 0.7 ml water to produce nitro-PhlP. The reaction mixture was stirred at room temperature for 60 min and 1.5 ml ice-cold water was added. After centrifugation, the precipitate was washed in water, centrifuged again and dissolved in 5% methanol in chloroform. Nitro-PhlP was purified by TLC mesh chromatography (Kieselgel 60 PF, methanol/chloroform 5:95 v/v) and evaporated to dryness under nitrogen. Nitro-PhlP was then reduced to N-hydroxy-PhIP by combining 2.5 mg 5% palladium on carbon with 0.5 mg nitro-PhlP in 1 ml tetrahydrofuran, cooling on ice, adding 5 y.\ of hydrazine hydrate and stirring for 45 min. The catalyst was removed by centrifugation and the resulting product was stored at — 80°C. The N-hydroxy-PhIP solution was evaporated under a stream of nitrogen and reconstituted in methanol immediately prior to use.
nmol/ml of P450 protein for all the purified forms The concentration of PhIP used in all reactions was 18 ^M. Analytical Metabolites were isolated by HPLC using a Nucleosil C ! 8 reverse-phase column (0.46x25 cm). Elution was with a linear gradient starting from 27% methanol/ water, 0.1% diethylamine, pH 4.0 to 60% methanol/water, pH 4.0 at 37 min. The flow rate was 1 ml/min and fractions were collected at 1 min intervals. One ml volumes of the terminated incubations were used for HPLC analysis. Scintillation counting was done on a Packard Tricarb Counter, model 3255 (Downers Grove, IL) using 15 ml Instagel (Packard Inst. Co.) for each 0 5 min fraction collected. Mass spectra of N-hydroxy-PhIP and PhIP were obtained under positive-ion fast atom bombardment (FAB) conditions on a VG ZAB-HS-2F mass spectrometer. Methanolic solutions (2 fd) of PhIP or HPLC-purified N-hydroxy-PhIP were mixed with 2 IJL\ 3-nitrobenzyl alcohol on the FAB target. Ionization was effected using a beam of xenon atoms (8 keV, 1 mA). Daughter ion spectra were obtained by introducing helium into the second field region of the mass spectrometer and CAD/MIKE scans were performed (32). Low-resolution mass spectrometry was performed with a Hewlett-Packard 5985A mass spectrometer utilizing the direct inlet probe with a source temperature of 200°C. Proton NMR spectra were obtained with a General Electric model NT-200 Fourier transform NMR spectrometer. The spectrometer frequency was 200.071 MHz and the high power 90° pulse width was 7 /is. A spectral width of 2450 Hz was used with a 20 s recycle delay following a free induction decay. Either 16K or 32K were used in data acquisition.
Metabolism of Phi P
3000
N-hydroiy-PNP
molecule, we examined the metabolite profiles generated by purified-rabbit P450 forms 2, 3c, 4 and 6 in a reconstitution assay. Rabbit forms 2 and 3c (Figure 1C and D respectively) were unable to metabolize PUP to any detectable degree. The two metabolites found in microsomal assays with PhIP were also detected after incubations with rabbit P450 form 4 (Figure IE) and rabbit P450 form 6 (Figure IF). In both cases, the metabolites had the same retention times as the microsomal metabolites and were shown to co-elute under isocratic conditions (27% methanol/ water, pH 4.0). Under these conditions - 2 0 % of the PhIP incubated with rabbit P450 form 6 was converted to the two major metabolites. Only 5 % of the PhIP was metabolized by rabbit P450 form 4. In both cases the ratio of peak 2 to peak 1 was — 1.5. Mutagenicity of microsomal metabolites To assess the mutagenic activity of the metabolites, fractions were collected by HPLC at 1 min intervals and tested for mutagenicity using Salmonella strain TA98 both with and without additional metabolic activation by rat Aroclor-induced S9 (2 mg protein/ plate). Figure 2A shows the radioactivity for each fraction. Mutagenicity of the parent compound was apparent only in the presence of S9 in the Ames/Salmonella test (Figure 2B). Figure 2 shows that the metabolite in frations 32-34 (peak 2 from Figure 1) was mutagenic to Salmonella without additional activation by rat S9. The metabolite in fractions 13 — 15 (peak 1 from Figure 1), however, was not mutagenic at the levels tested, either with or without added S9. An additional unidentified mutagenic peak eluted in fractions 17—22 (Figure 2B). Identification of PhIP microsomal metabolites The structural identity of peak 1 as 4'-hydroxy-PhIP was determined by spectral characterization of the HPLC-purified metabolite generated in microsomal incubations. The electronimpact mass spectrum of metabolite 1 (Figure 3) yielded a molecular ion at mlz 240 corresponding to the addition of 16 mass units of PhIP. NMR spectra (Table I) demonstrated that the 4' proton was missing, suggesting that substitution occurred at this position. The spectral data was consistent with hydroxylation of the parent compound at the 4' position of the phenyl ring. The presence of the hydroxyl proton at this position was tentatively assigned to a broad singlet peak at 7.84 p.p.m. but had an area equivalent to 0.5 protons. This area reduction may be due to a trace amount of D2O in the solvent exchanging with the hydroxyl proton. Since metabolite peak 2 was directly mutagenic to Salmonella strain TA98, it likely corresponded to N-hydroxy-PhlP. Synthetic N-hydroxy-PhIP was prepared and shown to be directly mutagenic to Salmonella strain TA98 (data not shown). The synthetic N-hydroxy-PhIP also tested positive for ferric-ion 240
80 60 -
20 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 FRACTION #
Fig. 2. Profiles of U C radioactivity (A) and mutagemcity (B,C) detected in microsomal incubations fractionated by reverse-phase HPLC. Mutagenicity was determined on 100 /il of each fraction with (B) and without (C) metabolic activation by rat hepatic S9 in the Ames;'Salmonella assay as described in Materials and methods.
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.
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Fig. 3. Electron impact mass spectrum of metabolite peak 1 (4'-hydroxyPWP) isolated from incubations of PhIP with mouse microsomes. The spectrum were obtained as described in Materials and methods.
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Incubation of PhIP with 3-MC-induced mouse microsomes and cofactors (Figure IB) resulted in formation of two metabolites (peaks 1 and 2). Unmetabolized PhIP was also present. Production of these metabolites was linearly dependent on protein concentration, substrate concentration and time (for up to 60 min). Both metabolites were produced in approximately equal ratios at all substrate and protein concentrations as judged by I4 C radioactivity in the peaks. Microsomal protein and substrate concentrations were chosen to maintain a linear rate of metabolite production for the hour-long incubations. Under the conditions shown in Figure 1, - 4 3 % of the PhIP was metabolized, with 36% of the recovered radioactivity eluting as the two major metabolites (16% in peak 1 and 20% in peak 2). Complete (100%) metabolism of PhIP was never achieved under the conditions used here. Metabolic profiles with 3-MC-induced rabbit hepatic microsomes (based on UV detection at 315 nm, data not shown) were similar to that observed with mouse microsomes (Figure IB). To determine which of the rabbit forms of cytochrome P450 were specific for the metabolism of PhIP, and whether cytochrome P450 forms exhibited regio-selectivities for the PhIP
K.W.Turtettaub et al.
reduction (data not shown) as described by Belanger et al (34), which is indicative of hydroxylamines. Structural confirmation of the synthesized N-hydroxy-PhIP was made by positive-ion Tabte I. NMR assignments and coupling constants for PhIP and 4'-hydroxy-PhIP Position
4'-Hydroxy-PhIP (p.p.m.)
PhIP (p.p.m.)
N-CH3 2',6'-H NH2 4'-H 3',5'-H 4'-OH 7-H 5-H
3 56 (s) 6.81 (d, y == 7.5 Hz) 6.93 (broad s) 746 (d, y == 7 5 Hz) 7.84 (broad s) 7.66 (broad s) 8.20 (broad s)
3• 6 ( s ) 7 .72 (d, y .3 Hz) 7 .0 (broad s) 7 35 (t, y = 7. 3 Hz) 7 .48 (d, y = 7 .3 Hz) 7 .71 (d, y = 2 .1 Hz) 8 .31 (d, y = 2 .1 Hz)
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m/z Fig. 4. FAB daughter ion spectra of synthetic N-hydroxy-PhIP (A) and PhIP (B) showing the molecular ion at m/z = 225 for PhIP and m/z = 241 for the arylhydroxyl amine metabolite FAB spectra were obtained as described in Materials and methods.
Discussion These data strongly demonstrate the role of the rabbit P450 form 6 in the metabolism of PhIP. This isozyme corresponds to the P450IA1 form of cytochrome P450. These data also suggest a minor role for the rabbit P450 form 4 (P450IA2) in the metabolism of PhIP. Rabbit P450IA1 was substantially more active in the metabolism of PhIP than P450IA2. None of the other P450 isozymes utilized in this study metabolized PhIP to an appreciable extent. These results support the mutagenicity data reported in an earlier study which demonstrated that rabbit liver P450IA1 and P450IA2 were more efficient in the activation of PhIP to Salmonella mutagens than rabbit P450 forms HB1, HC3, IIIA6 or IVB1 (26). It was shown that P450IA1 was 3.1-fold more active than P450IA2 in metabolizing PhIP to a Salmonella mutagen, which is similar to what we found for the generation of N-hydroxy-PhIP with these isozymes. Our data show that the lack of mutagenicity found with P450IIB1 and P450IIIA6 is not due to the metabolism of PhIP to non-mutagenic products, rather it is due to the lack of recognition of PhIP as a substrate by these isozymes. These data also demonstrate that the metabolism of PhIP by P450IA1 and P450IA2 results in formation of two major metabolites. N-Hydroxy-PhIP appears to be directly involved in the activation of PhIP as has been demonstrated for other heterocyclic amines (19,26,35,36). Identification of N-hydroxyPhlP was based on the mass spectrum, co-chromatography with synthetic N-hydroxy-PhIP, reduction of ferric iron to ferrous iron, and direct mutagenic activity toward Salmonella TA98. NHydroxy-PhlP has recently been reported to be formed from incubations with rat primary hepatocytes as well (16). 4'-Hydroxy-PhIP appears to be a detoxification product since it was not mutagenic either with or without S9 (see Figure 5). Identification of this metabolite was based on the mass spectrum, co-chromatography and NMR. 4'-Hydroxy-PhIP has also
Mutagenicity
PHIP
4'-hydroxy-PhIP Fig. S. Proposed scheme for cytochrome P45O-dependent metabolism of PhIP
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.
FAB mass spectrometry [Figure 4A; characteristic ions at m/z 241 (M +H) + and daughter ions at 226 (M + H ) + - C H 3 , 224 (M + H) + - O H and 223 (M + H) + -H 2 O]. The daughter ion spectrum of PhIP is shown for comparison (Figure 4B). Synthetic N-hydroxy-PhIP and the metabolically derived metabolite 2 co-eluted using HPLC under isocratic conditions (data not shown). This suggested that the metabolically derived peak 2 was N-hydroxy-PhIP.
Metabolism of PhlP
Acknowledgements The authors wish to thank Julie Avila for conducting the Ames/Salmonella assays, Dr A.Daniel Jones for FAB mass spectral characterization, and Kenneth Carroll and Chuck Morns for electron impact mass spectral characterizations. This work has been performed under the auspices of the US Department of Energy by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48 and supported by Interagency agreement no. 222Y-0I-ES-10I58 between the National Institutes of Environmental Health Services and the Department of Energy; and by the Australian National Health and Medical Council, and the Anti-Cancer Foundation of the Universities of South Australia.
References 1. FeltonJ.S., Knize.M.G., Shen.N.H., Lewis.P.R., Andresen.B.D., HappeJ. and Hatch,F.T. (1986) The isolation and identification of a new mutagen from fried ground beef: 2-amino-l-methyl-6-phenylimidazo[4,5-A]pyridine (PhlP) Cardnogenesis, 7, 1081-1086 2.Becher,G., Knize,M.G., Nes.I.F. and FeltonJ.S. (1988) Isolation and identification of mutagens from a fried Norwegian meat product. Cardnogenesis, 9, 247-253. 3.Alink,G.M., Knize.M.G., Shen.N.H., Hesse.S.P. and FeltonJ.S. (1988) Mutagenicity of food pellets from human diets in the Netherlands Mutat. Res., 206, 387-393. 4. Zhang,X.-M., Wakabayashi.K., Liu,Z.-C., Sugimura.T. and Nagao.M. (1988) Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods. Mutat. Res., 201, 181-188 5. Taylor.R.T., Fultz.E., Knize.M.G. and FeltonJ.S. (1987) Formation of the fried ground beef mutagens 2-ami™>3-rnethylimidazo(4,5:/]quinoline (IQ) and 2-amino-l-mcthyl-6-phenylimidazot4,5-*]pyridine (PhlP from L-phenylalanine (Phe) + Creatinine (Cre) (or creatine) Environ. Mutagenesis, 9, (suppl. 8), 106. 6. Shioya.M., Wakabayashi.K., Sato.S., Nagao.M. and Sugimura.T. (1987) Formation of a mutagen, 2-amino-l-methyl-6-phenylimidazo(4,5-6J-pyridine
(PhlP) in cooked beef, by heating a mixture containing creatinine, phenylalanine and glucose. Mutat. Res., 191, 133-138. 7. FeltonJ.S., Knize.M.G., Wu,R. and Becher.G. (1988) Mutagenic heterocyclic imidazoazaarenes in cooked foods. In King.C.M., Romano.L.G. and Schuetzle,D.D. (eds), Carcinogenic and Mutagenic N-substituted aryl compounds. Elsevier, New York, pp. 73 — 85. 8.Ohgaki,H., Hasegawa.H., Suenaga,M., Sato.S., Takayama.S. and Sugimura,T. (1987) Carcinogenicity in mice of a mutagenic compound, 2-amincH3,8Kumetriyumidazo[4,5-y)quinoxalinc (MelQx) from cooked foods. Cardnogenesis, 8, 665-668. 9. Kato,T., Ohgaki.H., Hasegawa.H., Sato,S., Takayama.S. and Sugimura.T. (1988) Carcinogenicity in rats of a mutagenic compound, 2-amino-3,8dimethylimidazo[4,5-/lquinoxaline. Cardnogenesis, 9, 71—73. 10. Ohgaki.H., Kusama.K., Matsukura.N., Morino.K., Hasegawa.H., Sato,S., Takayama,S. and Sugimura.T. (1984) Carcinogenicity in mice of a mutagenic compound 2-amino-3-methylimidazo[4,5-/lquinoline, from broiled sardine, cooked beef and beef extract. Cardnogenesis, 5, 921-924. ll.Esumi.H., Ohgaki.H., Kohzen.E., Takayama,S. and Sugimura.T. (1989) Induction of lymphoma in CDF1 mice by the food mutagen, 2-amino-lmethyl-6-phenylimidazo[4,5-A]pyridine. Gann, 80, 1176-1178. 12.Tanaka,T., Barnes.W.S., Williams.G.M. and Weisburger.J. (1985) Multipotential carcinogenicity of the fried fish mutagen 2-amino-3methylimidazo[4,5-/]quinoline in rats. Gann, 76, 570—576. 13. Sugimura.T. (1985) Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process. Mutat. Res., 233, 312-318. 14. FeltonJ.S., Knize.M.G., Shen,N.H., Andresen.B.D., Bjeldanes.L.F. and Hatch,F.T., (1986) Identification of the mutagens in cooked beef. Environ. Health Perspea., 67, 17-24. 15. Thompson.L.H., TuckerJ.D., Stewart.S.A., Christensen.M.L., Salazar.E.P., Carrano,A.V. and FeltonJ.S. (1987) Genotoxicity of compounds from cooked beef in repair-deficient CHO cells versus Salmonella mutagenicity. Mutagenesis, 2, 483-487. 16 Holme.J.A., Wallin.H., Brunborg.G., Soderlund.E.J., HongsloJ.K. and Alexander,J. (1989) Genotoxicity of the food mutagen 2-amino-l-methyl-6-phenylimidazo{4,5-i>]pyridine (PhlP): formation of 2-hydroxyamino-PhIP, a directly acting genotoxic metabolite. Carcmogenesis, 10, 1389-1396. 17.Tucker,J.D., Carrano.A V., Allen.N.A., Chnstensen.M.L., Knize.M.G., Strout.C.L. and FeltonJ.S. (1989) In vivo cytogenetk effects of cooked food mutagens. Mutat. Res., 224, 105-113. 18. Yamashita.K., Umemoto.A., Grivas.S., Kato.S. and Sugimura.T. (1988) In vitro reaction of hydroxyamino derivatives of MelQx, Glu-P-1 and Trp-P-1 with DNA. 32P-postlabelling analysis of DNA adducts formed in vivo by the parent amine and in vitro by their hydroxyamino derivatives. Mutagenesis, 3, 515-520. 19. Okamoto.T., Shudo.K., Hashimoto,Y., Kosuge.T., Sugimura.T. and Nishimura.S. (1981) Identification of a reactive metabolite of the mutagen, 2-amino-3-methylimidazolo[4,5-/)quinoline. Chem. Pharm. Bull., 29, 590-593. 20. Yamazoe.Y., Abu-Zeid.M., Manabe.S., Toyama.S. and Kato.R. (1988) Metabolic activation of a protein pyrolysate promutagen 2-amino-3,8dimethylimidazo{4,5-/]quinoxaline by rat liver microsomes and purified cytochrome P^tfO. Caranogenesis, 9, 105-109. 21. McManus.M.E., Burgess.W., Synderwine.E. and Stupans.I. (1988) Specificity of rabbit cytochrome P-450 isozymes involved in the metabolic activation of the food derived mdagen 2-amino-3-methylimidazo[4,5-/)quinoline. Cancer Res., 48, 4513-4519. 22. Yamazoe.Y., Abu-Zeid.M., Yamauchi.K. and Kato.R. (1988) Metabolic activation of pyrolysate arylamines by human liver microsomes; possible involvement of a P-448-H type cytochrome P^t50. Gann, 79, 1159-1167. 23. Paterson,P. and ChipmanJ.K. (1987) Activation of 2-amino-3-methyIimidazo(4,5-y)quinoline in rat and human hepsUxytdSalmonella mutagenicity assays: the contribution of hepatic conjugation. Mutagenesis, 2, 137-140. 24. Saito.K., Shmonara.A., Kamatati.T. and Kato.R. (1985) Metabolite activation of mutagenic AMiydroxyarylamines by O-acetyltransferase in Salmonella typhimurium TA98. Arch. Biochem. Biophys., 239, 286-295. 25.Snyderwine,E.G., Wirth.PJ., RolIer.P.P., Adamson.R.H., Sato.S. and Thorgeirsson.S.S. (1988) Mutagenicity and in vitro covalent DNA binding of 2-hydroxyamino-3-methylimidazoIo{4,5-/]quinoline. Cardnogenesis, 9, 411-419. 26. McManus.M.E., FeltonJ.S., Knize.M.G., Burgess.W.M., RobertsThomson.S., Pond.S.M., Stupans.I. and Veronese.M.E. (1989) Activation of the food-derived mutagen 2-amino-l-nxmyl-6^)herrylimidazo(4^-i>)pvridine by rabbit and human liver microsomes and purified forms of cytochrome P-450. Cardnogenesis, 10, 357-363. 27. Knize.M.G. and FeltonJ.S. (1986) The synthesis of the cooked-beef mutagen
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recently been found in incubations with isolated rat hepatocytes and rat liver microsomes (37). In contrast to our data with rabbit isozymes, rat liver P450IA2 has recently been shown to be more active than the P450IA1 in the metabolism of PhlP; however, metabolism of rabbit P450IA1 was similar to that observed by Alexander et al. (37) with their source of rabbit P450IA1. These data suggest that the results reported here are not an anomaly of the present study but reflect differences in the selectivity of P450 isozymes for the AIAs by these two species. Our data also contrast with results reported for another AIA mutagen, 2-amino-3-methylimidazo[4,5-/| quinoline. Cytochrome P450IA2 was 8- to 10-fold more efficient than the P450IA1 isozyme in the activation of this compound to a mutagen (26). In summary, we have shown that the polycyclic hydrocarbon inducible isozymes P450IA1 and P450IA2 are active in the metabolism of PhlP. P450IA1 appears to be the primary isozyme responsible for the generation of PhlP metabolites by rabbit liver microsomes. Two major metabolites are the products of this reaction, with one being the N-hydroxylated intermediate and the other being 4'-hydroxylated PhlP. The N-hydroxy-PhTP intermediate is a direct-acting metabolite, suggesting that it is a proximate mutagenic chemical species, while the 4'-hydroxyPrdP is most likely a detoxified metabolite since it is not mutagenic towards Salmonella, even with added S9. The ultimate mutagenic metabolite has yet to be identified. Further metabolism of the N-hydroxy-PhIP metabolite probably results in the production of reactive intermediates which can bind to DNA (see Figure 5 for scheme). Results in our laboratory suggest that acetylation is not requisite in the activation of N-hydroxy-PhIP and sulfation or other pathways may be more important in the activation of this compound (unpublished data). Additional studies to further characterize the activation and detoxification pathways of PhlP metabolism are currently underway in our laboratory.
K.W.Turteltaub et al.
Received on November 20, 1989; revised on March 6, 1990; accepted on March 16, 1990
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2-amino-6-phenylimidazo[4,5-i]pyridine and its 3-methyl isomer. Heterocycles, 24, 1815-1819. 28 Turteltaub.K.W , Knize.M G., Healy.S.K., Tucker.J.D. and Felton.J S. (1989) The metabolic disposition of 2-amino-6-phenylimidazo[4,5-fc]pyridine (PhIP) in the induced mouse. Food Chem. Toncol., 27, 667-673 29. FeltonJ.S. and Nebert,D.W. (1975) Mutagenesis of certain activated carcinogens in vitro: associated with genetically mediated increases in monooxygenase activity and cytochrome P|-450. /. Biol. Chem., 250, 6769-6778. 30. Grivas.S. (1988) Synthesis of 3,8-dimethyl-2-3//-imidazo[4,5-y]quinoxaline, the 2-nitro analogue of the food carcinogen MelQx. J. Chem. Res., 2, 84 31 WestraJ.G. (1981) A rapid and simple synthesis of reactive metabolites of carcinogenic aromatic amines in high yield. Carcinogenesis, 2, 347 — 357. 32. Winter.C.K., Segall.H.J. and Jones.A.D. (1987) Distribution of trans4-hydroxy-2-hexenal and tandem mass spectrometric detection of its urinary mercapturic acid in the rat. Drug Metab. Dispos., 15, 608—612. 33. Maron.D M. and Ames.B.N. (1983) Revised method for the Salmonella mutagemcity test. Muiat. Res., 113, 173-215 34. Belanger.P.M., Grech-Belanger.O. and Blouin.M. (1981) Colorimetric determination of M-hydroxylated metabolites in microsomal studies. Anal. Biochem., 118, 47-52. 35. Weisburgcr.E.K. (1978) Mechanisms of chemical carcinogenesis Annu. Rev. Pharmacol. Toxicol, 18, 395-415. 36 Kato.R. and Yamazoe.Y. (1987) Metabolic activation and covalent binding to nucleic acids of carcinogenic heterocyclic amines from cooked foods and amino acid pyrolysates. Gann, 78, 297-311. 37. Wallin.H., Mikalsen.A , Guengerich.F.P., Ingelman-Sundbcrg,M., Solberg.K.E., Rossland.O.S. and Alexander,J. (1990) Differential rates of metabolic activation and detoxication of the food mutagen 2-amino-l-methyl6-phenylimidazo{4,5-ft]pyridine by different cytochrome P450 enzymes Carcinogenesis, 11, 489-492.
Metabolism of 2-ainnLmo-l-metlhyl-6-pIhe]iiylliimdazo[4,S-Z>] pyridime (PMP) by liver microsomes and isolated rabbit cytochrome P4S© isozymes
K.W.Turteltaub, M.G.Knize, M.H.Buonarati, M.E.McManus', M.E.Veronese1, J.A.Mazrimas and J.S.Felton Biomedical Sciences Division, Lawrence Livermore National Laboratory, PO Box 5507, Livermore, CA 94550, USA and 'Department of Clinical Pharmacology, School of Medicine, Flinders University of South Australia, Bedford Park 5042, Australia
Introduction 2-Amino-I-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP*), a potent Salmonella and mammalian cell mutagen, was originally identified in fried beef and has since been reported in cooked pork, fish and sausage products (1 —4). PhlP is believed to be formed via the condensation of creatine and phenylalanine when meat is cooked at temperatures ranging from 150 to 25O°C (5,6). These temperatures approximate cooking conditions employed in the normal Western household, which suggests PhlP may be a potential risk factor in human carcinogenesis. To understand the cancer risk posed by exposure to PhlP, it is necessary to elucidate its activation and detoxification pathways in the animal models used to define the carcinogenicity of PhlP. The work presented here is an initial step towards that end. PhlP belongs to the amino-imidazoazaarene (AIA) family of compounds (7). Several members of this family, including PhlP, have been found to produce tumors in multiple species and at multiple sites (8—13). Thus the AIA mutagens may constitute •Abbreviations: PhJP, 2-amino-l-rnethyl-6-phenylimidazo{4,5-t]pyridine; ALA, amino-imidazoazaarene; TCDD, 2,3,7,8-tetrachlorodibenzo-/7-dioxin; 4'-hydroxyPWP, 2-amino-l-rnethyl-4'-hydroxy-6-phenylirrudazo(4,5-ft]pyridine; rutro-PhIP, 2-nitro-l-methyl-6-phenylimidaio[4,5-^]pyrkline; 3-MC, 3-methylcholanthrene; FAB, fast atom bombardment.
Materials and methods Chemicals [l4C]PhIP and PhlP were synthesized in our laboratory as previously described (27,28). Radiochemical punty was shown to be >98% by HPLC. 2-Nitro-lmethyl-6-phenylimidazo[4,5-6]pyridine (nitro-PhIP) standard was the kind gift of Dr S.Grivas, Swedish University of Agricultural Sciences. All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO). The Ames/'Salmonella tester strain TA98 was thelcind gift of Dr Bruce Ames, (University of California, Berkeley, CA).
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The cytochrome P450-dependent metabolism of the heterocyclic amine mutagen 2-amino-l-methyl-6-phenylimidazo [4,5-A]pyridine (PhlP) has been determined. We investigated the in vitro metabolism of PhlP by polycyclic hydrocarboninduced mouse and rabbit liver microsomes, and by purified rabbit liver P450 isozymes. Following a 60 min incubation, 3-methylcbolanthrene-induced mouse microsomes converted 36% of the PhlP to two major metabolites, N-hydroxy-PhIP and 4-hydroxy-PhIP, with 43% total metabolism. Rabbit P450 form 6 and form 4 produced the same two major metabolites (20 and 5% total metabolism respectively). Additional metabolites were produced in low yields and amounts varied depending on the isozyme used (1-5%). Metabolites were not detected in incubations of PhlP with P450 forms 2 and 3C. N-Hydroxy-PhIP was found to be directly mutagenic to Salmonella TA98, while the 4'-hydroxyPhlP was not mutagenic either with or without additional metabolic activation. These data suggest that the cytochrome P450IA isozymes are involved in the metabolism of PhlP by rabbit liver and that formation of N-hydroxy-PhIP is involved in the mutagenicity of PhlP.
a significant dietary risk for humans, particularly for cancers of the colon and gastrointestinal tract, since these tissues would have direct contact with these compounds. PhlP may be particularly important in this regard, since it has been reported to account for - 7 5 % , by mass, of the AIAs found in well-done cooked beef and 91% in fried fish (4,14). PhlP contains a phenylpyridine structure which differentiates it from its quinoline and quinoxaline AIA counterparts. PhlP is the least potent heterocyclic amine mutagen isolated from meat in Salmonella mutation assays, yet is the most potent clastogen in Chinese hamster ovary cells grown in culture (14,15). PhlP has recently been shown to cause DNA strand breaks in suspensions of rat hepatocytes, and is —4 times more potent than 2-amino-3,8-dimethylimidazo[4,5-/|quinoxaline in the induction of sister chromatid exchange in V79 cells (16). PhlP also has been shown to cause sister chromatid exchange in mouse bone marrow and in peripheral blood (17). These data suggest that a number of tissues may be susceptible to the genotoxic effects of PhlP. The genotoxic activity of PhlP, like other heterocyclic amine mutagens, presumably depends on metabolism of the compound to electrophilic intermediates. None of the AIAs are mutagenic in vitro without activation. Activation of 2-amino-3-methylimidazo[4,5-/|quinoline and 2-amino-3,8-dimethylimidazo[4,5-/] quinoxaline has been shown to require oxidation of the exocyclic amino group to the corresponding hydroxyamino derivative by cytochrome P450IA1 and P450IA2 (18-22). This is believed to be followed by esterification to N-acetoxy metabolites (23 -25). In an earlier report (26), PhlP was shown to be mutagenic to Salmonella in the presence of 2,3,7,8-tetrachlorodibenzo-/?-dioxin (TCDD)-induced rabbit liver and lung microsomes whereas control, acetone-induced, phenobarbital-induced and rifampicininduced rabbit liver homogenates were less effective. These data suggested the involvement of the P450IA family of monooxygenases in the activation of PhlP. Purified rabbit P450 forms 4 (P450IA2) and 6 (P450IA1) also were shown to be efficient in converting PhlP to a Salmonella mutagen, while forms 5 (P450IV1), 2 (P450ITB1), 3b (P450HC3) and 3c (P450IIIA6) were inactive (26). In the present paper we have isolated and identified the metabolic products of [ C]PhTP formed by mouse and rabbit cytochromes P450. The major metabolites produced were 2-amino-l-methyl-4'-hydroxy-6-phenylimidazo[4,5-£] pyridine (4'-hydroxy-PhIP) and 2-hydroxyamino-l-methyl-6phenylimidazo[4,5-fc]pyridine (N-hydroxy-PhIP), with the latter metabolite being the mutagenic species.
K.W.Turtdtaub et al. Animals C57BL/6N male mice were obtained from Simonsen Farms (Gilroy, CA). Animals were housed individually on hard-wood bedding in polystyrene cages with free access to food and water. Animals were induced with a single i.p. injection of 3-methylcholanthrene (3-MC; 80 mg/kg in corn oil) and killed 2 days later. Livers were harvested and immediately used to prepare microsomes (29). Preparation of rabbit microsomes and isolation of P450 isozymes were carried out as previously reported (26). Microsomes and purified P45Os were stored at —80°C until use.
In vitro metabolism PhIP was incubated with 3-MC-induced mouse hepatic microsomes, TCDDrnduced rabbit hepatic microsomes and highly purified rabbit cytochrome P45O forms 2, 3c, 4 and 6. Assays with mouse and rabbit microsomes were conducted in a buffer consisting of 8 mM MgCI2, 33 mM KC1 and 200 mM phosphate, pH 7.4. The protein concentration used for the mouse and rabbit TCDD-induced microsomes was 2 mg/ml. Reactions were initiated after a 3 min preincubation at 37°C by the addition of cofactor (5 mM glucose-6-phosphate, 5 mM NADPH, and 4 mM NADP final concentration). The total volume of each reaction mixture was 0 5 ml. Reactions were terminated with the addition of an equal volume of methanol Incubations using the isolated P450 forms were conducted in a similar fashion except that 0.5 unit of NADPH-cytochrome P450 reductase and 75 ng of dilauroylL-a-lecithin were added. P45O protein concentrations corresponded to 0.4
0
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Results Figure 1 shows representative PhIP metabolite profiles from mouse microsomes and purified rabbit P450 isozymes. Radioactivity for PhIP incubated with 3-MC-induced microsomes and no cofactors is shown (Figure 1 A) to be associated only with
40 44 4S
3-MCMouu
e
Mutageniaty testing Fractions taken directly from the HPLC were tested for mutagenicity using Salmonella TA98. Fractions (100 /J) were tested both with and without rat liver Aroclor-induced S9 (2 mg protein/plate). Ames assays were conducted as previously described (33).
4
A
1 1 1 1 1 1 0 2 4 2 1 1 2 1 1 4 0 4 4 4 1
Form 2
10 8 6 4 2 00
4
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16 20 24
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44
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Fraction (min) Fig. I. HPLC radiochromatograms of PhIP (A) and PhIP metabolites after I h of incubation with mouse 3-MC-induced hepatic microsomes (B). purified rabbit hepatic P450 forms 2 (C), 3c (D) 4 (E) and 6 (F). Incubations were conducted at 37°C for 1 h using 0.4 nmol/ml P450 protein or 2 mg/ml microsomes with 0.18 /iM PhIP. Peaks 1 and 2 on (B) were identified as 4'-hydroxy-PhIP and N-hydroxy-PhIP respectively.
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Synthesis of N-hydroxy-PhIP N-Hydroxy-PhIP was synthesized using a two-step procedure based upon modifications of previously descnbed methods for the synthesis of nitro-imidazoquinoxalines (30) and arylhydroxylamines (31). Briefly, 19 mg PhIP, dissolved in 1.5 ml of 5096 acetic acid in water, was added dropwise to a solution of 0.3 g NaNO^ in 0.7 ml water to produce nitro-PhlP. The reaction mixture was stirred at room temperature for 60 min and 1.5 ml ice-cold water was added. After centrifugation, the precipitate was washed in water, centrifuged again and dissolved in 5% methanol in chloroform. Nitro-PhlP was purified by TLC mesh chromatography (Kieselgel 60 PF, methanol/chloroform 5:95 v/v) and evaporated to dryness under nitrogen. Nitro-PhlP was then reduced to N-hydroxy-PhIP by combining 2.5 mg 5% palladium on carbon with 0.5 mg nitro-PhlP in 1 ml tetrahydrofuran, cooling on ice, adding 5 y.\ of hydrazine hydrate and stirring for 45 min. The catalyst was removed by centrifugation and the resulting product was stored at — 80°C. The N-hydroxy-PhIP solution was evaporated under a stream of nitrogen and reconstituted in methanol immediately prior to use.
nmol/ml of P450 protein for all the purified forms The concentration of PhIP used in all reactions was 18 ^M. Analytical Metabolites were isolated by HPLC using a Nucleosil C ! 8 reverse-phase column (0.46x25 cm). Elution was with a linear gradient starting from 27% methanol/ water, 0.1% diethylamine, pH 4.0 to 60% methanol/water, pH 4.0 at 37 min. The flow rate was 1 ml/min and fractions were collected at 1 min intervals. One ml volumes of the terminated incubations were used for HPLC analysis. Scintillation counting was done on a Packard Tricarb Counter, model 3255 (Downers Grove, IL) using 15 ml Instagel (Packard Inst. Co.) for each 0 5 min fraction collected. Mass spectra of N-hydroxy-PhIP and PhIP were obtained under positive-ion fast atom bombardment (FAB) conditions on a VG ZAB-HS-2F mass spectrometer. Methanolic solutions (2 fd) of PhIP or HPLC-purified N-hydroxy-PhIP were mixed with 2 IJL\ 3-nitrobenzyl alcohol on the FAB target. Ionization was effected using a beam of xenon atoms (8 keV, 1 mA). Daughter ion spectra were obtained by introducing helium into the second field region of the mass spectrometer and CAD/MIKE scans were performed (32). Low-resolution mass spectrometry was performed with a Hewlett-Packard 5985A mass spectrometer utilizing the direct inlet probe with a source temperature of 200°C. Proton NMR spectra were obtained with a General Electric model NT-200 Fourier transform NMR spectrometer. The spectrometer frequency was 200.071 MHz and the high power 90° pulse width was 7 /is. A spectral width of 2450 Hz was used with a 20 s recycle delay following a free induction decay. Either 16K or 32K were used in data acquisition.
Metabolism of Phi P
3000
N-hydroiy-PNP
molecule, we examined the metabolite profiles generated by purified-rabbit P450 forms 2, 3c, 4 and 6 in a reconstitution assay. Rabbit forms 2 and 3c (Figure 1C and D respectively) were unable to metabolize PUP to any detectable degree. The two metabolites found in microsomal assays with PhIP were also detected after incubations with rabbit P450 form 4 (Figure IE) and rabbit P450 form 6 (Figure IF). In both cases, the metabolites had the same retention times as the microsomal metabolites and were shown to co-elute under isocratic conditions (27% methanol/ water, pH 4.0). Under these conditions - 2 0 % of the PhIP incubated with rabbit P450 form 6 was converted to the two major metabolites. Only 5 % of the PhIP was metabolized by rabbit P450 form 4. In both cases the ratio of peak 2 to peak 1 was — 1.5. Mutagenicity of microsomal metabolites To assess the mutagenic activity of the metabolites, fractions were collected by HPLC at 1 min intervals and tested for mutagenicity using Salmonella strain TA98 both with and without additional metabolic activation by rat Aroclor-induced S9 (2 mg protein/ plate). Figure 2A shows the radioactivity for each fraction. Mutagenicity of the parent compound was apparent only in the presence of S9 in the Ames/Salmonella test (Figure 2B). Figure 2 shows that the metabolite in frations 32-34 (peak 2 from Figure 1) was mutagenic to Salmonella without additional activation by rat S9. The metabolite in fractions 13 — 15 (peak 1 from Figure 1), however, was not mutagenic at the levels tested, either with or without added S9. An additional unidentified mutagenic peak eluted in fractions 17—22 (Figure 2B). Identification of PhIP microsomal metabolites The structural identity of peak 1 as 4'-hydroxy-PhIP was determined by spectral characterization of the HPLC-purified metabolite generated in microsomal incubations. The electronimpact mass spectrum of metabolite 1 (Figure 3) yielded a molecular ion at mlz 240 corresponding to the addition of 16 mass units of PhIP. NMR spectra (Table I) demonstrated that the 4' proton was missing, suggesting that substitution occurred at this position. The spectral data was consistent with hydroxylation of the parent compound at the 4' position of the phenyl ring. The presence of the hydroxyl proton at this position was tentatively assigned to a broad singlet peak at 7.84 p.p.m. but had an area equivalent to 0.5 protons. This area reduction may be due to a trace amount of D2O in the solvent exchanging with the hydroxyl proton. Since metabolite peak 2 was directly mutagenic to Salmonella strain TA98, it likely corresponded to N-hydroxy-PhlP. Synthetic N-hydroxy-PhIP was prepared and shown to be directly mutagenic to Salmonella strain TA98 (data not shown). The synthetic N-hydroxy-PhIP also tested positive for ferric-ion 240
80 60 -
20 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 FRACTION #
Fig. 2. Profiles of U C radioactivity (A) and mutagemcity (B,C) detected in microsomal incubations fractionated by reverse-phase HPLC. Mutagenicity was determined on 100 /il of each fraction with (B) and without (C) metabolic activation by rat hepatic S9 in the Ames;'Salmonella assay as described in Materials and methods.
0
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.
200 220
—,-H—,—i—,—i—,—, 240 260 280 300 320 m/z
Fig. 3. Electron impact mass spectrum of metabolite peak 1 (4'-hydroxyPWP) isolated from incubations of PhIP with mouse microsomes. The spectrum were obtained as described in Materials and methods.
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Incubation of PhIP with 3-MC-induced mouse microsomes and cofactors (Figure IB) resulted in formation of two metabolites (peaks 1 and 2). Unmetabolized PhIP was also present. Production of these metabolites was linearly dependent on protein concentration, substrate concentration and time (for up to 60 min). Both metabolites were produced in approximately equal ratios at all substrate and protein concentrations as judged by I4 C radioactivity in the peaks. Microsomal protein and substrate concentrations were chosen to maintain a linear rate of metabolite production for the hour-long incubations. Under the conditions shown in Figure 1, - 4 3 % of the PhIP was metabolized, with 36% of the recovered radioactivity eluting as the two major metabolites (16% in peak 1 and 20% in peak 2). Complete (100%) metabolism of PhIP was never achieved under the conditions used here. Metabolic profiles with 3-MC-induced rabbit hepatic microsomes (based on UV detection at 315 nm, data not shown) were similar to that observed with mouse microsomes (Figure IB). To determine which of the rabbit forms of cytochrome P450 were specific for the metabolism of PhIP, and whether cytochrome P450 forms exhibited regio-selectivities for the PhIP
K.W.Turtettaub et al.
reduction (data not shown) as described by Belanger et al (34), which is indicative of hydroxylamines. Structural confirmation of the synthesized N-hydroxy-PhIP was made by positive-ion Tabte I. NMR assignments and coupling constants for PhIP and 4'-hydroxy-PhIP Position
4'-Hydroxy-PhIP (p.p.m.)
PhIP (p.p.m.)
N-CH3 2',6'-H NH2 4'-H 3',5'-H 4'-OH 7-H 5-H
3 56 (s) 6.81 (d, y == 7.5 Hz) 6.93 (broad s) 746 (d, y == 7 5 Hz) 7.84 (broad s) 7.66 (broad s) 8.20 (broad s)
3• 6 ( s ) 7 .72 (d, y .3 Hz) 7 .0 (broad s) 7 35 (t, y = 7. 3 Hz) 7 .48 (d, y = 7 .3 Hz) 7 .71 (d, y = 2 .1 Hz) 8 .31 (d, y = 2 .1 Hz)
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m/z Fig. 4. FAB daughter ion spectra of synthetic N-hydroxy-PhIP (A) and PhIP (B) showing the molecular ion at m/z = 225 for PhIP and m/z = 241 for the arylhydroxyl amine metabolite FAB spectra were obtained as described in Materials and methods.
Discussion These data strongly demonstrate the role of the rabbit P450 form 6 in the metabolism of PhIP. This isozyme corresponds to the P450IA1 form of cytochrome P450. These data also suggest a minor role for the rabbit P450 form 4 (P450IA2) in the metabolism of PhIP. Rabbit P450IA1 was substantially more active in the metabolism of PhIP than P450IA2. None of the other P450 isozymes utilized in this study metabolized PhIP to an appreciable extent. These results support the mutagenicity data reported in an earlier study which demonstrated that rabbit liver P450IA1 and P450IA2 were more efficient in the activation of PhIP to Salmonella mutagens than rabbit P450 forms HB1, HC3, IIIA6 or IVB1 (26). It was shown that P450IA1 was 3.1-fold more active than P450IA2 in metabolizing PhIP to a Salmonella mutagen, which is similar to what we found for the generation of N-hydroxy-PhIP with these isozymes. Our data show that the lack of mutagenicity found with P450IIB1 and P450IIIA6 is not due to the metabolism of PhIP to non-mutagenic products, rather it is due to the lack of recognition of PhIP as a substrate by these isozymes. These data also demonstrate that the metabolism of PhIP by P450IA1 and P450IA2 results in formation of two major metabolites. N-Hydroxy-PhIP appears to be directly involved in the activation of PhIP as has been demonstrated for other heterocyclic amines (19,26,35,36). Identification of N-hydroxyPhlP was based on the mass spectrum, co-chromatography with synthetic N-hydroxy-PhIP, reduction of ferric iron to ferrous iron, and direct mutagenic activity toward Salmonella TA98. NHydroxy-PhlP has recently been reported to be formed from incubations with rat primary hepatocytes as well (16). 4'-Hydroxy-PhIP appears to be a detoxification product since it was not mutagenic either with or without S9 (see Figure 5). Identification of this metabolite was based on the mass spectrum, co-chromatography and NMR. 4'-Hydroxy-PhIP has also
Mutagenicity
PHIP
4'-hydroxy-PhIP Fig. S. Proposed scheme for cytochrome P45O-dependent metabolism of PhIP
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.
FAB mass spectrometry [Figure 4A; characteristic ions at m/z 241 (M +H) + and daughter ions at 226 (M + H ) + - C H 3 , 224 (M + H) + - O H and 223 (M + H) + -H 2 O]. The daughter ion spectrum of PhIP is shown for comparison (Figure 4B). Synthetic N-hydroxy-PhIP and the metabolically derived metabolite 2 co-eluted using HPLC under isocratic conditions (data not shown). This suggested that the metabolically derived peak 2 was N-hydroxy-PhIP.
Metabolism of PhlP
Acknowledgements The authors wish to thank Julie Avila for conducting the Ames/Salmonella assays, Dr A.Daniel Jones for FAB mass spectral characterization, and Kenneth Carroll and Chuck Morns for electron impact mass spectral characterizations. This work has been performed under the auspices of the US Department of Energy by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48 and supported by Interagency agreement no. 222Y-0I-ES-10I58 between the National Institutes of Environmental Health Services and the Department of Energy; and by the Australian National Health and Medical Council, and the Anti-Cancer Foundation of the Universities of South Australia.
References 1. FeltonJ.S., Knize.M.G., Shen.N.H., Lewis.P.R., Andresen.B.D., HappeJ. and Hatch,F.T. (1986) The isolation and identification of a new mutagen from fried ground beef: 2-amino-l-methyl-6-phenylimidazo[4,5-A]pyridine (PhlP) Cardnogenesis, 7, 1081-1086 2.Becher,G., Knize,M.G., Nes.I.F. and FeltonJ.S. (1988) Isolation and identification of mutagens from a fried Norwegian meat product. Cardnogenesis, 9, 247-253. 3.Alink,G.M., Knize.M.G., Shen.N.H., Hesse.S.P. and FeltonJ.S. (1988) Mutagenicity of food pellets from human diets in the Netherlands Mutat. Res., 206, 387-393. 4. Zhang,X.-M., Wakabayashi.K., Liu,Z.-C., Sugimura.T. and Nagao.M. (1988) Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods. Mutat. Res., 201, 181-188 5. Taylor.R.T., Fultz.E., Knize.M.G. and FeltonJ.S. (1987) Formation of the fried ground beef mutagens 2-ami™>3-rnethylimidazo(4,5:/]quinoline (IQ) and 2-amino-l-mcthyl-6-phenylimidazot4,5-*]pyridine (PhlP from L-phenylalanine (Phe) + Creatinine (Cre) (or creatine) Environ. Mutagenesis, 9, (suppl. 8), 106. 6. Shioya.M., Wakabayashi.K., Sato.S., Nagao.M. and Sugimura.T. (1987) Formation of a mutagen, 2-amino-l-methyl-6-phenylimidazo(4,5-6J-pyridine
(PhlP) in cooked beef, by heating a mixture containing creatinine, phenylalanine and glucose. Mutat. Res., 191, 133-138. 7. FeltonJ.S., Knize.M.G., Wu,R. and Becher.G. (1988) Mutagenic heterocyclic imidazoazaarenes in cooked foods. In King.C.M., Romano.L.G. and Schuetzle,D.D. (eds), Carcinogenic and Mutagenic N-substituted aryl compounds. Elsevier, New York, pp. 73 — 85. 8.Ohgaki,H., Hasegawa.H., Suenaga,M., Sato.S., Takayama.S. and Sugimura,T. (1987) Carcinogenicity in mice of a mutagenic compound, 2-amincH3,8Kumetriyumidazo[4,5-y)quinoxalinc (MelQx) from cooked foods. Cardnogenesis, 8, 665-668. 9. Kato,T., Ohgaki.H., Hasegawa.H., Sato,S., Takayama.S. and Sugimura.T. (1988) Carcinogenicity in rats of a mutagenic compound, 2-amino-3,8dimethylimidazo[4,5-/lquinoxaline. Cardnogenesis, 9, 71—73. 10. Ohgaki.H., Kusama.K., Matsukura.N., Morino.K., Hasegawa.H., Sato,S., Takayama,S. and Sugimura.T. (1984) Carcinogenicity in mice of a mutagenic compound 2-amino-3-methylimidazo[4,5-/lquinoline, from broiled sardine, cooked beef and beef extract. Cardnogenesis, 5, 921-924. ll.Esumi.H., Ohgaki.H., Kohzen.E., Takayama,S. and Sugimura.T. (1989) Induction of lymphoma in CDF1 mice by the food mutagen, 2-amino-lmethyl-6-phenylimidazo[4,5-A]pyridine. Gann, 80, 1176-1178. 12.Tanaka,T., Barnes.W.S., Williams.G.M. and Weisburger.J. (1985) Multipotential carcinogenicity of the fried fish mutagen 2-amino-3methylimidazo[4,5-/]quinoline in rats. Gann, 76, 570—576. 13. Sugimura.T. (1985) Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process. Mutat. Res., 233, 312-318. 14. FeltonJ.S., Knize.M.G., Shen,N.H., Andresen.B.D., Bjeldanes.L.F. and Hatch,F.T., (1986) Identification of the mutagens in cooked beef. Environ. Health Perspea., 67, 17-24. 15. Thompson.L.H., TuckerJ.D., Stewart.S.A., Christensen.M.L., Salazar.E.P., Carrano,A.V. and FeltonJ.S. (1987) Genotoxicity of compounds from cooked beef in repair-deficient CHO cells versus Salmonella mutagenicity. Mutagenesis, 2, 483-487. 16 Holme.J.A., Wallin.H., Brunborg.G., Soderlund.E.J., HongsloJ.K. and Alexander,J. (1989) Genotoxicity of the food mutagen 2-amino-l-methyl-6-phenylimidazo{4,5-i>]pyridine (PhlP): formation of 2-hydroxyamino-PhIP, a directly acting genotoxic metabolite. Carcmogenesis, 10, 1389-1396. 17.Tucker,J.D., Carrano.A V., Allen.N.A., Chnstensen.M.L., Knize.M.G., Strout.C.L. and FeltonJ.S. (1989) In vivo cytogenetk effects of cooked food mutagens. Mutat. Res., 224, 105-113. 18. Yamashita.K., Umemoto.A., Grivas.S., Kato.S. and Sugimura.T. (1988) In vitro reaction of hydroxyamino derivatives of MelQx, Glu-P-1 and Trp-P-1 with DNA. 32P-postlabelling analysis of DNA adducts formed in vivo by the parent amine and in vitro by their hydroxyamino derivatives. Mutagenesis, 3, 515-520. 19. Okamoto.T., Shudo.K., Hashimoto,Y., Kosuge.T., Sugimura.T. and Nishimura.S. (1981) Identification of a reactive metabolite of the mutagen, 2-amino-3-methylimidazolo[4,5-/)quinoline. Chem. Pharm. Bull., 29, 590-593. 20. Yamazoe.Y., Abu-Zeid.M., Manabe.S., Toyama.S. and Kato.R. (1988) Metabolic activation of a protein pyrolysate promutagen 2-amino-3,8dimethylimidazo{4,5-/]quinoxaline by rat liver microsomes and purified cytochrome P^tfO. Caranogenesis, 9, 105-109. 21. McManus.M.E., Burgess.W., Synderwine.E. and Stupans.I. (1988) Specificity of rabbit cytochrome P-450 isozymes involved in the metabolic activation of the food derived mdagen 2-amino-3-methylimidazo[4,5-/)quinoline. Cancer Res., 48, 4513-4519. 22. Yamazoe.Y., Abu-Zeid.M., Yamauchi.K. and Kato.R. (1988) Metabolic activation of pyrolysate arylamines by human liver microsomes; possible involvement of a P-448-H type cytochrome P^t50. Gann, 79, 1159-1167. 23. Paterson,P. and ChipmanJ.K. (1987) Activation of 2-amino-3-methyIimidazo(4,5-y)quinoline in rat and human hepsUxytdSalmonella mutagenicity assays: the contribution of hepatic conjugation. Mutagenesis, 2, 137-140. 24. Saito.K., Shmonara.A., Kamatati.T. and Kato.R. (1985) Metabolite activation of mutagenic AMiydroxyarylamines by O-acetyltransferase in Salmonella typhimurium TA98. Arch. Biochem. Biophys., 239, 286-295. 25.Snyderwine,E.G., Wirth.PJ., RolIer.P.P., Adamson.R.H., Sato.S. and Thorgeirsson.S.S. (1988) Mutagenicity and in vitro covalent DNA binding of 2-hydroxyamino-3-methylimidazoIo{4,5-/]quinoline. Cardnogenesis, 9, 411-419. 26. McManus.M.E., FeltonJ.S., Knize.M.G., Burgess.W.M., RobertsThomson.S., Pond.S.M., Stupans.I. and Veronese.M.E. (1989) Activation of the food-derived mutagen 2-amino-l-nxmyl-6^)herrylimidazo(4^-i>)pvridine by rabbit and human liver microsomes and purified forms of cytochrome P-450. Cardnogenesis, 10, 357-363. 27. Knize.M.G. and FeltonJ.S. (1986) The synthesis of the cooked-beef mutagen
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recently been found in incubations with isolated rat hepatocytes and rat liver microsomes (37). In contrast to our data with rabbit isozymes, rat liver P450IA2 has recently been shown to be more active than the P450IA1 in the metabolism of PhlP; however, metabolism of rabbit P450IA1 was similar to that observed by Alexander et al. (37) with their source of rabbit P450IA1. These data suggest that the results reported here are not an anomaly of the present study but reflect differences in the selectivity of P450 isozymes for the AIAs by these two species. Our data also contrast with results reported for another AIA mutagen, 2-amino-3-methylimidazo[4,5-/| quinoline. Cytochrome P450IA2 was 8- to 10-fold more efficient than the P450IA1 isozyme in the activation of this compound to a mutagen (26). In summary, we have shown that the polycyclic hydrocarbon inducible isozymes P450IA1 and P450IA2 are active in the metabolism of PhlP. P450IA1 appears to be the primary isozyme responsible for the generation of PhlP metabolites by rabbit liver microsomes. Two major metabolites are the products of this reaction, with one being the N-hydroxylated intermediate and the other being 4'-hydroxylated PhlP. The N-hydroxy-PhTP intermediate is a direct-acting metabolite, suggesting that it is a proximate mutagenic chemical species, while the 4'-hydroxyPrdP is most likely a detoxified metabolite since it is not mutagenic towards Salmonella, even with added S9. The ultimate mutagenic metabolite has yet to be identified. Further metabolism of the N-hydroxy-PhIP metabolite probably results in the production of reactive intermediates which can bind to DNA (see Figure 5 for scheme). Results in our laboratory suggest that acetylation is not requisite in the activation of N-hydroxy-PhIP and sulfation or other pathways may be more important in the activation of this compound (unpublished data). Additional studies to further characterize the activation and detoxification pathways of PhlP metabolism are currently underway in our laboratory.
K.W.Turteltaub et al.
Received on November 20, 1989; revised on March 6, 1990; accepted on March 16, 1990
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2-amino-6-phenylimidazo[4,5-i]pyridine and its 3-methyl isomer. Heterocycles, 24, 1815-1819. 28 Turteltaub.K.W , Knize.M G., Healy.S.K., Tucker.J.D. and Felton.J S. (1989) The metabolic disposition of 2-amino-6-phenylimidazo[4,5-fc]pyridine (PhIP) in the induced mouse. Food Chem. Toncol., 27, 667-673 29. FeltonJ.S. and Nebert,D.W. (1975) Mutagenesis of certain activated carcinogens in vitro: associated with genetically mediated increases in monooxygenase activity and cytochrome P|-450. /. Biol. Chem., 250, 6769-6778. 30. Grivas.S. (1988) Synthesis of 3,8-dimethyl-2-3//-imidazo[4,5-y]quinoxaline, the 2-nitro analogue of the food carcinogen MelQx. J. Chem. Res., 2, 84 31 WestraJ.G. (1981) A rapid and simple synthesis of reactive metabolites of carcinogenic aromatic amines in high yield. Carcinogenesis, 2, 347 — 357. 32. Winter.C.K., Segall.H.J. and Jones.A.D. (1987) Distribution of trans4-hydroxy-2-hexenal and tandem mass spectrometric detection of its urinary mercapturic acid in the rat. Drug Metab. Dispos., 15, 608—612. 33. Maron.D M. and Ames.B.N. (1983) Revised method for the Salmonella mutagemcity test. Muiat. Res., 113, 173-215 34. Belanger.P.M., Grech-Belanger.O. and Blouin.M. (1981) Colorimetric determination of M-hydroxylated metabolites in microsomal studies. Anal. Biochem., 118, 47-52. 35. Weisburgcr.E.K. (1978) Mechanisms of chemical carcinogenesis Annu. Rev. Pharmacol. Toxicol, 18, 395-415. 36 Kato.R. and Yamazoe.Y. (1987) Metabolic activation and covalent binding to nucleic acids of carcinogenic heterocyclic amines from cooked foods and amino acid pyrolysates. Gann, 78, 297-311. 37. Wallin.H., Mikalsen.A , Guengerich.F.P., Ingelman-Sundbcrg,M., Solberg.K.E., Rossland.O.S. and Alexander,J. (1990) Differential rates of metabolic activation and detoxication of the food mutagen 2-amino-l-methyl6-phenylimidazo{4,5-ft]pyridine by different cytochrome P450 enzymes Carcinogenesis, 11, 489-492.
