Differential metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rat and human hepatocytes
Sophie Langouët,4,
Axel Paehler1,
Dieter H. Welti1,
Nathalie Kerriguy,
André Guillouzo and
Robert J. Turesky1,2,3,4
INSERM U456, Faculté de Pharmacie, Université de Rennes I, 35043 Rennes, France,
1 Nestlé Research Center, Nestec Ltd, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland and
2 Division of Chemistry, National Center for Toxicological Research, Jefferson, AR 72079, USA
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Abstract
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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 N2 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 N2 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 N2 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.
Abbreviations: CID, collision-induced dissociation; CYP, cytochrome P450; EROD, ethoxyresorufin O-deethylation; FCS, fetal calf serum; HAA, heterocyclic aromatic amine; LC-ESI-MS, liquid chromatography electrospray ionization mass spectrometry; 3-MC, 3-methylcholanthrene; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NOE, nuclear Overhauser enhancement; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 4'-HO-PhIP, 2-amino-4'-hydroxyl-1-methyl-6-phenylimidazo[4,5-b]pyridine; HONH-PhIP, 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine5-HO-PhIP, 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine; PhIP-4'-OSO3H, 4-(2-amino-1-methylimidazo[4,5-b]pyridin-6-yl)phenyl sulfate; HON-PhIP-N2-Gl, N2-ß-D-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine; PhIP-N2-Gl, N2-ß-D-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; HON-PhIP-N3-Gl, N3-ß-D-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine; PhIP-N3-Gl, N3-ß-D-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine; PhIP-4'-O-Gl; 4'-ß-D-glucosiduronyloxy-2-amino-4'-hydroxyl-1-methyl-6-phenylimidazo[4,5-b]pyridine.
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Introduction
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The heterocyclic aromatic amine (HAA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent bacterial mutagen and rodent carcinogen formed in cooked meats, fish and poultry (1), beer and wine (2) and cigarette smoke condensate (3). PhIP and several metabolites have been detected in urine of humans following consumption of grilled meats and PhIP has also been identified as a major mutagen in the urine of smokers (4,5). These findings reveal that human exposure to PhIP is extensive and that this carcinogen is readily absorbed and extensively metabolized in humans. Consequently, PhIP has been implicated as an etiological factor in some common forms of human cancers associated with consumption of cooked meats and cigarette smoking, including colorectal, prostate and breast cancer (68).
PhIP must undergo metabolism to exert its genotoxic effects and cytochrome P450 (CYP)1A2 is a key enzyme involved in the activation of PhIP to a genotoxin (9). Recent studies have shown that there are important catalytic differences between human and rodent CYPs in the oxidation of PhIP and other HAAs to produce the genotoxic N-hydroxy-HAA metabolites (10,11). The catalytic efficiency of the human ortholog is 19-fold greater than the rat enzyme in formation of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), suggesting that carcinogenicity data from the rat may underestimate the human health risk of this genotoxin (10). The biological potency of PhIP-induced carcinogenicity is strongly dependent upon its metabolism and a comprehensive understanding of the metabolism of PhIP in humans is essential for human risk assessment of this carcinogen. Primary hepatocytes are an excellent system to investigate diverse pathways of xenobiotic metabolism where cofactors are present at physiological concentrations and biotransformation pathways may closely simulate those which occur in vivo (12). Since liver is by far the most metabolically active tissue in the biotransformation of PhIP in rodents and humans (13), our objective was to analyze the balance between the bioactivation and detoxification reactions of PhIP in human and rat hepatocytes for interspecies extrapolations, which may aid in the risk assessment of this HAA.
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Materials and methods
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Chemicals
Culture media were obtained from Eurobio (Les Ulis, France) and fetal calf serum (FCS) from Dominique Dutcher (SA Brumath, France). Collagenase was purchased from Boehringer Mannheim (Indianapolis, IN). 3-Methylcholanthrene (3-MC), bovine serum albumin, bovine insulin and alamethicin were from Sigma Chemical Co. (St Louis, MO). Furafylline was purchased from Ultrafine Chemicals (Manchester, UK). PhIP and [2-14C]PhIP (10 mCi/mmol) were purchased from Toronto Research Chemicals (Ontario, Canada). 4-(2-Amino-1-methylimdazo[4,5-b]pyridin-6-yl)phenyl sulfate (PhIP-4'-OSO3H) and 4'-ß-D-glucosiduronyloxy-2-amino-4'-hydroxyl-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-4'-O-Gl) were kindly provided by Dr K.Turteltaub (Lawrence Livermore Laboratory, CA). Human liver sample HL G was kindly provided by Dr F.Kadlubar (National Center for Toxicological Research, Jefferson, AR). Rodent and human liver microsomes were prepared as previously described (10,14).
Cell isolation, culture and metabolism studies with PhIP
Human liver samples were obtained from four patients undergoing liver resection for primary or secondary hepatomas. Access to this material was in agreement with French laws and fulfilled the requirements of the local ethics committee. Hepatocytes were isolated by a two-step collagenase perfusion procedure as previously described (12,15). Cell viability was 7085%, as estimated by the trypan blue exclusion test. Hepatocytes of adult male SpragueDawley rats (150200 g) were prepared by the same procedure, with 8090% cell viability.
Both human and rat liver parenchymal cells were seeded at a density of 106 viable cells/35 cm2 dish in 2 ml of Williams' medium supplemented with 0.2% bovine insulin, 3.2% bovine serum albumin, 1% glutamine, 0.1% penicillin/streptomycin, 0.2% gentamycin and 10% FCS (all v/v). This medium supplemented with 7x105 M hydrocortisone hemisuccinate but lacking FCS was renewed daily. [2-14C]PhIP (at 10 mCi/mmol, dissolved in Me2SO) was added with medium renewal 3648 h after cell seeding for various times (248 h) at concentrations of 1, 10 or 50 µM without medium changes. Control cultures received the vehicle alone at a concentration of 0.1% (v/v), a concentration that had no detectable cytotoxic effect (data not shown). The incubations were terminated by collecting the culture medium. The cells were rinsed three times with phosphate-buffered saline and material was immediately stored at 80°C until analysis.
Experiments with the CYP1A2 inhibitor furafylline (16) were done 3648 h after cell seeding. Cells were treated with various concentrations of furafylline (0.1, 1 or 5 µM) for 24 h. [2-14C]PhIP (10 mCi/mmol, 1 µM) was then added and incubation was conducted for 24 h. Induction of CYP in rat hepatocytes was done by pretreatment with 3-MC (5 µM) prior to incubation with PhIP using the same protocol. There was no evidence of morphological alterations or cytotoxicity with these treatments.
Purification and analysis of metabolites
The cells were scraped with a spatula, combined with the culture medium and then added to 3 vol chilled acetonitrile, followed by removal of the precipitated material by centrifugation. After evaporation to dryness, extracts were analyzed by HPLC with a Varian Vista 5000 system (Basel, Switzerland) equipped with a Supelco C18 DB column (5 µm, 4.6x250 mm) connected to a Berthold LB 506 C-1 radioactivity monitor (Regensdorf, Switzerland). Metabolites were eluted at a flow rate of 1 ml/min using 20 mM diethylamine acetate buffer (pH 5.0) and methanol, with a linear gradient of 4070% methanol over 40 min, followed by 70100% methanol over 45 min, holding at 100% methanol for an additional 5 min (17). For incubations containing PhIP at 1 µM fractions, were collected (30 s) and measured by liquid scintillation counting. On-line acquisition of UV/visible spectra were acquired under the same HPLC conditions except that a Hewlett Packard 1090M HPLC (Geneva, Switzerland) equipped with a diode array UV/visible detection system was employed. Recovery of radioactivity from hepatocyte extracts and spiking experiments with 14C-labeled reference metabolites of PhIP was
85%. Estimation of metabolites was done in duplicate for incubations of PhIP and measurements were within ±15%.
Enzyme activity assays
Ethoxyresorufin O-deethylation (EROD) associated with CYP1A1/2 activity was measured in cultured living hepatocytes essentially as described by Burke and Mayer (18). The reaction rates were linear with time and proportional to protein concentration. Cellular protein content was estimated by the Bradford procedure (19). Values are means ± SD of triplicate measurements.
Biosynthetic and synthetic reactions
2-Amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-HO-PhIP) was prepared by incubation of PhIP (100 µM) and 1 mM NADPH with liver microsomal protein (2 mg/ml) of rats pretreated with polychlorinated biphenyls as previously reported (10). The enzymatic glucuronidation of PhIP and HONH-PhIP was done with human, rat or rabbit liver microsomes using 2 mg microsomal protein/ml (60 µg alamethicin/mg protein), 100 µM PhIP or HNOH-PhIP substrate, 5 mM UDP-glucuronic acid in 100 mM TrisHCl, pH 7.5, containing 5 mM MgCl2 as previously reported (20). Saccharolactone was omitted as it was found to inhibit the reaction (unpublished observations). These biosynthetic reactions with human and rodent liver microsomes resulted in formation of the isomeric N2- and N3-glucuronide conjugates of PhIP and HNOH-PhIP, which were distinguished by their characteristic UV spectra (20,21) and further characterized by mass spectrometry.
HONH-PhIP was prepared by reduction of 2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine as previously reported and the chemical purity exceeded 95% as determined by HPLC with UV detection (22). For preparation of the putative N-acetoxy-PhIP intermediate and its stable breakdown product 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-PhIP) (23,24), a 50-fold molar excess of acetic anhydride was added to HONH-PhIP (5 mg) in 10 ml of 100 mM phosphate buffer, pH 7.4, preincubated at 37°C. The solution was incubated for 1 h at 37°C and the reaction products isolated by applying the solution to a C-18 SepPak column prewashed with methanol followed by water. After washing the cartridge with water, 5-HO-PhIP was eluted with methanol. 5-HO-PhIP was further purified by HPLC on a Supelcosil C18 column (5 µm, 4.6x250 mm; Supelco, Buchs, Switzerland) with a linear gradient of 10% methanol in 20 mM diethylamine acetate at pH 7.0 to 100% methanol over 35 min at a flow rate of 1 ml/min. 5-HO-PhIP eluted at 18.5 min, showing a characteristic UV spectrum with an absorption maximum at 375 nm (23). A second chromatographic purification was performed applying a gradient from 10% methanol in 0.01% aqueous ammonia to 100% methanol over 30 min at a flow rate of 1 ml/min.
1H NMR and mass spectrometry
Proton NMR spectra were recorded on a DPX-360 spectrometer at 360.13 MHz with a selective 5-mm 1H probe head (Bruker AG, Fällanden, Switzerland). The parameters for basic 1-dimensional spectroscopy and nuclear Overhauser effect (NOE) difference spectra were similar to those previously described (25). The purified 5-HO-PhIP was placed under vacuum (0.02 mbar) in order to remove the HPLC solvents and organic acids and then the sample was prepared in Me2SO-d6 in a glove box under argon. Chemical shifts are reported in p.p.m. downfield from internal tetramethylsilane.
Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analyses were carried out in a Finnigan MAT TSQ-7000 mass spectrometer (Bremen, Germany) connected to a Finnigan electrospray API II interface and a Hewlett Packard 1100 pump (Geneva, Switzerland). The electrospray interface was operated with a high voltage of 3.5 kV and a capillary temperature of 280°C. Nitrogen was used as the carrier gas at a pressure of 80 p.s.i. The mass spectrometer operated in positive ionization mode, using full scan mode from 100 to 500 Da. Tandem MS/MS analyses of the metabolites were carried out after collision-induced dissociation (CID) of the protonated molecules of each of the metabolites. A collision energy of 2035 eV was used for metabolites and 35 eV for PhIP. Argon was used as the collision gas at a pressure of 2.7 mTorr. Metabolites were analyzed by infusion with a Harvard syringe pump (Harvard Apparatus, South Natick, MA) at a flow rate of 10 µl/min.
Statistical analyses
One-way ANOVA and post-test for linear trend were measured to determine statistical significance and conducted with GraphPad Software (San Diego, CA).
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Results
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PhIP biotransformation in human and rat hepatocytes and identification of major metabolites
The metabolism of PhIP was investigated at substrate concentrations of 1, 10 and 50 µM in four different human hepatocyte preparations, untreated rat hepatocytes and rat hepatocytes pretreated with 3-MC. Metabolite formation was analyzed by HPLC with UV and radioactive detection. Representative HPLC profiles of PhIP metabolites formed in hepatocytes are presented in Figure 1
. Products were identified by co-elution with authentic metabolites prepared biosynthetically and by their corresponding UV and LC-ESI-MS product ion spectra. This approach led to the identification of seven prominent metabolites and included: 4'-HO-PhIP, PhIP-4'-OSO3, PhIP-4'-O-Gl, N2-ß-D-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N2-Gl), N3-ß-D-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo [4,5-b]pyridine (HON-PhIP-N3-Gl), N2-ß-D-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-N2-Gl) and N3-ß-D-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-N3-Gl) (Figure 2
). As an example, the UV and LC-ESI-MS product ion spectra of the isomeric HON-PhIP-N2-Gl and HON-PhIP-N3-Gl metabolites are displayed in Figure 3
, and are consistent with previous spectral data supporting these proposed structures (17,20,21). In addition to these metabolites, three additional minor metabolites were formed in human hepatocytes but not in rat hepatocytes. The identities of these metabolites are unknown.

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Fig. 1. HPLC Radiochromatogram of PhIP metabolism in (A) rat hepatocytes, (B) rat hepatocytes pretreated with 3-MC and (C) human hepatocytes. Metabolites were analyzed 24 h after treatment with PhIP (10 µM).
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Fig. 3. On-line UV spectra and LC-ESI-MS product ion mass spectra of (A) NOH-PhIP-N2-Gl and (B) HON-PhIP-N3-Gl formed in human hepatocytes. The loss of 16 Da from the HONH-PhIP fragment derived from both conjugates may be attributed to loss of CH4 and requires further investigation.
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We examined the formation of 5-HO-PhIP, a novel oxidation product of PhIP, which was previously detected in rat liver S9 preparations and urine of rats treated with PhIP (24). 5-HO-PhIP is not formed by direct hepatic CYP-mediated oxidation of PhIP (10,13), but through a rearrangement of the reactive N-acetoxy or N-sulfonyloxy esters of HONH-PhIP (24). Thus, this product may be an indirect measure of the biologically effective dose. We prepared 5-HO-PhIP by reaction of HONH-PhIP in the presence of acetic anhydride to generate N-acetoxy-PhIP in situ, which underwent rearrangement into several products that were characterized by LC-ESI-MS/MS. Based upon the product ion spectra of the protonated molecules, the chemical structures of the reaction and breakdown products of HONH-PhIP were compatible with the hydroxamic acid derivative of PhIP (26), the deaminated 2-hydroxylated derivative of PhIP and several PhIP dimer products including hydrazino, hydrazo and azoxy linkages (data not shown). There was no evidence for the formation of 4'-HO-PhIP through rearrangement of N-acetoxy-PhIP, but 5-HO-PhIP was formed in
10% yield from this rearrangement reaction. This product was further characterized by 1H NMR.
1H NMR data for PhIP and 5-HO-PhIP
The 1H NMR spectral data for 5-HO-PhIP are presented along with the values for PhIP (Table I
), which are in good agreement with previously published data (27). The assignments of the 5-H and 7-H proton shift values were based upon 1-dimensional NOE difference experiments with pre-irradiation of the N-CH3 group, indicating proximity between the 7-H proton and the N-CH3 group of PhIP. A similar NOE effect was observed for the putative 5-HO-PhIP metabolite, with an effect on the unmodified 7-H proton upon pre-irradiation of the N-CH3 group. The NOE difference experiment, which was not reported previously (23), permitted unambiguous assignment of the site of hydroxylation. Consequently, the 5-H proton of PhIP is absent in the metabolite, with a new signal for the 5-HO group present at 5.40 p.p.m. appearing as a broad singlet. D2O exchange experiments revealed the presence of two D2O exchangeable signals, those of the 5-HO proton at 5.40 p.p.m. and of the free amino group at 6.73 p.p.m. The shift values of 5-HO-PhIP match those reported by Reistad et al. (23) except for the N-CH3 group; we observed a shift difference of
0.1 p.p.m., which might result from differences in sample preparation (i.e. concentration, pH or residual water content).
LC-ESI-MS product ion spectra for PhIP, 4'-HO-PhIP and 5-HO-PhIP
The isomeric oxidation products 5-HO-PhIP and 4'-HO-PhIP are readily distinguished by their LC-ESI-MS product ion spectra (Figure 4
). The protonated molecules [M+H]+ are present at m/z 241, 16 Da greater than PhIP [M+H]+ at m/z 225, indicating the presence of an oxygen atom. The major fragment ions of PhIP are observed at m/z 210 [M+HCH3·]+ and m/z 183 [M+HCH3·HCN]+. In the case of 5-HO-PhIP, the major fragment ion occurs through loss of water at m/z 223 [M+HH2O]+, with other ions detected at m/z 226 [M+HCH3·]+, 213 [M+HCO]+, 208 [M+HH2OCH3·]+ and 196 [M+HH2OHCN]+. The product ion mass spectrum of 4'-HO-PhIP under similar CID conditions reveals that the principal fragmentation occurs through cleavage of the N-methylimidazole moiety to produce the radical cation species m/z 226 [M+HCH3·]+. Under these tandem LC-ESI/MS/MS conditions there was little evidence for dehydration at m/z 223 [M+HH2O]+. Other notable fragment ions were detected at m/z 213 [M+HCO]+ and 198 [M+HCH3·CO]+, which are indicative of the phenolic group.

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Fig. 4. LC-ESI-MS product ion mass spectra of PhIP, 5-HO-PhIP and 4'-HO-PhIP. The precursor ion of m/z 196 in the spectrum of 5-HO-PhIP was observed at m/z 223, indicating rearrangement of a hydrogen atom to the C-5 atom of this species, followed by loss of HCN.
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We assayed for the formation of 5-HO-PhIP in hepatocytes by on-line radioactive monitoring and UV detection at 375 nm, a characteristic maximum for this PhIP oxidation product (23), and by tandem LC-ESI-MS/MS monitoring the transitions from [M+H]+ to [M+H18] and [M+H45]+. We confirmed the presence of 5-HO-PhIP in hepatocytes of rats pretreated with 3-MC (
0.1% of the dose), but not in untreated rat or any of the human hepatocyte populations.
The effect of concentration on PhIP metabolism in human and rat hepatocytes
The effect of PhIP concentration on metabolite formation in human and rat hepatocytes was investigated and the results are summarized in Table II
. N-Oxidation was the major pathway of PhIP metabolism in human hepatocytes at all concentrations, while 4'-hydroxylation of PhIP was the predominant pathway in rat hepatocytes. In the human hepatocyte populations the metabolism of PhIP was variable, which may be attributed in part to the different amounts of CYP1A2 expressed in these preparations. There were differences between the human and rat UDP-glucuronosyltransferases in preferred sites of conjugation to PhIP. Conjugation by the human enzyme(s) occurred largely at the N2 positions of PhIP and HONH-PhIP, while the N3 atom was the preferred site of conjugation for the rat enzyme(s). At a low PhIP concentration (1 µM) the three unknown metabolites were more prevalent than at a higher concentration of PhIP (50 µM) (Table II
). In rat hepatocytes 3-MC pretreatment led to a 10-fold induction of EROD activity while the overall percentage of biotransformed PhIP increased by 5-fold at 1 µM, 4.5-fold at 10 µM and only 1.5-fold at 50 µM. This increased metabolism was reflected in an increase in 4'-HO-PhIP and HONH-PhIP product formation, as well as by increased amounts of PhIP-N3-Gl.
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Table II. Percent Distribution of PhIP Metabolites Formed as a Function of Dose in Four Human Hepatocyte Preparations and Rat Hepatocytes after Incubation for 24 ha
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The contribution of CYP1A2 to PhIP metabolism in human hepatocytes
The contribution of CYP1A2 to the N-oxidation and 4'-hydroxylation of PhIP in human hepatocytes was assessed by preincubation of hepatocytes with the selective, mechanism-based CYP1A2 inhibitor furafylline (16). Furafylline resulted in a strong, concentration-dependent inhibition of HNOH-PhIP glucuronide conjugate formation with >90% inhibition of product formation at a concentration of 5 µM furafylline. In contrast to these N-hydroxylated metabolites, the amount of PhIP-4'-OSO3H remained unchanged, while the amount of PhIP-4'-O-Gl increased by >2-fold (Figure 5
). These results indicate that human CYP1A2 is the major enzyme involved in metabolic activation of PhIP to the genotoxic HONH-PhIP metabolite in human hepatocytes, while 4'-HO-PhIP formation is catalyzed by other enzymes. Unknown metabolites 1 and 3 increased as a function of furafylline pretreatment, while the amount of unknown metabolite 2 decreased by
25%.

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Fig. 5. Effect of furafylline on PhIP metabolism in human hepatocytes. Cells were pretreated with furafylline (0, 0.1, 1 or 5 µM) prior to treatment with PhIP (1 µM).Two-independent analyses were performed and the estimation of metabolite formation was within 15%. One-way ANOVA (P < 0.01) and the post-test linear trend (P < 0.05) were observed for PhIP-4'-O-Gl, PhIP-N3-Gl, PhIP-N2-Gl, HON-PhIP-N2-Gl, HON-PhIP-N3-Gl and PhIP.
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Discussion
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This is the first study that has examined metabolism of the dietary carcinogen PhIP in both human and rat hepatocytes and important interspecies differences were observed. Previous investigations have reported that rodent hepatocytes transform PhIP into a number of different products (13,28), many of which were also identified in this study. The lowest concentration used in this study (1 µM) approaches human exposure levels (1,29), where both PhIP and a number of these metabolites, including the isomeric glucuronide conjugates of HONH-PhIP, were detected in urine of humans exposed to a single dose of PhIP (30,31). The N2-glucuronide conjugates of PhIP and HONH-PhIP are the major metabolites formed in human hepatocytes and human urine, indicating that the metabolism of PhIP in vitro in hepatocytes may be extrapolated to in vivo in humans.
Human hepatocyte preparations were significantly more active than rat hepatocytes in conversion of PhIP to the genotoxic metabolite HNOH-PhIP, based upon the formation of the stable N2 and N3 isomeric glucuronide conjugates HON-PhIP-N2-Gl and HON-PhIP-N3-Gl. These two metabolites accounted for 3560% of the PhIP added in the three active human hepatocyte preparations exposed to 1 µM PhIP, while the amounts formed in untreated and 3-MC-pretreated rat hepatocytes ranged from 11 to 26% of the added PhIP. Moreover, three of the four human hepatocyte preparations examined were more active at N-oxidation of PhIP than rat hepatocytes pretreated with the potent CYP1A2 inducer 3-MC, although possible differences between human and rat UDP-glucuronsyltransferase activities cannot be excluded (20). The effect of drug pretreatment on CYP1A2 expression and activity in human hepatocytes cannot be readily ascertained since the basal enzyme levels in these subjects prior to medical treatment are unknown. However, previous studies in our laboratory have shown that EROD activity in hepatocytes of human subjects (n = 19) in primary culture under the same culture conditions varied from 0.2 to 8 pmol/min/mg protein (12), values which are within the range observed for these four hepatocyte populations.
We previously reported that the catalytic efficiency of N-oxidation of PhIP by human CYP1A2 was 19-fold greater than that of the rat ortholog (10,11). The higher efficiency of the human enzyme is attributed to a lower Km value; the Km for HONH-PhIP formation by recombinant human CYP1A2 is 12 µM, while the Km value is 160 µM for the rat enzyme (10). Comparable Km values were also measured in human and rat liver microsomes (10). Therefore, the differences in the rates of N-oxidation of PhIP observed between human and rat liver microsomes and purified CYP1A2 orthologs are also observed in primary hepatocyte cultures.
Whereas metabolic activation of PhIP through N-oxidation predominates in human hepatocytes, the detoxication of PhIP through 4'-hydroxylation is the principal biotransformation pathway in rat hepatocytes, where 4'-HO-PhIP and conjugated metabolites account for as much as 20 and 47% of the PhIP (1 µM), respectively, in untreated and 3-MC pretreated cells. These findings are consistent with previous observations on PhIP metabolism with rodent hepatocytes (28) and studies showed that 4'-HO-PhIP formation is catalyzed by rat CYP1A1 and CYP1A2 (32). 4'-HO-PhIP, as well as the sulfate and glucuronide conjugates, is also formed in human hepatocytes, but to a much smaller extent. However, 4'-HO-PhIP formation is not catalyzed by human CYP1A2, based upon inhibition experiments with furafylline (vide supra). This observation is in agreement with our previous study showing that 4'-HO-PhIP formation was not detected in human liver microsomes (n = 51) fortified with NADPH or purified recombinant human CYP1A2 and suggests that CYP enzymes in human liver are not involved in this oxidation process (10).
The oxidation product 5-HO-PhIP has been proposed as a biomarker to indirectly measure the amount of reactive genotoxic N-acetoxy and N-sulfonyloxy esters of HONH-PhIP (24). We confirmed the formation of 5-HO-PhIP through the decomposition of N-acetoxy-PhIP synthesized in situ. However, the amount of this product present in hepatocytes is very low, suggesting that the reactive esters of HONH-PhIP are effectively scavenged by glutathione S-transferases (33). Moreover, the decomposition of N-acetoxy-PhIP in situ did not result in generation of 4'-HO-PhIP, further reinforcing the notion that 4'-HO-PhIP formation is not catalyzed by CYP1A2 directly or indirectly through solvolysis of reactive esters of HONH-PhIP.
In contrast to PhIP, the contribution of CYP1A2-mediated N-oxidation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), another HAA, in human hepatocytes appeared to be less important, where the N2-glucuronide conjugate of hydroxyamino-MeIQx accounted for 510% of the MeIQx added under comparable incubation conditions (14), although differences in rates of UDP-glucuronosyltransferase conjugation of the respective N-hydroxy HAAs cannot be excluded. In the case of MeIQx, CYP1A2 catalyzes formation of the 8-carboxylic acid derivative, a major biotransformation pathway of detoxification in both hepatocytes and in humans exposed to MeIQx (14). We observed little evidence of human CYP1A2-mediated detoxification products of PhIP in human hepatocytes or human liver microsomes (10).
Hepatocytes in primary culture represent a suitable model for metabolism studies to elucidate competing pathways of metabolism under physiological conditions and to aid in interspecies extrapolation of biochemical mechanisms of genotoxicity. The target organs of PhIP-induced cancer in humans are believed to be colon, prostate and breast (68). The capacity of these organs to activate PhIP to the genotoxic HONH-PhIP species is considerably lower than liver and a significant portion of the biologically effective dose of PhIP may be formed in liver and then undergo circulation to these target organs (13). Thus, human hepatocytes are an ideal system to elucidate HAA metabolism and also to assess the potential of chemoprotective agents to modulate HAA genotoxicity in liver and extrahepatic tissues. The effects of various chemoprotective agents on HAA activity in hepatocytes are under current investigation.
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Notes
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3 Present address: National Center for Toxicological Research, Jefferson, AR 72079-9502, USA 
4 To whom correspondence should be addressed Email: sophie.langouet{at}rennes.inserm.fr; rturesky{at}nctr.fda.gov 
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Acknowledgments
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We thank Mme J.Markovic, Nestlé Research Center, for excellent technical assistance, and Dr F.Kadlubar, NCTR, for valuable comments on this manuscript. This work was supported in part by the Institut National de la Santé et de la Recherche Médicale and the Association pour la Recherche contre le Cancer (S.L., N.K. and A.G.). S.L. was the recipient of a fellowship from the Association pour la Recherche contre le Cancer and N.K. was supported by EEC contract EUROCYP, BM-CT96-0254.
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References
|
---|
-
Felton,J.S. and Knize,M.G. (1990) Heterocyclic amine mutagens/carcinogens in foods. In Cooper,C.S. and Grover,P.L. (eds) Handbook of Experimental Pharmacology. Springer-Verlag, Berlin, Germany, Vol. 94/I, pp. 471502.
-
Manabe,S., Suzuki,H., Wada,O. and Ueki,A. (1993) Detection of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in beer and wine. Carcinogenesis, 14, 899901.[Abstract]
-
Manabe,S., Tohyama,K., Wada,O. and Aramaki,T. (1991) Detection of a carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, in cigarette smoke condensate. Carcinogenesis, 12, 19451947.[Abstract]
-
Peluso,M., Castegnaro,M., Malaveille,C., Friesen,M., Garren,L., Hautefeuille,A., Vineis,P., Kadlubar,F. and Bartsch,H. (1991) 32P postlabelling analysis of urinary mutagens from smokers of black tobacco implicates 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as a major DNA-damaging agent. Carcinogenesis, 12, 713717.[Abstract]
-
Bartsch,H., Malaveille,C., Friesen,M., Kadlubar,F.F. and Vineis,P. (1993) Black (air-cured) and blond (flue-cured) tobacco cancer risk. IV: Molecular dosimetry studies implicate aromatic amines as bladder carcinogens. Eur. J. Cancer, 29A, 11991207.
-
Willett,W.C. (1995) Diet, nutrition, and avoidable cancer. Environ. Health Perspect., 103 (suppl. 8), 165170.[Medline]
-
Sugimura,T. (2000) Nutrition and dietary carcinogens. Carcinogenesis, 21, 387395.[Abstract/Free Full Text]
-
Shirai,T., Sano,M., Tamano,S., Takahashi,S., Hirose,M., Futakuchi,M., Hasegawa,R., Imaida,K., Matsumoto,K., Wakabayashi,K., Sugimura,T. and Ito,N. (1998) The prostate: a target for carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) derived from cooked foods. Cancer Res., 57, 195198.[Abstract]
-
Kato,R. (1986) Metabolic activation of mutagenic heterocyclic aromatic amines from protein pyrolysates. CRC Crit. Rev. Toxicol., 16, 307348.[ISI]
-
Turesky,R.J., Constable,A., Richoz,J., Varga,N., Markovic,J., Martin,M.V. and Guengerich,F.P. (1998) Activation of heterocyclic aromatic amines by rat and human liver microsomes and by purified rat and human cytochrome P450 1A2. Chem. Res. Toxicol., 11, 925936.[ISI][Medline]
-
Turesky,R.J., Constable,A., Fay,L.B. and Guengerich,F.P. (1999) Interspecies differences in metabolism of heterocyclic aromatic amines by rat and human P450 1A2. Cancer Lett., 143, 109112.[ISI][Medline]
-
Guillouzo,A. (1998) Liver cell models in in vitro toxicology. Environ. Health Perspect., 106 (suppl.), 511532.[ISI][Medline]
-
King,R.S., Kadlubar,F.F. and Turesky,R.J. (2000) In vivo metabolism of heterocyclic amines. In Nagao,M. and Sugimura,T. (eds) Food Borne Carcinogens: Heterocyclic Amines. John Wiley & Sons, Chichester, UK.
-
Langouët,S., Welti,D.H., Kerriguy,N., Fay,L.B., Huynh-Ba,T., Markovic,J., Guengerich,F.P., Guillouzo,A. and Turesky,R.J. (2001) Metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in human hepatocytes: 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid is a major detoxification pathway catalyzed by cytochrome P450 1A2. Chem. Res. Toxicol., 14, 211221.[ISI][Medline]
-
Guguen-Guillouzo,C., Campion,J.P., Brissot,P., Glaise,D., Launois,B., Bourel,M. and Guillouzo,A. (1982) High yield preparation of isolated human adult hepatocytes by enzymatic perfusion of the liver. Cell Biol. Int. Rep., 6, 625628.[ISI][Medline]
-
Kunze,K.L. and Trager,W.F. (1993) Isoform-selective mechanism-based inhibition of human cytochrome P450 1A2 by furafylline. Chem. Res. Toxicol., 6, 649656.[ISI][Medline]
-
Kaderlik,K.R., Minchin,R.F., Mulder,G.J., Ilett,K.F., Daugaard-Jenson,M., Teitel,C.H. and Kadlubar,F.F. (1994) Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues. Carcinogenesis, 15, 17031709.[Abstract]
-
Burke,M.D. and Mayer,R.T. (1983) Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450 binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins). Chem. Biol. Interact., 45, 243258.[ISI][Medline]
-
Bradford,M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248254.[ISI][Medline]
-
Nowell,S.A., Massengill,J.S., Williams,S., Radominska-Pandya,A., Tephly,T.R., Cheng,Z., Strassburg,C.P., Tukey,R.H., MacLeod,S.L., Lang,N.P. and Kadlubar,F.F. (1999) Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved. Carcinogenesis, 20, 11071114.[Abstract/Free Full Text]
-
Styczynski,P.B., Blackmon,R.C., Groopman,J.D. and Kensler,T.W. (1993) The direct glucuronidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human and rabbit liver microsomes. Chem. Res. Toxicol., 6, 846851.[ISI][Medline]
-
Turesky,R.J., Lang,N.P., Butler,M.A., Teitel,C.H. and Kadlubar,F.F. (1991) Metabolic activation of carcinogenic heterocyclic aromatic amines by human liver and colon. Carcinogenesis, 12, 18391845.[Abstract]
-
Reistad,R., Frandsen,H., Grivas,S. and Alexander,J. (1994) In vitro formation and degradation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) protein adducts. Carcinogenesis, 15, 25472552.[Abstract]
-
Frandsen,H. and Alexander,J. (2000) N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Carcinogenesis, 21, 11971203.[Abstract/Free Full Text]
-
Turesky,R.J., Bracco-Hammer,I., Markovic,J., Richli,U., Kappeler,A.-M. and Welti,D.H. (1990) The contribution of N-oxidation to the metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in rat hepatocytes. Chem. Res. Toxicol., 3, 524535.[ISI][Medline]
-
Frandsen,H., Grivas,S., Andersson,R., Dragsted,L. and Larsen,J.C. (1992) Reaction of the N2-acetoxy derivative of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with 2'-deoxyguanosine and DNA. Synthesis and identification of N2-(2'-deoxyguanosin-8-yl)-PhIP. Carcinogenesis, 13, 629635.[Abstract]
-
Knize,M.G. and Felton,J.S. (1986) The synthesis of the cooked-beef mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its 3-methyl isomer. Heterocycles, 24, 18151819.[ISI]
-
Alexander,J., Heidenreich,B., Reistad,R. and Holme,J.A. (1995) Metabolism of the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the rat and other rodents. In Adamson,R.H., Gustafsson,J.-A., Ito,N., Nagao,M., Sugimura,T., Wakabayashi,K. and Yamazoe,Y. (eds) Heterocyclic Amines in Cooked Foods: Possible Human Carcinogens. 23rd Proceedings of the Princess Takamatusu Cancer Society. Princeton Scientific Publishing, Princeton, NJ, pp. 5968.
-
Sinha,R., Rothman,N., Brown,E.D., Mark,S.D., Hoover,R.N., Caporaso,N.E., Levander,O.A., Knize,M.G., Lang,N.P. and Kadlubar,F.F. (1994) Pan-fried meat containing high levels of heterocyclic aromatic amines but low levels of polycyclic aromatic hydrocarbons induces cytochrome P4501A2 activity in humans. Cancer Res., 54, 61546159.[Abstract]
-
Malfatti,M.A., Kulp,K.S., Knize,M.G., Davis,C., Massengill,J.P., Williams,S., Nowell,S., MacLeod,S., Dingley,K.H., Turteltaub,K.W., Lang,N.P. and Felton,J.S. (1999) The identification of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans. Carcinogenesis, 20, 705713.[Abstract/Free Full Text]
-
Kulp,K.S., Knize,M.G., Malfatti,M.A., Salmon,C.P. and Felton,J.S. (2000) 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. Carcinogenesis, 21, 20652072.[Abstract/Free Full Text]
-
Wallin,H., Mikalsen,A., Guengerich,F.P., Ingelman-Sundberg,I., Solberg,K.E., Rossland,O.J. and Alexander,J. (1990) Differential rates of metabolic activation and detoxification of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by different cytochrome P450 enzymes. Carcinogenesis, 11, 489492.[Abstract]
-
Lin,D.-X., Meyer,D.J., Ketterer,B., Lang,N.P. and Kadlubar,F.F. (1994) Effects of human and rat glutathione-S-transferase on the covalent binding of the N-acetoxy derivatives of heterocyclic amine carcinogens in vitro: a possible mechanism of organ specificity in their carcinogenesis. Cancer Res., 54, 49204926.[Abstract]
Received June 19, 2001;
revised August 20, 2001;
accepted September 17, 2001.