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

Henrik Frandsen2 and Jan Alexander1

Institute of Food Safety and Toxicology, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark and
1 Department of Environmental Medicine, National Institute of Public Health, PO Box 4404, N-0403 Oslo, Norway


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. PhIP is metabolically activated to the ultimate mutagenic metabolite by CYP P450-mediated N-hydroxylation followed by phase II esterification. Incubation of N-hydroxy-PhIP (N-OH-PhIP) with cytosol, acetyl coenzyme A (AcCoA) and 2'-deoxyguanosine for 24 h resulted in the formation of three different adducts:N2-(deoxyguanosin-8-yl)-PhIP, N2-(guanosin-8-yl)-PhIP and PhIP-xanthine. One additional product, 5-hydroxy-PhIP (5-OH-PhIP), was also identified in the incubation mixtures. 5-hydroxy-PhIP is formed as a degradation product of conjugates formed from N-acetoxy-PhIP and protein, glutathione or buffer constituents. A similar spectrum of products was obtained using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) instead of acetyl CoA. Addition of glutathione (3 mM) to the incubation mixture resulted in a 50% reduction in both adducts and 5-hydroxy-PhIP formation in liver cytosol. The main product detected was PhIP, suggesting glutathione-dependent reduction of the N-acetoxy-PhIP. Addition of glutathione to incubation mixtures from the other cytosolic preparations had less dramatic effects. In addition, increasing the amount of N-OH-PhIP in the incubation mixture resulted in proportional increased amounts of total adducts and 5-OH-PhIP. Incubation of rat and human S9 with PhIP resulted in the formation of only traces of 5-OH-PhIP. Fortification with AcCoA clearly increased the formation of 5-OH-PhIP. Addition of the CYP 450 1A2 inhibitor, furafylline, completely inhibited the formation of 5-OH-PhIP in incubations with human S9. These results indicate that both PhIP adducts and 5-OH-PhIP are formed by similar routes of activation of N-OH-PhIP. 5-OH-PhIP may therefore serve as a biomarker for the formation of the ultimate mutagenic metabolite of PhIP. A rat dosed orally with PhIP excreted 1% of the dose as 5-OH-PhIP in the urine at 24 h and 0.05 and 0.01% at 48 and 72 h, respectively. This shows that 5-OH-PhIP is also formed in vivo and indicates the possible use of 5-OH-PhIP as a urinary biomarker.

Abbreviations: AcCoA, acetyl coenzyme A; dG, 2'-deoxyguanosine; dG-C8-PhIP, N2-(deoxyguanosin-8-yl)-PhIP; DMF, dimethylformamide; GSH, glutathione (reduced); G-C8-PhIP, N2-(guanosin-8-yl)-PhIP; N-OH-PhIP, N-hydroxy-PhIP, hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 4'-OH-PhIP, 2-amino-1-methyl-b-(4-hydroxyphenyl)imidazo[4,5-b]pyridine; 5-OH-PhIP, 5-hydroxy-PhIP, 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine; PAPS, 3'-phosphoadenosine-5'-phosphosulphate; PCB, Aroclor 1254; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of a number of mutagenic and carcinogenic heterocyclic amines formed in the crust of meat during ordinary frying (1,2). PhIP induces lymphomas in mice, mammary carcinomas in female rats and colon carcinomas in male rats (3,4). PhIP is metabolically activated to a proximate mutagenic metabolite mainly by cytochrome P450 1A2-dependent oxidation of the exocyclic amino group (5). Further activation by esterifying enzymes such as acetyltransferase and sulphotransferase is needed in order to obtain products which can react with DNA. This results in adducts where the exocyclic amino group of PhIP is attached to C8 of guanine (69).

N-acetoxy-PhIP is also able to react with proteins, reduced glutathione (GSH) and other cellular constituents resulting in unstable products, which spontaneously degrade to 5-hydroxy-PhIP (5-OH-PhIP) (10). 5-OH-PhIP and its glucuronyl derivative have been found in incubations of PhIP with hepatocytes from Aroclor 1254 (PCB)-treated rats (11). Glucuronyl and sulphate derivatives of 5-OH-PhIP were also found in the bile from PCB-treated rats dosed with PhIP. Rat liver microsomes apparently did not catalyse the oxidation of PhIP to 5-OH-PhIP directly (11). The route of formation of 5-OH-PhIP from PhIP was suggested to proceed through N-hydroxylation, followed by esterification, reaction with thiols and hydrolysis (10,11).

Metabolic pathways of activation and detoxification of chemical carcinogens vary among species and tissues and also enzymes involved in the metabolism of chemical carcinogens exhibit genetic polymorphisms that affect their expression or activity. Extrapolation of cancer risk data from one species to another, as well as evaluation of individual risk within humans, therefore requires the use of biomarkers that reflect the bioactive dose of a chemical carcinogen (12,13).

In the present study we wanted to further explore whether 5-OH-PhIP could be an indicator of the bioactive or genotoxic dose of PhIP. The questions asked were whether the formation of 5-OH-PhIP parallels that of PhIP–2'-deoxyguanosine (dG) adducts and whether formation proceeds through the same activation pathway.

Acetyl coenzyme A (AcCoA)-dependent activation of N-hydroxy-PhIP (N-OH-PHIP) in vitro was investigated in cytosols from various organs of the rat. dG was included in the incubation mixture, which made monitoring of formation of both adducts and 5-OH-PhIP possible. The route of 5-OH-PhIP formation was further investigated in rat and human S9 mix with and without fortification with AcCoA. Finally, urinary excretion of 5-OH-PhIP was studied in a rat orally dosed with PhIP.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
PhIP was obtained from Toronto Research Chemicals (Toronto, Canada). Non-specifically labelled [3H]PhIP was made from PhIP by Amersham UK. N-OH-PhIP, N-acetoxy-PhIP and N2-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) were synthesized as previously described (7). N2-(guanosin-8-yl)-PhIP (G-C8-PhIP) was obtained by hydrolysis of dG-C8-PhIP in 0.1 M HCl at 95°C for 1 h. 5-OH-PhIP was obtained by addition of N-acetoxy-PhIP to 0.1 M sodium phosphate buffer, pH 7.6, at 37°C followed by overnight incubation at room temperature as previously described (10). AcCoA, GSH, dG, xanthine, 3'-phosphoadenosine-5'-phosphosulphate (PAPS) and furafylline were obtained from Sigma (St Louis, MO). ß-Glucuronidase/arylsulphatase was obtained from Roche Diagnostic (Switzerland). Human hepatic S9 (pool of 15 donors) was obtained from In Vitro Technologies (Baltimore, MD). HPLC grade acetonitrile was obtained from Fisher Scientific (Loughborough, UK). Bond Elut columns (C18 and 2OH) were from Varian (Harbor City, CA). All other chemicals were obtained from Merck (Darmstadt, Germany) and were of analytical purity.

Analytical
High performance liquid chromatography analyses were performed on a Hewlett Packard model 1090 B liquid chromatograph equipped with a photodiode array detector (Hewlett Packard, Wallbronn, Germany). Chromatographic conditions were as specified in the figure legends. The oven temperature was 40°C.

Positive ion electrospray mass spectra were obtained with a Hewlett Packard MSD 1100 mass spectrometer equipped with an electrospray interface (Hewlett Packard). The following interphase settings were used: nebulizer pressure 40 p.s.i.; drying gas 10 l/min, 350°C; capillary voltage 4000 V; fragmentor voltage 70 V.

Liquid scintillation counting was performed on a Tri Carb 2500TR using a Hionic-Flour scintillation cocktail with external standardization (Packhard, Meriden, CT).

Preparation of S9 mix and cytosol
Untreated male Wistar rats (age 7–8 weeks) from Møllegård Breeding Center (Lille Skensved, Denmark) were used for preparation of S9 and cytosolic fractions. S9 from liver and cytosol from liver, intestine, colon, lung, kidney and heart were prepared in 0.1 M sodium phosphate buffer, pH 7.6, essentially as described (14). Protein concentrations were determined on a Cobas Mira S by use of Unimate kits (Roche).

AcCoA- and PAPS-dependent activation of N-OH-PhIP in cytosol
AcCoA-dependent activation of N-OH-PhIP was performed by a modification of the method of Davis et al. (15). The incubation mixtures contained 1mg cytosolic protein, 7.5 mM dG and 2.5 mM AcCoA in 0.5 ml of 0.1 M sodium phosphate buffer, pH 7.6, with or without 3 mM GSH. The incubations were carried out in sealed tubes at 37°C after purging with argon. The reaction was initiated by the addition of N-OH-PhIP at a concentration of 20 µM (dissolved in 10 µl methanol).

Samples of 100 µl were taken at 1, 24 and 48 h of incubation and added to 100 µl of argon-purged –20°C ethanol. The mixtures were stored at – 20°C for 30 min and, after centrifugation, the supernatants were analysed by HPLC. The biotransformation products were quantitated by comparison of UV absorbance areas with UV absorbance areas of tritium-labelled standards, where relevant peaks had been collected and analysed by liquid scintillation counting.

PAPS-dependent activation of N-OH-PhIP was also studied in liver cytosol by the same procedure as above by substituting AcCoA with PAPS.

AcCoA-dependent formation of 5-OH-PhIP in S9
The incubation mixtures contained 4 mg rat hepatic S9 protein/ml or 2 mg human hepatic S9 protein/ml incubation mixture, 1 U/ml DL-isocitrate dehydrogenase, 0.5 mM NADP+, 10 mM sodium DL-isocitrate and 5 mM magnesium chloride in 1 ml of 0.1 M sodium phosphate buffer, pH 7.6. The formation of 5-OH-PhIP was also investigated in the presence of 2.5 mM AcCoA and, for human S9, the presence of 2.5 mM AcCoA together with 50 µM furafylline. The mixtures were preincubated for 5 min at 37°C before addition of 10 µg PhIP [dissolved in 10 µl of dimethylformamide (DMF)]. The mixtures were incubated at 37°C overnight. The reaction was terminated by addition of 1 vol of ice-cold, argon-purged ethanol. The mixtures were stored at –20°C for 30 min and, after centrifugation, the supernatants were analysed by HPLC.

Synthesis of PhIP–xanthine
Xanthine (2.2 mg) was dissolved in 2 ml of 0.1 M phosphate buffer, pH 7.6, by addition of 100 µl of 0.5 M NaOH. The solution was heated to 37°C and ~0.25 mg N-acetoxy-PhIP in 300 µl of 50% DMF was added dropwise under vigorous stirring. Incubation at 37°C was continued for 30 min. PhIP–xanthine was purified by HPLC and characterized by UV spectroscopy and mass spectrometry.

Urinary excretion of 5-OH-PhIP
One male Wistar rat (weight ~200 g) was injected with Aroclor (500 mg/kg dissolved in corn oil) i.p. After 5 days the animal received an oral dose of 1.6 mg PhIP dissolved in 0.25 ml 50% DMF. The animal was placed in a metabolism cage and urine was collected at 24, 48 and 72 h post-dosing. Urine was stored at –20°C until analyses.

Urine (0.5 ml) was diluted with 1 ml of 50 mM sodium acetate, pH 5.5, and added to 200 µl of ß-glucuronidase/arylsulphatase and incubated overnight at ambient temperature. The sample was applied to an activated Bond Elut C18 column equilibrated with 0.1 M Tris (pH ~10). The column was washed with 2 ml of 0.1 M Tris/acetonitrile (15:85) followed by 2 ml of water and eluted with 2 ml of 50 mM ammonium formate, pH 3.5/acetonitrile (2:8). The eluate was placed in a 50°C water bath and evaporated to dryness under a stream of nitrogen.

The residue was dissolved in 2 ml of DMF/ethyl acetate (1:4) and applied to a Bond Elut 2OH column equilibrated with ethyl acetate. The column was washed with 2 ml of formic acid/methanol/ethyl acetate (1:100:900) and eluted with 2 ml of formic acid/methanol (1:100). The eluate was evaporated to dryness under a stream of nitrogen, redissolved in 50 µl of 0.1% formic acid/DMF (1:1) and analysed by HPLC. The recovery of 5-OH-PhIP during column purification (C18 and 2OH) was 85%.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
The AcCoA-dependent metabolism of N-OH-PhIP by cytosolic enzymes from various organs of the rat was investigated in vitro in the presence of dG. After 1 h incubation several metabolic products, including PhIP, dG-C8-PhIP and G-C8-PhIP, were detected in the incubation mixture, together with some unknown products. A large fraction of the N-OH-PhIP was still unmetabolized after 1 h incubation, the amount varying between cytosols from various organs. Continuing the incubation for 24 and 48 h resulted in almost complete consumption of the N-OH-PhIP in the incubation mixture and increasing amounts of metabolic products, but also in the formation of one additional metabolite, 5-OH-PhIP. Chromatograms obtained after 48 h incubation were almost identical to chromatograms obtained after 24 h incubation.

Figure 1Go shows a chromatogram of an incubation mixture of hepatic cytosol with N-OH-PhIP, AcCoA and dG after 24 h. Four products were readily identified by comparison of HPLC retention times and UV spectra of authentic standards as PhIP, 5-OH-PhIP, G-C8-PhIP and dG-C8-PhIP. One major unknown product eluting at 17.5 min (PhIP-X) and three minor unknowns eluting at 15, 16 and 17 min were also present in the incubation mixture.



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Fig. 1. Chromatogram of the incubation mixture of hepatic cytosol with N-OH-PhIP, AcCoA and dG after 24 h. Products were separated on a Purospher C18, 5 µm, 250x4 mm column obtained from Hewlett Packard. The flow rate was 0.75 ml/min. Solvents were: A, 0.1% formic acid; B, 0.1% formic acid in acetonitrile. Solvent programming was isocratic 2% B for 3 min followed by a linear gradient to 80% by 22 min. (Upper inset) Merged spectra of dG-C8-PhIP and the library spectrum; (lower inset) merged spectra of G-C8-PhIP and the library spectrum.

 
Control incubation with heat-denatured hepatic cytosol, as well as incubation without addition of AcCoA, did not result in any biotransformation of N-OH-PhIP, which was stable for at least 48 h. Incubation without dG resulted in only two products, PhIP and 5-OH-PhIP. Incubation of hepatic cytosol with AcCoA and dG, guanosine, 5-OH-PhIP, dG-C8-PhIP or G-C8-PhIP did not result in formation of any of the unknown metabolites; likewise, incubation with dG-C8-PhIP did not result in formation of G-C8-PhIP.

The major unknown product, PhIP-X, was purified by HPLC and analysed by electrospray mass spectrometry giving a molecular ion [M+H]+ at m/z 375. PhIP-X was finally identified as an adduct between PhIP and xanthine, a catabolic product of guanine, by comparison of HPLC retention times, UV spectra and mass spectra with a product obtained after reaction of N-acetoxy-PhIP and xanthine (Figures 2 and 3GoGo). Further structural characterization of this product was not done.



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Fig. 2. Merged UV spectra of PhIP-X, eluting at 17.5 min, and PhIP–xanthine.

 


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Fig. 3. Mass spectrum of PhIP–xanthine.

 
Re-analyses of the chromatograms, constructing profiles at 280 nm using stored data obtained with the diode array detector, revealed that extensive degradation of dG took place during the incubations; >90% had disappeared after 24 h. The catabolism of dG by cytosolic enzymes was faster in some organs than in others.

As dG-C8-PhIP and G-C8-PhIP were not metabolized by cytosolic enzymes, the appearance of three adducts in the incubation mixture, dG-C8-PhIP, G-C8-PhIP and PhIP– xanthine, seems to originate from the reaction of N-acetoxy-PhIP with dG and two of its degradation products, guanine and xanthine.

The relative amounts of the reaction products formed differed between cytosols from different organs. This reflects the different rates of AcCoA-dependent activation of N-OH-PhIP and degradation of dG. The amount of PhIP formed was higher in liver cytosol than in cytosols from other organs (Table IGo), indicating more efficient reduction of the N-acetoxy-PhIP formed in liver cytosol. The amount of 5-OH-PhIP formed was fairly constant, varying between 7.3 and 10.2% of the amount of N-OH-PhIP metabolized (8.5 ± 1.2%, mean ± SD). The amounts of the three adducts G-C8-PhIP, dG-C8-PhIP and PhIP–xanthine formed varied between the cytosolic preparations, with PhIP–xanthine accounting for the highest amounts. The sum of adducts (G-C8-PhIP + dG-C8-PhIP + PhIP–xanthine) was highest in cytosols from organs where the amount of PhIP–xanthine was high, indicating that N-acetoxy-PhIP reacts more efficiently with xanthine than with dG (Table IGo).


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Table I. AcCoA-dependent formation of products of N-OH-PhIP by cytosol from various organs of the rat
 
Addition of 3 mM GSH to the incubation mixtures resulted in increased formation of PhIP and in an ~50% reduction in the formation of both adducts and 5-OH-PhIP in liver cytosol (Table IIGo). Addition of GSH to cytosolic incubations from other organs had much less effect on the formation of PhIP, as well as adducts and 5-OH-PhIP. Levels of adducts and 5-OH-PhIP ranged from 67 to 100% of levels without GSH. The addition of GSH to incubations of extrahepatic cytosol also resulted in some inhibition of N-OH-PhIP metabolism, leaving up to 40% unmetabolized after 24 h. Lower levels of adducts and 5-OH-PhIP were found in cytosol from organs where GSH-dependent inhibition of N-OH-PhIP metabolism was highest.


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Table II. AcCoA-dependent formation of products of N-OH-PhIP by cytosol from various organs of the rat in the presence of 3 mM GSH
 
The results show that both nucleotide/nucleoside adducts of PhIP and 5-OH-PhIP are formed by AcCoA-dependent activation of N-OH-PhIP in cytosolic incubations containing dG. In hepatic cytosol the presence of GSH caused a reduction in the amounts of both adducts and 5-OH-PhIP, formed as well as extensive formation of PhIP. Cytosolic incubates from other organs were less affected by the presence of GSH.

The relationship between formation of adducts and 5-OH-PhIP indicates that similar routes of reaction form these products. 5-OH-PhIP, therefore, may serve as a biomarker for the formation of the ultimate mutagenic metabolite of PhIP. In order to investigate this further, hepatic cytosol was incubated with AcCoA, dG and increasing amounts of N-OH-PhIP. The amounts of both adducts and of 5-OH-PhIP formed in the incubation mixtures increased linearly with the amount of N-OH-PhIP added to the incubation mixture (Figure 4Go).



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Fig. 4. Relation between the formation of 5-OH-PhIP and the dose of N-OH-PhIP (left) and the formation of adducts (G-C8-PhIP + dG-C8-PhIP + PhIP–xanthine) and the dose of N-OH-PhIP (right).

 
We also examined whether the formation of adducts and 5-OH-PhIP were independent of the ultimate reaction pathway. PAPS-dependent activation of N-OH-PhIP was studied in rat hepatic cytosol containing dG. HPLC analysis of the incubation mixture after 24 h showed that only half of the N-OH-PhIP was consumed, indicating a lower sulphotransferase activity in this cytosol compared with acetyltransferase activity. The extracted ion chromatograms showed the presence of PhIP (m/z 225), N-OH-PhIP and 5-OH-PhIP (m/z 241), dG-C8-PhIP (m/z 490), G-C8-PhIP (m/z 374) and PhIP–xanthine (m/z 375) in the incubation mixture, indicating that PAPS-dependent activation of N-OH-PhIP results in the same products as AcCoA-dependent activation (Figure 5Go).



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Fig. 5. Chromatogram of the incubation mixture of hepatic cytosol with N-OH-PhIP, PAPS and dG after 24 h. Products were separated on a Zorbax SB-C3, 5 µm, 150x3 mm column obtained from Hewlett Packard. The flow rate was 0.4 ml/min. Solvents were: A, 0.1% formic acid; B, acetonitrile. Solvent programming was isocratic 2% B for 2 min followed by a linear gradient to 60% by 20 min. Chromatograms shown are extracted ion chromatograms of PhIP (m/z 225), N-OH-PhIP and 5-OH-PhIP (m/z 241), dG-C8-PhIP (m/z 490), G-C8-PhIP (m/z 374) and PhIP–xanthine (m/z 375).

 
Incubation of PhIP with hepatic S9 from rats for 24h resulted in formation of trace amounts of 5-OH-PhIP together with N-OH-PhIP and 4'-hydroxy-PhIP (4'-OH-PhIP). Addition of AcCoA to the incubation mixture resulted in formation of approximately three times more 5-OH-PhIP (not shown). Incubation of PhIP with human hepatic S9 resulted in formation of N-OH-PhIP; formation of 5-OH-PhIP was hardly detectable. Addition of AcCoA to the incubation mixture resulted in formation of 5-OH-PhIP in amounts at least seven times higher than without AcCoA. The presence of furafylline, a specific inhibitor of human CYP 450 1A2, in the incubation mixture completely inhibited formation of both N-OH-PhIP and 5-OH-PhIP, confirming that both N-hydroxylation and AcCoA-dependent phase II biotransformation are important for the formation of 5-OH-PhIP (16; Figure 6Go).



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Fig. 6. Chromatogram of the incubation mixture of human hepatic cytosol with PhIP (A), PhIP and AcCoA (B) and PhIP, AcCoA and furafylline (C) after 24 h. Chromatographic conditions were as in Figure 1Go.

 
In a preliminary in vivo experiment we examined whether 5-OH-PhIP and PhIP adducts were excreted in the urine of a PhIP-exposed rat. One male rat was dosed orally with PhIP and urine was collected after 24, 48 and 72 h. Urine samples were treated with ß-glucuronidase/arylsulphatase to release conjugated metabolites and, following a two-step solid phase extraction, the samples were analysed by LCMS. Figure 7Go shows a chromatogram of a 24 h urine sample. Three compounds, 4'-OH-PhIP, PhIP and 5-OH-PhIP were identified in the sample by comparison of HPLC retention times, UV spectra and mass spectra with standards. The amounts of 5-OH-PhIP excreted in the urine were 71.3 nmol at 24 h, 3.2 nmol at 48 h and 1.0 nmol at 78 h, corresponding to 1.0, 0.05 and 0.01% of the dose of PhIP. Urinary excretion of G-C8-PhIP and dG-C8-PhIP, however, was below the limit of detection, even with the use of single ion monitoring.



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Fig. 7. Chromatogram of a urine sample 24 h post-dosing with PhIP. Products were separated on a Zorbax SB-C3, 5 µm, 150x3 mm column obtained from Hewlett Packard. The flow rate was 0.4 ml/min. Solvents were: A, 0.1% formic acid; B, acetonitrile. Solvent programming was isocratic 2% B for 2 min followed by a linear gradient to 60% by 20 min. (Upper) UV trace monitored at 315 nm; (lower) extracted ion chromatogram of m/z 241 showing the presence of 4'-OH-PhIP and 5-OH-PhIP.

 

    Discussion
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 Abstract
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 Materials and methods
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 References
 
Previously N-OH-PhIP was shown to be activated to DNA-binding intermediates by cytosolic acetyltransferase, sulphotransferase, aminoacyl-tRNA synthetase and phosphatase from humans, rats, mice and monkeys (6,15). In the present study we examined 5-OH-PhIP as a possible biomarker of the ultimate reactive dose of PhIP. This is based on previous information on 5-OH-PhIP formed as a degradation product after reaction of N-acetoxy-PhIP with proteins, thiols and other cellular constituents (10,11). Formation of PhIP nucleotide/nucleoside adducts in parallel with 5-OH-PhIP following AcCoA-dependent activation of N-OH-PhIP was monitored in an in vitro system using cytosols from various organs of the rat. Evidence for a possible relevance of 5-OH-PhIP in vivo was provided in a preliminary study of urine from a rat dosed with PhIP.

Incubation of N-OH-PhIP with cytosol from various organs, AcCoA and dG resulted in the formation of five compounds: PhIP, dG-C8-PhIP, G-C8-PhIP, PhIP–xanthine and 5-OH-PhIP; the latter was released after 24 h incubation. The appearance of substantial amounts of G-C8-PhIP and PhIP–xanthine in comparison with dG-C8-PhIP reflected extensive degradation of dG by cytosolic enzymes during the 24 h incubation. Such extensive degradation of dG would not be considered to occur in vivo, therefore further structural characterization of PhIP–xanthine was not attempted.

Control incubations showed that the presence of both enzymatic activity and AcCoA were necessary for the formation of 5-OH-PhIP and additionally the presence of dG was necessary for the formation of adducts. The experiment substituting AcCoA with PAPS clearly showed that the formation of 5-OH-PhIP and adducts were independent of the esterification pathway of N-OH-PhIP.

Addition of GSH to the incubation mixture from liver cytosol resulted in a 50% reduction in the amounts of adducts and 5-OH-PhIP formed. PhIP was the major product (67%), indicating a GSH-dependent reduction in N-acetoxy-PhIP, which is in accordance with previous studies (17,18). The simultaneously reduced formation of both 5-OH-PhIP and adducts indicates that these compounds are formed from the same precursor. Less effect of GSH addition to cytosolic incubations from extrahepatic organs indicates less active glutathione S-transferase-mediated detoxification of N-acetoxy-PhIP in these organs.

Addition of increasing amounts of N-OH-PhIP to incubations of rat hepatic cytosol fortified with AcCoA and dG gave a linear increase in both the amounts of adducts formed and in the amounts of 5-OH-PhIP formed (Figure 4Go).

Whereas 5-OH-PhIP was formed only in trace amounts in incubations of PhIP with rat or human hepatic S9, addition of AcCoA to the incubation mixture clearly increased the formation of 5-OH-PhIP. Addition of the CYP 450 1A2 inhibitor furafylline completely inhibited formation of both N-OH-PhIP and 5-OH-PhIP in human hepatic S9.

Taken together, these results indicate that 5-OH-PhIP and PhIP adducts are formed by similar routes of reaction and in proportional amounts. O-acetylation of N-OH-PhIP results in formation of N-acetoxy-PhIP, followed by heterolytic fission of the N–O bond, giving rise to a nitrenium ion, which is considered as the ultimate carcinogenic metabolite (19). This nitrenium ion can react either with dG, giving rise to adducts, or with thiols, proteins or other compounds, giving rise to unstable products that are degraded to 5-OH-PhIP (Figure 8Go). N-acetoxy-PhIP was previously shown to form unstable products with GSH and proteins, which degraded to 5-OH-PhIP (11).



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Fig. 8. Proposed reaction scheme for the formation of adduct, 5-OH-PhIP and PhIP following AcCoA-dependent activation of N-OH-PhIP.

 
Rats exposed to PhIP excrete a relatively constant proportion unmetabolized in the urine. Measurement of urinary PhIP has therefore been suggested as a marker of dietary exposure (20). In humans, however, the percentage of ingested PhIP excreted unchanged in the urine seems to vary from person to person, due to interindividual variations in the enzyme activities involved in metabolism (21). Recently, N-hydroxy-PhIP-N2-glucuronide was identified as a major urinary metabolite in humans exposed to PhIP and this metabolite was suggested as a biomarker for both exposure and activation (22). It appears, however, that the second step in metabolic activation is the rate limiting step determining the level of DNA adduct formation (23). A rat dosed orally with PhIP excreted 1% of the dose as 5-OH-PhIP in the urine at 24 h and only 0.06% from 24 to 72 h. Notably, dG-C8-PhIP was not detectable in rat urine in this study. Together our results indicate that 5-OH-PhIP may represent an in vivo urinary biomarker for the ultimate genotoxic dose of ingested PhIP, including both phase I and phase II activation. Further evaluation of 5-OH-PhIP as a biomarker for the active dose of PhIP in vivo is in progress.


    Notes
 
2 To whom correspondence should be addressed Email: hf{at}fdir.dk Back


    Acknowledgments
 
The authors thank Joan Frandsen for skilful technical assistance. This work was supported by a grant from the European Commission.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 6, 1999; revised February 23, 2000; accepted February 24, 2000.