The identification of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans
Michael A. Malfatti1,
Kristen S. Kulp1,
Mark G. Knize1,
Cindy Davis3,
Joyce P. Massengill3,
Suzanne Williams3,
Susan Nowell2,
Stewart MacLeod2,
Karen H. Dingley1,
Kenneth W. Turteltaub1,
Nicholas P. Lang2,3 and
James S. Felton1,4
1 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, PO Box 808, L-452, Livermore, CA 94551,
2 Arkansas Cancer Research Center, University of Arkansas for Medical Sciences and
3 John L.McClellan Memorial Veterans Administration Medical Center, Little Rock, AR 72205, USA
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Abstract
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[2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 7084 µg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, ~90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuronide (N-hydroxy-PhIP-N2-glucuronide), accounting for 4760% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 5617% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.
Abbreviations: CID, collision induced dissociation; CYP1A2, cytochrome P4501A2; HA, heterocyclic amine; 4'-hydroxy-PhIP, 2-amino-1-methyl-6-(4'-hydroxy)phenylimidazo[4,5-b]pyridine; NAT2, N-acetyltransferase; N-hydroxy-PhIP, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; rt, retention time; 4'-PhIP-sulfate, 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl) phenyl sulfate; ST, sulfotransferase; UDPGT, UDPglucuronosyltransferase.
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Introduction
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Exposure to heterocyclic amines (HA) derived from cooked foods has been implicated as an important dietary risk factor in the etiology of certain human cancers (1). These compounds can be found in concentrations of up to 500 p.p.b. in foods commonly consumed in a typical Western diet. All of the HAs thus far examined exhibit mutagenic activity and have produced cancers in rodent tumor bioassays (2). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant HA found in well-done cooked beef and chicken (3,4), has been shown to cause mammary, colon and prostate tumors in rats (5,6), lymphomas in mice (7) and hepatic adenomas in neonatal mice (8). PhIP has also been shown to induce DNA strand breaks and sister-chromatid exchanges, and to form DNA adducts in vitro and in vivo (913). These findings, together with the relative abundance of PhIP in cooked foods, suggest that PhIP may pose a significant carcinogenic risk to humans.
The metabolism of PhIP involves both phase I and phase II pathways for bioactivation and/or detoxification. PhIP bioactivation is highly dependent upon the cytochrome P4501A2 (CYP1A2)-mediated N-hydroxylation of the parent amine to the corresponding 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) (14,15). N-hydroxy-PhIP is subsequently esterified by sulfotransferases and/or acetyltransferases which generate the highly electrophilic O-sulfonyl and O-acetyl esters, respectively. These esters are capable of covalently binding DNA (10,16). N-hydroxy-PhIP can also form stable glucuronide conjugates at the N2 and N3 positions, which can be excreted through the urine or bile, or can be transported to extrahepatic tissue where further metabolism can occur (17,18). PhIP can also form a non-reactive 2-amino-1-methyl-6-(4'-hydroxy)phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP), which can undergo conjugation by sulfation and glucuronidation producing more polar unreactive compounds, which are readily excreted (19,20). Furthermore, PhIP has been shown to directly form non-reactive glucuronides at the N2 and N3 positions (21).
Due to the complexities of human in vivo studies, PhIP metabolism studies in humans has mainly been limited to in vitro studies. Human hepatic microsomes have been shown to N-hydroxylate PhIP via CYP1A2 to the reactive N-hydroxy-PhIP intermediate, but have little or no capacity to convert PhIP to the 4'-hydroxy-PhIP intermediate (2224). Human liver microsomes have also been shown to catalyze the glucuronidation of N-hydroxy-PhIP (18). Other studies have demonstrated that N-hydroxy-PhIP can be further activated by human cytosolic hepatic O-acetyltransferases, as well as hepatic and colonic sulfotransferases (22,25,26).
The in vivo metabolism of PhIP has been well characterized in rodents and non-human primates (19,27,28); however, only a few studies have investigated the disposition of PhIP in humans (15,2931). The goal of the present study was to discern the in vivo metabolism of PhIP in humans. The identification of the major PhIP metabolites in urine and plasma, and the variation in metabolism between three human subjects is reported. The characterization of the major PhIP metabolites will determine the biological fate of PhIP in humans leading to an understanding of the balance between bioactivation and detoxification of this carcinogen. Additionally, identifying PhIP metabolites may provide a reliable biomarker to indicate PhIP exposure and bioactivation.
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Materials and methods
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Chemicals
[2-14C]PhIP was obtained from Toronto Research Chemicals (Ontario, Canada) and was found to be >95% chemically and radio-pure. ß-Glucuronidase type VII-A from Escherichia coli and aryl-sulfatase type H-5 from Helix pomatia were purchased from Sigma (St Louis, MO). Dog urine from [14C]PhIP-exposed dogs was kindly provided by Dr F.F.Kadlubar (National Center for Toxicological Research, Jefferson, AR). All reagents and HPLC solvents used were of the highest grade available.
Human study
The study protocol was independently reviewed and approved by the Institutional Review Boards for Human Research at the Lawrence Livermore National Laboratory and the Arkansas Cancer Research Center, the Radioactive Drug Research Committee and the Radiation Safety Committee. Informed consent was obtained from each subject prior to the start of the study. [14C]PhIP was packaged in gelatin capsules containing lactose as filler and administered orally to three human subjects undergoing a partial colonectomy as treatment for colon cancer. The patient profiles are listed in Table I
. At 4872 h prior to surgery, subjects 1, 2 and 3 received a [14C]PhIP dose of 0.93, 0.53 or 1.01 µg/kg body wt, respectively (7084 µg/person). In subjects 1 and 2 the specific activity was 56.0 mCi/mmol whereas in subject 3 it was 41.8 mCi/mmol. The difference in the specific activity of [14C]PhIP was a result of preparing the PhIP capsules on two different occasions from two batches of [14C]PhIP. The PhIP dose each subject received was equivalent to the amount of PhIP found in two well-done broiled chicken breasts (4,32). The radioactive dose received was 0.011 mSv, which is the energy equivalent of 1/29 of the energy received from an average chest X-ray. After administration of [14C]PhIP, urine and blood was collected and analyzed for PhIP and PhIP metabolites.
Separation of urinary PhIP metabolites
Urine was collected at various time intervals for up to 72 h post-ingestion of the PhIP dose, and immediately frozen and stored at 20°C. Ten fractions were collected from subject 1, seven from subject 2 and six from subject 3 (see Figure 1
for specific time intervals). Prior to HPLC analysis each urine sample was thawed and the total volume of each fraction recorded. A 1.0 ml aliquot from each fraction was analyzed by liquid scintillation counting to determine the 14C content. Each sample was then analyzed by reversed-phase HPLC for PhIP and PhIP metabolites. Approximately 60008000 disintegrations per minute (d.p.m.) of each urine sample was concentrated under nitrogen to 1.0 ml, filtered using a 0.45 µm nylon centrifuge filter (MSI, Westborough, MA), then directly injected onto a Rainin HPLC system (Varian, Walnut Creek, CA) equipped with a 5 µm, 4.6x220 mm TSK-GEL ODS-80 TM column (TosoHaas, Montgomeryville, PA), and monitored at 315 nm. Metabolites were eluted at 1.5 ml/min initially using a solvent of 10% (v/v) methanol/0.1% (v/v) triethylamine, pH 6, for 5 min. This was followed by a gradient to 33% (v/v) methanol/0.1% triethylamine at 45 min, followed by a final gradient to 55% (v/v) methanol/0.1% triethylamine at 60 min. The methanol concentration was maintained at 55% (v/v) from 60 to 70 min. The column eluate was collected at 1 min intervals and radioactivity was quantified by scintillation counting (Wallic, Gaithersburg, MD). Approximately 90% of the radioactivity was recovered after sample preparation and analysis.

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Fig. 1. Radioactivity levels in urine fractions collected over time from three human subjects dosed with [14C]PhIP. (A) Subject 1; (B) subject 2; (C) subject 3. Data are expressed as percentages of the total excreted radioactivity.
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Purification and concentration of PhIP metabolites
PhIP metabolites were concentrated from 1.5 l of urine by passing the crude material through an Amberlite XAD-2 column. The column was washed with water, followed by 10% (v/v) methanol/water and the metabolites were eluted with two 100 ml aliquots of methanol followed by 100 ml of methanol/ammonia, 9:1 (v/v). The elution aliquots were pooled and evaporated by rotary evaporation under reduced pressure.
HPLC purification step I
The concentrated material was re-suspended in 5% (v/v) methanol and the metabolites were separated using a Waters Millennium 2010 HPLC system equipped with a 5 µm, 4.6x220 mm TSK-GEL ODS-80 TM column and the products detected with a Waters model 996 Photodiode Array Detector (Milford, MA), a HewlettPackard model 1046A programmable Fluorescence Detector (Palo Alto, CA) and a Radiomatic radioactivity flow detector (Tampa, FL). The solvent system consisted of 0.01 M triethylamine phosphate, pH 6.0, and methanol, operating with a linear gradient at 1 ml/min. The gradient used was as follows: 5% (v/v) methanol for 5 min; 530% (v/v) methanol from 540 min; 3040% (v/v) methanol from 4070 min; 4070% (v/v) methanol from 7075 min; and held at 70% (v/v) methanol from 7580 min. Fractions were collected at 1 min intervals.
HPLC purification step II
Radioactive fractions were evaporated to dryness under nitrogen, re-suspended in 4% (v/v) acetonitrile and re-injected onto the TSK-GEL column, using a solvent system consisting of 0.01M triethylamine phosphate, pH 3.0, and acetonitrile. The gradient used was as follows: 425% (v/v) acetonitrile for 90 min; 2540% (v/v) acetonitrile for 90110 min and 4050% (v/v) acetonitrile for 110115 min. One-minute fractions were collected and the radioactive fractions were again evaporated to dryness.
HPLC purification step III
The radioactive fractions were re-suspended in 5% (v/v) methanol and re-injected using a solvent system consisting of 0.01 M triethylamine phosphate, pH 6.0, and methanol. The gradient was as follows: 5% (v/v) methanol for 5 min, 530% (v/v) methanol for 545 min, 3040% (v/v) methanol for 4580 min, 4070% (v/v) methanol for 8085 min; and held at 70% (v/v) methanol for 10 min. One-minute fractions were collected, and the radioactive fractions were evaporated to dryness.
HPLC purification step IV
The fractions were resuspended in the starting mobile phase and re-injected onto the TSK-gel column for further purification. The solvent system consisted of 0.01 M triethylamine phosphate, pH 3.0, acetonitrile and methanol. The gradient was as follows: 220% (v/v) methanol and 320% (v/v) acetonitrile for 90 min, 2025% (v/v) methanol and 2025% (v/v) acetonitrile for 90100 min, 2540% (v/v) methanol and 2530% (v/v) acetonitrile for 100105 min. One-minute fractions were collected and the radioactive fractions were evaporated to dryness. The metabolites were then each resuspended in 2% (v/v) methanol for analysis by mass spectrometry.
Mass spectrometry
Each isolated metabolite was directly injected into a Michrom µLC system (Michrom Bioresources, Auburn, CA) coupled to a Finnigan MAT TSQ-700 triple quadrupole mass spectrometer through a Finnigan electrospray interface (Finnigan MAT, San Jose, CA). Samples were loaded onto a Zorbax C18 SB column (0.2 x150 mm) (Michrom Bioresources, Auburn, CA) and eluted at a flow rate of 5.0 µl/min initially using a solvent of 95% A [solvent A: 2.0% (v/v) methanol/1.0% acetic acid; solvent B: 95% (v/v) methanol/1.0% acetic acid] for 1.0 min. This was followed by a linear gradient up to 95% B at 16 min. The methanol concentration was held at 95% B for 5 min, then re-equilibrated to 95% A over 10 min. Full scan and collision induced dissociation (CID) mass spectra were obtained for each metabolite, using previously described parameters (33).
Enzymatic and chemical analysis
To determine the presence of conjugated PhIP metabolites, individual metabolite peaks or whole urine samples were treated with ß-glucuronidase, aryl-sulfatase or HCl to determine the susceptibility of the metabolites to enzymatic cleavage or acid hydrolysis. Individual metabolite peaks were treated with 3000 U/ml ß-glucuronidase in 25 mM potassium phosphate buffer, pH 7.0, and incubated for 1 h at 37°C. To determine susceptibility to aryl-sulfatase, 250 U/ml aryl-sulfatase in 50 mM sodium acetate buffer, pH 5.0, was added to 1 ml of whole urine, and incubated for 5 h at 37°C. Samples were placed on dry-ice to stop the reaction. For acid hydrolysis treatment, 200 µl of 6 N HCl was added to 1.0 ml of urine and incubated for 5 h at 60°C. After the incubation time, the acidic samples were neutralized with potassium hydroxide, then all samples were analyzed by HPLC as described above in Separation of urinary PhIP metabolites.
Plasma metabolites
Blood collected at 1, 2 and 4 h post PhIP exposure was separated into plasma and red blood cells by centrifugation. The plasma was removed and analyzed for PhIP metabolites. Approximately 2 ml of plasma was used per time point. Prior to HPLC analysis, 2 vol of ice-cold methanol was added to the plasma samples to precipitate the proteins. The samples were then centrifuged at 700 g (Sorvall) for 20 min at 4°C. The supernatant was removed and concentrated to ~0.5 ml under a steady stream of nitrogen. Each sample was then analyzed for PhIP and PhIP metabolites by HPLC, as described above in Separation of urinary PhIP metabolites.
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Results
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Recovery of urinary radioactivity
Since the urine collection time intervals were not consistent among subjects, it was not possible to accurately compare the excretion kinetics of the three individuals. Certain trends in excretion patterns, however, were evident. In subjects 1 and 2, ~90% of the administered [14C]PhIP dose was recovered in the urine; however, in subject 3, only 50% of the administered dose could be accounted for in the urine over the time span of the study. In all three subjects, 8090% of the recovered radioactivity was detected in the first 24 h of urine collected (Figure 1
). In subjects 1 and 3, nearly 90% of the excreted radiolabel was present in the first 24 h of collection, whereas in subject 2, 80% was detected after 33 h of urine collection. In subject 1, ~50% of the excreted radioactivity was detected in the first 4 h of urine collection. In subjects 2 and 3, the first 4 h of urine contained only 14 and 18% of the excreted dose, respectively. Due to the relatively fast excretion rate of PhIP, evidenced by the low radioactivity in the later time points, only samples collected in the first 24 h after dosing were analyzed for PhIP metabolites.
Separation and identification of urinary PhIP metabolites
Chromatographic analysis of the urine samples revealed 12 radioactive metabolite peaks associated with PhIP from the first 24 h of collected urine. Metabolite numbers were assigned based on the order of HPLC elution (Figure 2A
). The relative amount of each metabolite varied between subjects (Figure 3
). In subject 1, metabolites 1, 2, 3 and 5 were detected at levels just above background, whereas in subjects 2 and 3 these metabolites were much more prevalent. Subject 3 possessed the highest concentration of metabolite 6 among the three subjects. In all three subjects, metabolite 9 was the most abundant metabolite, accounting for up to 60, 48 and 47% of the recovered radiolabel from subjects 1, 2 and 3, respectively (Table II
). Within each subject, the amounts of each metabolite changed over time. In subject 1, each metabolite decreased over time; whereas in subjects 2 and 3 the levels of metabolites 19 increased over time (Figure 3
).

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Fig. 2. Comparison of human (A), dog (B) and mouse (C) urinary PhIP metabolic HPLC radio-profiles. Chromatogram A was derived from subject 2, urine fraction 3 (17.533 h). Numbers represent PhIP metabolite peaks as described in Table III .
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Fig. 3. Change in [14C]PhIP urinary metabolites over time from three human subjects dosed with [14C]PhIP. Figure legends correspond to the time urine fractions were collected post-PhIP dose. (A) Subject 1; (B) subject 2; (C) subject 3. Data are expressed as percentages of total recovered radioactivity up to 24 h of collection for (A) and (C), and 33 h of collection for (B). Each urine fraction was assayed three times and the means ± SD are reported.
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Based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis, and susceptibility to enzymatic cleavage, several of the metabolites have been identified (Table III
). Metabolites 1, 2 and 3 have been tentatively identified as isomers of 4'-hydroxy-PhIP-glucuronide. ESIMS analysis of all three metabolites produced molecular ions at m/z 419 [M + H]+ and, upon CID, produced fragment ions at m/z 243 [M + H]+ and m/z 227 [M + H]+, which correspond to 4'-hydroxy-PhIP and PhIP, respectively (note: due to the 14C-label, a mass increase of 2 atomic mass units (amu) was accounted for in all the mass spectral determinations). All three metabolites were susceptible to ß-glucuronidase treatment forming a product with an HPLC retention time (rt) similar to that of 4'-hydroxy-PhIP. Metabolite 1 also produced a molecular ion at m/z 403 [M + H]+ and fragmented to m/z 227 [M + H]+, which indicates a direct PhIP-glucuronide. It is possible that metabolite 1 is actually two metabolites that could not be separated with the HPLC conditions used. Further analysis is needed to fully characterize these metabolites.
Metabolite 6, which had an HPLC rt of ~41 min was identified as 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate). Mass spectral analysis revealed a primary molecular ion at m/z 323 [M + H]+ and upon CID a fragment ion at m/z 243 [M + H]+ was observed, indicating the loss of the SO3 group, revealing 4'-hydroxy-PhIP. Upon incubation of whole urine with aryl-sulfatase or 6 N HCl, this metabolite was quite susceptible to hydrolysis. Furthermore, this metabolite co-chromatographed with an authentic 4'-PhIP-sulfate standard derived from the urine of PhIP-exposed mice (19).
Metabolite 8, which eluted at 50 min, produced a molecular ion at m/z 403 [M + H]+, and a CID fragment ion at m/z 227 [M + H]+. The loss of a 176 Da fragment indicates the cleavage of the dehydrogenated glucuronic acid. This metabolite was resistant to bacterial ß-glucuronidase treatment and co-chromatographed with the previously characterized PhIP-N2-glucuronide (34), which was isolated from the urine of PhIP-exposed dogs (Figure 4
).

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Fig. 4. HPLC radio-profile of urine from humans dosed with [14C]PhIP, and urine spiked with 14C-labeled PhIP-N2-glucuronide and N-OH-PhIP-N2-glucuronide, isolated from dog urine. ( . . . . .) Human urine; () human urine spiked with metabolites isolated from dogs.
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Metabolite 9, the most abundant urinary metabolite, with an HPLC rt of ~52 min, was identified as N-hydroxy-PhIP-N2-glucuronide. Mass spectral analysis revealed a primary molecular ion at m/z 419 [M + H]+ and, upon CID, produced fragment ions at m/z 243 [M + H]+ and 227 [M + H]+, which is indicative of N-hydroxy-PhIP and PhIP, respectively (Figure 5
). This metabolite was resistant to both acid hydrolysis, (0.1 N HCl at 37°C for 1 h) and bacterial ß-glucuronidase treatment. In addition, the chromatographic properties were identical to an N-hydroxy-PhIP-N2-glucuronide, which was isolated from the urine of PhIP-exposed dogs and previously characterized by Kadlubar et al. (34) (Figure 4
).

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Fig. 5. Full scan mass spectra (A) and collision-induced mass spectra (B) of the human PhIP metabolite N-OH-PhIP-N2-glucuronide.
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Metabolite 10, which eluted at ~ 57 min, was identified as N-hydroxy-PhIP-N3-glucuronide. Unlike the N-hydroxy-PhIP-N2-glucuronide this metabolite was susceptible to ß-glucuronidase treatment, producing a product with the same retention time as N-hydroxy-PhIP, which is indicative of an N3-glucuronide (18). Furthermore, this metabolite co-eluted with an N-hydroxy-PhIP-N3-glucuronide standard, derived from the urine of PhIP exposed mice (19). Due to an insufficient amount of this metabolite, mass spectral data were not available.
Acid hydrolysis of whole urine resulted in a dramatic increase in metabolite 12 (rt 65 min) which was determined to be the parent compound PhIP, due to its co-elution with an authentic PhIP standard. PhIP accounted for <1% of the excreted radiolabel in all three subjects. The remaining metabolites, 4, 5, 7 and 11, are less abundant (<10% of recovered radioactivity) than the other metabolites and have yet to be fully characterized.
Blood plasma metabolites
HPLC analysis of the plasma revealed four major radioactive metabolite peaks in all three subjects. The four metabolites detected had chromatographic properties identical to 4'-PhIP-sulfate, N-hydroxy-PhIP-N2-glucuronide, N-hydroxy-PhIP-N3-glucuronide and the parent compound PhIP, as seen in the urine samples. In subject 1, the most abundant metabolite was N-hydroxy-PhIP-N2-glucuronide, which accounted for nearly 60% of the recovered radioactivity in the plasma at 1 h post-PhIP dose. Seventeen percent of the radioactivity in the plasma was unchanged PhIP (Figure 6
). In subject 2, PhIP was the most abundant compound accounting for ~56% of the radiolabel at 1 h, whereas N-hydroxy-PhIP-N2-glucuronide accounted for only 18%. In subject 3, the overall metabolite levels were much lower than those seen in subjects 1 and 2. PhIP was the major compound representing 37% of the radioactivity at 1 h post-PhIP dose and N-hydroxy-PhIP-N2-glucuronide comprised 20% of the detected radioactivity. In all three subjects, as expected, the level of PhIP decreased over time. In subjects 2 and 3, N-hydroxy-PhIP-N2-glucuronide increased over time, whereas in subject 1 the level slightly decreased (Figure 6
). 4'-PhIP-sulfate and N-hydroxy-PhIP-N3-glucuronide accounted for <10% of the recovered dose at each time point, in all three subjects. Peak 11 was detected in small quantities in subject 1. In subjects 2 and 3, peak 2 was detected at low levels in the 2 and 4 h time points, and PhIP-N3-glucuronide was detected in subject 3 (data not shown).

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Fig 6. [14C]PhIP plasma metabolites at various time points post [14C]PhIP exposure. Figure legends correspond to the time blood was drawn post PhIP dose. (A) Human subject 1; (B) human subject 2; (C) human subject 3. Data are expressed as percentages of recovered plasma radioactivity at each time point.
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Comparison of human, dog and mouse urinary metabolite profiles
The urinary PhIP metabolite profiles from the current human study were compared with urinary metabolite profiles from mice and dogs that had been dosed with PhIP. The major metabolite in the mouse was 4'-PhIP-sulfate (peak at 41 min), whereas in the human and the dog this was a minor metabolite (Figure 2
). The most abundant metabolite in the human (N-hydroxy-PhIP-N2-glucuronide; rt 52 min) was also a major metabolite in the dog urine. The dog urine also contained a relatively large amount of PhIP-N2-glucuronide (rt 50 min). This compound was also detected in the human urine but at much lower levels. Neither the N-hydroxy-PhIP-N2-glucuronide nor the PhIP-N2-glucuronide were detected in the mouse urine. The presence of the large amounts of unmetabolized PhIP excreted from the mouse and dog is most likely due to the much higher PhIP dose administered to the animals (10 mg/kg) than the dose administered to the humans. The enzymes involved in PhIP metabolism were presumably saturated and unable to fully metabolize all the PhIP present. The large number of PhIP-glucuronides in the human and dog seems to indicate that humans and dogs have a higher capacity for glucuronidation than the mouse. However, the mouse appears to have a higher sulfation rate, as evidenced by the large concentration of 4'-PhIP-sulfate. The human urinary profile contains more metabolites than the mouse and dog profiles do, suggesting a more complicated metabolic route is involved in PhIP metabolism in humans, than in the dog or mouse.
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Discussion
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The metabolism of PhIP has been well characterized in several animal species (19,28,35); however, little has been reported about the disposition of PhIP in humans. The present study is the first to use radiolabeled PhIP in a comprehensive examination of the metabolism of PhIP in humans. The variation in PhIP metabolism between three human subjects and the characterization and identification of the major urinary and plasma metabolites of PhIP is reported.
Interpretation of the results from this study must take into account that the human subject volunteers were elderly cancer patients undergoing colorectal cancer surgery. Each subject was taking numerous medications for their conditions prior to surgery. Furthermore, the PhIP dose they received was administered in capsule form and was not part of the diet. These factors could have influenced the metabolism of PhIP in these subjects.
An inter-individual variation in PhIP metabolism, over time, between the three subjects was evident from the recovery of the radiolabel in the urine of each subject. Because the urine fractions were collected at different times in each of the three subjects, direct comparisons with regard to excretion kinetics of PhIP metabolites could not be made. The detection of nearly 50% of the recovered radioactivity within the first 4 h of urine collected from subject 1 was ~3 times greater than that detected in the first 4 h from subjects 2 and 3. This indicates that subject 1 had an overall faster uptake and/or clearance rate than the other two subjects. The low levels of PhIP in the plasma of subject 1 compared with the other two subjects also suggests a more rapid clearance rate. Subject 1 also differed from subjects 2 and 3 in that almost none of the more polar metabolites (metabolites 1, 2, 3) were excreted, whereas these metabolites were readily detected in subjects 2 and 3. These differences may be attributed to a difference in the levels of enzymes responsible for PhIP metabolism present in each individual. It cannot be discounted, however, that the medical condition of each subject (i.e. medication, health status) could have influenced PhIP metabolism.
All three subjects were phenotyped for levels of the specific enzymes, cytochrome P4501A2 (CYP1A2), N-acetyltransferase (NAT2) and sulfotransferase (ST) (36,37). All of which are known to be involved in the biotransformation of PhIP. The observed differences in the metabolism among the three subjects correlated with the reported differences in the CYP1A2 phenotypes of each subject. Subject 1 had the highest CYP1A2 activity, the fastest excretion of the PhIP radiolabel and the highest level of N-hydroxy-PhIP-N2-glucuronide in the urine and plasma. There was no observed correlation between metabolism differences and NAT2 or ST levels. Because of the low number of test subjects these results may be anecdotal. More studies are needed to accurately determine the significance of the differences in enzyme activity with regards to PhIP metabolism.
Studies in our laboratory and others have shown that sulfotransferase may be involved in both the bioactivation and detoxification of PhIP (10,26,38,39). Most recently, when the sulfotransferase gene from human liver (HAST1) was transfected into the UV5P3 Chinese hamster ovary cell line, an increase in cytotoxicity over controls was observed. The addition of the sulfotransferase inhibitor 2,6-dichloro-4-nitrophenol effectively inhibited the cytotoxicity (R.W.Wu et al., unpublished data). Due to the instability of the sulfoxy metabolite of N-hydroxy-PhIP it was not possible to assess bioactivation of PhIP by sulfotransferase in the present studies. However, the presence of 4'-PhIP-sulfate (a detoxification metabolite) in human urine is an indication that sulfotransferases may play a role in PhIP metabolism in humans.
The identification of N-hydroxy-PhIP-N2-glucuronide, N-hydroxy-PhIP-N3-glucuronide, PhIP-N2-glucuronide and the tentative assignment of metabolites 1, 2 and 3 as PhIP-glucuronides suggests that, in humans, glucuronidation is a major metabolic pathway for PhIP metabolism. The finding of N-hydroxy-PhIP-N2-glucuronide as the major human urinary metabolite is in agreement with previous studies showing this compound as a major metabolite from human hepatic microsomes fortified with uridine-5'-diphosoglucuronic acid and treated with N-hydroxy-PhIP (18). This compound was also a major metabolite produced by dog hepatic microsomes, and was found in the urine of dogs that have been dosed with PhIP (34). The N-hydroxy-PhIP-N3-glucuronide, which was a relatively minor metabolite in the human and dog urines, was a major metabolite in the rat and mouse (18,19,40). In the present study, the N2-glucuronide was preferentially formed over the N3-glucuronide by a ratio of 6:1. It is unclear why humans preferentially form the N2-glucuronide over the N3-glucuronide but it could be due to substrate specificity of a particular UDPglucuronosyltransferase (UDPGT) involved in the formation of these compounds. Also plausible, is that different UDPGT isozymes are responsible for the formation of each glucuronide and the relative amounts of each isozyme would dictate the level of each N-glucuronide formed. If this were the case, the present results suggest that humans have a higher level of the UDPGT that catalyzes the formation of the N2-glucuronide over formation of the N3-glucuronide. This is important since the N3 glucuronides can be de-conjugated by bacterial ß-glucuronidase (found in human intestine and colon) back to N-hydroxy-PhIP where re-conjugation can occur to possibly form highly reactive compounds capable of binding DNA (17). Conversely, the N2-glucuronides are resistant to ß-glucuronidase and are readily excreted into the urine. At present it is unknown which UDPGTs catalyze the formation of the N2- or N3-glucuronides. Additional studies are needed to determine which UDPGT isozymes are involved in these conjugation reactions.
Due to the instability of the bioreactive PhIP metabolites it was not possible to directly assess the bioactivation of PhIP in these human subjects. The presence of the detoxification metabolite, N-hydroxy-PhIP-N2-glucuronide, as well as the N-hydroxy-PhIP-N3-glucuronide, however, serves as an indirect indicator of bioactivation. The formation of the CYP1A2-mediated, reactive intermediate N-hydroxy-PhIP is prerequisite to conjugation with the glucuronide (17,18). Therefore, detection of N-hydroxy-PhIP-glucuronides in the urine indicates the formation of the reactive intermediate, N-hydroxy-PhIP, earlier in the metabolic process. N-hydroxy-PhIP is readily formed in the liver of humans via CYP1A2 N-hydroxylation (22,24). It can then be further conjugated to form highly reactive compounds that bind DNA. Alternatively, N-hydroxy-PhIP can form the detoxification products, N-hydroxy-PhIP-N2-glucuronide and N-hydroxy-PhIP-N3-glucuronide, which are readily excreted into the urine. It is unclear at this time whether N-hydroxy-PhIP has a higher binding affinity for conjugation by a glucuronide or by a mechanism that will make it more reactive (i.e. sulfotransferase, acetyltransferase). Additional studies are needed to characterize the substrate specificities for N-hydroxy-PhIP conjugation reactions. Furthermore, it is unknown where in the metabolic pathway glucuronidation occurs. UDPGTs are known to be present in the liver, as well as in extrahepatic tissue, and previous studies have shown glucuronidation of N-hydroxy-PhIP to occur in both the liver and in extrahepatic tissue (17,18,41). The presence of the relatively large concentration of N-hydroxy-PhIP-N2-glucuronide in the plasma of the three subjects leads to speculation that glucuronidation of N-hydroxy-PhIP may occur at numerous sites in humans, and that N-hydroxy-PhIP maybe stable enough to be transported via the blood to other tissues where it can be further metabolized. The presence of N-hydroxy-PhIP-N2-glucuronide in such a high concentration in the urine and plasma, and the fact that it is an indicator of bioactivation, makes this metabolite a good candidate as a biomarker for PhIP exposure and PhIP bioactivation.
The urinary PhIP metabolic profiles of humans, dogs and mice were significantly different. Although the PhIP dose received by the dog and mouse were higher than that received by the humans, the dramatic differences in the metabolic profiles need to be considered. The presence of 4'-PhIP-sulfate as the major metabolite in the mouse and the absence of the N-hydroxy-PhIP-N2-glucuronide leads to the conclusion that the mouse may not be a suitable model for studying PhIP metabolism in humans. The dog has a metabolic profile more similar to that of humans; however, the dog produced much more of the PhIP-N2-glucuronide than the humans. The human metabolite profile contained the highest levels of the N-hydroxy-PhIP-N2-glucuronide when compared with the other species. These results reflect the observations from previous studies, which report that humans have the greatest capacity to form glucuronides, followed by dogs then rats (42,43). The differences observed in the human, dog and rodent PhIP urinary metabolic profiles will make it difficult to extrapolate from animal data to humans, especially when making risk assessment determinations from PhIP exposure.
This study has shown that individual variation in metabolism can be measured. These metabolic differences could be attributed to variations in certain metabolizing enzymes, as shown by different CYP1A2 phenotypes in the three subjects tested. More experiments are needed, using a larger population, to make an accurate assessment of the significance of the inter-individual variation in PhIP metabolism. The identification of N-hydroxy-PhIP-N2-glucuronide as a potential biomarker has implications that are 2-fold. The abundance of this compound and its stability in the urine makes it a possible indicator of PhIP exposure, as well as an indicator of bioactivation. In parallel experiments on the same human subjects, DNA and protein adducts are being analyzed by accelerator mass spectrometry. These results should help elucidate the significance of this metabolite as a biomarker. By characterizing all the major PhIP metabolites and the enzymes involved in the biotransformation of PhIP in humans, and determining the relative differences in PhIP metabolites among individuals, it is hoped that a prediction of individual susceptibility to carcinogenic risk from PhIP exposure can be achieved. Currently, efforts are under way to analyze more human subjects exposed to PhIP, and to develop sensitive mass spectrometry methods to identify PhIP metabolites in human urine so ultimately human exposure to radiolabeled compounds will not be needed.
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Acknowledgments
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The authors would like to thank Dr Fred F.Kadlubar, National Center for Toxicological Research, Jefferson, AR, for supplying the dog urine containing 14C-labeled PhIP metabolites and for his helpful discussions, and Dr Hugh Gregg, Lawrence Livermore National Laboratory, Livermore, CA, for his assistance with the mass spectral determinations. This work was performed under the auspices of the US DOE by LLNL under contract #W-7405-ENG-48 and supported by NCI grants CA55861, CA55751, CA58697, EPA grant R825280, National Institute on Aging grant AG15722 and UC Breast Cancer Research grant 2RB-0126.
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Notes
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4 To whom correspondence should be addressed Email: felton1{at}llnl.gov 
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References
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Received September 10, 1998;
revised November 19, 1998;
accepted December 4, 1998.