The impact of glucuronidation on the bioactivation and DNA adduction of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in vivo
Michael A. Malfatti,
Esther A. Ubick and
James S. Felton *
Biosciences Directorate, Lawrence Livermore National Laboratory, PO Box 808, L-452, Livermore, CA 94551, USA
* To whom correspondence should be addressed. Tel: 925 422 5656; Fax: 925 422 2282; Email: felton1{at}llnl.gov
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Abstract
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UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of many different chemicals. Glucuronidation is especially important for detoxifying reactive intermediates from metabolic reactions, which otherwise can be biotransformed into highly reactive cytotoxic or carcinogenic species. Detoxification of certain food-borne-carcinogenic heterocyclic amines (HAs) is highly dependent on UGT1A-mediated glucuronidation. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant carcinogenic HA found in well-done cooked meat, is extensively glucuronidated by UGT1A proteins. In humans, CYP1A2 catalyzed N-hydroxylation and subsequent UGT1A-mediated glucuronidation is a dominant pathway in the metabolism of PhIP. Therefore, changes in glucuronidation rates could significantly alter PhIP metabolism. To determine the importance of UGT1A-mediated glucuronidation in the biotransformation of PhIP, hepatic UGT1A deficient Gunn and UGT1A proficient Wistar rats were exposed to a 100 µg/kg oral dose of [14C]PhIP. Urine was collected over 24 h and the PhIP urinary metabolite profiles were compared between the two strains. After the 24 h exposure, livers and colons were removed and analyzed for DNA adduct formation by accelerator mass spectrometry. Wistar rats produced several PhIP and N-hydroxy-PhIP glucuronides that accounted for
25% of the total amount of recovered urinary metabolites. In the Gunn rats, PhIP and N-hydroxy-PhIP glucuronides were reduced by 6892%, compared with the Wistar rats. PhIPDNA adduct analysis from the Gunn rats revealed a correlation between reduced urinary PhIP and N-hydroxy-PhIP glucuronide levels and increased hepatic DNA adducts, compared with the Wistar rats. In the colon, DNA adduct levels were lower in the Gunn rats compared with the Wistar rats, suggesting deficient hepatic UGT1A activity provides protection against DNA adduct formation in peripheral tissue. Due to differences in PhIP metabolism between humans and rodents, extrapolation of these results to the human situation must be done with caution. These results indicate that UGT1A-mediated glucuronidation of PhIP and N-hydroxy-PhIP is an important pathway for PhIP detoxification, and demonstrate the importance of tissue-specific metabolism. Tissues with reduced UGT1A activity can have a higher rate of PhIP activation and be more inclined to form DNA adducts compared with tissues with normal UGT1A activity.
Abbreviations: AMS, accelerator mass spectrometry; BaP, benzo[a]pyrene; BNF, ß-naphthoflavone; CYP 450, cytochrome P450; HA, heterocyclic amine; NADP, nicotinamide adenine dinucleotide phosphate; N-hydroxy-PhIP, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; UDPGA, uridine diphosphate glucuronic acid; UGT, uridine diphosphate-glucuronosyltransferase
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Introduction
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UDP-glucuronosyltransferase (UGT)-mediated glucuronidation is rapidly gaining recognition as an important pathway in the metabolism of certain food-borne carcinogenic heterocyclic amines (HAs) (1,2). The UGTs are a superfamily of membrane-bound enzymes that catalyze the glucuronidation of many endogenous and xenobiotic compounds. These conjugation reactions are catalyzed by numerous isoforms that have a broad and overlapping substrate selectivity and tissue distribution (reviewed in ref. 3). Glucuronidation is an especially important pathway for detoxifying reactive intermediates from metabolic reactions, which otherwise can be biotransformed into highly reactive cytotoxic or carcinogenic species (4). The UGT proteins are divided into two families based on sequence homologies, designated UGT1 and UGT2 (5). These families are further divided into three subfamilies, UGT1A, UGT2A and UGT2B. Studies have shown that the detoxification of certain cooked-food-derived carcinogenic HAs is highly dependent on UGT1A-mediated glucuronide conjugation (1,68). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most mass abundant carcinogenic HA found in well-done cooked meat and is extensively glucuronidated by UGT1A proteins (911). A more recent study, using microsomal preparations from baculovirus infected insect cells containing all the functional UGT1A proteins, has implicated UGT1A1 as the primary UGT1A isoform responsible for glucuronidation of the reactive intermediate of PhIP (8).
PhIP has been shown to be carcinogenic in rodents, producing lymphomas in mice, and colon, mammary and prostate tumors in rats (1214). In humans, PhIP forms DNA adducts in several tissues (15,16). Furthermore, intake of PhIP from well-done red meat consumption has been associated with an increased risk for colon and breast cancer (17,18). These findings, together with the relative abundance of PhIP in cooked foods, suggest that PhIP may pose a significant risk to the development of certain human cancers.
Bioactivation of PhIP is highly dependent upon the hepatic cytochrome P4501A2 (CYP1A2)-mediated N-hydroxylation to the corresponding 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) (19,20). N-Hydroxy-PhIP is subsequently esterified by phase II sulfotransferases and/or acetyltransferases that generate the highly electrophilic O-sulfonyl and O-acetyl esters, respectively. These esters are capable of heterolytic cleavage to generate the reactive nitrenium ion which is considered the ultimate carcinogenic species (21). The reactive nitrenium ion has been shown to form DNA adducts in multiple tissues (2224). UGT1A-mediated N-glucuronidation can compete with these activation reactions resulting in the formation of the less reactive N-hydroxy-PhIP-N2-glucuronide and N-hydroxy-PhIP-N3-glucuronide. These compounds can be excreted through the urine or bile, or can be transported to extrahepatic tissue where deconjugation by ß-glucuronidase can occur, leading to the regeneration of the reactive intermediate N-hydroxy-PhIP (6,25).
In humans, studies have indicated that CYP1A2 catalyzed N-hydroxylation and subsequent UGT1A1-mediated glucuronidation is quantitatively the most important pathway in the metabolism of PhIP. When human volunteers were exposed to PhIP, N-hydroxy-PhIP glucuronide conjugates accounted for
60% of the total PhIP urinary metabolites (2). N-hydroxy-PhIP-N2-glucuronide was also the major metabolite in human urine after consumption of a single cooked chicken meal (26). Since glucuronidation is such a major contributor to PhIP metabolism, it can be reasoned that changes in glucuronidation rates could significantly alter PhIP metabolism and bioactivation. This concept was reinforced when Chinese hamster ovary cells, that were transfected with the human UGT1A1 gene, were shown to provide a protective effect against PhIP-induced cytotoxicity and mutation induction when compared with control cells that did not contain the UGT1A1 gene (27). Similar results were seen when Chinese hamster ovary cells were transfected with the rat UGT1A1 gene (unpublished data).
Most of the studies investigating the differential expression of UGT1A on PhIP metabolism have used in vitro techniques. Little has been done, however, to investigate how UGT1A expression affects PhIP bioactivation in vivo. The use of the Gunn rat as an in vivo model for hepatic UGT1A deficiency has been routinely used to study the effects of reduced hepatic UGT1A activity in the metabolism of various chemicals. The Gunn rat is a mutant strain of the Wistar rat that contains a frame-shift deletion of a single guanosine residue that renders the entire hepatic UGT1A gene locus inactive (28,29). By using the Gunn rat in this current study, it was possible to determine the effect reduced UGT1A activity has on PhIP metabolism in vivo. Furthermore, by using accelerator mass spectrometry (AMS) to analyze DNA adducts, the effects of PhIP exposure can be assessed at PhIP concentrations that are 10100 times lower than what is typically used for in vivo studies using traditional detection techniques. This technique allows for the use of dose levels that approach human dietary relevancy. These studies should help provide a better understanding of the overall mechanisms of PhIP biotransformation. Furthermore, understanding how differential UGT1A activity affects PhIP bioactivation may help in evaluating the individual susceptibility to potential cancer risks from PhIP exposure.
The goal of the current study is to determine what effects reduced UGT1A expression has on PhIP metabolism and DNA adduct formation in vivo and to correlate UGT1A expression levels with DNA adduct levels in certain tumor target tissues. Since the Gunn rat is deficient in the expression of all the hepatic UGT1A isozymes, this study focused on overall UGT1A activity toward PhIP and not on any one specific isoform. A decrease in urinary PhIP glucuronide levels in the Gunn rat correlated with an increase in hepatic DNA adducts, compared with the control rats. These findings suggest that diminished UGT1A activity in specific tissues could pose a significant risk for the development of certain cancers from exposure to PhIP. Since the metabolism of PhIP is known to be different in humans than in rodents (2), the extrapolation of the data from these studies to the human situation must be done with caution.
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Materials and methods
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Chemicals
[14C]PhIP was purchased from Toronto Research Chemicals (North York, Ontario, Canada). N-hydroxy-PhIP was obtained from SRI International (Palo Alto, CA). The radiochemical purity of [14C]PhIP and the chemical purity of N-hydroxy-PhIP were assessed by HPLC (isocratic at 40% methanol) and were determined to be >98% pure for both compounds. Nicotinamide adenine dinucleotide phosphate (NADP), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, ß-naphthoflavone, urea, Triton X-100, ribonuclease A and T1, dithiothreitol and proteinase K were purchased from Sigma (St Louis, MO). DNA isolation columns were obtained from Qiagen (Valencia, CA). UDP-glucuronic acid (UDPGA), alamethicin, and all other microsomal reaction buffer components were obtained as a mix from BD Gentest, (Bedford, MA). All immunoblotting reagents were obtained from BioRad (Hercules, CA). Antibodies for immunoblotting were obtained from BD Gentest and Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were of analytical grade or better.
Animals
Male Wistar and Gunn rats weighing 200250 g were obtained from Harlan (Indianapolis, IN). All animals were housed in polystyrene cages containing hardwood bedding and kept on a 12 h light/dark cycle in a ventilated room maintained at 24°C. Animals were allowed to acclimate to their quarters for 7 days prior to use. During this time, food (standard lab chow) and water was provided ad libitum. In experiments requiring cytochrome P450 and UGT1A induced animals, rats were injected intraperitoneally with ß-naphthoflavone (BNF) at 80 mg/kg in corn oil once a day for 3 days prior to dosing with [14C]PhIP.
Metabolite and tissue collection
Uninduced and BNF-nduced animals were administered a single oral gavage dose of 100 µg/kg [14C]PhIP in corn oil containing 1% DMSO v/v (sp. act. 14.2 mCi/mmol). The animals were placed in metabolism cages and urine was collected over dry ice for 24 h in 04 h and 424 h fractions. At 24 h post [14C]PhIP dose, the animals were killed by CO2 asphyxiation and their livers and colons were removed and stored at 80°C until DNA isolation.
Separation of PhIP metabolites from rat urine
Collected urine was thawed and the total volume of each fraction recorded. A 0.5 ml aliquot from each fraction was analyzed for carbon-14 content by liquid scintillation counting (Wallic, Gaithersburg, MD). A 0.5 ml aliquot of each urine sample was then analyzed by reverse-phase HPLC for PhIP and PhIP metabolites by directly injecting the samples onto a Rainin HPLC system (Varian, Walnut Creek, CA) equipped with a 5 µm, 4.6 mm x 150 mm TSK-GEL ODS-80 TM column (Toso Bioscience, Montgomeryville, PA) and monitored at 315 nm. Metabolites were eluted at 0.75 ml/min initially using a solvent of 10% (v/v) methanol/0.1% (v/v) triethylamine (pH 6), for 2 min. This was followed by a gradient to 33% (v/v) methanol/0.1% triethylamine at 32 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.
Microsomal incubations
Microsomes were prepared from both uninduced and BNF-induced Wistar and Gunn rat livers by differential centrifugation (30). Briefly, livers were removed from the animals and homogenized using a Polytron tissue homogenizer (Brinkmann, Westbury, NY) in 3 volumes of ice-cold 0.02 M TrisHCl buffer containing 1.15% KCl (pH 7.4). The homogenate was centrifuged at 9000 g at 4°C for 20 min. The supernatant was removed and centrifuged again at 100 000 g at 4°C for 1 h. The supernatant was removed and the microsomal pellet was gently resuspended in 0.1 M TrisHCl (pH 7.4). The protein concentration was determined by the Bradford method (31). Microsomal incubations were prepared on ice in 1.5 ml conical plastic tubes. To determine CYP450-mediated PhIP hydroxylation rates, samples consisted of 2.0 mg/ml microsomal protein, 15 mM MgCl2, an NADPH regenerating system (1 mM NADP, 15 mM glucose-6-phosphate and 1 U/ml glucose-6-phosphate dehydrogenase) and 10100 µM PhIP (dissolved in DMSO delivered in 5 µl) in 0.1 M sodium phosphate buffer (pH 7.4), in a total volume of 500 µl. Samples were incubated for 30 min at 37°C. To determine UGT-catalyzed N-hydroxy-PhIP glucuronidation rates, samples consisted of 2.0 mg/ml microsomal protein, 8.0 mM MgCl2, 0.5 mM EDTA, 2.0 mM UDPGA, 25 µg/ml alamethicin and 10100 µM N-hydroxy-PhIP (dissolved in DMSO delivered in 5 µl) in 50 mM Tris-HCl buffer (pH 7.4), in a total volume of 200 µl. Samples were incubated for 3 h at 37°C. After the incubation times, 2 volumes of ice-cold methanol were added to each sample to precipitate the proteins and terminate the reaction. The samples were then allowed to stand at 20°C for 30 min. The protein was then removed by centrifugation in a microcentrifuge at maximum speed for 5 min. The methanolic extracts containing the reaction products were placed in clean plastic tubes and stored at 80°C until HPLC analysis.
HPLC analysis of microsomal products
The aqueousmethanol extracts from the microsomal incubations were evaporated to dryness under nitrogen and then reconstituted in 60 µl of HPLC starting mobile phase. The samples were centrifuged in a microcentrifuge for 1 min at maximum speed and 50 µl of supernatant was injected into an Alliance HPLC system (Waters, Milford, MA) equipped with a 5 µm, 4.6 mm x 150 mm TSK-GEL ODS-80 TM column (Toso Bioscience, Montgomeryville, PA) and a Waters 990 photodiode array detector. The metabolites were eluted at 0.75 ml/min using a gradient starting at 30% methanol/0.1% triethylamine (pH 6.0), up to 55% methanol/0.1% triethylamine (pH 6.0), at 8 min. The methanol concentration was maintained at 55% from 8 to 20 min. The identities of the CYP450-mediated PhIP-hydroxy intermediates and the UGT-mediated N-hydroxy-PhIP-glucuronide conjugates were confirmed by comparing the HPLC retention time and UV spectra with known metabolite standards. Quantification of each metabolite was based on the molar extinction coefficient of PhIP (19 440 at 315 nm) (30).
Immunoblotting
Western blot analysis was used to confirm the presence/absence of UGT1A proteins in the two rat strains. Immunoblotting was performed according to the manufacturer's recommendations (BD Gentest). Briefly, 2100 µg of microsomal protein from each microsomal preparation was heated at 95°C for 4 min in loading buffer (62.5 mM TrisHCl (pH 6.8), 2% SDS, 25% glycerol and 0.01% Bromophenol Blue) containing 2% 2-mercaptoethanol and then separated on a 10% SDS-polyacrylamide gel. The separated proteins were electrotransferred onto a nitrocellulose membrane. The membrane was blocked in 5% non-fat powdered milk in 25 mM TrisHCl (pH 7.5) and 150 mM NaCl (solution A) for 1 h, and then washed three times with solution A containing 0.1% Tween-20. The membranes were then incubated for 1 h in 0.5% non-fat powdered milk in solution A containing an antibody (1:500) prepared from a rabbit immunized with a peptide specific for the conserved C-terminal region of all UGT1A isoforms (WBUGT1A; BD Gentest). The membrane was washed three more times and was then incubated for 1 h in 0.5% non-fat powdered milk in solution A containing an HRPconjugated goat anti-rabbit IgG secondary antibody (1:5000) (Santa Cruz Biotechnology, Inc.), followed by three more washings. Visualization was performed using the Immun-Star HP substrate detection kit (BioRad). The identity of the UGT1A protein was based on comparison of the chemiluminescence band with a known UGT1A protein standard (BD Gentest).
DNA isolation and adduct analysis
DNA isolation from rat liver and colon, and sample preparation for the quantification of DNA adduct levels by AMS has been reported elsewhere (32). Briefly, tissues were homogenized then digested in lysis buffer (4 M urea, 1.0% Triton X-100, 10 mM EDTA, 100 mM NaCl, 10 mM DTT and 10 mM TrisHCl, pH 8.0) containing 0.8 mg/ml proteinase K overnight at 37°C. Undigested tissue was removed by centrifugation, and the supernatant was treated for 1 h at room temperature with RNase A (0.5 mg/ml) and RNase T1 (5 µg/ml). DNA was extracted using Qiagen column chromatography (Qiagen, Valencia, CA) according to the manufacturer's instructions. DNA purity was determined by the A260 nm/A280 nm ratio. A ratio between 1.6 and 1.8 was considered pure. Pure DNA samples were then submitted for adduct analysis by AMS.
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Results
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Recovery of urinary metabolites
The total amount of radioactivity recovered in the urine over the 24 h collection period was similar in the two rat strains. Over 24 h,
14% of the administered dose was recovered in the urines of both the Wistar and Gunn rats. Induction with BNF did not change the amount of overall radioactivity excreted; however, the rate of excretion was different in the two strains. In the Wistar rats
50% of the recovered radioactivity was excreted in the first 4 h of collection in both the induced and uninduced animals (Figure 1). In the uninduced Gunn rats 60% of the recovered radioactivity was excreted in the first 4 h, and in the BNF-induced Gunn rats >70% of the recovered radioactivity was excreted in the first 4 h.

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Fig. 1. Urinary excretion of [14C]PhIP in BNF-induced and uninduced Wistar and Gunn rats over time. Urine was collected over 24 h, divided into two fractions: grey column, 04 h fraction; black column, 424 h fraction. Data are the mean of four animals ± SD.
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Chromatographic analysis of the urine from BNF-induced Wistar rats revealed eight relevant radioactive peaks (Figure 2A). Based on co-elution with authentic PhIP metabolite standards five of the peaks have been positively identified. The major peak, at a retention time of 33 min, co-eluted with a 4'-PhIP-sulfate authentic standard. The peaks at 31 and 52 min were determined to be 4'-O-PhIP-glucuronide and N-hydroxy-PhIP-N3-glucuronide, respectively. The peak at 60 min co-eluted with an N-hydroxy-PhIP standard. This peak was observed only in the 04 h urine fraction of the Wistar rat. The parent compound PhIP was detected at very low levels at 62 min. Based on electrospray ionization-mass spectral characterization, the peak at 22 min produced molecular ions at m/z 401 [M+H]+ and upon collision-induced dissociation produced fragment ions at m/z 225 [M+H]+ which is consistent with a direct PhIP-glucuronide. The radioactivity between 43 and 47 min was unresolvable by HPLC; however, MS/MS analysis of these peaks revealed molecular ions at m/z 417 [M+H]+ and fragment ions at m/z 241 [M+H]+ and m/z 225 [M+H]+. The fragmentation pattern was identical to what has been seen for N-hydroxy-PhIP-N2-glucuronide (6). Molecular parent ions of m/z 241 [M+H]+ were also detected indicating the presence of a hydroxy-PhIP metabolite. Both N-hydroxy-PhIP-N2-glucuronide and 4'-hydroxy-PhIP standards co-eluted at
44 min on the HPLC. The peak that eluted at 3637 min was not identified. In the uninduced Wistar rats, the levels of all detectable metabolites were decreased by
10% compared with the induced animals (data not shown).

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Fig. 2. HPLC radio-profile of urine from BNF-induced rats exposed to a single oral dose of 100 µg/kg [14C]PhIP. Urine was collected over 24 h. (A) UGT1A proficient Wistar rats; (B) UGT1A deficient Gunn rats.
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The urinary PhIP metabolic profile in the Gunn rats was significantly different from what was observed in the Wistar rats (Figure 2B). The levels of all the PhIP and N-hydroxy-PhIP glucuronide conjugates were dramatically reduced in the Gunn rat compared with the Wistar rat. In the BNF-induced Gunn rats N-hydroxy-PhIP-N3-glucuronide, the direct PhIP-glucuronide, and 4'-O-PhIP-glucuronide were reduced by 92, 75 and 68%, respectively, compared with the glucuronide conjugate levels in the Wistar rats (Figure 3). Similar reductions were also seen in the uninduced animals. In the Wistar rats all the PhIP and N-hydroxy-PhIP glucuronide conjugates accounted for 18 and 25% of the urinary metabolites in the uninduced and induced animals, respectively. Whereas, in the Gunn rats the PhIP and N-hydroxy-PhIP glucuronides accounted for only 2.54.0% of the total amount of recovered metabolites (Figure 3). Interestingly, the reduction of glucuronide conjugate formation in the Gunn rats did not result in the increase in any of the other known PhIP metabolites. There were, however, small increases in radioactivity at HPLC retention times of 19, 2526, 2830 and 3841 min compared with the Wistar rats (Figure 2). These retention times are not associated with any known PhIP metabolites. The cumulative increase in radioactivity associated with the unknown peaks observed in the Gunn rat, accounted for
18% of the total amount of recovered radioactivity. In the Wistar rats the radioactivity associated with the same retention times accounted for only 6% of the total recovered radioactivity. These results suggest that other pathways may be involved in the metabolism of PhIP in the Gunn rats.

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Fig. 3. Difference in urinary PhIP metabolite levels from BNF-induced UGT1A proficient Wistar rats and UGT1A deficient Gunn rats exposed to a single oral dose of 100 µg/kg [14C]PhIP. Grey column, Wistar rat; black column, Gunn rat. Data are the mean of four animals ± SD.
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Microsomal metabolism
To ensure cytochrome P450 hydroxylation rates were consistent between the two rat strains, hepatic microsomes were prepared from each strain and exposed to PhIP. NADPH-dependent metabolism of PhIP was assessed in each microsomal preparation. There was no difference in CYP450 mediated hydroxylation rates of PhIP between the two rat strains (Figure 4). Microsomes from both the Wistar and the Gunn rats produced both 4'-hydroxy-PhIP and N-hydroxy-PhIP. 4'-Hydroxy-PhIP was formed at a rate approximately two times faster than N-hydroxy-PhIP.

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Fig. 4. Cytochrome P450-mediated hydroxylation of PhIP from hepatic microsomes prepared from BNF-induced Wistar (solid line) and Gunn (dashed line) rats. Data are the mean of three incubations ± SD. Similar results were seen from the microsomal preparations from the uninduced animals.
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To assess hepatic UDP-glucuronosyltransferase rates, N-hydroxy-PhIP was exposed to both Wistar and Gunn liver microsomes that were fortified with UDPGA. The microsomes prepared from the Wistar rats produced both N-hydroxy-PhIP-N2-glucuronide and N-hydroxy-PhIP-N3-glucuronide (Figure 5). N-hydroxy-PhIP-N3-glucuronide was produced at a rate seven times faster than N-hydroxy-PhIP-N2-glucuronide. In the microsomes prepared from the Gunn rats the formation of N-hydroxy-PhIP-N2-glucuronide was reduced
10-fold compared with the rate of formation observed in the microsomes derived from the Wistar rats. Unexpectedly, the rate of formation of N-hydroxy-PhIP-N3-glucuronide was similar to the rate seen in the Wistar rat-derived microsomes (Figure 5).

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Fig. 5. UDP-glucuronosyltransferase-mediated glucuronidation of N-hydroxy-PhIP from hepatic microsomes prepared from Wistar (solid line) and Gunn (dashed line) rats. Data are the mean of three incubations ± SD. Similar results were seen from the microsomal preparations from the uninduced animals.
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Immunoblotting
To confirm the presence/absence of hepatic UGT1A protein expression in each rat strain, western blot analysis was performed on the microsomal extracts from both the Wistar and Gunn rats. Two micrograms of microsomal protein was analyzed from the BNF-induced Wistar rats. All other analyses used 100 µg of microsomal protein. The antibody used was a polyclonal anti-human UGT1A peptide raised in a rabbit that is specific for the carboxyl terminus of the protein, whose sequence is conserved for human and rat for all UGT1A proteins (33). Since this peptide will react with all of the UGT1A isoforms it was not possible to determine the expression levels of the individual UGT1A isozymes. The microsomes isolated from the BNF-induced Wistar rats revealed the presence of a protein with a molecular mass of
54 kDa, which was identical to the UGT1A protein standard (Figure 6). No protein band could be detected from the samples from the uninduced animals. These findings indicate there was an increase in UGT1A protein expression due to BNF treatment. There was no evidence of UGT1A protein expression from the microsomes derived from the Gunn rats (Figure 6).

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Fig. 6. Western blot of UGT1A from hepatic microsomal preparations from Wistar and Gunn rats. Lanes 3 and 4 contained 2 µg of microsomal protein. All other lanes contained 100 µg of micosomal protein. The antibody is specific for the C-terminal region of the protein, which is conserved for all UGT1A proteins. The protein bands at 52 kDa represent all UGT1A subfamily members.
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DNA adduct analysis
DNA adducts were detected, by AMS analysis, in the livers and colons of all animals that were treated with a single oral dose of [14C]PhIP (Table I). In the liver, adducts were detected in the BNF-induced UGT1A proficient Wistar rats at 268.6 ± 63.8 adducts/1012 nucleotides. Adduct levels from the hepatic UGT1A deficient Gunn rats with similar treatment were five times higher at 1051.8 ± 251.9 adducts/1012 nucleotide. In the uninduced animals adduct levels in the livers of the Wistar and Gunn rats were similar at 847.8 ±114.5 and 996.1 ± 205.9 adducts/1012 nucleotide, respectively. These results suggest that inducible UGT1A proteins may play a role in the detoxification of PhIP in the liver since the animals that were deficient in hepatic UGT1A produced more liver DNA adducts than the BNF-induced UGT1A proficient animals, whereas the uninduced animals had similar adduct levels.
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Table I. DNA adducts from hepatic UGT1A deficient Gunn and UGT1A proficient Wistar rats that were exposed to a single oral dose of [14C]PhIPa
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In the colon, DNA adducts in the BNF-induced Wistar and Gunn rats were seven and eight times lower than what was observed in the colons of the uninduced animals, respectively (Table I). The lower adduct levels in the induced animals compared with the uninduced animals indicates that inducible UGT1A activity in the colon of both strains was capable of detoxifying the N-hydroxy-PhIP reactive intermediate. In both the uninduced and BNF-induced animals, adduct levels in the colon of the Gunn rats were significantly lower than adduct levels in the Wistar rats, suggesting that deficient hepatic UGT1A activity in the Gunn rats may provide protection against PhIP-induced DNA adducts in the colon.
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Discussion
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The use of the Gunn rat as a model for hepatic UGT1A deficiency has allowed for comparisons with the UGT1A proficient Wistar rats on the impact UGT1A expression has on PhIP bioactivation and DNA adduct formation. The use of a rodent model to study PhIP metabolism, however, does have some limitations. It has been well established that the metabolism of PhIP in humans and rodents is quite different (2). In humans N-hydroxy-PhIP-N2-glucuronide is the major metabolite detected in the urine of PhIP exposed individuals. In rats 4'-PhIP-sulfate is the predominant metabolite formed. In humans, formation of N-hydroxy-PhIP-N2-glucuronide is favored over N-hydroxy-PhIP-N3-glucuronide, whereas in rodents the opposite is seen. Because of these differences, extrapolation of the data from the present study to humans must be done with caution. Nevertheless, the importance of N-glucuronidation of N-hydroxy-PhIP in humans cannot be overlooked and at the present time the rodent model is the best that is available. Furthermore, in both humans and rodents N-glucuronidation is the predominant pathway involved in the detoxification of the reactive intermediate N-hydroxy-PhIP. It is hypothesized that glucuronidation in rats makes sufficient contributions to the detoxificaton of N-hydroxy-PhIP, and that diminished UGT1A activity will cause significant changes in the overall metabolism of PhIP. Therefore, by determining the effect of glucuronidation on PhIP biotransformation in rodents, inferences can be made as to what might occur in humans. Since UGT-mediated glucuronidation is such a predominant pathway for PhIP bioransformation in humans, the effects of UGT activity on PhIP metabolism may be more dramatic compared with what has been observed in these rat studies.
By using AMS to detect DNA adducts, the effects from PhIP exposure could be assessed at a relatively small dose. Although AMS can detect DNA adducts at human dietary dose levels, the dose used in these present experiments was about 10 times higher that what a person might receive by consuming one well-done cooked chicken breast (34). This dose level allowed for the detection of PhIP metabolites in the urine of the animals used in these experiments.
The dramatic decrease in all the PhIP and N-hydroxy-PhIP glucuronides in the urine of the Gunn rats clearly demonstrates that glucuronidation plays a major role in the metabolism of PhIP. In the BNF-induced Wistar rats, PhIP and N-hydroxy-PhIP glucuronides accounted for
25% of all the recovered metabolites. Whereas, in the Gunn rats only 4% of the metabolites were glucuronide conjugates. Interestingly, this 84% reduction in glucuronides did not cause an increase in any of the other known PhIP urinary metabolites. However, the 12% increase in radioactivity, in the Gunn rat over the Wistar rat, at HPLC retention times not associated with known PhIP metabolites suggests that other pathways maybe involved in metabolizing PhIP. These findings are in contrast to a previous study that reported increases in several PhIP metabolites in the urine of Gunn rats after an intravenous (i.v.) PhIP exposure and a 2 h urine collection (35). In that study the only metabolite that was decreased was N-hydroxy-PhIP-N3-glucuronide. The discrepancies between the previous and this current study could be due to the differences in dosing methods and urine collection times. By using an i.v. exposure route the distribution of PhIP would be much quicker compared with an oral exposure; however, an i.v. exposure would also bypass first pass metabolism. Since human exposure to PhIP is predominantly oral, first pass metabolism could be a significant contributor to the overall biotransformation of PhIP.
The lack of compensatory changes in non-glucuronide PhIP metabolites in the Gunn rats despite decreases in the PhIP and N-hydroxy-PhIP glucuronide conjugates was somewhat unexpected. However, since the greatest metabolite reduction was from N-hydroxy-PhIP-N3-glucuronide it is possible that increases could have occurred in N-hydroxy-PhIP and its subsequent bioreactive acetoxy and sulfoxy derivatives. Due to their high reactivity, these compounds would not have been detected in the urine. Evidence for this concept was observed from the DNA adduct data. The increase in DNA adducts in the livers of the BNF-induced Gunn rats positively correlated with a decrease in N-hydroxy-PhIP-N3-glucuronide levels in the urine indicating an increase in PhIP bioactivation compared with the Wistar rats. Similar results were obtained from Gunn rats exposed to benzene[a]pyrene (B[a]P) (36). Enhanced covalent binding of B[a]P to hepatic DNA and microsomal protein was correlated with a deficiency in B[a]P glucuronidation compared with control rats with normal UGT activity.
In contrast to the adduct levels in the liver, colon DNA adduct levels were significantly higher in the Wistar rats compared to the Gunn rats, in both the BNF-induced and uninduced treatment groups. The higher adduct levels in the Wistar rats could be due to the observation that the excretion of urinary PhIP metabolites was slower in the Wistar rats compared with the Gunn rats. A slower excretion rate would allow for more N-hydroxy-PhIP to be transported to the colon via entero-hepatic circulation as well as reabsorption through the gut. In addition, de-conjugation of the N-hydroxy-PhIP-glucuronides by bacterial ß-glucuronidase could also be contributing to the higher adduct levels by providing more reactive intermediate at the target site. In contrast, in the Gunn rats, entero-hepatic circulation could be bypassed due to the faster excretion rate, which would result in less reactive compound getting to the colon, thus reducing DNA adducts. Furthermore, the absence of UGT1A activity in the liver would limit the amount of N-hydroxy-PhIP-glucuronides being transported to the colon, thereby limiting the effect of de-conjugation by bacterial ß-glucuronidase. These results suggest that the absence of UGT1A activity in the liver may provide protection against DNA adduct formation in the colon because the amount of potential reactive substrate being transported to the colon will be diminished.
Induction with BNF had a significant effect on DNA adduct levels in both rat strains. The 3-fold decrease in DNA adducts observed in the liver of the BNF-induced Wistar rats compared with the uninduced animals suggests that inducible UGT1A activity in the liver contributes to the detoxification of N-hydroxy-PhIP. As expected, there was no difference in liver DNA adduct levels in the BNF-induced and uninduced Gunn rats. This was presumably due to the absence of UGT1A activity in the liver of the Gunn rat. Since UGT1A activity is present in the colon of both rat strains (28,29), BNF induction caused a 7-fold and 8-fold decrease in colon DNA adduct levels in the Wistar and Gunn rats, respectively, indicating that inducible UGT1A activity in the colon of both strains is capable of detoxifying N-hydroxy-PhIP.
Treatment with BNF also induces CYP450 enzymes. It is unlikely, however, that differential Phase I hydroxylation of PhIP contributed to the observed differences in DNA adduct levels because the PhIP hydroxylation rates were similar in the two rat strains. Nevertheless, because of the complexities of multiple enzyme induction by BNF and the presence of competing metabolic pathways, the contributions of other metabolic pathways affecting the disposition of PhIP cannot be discounted.
Based on these results, Figure 7 represents a proposed schematic as to the metabolic fate of PhIP and the effect of UGT1A activity on PhIP bioactivation in both the Wistar and Gunn rats. After oral ingestion, PhIP enters the gut where it is transported to the liver and converted via CYP1A2 to N-hydroxy-PhIP. In the BNF-induced Wistar rat the increase in hepatic UGT activity would be capable of forming N-hydroxy-PhIP glucuronides, which are readily excreted through the urine or transported to the colon and excreted. The remaining N-hydroxy-PhIP would be further bioactivated to form low levels of DNA adducts in both the liver and colon. In the uninduced Wistar rat, where the UGT1A levels are lower, N-hydroxy-PhIP would be the predominant metabolite, which would result in the formation of DNA adducts in the liver and colon at higher levels than what was seen in the induced animals (Figure 7A). In the BNF-induced Gunn rat the fate of PhIP would be different (Figure 7B). Since the Gunn rat is deficient in hepatic UGT1A activity, N-hydroxy-PhIP would predominate in the liver leading to increased DNA adduct levels due to the inability to form N-hydroxy-PhIP-glucuronides. In the colon, however, induced UGT1A activity would be sufficient to glucuronidate N-hydroxy-PhIP leading to detoxification over bioactivation (Figure 7B). In the uninduced Gunn rat, colon DNA adduct will be higher compared with the induced animals because the level of UGT activity will be lower, thereby reducing the capacity to form glucuronide conjugates. These proposed pathways demonstrate the importance of UGT1A activity in the biotransformation of PhIP.

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Fig. 7. Proposed schematic of the metabolic fate of PhIP and the effect of UGT1A activity on PhIP bioactivation in (A) Wistar, and (B) Gunn rats. Thick lines represent the predominant pathway. See text for further explanation.
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The data obtained from the microsomal incubations was contrary to what was observed in the in vivo situation. The formation of N-hydroxy-PhIP-N3-glucuronide in the microsomal incubations derived from the UGT1A-deficient Gunn rats at levels comparable to the UGT1A proficient Wistar rats was unexpected. This finding was significantly different from what was observed in the urinary metabolic profile where N-hydroxy-PhIP-N3-glucuronide was decreased by 92% in the UGT1A-deficient rats compared with the control animals. It is unclear why the in vivo data were so different from the microsomal data. These differences could be due to the involvement of other UGT isozymes and/or competition from other enzymatic pathways. This idea was reinforced from the western blot analysis of the hepatic microsomal fractions that showed no presence of UGT1A proteins in the Gunn rats. Further studies are needed to investigate these findings.
The results from these studies indicate that UGT1A-mediated glucuronidation of PhIP and N-hydroxy-PhIP is an important pathway in the detoxification of PhIP. The failure to form glucuronide conjugates can lead to increases in PhIP bioactivation and DNA adduct formation, as evidenced by the increase in hepatic DNA adducts in the Gunn rats. In the colon of the Gunn rats DNA adducts were actually lower than what was observed in the Wistar rats. These findings suggest that diminished UGT activity in the liver may provide a degree of protection against DNA adduct formation in the colon. This can be attributed to lower levels of potential reactive substrate being transported to the colon. The results from these studies demonstrate the importance of tissue-specific metabolism of PhIP. Since the colon is a tumor target tissue for PhIP, differential UGT1A expression not only at the target site but in the liver as well could affect the rate of PhIP bioactivation and DNA adduuct formation. Based on these results it appears that tissues with reduced UGT1A activity would have a higher rate of PhIP activation and be more inclined to form DNA adducts compared with tissues with normal UGT1A activity. However, low UGT1A activity in the liver can provide protection against PhIP-induced DNA adducts in peripheral tissue. Therefore, diminished UGT1A activity in tumor target tissues could pose a significant risk for the development of certain cancers from exposure to PhIP whereas diminished UGT1A activity in the liver may decrease the risk of tumor development in peripheral tissue. It is hoped that these findings will serve as a basis for future studies to better assess individual susceptibility to potential cancer risk from exposure to PhIP, as well as other heterocyclic amines.
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Acknowledgments
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The authors thank Mark Knize for his analysis of the PhIP metabolites by LC/MS. This work was performed under the auspices of the US Department of Energy by the University of California, Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48, and supported by NCI Grant CA55861 and NIH grant RR13461. Conflict of Interest Statement: None declared.
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Received April 1, 2005;
revised June 2, 2005;
accepted June 6, 2005.