Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved
Susan A. Nowell1,7,
Joyce S. Massengill1,
Suzanne Williams1,
Anna Radominska-Pandya2,
Thomas R. Tephly3,
Ziqiang Cheng3,
Christian P. Strassburg4,
Robert H. Tukey4,
Stewart L. MacLeod1,
Nicholas P. Lang1,5 and
Fred F. Kadlubar6
1 University of Arkansas for Medical Sciences, Surgical Oncology Department, 4301 West Markham Street, VA Research Slot 151 and
2 University of Arkansas for Medical Sciences, Department of Biochemistry, Little Rock, AR 72205,
3 Department of Pharmacology, The University of Iowa, Iowa City, IA 52242,
4 Department of Pharmacology, UCSD Cancer Center, University of California-San Diego, La Jolla, CA 92093,
5 John L.McClellan Memorial Veterans Administration Medical Center, Little Rock, AR 72205 and
6 National Center for Toxicological Research, Jefferson, AR 72079, USA
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Abstract
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.
Abbreviations: AIA, amino-imidazoazaarene; N-OH-PhIP, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; UDP-GlcUA, UDP-glucuronic acid; UGTs, UDP-glucuronosyltransferases.
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Introduction
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Colon cancer is the fourth most common cancer in the world, accounting for 9.7% of all new cancers diagnosed (1). Although the etiology of this disease is not fully understood, epidemiological studies demonstrate that the consumption of cooked meat is the factor most consistently associated with colon cancer. A number of heterocyclic aromatic amines are formed in meat during normal cooking processes and have been implicated in the initiation of colon cancer (2). Of these compounds, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most mass abundant (3,4). PhIP is thought to be formed by the condensation of creatine and phenylalanine in meat and fish cooked at high temperatures (5), but has also been detected in cigarette smoke (6). Animal studies have shown that PhIP can induce colon and mammary tumors in rats (7). PhIP has also been shown to cause DNA strand breaks in rat hepatocyte suspensions (8) and can cause sister chromatid exchange in cells from mouse bone marrow and peripheral blood (9). Given the abundance of PhIP in the diet and its potential role in carcinogenesis, elucidation of the metabolic pathways involved in the activation and detoxification of PhIP are crucial.
PhIP is a member of the amino-imidazoazaarene (AIA) family of compounds (10) and, in common with other AIAs, requires metabolic activation to exert genotoxic effects. Bioactivation of PhIP to a carcinogenic species in vivo is initiated by the N-oxidation of PhIP to 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). This reaction occurs primarily in the liver and, depending on the species, is catalyzed by cytochrome P4501A2 or P4501A1 (5,1113). N-OH-PhIP is capable of forming DNA adducts, but additional metabolism by acetyltransferases and sulfotransferases generates N-acetoxy and N-sulfonyloxy esters, electrophiles that are much more reactive with DNA (14,15).
Previous models of PhIP-induced carcinogenesis suggested that PhIPDNA adduct formation in extrahepatic tissue occurs via the sequential N-oxidation and N-glucuronidation of PhIP in the liver, followed by the biliary transport of the glucuronide conjugate to other sites, such as the colon, where ß-glucuronidases cleave the glucuronic acid moiety to regenerate N-OH-PhIP (16). Acetyltransferases and sulfotransferases in target tissues could then metabolize N-OH-PhIP to the corresponding reactive species capable of binding to DNA and ultimately leading to carcinogenesis. However, it was subsequently shown by Kaderlik et al. (17) that bile duct ligation had no effect on PhIPDNA adduct formation in rat colon, suggesting that N-OH-PhIP or N-acetoxy-PhIP are transported through the circulation to the target tissue. Moreover, recent studies in our laboratory indicate that, in humans, the major glucuronide formed is the N-OH-PhIP-N2-glucuronide, a metabolite that is resistant to both ß-glucuronidase cleavage and acid hydrolysis (18). Consequently, glucuronidation of N-OH-PhIP in the liver and in extrahepatic tissues can be considered a detoxification mechanism in humans. Although the significant role of glucuronidation in the detoxification of N-OH-PhIP has long been recognized, the specific isoforms responsible were unknown.
The UDP-glucuronosyltransferases (UGTs, EC 2.4.1.17) are members of a gene superfamily divided into two families based on evolutionary divergence (19,20). The UGT1A locus is on chromosome 2 and can potentially encode nine functional isoforms (21). The UGT1A gene complex is composed of multiple tandem first exons that encode the variable N-terminal part of the enzyme and are linked by differential splicing to common exons that encode the C-terminal region (2224). Members of the UGT2B family, on the other hand, are unique gene products and preferentially glucuronidate steroids and bile acids, in addition to xenobiotics (19).
Traditionally, UGT studies have focused on the hepatic isoforms, but recent studies indicate that extrahepatic tissues, particularly the intestinal tract, express significant levels of UGT protein (2527). Therefore, it is likely that the intestinal tract contributes more to the detoxification of orally administered drugs and food-borne carcinogens than was previously recognized. In this study, we show that UGTs from intestinal mucosa glucuronidate N-OH-PhIP, with the colon having catalytic activity comparable with that found in the liver. Additional studies revealed that at least four UGT1A isoforms exhibit catalytic activity toward N-OH-PhIP, specifically UGT1A3, UGT1A8, UGT1A9 and UGT1A10. In humans, UGT1A8 and UGT1A10 are expressed predominantly in the colon (28), whereas UGT1A3 and UGT1A9 are expressed in both liver and colon (21,29). The presence of these enzymes in both the liver, where PhIP is activated, and in the colon, where tumors can occur, underscores the importance of glucuronidation as a protective mechanism in respect to PhIP-induced carcinogenesis.
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Materials and methods
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Materials
Radiolabeled [ring-3H]PhIP (88 mCi/mmol) was purchased from Chem-Syn Science Laboratories (Lenexa, KS) and diluted with unlabeled PhIP (Toronto Research Chemicals, Toronto, Canada). The nitro derivative of PhIP was synthesized according to the method of Grivas (30), with the modifications made by Turesky et al. (13). UDP-glucuronic acid was purchased from the Sigma Chemical Co. (St Louis, MO). Recombinant UGT1A9, expressed in lymphoblastoid cells, and human liver microsomes were purchased from Gentest Corp. (Woburn, MA). Liver microsomes from 3-methylcholanthrene-induced female SpragueDawley rats and recombinant UGT2B10, expressed in lymphoblastoid cells, were a generous gift from Dr Julian Leakey (National Center for Toxicological Research, Jefferson, AR). Human kidney microsomes were purchased from the International Institute for the Advancement of Medicine (Exton, PA). Anti-peptide anti-UGT1 antibody was provided by Dr Chris Patton (Gentest Corp., Woburn, MA). Electrophoresis and western blot reagents were from Novex (San Diego, CA). Detection of immunoreactive bands was performed using the ECL chemiluminescence method from Amersham Life Science (Arlington Heights, IL).
Intestinal microsome preparation
Human intestine was harvested from organ donors under a protocol approved by the institutional review board. The intestine was received from the surgeons in saline on ice. Tissue from jejunum to rectum was included for all subjects, except for donor H-1, from whom only ileum and colon were received. Working at 4°C, small intestine was divided into four segments of 80100 cm in length. Each segment of intestine was opened, the contents were removed and the tissue was rinsed in cold 0.9% NaCl. Mucosa was removed from each segment of small intestine and from colon by scraping with a glass slide. Mucosal samples were weighed and transferred to Dounce homogenizers kept on ice. Microsomes were prepared as previously described (31), except that two, rather than three, volumes of buffer were used for homogenization and trypsin inhibitor (2 mg/g mucosa) was added to the buffer prior to homogenization.
Expression of human UGT isoforms
The heterologous expression of human UGTs 1A1 (32), 1A3 (33), 1A4 (34), 1A8 (35), 2B7 (36) and 2B15 (37) in HK293 cells have been described previously. UGT1A10 was cloned and expressed as described by Strassburg et al. (21).
Western blot analysis
Equal amounts of protein from each sample (30 µg) were separated by SDSPAGE using Novex 412% NuPAGE gradient gels and transferred to nitrocellulose by the method of Towbin et al. (38). Prior to immunostaining, the nitrocellulose was stained with Ponceau S solution, a reversible protein stain, to ensure equal protein loads between lanes. Blotted proteins were exposed to the appropriate antibody and visualized by alkaline phosphatase or by chemiluminescence using Amersham reagents (Amersham Life Science).
Enzyme assays
Enzymatic N-OH-PhIP glucuronidation activities in human liver and intestinal microsomes and recombinant human UGTs were assessed by TLC using a modification of the method of Bansal and Gessner (39). All solutions were argon-saturated prior to use and incubations were also carried out under argon. Membranes were activated by preincubation with alamethicin (60 µg/mg protein) as described by Little et al. (40). Incubations were performed at 37°C for 30 min and consisted of 100 mM TrisHCl, pH 7.5, 5 mM MgCl2, 8.5 mM saccharolactone, 50 µg microsomal or recombinant protein, 5 mM UDP-glucuronic acid and 100 µM [3H]N-OH-PhIP in a final volume of 50 µl. Reactions were terminated by the addition of 20 µl ethanol. Aliquots of 50 µl of the samples were spotted onto the preabsorbent layer of channeled silica gel TLC plates [Baker 250Si-PA (19C); VWR Scientific, Suwanee, GA] and developed in chloroform/methanol/glacial acetic acid/water (65:25:2:4 v/v). After development, the plates were dried and subjected to autoradiography for ~3 days at 80°C. Metabolite-containing silica gel (localized by autoradiography) and silica gel from corresponding areas in control lanes were scraped into scintillation vials. Radioactivity was determined by scintillation counting (Tri-Carb 2100TR Liquid Scintillation Analyzer; Packard Co., Downer's Grove, IL) in Opti-Fluor scintillant.
HPLC analysis of glucuronides
The structural nature of the glucuronide conjugate was identified by HPLC as previously described (18,41). Briefly, enzymatic assays were performed as described above. After termination of the incubation, proteins were precipitated by centrifugation at 18 000 g for 10 min and the supernatant was used for HPLC analysis. The HPLC system consisted of a Waters Alliance Model 2690 Separations Module with a photodiode array detector (Model 996) in conjunction with a Radiomatics Model 500TR Flow Scintillation Analyzer. The column was a Supelco C18 (25x0.46 cm, 5 µm particle size) and the mobile phase was (A) 20 µM diethylamine acetate, pH 5.0, and (B) methanol. A flow rate of 1 ml/min with a gradient of 4070% B over 40 min was used to separate the metabolites.
Immunoinhibition of enzymatic activity toward N-OH-PhIP
Immunoinhibition was assessed using the anti-UGT1A-anti-peptide antibody generously provided by Drs Chris Patton and Charles Crespi (Gentest Corp.). The assays were performed essentially according to Gentest instructions for CYP1A1 inhibition studies. Briefly, microsomes from human liver or human colon were incubated at room temperature for 30 min in the presence of increasing amounts of antibody. Normal rabbit serum was used in control incubations and to normalize the amount of serum/antiserum for each concentration. Enzymatic activity toward N-OH-PhIP was performed as described above. The glucuronides were separated by TLC and the radioactive products were visualized by autoradiography and scraped into scintillation vials for quantitation by scintillation counting.
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Results
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Western blot analysis of intestinal samples
Entire intestinal tracts were removed by the surgeons from the organ donors and prepared as described in Materials and methods. Table I
provides tissue donor information, including age, sex, cause of death and drug status. Western blot analysis of microsomal proteins isolated from the intestinal mucosa revealed a significant level of expression of UGT1A immunoreactive protein. Figure 1
shows a representative blot of intestinal mucosa microsomes from a single donor. Quantities of immunoreactive proteins found in the duodenum (Figure 1
, lane 2) and the colon (Figure 1
, lane 7) are comparable with levels found in the liver (Figure 1
, lane 1), while levels from the proximal jejunum to the terminal ileum (Figure 1
, lanes 36) are less pronounced. However, the electrophoretic mobilities of immunoreactive proteins from liver and intestinal microsomes differ slightly.

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Fig. 1. Western blot analysis of human liver and intestinal microsomes. Microsomes (30 µg) from human liver and different intestinal sites were separated by SDSPAGE, transferred to nitrocellulose and exposed to an antibody directed against a common exon of the UGT1A family as described in Materials and methods. Lane 1, human liver microsomes; lane 2, intestinal microsomes from duodenum; lane 3, intestinal microsomes from proximal jejunum; lane 4, intestinal microsomes from upper small bowel; lane 5, intestinal microsomes from lower small bowel; lane 6, intestinal microsomes from terminal ileum; lane 7, intestinal microsomes from colon.
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N-OH-PhIP glucuronidation by intestinal microsomes
Microsomes from different sections of intestine were evaluated for their ability to glucuronidate N-OH-PhIP. Figure 2
compares the glucuronidation activities throughout the intestinal tract of two different donors. Incubations were carried out as described in Materials and methods and the resulting glucuronides were separated by TLC. In both cases, glucuronidation of N-OH-PhIP occurred at the highest levels in the colon, despite the fact that there were comparable amounts of immunoreactive protein in the duodenum. TLC separation of the products revealed two distinct UDP-glucuronic acid (UDP-GlcUA)-dependent radioactive products. Identification of the glucuronide conjugate was subsequently performed by HPLC. As shown in Figure 3A
, incubations performed using microsomal proteins without the co-substrate, UDP-GlcUA, results in a radioactive trace with only two peaks with retention times of ~17 and 20 min, respectively. Spectral analysis performed in a previous study in our laboratory (18,41) determined that the two peaks correspond to N-OH-PhIP (retention time 17 min) and 2-OH-PhIP (retention time 20 min), a by-product of the synthesis of N-OH-PhIP. When incubations were carried out using microsomal protein from liver (Figure 3B
) or colon (Figure 3C
) in the presence of UDP-GlcUA, two UDP-GlcUA-dependent radioactive peaks with retention times of ~8 and 10 min were apparent. The UV absorbance profiles of the two peaks were identical to the profiles reported previously for N-OH-PhIP-N2-glucuronide (retention time 8 min) and N-OH-PhIP-N3-glucuronide (retention time 10 min).

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Fig. 2. Comparison of glucuronidation of N-OH-PhIP by intestinal microsomes from two individual donors. Enzymatic activity was assessed as described in Materials and methods. Incubations were separated by TLC, subjected to autoradiography and the localized radioactive bands were scraped into scintillation vials and counted. Each point is the mean (n = 4) of separate determinations ± the standard deviation.
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Fig. 3. HPLC separation of glucuronides. N-OH-PhIP conjugates formed by incubation of microsomes with [3H]N-OH-PhIP were separated by HPLC as described in Materials and methods. (A) The radioactive trace of an incubation carried out using human liver microsomes, omitting UDP-GlcUA. (B) The radioactive trace of an incubation of human liver microsomes including UDP-GlcUA. Spectral analysis revealed that the first radioactive peak (retention time ~8 min) corresponds to the N-OH-PhIP-N2-glucuronide while the second (retention time ~10 min) was identified as the N-OH-PhIP-N3-glucuronide. (C) A representative radioactive trace of an incubation using human colon microsomes in the presence of UDP-GlcUA. Peaks at ~8 and 10 min represent the N-OH-PhIP-N2-glucuronide and N-OH-PhIP-N3-glucuronide, respectively.
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Since UGTs have been reported in the kidney, microsomes from this organ were assayed for N-OH-PhIP glucuronidation activity. As shown in Table II
, however, kidney microsomes did not catalyze N-OH-PhIP glucuronidation under the assay conditions used for the other tissues. However, when the pH of the incubation mixture was raised to 8.0 and 8.5, glucuronidation by kidney microsomes could be detected (data not shown). In both human liver and colon, the predominant glucuronide of N-OH-PhIP is the N-OH-PhIP-N2-glucuronide. This is in contrast to rat liver, where the major product of this reaction is the N-OH-PhIP-N3-glucuronide (Table II
).
Subsequent studies were performed to assess inter-individual variability in N-OH-PhIP glucuronidation in the colon. Figure 4
shows the glucuronidation activity of colon microsomal protein from the intestinal samples detailed in Table I
. The values obtained showed considerable variation, although in each case, the N-OH-PhIP-N2-glucuronide was the predominant product. This pattern was also observed when human liver microsomes were analyzed for N-OH-PhIP glucuronidation activity. However, when rat liver microsomes were used for the assay, the major product formed was the N-OH-PhIP-N3-glucuronide (Figure 4
, last column).

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Fig. 4. Glucuronidation of N-OH-PhIP by human colon microsomes from different individuals. N-OH-PhIP glucuronide formation was examined using microsomal protein from the colon of individual donors detailed in Table I and compared with activities from human and rat liver microsomes. Enzymatic activity was assessed as described in Materials and methods. Incubations were separated by TLC, subjected to autoradiography and the localized radioactive bands were scraped into scintillation vials and counted. Each point is the mean (n = 4) of separate determinations ± the standard deviation.
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Identification of UGT isoforms involved in N-OH-PhIP glucuronidation
Several recombinant UGTs from both subfamilies were assayed to determine the specific isoforms responsible for the glucuronidation of N-OH-PhIP. Table III
details the recombinant isoforms that were screened for catalytic activity. Of the UGT1A family isoforms analyzed, UGT1A3, UGT1A8, UGT1A9 and UGT1A10 all showed activity toward the substrate, while UGT1A1, UGT1A4 and UGT1A6 did not. None of the UGT2B isoforms available for this study, namely UGT2B7, UGT2B10 and UGT2B15, were active toward N-OH-PhIP. However, there was a striking difference in the predominant glucuronide formation catalyzed by the specific isoforms. Figure 5
shows that UGT1A3 and UGT1A9, both of which are expressed in the liver, catalyze the formation of the N-OH-PhIP-N3-glucuronide, while intestine-specific UGT1A8 and UGT1A10 preferentially produce the N-OH-PhIP-N2-glucuronide.

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Fig. 5. Glucuronidation of N-OH-PhIP by human recombinant UGTs. Recombinant human UGTs were analyzed for their ability to glucuronidate N-OH-PhIP as described in Materials and methods. UGT1A3 and UGT1A9 were shown to preferentially produce the N3-glucuronide, whereas UGT1A8 and UGT1A10 mainly produce the N2-glucuronide. Each point is the mean (n = 4) of separate determinations ± the standard deviation.
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Immunoinhibition studies
The anti-UGT1A antibody used in this study was raised against a peptide common to all UGT1A family members and does not cross-react with UGT2B isoforms (data not shown). Immunoinhibition studies were performed to determine the relative contribution of UGT1A family isoforms to the glucuronidation of N-OH-PhIP. As shown in Figure 6A
, the presence of the antibody did not affect the formation of N-OH-PhIP-N2-glucuronide in human liver, but in colon, product formation was significantly reduced in a concentration-dependent manner. However, the formation of the N-OH-PhIP-N3-glucuronide showed similar concentration-dependent inhibition in both the liver and the colon (Figure 6B
). Since this antibody is specific for the UGT1A family, this suggests the involvement of an unidentified isoform, possibly from the UGT2B family, in the N2-glucuronidation of N-OH-PhIP in the liver.


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Fig. 6. Immunoinhibition of N-OH-PhIP glucuronidation by human liver and human colon microsomes. Microsomes from human liver and colon were incubated with increasing amounts of anti-UGT1A anti-peptide antibody or preimmune serum. Glucuronidation activity toward N-OH-PhIP was subsequently analyzed. Each point represents the average of three determinations. (A) Inhibition of the formation of the N-OH-PhIP-N2-glucuronide. (B) Inhibition of the formation of the N-OH-PhIP-N3-glucuronide.
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Discussion
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Recent studies on the metabolic disposition of PhIP in humans, using [2-14C]PhIP, revealed that glucuronide conjugates account for >70% of the recovered radiolabeled urinary metabolites (42). Of the glucuronide conjugates identified, N-OH-PhIP-N2-glucuronide was the most abundant. While traditional studies of glucuronidation have focused on the metabolism occurring in the liver, more recent work, at both the protein and mRNA level, indicates that the intestinal tract may in fact play a significant role in glucuronidation, particularly of ingested drugs and dietary carcinogens (21,28,35). Access to gastrointestinal tissue allowed us to determine that in the case of N-OH-PhIP, the intestinal tract does contribute to the detoxification of this compound. Western blot analysis of expression of the UGT1A locus throughout the intestinal tract showed that expression of UGT1A immunoreactive protein is highest in the duodenum and colon, with these two sites having levels of protein comparable with that found in the liver. There were, however, slight differences in the electrophoretic mobility of the immunoreactive protein found in the liver and throughout the intestinal tract. The observed differences are likely due to different amounts of glycosylation of the enzyme.
Glucuronidation activity toward N-OH-PhIP is present in every section of the intestine, but is significantly higher in the colon than in the other sites. UGT isoforms are differentially expressed (21,43) and expression levels of specific isoforms could account for the discrepancy between high levels of UGT immunoreactive protein in the duodenum and low catalytic activity toward N-OH-PhIP at this site.
Due to the relatively high level of activity found in the colon as compared with the rest of the intestinal tract, assays were then performed using colon microsomes from six individual donors. There was considerable interindividual variability between donors in the ability to glucuronidate N-OH-PhIP, although the predominant conjugate formed in each sample was the N-OH-PhIP-N2-glucuronide. This pattern of conjugate formation is also found in human liver. In contrast, rat liver microsomes preferentially produce the N-OH-PhIP-N3-glucuronide, which is also the major urinary glucuronide produced by rats (41). The N-OH-PhIP-N2-glucuronide is resistant to ß-glucuronidase treatment and, therefore, will not undergo hydrolysis in the colon. The N-OH-PhIP-N3-glucuronide is susceptible to ß-glucuronidase and is formed in the liver, although to a lesser extent than the N2-glucuronide. Expression of UGT isoforms in the colon would allow for the reconjugation of hydrolyzed N-OH-PhIP. UGT activity is known to be inducible (44); therefore, it is possible that some of the observed variability could be attributed to induction of the enzymes by drugs. Another factor that could contribute to differences in activity is tissue ischemia. Since tissue from each donor was removed by the transplant surgeon, placed on ice and immediately processed for microsome and cytosol isolation, this is probably not a significant contributor to differences observed in catalytic activity. The finding of variability in the in vivo data on urinary metabolites of PhIP among individuals (42) suggests that their different activity levels are due to differential expression of the enzymes or, alternatively, genetic polymorphisms in the UGT isoforms.
Recombinant UGTs were subsequently screened in an attempt to identify the specific UGT isoforms involved in N-OH-PhIP glucuronidation. Of the isoforms examined, listed in Table III
, only UGT1A3, UGT1A8, UGT1A9 and UGT1A10 exhibited catalytic activity toward this substrate. UGT1A8 and UGT1A10 are expressed in extrahepatic tissue and are not expressed in the liver (21,28,35), while UGT1A3 and UGT1A9 are found in both the liver and the colon. However, the pattern of glucuronide formation is different between the isoforms. UGT1A8 and UGT1A10 both predominantly produce the N-OH-PhIP-N2-glucuronide, but, surprisingly, UGT1A3 and UGT1A9 preferentially conjugate N-OH-PhIP at the N3 position. Comparison with glucuronidation by human liver microsomes shows that human liver produces the N-OH-PhIP-N2-glucuronide in amounts approximately six times higher than they produce the N3-glucuronide. Therefore, the results of the assays with recombinant UGTs indicate that UGT1A3 and UGT1A9 are not exclusively responsible for the overall pattern of N-OH-PhIP glucuronide formation observed with human liver microsomes. Subsequent immunoinhibition studies revealed that the anti-peptide antibody did not inhibit formation of the N-OH-PhIP-N2-glucuronide by human liver microsomes, but did inhibit formation in human colon microsomes in a concentration-dependent manner. However, formation of the N3-glucuronide was inhibited by the antibody in microsomes from both tissues. Since this antibody was directed against a common exon of the UGT1 family, this strongly suggests the contribution of an unrecognized isoform to the glucuronidation of N-OH-PhIP in the liver, although none of the UGT2B isoforms available for this study had catalytic activity towards N-OH-PhIP. Studies will continue, as human recombinant UGT2B isoforms become available, to elucidate the contribution of all UGT isoforms to the detoxification of this food-borne carcinogen.
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Acknowledgments
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This work was supported by NIH grants CA55751 and CA58697, EPA grant R825280 and NIA grant AG15722.
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Notes
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7 To whom correspondence should be addressed Email: nowellsusana{at}exchange.uams.edu 
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Received November 20, 1998;
revised January 25, 1999;
accepted February 5, 1999.