Metabolic Activation of 2-Hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Syrian Hamsters Congenic at the N-Acetyltransferase 2 (NAT2) Locus

Adrian J. Fretland, Uday S. Devanaboyina, Mark A. Doll, Shuang Zhao, Gong H. Xiao and David W. Hein1

Department of Pharmacology and Toxicology and James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky 40292

Received February 24, 2003; accepted April 24, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine carcinogen prevalent in the human diet. To exert its mutagenic and carcinogenic effects, PhIP undergoes bioactivation to N-hydroxy-PhIP followed by O-esterification via cytosolic acetyltransferases or sulfotransferases to form DNA adducts. We investigated the role of cytosolic acetyltransferases and sulfotransferases and the role of the N-acetyltransferase 2 genetic polymorphism on PhIP DNA-adduct levels in a congenic Syrian hamster model. DNA adduct levels were detected in all hepatic and extrahepatic tissues tested following administration of PhIP (4 x 100 mg/kg) or N-hydroxy-PhIP (1 x 50 mg/kg), with the highest levels in pancreas. DNA-adduct levels were higher in the gastrointestinal tract of rapid and slow acetylator hamsters administered N-hydroxy-PhIP. N-hydroxy-PhIP O-acetyltransferase and O-sulfotransferase activities were detected in most hepatic and extrahepatic cytosols derived from rapid and slow acetylator congenic hamsters. N-hydroxy-PhIP O-acetyltransferase activity was significantly higher (p < 0.05) in liver, small intestine, and esophagus in rapid than in slow acetylator congenic hamsters. N-hydroxy-PhIP O-acetyltransferase activities correlated significantly with N-acetyltransferase 2 activities across tissues in rapid (r = 0.83; p = 0.0004) but not in slow (r = 0.46; p = 0.1142) acetylator congenic hamsters, suggesting catalysis primarily by NAT2 in rapid acetylators but NAT1 in slow acetylators. N-hydroxy-PhIP O-sulfotransferase activities did not vary with acetylator genotype. DNA-adduct levels following administration of PhIP or N-hydroxy-PhIP did not correlate with either N-hydroxy-PhIP O-acetyltransferase or O-sulfotransferase catalytic activities.

Key Words: N-acetyltransferase 2 (NAT2); 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP); acetylator genotype; DNA adducts; Syrian hamster.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine carcinogen prevalent in the human environment, primarily through the consumption of well-done meat (Sinha, 2002Go). PhIP induces prostate, mammary gland, and colon tumors in rats; cancer sites associated with a Western diet in humans (Weisburger, 2002Go). Epidemiological studies suggest that heterocyclic amines present in well-done meats are putative carcinogens for cancers in humans (Sinha, 2002Go).

To exert its mutagenic and carcinogenic effects, PhIP undergoes host-mediated biotransformation to electrophilic metabolites that form DNA adducts (Schut and Snyderwine, 1999Go). The major metabolic activation pathway includes N-hydroxylation by hepatic CYP1A2 followed by O-esterification by N-acetyltransferases and sulfotransferases (Hein, 2002Go; Kaderlik et al., 1994aGo; Lin et al., 1995Go). Genetic polymorphisms in enzymes that bioactivate PhIP and/or N-hydroxy-PhIP may modify DNA-adduct levels and cancer risk. Since PhIP does not undergo N-acetylation (deactivation), phenotypic differences in O-acetyltransferase-catalyzed metabolic activation of N-hydroxy-PhIP may lead to differences in DNA-adduct formation. Two isozymes of N-acetyltransferase, NAT1 and NAT2, have been identified in humans and Syrian hamsters and both are subject to genetic polymorphism (Hein et al., 2000Go). Previous studies have shown polymorphic expression of NAT2 in hepatic and extrahepatic tissues (Hein et al., 1991Go). In the slow acetylator congenic strains (NAT2*16A/NAT2*16A), three single nucleotide polymorphisms are present in the 870 base pair NAT2 open reading frame compared to the rapid (NAT2*15/NAT2*15) acetylator strains (Ferguson et al., 1994aGo). Two (T36C and A633G) are silent, but the third (C727T) encodes a stop codon resulting in a truncated NAT2 protein with reduced N- and O-acetylation activity (Ferguson et al., 1996Go). Rapid and slow acetylator hamster NAT1 differ only in a silent single-nucleotide polymorphism (T60C) in the 870 base pair open reading frame that has no functional effect on N- or O-acetyltransferase activity (Ferguson et al., 1994bGo). Thus, genetic polymorphism in NAT2 is solely responsible for phenotypic differences in N-acetyltransferase activity in the Syrian hamster.

A rapid and slow acetylator hamster model congenic at the NAT2 locus has been described previously (Ferguson et al., 1996Go; Hein, 1991Go). Although humans and hamsters differ in N-acetyltransferase substrate specificity, this model is very useful for studying the specific effect of acetylator genotype since it eliminates phenotypic differences in other metabolic pathways and DNA repair. Previous studies did not find differences in DNA adduct levels between rapid and slow acetylator congenic hamsters administered PhIP (Fretland et al., 2001aGo,bGo). In this study, we investigated the role of NAT2 acetylator genotype on N-hydroxy-PhIP O-acetyltransferase activity and DNA-adduct formation in hepatic and extrahepatic tissues of the congenic Syrian hamster model.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Homozygous rapid and slow acetylator Bio 82.73/H congenic Syrian hamsters were bred and maintained at the University of Louisville School of Medicine. All experiments were performed under protocols approved by the University of Louisville Institutional Animal Care and Use Committee.

Preparation of tissue cytosols.
Twelve-week-old rapid and slow acetylator male congenic hamsters were sacrificed by CO2 asphyxiation and various tissues were homogenized in 50 µM sodium phosphate (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 100 µM phenylmethanesulfonyl fluoride, and 10 µM leupeptin. Homogenates were centrifuged at 100,000 x g for one h and supernatants collected for enzymatic and protein determinations. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA).

p-Aminobenzoic acid (PABA) N-acetyltransferase assays.
Tissue cytosols from rapid and slow acetylator congenic hamsters were assayed for the level of NAT2 activity, using the NAT2-selective substrate PABA as described previously (Leff et al., 1999aGo). Briefly, reactions containing tissue cytosol (<2 mg/ml), 100 µM PABA, and 1 mM acetyl coenzyme A were incubated at 37°C for 10 min in a total reaction volume of 300 µl. Reactions were terminated by the addition of 30 µl of 1 M perchloric acid, pH was adjusted by the addition of 22 µl of 1 M sodium hydroxide, and proteins pelleted by centrifugation at 13,000 x g for five min. Reactants and products were separated and identified by comigration with authentic standards, using high-performance liquid chromatography (HPLC). Absorbance was measured at 280 nm and the amount of product was determined by comparison to a standard curve.

N-Hydroxy-PhIP O-acetyltransferase assays.
Tissue cytosols from rapid and slow acetylator congenic hamsters were assayed for N-hydroxy-PhIP O-acetyltransferase activity, as previously described, with minor modifications (Leff et al., 1999bGo). Briefly, cytosols (<2.5 mg protein/ml) were incubated at 37°C in a reaction containing N-hydroxy-PhIP (200 µM; NCI Chemical Carcinogen Reference Standard Repository) and 1 mM acetyl coenzyme A for 30 min. Control reactions substituted water for acetyl coenzyme A. Reactions were terminated by the addition of 1 M acetic acid. Proteins were precipitated by centrifugation at 13,000 x g for 10 min. Reactants and products were separated and identified by comigration with authentic standards, using HPLC as previously described (Leff et al., 1999bGo). Absorbance was measured at 280 nm and the amount of product was determined by comparison to a standard curve.

N-Hydroxy-PhIP O-sulfotransferase assays.
Tissue cytosols from rapid and slow acetylator congenic hamsters were assayed for N-hydroxy-PhIP O-sulfotransferase activity as previously described (Purewal et al., 2000bGo) with minor modifications. Briefly, cytosols (<2.5 mg protein/ml) were incubated at 37°C in a reaction containing N-hydroxy-PhIP (400 µM; NCI Chemical Carcinogen Reference Standard Repository) 20 µM 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and 5 mM 4-nitrophenyl sulfate (Sigma, St. Louis, MO) for 20 min. Control reactions substituted water for PAPS. Reactions were terminated by the addition of 1 M acetic acid. Proteins were precipitated by centrifugation at 13,000 x g for 10 min. Reactants and products were separated by HPLC and identified by comigration with authentic standards, using an EM Science (Gibbstown, NJ) LichroCART C18 (125 x 4 mm) column eluted with 35% methanol:65% diethylamine acetate (pH 4.0). PhIP and N-hydroxy-PhIP eluted at approximately 6 and 8 min, respectively. Absorbance was measured at 317 nm and the amount of product was determined by comparison to a standard curve.

DNA adduct assays.
PhIP was dissolved in a solution of 0.1 M HCl/dimethylsulfoxide (3:2, v/v) at a concentration of 10 mg/ml. Twelve-week-old male rapid and slow acetylator congenic hamsters were administered four daily doses of 100 mg/kg body weight PhIP by oral gavage. Hamsters were sacrificed 6 h after the last dose, and various tissues were collected and snap frozen in liquid nitrogen. One rapid and one slow acetylator congenic hamster received vehicle alone as a control. N-Hydroxy-PhIP was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mg/ml. Since N-hydroxy-PhIP is the proximate carcinogenic metabolite and does not require hepatic N-hydroxylation, 8-week-old male rapid and slow acetylator congenic hamsters received a single dose of N-hydroxy-PhIP of 50-mg/kg body weight, ip, in the lower right quadrant. One rapid and one slow acetylator congenic hamster received DMSO alone as a control. Hamsters were sacrificed 6 h after dosing, and various tissues were collected and snap frozen in liquid nitrogen.

Analysis of PhIP-DNA adducts.
PhIP-DNA-adduct levels were measured by 32P-postlabeling as previously described (Leff et al., 1999aGo). Briefly, genomic DNA was isolated by digestion with proteinase K followed by phenol:chloroform extraction. Ten µg of DNA was digested to 3'-nucleotide monophosphates, using spleen phosphodiesterase (Sigma, St. Louis, MO) and micrococcal nuclease (Sigma). The adducted nucleotides were enriched by n-butanol extraction. The PhIP-DNA adducts were labeled in the presence of [{gamma}-32P]-ATP (ICN, Costa Mesa, CA) and T4 polynucleotide kinase (United States Biochemical, Cleveland, OH). PhIP-DNA adducts were resolved by thin layer chromatography on PEI-cellulose F plates (Alltech, Deerfield, IL). Adducts were quantified using the Instant Imager electronic autoradiography system (Packard Instruments, Chicago, IL) and compared with the synthetic PhIP-DNA-adduct standard, N-(deoxyguanosin-8-yl)-PhIP, the major adduct formed following metabolic activation of PhIP (Lin et al., 1992Go).

Statistical analyses.
Differences in enzymatic rates or DNA-adduct levels between rapid and slow acetylators were tested for significance by Student’s t-test (two-tailed). Differences were considered significant at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PABA N-Acetyltransferase Activity
PABA N-acetyltransferase activity (selective for NAT2) was significantly (p < 0.001) higher in rapid than slow acetylator congenic hamsters in all tissues tested (data not shown).

N-Hydroxy-PhIP O-Acetyltransferase Activity
N-hydroxy-PhIP O-acetyltransferase activity was detected in cytosols of all hepatic and extrahepatic tissues tested (Fig. 1Go). The activity was highest in liver and then generally decreased along the gastrointestinal tract. Activity levels were lowest in pancreas, prostate, and heart. N-hydroxy-PhIP O-acetyltransferase activities were generally equivalent in rapid and slow acetylators except for liver, small intestine, and esophagus where levels were significantly (p < 0.05) higher in rapid versus slow acetylator congenic hamsters. N-hydroxy-PhIP O-acetyltransferase and PABA NAT2 activities were significantly correlated in rapid (r = 0.83; p = 0.0004) but not in slow (r = 0.46; p = 0.1142) acetylator congenic hamsters (Fig. 2Go).



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FIG. 1. N-hydroxy-PhIP O-acetyltransferase activities in rapid and slow acetylator congenic hamster tissue cytosols. Data represent mean ± SE for three to eight individual hamsters. Solid bars represent homozygous rapid acetylators; open bars represent homozygous slow acetylators. *N-hydroxy-PhIP O-acetyltransferase activity was significantly higher (p < 0.05) in rapid versus slow acetylator congenic hamsters. Abbreviations: LV, liver; SI, small intestine; CE, cecum; AC, ascending colon; TC, transverse colon; DC, descending colon; RT, rectum; PS, pancreas; PT, prostate; HT, heart; ST, stomach; ES, esophagus.

 


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FIG. 2. Correlation of PABA NAT and N-hydroxy-PhIP OAT activities in various tissues of rapid (top panel) and slow (bottom panel) acetylator congenic hamsters. The Pearson correlation coefficient was 0.83 (p = 0.0004) in rapid acetylators and 0.46 (p = 0.1142) in slow acetylators.

 
N-Hydroxy-PhIP O-Sulfotransferase Activity
N-hydroxy-PhIP O-sulfotransferase activities were measurable in cytosols from hepatic and extrahepatic tissues in the general order liver > small intestine > rectum > cecum, colon, prostate, esophagus > pancreas (Fig. 3Go). Levels were undetectable in stomach and heart. No differences in activity were observed between rapid and slow acetylator congenic hamsters in any tissue.



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FIG. 3. N-hydroxy-PhIP O-sulfotransferase activities in rapid and slow acetylator congenic hamster tissue cytosols. Data represent mean ± SE for three to eight individual hamsters. Solid bars represent homozygous rapid acetylators; open bars represent homozygous slow acetylators. Abbreviations: LV, liver; SI, small intestine; CE, cecum; CN, total colon; RT, rectum; PS, pancreas; PT, prostate; HT, heart; ST, stomach; ES, esophagus. Levels were undetectable in heart and stomach.

 
DNA Adducts following Administration of PhIP or N-hydroxy-PhIP
A single DNA adduct spot was observed in all hamsters treated with PhIP or N-hydroxy-PhIP after resolution by thin layer chromatography that comigrated with synthetic N-(deoxyguanosin-8-yl)-PhIP. No adduct spots were observed in hamsters treated with vehicle. Following administration of 50 mg/kg N-hydroxy-PhIP, DNA adduct was detected in all hepatic and extrahepatic tissues examined (Fig. 4Go). Levels were highest in the pancreas followed by the esophagus, stomach, and gastrointestinal tract, and lowest levels were in liver, prostate, and heart. Levels were slightly and consistently higher in rapid than in slow acetylators in the gastrointestinal tract, but these differences did not reach significance (p > 0.05). DNA adduct levels did not correlate with either N-hydroxy-PhIP O-acetyltransferase or O-sulfotransferase activities. DNA adducts also were detected in all hepatic and extrahepatic tissues examined following administration of PhIP (Fig. 5Go). The DNA adduct levels were similar across tissues except for high levels in pancreas. DNA adduct levels did not differ significantly between rapid and slow acetylators and did not correlate with N-hydroxy-PhIP O-acetyltransferase or O-sulfotransferase activities. DNA adduct levels following administration of PhIP (4 x 100 mg/kg) or its proximate metabolite N-hydroxy-PhIP (1 x 50 mg/kg) are compared in Figure 6Go. DNA adduct levels following administration of N-hydroxy-PhIP were higher than PhIP in the lower gastrointestinal tract (cecum through rectum) but lower in liver, prostate, and heart. DNA adduct levels were highest in pancreas following administration of both PhIP and N-hydroxy-PhIP.



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FIG. 4. DNA-adduct distribution in rapid and slow acetylator congenic hamsters administered N-hydroxy-PhIP (50 mg/kg). Data represent mean ± SE in pmol PhIP adduct/mg DNA of eight (except ES, n = 5) individual hamsters. Solid bars represent homozygous rapid acetylators; open bars represent homozygous slow acetylators. Abbreviations: LV, liver; SI, small intestine; CE, cecum; AC, ascending colon; TC, transverse colon; DC, descending colon; RT, rectum; PS, pancreas; ST, stomach; PT, prostate; ES, esophagus; HT, heart.

 


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FIG. 5. DNA-adduct distribution in rapid and slow acetylator congenic hamsters administered PhIP (4 x 100 mg/kg). Data represent mean ± SE in pmol PhIP adduct/mg DNA of eight individual hamsters. Solid bars represent homozygous rapid acetylators; open bars represent homozygous slow acetylators. Abbreviations: LV, liver; SI, small intestine; CE, cecum; AC, ascending colon; TC, transverse colon; DC, descending colon; RT, rectum; PS, pancreas; PT, prostate; HT, heart.

 


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FIG. 6. DNA-adduct distribution in rapid acetylator congenic hamsters administered 50 mg/kg N-OH-PhIP (solid bars; n = 8) or 4 x 100 mg/kg PhIP (open bars; n = 8). Data represent mean ± SE in pmol PhIP adduct/mg DNA. Abbreviations: LV, liver; SI, small intestine; CE, cecum; AC, ascending colon; TC, transverse colon; DC, descending colon; RT, rectum; PS, pancreas; PT, prostate; HT, heart.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
N-hydroxy-PhIP O-acetyltransferase activity was detected in cytosols of all hepatic and extrahepatic tissues tested. The activity was highest in liver and lowest in pancreas, prostate, and heart. This is consistent with recent studies in the rat, wherein N-hydroxy-PhIP O-acetyltransferase activity was detected in all tissue cytosols tested, with highest levels in liver and lowest in heart (Dubuisson et al., 2001Go). N-hydroxy-PhIP O-acetyltransferase activities were significantly higher (p < 0.05) in liver, small intestine, and esophagus of rapid as compared with slow acetylator hamsters. Recent studies have shown that about two-fold higher levels of N-hydroxy-PhIP O-acetyltransferase were observed in rapid than in slow acetylator prostate cytosol when using a higher substrate concentration of N-hydroxy-PhIP (Hein et al., 2003Go). N-hydroxy-PhIP O-acetyltransferase and PABA NAT2 activities were significantly correlated in rapid (r = 0.83; p = 0.0004) but not in slow (r = 0.46; p = 0.1142) acetylator congenic hamsters. N-hydroxy-PhIP is bioactivated by both hamster NAT1 and NAT2 (Hein et al., 2003Go). The current study suggests that O-acetylation occurs primarily by NAT2 in rapid acetylator hamsters but almost exclusively by NAT1 in slow acetylator hamsters. The extrapolation of these results should be interpreted with caution in light of differences in N-acetyltransferase substrate specificity between hamsters and humans.

We observed higher levels of N-hydroxy-PhIP O-acetyltransferase activity in the proximal gastrointestinal tract (i.e., small intestine and cecum) with decreased levels in the colon. This trend was dependent somewhat on tissue and acetylator genotype but is consistent with previous studies showing a general decrease in NAT2 activity along the gastrointestinal tract in rodents (Ware and Svensson, 1996Go) and humans (Hickman et al., 1998Go).

Since sulfotransferases also activate N-hydroxy-PhIP to DNA adducts (Kaderlik et al., 1994aGo; Lin et al., 1995Go), tissue-dependent differences in DNA adduct formation may reflect tissue-dependent differences in N-hydroxy-PhIP O-sulfotransferase activities. Although there was some variation with tissue and acetylator genotype, higher levels of N-hydroxy-PhIP O-acetyltransferase than O-sulfotransferase activity were observed across hamster tissue cytosols. This is consistent with previous studies in the rat (Dubuisson et al., 2001Go; Lin et al., 1995Go) although the latter study reported levels of N-hydroxy-PhIP O-acetyltransferase that were considerably higher than O-sulfotransferase activity across rat tissues.

Intraperitoneal injection of the proximate carcinogen N-hydroxy-PhIP to the congenic hamsters enabled us to bypass the hepatic N-hydroxylation step and to directly assess the effects of genetic polymorphism in O-acetylation in each target tissue. Adduct levels were detected in all tissues examined, suggesting that N-hydroxy-PhIP is sufficiently stable for widespread distribution to extrahepatic tissues of Syrian hamsters, as previously described in rats (Kaderlik et al., 1994aGo). The lowest level of DNA adducts in congenic hamsters administered N-hydroxy-PhIP was observed in the liver. This was consistent with low hepatic DNA adduct levels following administration of PhIP and may reflect higher levels of glutathione or glucuronide conjugation of N-hydroxy-PhIP or N-acetoxy-PhIP in the liver (Kaderlik et al., 1994aGo,bGo; Lin et al., 1993Go; Malfatti and Felton, 2001).

The highest level of DNA adducts following administration of N-hydroxy-PhIP was observed in the pancreas, a finding consistently observed following administration of PhIP to Syrian hamsters (Fretland et al., 2001aGo,bGo) and rats (Kaderlik et al., 1994aGo; Pfau et al., 1997Go). Both PhIP and N-hydroxy-PhIP are readily taken up into pancreatic acini (Butcher et al., 1996Go). Despite the high DNA adduct levels in pancreas, PhIP does not appear to induce pancreatic tumors in rats (Yoshimoto et al., 1999Go) or Syrian hamsters (Fretland et al., 2001aGo,bGo). However, epidemiological studies suggest a role for well-done meat consumption in human pancreatic cancer (Anderson et al., 2002Go). Human (Anderson et al., 1997Go) and Syrian hamster (this study) pancreas have been shown to catalyze O-acetyltransferase activity and human pancreatic activity appears to be catalyzed primarily by NAT1 (Anderson et al., 1997Go). In our study in the Syrian hamster, both N-hydroxy-PhIP O-acetyltransferase and O-sulfotransferase activities in pancreas were detectable but modest compared to hepatic levels. Thus, tissue distributions of neither activity correlated with tissue distributions of DNA adduct levels following administration of PhIP or N-hydroxy-PhIP. These results suggest the importance of other activation and/or deactivation pathways in the generation of DNA adducts in the pancreas that were not investigated in this study.

DNA adduct levels in the gastrointestinal tract following administration of N-hydroxy-PhIP were generally highest in the proximal colon and were lower in the more distal portions of the colon. The descending colon did not follow this trend, however, as the highest level of DNA adducts were observed. This contrasts to what was observed following administration of PhIP in the current (Fig. 4Go) and previous (Steffensen et al., 2000Go) studies. Route of administration also has been shown to affect DNA adduct levels following administration of aromatic amines in mice (Schurdak and Randerath, 1989Go) and rats (DeBord et al., 1996Go).

Previous studies have documented detection of PhIP DNA adducts in human colon following dietary relevant doses of PhIP (Dingley et al., 1999Go). DNA adduct levels were consistently higher in rapid versus slow acetylator congenic hamsters in the gastrointestinal tract. This is consistent with previous results showing NAT2-dependent O-acetylation in hamster intestine and colon (Hein et al., 1993Go; Ogolla et al., 1990Go) and with higher levels of DNA adducts in rapid than in slow acetylator rats administered PhIP (Purewal et al., 2000aGo). The results support human epidemiological studies reporting that consumption of well-done meat increases colorectal cancer risk in the presence of high CYP1A2 (which catalyzes the N-hydroxylation of PhIP) and rapid NAT2 (Lang et al., 1994Go; Le Marchand et al., 2001Go) acetylator genotypes.

DNA adduct levels in the gastrointestinal tract (cecum through rectum) were higher following administration of 50 mg/kg N-hydroxy-PhIP than following 4 x 100 mg/kg PhIP. This is consistent with previous studies in the rat (Kaderlik et al., 1994aGo) and with the relative lack of PhIP N-hydroxylation activity in rat colon (Kaderlik et al., 1994aGo; Purewal et al., 2000bGo). When considered together with the high N-hydroxy-PhIP O-acetyltransferase activity in the gastrointestinal tract, these findings support the general metabolic scheme of N-hydroxylation in the liver followed by O-acetylation in the gastrointestinal tract as relevant for formation of colon DNA adducts from PhIP. As noted above, the relatively low DNA adduct levels in the liver, despite high levels of O-acetyltransferase activity, may reflect higher levels of glutathione or glucuronide conjugation of N-hydroxy-PhIP or N-acetoxy-PhIP in the liver (Kaderlik et al., 1994aGo,bGo; Lin et al., 1993Go; Malfatti and Felton, 2001).

Previous human epidemiological studies have shown associations between intake of barbecued/grilled meat and stomach cancer (Ward et al., 1997Go). However, we did not observe differences in N-hydroxy-PhIP O-acetyltransferase activity in stomach cytosols of rapid and slow acetylator hamsters. This is consistent with a recent study reporting no apparent association between NAT2 acetylator genotype and human stomach cancer (Lan et al., 2003Go). Previous human epidemiological studies have shown associations between intake of barbecued/grilled meat and esophageal cancer (Castelletto et al., 1994Go; Ward et al., 1997Go). Our study is the first to report NAT2 acetylator genotype-dependent metabolic activation of N-hydroxy-PhIP in the esophagus and suggests that NAT2 acetylator genotype may modify this esophageal cancer risk in humans. Two previous studies have reported that NAT2 genotype does (Morita et al., 1998Go) or does not (Lee et al., 2000Go) modify esophageal cancer risk. Additional NAT1 and NAT2 genotype studies in subjects with documented dietary exposures to heterocyclic amine carcinogens are warranted.


    ACKNOWLEDGMENTS
 
Portions of this work constituted partial fulfillment by Adrian J. Fretland for the Ph.D. in Pharmacology and Toxicology at the University of Louisville. We thank Dr. Fred F. Kadlubar (National Center for Toxicological Research, Jefferson, AR) for his generous donation of the synthetic PhIP-DNA adduct standard, N-(deoxyguanosin-8-yl)-PhIP.

This study was supported in part by United States Public Health Service grant CA-34627 from the National Cancer Institute.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292. Fax: (502) 852-7868. E-mail: d.hein{at}louisville.edu. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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