pyridine (PhIP) - American Chemical Society

Chem. Res. Toxicol. 2005, 18, 1471-1478

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Differential Metabolism of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Mice Humanized for CYP1A1 and CYP1A2 Connie Cheung,† Xiaochao Ma,† Kristopher W. Krausz,† Shioko Kimura,† Lionel Feigenbaum,‡ Timothy P. Dalton,§ Daniel W. Nebert,§ Jeffrey R. Idle,# and Frank J. Gonzalez*,† Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, Laboratory Animal Science Program, SAIC, National Cancer Institute, Frederick, Maryland 21702, Department of Environmental Health and the Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056, and Institute of Pharmacology, 1st Faculty of Medicine, Charles University, 128 00 Praha 2, Czech Republic Received May 25, 2005

The procarcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine formed during the cooking of foods. Metabolism of PhIP by CYP1A2 differs substantially between humans and rodents, with more N2-hydroxylation (activation) and less 4′-hydroxylation (detoxication) in humans. Therefore, the human response to PhIP and other heterocyclic amine exposure may not be accurately reflected in the laboratory rodent. By generating mouse models expressing the human genes, species differences in heterocyclic amine metabolism can be addressed. Two transgenic mouse lines were developed, one expressing the human CYP1A1 CYP1A2 transgene in a mouse Cyp1a1-null background (hCYP1A1) and another expressing human CYP1A1 CYP1A2 in a mouse Cyp1a2-null background (hCYP1A2). Expression of human CYP1A2 protein was detected in the liver and also at considerably lower levels in extrahepatic tissues such as lung, kidney, colon, and heart. In the hCYP1A1 and hCYP1A2 mice, 3-methylcholanthrene (3-MC) induced both human CYP1A1 and CYP1A2 protein in the liver. Differences in the metabolism of the heterocyclic amine PhIP were observed between wild-type and hCYP1A2 mice. PhIP was preferentially metabolized by N 2-hydroxylation in hCYP1A2 mice, whereas in wild-type mice, 4′-hydroxylation was the predominant pathway. Since the N2-hydroxylation pathway for PhIP metabolism has been reported to be predominant in humans, these results illustrate the potential effectiveness of using these transgenic, humanized mice as models for determining human health risks to PhIP and other heterocyclic amines instead of wild-type mice.

Introduction The human cytochrome P450 1 (CYP1)1 family members consist of CYP1A1, CYP1A2, and CYP1B1. The human CYP1A1 and CYP1A2 genes lie in the opposite orientation on chromosome 15q22-qter separated by a 23 kb segment that contains no other open reading frames (1). Both CYP1A1 and CYP1A2 are highly inducible upon exposure to xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene (3-MC), polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and * Corresponding author: Frank J. Gonzalez, Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Building 37, Room 3106, Bethesda, MD 20892. Telephone: +301 496 9067. Fax: +301 496 8419. E-mail: [email protected]. † Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health. ‡ Laboratory Animal Science Program, SAIC, National Cancer Institute. § University of Cincinnati Medical Center. # Charles University. 1 Abbreviations: PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; N-OH-PhIP, N2-hydroxy-PhIP; 4-OH-PhIP, 4′-hydroxy-PhIP; 3-MC, 3-methylcholanthrene; DMSO, dimethyl sulfoxide; CYP, cytochrome P450; AHR, aryl hydrocarbon receptor; hCYP1A1, human CYP1A1/2 transgene in a mouse Cyp1a1-null background; hCYP1A2, human CYP1A1/2 transgene in a mouse Cyp1a2-null background.

β-naphthoflavone (2, 3). Many of these xenobiotics are ligands for the aryl hydrocarbon receptor (AHR), which activates expression of genes in the arylhydrocarbon [Ah] gene battery including Cyp1a1, Cyp1a2, NAD(P)H: quinone oxidoreductase-1 (Nqo1), aldehyde dehydrogenase 3A1 (Aldh3a1), UDP glucuronosyltransferase 1A6 abd 1A7 (Ugt1a6, Ugt1a7), and glutathione transferase A1 (Gsta1) (4). CYP1 enzymes are primarily involved in the metabolic activation of numerous procarcinogens, including aflatoxin B1, arylamines, heterocyclic amines, and polycyclic aromatic hydrocarbons (5). PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine, is the most abundant heterocyclic amine in food formed during the cooking of meats and fish (6). Metabolic activation of PhIP is principally mediated by CYP1A2, followed by esterification with N-acetyltransferase or sulfotransferase to produce the activated esters (N2-acetoxy-PhIP and N2sulfonyloxy-PhIP) (Figure 1) that bind covalently with DNA to yield N2-(2-deoxyguanosin-8-yl)-PhIP as the major adduct (7). These activated esters can also bind with proteins and other cellular constituents, and these adducts spontaneously degrade to 5-hydroxy-PhIP (8). PhIP-DNA adducts have been detected in the colon,

10.1021/tx050136g CCC: $30.25 © 2005 American Chemical Society Published on Web 08/05/2005

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Figure 1. Metabolism of PhIP (I) to 4′-hydroxy-PhIP (II) and its sulfate conjugate (III), to PhIP N2-glucuronide (IV), to PhIP N3-glucuronide (V), and to N2-hydroxy-PhIP (VI). (VI) forms two glucuronides, N2-hydroxy-PhIP-N2-glucuronide (VII) and N2-hydroxyPhIP-N3-glucuronide (VIII), and two chemically reactive esters, N2-acetoxy-PhIP (IX) and N2-sulfonyloxy-PhIP (X), which form macromolecular adducts.

heart, and lung of rats treated with the carcinogen (7, 9, 10) and in the liver, colon, and mammary gland of mice (11). PhIP-DNA adducts have also been detected in colonic tissue of humans (12, 13). Formation of DNA adducts are considered a necessary initial step in chemical carcinogenesis. Interindividual differences in the levels of expression of CYP1A2 have been reported in humans (14). A higher CYP1A2 activity in combination with higher N-acetyltransferase activity has been associated with an elevated risk for colorectal cancer in individuals eating well-cooked meats, a rich source of heterocyclic amines (15, 16). Species differences in the metabolism of PhIP have been observed between humans and rodents (17). PhIP can generally be metabolized by N2-hydroxylation or by 4′-hydroxylation, with both reactions occurring primarily in the liver. In rodents, metabolism of PhIP occurs predominantly by oxidation in the ring system (4′hydroxylation) followed by phase II conjugation. Oxidation of the exocyclic amino group to the proximate mutagen N2-hydroxy-PhIP (N-OH-PhIP) appears to be the minor metabolic pathway in rodents (8). However, in humans, oxidation of the exocyclic amino group (N2hydroxylation) is the major metabolic pathway for PhIP, followed by glucuronidation (8, 17) (Figure 1). Since large differences exist in the metabolism of PhIP between humans and rodents (17), as well as within the human population itself, appropriate extrapolation of cancer risk from experimental animals to humans is of concern, particularly in establishing safe thresholds for human exposure to PhIP. In this study, the generation of transgenic mice expressing the human CYP1A1 and CYP1A2 genes, in either the Cyp1a1-null or Cyp1a2-null background, is

described. A recent report described a similar model using another BAC clone (26). The humanized mouse models described herein were found to accurately express CYP1A1 and CYP1A2 protein reflective of their expression in humans. The hCYP1A2 mouse line was further used to demonstrate that PhIP is preferentially N2hydroxylated by human CYP1A2 when compared to wildtype mice, a pathway for PhIP metabolism that in vitro studies revealed was predominant with the human ortholog (18-20). These results illustrate the potential effectiveness of using transgenic, humanized mice as models for determining human health risks to PhIP and other heterocyclic amines.

Materials and Methods Caution: N2-hydroxy-PhIP is carcinogenic to rodents and should be handled with care. Chemicals. PhIP and N2-hydroxy-PhIP (N-OH-PhIP) were obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repository at the Midwest Research Institute (Kansas City, MO). 4′-Hydroxy-PhIP (4-OH-PhIP) was kindly provided by Dr M. Nagao (National Cancer Research Center, Tokyo, Japan). NADP(H), 3-MC, 6-chloromelatonin, β-glucuronidase (EC 3.2.1.31), and arylsulfatase (EC 3.1.6.1) were purchased from Sigma-Aldrich (St. Louis, MO). Animals and Treatments. Mice were maintained under a standard 12 h light/12 h dark cycle with water and chow provided ad libitum. Handling was in accordance with animal study protocols (LM-016 and LM-036) approved by the National Cancer Institute Animal Care and Use Committee. Three male mice, age 7-8 weeks, were used for each treatment group. For examining CYP1A1/2 induction, mice were injected ip with

PhIP Metabolism in Humanized CYP1A1 and CYP1A2 Mice

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Figure 2. Generation of hCYP1A1 and hCYP1A2 mice. (A) Typical genotyping results for detection of the human CYP1A1/2 transgene by Southern blotting. (B) Typical example of PCR genotyping for the human CYP1A2 transgene using Ephx1 as an internal control. (C) Testing founder lines (J1, E6, and B10) for human CYP1A2 protein expression in liver microsomes (10 µg) by western blot analysis using monoclonal antibodies specific to human CYP1A2 and anti-rat CYP1A1 polyclonal antibody which detects both human and mouse CYP1A1 and CYP1A2. (D) Typical PCR genotyping results for detecting the mouse Cyp1a2 wild-type and null alleles; Ephx1 was used as an internal control. 100 mg/kg of 3-MC or corn oil (vehicle) and sacrificed 72 h later. For the in vivo PhIP metabolism study, mice were treated by oral gavage with 27 mg/kg of PhIP (dissolved in 20% dimethyl sulfoxide (DMSO) as a vehicle) or vehicle alone and immediately housed in metabolic chambers (Jencons, Leighton Buzzard, U.K.). The pooled urine from each group of mice was collected over a 24 h period following PhIP administration; mice were then sacrificed, and tissues were collected and snap-frozen in liquid nitrogen before storage at -80 °C. Generation of CYP1A1 and CYP1A2 Humanized, Transgenic Mice. A bacterial artificial chromosome (BAC) was previously identified containing the human CYP1A1 and CYP1A2 genes (1). The BAC clone was linearized using restriction enzyme digestion and purified before microinjection into fertilized FVB/N mouse eggs. Transgenic founders were screened by Southern blot analysis and bred with C57BL/6 wild-type mice. From this breeding, mice positive for the human CYP1A1 CYP1A2 transgene were identified by Southern blotting and further mated to Cyp1a1-null or Cyp1a2-null mice. Mice containing the human CYP1A1 CYP1A2 transgene in either the Cyp1a1-null or Cyp1a2-null background were bred together to generate homozygous mice and designated hCYP1A1 or hCYP1A2 mice, respectively. Genotyping by Southern Hybridization and PCR. For genotyping mice by Southern hybridization, tail genomic DNA was digested with BamH1. Electrophoresis and Southern hybridization conditions were as described previously (21). To confirm the presence of the human CYP1A1/2 BAC transgene, initial screening of mice was carried out using a 1.7 kb Apa I/Nco I fragment of the pUC18-human CYP1A1 cDNA plasmid as a DNA probe, which was 32P-labeled using random primers for Southern hybridization (Figure 2A). For additional breeding steps, the presence of the CYP1A1 CYP1A2 transgene was verified using the following primers: Hum1A2 F, 5′-AAT CAG GAG TGG CTG GAA CAC G-3′, and Hum1A2 R, 5′-TTG GCA GGG TTG TAA TGG CTG GTG-3′, giving a PCR product of 503 bp in only the samples positive for this transgene (Figure 2B). Mouse epoxide hydrolase 1 gene (Ephx1) primers served as an internal positive control for amplification, yielding a fragment of 341 bp in all samples (22). The following primers were used to identify the mouse Cyp1a2 wild-type allele: mCYP1A2#1, 5′CTA CAG CTT CAC ACT TAT CAC T-3′, and mCYP1A2#2, 5′GGA AGT TCT TCC CAA AGC ACA TG-3′, giving a PCR

product of ∼300 bp. Ephx1 primers were used an internal control, while additional bands of ∼300 bp were amplified exclusively in mice positive for the Cyp1a2 wild-type allele or the null allele (Figure 2D). Mouse Cyp1a1 wild-type and null alleles were identified by PCR using primers as described previously (23). Microsome Preparation. Tissues were homogenized and microsomes were prepared as described previously (24). Western Blot Analysis. Microsomal protein (1-30 µg) from each sample was subjected to SDS-PAGE in 10% polyacrylamide gels, electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH), and probed using anti-human CYP1A1 (clone 1-599-16), anti-human CYP1A2 (clone 26-7-5), anti-rat CYP1A2 (clone 22-341) (25), goat antirat CYP1A1 (cat. no. 458124, BD Gentest Woburn, MA), and anti-GAPDH (Chemicon International, Temecula, CA). Human CYP1A2 cDNA expressed protein (BD Gentest, BD Biosciences, Bedford, MA) or human liver microsomes (BD Gentest, BD Biosciences, Bedford, MA) were used as positive controls for human CYP1A2 detection. The relative amount of the human CYP1A2 in liver microsomes of hCYP1A2 mouse was determined by comparing with recombinant human CYP1A2 electrophoresed concurrently on a Western blot. A series of recombinant human CYP1A2 (2, 5, 10, 20, and 30 pmol) and 0.5 µg liver micrsomes were loaded in the same gel for Western blotting analysis. The intensity of each band was compared to determine the relative amount of the human CYP1A2 in liver microsomes of hCYP1A2 mouse. In Vitro PhIP Metabolism in Mouse Liver Microsomes. Incubation reactions were carried out in a final volume of 200 µL, containing 100 mM potassium phosphate, pH 7.4, 25 µL of liver microsomes, 25 µM PhIP, and 1 mM NADP(H). The reactions were initiated by the addition of NADP(H) after preincubation for 5 min at 37 °C. Following a 10 min incubation at 37 °C, the reactions were terminated by the addition of 0.5 mL ethyl acetate and 0.5 mL methyl tert-butyl ether. A volume of 5 µL of 100 µM 6-chloromelatonin was added as an internal standard. The samples were centrifuged at 3000 rpm for 5 min at 4 °C. The organic layer was transferred to a new tube, dried by N2 gas, and reconstituted with 70% methanol. The sample was reconstituted with 100 µL of 70% methanol, and a volume

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of 6 µL was injected for LC/MS/MS analysis. The recoveries for N-OH-PhIP and 4-OH-PhIP were 72.6 ( 10.2% and 77.7 ( 12.4%, respectively. All reactions were performed in duplicate. In Vivo PhIP Metabolism in hCYP1A2 Mice. Following PhIP or DMSO treatment to the wild-type and hCYP1A2 mice, the pooled urine collected over 24 h was analyzed. N2-OH-PhIPN3-Glucuronide and N2-OH-PhIP-N2-glucuronide, two major phase II metabolites in the urine of mice administered PhIP, were directly measured. Urine (100 µL) was diluted in 900 µL of H2O and centrifuged at 14 000 rpm for 5 min, 4 °C, and a total of 6 µL of each sample was injected for N2-OH-PhIP-N3glucuronide and N2-OH-PhIP-N2-glucuronide analysis by LC/ MS/MS. Free and total N-OH-PhIP in the urine were also detected. For free N-OH-PhIP, 100 µL of urine was diluted in 100 µL of H2O, and then extracted by 0.5 mL ethyl acetate and 0.5 mL methyl tert-butyl ether. For total N-OH-PhIP, 100 µL of urine was diluted in 900 µL of 20 mM phosphate-buffered saline containing 200 units β-glucuronidase and incubated at 37 °C for 6 h with shaking. For total 4-OH-PhIP detection, 100 µL of urine was diluted in 900 µL of 20 mM phosphate-buffered saline containing 100 units arylsulfatase and incubated at 37 °C for 6 h with shaking. The incubation was terminated by the addition of 1 mL ethyl acetate and 2 mL methyl tert-butyl ether. A total of 5 µL of 100 µM 6-chloromelatonin was added as an internal standard. The samples were centrifuged at 3000 rpm for 5 min at 4 °C. The organic layer was transferred to a new tube, dried by N2 gas, and reconstituted with 100 µL of 70% methanol, and 6 µL of the reconstituted sample was injected for LC/MS/MS analysis. The recovery was below 30% for N-OHPhIP and about 50% for 4-OH-PhIP from urine after a 6 h enzymatic hydrolysis by β-glucuronidase or arylsulfatase. PhIP and Its Metabolites Analysis by LC/MS/MS. A PE SCIEX API 2000 ESI triple quadrupole mass spectrometer (PerkinElmer/ABI, Foster City, CA) was used in this study. A synergi 4 µm Polar-RP 50 mm × 2 mm i.d. column (Phenomenex, Torrance, CA) was used for PhIP, N-OH-PhIP, 4-OH-PhIP, and 6-chloromelatonin analysis. A Luna C18 50 mm × 4.6 mm i.d. column (Phenomenex, Torrance, CA) was used for N2-OHPhIP-N3-glucuronide and N2-OH-PhIP-N2-glucuronide analysis. The flow rate through the column at ambient temperature was 0.25 mL/min with 70% methanol and 30% water containing 0.1% formic acid. The mass spectrometer was equipped with a turbo ion spray source and run in the positive ion mode. The turbo ion spray temperature was maintained at 350 °C, and a voltage of 5.5 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas. The detection of PhIP, its metabolites, and the internal standard was accomplished by multiple reaction monitoring (MRM) with the transitions m/z 225.2/210.2 for PhIP, 241.2/223.2 for N-OH-PhIP, 241.2/226.2 for 4-OH-PhIP, 267.0/208.4 for 6-chloromelatonin, 416.8/241.1 for N2-OH-PhIP-N2-glucuronide, and 416.8/225.1 for N2-OHPhIP-N3-glucuronide. Statistical Analysis. All values are expressed as the mean ( SD or mean ( SE (n ) 3-6). All data were analyzed by paired or unpaired Student’s t test for significant differences between the mean values of each group.

Results Generation of hCYP1A1 and hCYP1A2 Transgenic Mice. Transgenic mice carrying the human CYP1A1 and CYP1A2 genes were generated as described in Materials and Methods, with the transgene initially genotyped by Southern blot analysis and then, at later breeding steps, by PCR genotyping (Figure 2A and Figure 2B). Western blot analysis of liver microsomes isolated from the three different founder lines identified two of these lines that express human CYP1A2 protein (J1 and B10) (Figure 2C). One founder (J1) was selected to breed further to generate transgenic mice carrying the human CYP1A1 CYP1A2 genes in the absence of the mouse

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Figure 3. Expression analysis of hCYP1A1 and hCYP1A2 mice. Western blot analysis of liver microsomes (10 µg) from untreated wild-type, Cyp1a1-null, Cyp1a2-null, hCYP1A1, and hCYP1A2 mice detected using monoclonal antibodies specific to human CYP1A2 and rat CYP1A2.

Cyp1a1 gene (designated as hCYP1A1) or the mouse Cyp1a2 gene (designated as hCYP1A2). Confirmation of transgenic mice in a Cyp1a1-null or Cyp1a2-null background was carried out by PCR genotyping (Figure 2D). Expression Analysis of hCYP1A1 and hCYP1A2 Transgenic Mice. Western blot analysis of liver microsomes from hCYP1A2 mice revealed the constitutive expression of human CYP1A2 protein (Figure 3). Analysis of major tissues from hCYP1A2 mice revealed expression of human CYP1A2 protein predominantly in the liver with lower amounts present in most of the extrahepatic tissues examined (Figure 4A). By comparing the amount of human CYP1A2 detected in the liver of hCYP1A2 mice to that of recombinant human CYP1A2, we established that the amount of human CYP1A2 protein in 0.5 µg of liver microsomes from this transgenic mouse was approximately equivalent to 20 pmol of recombinant human CYP1A2 protein (Figure 4A). Confirmation that the native mouse CYP1A2 in wild-type mice was constitutively expressed only in the liver was also demonstrated (Figure 4B). The inducibility of the human CYP1A1 and CYP1A2 proteins in these transgenic mouse models was investigated by treatment with 3-MC. In the liver of hCYP1A1 and hCYP1A2 mice, human CYP1A2 protein was induced by approximately 3-fold with 3-MC treatment (Figure 5). Human CYP1A1 protein was undetectable in the untreated hCYP1A1 and hCYP1A2 mice; however, upon 3-MC treatment, robust expression of human CYP1A1 protein was observed in the liver (Figure 5). Similar profiles of human CYP1A1 and human CYP1A2 protein expression were observed between hCYP1A1 and hCYP1A2 mice, since the incorporated transgene contained both genes. Using a monoclonal antibody against rat CYP1A2, the expression of mouse CYP1A2 was examined in untreated and 3-MC-treated mice, and the expected induction was observed (Figure 5). In Vitro PhIP Metabolism. In liver microsomes isolated from hCYP1A1 and hCYP1A2 mice, the amount of N-OH-PhIP detected was significantly higher than that observed in wild-type mice (88.9 ( 5.69 (pmol/min)/mg protein). 4′-Hydroxylation of PhIP in liver microsomes isolated from hCYP1A1 and hCYP1A2 mice was signifi-



Figure 4. Tissue specific expression in hCYP1A2 and wild-type mice. Analysis of extrahepatic tissues for human CYP1A2 protein expression by western blotting of microsomes (0.5-30 µg) isolated from untreated hCYP1A2 and wild-type mice detected using monoclonal antibodies specific to human CYP1A2, rat CYP1A2, and GAPDH. Recombinant human CYP1A2 (2-30 pmol) was used to compare human CYP1A2 expression levels in the liver to extrahepatic tissues.

Figure 5. Induction of hepatic human CYP1A1 and CYP1A2 proteins following 3-MC treatment of hCYP1A1 and hCYP1A2 mice. Western blot analysis of liver microsomes (10 µg) from wild-type, hCYP1A1, and hCYP1A2 mice treated with 3-MC or corn oil (control; CON), detected using monoclonal antibodies specific to human CYP1A1, human CYP1A2, rat CYP1A2, and GAPDH.

cantly lower than in wild-type mice (46.1 ( 4.68 (pmol/ min)/mg protein). The levels of 4-OH-PhIP detected in hCYP1A1 mice were significantly higher than that in hCYP1A2 mice because of the contribution of mouse CYP1A2 to PhIP metabolism. In Cyp1a1-null mice, both N-OH-PhIP and 4-OH-PhIP detection was similar to that seen in wild-type mice as a result of the contribution of mouse CYP1A2 to PhIP metabolism. Significantly lower N-OH-PhIP and 4-OH-PhIP were detected in liver microsomes from Cyp1a2-null mice, demonstrating the importance of mouse CYP1A2 in the metabolism of PhIP (Figure 6). In Vivo PhIP Metabolism. To confirm that the differences in PhIP metabolism observed between wildtype and hCYP1A2 mice in vitro could also have been observed in the whole animal, mice were treated with an oral dose of PhIP and the urine was analyzed. In mice treated with the vehicle DMSO, no PhIP or metabolites were detected in the urine. In hCYP1A2 mice treated with PhIP, free N-OH-PhIP in the urine was 1.1 µM, and

total N-OH-PhIP (free and de-conjugated) in the urine was 28.4 µM. Because of the low percentage of free N-OHPhIP (below 5%) in the urine and the low recovery (below 30%) of N-OH-PhIP after enzymatic hydrolysis, the relative total N-OH-PhIP in the urine was presented as the total of N2-OH-PhIP-N2-glucuronide and N2-OHPhIP-N3-glucuronide. The content of N2-OH-PhIP-N3glucuronide was about 5 times more than N2-OH-PhIPN2-glucuronide in wild-type and hCYP1A2 mice (Figure 7). The relative level of total N-OH-PhIP, detected in the urine of hCYP1A2 mice, was over 4-fold higher than that in PhIP-treated wild-type mice (Figure 8). Similar to the in vitro data for 4-OH-PhIP, 4′-hydroxylation of PhIP was 50% lower in the PhIP-treated hCYP1A2 mice as compared to PhIP-treated wild-type mice (Figure 8).

Discussion A transgenic mouse line expressing the human CYP1A1 and CYP1A2 genes in a mouse Cyp1a2-null background

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Figure 6. PhIP metabolism in vitro using liver microsomes. N2-Hydroxylation and 4′-hydroxylation of PhIP in liver microsomes isolated from wild-type (Reaction 1), Cyp1a2-null (Reaction 2), Cyp1a1-null (Reaction 4), hCYP1A2 (Reaction 3), and hCYP1A1 (Reaction 5) mice as measured by the relative formation of the metabolites N-OH-PhIP and 4-OH-PhIP. The relative percentage of metabolite formed was calculated based on the mean value of the wild-type liver sample (defined as 100%). Values are mean ( SD; n ) 3-6; *, p < 0.05 compared to wild-type (Reaction 1).

Figure 7. Analysis of N2-OH-PhIP-N3-glucuronide and N2OH-PhIP-N2-glucuronide in the urine of wild-type and hCYP1A2 mice after PhIP treatment, detected by LC/MS/MS (MRM), m/z 416.80/241.06 for N2-OH-PhIP-N2-glucuronide and m/z 416.80/ 225.13 for N2-OH-PhIP-N3-glucuronide. Chromatograms A and B show N2-OH-PhIP-N2-glucuronide and N2-OH-PhIP-N3glucuronide in the urine of PhIP treated hCYP1A2 mice. Chromatograms C and D show N2-OH-PhIP-N2-glucuronide and N2-OH-PhIP-N3-glucuronide in the urine of PhIP treated wild-type mice. The units on the y-axis represent ion counts per second.

(hCYP1A2 mouse) was developed that metabolizes the heterocyclic amine PhIP in a manner quantitatively and qualitatively more similar to man than to mouse. Both in vitro and in vivo, the hCYP1A2 mouse switches the balance of PhIP hydroxylation from the predominant

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Figure 8. PhIP metabolism in vivo in hCYP1A2 mice. N2Hydroxylation and 4′-hydroxylation of PhIP was examined in the urine of wild-type and hCYP1A2 mice which had been treated with an oral dose of PhIP. The relative amount of N-OH-PhIP include the N2-OH-PhIP-N2-glucuronide and N2OH-PhIP-N3-glucuronide. The relative amount of 4-OH-PhIP was present by total of free 4-OH-PhIP and de-conjugated 4-OHPhIP. The relative percentage of metabolite detected was calculated based on the mean value of the wild-type mice (defined as 100%). The pooled urine samples from three mice were analyzed in triplicate (values are mean ( SE). *, p < 0.05 compared to wild-type.

murine pathway of 4′-hydroxylation to the principal human hydroxylation pathway of N2-hydroxylation. This latter pathway can lead to chemically reactive intermediates formed by either sulfation or acetylation (Figure 1) and thus the possibility of macromolecular adduct formation. In this regard, the hCYP1A2 mouse provides a more appropriate animal model than other rodents with which to perform human risk evaluations and the estimation of safe levels of exposure to this ubiquitous mutagen. Two novel transgenic mouse lines, both expressing the human CYP1A1 and CYP1A2 genes, were produced using a BAC clone as a transgene. This BAC clone was previously characterized by DNA sequencing and was the first demonstration that the two genes are arranged in a head-to-head orientation with a 23 kb spacer containing the regulatory elements for the AHR and other liver transcription factors (1). This orientation, which was later confirmed by others (26) and the human genome sequence, allows the possibility of regulation of both genes by the same cis-acting gene control elements. The first of these lines expresses both the CYP1A1 and CYP1A2 genes on a Cyp1a1-null background and thus expresses both mouse and human CYP1A2. Moreover, as expected, constitutive expression of the CYP1A1 transgene was found, and the protein was induced by treatment with 3-MC. This mouse line, designated the hCYP1A1 mouse, was not further investigated here but will undoubtedly prove of value in further investigations of the role of human CYP1A1 in chemical activation and detoxication and their consequences. The second transgenic mouse line expresses human CYP1A2 against a Cyp1a2-null background and therefore


does not express the mouse CYP1A2 enzyme. This mouse is referred to here as the hCYP1A2 mouse. In this line, human CYP1A2 is incorporated into the mouse genome in the absence of the mouse Cyp1a2 gene. Consequently, the human CYP1A2 protein, but not the mouse CYP1A2 protein, is expressed in liver microsomes. In addition, various extrahepatic tissues in hCYP1A2 and wild-type mice are positive for the expression of CYP1A2 protein. The human CYP1A2 was strongly expressed in liver, as expected, but was also detected in kidney, spleen, lung, heart, ovary, colon, and testis, albeit at levels of about 1% or less than those found in liver; it was not detected in duodenum or brain. 3-MC treatment induced human CYP1A2 in the hCYP1A2 mouse liver and, as expected, did not induce any mouse CYP1A2 protein in these animals. The hCYP1A2 mice were employed to investigate PhIP hydroxylation, specifically, the balance between the detoxication pathway of 4′-hydroxylation and the activation pathway of N2-hydroxylation (Figure 1). As shown in Figure 6, liver microsomes from Cyp1a2-null mice (Reaction 2) have a statistically significantly reduced production of both 4-OH-PhIP and N-OH-PhIP. In liver microsomes from hCYP1A2 mice (Reaction 3), however, 4-OH-PhIP production is also significantly reduced, but N-OH-PhIP production is statistically significantly increased about 2.5-fold over the wild-type. The Cyp1a1null mouse microsomes (Reaction 4) behaved like the wild-type microsomes (Reaction 1). Interestingly, the hCYP1A1 mouse liver microsomes (Reaction 5) behaved like the hCYP1A2 mouse microsomes (Reaction 3). hCYP1A1 mice express both human and mouse CYP1A2 in the liver. Thus, in vitro, hCYP1A2 mouse microsomes display an increased preference for N2-hydroxylation and a markedly reduced 4′-hydroxylation of PhIP. This phenomenon is more clearly observed in vivo after PhIP treatment; there was a 2-fold reduction in the excretion of 4-OH-PhIP and a 4-fold increase in the excretion of N-OH-PhIP in the hCYP1A2 mouse when compared with that of wild-type mouse. The data presented herein suggest strongly that CYP1A2 is largely responsible for both the 4′- and N 2hydroxylation of PhIP but that the human form shows a clear preference for N2-hydroxylation relative to the mouse homologue. This phenomenon was observed both in vitro and in vivo. This confirms earlier work (10) that first showed the human CYP1A2 preference for N2hydroxylation relative to 4′-hydroxylation (ratio of 97:1) compared with the mouse ratio of 1.7:1 for these pathways. In addition, human recombinant CYP1A2 has been reported to possess 10-19-fold higher PhIP N2-hydroxylation than rat CYP1A2 (18). These workers also reported that 4′-hydroxylation of PhIP cannot be detected with recombinant human CYP1A2 but that N2-hydroxylation is massively increased in parallel to the induction of CYP1A2 by treatment of rats with 3-MC, β-naphthoflavone, or Arochlor 1254 (19). They also reported a strong correlation between human hepatic CYP1A2 content and PhIP N2-hydroxylation rates (19). The difference between hCYP1A2 mouse and wild-type mouse metabolism of PhIP reported here is entirely consistent with these earlier reports (10, 18, 19). It has been reported both in vitro and in vivo that PhIP is mainly metabolized by glucuronidation. When PhIP was added to human hepatocyte cultures, 60% of the metabolites recovered comprised the stable and nontoxic


N2- and N3-glucuronides of N2-hydroxy-PhIP (metabolites 7 and 8 in Figure 1). These metabolites were formed in substantially lower amounts in rat hepatocyte cultures (20). In addition, PhIP itself underwent direct N-glucuronidation to yield both the N2- and N3-glucuronides (metabolites 4 and 5 in Figure 1) in both rat and human hepatocyte cultures (20). A study performed in three human patients administered 14C-labeled PhIP orally has been reported (27), in which 47-60% of the radioactivity in the 24 h urine was recovered as N2-hydroxy-PhIP N2glucuronide (metabolite 7 in Figure 1). PhIP N2-glucuronide (metabolite 4 in Figure 1), N2-hydroxy-PhIP N3glucuronide (metabolite 8), and 4′-hydroxy-PhIP sulfate (metabolite 3) were the other major urinary metabolites reported (27). In this hCYP1A2 mice model, the amount of N2-OH-PhIP-N3-glucuronide was about 5 times that of the N2-OH-PhIP-N2-glucuronide, which is comparable to the rodent with majority of N2-OH-PhIP-N3-glucuronide formation for N-hydroxy heterocyclic amines (28). A study using recombinant UDP-glucuronosyltransferases (UGTs) suggest that the formation of the major urinary metabolite N2-hydroxy-PhIP N2-glucuronide is mediated almost exclusively by UGT1A1 at a substrate concentration of 5 µM (29). In contrast, the minor N2hydroxy-PhIP N3-glucuronide is formed almost equally by UGT1A1 and UGT1A9. However, when a substrate concentration of 100 µM was employed, the isozyme selectivity in the formation of both glucuronides was less obvious, with UGT1A1, UGT1A3, UGT1A4, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 all playing a role, but with UGT1A1 still the dominant isozyme (29). These findings on N-OH-PhIP glucuronidation are reminiscent of a study of the role of various P450s in the activation of PhIP to mutagenic metabolites (30). It was reported in this study, which used a concentration of 220 µM PhIP, that recombinant CYP1A2 was the principal isozyme responsible for the conversion of PhIP to mutagenic metabolites but that CYP1A1, CYP1B1, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 also displayed about 10-30% of the activity of CYP1A2. At the estimated daily human dietary intake of PhIP of 0.1-13.8 µg (31), the concentration of PhIP or its metabolites in tissues, including the liver, is unlikely to exceed 1 µM. Therefore, the role of CYP forms other than CYP1A2 in the metabolism of PhIP in humans is questionable. The hCYP1A2 mouse should assist in the assessment of risks to human populations of chemical exposures from substances that are either detoxicated or activated largely by human CYP1A2.

Acknowledgment. We thank John R. Buckley for technical assistance. J.R.I. is grateful to U.S. Smokeless Tobacco Company for a grant for collaborative research. Funded, in part, by NIH Grants R01-ES08147 and P30ES06096 and by the NCI Intramural Research Program of the NIH.

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TX050136G

pyridine (PhIP) - American Chemical Society

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