Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892,
1 Veterinary and Tumor Pathology Section, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702
2 National Center for Toxicological Research, Jefferson, AR 72079, USA
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Abstract |
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Abbreviations: 4-ABP, 4-aminobiphenyl; AHR, aryl hydrocarbon receptor; APMSF, 4-amidinophenylmethanesulfonyl fluoride; DMSO, dimethylsulfoxide; MRM, multiple reactions monitoring; N2-OH-PhIP, N2-hydroxy-PhIP; 4'-OH-PhIP, 4'-hydroxy-PhIP; P450s, cytochromes P450; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
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Introduction |
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P450s consist of a large superfamily of heme-containing proteins (http://drnelson.utmem.edu/CytochromeP450.html) that includes, among others, four families, CYP1, CYP2, CYP3 and CYP4, that appear to be primarily involved in the metabolism of xenobiotics. However, only a rather limited group of enzymes primarily carry out the metabolic activation of toxins and carcinogens (3). CYP1A1 and CYP1B1 metabolically activate polycyclic aromatic hydrocarbons and CYP2E1 metabolizes a large number of low molecular weight toxins, carcinogens and suspected cancer-causing agents. CYP1A2 carries out the N2-hydroxlation of arylamine and heterocyclic amine carcinogens. The N2-hydroxy metabolites are further metabolized by O-esterification by the phase II enzymes such as N-acetyltransferase and sulfotransferase.
CYP1A2 is constitutively expressed in the liver of mice, rats and humans and is inducible by ligands of the aryl hydrocarbon receptor (AHR), the prototypes of which include 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene (4,5). Upon ligand binding, activated AHR dimerizes with ARNT and translocates to the nucleus where it activates the expression of target genes, including CYP1A1, CYP1A2 and CYP1B1, phase II enzymes, such as UDP-glucuronosyl transferase, and growth factors, such as epidermal growth factor.
Interindividual differences in levels of expression of CYP1A2 have been found in humans (6) and a higher CYP1A2 activity in conjunction with higher N-acetyltransferase activity has been associated with elevated risk for colorectal cancer in individuals eating well-done meat, a rich source of heterocyclic amines (7,8). This makes CYP1A2 a potential susceptibility factor for environmental carcinogens activated by this enzyme. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is among the most abundant of the heterocyclic amines found in cooked meat (9). Metabolism of PhIP by N2-hydroxylation followed by O-acetylation occurs primarily in the liver of rodents. CYP1A2 appears to be the primary P450 responsible for the N2-hydroxylation of heterocyclic amine food mutagens in humans (1012). However, the role of CYP1A2 in the carcinogenesis of food mutagens is largely assumed based on in vitro metabolic studies using liver microsomes and recombinant P450s. We previously compared the sensitivities of Cyp1a2-null and wild-type mice to 4-aminobiphenyl (4-ABP) arylamine-induced liver carcinogenesis. These results demonstrated that CYP1A2 is not the primary P450 responsible for 4-ABP metabolic activation in mice when the neonatal mouse bioassay was used (13).
In the present study, the neonatal carcinogen bioassay was used to determine the role of CYP1A2 in PhIP carcinogenesis by analyzing Cyp1a2-null and wild-type mice. The neonatal bioassay is known to cause liver tumors in mice administered PhIP (14). These results, together with the in vitro data, suggest the existence of a pathway not involving CYP1A2 that is likely to be responsible for PhIP carcinogenesis in mice.
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Materials and methods |
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Animals
Characterization of Cyp1a2-null (15) and Ahr-null mice (16) was previously described. They are derived from a mixed background of 129/Sv and C57BL/6 mouse strains, termed B6;129. Animals were maintained in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, 1996) under an animal study proposal approved by the NCI Animal Care and Use Committee.
Animal treatment
PhIP was dissolved in dimethylsulfoxide (DMSO) at 20 and 40 mM, which we refer to as the low and high dose groups, respectively. Mice were injected i.p. with 10 and 20 µl of PhIP solution on 8 and 15 days of age, respectively. Thus, the mice received 11 (low dose) and
22 (high dose) mg/kg PhIP per injection, assuming that 8- and 15-day pups weigh
4 and
8 g, respectively. This corresponds to cumulative amounts of 600 and 1200 nmol PhIP for the low and high dose groups, respectively. Control animals were injected with an equivalent volume of DMSO. The mice were killed at 1921 months of age and various tissues were subjected to histological examination.
Pathology
Mice received a complete necropsy. All organs were examined and all grossly visible lesions in liver, lung and spleen were routinely fixed in 10% neutral buffered formalin, embedded in paraffin and sections, 46 µm in thickness, were prepared and stained with hematoxylin and eosin.
Microsomes
Liver microsomes used for western blotting were prepared from Cyp1a2-null, Ahr-null and wild-type mice that had been treated with PhIP or DMSO (as a control) on 8 and 15 days of age, using the same regimen as the high dose PhIP neonatal bioassay, and killed on day 16. The Ahr-null mice were used as a control for no expression of CYP1A1 in both metabolism assays and western blotting analyses. Liver microsomes used for metabolite assays were prepared from adult (2- to 3-month-old) and 16- to 17-day-old Cyp1a2-null and wild-type mice. Briefly, immediately after excision the liver was washed with ice-cold washing solution (250 mM sucrose, 10 mM potassium phosphate, 1 mM EDTA and 1 mM APMSF, pH 7.4), minced with scissors and homogenized using a motor driven, Teflon tipped pestle. The homogenates were centrifuged at 9000 g for 20 min at 4°C and the resultant supernatants were twice centrifuged at 100 000 g for 60 min at 4°C. The microsomal pellets were resuspended in ice-cold freezing solution (100 mM potassium phosphate, 20% v/v glycerol, 1 mM EDTA, 1 mM APMSF, pH 7.4), aliquoted and stored at 80°C until use. Protein concentrations were determined using the BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL), following the manufacturers instructions.
Resolution of PhIP metabolites
Generally, incubation reactions were carried out in 100 mM potassium phosphate, pH 7.4, containing mouse liver microsomes (with 2060 µg protein), recombinant CYP1A2 enzyme (3 pmol, positive control) or insect control (3 µl, negative control) and substrate in a final volume of 200 µl. The reactions were initiated by the addition of 20 µl of 10 mM NADPH after 5 min preincubation at 37°C. PhIP concentration was fixed at 10 µM to compare its N2-hydroxylation activity in individual mouse liver microsomes. For the kinetic analysis, pooled liver microsomes from three adult mice were used and PhIP concentrations ranged from 1.0 to 800 µM. After a 10 min incubation, the reactions were terminated by the addition of 50 µl of ice-cold 100 mM sodium hydroxide solution. The mixtures were cooled on ice for 10 min and 15 µl of 10 µM acetanilide (internal standard) was added. Samples were extracted with ethyl acetate (1 ml) and methyl t-butyl ether (2 ml) mixture and reconstituted with 50% methanol containing 0.2% formic acid. Aliquots of 5 µl of sample were injected for LC-MS/MS analysis. All reactions were performed in duplicate.
Identification and quantitation of metabolites by LC-MS/MS
LC-MS/MS analysis was performed on a PE SCIEX API2000 ESI triple-quadrupole mass spectrometer (PerkinElmer/ABI, Foster City, CA) controlled by Analyst software. A Phenomenex Synergi 4µ Polar-RP 50 mmx2 mm i.d. column (Torrance, CA) was used to separate the substrate and its metabolites. The flow rate through the column at ambient temperature was 0.2 ml/min with 50% methanol and 50% water containing 0.1% formic acid. The mass spectrometer was operated in the turbo ion spray mode with positive ion detection. The turbo ion spray temperature was maintained at 350°C and a voltage of 4.8 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas. The detection and quantification of PhIP, its metabolites and the internal standard were accomplished by multiple reactions monitoring (MRM) with the transitions m/z 225.2/210.2 for PhIP, 241.2/223.2 for N2-OH-PhIP, 241.2/226.2 for 4'-OH-PhIP and 135.9/94.1 for acetanilide. MS/MS conditions were optimized automatically for each analyte and raw data were processed using Analyst Software. The calibration curve was linear for PhIP and N2-OH-PhIP concentrations ranging from 10 to 200 µM. The recoveries of these compounds ranged from 85 to 110%. Intra-day and inter-day coefficients of variation were <10%.
Data analysis
Estimated parameters were expressed as means ± SEM. Enzyme MichaelisMenten parameters (Km and Vmax values) were estimated by non-linear regression (GraphPad PrismTM 3.02; San Diego, CA). Values were compared with the unpaired t-test and the difference was considered significant if the probability (P value) was <5%.
Western blotting
Liver microsomal protein (13 µg) was subjected to 8% SDSPAGE and proteins were transferred to nitrocellulose membranes for western blotting (Schleicher & Schuell, Keene, NH). The blots were developed using rabbit anti-rat CYP1A1, CYP2B1 and CYP2C11 (BD Gentest, Woburn, MA) or anti-mouse CYP1B1 antibodies (kindly provided by Dr C.Jefcoate, Madison, WI) and secondary antibody coupled with horseradish peroxidase for enhanced chemiluminescence detection (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL). The anti-rat CYP1A1 antibody cross-reacts with mouse CYP1A1 and CYP1A2. The anti-rat CYP2B1 and CYP2C11 antibodies cross-react with mouse P450s in the CYP2B and CYP2C families, respectively.
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Results |
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Various other tumors were found in some mice at low incidences without obvious significant group differences due to low numbers (histiocytic sarcoma, hemangioma/hemangiosarcoma, hepatoblastoma, endometrial sarcoma, renal cell carcinoma, transitional cell carcinoma, intestinal carcinoma, pheochromocytoma, Harderian gland carcinoma and pituitary adenoma).
LC-MS/MS analysis of PhIP and its metabolites
A sensitive and selective LC-MS/MS method was developed for the analysis of PhIP and its two hydroxylated metabolites. This method offers good separation of PhIP (2.24 min) and its metabolites, 4'-OH-PhIP (1.46 min) and N2-OH-PhIP (2.36 min) (Figure 1). The assay takes <5 min with isocratic elution and thus could be used for high throughput analysis. Hydroxylated PhIP was detected in the reactions with wild-type and Cyp1a2-null mouse liver microsomes and recombinant CYP1A2, but not in the incubations with insect control or the reactions in the absence of NADPH.
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Discussion |
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The in vitro PhIP metabolite analysis demonstrated that PhIP N2-hydroxylation activity in wild-type mouse liver microsomes was 8-fold higher than that in Cyp1a2-null mouse liver, suggesting that in the presence of CYP1A2, PhIP is preferentially metabolized by CYP1A2 to N2-OH PhIP, the initial metabolite that is converted to the ultimately proximal carcinogenic derivative of PhIP. Western blotting results revealed no detectable expression of either CYP1A1, CYP1A2 or CYP1B1 in the Cyp1a2-null mouse livers. However, surprisingly, in the PhIP carcinogenesis study Cyp1a2-null mice appeared to have a higher incidence of tumors after treatment with PhIP as compared with wild-type mice. These data suggest the existence of another pathway unrelated to CYP1A2 that is responsible for PhIP activation and carcinogenesis. In fact, CYP1A2 appears to be inactivating the carcinogenic effect of the unknown pathway, especially in females. Another pathway of metabolic activation of PhIP having higher activity in the absence of CYP1A2 could account for greater tumorigenicity in Cyp1a2-null mice. This second pathway of PhIP metabolic activation is not known. It is noteworthy that an unknown P450 of
51 kDa, together with CYP1A1 and CYP1A2, has been reported to be induced by PhIP in rat liver (20). However, none of these P450s are induced by PhIP in mice, hamsters and guinea pigs (20). It is possible that this unidentified P450 of
51 kDa or other P450s may be involved in PhIP carcinogenesis. However, it remains to be determined which pathway is responsible for the metabolic activation of PhIP, leading to tumorigenesis in mice.
Recently, DNA adduct levels were determined using 32P-post-labeling in the Cyp1a2-null and respective wild-type adult mice treated with PhIP (21). No PhIPDNA adducts were detected in either liver or kidney of Cyp1a2-null mice, whereas the levels of adducts in mammary gland and colon were <10% of those found in the wild-type mice. In this report, mice were treated with a single oral dose of 150 mg/kg PhIP by gavage at the age of 35 months and PhIPDNA adduct levels were determined 3 h after treatment. This finding raised the possibility that extrahepatic P450 expressed in mammary gland and colon contributed to the formation of adducts in these tissues. Alternatively, a hepatic P450 other than CYP1A2 may have contributed to the N2-hydroxylation of PhIP in Cyp1a2-null mice and N2-OH-PhIP was carried via the bloodstream to extrahepatic tissues. It should be noted that higher glutathione S-transferase activity has been found in liver and kidney (22), which may have been partly the reason for the lack of detectable PhIPDNA adducts in liver and kidney. The reason for the discrepancy in liver between these results and the current study is not known, however, it could be due to different experimental protocols. The dose, route and time when mice receive PhIP may be very critical in PhIP carcinogenesis bioassays. It is possible that PhIP may be mainly metabolically activated through a non-P450 pathway in liver, whereas it is metabolically activated by P450 in mammary gland and colon, resulting in DNA adduct formation. It is also possible that a hepatic P450 other than CYP1A2 is involved in both PhIPDNA adduct formation in mammary gland and colon, if that is the case, and the N2-hydroxylation of PhIP in the current study. In fact, a member of the CYP3A family was suggested to be a candidate P450 involved in PhIP metabolism (21).
Our results demonstrate that PhIP carcinogenesis in mice is likely to be independent of CYP1A2 when examined using the neonatal carcinogen bioassay. Based on in vitro studies using rats and humans, CYP1A2 is known to be the primary P450 required for arylamine/heterocyclic amine hydroxylation and their induced carcinogenesis (12). Our data demonstrate that species differences in arylamine/heterocyclic amine metabolic activation among rats, humans and mice and the route and timing of administration of chemicals may be critical factors to be considered when extrapolating mouse carcinogenesis bioassay data to humans.
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Notes |
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4 Present address: Takeda Chemical Industries Ltd, Yodogawa-ku, Osaka 532-8686, Japan
5 Present address: Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06080 Badajoz, Spain
6 To whom correspondence should be addressed at: Building 37, Room 2A19, National Institutes of Health, Bethesda, MD 20892, USA Email: shioko{at}helix.nih.gov
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Acknowledgments |
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References |
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