Carcinogenesis of the food mutagen PhIP in mice is independent of CYP1A2

Shioko Kimura6, Mayumi Kawabe1,3, Aiming Yu, Hideki Morishima1,4, Pedro Fernandez-Salguero5, George J. Hammons2, Jerrold M. Ward1, Fred F. Kadlubar2 and Frank J. Gonzalez

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the heterocyclic amines found in cooked meat. Based on in vitro studies with rats and humans, CYP1A2 is believed to be the primary enzyme responsible for N2-hydroxylation, the initial step in the metabolic activation of PhIP. To determine whether CYP1A2 is the primary P450 responsible for metabolic activation of PhIP in mice that leads to tumor formation, neonatal Cyp1a2-null and wild-type mice were treated with ~11 (low dose) and ~22 (high dose) mg/kg PhIP at days 8 and 15, corresponding cumulatively to 600 and 1200 nmol PhIP, and analyzed at 19–21 months of age. Three major induced tumors were found; lymphomas and tumors in lung and liver. The incidence of lymphoma was higher in Cyp1a2-null females than wild-type females treated with low dose (600 nmol) PhIP whereas no significant differences were observed in other treatment groups of mice. Overall differences in incidences of lung adenoma/adenocarcinoma were in general not consistent among sexes, genotypes and PhIP doses used, although reduced incidences of lung tumors were found in Cyp1a2-null males with low dose (600 nmol) and null females with high dose (1200 nmol) PhIP. Higher incidences of hepatocellular adenoma were observed in Cyp1a2-null female and male mice as compared with wild-type mice. In vitro studies using Cyp1a2-null and wild-type mouse liver microsomes revealed that CYP1A2 is the major enzyme required for PhIP N2-hydroxylation in mouse, the initial metabolic activation of PhIP that is thought to lead to tumor formation. These in vivo and in vitro results suggest that although the metabolic activation of PhIP is carried out primarily by CYP1A2, an unknown pathway unrelated to CYP1A2 appears to be responsible for PhIP carcinogenesis in mouse when examined in the neonatal bioassay. In fact, CYP1A2 may even be protective against all transformation, especially in females.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arylamines and heterocyclic amines are believed to be carcinogenic in humans (1,2). These chemicals require metabolic activation by xenobiotic-metabolizing enzymes to produce reactive electrophilic derivatives that can form DNA adducts, ultimately leading to mutagenicity and carcinogenicity. Xenobiotic-metabolizing enzymes are responsible for either mediating the toxicity of chemicals or protecting the organism by rapidly detoxifying chemicals to inert derivatives that can be eliminated (3). Cytochromes P450 (P450s) are the most well studied of the xenobiotic-metabolizing enzymes that are responsible for oxidative metabolism.

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 (10–12). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and enzymes
PhIP and N2-hydroxy-PhIP (N2-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). NADPH, 4-amidinophenylmethanesulfonyl fluoride (APMSF), acetanilide, EDTA and 60% perchloric acid were purchased from Sigma (St Louis, MO). Recombinant human CYP1A2 SupersomesTM and CYP insect cell control microsomes were bought from BD Gentest (Woburn, MA).

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 19–21 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, 4–6 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 manufacturer’s instructions.

Resolution of PhIP metabolites
Generally, incubation reactions were carried out in 100 mM potassium phosphate, pH 7.4, containing mouse liver microsomes (with 20–60 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 Michaelis–Menten 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% SDS–PAGE 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumors
The final mean body weights were not significantly different among all mouse groups killed at 19–21 months (data not shown). Tumors were found most commonly in spleen, lymph nodes, liver and lung (Table IGo). The incidences of lymphoma were higher in the Cyp1a2-null female mice than wild-type females treated with the low dose (600 nmol) PhIP. No differences, however, were observed between the high dose (1200 nmol) females or male groups treated with either high or low dose PhIP. Lymphomas were mostly follicular (primarily mixed cell type but lesser numbers of small or large cell type) and found in spleen, mesenteric lymph nodes, liver, lung and occasionally other sites. They were mostly typical of B cell lymphomas arising in spleen or mesenteric nodes of mice and in wild-type (+/+) B6;129 mice (17). A few lymphoblastic lymphomas were found in low dose null or wild-type mice.


View this table:
[in this window]
[in a new window]
 
Table I. Tumor incidences in Cyp1a2 –/– and +/+ mice treated with PhIP neonatally and analyzed at 19–21 months of age
 
The incidence of adenoma/adenocarcinoma in lung was lower in Cyp1a2-null males at low dose and Cyp1a2-null females at high dose PhIP as compared with wild-type mice treated with the same PhIP dose, although in general no consistent differences in lung tumor incidences were found between the two genotypes with either sex or PhIP dose. The lung tumors found were alveolar Type II cell neoplasms, the typical induced and spontaneous alveolar–bronchiolar lung tumor found in mice. The majority were adenomas; a few carcinomas were found. As for liver, most tumors were hepatocellular adenomas and only one carcinoma was found (in a low dose Cyp1a2-null male). The higher incidence of hepatocellular adenomas was generally found in male than female mice. Of interest was that the incidence of hepatocellular adenoma was higher in high dose Cyp1a2-null females and males as compared with wild-type females and males, respectively. Especially notable is the incidence in females, in which wild-type mice did not develop any liver tumors in both low and high dose PhIP-treated groups.

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 1Go). 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.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1. LC-ESI-MS/MS analysis of PhIP, N2-OH-PhIP, 4'-OH-PhIP and acetanilide (internal standard) standard compounds. The detection was accomplished by 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.

 
Hydroxylation of PhIP in Cyp1a2-null and wild-type mouse liver microsomes
Both the Cyp1a2-null and wild-type mouse liver microsomes produced the carcinogenic metabolite, N2-OH-PhIP, from PhIP. However, the activity was significantly decreased in microsomes prepared from Cyp1a2-null adult mice as well as pups (P < 0.0001). PhIP N2-hydroxylation activity in wild-type mouse liver microsomes was ~8-fold higher than that in Cyp1a2-null (Figure 2Go). Interestingly, <10% 4'-OH-PhIP was formed in Cyp1a2-null mouse livers as compared with wild-type (data not shown). In addition, Michaelis–Menten kinetic parameters were estimated for the formation of N2-OH-PhIP from PhIP in pooled adult mouse liver microsomes (Table IIGo). Consistently, Cyp1a2-null mice exhibited lower Vmax and relatively higher Km, resulting in much lower intrinsic clearance (~5%) compared to wild-type mice.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Relative activity of N2-OH-PhIP formation from PhIP in wild-type and Cyp1a2-null mouse liver microsomes. The P value was calculated using the unpaired t-test.

 

View this table:
[in this window]
[in a new window]
 
Table II. Michaelis–Menten kinetic parameters for PhIP N2-hydroxylation in pooled wild-type and Cyp1a2-null mouse liver microsomes
 
Analysis of CYP1A2 expression
Expression levels of CYP1A2 in Cyp1a2-null and Ahr-null mice as compared with wild-type mice were examined by western blotting with liver microsomes prepared from PhIP- or DMSO-treated pups (Figure 3Go). Ahr-null mice were used as a negative control since the AHR is required for both CYP1A1 and CYP1A2 expression. Both strains demonstrated no detectable expression of CYP1A2 as well as CYP1A1. Anti-mouse CYP1B1 antibody also did not reveal any CYP1B1 protein in these microsomes (data not shown). The anti-rat CYP2B1 and CYP2C11 antibodies were also used for western blotting and similar intensity bands were observed in all the microsomal samples (data not shown).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3. Western blotting analysis of CYP1A1 and CYP1A2 in wild-type (WT), Cyp1a2-null and Ahr-null mice administered PhIP and DMSO as the control vehicle. Recombinant mouse CYP1A1 and CYP1A2 were used as positive controls.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
By use of the Cyp1a2-null mouse line, CYP1A2 was found not to be the primary P450 responsible for 4-ABP carcinogenesis using the neonatal mouse bioassay (13). Using this model, the role of CYP1A2 in PhIP activation and tumorigenesis was evaluated. PhIP was shown to be metabolically activated primarily by CYP1A2 in rat and human livers (12). Interestingly, the human enzyme had 10- to 19-fold higher N2-hydroxylation activity than the corresponding rat P450. Treatment of neonatal mice with PhIP primarily induced three types of tumors; lymphomas, lung tumors and liver tumors. These three organs were reported to be the major sites that develop tumors in aging wild-type B6;129 mice (maximum 104–105 weeks) (17). In particular, a high incidence of lymphomas (male 42%, female 67%) was reported, but in 2-year-old mice. In the current study, a higher incidence of hepatocellular adenomas was generally found in male than female mice. The higher susceptibility of hepatocarcinogenesis in males as compared to females was well documented in mice (18,19). Further, a higher incidence of hepatocellular adenomas was observed in Cyp1a2-null female and male mice as compared with wild-type mice, but only in mice up to 21 months of age. Of interest is the data for females, where the wild-type mice developed no liver tumors with either PhIP dosage. The incidence of adenoma/adenocarcinoma in lung was lower in Cyp1a2-null males at low dose and Cyp1a2-null females at high dose PhIP as compared with wild-type mice treated with the same PhIP dose. Also, the incidence of lymphomas was higher in low dose Cyp1a2-null females than the respective wild-type group. It is uncertain why the low dose group of females developed more lymphomas than the high dose group. These results indicate that higher tumor incidences were generally found in Cyp1a2-null mice and particularly in females. The mechanism for the gender-specific response is currently unknown.

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 PhIP–DNA 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 3–5 months and PhIP–DNA 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 PhIP–DNA 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 PhIP–DNA 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.


    Notes
 
3 Present address: Department of Pathology, Nagoya City University Medical School, Nagoya 467-8601, Japan Back

4 Present address: Takeda Chemical Industries Ltd, Yodogawa-ku, Osaka 532-8686, Japan Back

5 Present address: Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06080 Badajoz, Spain Back

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 Back


    Acknowledgments
 
We would like to thank Deborah E.Devor-Henneman for mouse necropsy and Dr Elizabeth Snyderwine for critical review of the manuscript. This work was supported in part by NCI contract NO1-CO-56000 to SAIC Frederick.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Sugimura,T. (1997) Overview of carcinogenic heterocyclic amines. Mutat. Res., 376, 211–219.[ISI][Medline]
  2. Kadlubar,F.F. (1990) Carcinogenic aromatic amine metabolism and DNA adduct detection in humans. Princess Takamatsu Symp., 21, 329–338.[Medline]
  3. Guengerich,F.P. (2001) Forging the links between metabolism and carcinogenesis. Mutat. Res., 488, 195–209.[ISI][Medline]
  4. Whitlock,J.P.Jr (1999) Induction of cytochrome P4501A1. Annu. Rev. Pharmacol. Toxicol., 39, 103–125.[CrossRef][ISI][Medline]
  5. Safe,S. (2001) Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol. Lett., 120, 1–7.[CrossRef][ISI][Medline]
  6. Nakajima,M., Yokoi,T., Mizutani,M., Shin,S., Kadlubar,F.F. and Kamataki,T. (1994) Phenotyping of CYP1A2 in Japanese population by analysis of caffeine urinary metabolites: absence of mutation prescribing the phenotype in the CYP1A2 gene. Cancer Epidemiol. Biomarkers Prev., 3, 413–421.[Abstract]
  7. Lang,N.P., Butler,M.A., Massengill,J., Lawson,M., Stotts,R.C., Hauer-Jensen,M. and Kadlubar,F.F. (1994) Rapid metabolic phenotypes for acetyltransferase and cytochrome P4501A2 and putative exposure to food-borne heterocyclic amines increase the risk for colorectal cancer or polyps. Cancer Epidemiol. Biomarkers Prev., 3, 675–682.[Abstract]
  8. Le Marchand,L., Hankin,J.H., Wilkens,L.R. et al. (2001) Combined effects of well-done red meat, smoking and rapid N-acetyltransferase 2 and CYP1A2 phenotypes in increasing colorectal cancer risk. Cancer Epidemiol. Biomarkers Prev., 10, 1259–1266.[Abstract/Free Full Text]
  9. Sugimura,T. (2000) Nutrition and dietary carcinogens. Carcinogenesis, 21, 387–395.[Abstract/Free Full Text]
  10. Kato,R., Kamataki,T. and Yamazoe,Y. (1983) N-hydroxylation of carcinogenic and mutagenic aromatic amines. Environ. Health Perspect., 49, 21–25.[ISI][Medline]
  11. Butler,M.A., Iwasaki,M., Guengerich,F.P. and Kadlubar,F.F. (1989) Human cytochrome P-450PA (P-450IA2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines. Proc. Natl Acad. Sci. USA, 86, 7696–7700.[Abstract]
  12. Turesky,R.J., Constable,A., Fay,L.B. and Guengerich,F.P. (1999) Interspecies differences in metabolism of heterocyclic aromatic amines by rat and human P450 1A2. Cancer Lett., 143, 109–112.[CrossRef][ISI][Medline]
  13. Kimura,S., Kawabe,M., Ward,J.M., Morishima,H., Kadlubar,F.F., Hammons,G.J., Fernandez-Salguero,P. and Gonzalez,F.J. (1999) CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice. Carcinogenesis, 20, 1825–1830.[Abstract/Free Full Text]
  14. Dooley,K.L., Von Tungeln,L.S., Bucci,T., Fu,P.P. and Kadlubar,F.F. (1992) Comparative carcinogenicity of 4-aminobiphenyl and the food pyrolysates, Glu-P-1, IQ, PhIP and MeIQx in the neonatal B6C3F1 male mouse. Cancer Lett., 62, 205–209.[ISI][Medline]
  15. Buters,J.T., Tang,B.K., Pineau,T., Gelboin,H.V., Kimura,S. and Gonzalez,F.J. (1996) Role of CYP1A2 in caffeine pharmacokinetics and metabolism: studies using mice deficient in CYP1A2. Pharmacogenetics, 6, 291–296.[ISI][Medline]
  16. Fernandez-Salguero,P., Pineau,T., Hilbert,D.M., McPhail,T., Lee,S.S., Kimura,S., Nebert,D.W., Rudikoff,S., Ward,J.M. and Gonzalez,F.J. (1995) Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science, 268, 722–726.[ISI][Medline]
  17. Haines,D.C., Chattopadhyay,S. and Ward,J.M. (2001) Pathology of aging B6;129 mice. Toxicol. Pathol., 29, 653–661.[CrossRef][ISI][Medline]
  18. Ward,J.M., Shibata,M. and Devor,D.E. (1996) Emerging issues in mouse liver carcinogenesis. Toxicol. Pathol., 24, 129–137.[ISI][Medline]
  19. Kemp,C.I. and Drinkwater,N.R. (1990) The androgen receptor and liver tumor development in mice. In Stevenson,D.E., Popp,J.A., Ward,J.M., McClain,R.M., Slaga,T.J. and Pitot,H.C. (eds) Mouse Liver Carcinogenesis. Mechanisms and Species Comparisons. Wiley-Liss, New York, NY, Vol. 331, pp. 203–214.
  20. Degawa,M., Kobayashi,K., Miura,S., Arai,H., Esumi,H., Sugimura,T. and Hashimoto,Y. (1992) Species difference among experimental rodents in induction of P450IA family enzymes by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Jpn. J. Cancer Res., 83, 1047–1051.[ISI][Medline]
  21. Snyderwine,E.G., Yu,M., Schut,H.A.J., Knight-Jones,L. and Kimura,S. (2002) Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice. Food Chem. Toxicol., 40, 1529–1533.[CrossRef][ISI][Medline]
  22. Howie,A.F., Forrester,L.M., Glancey,M.J., Schlager,J.J., Powis,G., Beckett,G.J., Hayes,J.D. and Wolf,C.R. (1990) Glutathione S-transferase and glutathione peroxidase expression in normal and tumour human tissues. Carcinogenesis, 11, 451–458.[Abstract]
Received December 5, 2002; accepted December 15, 2002.