Mouse lung CYP1A1 catalyzes the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis is initiated by N(2)-hydroxylation, mediated by several cytochromes P450, including CYP1A1. However, the role of CYP1A1 in PhIP metabolic activation in vivo is unclear. In this study, Cyp1a1-null and wild-type (WT) mice were used to investigate the potential role of CYP1A1 in PhIP metabolic activation in vivo. PhIP N(2)-hydroxylation was actively catalyzed by lung homogenates of WT mice, at a rate of 14.9 +/- 5.0 pmol/min/g tissue, but <1 pmol/min/g tissue in stomach and small intestine, and almost undetectable in mammary gland and colon. PhIP N(2)-hydroxylation catalyzed by lung homogenates of Cyp1a1-null mice was approximately 10-fold lower than that of WT mice. In contrast, PhIP N(2)-hydroxylation activity in lung homogenates of Cyp1a2-null versus WT mice was not decreased. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased lung Cyp1a1 mRNA and lung homogenate PhIP N(2)-hydroxylase activity approximately 50-fold in WT mice, where the activity was substantially inhibited (70%) by monoclonal antibodies against CYP1A1. In vivo, 30 min after oral treatment with PhIP, PhIP levels in lung were similar to those in liver. After a single dose of 0.1 mg/kg [(14)C]PhIP, lung PhIP-DNA adduct levels in Cyp1a1-null mice, but not in Cyp1a2-null mice, were significantly lower (P = 0.0028) than in WT mice. These results reveal that mouse lung has basal and inducible PhIP N(2)-hydroxylase activity predominantly catalyzed by CYP1A1. Because of the high inducibility of human CYP1A1, especially in cigarette smokers, the role of lung CYP1A1 in PhIP carcinogenesis should be considered. (237 words).     

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

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 N(2)-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 approximately 11 (low dose) and approximately 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.     

Colonic adenocarcinomas rapidly induced by the combined treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and dextran sodium sulfate in male ICR mice possess beta-catenin gene mutations and increases immunoreactivity for beta-catenin, cyclooxygenase-2 and inducible nitric oxide synthase

Heterocyclic amines are known to be important environmental carcinogens in several organs including the colon. The aim of this study was to induce colonic epithelial malignancies within a short-term period and analyze the expression of cycooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and beta-catenin, and mutations of beta-catenin gene in induced tumors. Male Crj: CD-1 mice were given a single i.g. administration (200 mg/kg body wt) of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) followed by 2% dextran sodium sulfate (DSS) in the drinking water for a week. The expression of beta-catenin, COX-2 and iNOS was immunohistochemically assessed in colonic epithelial lesions and the beta-catenin gene mutations in colonic adenocarcinomas induced were analyzed by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. At week 16, a high incidence of colonic neoplasms with dysplastic lesions developed in mice that received PhIP and DSS, but only a few developed in those given MeIQx and DSS. Immunohistochemically, the adenocarcinomas induced were all positive for three proteins. All seven adenocarcinomas induced by PhIP and DSS have mutations. The findings suggest that DSS exerts powerful tumor-promoting effects on PhIP-initiated colon carcinogenesis in mice and this mouse model is useful for investigating environment-related colon carcinogenesis within a short-term period.     

Dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced prostate carcinogenesis in CYP1A-humanized mice

To develop a relevant mouse model for prostate cancer prevention research, we administered a dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to CYP1A-humanized mice. In comparison with mouse Cyp1a2, human CYP1A2 preferentially activates PhIP to a proximate carcinogen. Following a single oral dose of PhIP (200 mg/kg body weight), we observed inflammation, atrophy of acini, low-grade prostatic intraepithelial neoplasia (PIN; after 20 weeks), and high-grade PIN (HgPIN; after 30 to 50 weeks) in dorsolateral, ventral, and coagulating anterior prostate glands of these mice. These lesions were androgen receptor positive and featured the loss of expression of the basal cell marker p63 and the tumor suppressor PTEN. Similar to human prostate carcinogenesis, glutathione S-transferase P1 (GSTP1) expression was lost or partially lost in HgPIN. E-Cadherin expression was also lost in HgPIN. The expression of DNA methyltransferase 1 was elevated, possibly to enhance promoter hypermethylation for the silencing of GSTP1 and E-cadherin. Prostate carcinogenesis was promoted by a high-fat stress diet, resulting in HgPIN that developed earlier and in advanced lesions displayed features consistent with carcinoma in situ. This dietary carcinogen-induced prostate cancer model, recapitulating important features of early human prostate carcinogenesis, constitutes a new experimental system for prostate cancer research.     

大白菜抑制大鼠体内致癌物PhIP-DNA加合物形成及其可能的作用机理

目的研究大白菜（ｂｒａｓｓｉｃａｃｈｉｎｅｎｓｉｓ）对结肠致癌物２－氨基－１－甲基－６－苯基咪唑［４,５－ｂ］吡啶（ＰｈＩＰ）致癌的预防作用及机理。方法雄性ＳＤ大鼠喂饲基础饲料或掺有大白菜粉（２０％）混合饲料１０天后,经口摄入ＰｈＩＰ（１０ｍｇ／ｋｇ）。以３２Ｐ－后标记方法分析动物结肠粘膜、心、肺和肝中ＰｈＩＰ－ＤＮＡ加合物含量,并测定参与ＰｈＩＰ代谢的细胞色素Ｐ４５０（ＣＹＰ）１Ａ１和１Ａ２以及谷胱甘肽转硫酶（ＧＳＴ）活性。结果经喂饲含大白菜饲料的大鼠,其结肠、心、肺、肝等器官中,ＰｈＩＰ－ＤＮＡ加合物含量显著低于喂饲基础饲料的动物（Ｐ＜０．０１）,抑制率结肠为８２．３％、心脏为６０．６％、肺为４８．４％、肝脏为４８．９％。大白菜对参与ＰｈＩＰ解毒的ＣＹＰ１Ａ１和ＧＳＴ有显著的诱导作用。与对照组比较,喂饲大白菜大鼠的肝ＣＹＰ１Ａ１活性增加８０．６％（Ｐ＜０．０１）,肝和肺细胞浆ＧＳＴ活性分别增加１８．２％和３５．６％（Ｐ＜０．０５）。结论食用大白菜可有效抑制ＰｈＩＰ－ＤＮＡ加合物形成,其作用机理可能是诱导解毒酶     

The urinary metabolite profile of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is predictive of colon DNA adducts after a low-dose exposure in humans

Epidemiologic evidence indicates that exposure to heterocyclic amines in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of heterocyclic amines, which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of heterocyclic amine bioactivation in humans, the most mass abundant heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of [(14)C]PhIP 48 to 72 hours before surgery to remove colon tumors. Urine was collected for 24 hours after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All 10 subjects were phenotyped for cytochrome P4501A2 (CYP1A2), N-acetyltransferase 2, and sulfotransferase 1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N(2)-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N(2)-glucuronide had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N(2)-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.     

A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate

To develop an efficient animal model for colitis-related carcinogenesis, male Crj: CD-1 (ICR) mice were given a single intraperitoneal administration (10 mg/kg body weight) of a genotoxic colonic carcinogen, azoxymethane (AOM), and a 1-week oral exposure (2% in drinking water) to a non-genotoxic carcinogen, dextran sodium sulfate (DSS), under various protocols. At week 20, colonic neoplasms (adenocarcinomas, 100% incidence with 5.60±2.42 multiplicity; and adenomas, 38% incidence with 0.20±0.40 multiplicity) with dysplastic lesions developed in mice treated with AOM followed by DSS. Protocols in which AOM was given during or after DSS administration induced a few tubular adenomas or no tumors in the colon. Immunohistochemical investigation of such dysplasias and neoplasms revealed that all lesions were positive for β-catenin, cyclooxygenase-2 and inducible nitric oxide synthase, but did not show p53 immunoreactivity. The results indicate that 1-week administration of 2% DSS after initiation with a low dose of AOM exerts a powerful tumor-promoting activity in colon carcinogenesis in male ICR mice, and may provide a novel mouse model for investigating colitis-related colon carcinogenesis and for identifying xenobiotics with modifying effects.     

beta-catenin is strongly elevated in rat colonic epithelium following short-term intermittent treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and a high-fat diet

Colon tumors expressing high levels of β-catenin and c-myc have been reported in male F344 rats given three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) alternating with a high-fat (HF) diet. Using the same experimental protocol, rats were euthanized 24 h after the last dose of PhIP so as to examine early changes in colonic crypt homeostasis and β-catenin expression, before the onset of frank tumors. PhIP/HF dosing caused a significant increase in the bromodeoxyuridine labeling index throughout the entire colon, and within the colonic crypt column cleaved caspase-3 was elevated in the basal and central zones, but reduced in the luminal region. In vehicle/HF controls, β-catenin was immunolocalized primarily at the border between cells at the top of the crypt, whereas in rats given PhIP/HF diet there was strong cytoplasmic staining, which appeared as a gradient of increased β-catenin extending from the base of the crypt column to the luminal region. Quantitative real-time PCR and immunoblot analyses confirmed that β-catenin and c-myc were increased significantly in the colonic mucosa of rats given PhIP/HF diet. Collectively, these findings suggest that PhIP/HF cycling alters β-catenin and c-myc expression in the colonic mucosa, resulting in expansion of the proliferative zone and redistribution of apoptotic cells from the lumen to the central and basal regions of the colonic crypt. Thus, during the early stages of colon carcinogenesis, alternating exposure to heterocyclic amines and a high-fat diet might facilitate molecular changes resulting in dysregulated β-catenin and c-myc expression. ( Cancer Sci 2008; 99: 1754–1759)     

Mechanistic approach of contrasting modifying effects of caffeine on carcinogenesis in the rat colon and mammary gland induced with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Caffeine exerts potent chemopreventive action against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland carcinogenesis, but acts as a co-carcinogen in the colon. The present work was performed to clarify mechanisms underling these organ dependent actions. Female F344 rats were given PhIP and caffeine, PhIP alone, caffeine alone or no treatment for 4 weeks. PhIP-DNA adduct formation in the colon was significantly higher in the PhIP+caffeine than in the PhIP group, but levels in the mammary glands showed no inter-group differences. CYP1A2 mRNA expression in the livers of the PhIP+caffeine group tended to be higher than in either the PhIP or the caffeine alone groups. High mRNA expression for both N-acetyltransferase (NAT) 1 and NAT2 was observed in the colon, with less expression in the mammary gland. The levels of four DNA-repair enzymes were not influenced by the caffeine treatment. In conclusion, only increased level of DNA adducts in the colon partially related to the modifying effects of caffeine on PhIP-induced rat carcinogenesis. Thus, other unknown factors must be contributory.     

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human cytochrome P4501B1

Cytochrome P4501B1 (CYP1B1) is the most recently identified member of the dioxin-inducible CYP1 family. CYP1B1 is constitutively expressed in most human tissues, including colon and breast, and can activate numerous chemically diverse carcinogens. We evaluated the metabolism of the dietary heterocyclic amine carcinogen 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP) by microsomes from yeast expressing the human CYP1B1 protein. PhIP metabolites were analysed by HPLC with fluorescence and absorbance detection. We found that human CYP1B1 metabolizes PhIP to three products: N2-OH-PhIP, a mutagenic activation product; 4'-OH-PhIP, a detoxification product; and 2-OH-PhIP, the mutagenic potential of which is unknown. Metabolite identity was confirmed by co-elution with authentic standards and synchronous fluorescence spectroscopy. The identity of the 2-OH-PhIP standard was additionally confirmed by mass spectrometry. Kinetic studies of the formation of N2-OH-PhIP, 4'-OH-PhIP and 2-OH-PhIP by CYP1B1 indicated apparent Km values of 5.7 +/- 1.3, 2.2 +/- 0.5 and 1.3 +/- 0.2 microM, respectively. Apparent turnover rates were 0.40 +/- 0.03, 0.93 +/- 0.02 and 0.04 +/- 0.00 nmol product/min nmol P450, respectively. At saturating levels of substrate, CYP1B1-mediated formation of the non- mutagenic metabolite 4'-OH-PhIP was favored two-fold over that of the mutagenic metabolite, N2-OH-PhIP and >10-fold over that of 2-OH-PhIP. The formation of N2-OH-PhIP, a potent mutagen implicated in the etiology of human colon and breast cancer, indicates that CYP1B1 may play an important role in PhIP-mediated carcinogenesis.      

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human cytochrome P4501A1, P4501A2 and P4501B1

While the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) by N-hydroxylation has been well documented, the relative roles of the human cytochrome P450 (CYP) enzymes that catalyze this reaction have not been established. Previous studies indicated that the mutagenic activation product, 2-hydroxyamino-PhIP (N2-OH- PhIP), is produced primarily by CYP1A2, and to a lesser extent by CYP1A1. We recently reported that human CYP1B1 also produces N2-OH-PhIP (Carcinogenesis, 18, 1793-1798, 1997). In the present study, we examined PhIP metabolism by microsomes containing recombinant human CYP1A1, 1A2 or 1B1 expressed in Sf9 insect cells and compared the kinetic values for PhIP metabolite formation. PhIP metabolites were analyzed by high pressure liquid chromatography with fluorescence and absorbance detection. Vmax values for N2-OH-PhIP formation were 90, 16 and 0.2 nmol/min/nmol P450, and the apparent Km values were 79, 5.1 and 4.5 microM for human CYP1A2, 1A1 and 1B1, respectively. The non- mutagenic metabolite, 4'-hydroxy-PhIP, was also formed by all three CYP enzymes with Vmax values of 1.5, 7.8 and 0.3 nmol/ min/nmol P450 and apparent Km values of 43, 8.2 and 2.2 microM for human CYP1A2, 1A1 and 1B1, respectively. Although the Vmax for N2-OH-PhIP production was highest for CYP1A2, the catalytic efficiency (Vmax/Km) of CYP1A1 was greater than that of CYP1A2. These results suggest that, for humans, extrahepatic CYP1A1 may be more important than previously thought for the metabolic activation of the dietary carcinogen PhIP.      

Enhanced colitis-associated colon carcinogenesis in a novel Apc mutant rat

To establish an efficient rat model for colitis-associated colorectal cancer, azoxymethane and dextran sodium sulfate (AOM/DSS)-induced colon carcinogenesis was applied to a novel adenomatous polyposis coli ( Apc ) mutant, the Kyoto Apc Delta (KAD) rat. The KAD rat was derived from ethylnitrosourea mutagenesis and harbors a nonsense mutation in the Apc gene (S2523X). The truncated APC of the KAD rat was deduced to lack part of the basic domain, an EB1-binding domain, and a PDZ domain, but retained an intact β-catenin binding region. KAD rats, homozygous for the Apc mutation on a genetic background of the F344 rat, showed no spontaneous tumors in the gastrointestinal tract. At 5 weeks of age, male KAD rats were given a single subcutaneous administration of AOM (20 mg/kg, bodyweight). One week later, they were given DSS (2% in drinking water) for 1 week. At week 15, the incidence and multiplicity of colon tumors developed in the KAD rat were remarkably severe compared with those in the F344 rat: 100 versus 50% in incidence and 10.7 ± 3.5 versus 0.8 ± 1.0 in multiplicity. KAD tumors were dominantly distributed in the rectum and distal colon, resembling human colorectal cancer. Accumulation of β-catenin protein and frequent β-catenin mutations were prominent features of KAD colon tumors. To our knowledge, AOM/DSS-induced colon carcinogenesis using the KAD rat is the most efficient to induce colon tumors in the rat, and therefore would be available as an excellent model for human colitis-associated CRC.     

Distribution of preneoplastic lesions and tumors, and beta-catenin gene mutations in colon carcinomas induced by 1,2-dimethylhydrazine plus dextran sulfate sodium

Mucin-depleted foci (MDF) are considered as useful biomarkers in rat colon carcinogenesis. The purpose of the present study was to examine the mechanism(s) underlying rat colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus 1% Dextran Sulfate Sodium (DSS). Twelve male F344 rats were given subcutaneous injections (40mg/kg body) of DMH twice a week. They received DSS in the drinking water for 1 week after the first injection of DMH and then were maintained on tap water. The rats were sacrificed at 10 and 14 weeks after the first injection of DMH. Colon tissues were divided into 10 segments from anus to cecum (A/J) and stained with Alcian blue (AB) to identify MDF. We found that MDF and tumors were induced in the rat colon after treatment with DMH plus DSS and that the number of MDF in each segment of the colon was significantly correlated with that of tumors (p=0.006). In addition, we found that the beta-catenin protein was accumulated in cytoplasm and nuclei of MDF and the frequent beta-catenin gene mutations in the colon tumors. These results suggest that MDF is closely related to rat colon carcinogenesis induced by DMH plus DSS.     

人细胞色素P4502E1基因在胚胎鼻咽组织、鼻咽癌细胞和组织中的表达

背景与目的有研究表明,亚硝胺类化学物质能诱导体内外鼻咽细胞癌变,但作为间接致癌物,其活化主要依赖细胞色素P4502E1(CYP2E1)的代谢.本研究旨在探讨人细胞色素P4502E1(hCYP2E1)基因在化学物质二亚硝基哌嗪(N,N-dinitrosopiperazine,DNP)致鼻咽癌变中的可能作用,为鼻咽癌病因和发病学机制提供新的证据.方法采用RTPCR方法检测8例胚胎鼻咽组织,10例鼻咽癌活检组织,5株鼻咽癌细胞系(CNE1,CNE2,HNE1,HNE2,HNE3)和1株体外恶性转化的鼻咽细胞系(7429)CYP2E1mRNA水平的表达.用不同浓度(200,250,300μg/ml)化学致癌物DNP作用于体外培养的正常鼻咽细胞后,也用RTPCR检测其CYP2E1mRNA表达.结果(1)100%胚胎鼻咽组织(8/8)和鼻咽癌细胞系(6/6,含7429细胞系)、80%(8/10)的鼻咽癌活检组织均存在CYP2E1的表达;(2)经DNP处理的胚胎鼻咽细胞CYP2E1表达增高.结论鼻咽部存在可诱导的CYP2E1基因表达,提示该基因在亚硝胺类间接致癌物(如DNP)致鼻咽癌变过程中可能发挥重要作用.     

Heterocyclic amine mixture carcinogenesis and its enhancement by caffeine in F344 rats.

In order to elucidate whether mixed exposure to environmental carcinogens and caffeine increases the risk of cancer induction, we investigated the relationship between preneoplastic lesion development in the liver and colon and drug metabolizing enzyme induction and DNA adduct formation, in rats treated with a mixture of heterocyclic amines (HCAs) and caffeine. In Experiment 1, male F344 rats were administered 3 different HCAs, the food carcinogens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), alone or in combinations of 2 or 3 at 50 ppm in the diet for 16 weeks. The numbers of hepatic glutathione-S-transferase P form positive (GST-P+) foci and colonic aberrant crypt foci (ACF) were greater in the IQ + MeIQx group than expected from simple summation and increased levels of HCA-DNA adducts were noted. However, no summation was obtained when combined with PhIP, which rather caused inhibition. In Experiment 2, the effects of concurrent caffeine administration on the PhIP carcinogenicity were assessed. Caffeine at 1000 and 500 ppm in the drinking water for 2 weeks significantly increased levels of CYP1A2. Ten weeks concurrent administration of caffeine (1000 ppm) and PhIP (400 ppm) resulted in significant increase of colon ACFs and CYP1A2 expression. Thus, concurrent administration of IQ and MeIQx caused elevation of their carcinogenicity but other mixtures with PhIP did not enhance carcinogenicity. However, a non-carcinogen, caffeine, enhanced PhIP colon carcinogenesis, possibly due to induction of CYP1A2.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine induces a higher number of aberrant crypt foci in Fischer 344 (rapid) than in Wistar Kyoto (slow) acetylator inbred rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine carcinogen in the human diet and is a colon carcinogen in the rat. N-Acetyltransferase-2 (NAT2) catalyzes the conversion of PhIP and other heterocyclic amines to a DNA-reactive form. NAT2 has a polymorphic distribution in humans and other mammals, including rats. The rapid NAT2 genotype has been shown to be associated with increased colorectal cancer risk in some, but not all, human epidemiological studies. This investigation was designed to study the role of acetylator genotype in PhIP-induced colon carcinogenesis using aberrant crypt foci (ACF) as an intermediate biomarker. Five-week-old male, rapid-acetylator Fischer 344 (F344) rats and slow-acetylator Wistar-Kyoto (WKY) rats were fed the semipurified AIN76A diet with 0.01% PhIP, 0.04% PhIP, or no PhIP (control) for 8 weeks. PhIP induced ACF in both rapid- and slow-acetylator rats; 0.04% PhIP induced more ACF than 0.01% PhIP. There was no difference in the number of ACF between rapid- and slow-acetylator rats that were fed 0.01% PhIP. However, 0.04% PhIP induced 2-fold higher ACF and a greater dose-dependent increase in PhIP-induced ACF in the rapid-acetylator F344 rats compared with the slow-acetylator WKY rats. The results support human epidemiological studies showing higher risk for colorectal cancer in rapid acetylators who frequently consume meat that is very well done.     

The cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates the extracellular signal regulated kinase mitogen-activated protein kinase pathway

During the cooking of meat, mutagenic and carcinogenic heterocyclic amines are formed, the most abundant of which, 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP), induces tumors of the prostate, colon, and mammary gland in rats. Humans consuming cooked meat are exposed to PhIP on a daily basis, yet few studies have assessed the effects of PhIP at dietary relevant concentrations. In addition to its genotoxic properties, recent studies have shown that PhIP can activate estrogen receptor-mediated signaling pathways at doses that are similar to those that may be present in the body following consumption of a cooked meat meal. In the present study, we examined whether such doses of PhIP can affect estrogen receptor-independent signal transduction via the mitogen-activated protein kinase (MAPK) extracellular signal-related kinase (ERK) pathway to influence proliferation and migration in the human mammary epithelial cell line MCF10A and the prostate cancer cell line PC-3. At doses shown to have a proliferative effect on MCF10A cells (10(-11)-10(-7) mol/L), PhIP induced a rapid, transient increase in phosphorylation of both MAPK/ERK kinase 1/2 and ERKs. Inhibition of this pathway significantly reduced the PhIP-induced proliferation of MCF10A cells and the migration of PC-3 cells. The data presented here show that levels of PhIP that approximate to human dietary exposure stimulate cellular signaling pathways and result in increased growth and migration, processes linked to the promotion and progression of neoplastic disease. These findings provide strong evidence that PhIP acts as a tumor initiator and promoter and that dietary exposure to this compound could contribute to carcinogenesis in humans.     

Msh2 DNA mismatch repair gene deficiency and the food-borne mutagen 2-amino-1-methy1-6-phenolimidazo [4,5-b] pyridine (PhIP) synergistically affect mutagenesis in mouse colon

Msh2 deficiency and food-borne carcinogen PhIP have been implicated as genetic and environmental factors, respectively, in human colon carcinogenesis. It is not clear whether loss of one or both alleles of Msh2 gene increases the mutational sensitivity in colon when exposed to environmental carcinogens. In the current study, Msh2(+/-)/lacI and Msh2(-/-)/lacI double transgenic mice were treated with PhIP and mutations in the lacI gene were studied in the colon. The spontaneous mutation frequency (MF) is approximately eightfold higher in Msh2(-/-) mice than in Msh2(+/+) mice, while Msh2(+/-) mice display similar levels of spontaneous mutation as the Msh2 wild type mice. PhIP induced a significant increase in MF in all genotypes of mice. However, induced MF is much higher in Msh2(-/-) mice compared to Msh2(+/+) and Msh2(+/-) mice. Msh2(+/-) mice displayed an increased level of G:C>T:A transversions and -1 frameshifts upon PhIP treatment. In contrast, loss of both Msh2 alleles mainly results in increased frequency of G:C>A:T transitions when exposed to PhIP. These results suggest that a defect in mismatch repair may result in an enhanced sensitivity from exposure to a dietary carcinogen. It also provides insight into interaction between genetic and environmental factors in human carcinogenesis.     

Reduction in formation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP)-induced aberrant crypt foci in the rat colon by docosahexaenoic acid (DHA)

Docosahexaenoic acid (DHA), a major component of fish oil, suppresses the formation and growth of aberrant crypt foci induced by 1,2- dimethylhydrazine and azoxymethane. In the present study we examined the effects of intragastric gavage administration of DHA on the yield of rat colonic aberrant crypt foci due to treatment with a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induces colon cancer in male F344 rats and is considered to be a possible human colon carcinogen. Male F344 rats were given a standard diet (AIN-76A) and received 10 doses of PhIP (75 mg/kg body wt, by intragastric intubation, on days 1-5 and 8-12) with or without intragastric application of 1 ml DHA 4 h prior to each carcinogen treatment, followed by further DHA dosing. The numbers of PhIP-induced aberrant crypt foci per colon after 4 and 12 weeks DHA administration were significantly reduced to 47 and 38% respectively of the values obtained when PhIP alone was used. The mean number of aberrant crypts per focus was also decreased by DHA treatment. At week 4 the PhIP-DNA adduct levels in the colon of rats from the PhIP+DHA group were approximately two thirds of the PhIP group value. The results thus suggest that DHA exerts a preventive effect on PhIP-induced colon carcinogenesis.