DNA adduct levels and intestinal lesions in congenic rapid and slow acetylator syrian hamsters admi food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)

Epidemiological studies indicate that rapid acetylators with a high intake of well-done red meat have an increased risk of colorectal cancer. Arylamine N-acetyltransferase enzymes (E.C. 2.3.1.5) activate carcinogenic heterocyclic amines found in the crust of fried meat via O-acetylation of their N-hydroxylamines to reactive intermediates that bind covalently to DNA and produce mutations. Syrian hamsters as well as humans express two N-acetyltransferase isozymes (NAT1 and NAT2) which differ in substrate specificity and genetic control. Nucleic acid substitutions in the NAT2 gene segregate individuals into rapid, intermediate and slow acetylator phenotypes. In the present paper, we examined the role of the polymorphic NAT2 acetylator genotype in carcinogenesis induced by the food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by comparing Syrian hamster lines congenic at the NAT2 locus. No differences were found between rapid and slow acetylator congenic hamsters in levels of intestinal PhIP-DNA adducts. In contrast to previous studies in rats, no carcinogen-related induction of the preneoplastic lesions aberrant crypt foci or tumors was found in the intestines of rapid and slow acetylator congenic Syrian hamsters administered PhIP or IQ.     

Effect in vitro of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the hepatic activation of dietary genotoxins by rat post-mitochondrial fractions.

The effect of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the conversion of the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay. The assay used Salmonella typhimurium TA98 as an indicator of the mutagenicity and hepatic post-mitochondrial fractions (S-9) from male Sprague-Dawley rats as the activating system. All three fatty acids inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA repair processes within the bacterial cell. The activation of three other food mutagens, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and aflatoxin B1 (AFB1) was also inhibited by these fatty acids.     

Effects of soy sauce and sugar on the formation of heterocyclic amines in marinated foods.

The effects of soy sauce and sugar on the formation of heterocyclic amines (HAs) in marinated pork, eggs, and bean cakes were studied. Food samples were immersed in water in the presence of various levels of soy sauce and sugar, and the mixtures were subjected to simmering at 98+/-2 degrees C for 1 h in a closed saucepan. The various HAs in marinated food samples were analyzed by HPLC with photodiode-array detection. Results showed that seven HAs: 2-amino-3-methylimidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx); 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1); 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine (PhIP); and 2-amino-9H-pyrido[2,3,-b]indole (AalphaC) were detected in marinated pork, while five HAs: IQ, MeIQx; 4,8-DiMeIQx; PhIP; and AalphaC in bean cakes, as well as four HAs, MeIQx, 4,8-DiMeIQx, Trp-P-1 and PhIP in eggs. In most samples PhIP was formed in largest amount, followed by MeIQx, 4,8-DiMeIQx, IQ, AalphaC, Trp-P-1 and MeIQ. The amounts of HAs produced in marinated food samples followed an increased order for each increasing level of soy sauce or sugar. Marinated juice was found to contain a higher content of HAs than marinated foods.     

Formation of a mutagenic heterocyclic aromatic amine from creatinine in urine of meat eaters and vegetarians

Liquid chromatography electrospray ionization mass spectrometry (MS) with a triple quadrupole MS was used to identify known and novel heterocyclic aromatic amines (HAAs) in human urine. The identities of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were confirmed by their product ion spectra. The constant neutral loss scan mode was employed to probe for other analytes in urine that display the transition [M+H]+-->[M+H-CH3*]+*, which is common to HAAs containing an N-methylimidazo moiety, and led to the detection of a previously unreported isomer of 8-MeIQx [Holland, R., et al. (2004) Chem. Res. Toxicol. 17, 1121-1136]. We now report the identification of another novel HAA, 2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]), an isomer of the powerful animal carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The amounts of IQ[4,5-b] measured in the urine of human volunteers who consumed grilled beef ranged from 15 to 135% of the ingested dose, while the amounts of 8-MeIQx and PhIP excreted in urine were on average <2% of the ingested dose. Base treatment of urine at 70 degrees C increased the concentrations of 8-MeIQx and PhIP by as much as 6-fold, indicating the presence of phase II conjugates; however, the amount of IQ[4,5-b] increased by more than 100-fold. IQ[4,5-b] was also detected in the urine of vegetarians following base hydrolysis. The formation of IQ[4,5-b], but not IQ, 8-MeIQx, or PhIP, also occurred in urine incubated at 37 degrees C. Creatinine and 2-aminobenzaldehyde are likely precursors of IQ[4,5-b]. The detection of IQ[4,5-b] in the urine of both meat eaters and vegetarians suggests that this HAA may be present in nonmeat staples or that IQ[4,5-b] formation may occur endogenously within the urinary bladder or other biological fluids.     

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of protein-rich foods. HCA residues adducted to blood proteins have been postulated as biomarkers of HCA exposure. However, the viability of quantifying HCAs following hydrolytic release from adducts in vivo and correlation with dietary intake are unproven. To definitively assess the potential of labile HCA-protein adducts as biomarkers, a highly sensitive UPLC-MS/MS method was validated for four major HCAs: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Limits of detection were 1–5 pg/ml plasma and recoveries 91–115%. Efficacy of hydrolysis was demonstrated by HCA-protein adducts synthesised in vitro. Plasma and 7-day food diaries were collected from 122 fasting adults consuming their habitual diets. Estimated HCA intakes ranged from 0 to 2.5 mg/day. An extensive range of hydrolysis conditions was examined for release of adducted HCAs in plasma. HCA was detected in only one sample (PhIP, 9.7 pg/ml), demonstrating conclusively for the first time that acid-labile HCA adducts do not reflect dietary HCA intake and are present at such low concentrations that they are not feasible biomarkers of exposure. Identification of biomarkers remains important. The search should concentrate on stabilised HCA-peptide markers and use of untargeted proteomic and metabolomic approaches.     

Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells

Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24 h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.     

A comparison of whole wheat, refined wheat and wheat bran as inhibitors of heterocyclic amines in the Salmonella mutagenicity assay and in the rat colonic aberrant crypt focus assay.

Refined wheat, unrefined whole wheat, and wheat bran were studied for their ability to protect against heterocyclic amines (HCAs) in vitro and in vivo. Wheat bran, which binds HCAs in vitro, as well as refined wheat and unrefined whole wheat, inhibited the mutagenic activities of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) when they were co-incubated and the supernatant (minus grain) was added to the Salmonella assay. The water-soluble fraction alone from refined and unrefined wheat, but not bran, also inhibited against these mutagens in vitro. In vivo, AIN-93G diets containing refined wheat or unrefined wheat were examined for their ability to inhibit IQ-induced colonic aberrant crypt foci (ACF) in the Fischer 344 rat. A slight increase in the number of AC/ACF (aberrant crypts/ACF) was seen after 16 weeks in rats treated post-initiation with refined wheat (P < 0.05), and fewer foci with two or three aberrant crypts (ACF-2) were found in rats given unrefined whole wheat post-initiation compared with animals treated with the same diet during the initiation phase (P < 0.05). There was no significant difference in the profile of IQ urinary metabolites or excretion of promutagens 0-48 h after carcinogen dosing, and grains had no effect on hepatic cytochrome P4501A1 (CYP1A1), CYP1A2, aryl sulfotransferase or N-acetyltransferase activities; however, a slightly higher UDP-glucuronosyl transferase activity was observed in rats fed unrefined wheat compared with refined wheat diets (P < 0.05). Thus, despite their antimutagenic activities in vitro, only marginal effects were seen with refined and unrefined wheat in vivo with respect to hepatic enzyme activities, carcinogen metabolism and IQ-induced ACF in the rat colon.     

Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice.

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20-30% of the levels in WT mice, a difference that was statistically significant (Student's t-test, P < 0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6+/-0.6 vs 4.6+/-0.7, respectively, relative adduct labeling (RAL) x 10(8), mean+/-S.E.M., n = 3-5 mice). In the mammary gland, however, there was no difference in IQ-DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP-DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP-DNA adducts in liver and kidney of WT mice were 9.9+/-1.1 and 22.5+/-6.9, respectively, whereas no PhIP-DNA adducts were detected in either organ of KO mice (limit of detection, 1.4-2.8 x 10(9)). PhIP-DNA adduct levels in mammary gland and colon of WT mice were 47.1+/-9.5 and 58.0+/-21.7, respectively, but accordingly only 3.8+/-0.7 and 5.4+/-0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.     

Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat

1. Gavage administration of the natural flavonoids tangeretin, chrysin, apigenin, naringenin, genistein and quercetin for 2 consecutive weeks to the female rat resulted in differential effects on selected phase 1 and 2 enzymes in liver, colon and heart as well as antioxidant enzymes in red blood cells (RBC). 2. Glutathione transferase (GST) activity assayed by use of the substrate 1-chloro-2,4-dinitrobenzene was significantly induced by apigenin, genistein and tangeretin in the heart but not in colon or liver. 3. In RBC chrysin, quercetin and genistein significantly decreased the activity of glutathione reductase (GR), catalase (CAT) and glutathione peroxidase (GPx), whereas superoxide dismutase (SOD) was only significantly decreased by genistein. 4. The oxidative status of the animal, measured as plasma malondialdehyde, revealed that chrysin, quercetin, genistein, and beta-naphthoflavone (BNF) significantly protected against, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced oxidative stress. Hepatic PhIP-DNA adduct formation was not affected by any of the administered flavonoids, whereas PhIP-DNA adduct formation in colon was slightly, but significantly, inhibited by quercetin, genistein, tangeretin and BNF. 5. The observed effects of chrysin, quercetin and genistein on antioxidant enzymes, concurrently with a protection against oxidative stress, suggest a feedback mechanism on the antioxidant enzymes triggered by the flavonoid antioxidants. 6. Despite the use of high flavonoid doses, which by far exceed the human exposure levels, the effect on drug metabolizing and antioxidant enzymes was still very minor. The role of singly administered flavonoids in the protection against cancer and heart disease is thus expected to be limited.     

Dietary Chlorella protects against heterocyclic amine-induced aberrant gene expression in the rat colon by increasing fecal excretion of unmetabolized PhIP

The food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines formed by cooking meat and fish at high temperature. PhIP induces colorectal adenoma risk in male rats when administered orally. This study used male Fisher 344 rats to investigate the impact of dietary Chlorella on PhIP metabolism and aberrant colonic gene expression following short-term PhIP treatment. High-performance liquid chromatography analysis revealed that fecal excretion of unmetabolized PhIP was significantly increased in rats whose diets were supplemented with Chlorella compared to rats in a PhIP-only group (P < 0.001). Quantitative realtime PCR confirmed that the increase in beta-catenin and cyclin D1 mRNA in the colon induced by PhIP was ameliorated in rats pre-fed with Chlorella (P = 0.052 for beta-catenin; P = 0.005 for cyclin D1). The increase in DNA shearing that is a hallmark of caspase-8-mediated apoptosis by PhIP was also significantly diminished in the colons of rats pre-fed Chlorella (P = 0.012). These results suggested that administering dietary Chlorella with a Western-style diet concomitantly or immediately before mutagen exposure might be beneficial in blocking the absorption of food mutagens such as PhIP.     

Incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into fur and correlation with intestinal tumourigenesis in Min/+ mice

The purpose of this work was to correlate the amount of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) determined in mouse fur with the number of intestinal tumours induced by PhIP, to further evaluate incorporation of PhIP into hair as a putative exposure biomarker for food mutagens. We have previously shown that PhIP increases intestinal tumourigenesis in C57BL/6J-Min/+ (Multiple Intestinal Neoplasia) mice. Fur was sampled from mice exposed according to various PhIP protocols, and the amount of PhIP in the fur was quantitated by high performance liquid chromatography - mass spectrometry (HPLC-MS). A quantitative incorporation of PhIP in the fur was demonstrated after a single subcutaneous injection of PhIP, and between one and eight PhIP exposures either via direct subcutaneous injections or through breast milk from PhIP-injected dams. However, after higher exposures, the amount of PhIP in the fur appeared to reach saturation. After low exposures to PhIP, the number of intestinal tumours correlated with the amount of PhIP in the fur of individual mice, whereas after higher exposures, the number of tumours was relatively higher than the amounts of incorporated PhIP in the fur. Other factors, e.g. amounts of reactive PhIP metabolites formed, are also determining the number of intestinal tumours. The demonstrated quantitative incorporation of PhIP into mice fur and the correlation with number of intestinal tumours at the lower exposures, indicate that determination of PhIP in human hair may be a suitable biomarker for exposure to dietary PhIP, which is found in human hair in 10-3 lower amounts than in these mice.     

Carcinogenic potential of cooked food mutagens (IQ and MeIQ) in Wistar rats after short-term exposure

Two potent cooked food mutagens, 2-amino-3-methylimidazo/4,5-f/quinoline (IQ) and 2-amino-3,4-dimethylimidazo/4,5-f/quinoline (MeIQ), were examined in an initiation-promotion assay in the male wistar rat. Fourteen doses of 10 mg IQ or 10 mg MeIQ/kg b.wt. were given during initiation, followed by promotion with 500 p.p.m. phenobarbital sodium (PB) in the drinking water up to week 58. A small number of tumours of Zymbal's gland were seen in all groups treated with IQ or MeIQ, irrespective of PB-treatment. Though the promotional regimen failed to produce the expected number of liver tumours, it did induce a significant amount of gamma-glutamyltranspeptidase (GGT) activity. These results suggest that even short exposures to low doses of IQ or MeIQ produce persistent procarcinogenic lesions in the rat, and that secondary factors, e.g. promoters or high cell turnover, may over time develop these lesions into cancer.     

Lactoperoxidase-catalyzed activation of carcinogenic aromatic and heterocyclic amines

Lactoperoxidase, an enzyme secreted from the human mammary gland, plays a host defensive role through antimicrobial activity. It has been implicated in mutagenic and carcinogenic activation in the human mammary gland. The potential role of heterocyclic and aromatic amines in the etiology of breast cancer led us to examination of the lactoperoxidase-catalyzed activation of the most commonly studied arylamine carcinogens: 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), benzidine, 4-aminobiphenyl (ABP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). In vitro activation was performed with lactoperoxidase (partially purified from bovine milk or human milk) in the presence of hydrogen peroxide and calf thymus DNA. Products formed during enzymatic activation were monitored by HPLC with ultraviolet and radiometric detection. Two of these products were characterized as hydrazo and azo derivatives by means of mass spectrometry. The DNA binding level of 3H- and 14C-radiolabeled amines after peroxidase-catalyzed activation was dependent on the hydrogen peroxide concentration, and the highest levels of carcinogen binding to DNA were observed at 100 microM H2O2. Carcinogen activation and the level of binding to DNA were in the order of benzidine > ABP > IQ > MeIQx > PhIP. One of the ABP adducts was identified, and the level at which it is formed was estimated to be six adducts/10(5) nucleotides. The susceptibility of aromatic and heterocyclic amines for lactoperoxidase-catalyzed activation and the binding levels of activated products to DNA suggest a potential role of lactoperoxidase-catalyzed activation of carcinogens in the etiology of breast cancer.     

Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells

In the present study the chemopreventive effects of water soluble AquaROX^® 15 and oil soluble VivOX^® 40 rosemary extracts against 4-nitroquinoline-N-oxide (NQNO) and 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline (IQ) induced mutagenicity in the reverse mutation assays with Salmonella typhimurium TA98 and against t-butyl hydroperoxide (t-BOOH), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced DNA damage in HepG2 cells were studied, applying the comet assay. The results showed comparable protective effect of AquaROX and VivOX against oxidative DNA damage, whereas protection against indirect active genotoxic carcinogens was more efficient by VivOX.     

Effects of roselle (Hibiscus sabdariffa Linn.), a Thai medicinal plant, on the mutagenicity of various known mutagens in Salmonella typhimurium and on formation of aberrant crypt foci induced by the colon carcinogens azoxymethane and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in F344 rats.

The 80% ethanol extract of roselle (Hibiscus sabdariffa Linn.), a medicinal plant in Thailand, was examined for antimutagenic and chemopreventive activity in a colon carcinogenesis model. It reduced about 60-90% of the mutagenicity induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and other heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MelQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline(MelQx),3-amino-1,4-dimet hyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2),2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1),2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), at a concentration of 12.5 mg/plate in the Salmonella mutation assay. The extract showed no mutagenicity and no antibacterial activity below this dose. Mutagenicity of methylazoxymethanol (MAM) acetate, which, like PhIP, is a colon carcinogen,was also efficiently inhibited by the roselle extract. To investigate chemoprevention by roselle in a colon carcinogenesis model, we examined the inhibitory effects of the roselle extract in F344 rats in which aberrant crypt focus (ACF) formation was induced by azoxymethane (AOM) and PhIP. In the initiation stage, the number of AOM-induced ACF in the colon was significantly decreased by roselle (17-25%) compared with that in rats treated with AOM alone. The amount of O6-methylguanine in the colonic mucosa tended to be decreased in the roselle-treated rats. The number of PhIP-induced ACF was also significantly decreased by roselle treatment (22%) at a concentration of 1.0 g/kg body weight in the initiation stage. However, in the post-initiation stage of AOM-induced ACF formation, roselle increased the number of ACF, especially the number of foci which had more than three crypts/focus. These results indicate that roselle has antimutagenic activity against MAM acetate and heterocyclic amines and that it decreases the number of AOM- and PhIP-induced ACF in the initiation stage, although it rather increased the number of ACF in the post-initiation stage.     

DNA Adduct Levels and Intestinal Lesions in Congenic Rapid and Slow Acetylator Syrian Hamsters Administered the Food Mutagens 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) or 2‐Amino‐3‐methylimidazo[4,5‐f]quinoline (IQ

Epidemiological studies indicate that rapid acetylators with a high intake of well‐done red meat have an increased risk of colorectal cancer. Arylamine N‐acetyltransferase enzymes (E.C. 2.3.1.5) activate carcinogenic heterocyclic amines found in the crust of fried meat via O‐acetylation of their N‐hydroxylamines to reactive intermediates that bind covalently to DNA and produce mutations. Syrian hamsters as well as humans express two N‐acetyltransferase isozymes (NAT1 and NAT2) which differ in substrate specificity and genetic control. Nucleic acid substitutions in the NAT2 gene segregate individuals into rapid, intermediate and slow acetylator phenotypes. In the present paper, we examined the role of the polymorphic NAT2 acetylator genotype in carcinogenesis induced by the food mutagens 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) or 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) by comparing Syrian hamster lines congenic at the NAT2 locus. No differences were found between rapid and slow acetylator congenic hamsters in levels of intestinal PhIP‐DNA adducts. In contrast to previous studies in rats, no carcinogen‐related induction of the preneoplastic lesions aberrant crypt foci or tumors was found in the intestines of rapid and slow acetylator congenic Syrian hamsters administered PhIP or IQ.     

Impact of environmental exposures on the mutagenicity/carcinogenicity of heterocyclic amines

Carcinogenic heterocyclic amines are produced from overcooked foods and are highly mutagenic in most short-term test systems. One of the most abundant of these amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast, colon and prostate tumors in rats. Human dietary epidemiology studies suggest a strong correlation between either meat consumption or well-done muscle meat consumption and cancers of the colon, breast, stomach, lung and esophagus. For over 20 years our laboratory has helped define the human exposure to these dietary carcinogens. In this report we describe how various environmental exposures may modulate the risk from exposure to heterocyclic amines, especially PhIP. To assess the impact of foods on PhIP metabolism in humans, we developed an LC/MS/MS method to analyze the four major PhIP urinary metabolites following the consumption of a single portion of grilled chicken. Adding broccoli to the volunteers' diet altered the kinetics of PhIP metabolism. At the cellular level we have found that PhIP itself stimulates a significant estrogenic response in MCF-7 cells, but even more interestingly, co-incubation of the cells with herbal teas appear to enhance the response. Numerous environmental chemicals found in food or the atmosphere can impact the exposure, metabolism, and cell proliferation response of heterocyclic amines.     

Protective effects of xanthohumol against the genotoxicity of heterocyclic aromatic amines MeIQx and PhIP in bacteria and in human hepatoma (HepG2) cells

Previous studies showed that xanthohumol (XN), a hop derived prenylflavonoid, very efficiently protects against genotoxicity and potential carcinogenicity of the food borne carcinogenic heterocyclic aromatic amine (HAA) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In this study, we showed that XN was not mutagenic in Salmonella typhimurium TA98 and did not induce genomic instability in human hepatoma HepG2 cells. In the bacteria XN suppressed the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8 dimethylimidazo[4,5-f]quinoxaline (MeIQx) induced mutations in a dose dependent manner and in HepG2 cells it completely prevented PhIP and MeIQx induced DNA strand breaks at nanomolar concentrations. With the QRT-PCR gene expression analysis of the main enzymes involved in the biotransformation of HAAs in HepG2 cells we found that XN upregulates the expression of phase I (CYP1A1 and CYP1A2) and phase II (UGT1A1) enzymes. Further gene expression analysis in cells exposed to MeIQx and PhIP in combination with XN revealed that XN mediated up-regulation of UGT1A1 expression may be important mechanism of XN mediated protection against HAAs induced genotoxicity. Our findings confirm the evidence that XN displays strong chemopreventive effects against genotoxicity of HAAs, and provides additional mechanistic information to assess its potential chemopreventive efficiency in humans.     

Induction of colon tumors in C57BL/6J mice fed MeIQx, IQ, or PhIP followed by dextran sulfate sodium treatment.

Heterocyclic amines (HCAs) have been shown to induce tumors in several organs of rodents, but except for MeIQ and PhIP, other HCAs such as MeIQx and IQ consistently failed to induce colon tumors in mice, whereas MeIQ, IQ, and PhIP exerted colon tumorigenicity in rats. Recently, we found that dietary MeIQx induces genotoxicity in the colon as well as the liver of two different types of reporter gene transgenic mice at subcarcinogenic doses such as 300 ppm. However, in the present study, dietary MeIQx did not significantly induce any tumors in C57BL/6J mice or gpt delta mice even when fed at 300 ppm for 78 weeks, suggesting that the treatment of MeIQx alone was not sufficient to promote colon tumors. In order to clarify a possibility whether such HCAs can induce colon tumors, C57BL/6J mice were fed MeIQx, IQ, or PhIP at a dose of 300 ppm for 12 weeks and, thereafter, twice received 1-week treatment with dextran sulfate sodium (DSS), 2 weeks apart. After 20 weeks, colon tumors including adenocarcinomas were found at incidences of 22%, 24%, and 45% in the groups receiving MeIQx, IQ, and PhIP, respectively, which were significantly (p < 0.05 or 0.01) different from the DSS alone value (0%). Thus our results clearly indicate that, in addition to PhIP, MeIQx and IQ can induce colon tumors in mice under an experimental condition promoting colon tumors.