Effects of Seasoning and Heating Device on Mutagenicity and Heterocyclic Amines in Cooked Beef

Pan-roasted beef showed a lower mutagenicity after various degrees of cooking than charcoaled one. The high mutagenicity of charcoaled beef was due to the formation of more heterocyclic amines, especially AαC (2-amino-9 H-pyrido- [2,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) because of rapid and direct heating on the surface of the meat at a high temperature. Seasoning decreased mutagenicity of pan-roasted beef except the very well done sample with unchanged heterocyclic amine contents, but increased mutagenicity of charcoaled beef with decreased levels of AαC and PhIP, probably due to the change of heterocyclic amine precursors or alternatively to the occurrence of other mutagens.     

Mutagenic and carcinogenic heterocyclic amines as affected by muscle types/skin and cooking in pan-roasted mackerel

The dependence on muscle types/skin and on degrees of cooking in the formation of heterocyclic amines (HCAs) of pan-roasted mackerel was studied. High levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were found in very well done skin and ordinary muscle, being 4.2 and 5.3 ng/g, followed by 2-amino-3,4-dimethylimidazo[4,5-f] quinoxaline (MeIQx), being 1.8 and 2.1 ng/g and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), being 1.2 and 2.8 ng/g, respectively. In pan-roasted mackerel, ordinary muscles contributed much more greatly to the formation of HCAs than skins due to its higher HCA contents and composition (76.1%).     

Identification of mutagenic heterocyclic amines (IQ, Trp-P-1 and AalphaC) in the water of the Danube river

Three mutagenic heterocyclic amines, 2-amino-3-methylimidazo-[4, 5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), were isolated and identified in water from the Danube River in Vienna. Heterocyclic amines were extracted from river water by the blue rayon hanging method, and analyzed by gas chromatography with a nitrogen-phosphorous detector (GC-NPD) and GC-mass spectrometry (GC-MS) after conversion into their N-dimethylaminomethylene derivatives. Identity of IQ, Trp-P-1 and AalphaC in the river water was confirmed by GC-MS. The contents of IQ, Trp-P-1 and AalphaC were estimated by GC-NPD at 1.78+/-0.17, 0.14+/-0.02 and 0.44+/-0.02 ng/g blue rayon equivalent (n=3), respectively. The total amounts of these amines accounted for 26% of the mutagenicity of blue rayon extracts evaluated by the Ames test using TA98 with metabolic activation.     

Inhibition of the metabolism of mutagens occurring in food by arachidonic acid.

Hepatic microsomal fractions (microsomes) were prepared from male Sprague-Dawley rats. The effect of arachidonic acid on the conversion of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay (indicator: Salmonella typhimurium TA98). Arachidonic acid 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 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 this polyunsaturated fatty acid.     

Anti-genotoxic activity of Vitis coignetiae Pulliat towards heterocyclic amines and isolation and identification of caftaric acid as an antimutagenic component from the juice

Our study demonstrated that the formation of DNA adducts in liver, lungs, colon and kidneys of mice given a carcinogenic heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in the diet significantly decreased following the administration of the juice of Vitis coignetiae, purple berries from a vine tree. The juice of V. coignetiae significantly inhibited the clastogenicity and mutagenicity of heterocyclic amines in the micronucleus assay and the Ames test, and was an effective inhibitor of the activities of phase I enzymes (cytochrome P450 1A1 and cytochrome P450 1A2) and enhancer of the activities of phase II enzymes (uridine 5'-diphospho-glucuronosyltransferase and glutathione S-transferase). We investigated the purification and isolation of an active compound in the juice of V. coignetiae using antimutagenicity as a separation marker. Caftaric acid, a polyphenolic compound, was identified as a component responsible for antimutagenicity in the juice of V. coignetiae towards the carcinogenic heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). This is the first report of antimutagenicity of caftaric acid. Caftaric acid was reported as an inhibitor of the protein-protein interactions mediated by the Src-family kinases. The impact of the juice of V. coignetiae and its constituents on tumor initiation and promotion thus warrants further study.     

Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Resveratrol is a phytoalexin, that belongs to a family of naturally occurring stilbenes. It has been reported that resveratrol can inhibit chemical carcinogenesis in experimental animals and although the mechanisms involved are unknown, an anti-mutagen mechanism has been proposed. We have explored this hypothesis using mutagenicity assays based on bacterial (Salmonella typhimurium) and eukaryotic cells (Chinese hamster V79 cells). We found resveratrol to be potent in both systems, blocking the mutagenicity of the food-derived heterocyclic amines (HA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at micromolar concentrations. Furthermore, in cells capable of activating 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine to cytotoxic derivatives, resveratrol was able to attenuate cytotoxicity. Paradoxically, in cells lacking the ability to activate PhIP, resveratrol itself was toxic and co-incubation with PhIP reduced this toxicity. Our data confirm the potent anti-mutagenic activity of resveratrol and support its potential as a chemopreventative.     

DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AalphaC), analysed by 32P_HPLC.

Heterocyclic aromatic amines (HAAs) are produced during cooking of proteinaceous food such as meat and fish. Humans eating a normal diet are regularly exposed to these food-borne substances. HAAs have proved to be carcinogenic in animals and to induce early lesions in the development of cancer. DNA adduct levels in mouse liver have been measured by 32P-HPLC after oral administration each of 14 different HAAs. The highest DNA adduct levels were detected for 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), respectively. To assess a relative risk in a human population, a relative risk index was calculated by combining the DNA adduct levels in mouse liver with human daily intake of heterocyclic amines in a US and in a Swedish population. Such calculations suggest that AalphaC presents the highest risk for humans, e.g. nine-fold higher compared with the most abundant amines in food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP). Therefore, the distribution of DNA adducts in different tissues of mouse was investigated after oral administration of AalphaC. The highest AalphaC-DNA adduct levels were found in liver (137 adducts/10(8) normal nucleotides) followed by heart, kidney, lung, large intestine, small intestine, stomach and spleen, in descending order. To characterize the chemical structure of the major DNA adduct, chemical synthesis was performed. The major DNA adduct from the in vivo experiments was characterized by five different methods. On the basis of these results, the adduct was characterized as N2-(deoxyguanin-8-yl)-2-amino-9H-pyrido [2,3-b]indole. Considering the abundance of AalphaC not only in grilled meat, but also in other products like grilled chicken, vegetables and cigarette smoke and in light of the results of the present study, it is suggested that the human cancer risk for AalphaC might be underestimated.     

Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods

Samples of 7 foods commonly eaten in the Northeast of China (i.e. fried and broiled fishes and broiled meat) were tested for mutagenicity on Salmonella typhimurium TA98 with S9 mix. The basic fractions of the samples were mutagenic, inducing 33-2930 revertants/g of cooked food. Fried walleye pollack (a kind of cod fish heated on a stainless steel pan) showed the highest mutagenicity, so attempts were made to isolate mutagens from the basic fraction of this food. The mutagens were purified by treatment with blue cotton and HPLC on a semi-preparative ODS column and analytical cation exchange and ODS columns. 5 mutagens were isolated and identified as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 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-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). 1 g of fried fish was estimated to contain 0.16 ng of IQ, 0.03 ng of MeIQ, 6.44 ng of MeIQx, 0.10 ng of 4,8-DiMeIQx and 69.2 ng of PhIP. MeIQx and PhIP accounted for 24% and 4.7%, respectively, of the total mutagenicity. The other 3 heterocyclic amines were each responsible for only 0.3-1.2% of the total mutagenicity.     

Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding its antimutagenic properties against cooked food mutagens. Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent.     

Mutagenic activity and heterocyclic amine carcinogens in commercial pet foods

Twenty-five commercial pet foods were analyzed for mutagenic activity using the Ames/Salmonella test with strain TA98 and added metabolic activation. All but one gave a positive mutagenic response. Fourteen of these samples were analyzed for heterocyclic amine mutagens/carcinogens and all but one contained 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 10 of 14 contained 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as analyzed by HPLC and confirmed by photodiode array peak matching. From these findings it is hypothesized that there is a connection between dietary heterocyclic amines and cancer in animals consuming these foods.     

Regional mutagenicity of heterocyclic amines in the intestine: mutation analysis of the cII gene in lambda/lacZ transgenic mice

Transgenic mouse assays have revealed that the mouse intestine, despite its resistance to carcinogenesis, is sensitive to the mutagenicity of some heterocyclic amines (HCAs). Little is known, however, about the level and localization of that sensitivity. We assessed the mutagenicity of four orally administered (20 mg/kg per day for 5 days) HCAs-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) hydrochloride, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate-in the intestine of male MutaMice. Two weeks after the last administration, we isolated epithelium from the small intestine, cecum, and colon and analyzed lacZ and cII transgene mutations. PhIP increased the lacZ mutant frequency (MF) in all the samples, and in the small intestine, cII and lacZ MFs were comparable. In the cII gene, G:C to T:A and G:C to C:G transversions were characteristic PhIP-induced mutations (which has also been reported for the rat colon, where PhIP is carcinogenic). In the small intestine, PhIP increased the cII MF to four-fold that of the control, but IQ, MeIQ, and Trp-P-2 did not have a significant mutagenic effect. In the cecum, cII MFs induced by IQ and MeIQ were 1.9 and 2.7 times those in the control, respectively. The MF induced by MeIQ in the colon was 3.1 times the control value. Mutagenic potency was in the order PhIP>MeIQ>IQ; Trp-P-2 did not significantly increase the MF in any tissue. The cecum was the most susceptible organ to HCA mutagenicity.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.     

Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane--a constituent of broccoli

Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines). These include imidazoazaarenes such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9. Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.     

Effects of industrially produced flavours with pro- and antioxidative properties on the formation of the heterocyclic amine PhIP in a model system

PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is a heterocyclic aromatic amine belonging to a class of mutagens found in food. This project studied the effects of commercially available flavours of spices on the formation of PhIP, one of the most common heterocyclic aromatic amines in heated meat and fish products. The model reactions were carried out in diethylene glycol. Highest amounts of PhIP were obtained at 200 degrees C, a heating time of 60 min and an equivalent molar ratio of phenylalanine and creatinine. With this model system, the influence of Monascus red and flavours extracted from thyme, marjoram and rosemary on the formation of PhIP was tested. The flavours were added to the model system in different amounts. The oxidative properties were determined with the rancimat method. It was shown that all tested products, independent of their pro- or antioxidative properties, increased PhIP in the model system.     

Inhibitory effect of curcumin and its natural analogues on genotoxicity of heterocyclic amines from cooked food

Curcumin (C) and its natural analogues demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC), known for their potent anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic effects, were tested for their possible inhibitory effects against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor induced rat liver S9 homogenate. In the present investigations, curcumin as well as its two natural analogues i.e., dmC and bdmC were found to be highly effective in suppressing genotoxicity of all the tested cooked food mutagens in a dose-dependent manner, in both the frame shift (TA98) as well as base pair mutation sensitive (TA100) strains of S. typhimurium. However, bdmC appeared to be a relatively less active antimutagen compared to C and dmC. More than 80% inhibition of mutagenicity was observed at 200 microg/plate in case of C and dmC in both TA98 and TA100 against all tested cooked food mutagens. Where as, bdmC showed 39-79% inhibition in TA100 and 60-80% inhibition in TA98, at a dose of 200 microg/plate. These findings warrant further biochemical, enzymatic and in vivo investigations in animal models as well as in humans to establish the chemoprotective effect of these agents against mutagenic heterocyclic amines found in cooked food.     

Formation of a mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) in cooked beef, by heating a mixture containing creatinine, phenylalanine and glucose

When a mixture of creatinine, phenylalanine and glucose in diethylene glycol-water solution was heated for 2 h at 128 degrees C, a mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was found to be produced, identified by high-performance liquid chromatography, and UV-visible and mass spectrometries. The yield of PhIP was 3.6 nmoles/mmole original creatinine. PhIP was originally isolated from cooked beef, and this study shows creatinine, phenylalanine and glucose to be the probable precursors of PhIP in beef.     

Inhibitory effects of beer on heterocyclic amine-induced mutagenesis and PhIP-induced aberrant crypt foci in rat colon

Anti-mutagenic and anti-carcinogenic effects of beer on heterocyclic amine (HCA)-induced carcinogenesis were studied in vitro and in vivo. Four commercial beers (two pilsner-type, black, and stout) showed inhibitory effects against five HCAs, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 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) and 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), in the Ames assay using Salmonella typhimurium TA98 in the presence of rat S9 mix. The inhibitory effects of dark-colored beers (stout and black beer) were greater than those of pilsner-type beers. Dark-colored beers suppressed CYP1A2 activity in a dose-dependent manner, suggesting that inhibition of HCA activation is partly responsible for their strong anti-mutagenic effects. Anti-mutagenic effects were also observed when the pooled human S9 mix or activated IQ was used in the assay. The micronucleus test using Chinese hamster lung CHL/IU cells showed that the addition of freeze-dried samples of pilsner-type and stout beer to the culture medium significantly reduced the number of cells with micronuclei induced with PhIP or Trp-P-2. Single-cell gel electrophoresis assay (comet assay) revealed that oral ingestion of pilsner-type and stout beers for 1 week significantly inhibited DNA damage in the liver cells of male ICR mice exposed to MeIQx (13 mg/kg, i.p.). A decrease in the formation of DNA adducts was also observed using a 32P-postlabeling method. Male Fischer 344 rats orally received PhIP (75 mg/kg, five times a week for 2 weeks) and aberrant crypt foci (ACF) formation in the colon was analyzed after 5 weeks. The number of ACF was significantly reduced in rats fed a diet containing freeze-dried beer. These results suggest that beer inhibits the genotoxic effects of HCAs and may reduce the risk of carcinogenesis caused by food borne carcinogens.     

Determination of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and its metabolite 2-hydroxyamino-PhIP by liquid chromatography/electrospray ionization-ion trap mass spectrometry.

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic aromatic amine found in cooked meat. It is metabolically activated by the human cytochrome P450 enzymes to form the carcinogenic metabolite N2-OH-PhIP. PhIP has been found to induce tumors in rats and is a suspected human carcinogen. In the present work, we have developed and validated a liquid chromatography-electrospray ionization/ion trap mass spectrometry (LC-ESI/ITMS) method for the determination of PhIP and N2-OH-PhIP. PhIP was incubated with microsomes prepared from the human liver; the PhIP and N2-OH-PhIP formed were isolated from the biomatrices by solid-phase extraction using C18 cartridges, with recoveries greater than 86%. Subsequently, the products were separated on a microbore reversed-phase C18 liquid chromatograph coupled to an ESI-ITMS. The ESI interface and the ITMS were tuned for various parameters, and data acquisition was performed in selective ion monitoring mode. The detection limit of PhIP and N2-OH-PhIP was 1 and 10 pg, respectively. The method is highly sensitive and selective, has simple sample preparation protocols, and should be applicable to the study of the metabolic activation of PhIP in various human tissues.     

DNA adduct levels and absence of tumors in female rapid and slow acetylator congenic hamsters administered the rat mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine

N-acetyltransferases (EC 2.3.1.5) catalyze O-acetylation of heterocyclic amine carcinogens to DNA-reactive electrophiles that bind and mutate DNA. An acetylation polymorphism exists in humans and Syrian hamsters regulated by N-acetyltransferase-2 (NAT2) genotype. Some human epidemiological studies suggest a role for NAT2 phenotype in predisposition to cancers related to heterocyclic amine exposures, including breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine carcinogen prevalent in the human environment and induces a high incidence of mammary tumors in female rats. PhIP-induced carcinogenesis was examined in female rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus. In both rapid and slow acetylators, PhIP-DNA adduct levels were highest in pancreas, lower in heart, small intestine, and colon, and lowest in mammary gland and liver. Metabolic activation of N-hydroxy-PhIP by O-acetyltransferase was highest in mammary epithelial cells, lower in liver and colon, and lowest in pancreas. Metabolic activation of N-hydroxy-PhIP by O-sulfotransferase was low in liver and colon and below the limit of detection in mammary epithelial cells and pancreas. Unlike the rat, PhIP did not induce breast or any other tumors in female rapid and slow acetylator congenic hamsters administered high-dose PhIP (10 doses of 75 mg/kg) and a high-fat diet.     

Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites

People are commonly exposed to organophosphorus ester (OP) insecticides through the treatment of pets, homes, lawns, gardens, workplaces and in commercial agriculture. Aromatic amines are another chemical class with wide human exposure particularly dietary heterocyclic aromatic amines (HAAs). Previously, we reported that specific aromatic amines and ethyl paraoxon (the metabolite of the insecticide ethyl parathion) induced enhanced mutagenic responses in Salmonella typhimurium. In the present study, we demonstrated that the mutagenicity of 2-acetoxyacetylaminofluorene (2AAAF) and the heterocyclic dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) was enhanced in the presence of the OP insecticides, ethyl parathion or methyl parathion or a metabolite (methyl paraoxon). The mutagenicity of 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) was increased by methyl parathion and methyl paraoxon but not by ethyl parathion. This mutagenic synergy was expressed in S. typhimurium strain YG1024. Mammalian microsomal activation was required for PhIP and IQ to express mutagenic synergy. Synergistic responses are rarely incorporated in risk assessment models, yet such responses are important in establishing accurate toxicological characteristics of agents. Under real world conditions where people are exposed to a multitude of agents, the results of this study raise a concern about the environmental and public health impacts of OP insecticides.