Screening for heterocyclic amines in chicken cooked in various ways.

Chicken cooked under well-controlled conditions and commercial chicken products were screened for heterocyclic amines (HAs). Chicken samples were boiled, deep-fried, pan-fried, oven-roasted, cooked in an unglazed clay pot or in a roasting bag in the oven, and oven broiled. 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 1-methyl-9H-pyrido[3,4-b]indole (harman) and 9H-pyrido[3,4-b]indole (norharman) were identified in several samples. Chicken cooked at low temperatures contained low amounts of HAs. In pan-fried chicken breasts, MeIQx was detected in amounts below 2 ng/g, 4,8-DiMeIQx below 0.6 ng/g, and PhIP in amounts up to 38 ng/g. Harman and norharman were detected in almost all samples (below 15 ng/g). In skin from a commercially barbecued chicken, MeIQx, 4,8-DiMeIQx and PhIP were detected, while only traces of MeIQx were detected in the meat. MeIQx was detected in a commercial chicken flavour, 0.1 ng/ml. No HAs were detected in pan-fried chicken liver. The results show that the content of HAs in chicken cooked in various ways is low if prepared at low temperatures, and increases with increasing cooking temperature. PhIP formation seems to start accelerating at cooking temperatures around or above 200 degrees C. Colour development increases with cooking temperature, but no correlation with HA content was observed.     

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.     

Carcinogenic risk of heterocyclic amines in combination – Assessment with a liver initiation model

Carcinogenic potential of heterocyclic amines (HCAs) was investigated using an in vivo 5-week initiation assay with quantitative evaluation of glutathione S-transferase placental form (GST-P) positive foci in rat liver. Numbers of GST-P positive foci were significantly increased with individual administration of six different HCAs, indicating utility of the assay. It was therefore applied to investigate risk with multiple HCAs in combination. Unexpectedly, concomitant treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) did not result in any additive carcinogenicity. In the rats taking MeIQx prior to PhIP the value was almost equal to the sum total of individual data, indicating additive initiation activities. In contrast, simultaneous or prior administration of PhIP rather exerted inhibitory effects on the carcinogenic potential of MeIQx. Moreover, microarray and quantitative RT-PCR assessment revealed that PhIP induced cytochrome P450 1A1, responsible for both activation and detoxification of HCAs, more strongly than MeIQx. It is noteworthy that complex exposure to multiple HCAs is not necessarily associated with increased risk of carcinogenesis because they are simultaneously and continuously ingested under normal circumstances.     

PhIP carcinogenicity in breast cancer: computational and experimental evidence for competitive interactions with human estrogen receptor

Many carcinogens have been shown to cause tissue specific tumors in animal models. The mechanism for this specificity has not been fully elucidated and is usually attributed to differences in organ metabolism. For heterocyclic amines, potent carcinogens that are formed in well-done meat, the ability to either bind to the estrogen receptor and activate or inhibit an estrogenic response will have a major impact on carcinogenicity. Here, we describe our work with the human estrogen receptor alpha (ERalpha), the mutagenic/carcinogenic heterocyclic amines PhIP, MeIQx, and IFP, and the hydroxylated metabolite of PhIP, N2-hydroxy-PhIP. We demonstrate both by computational docking and NMR analysis that PhIP binds with the ligand binding domain (LBD). This binding competes with estradiol (E2) in the native E2 binding cavity of the receptor. In vitro assays show that PhIP, in contrast to the other heterocyclic amines, increases cell proliferation in MCF-7 human breast cancer cells and activates the ERalpha receptor. We also find that other heterocyclic amines and N2-hydroxy-PhIP inhibit ERalpha activation. We propose that the mechanism for the tissue-specific carcinogenicity seen in the rat breast tumors and the presumptive human breast cancer associated with the consumption of well-done meat maybe mediated by this receptor activation.     

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.     

The cooked meat-derived mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine promotes invasive behaviour of breast cancer cells

The cooked meat derived genotoxic carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces cancer of the colon, prostate and mammary gland when fed to rats. Epidemiology studies link these tumours to a Western diet and exposure to heterocyclic amines such as PhIP. We have shown that PhIP is also potently estrogenic and have proposed that this hormonal activity contributes to its target site carcinogenicity. We now postulate that the estrogenic properties of PhIP influence metastatic potential. We have used an in vitro assay for cell invasion based upon digestion and migration through a reconstituted basement membrane model. Zymography and immunoblotting were used to confirm PhIP-mediated changes associated with induction of the invasive phenotype. Treatment of the mammary cancer cell lines MCF-7 and T47D with PhIP induces cells to digest and migrate through a reconstituted basement membrane. The response was dose dependent, observed at sub-nanomolar concentrations of PhIP and was inhibited by the antiestrogen ICI 182,780. The PhIP-induced invasive phenotype was associated with expression of cathepsin D, cyclooxygenase-2 and matrix metalloproteinase activity. These findings emphasise the range and potency of the biological activities associated with this cooked meat product and mechanistically support the tissue-specific carcinogenicity of the chemical.     

Preferential induction of the AhR gene battery in HepaRG cells after a single or repeated exposure to heterocyclic aromatic amines

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are two of the most common heterocyclic aromatic amines (HAA) produced during cooking of meat, fish and poultry. Both HAA produce different tumor profiles in rodents and are suspected to be carcinogenic in humans. In order to better understand the molecular basis of HAA toxicity, we have analyzed gene expression profiles in the metabolically competent human HepaRG cells using pangenomic oligonucleotide microarrays, after either a single (24-h) or a repeated (28-day) exposure to 10 μM PhIP or MeIQx. The most responsive genes to both HAA were downstream targets of the arylhydrocarbon receptor (AhR): CYP1A1 and CYP1A2 after both time points and CYP1B1 and ALDH3A1 after 28 days. Accordingly, CYP1A1/1A2 induction in HAA-treated HepaRG cells was prevented by chemical inhibition or small interference RNA-mediated down-regulation of the AhR. Consistently, HAA induced activity of the CYP1A1 promoter, which contains a consensus AhR-related xenobiotic-responsive element (XRE). In addition, several other genes exhibited both time-dependent and compound-specific expression changes with, however, a smaller magnitude than previously reported for the prototypical AhR target genes. These changes concerned genes mainly related to cell growth and proliferation, apoptosis, and cancer. In conclusion, these results identify the AhR gene battery as the preferential target of PhIP and MeIQx in HepaRG cells and further support the hypothesis that intake of HAA in diet might increase human cancer risk.     

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.     

Quantification of the neurotoxic beta-carboline harmane in barbecued/grilled meat samples and correlation with level of doneness

Harmane, one of the heterocyclic amines (HCAs), is a potent neurotoxin linked to human diseases. Dietary exposure, especially in cooked meats, is the major source of exogenous exposure for humans. However, knowledge of harmane concentrations in cooked meat samples is limited. Our goals were to (1) quantify the concentration of harmane in different types of cooked meat samples, (2) compare its concentration to that of other more well-understood HCAs, and (3) examine the relationship between harmane concentration and level of doneness. Thirty barbecued/grilled meat samples (8 beef steak, 12 hamburger, 10 chicken) were analyzed for harmane and four other HCAs (2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine [PhIP], amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [DiMeIQx], and 2-amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-b]pyridine [IFP]). Mean (+/- SD) harmane concentration was 5.63 (+/- 6.63) ng/g; harmane concentration was highest in chicken (8.48 +/- 9.86 ng/g) and lowest in beef steak (3.80 +/- 3.6 ng/g). Harmane concentration was higher than that of the other HCAs and significantly correlated with PhIP concentration. Harmane concentration was associated with meat doneness in samples of cooked beef steak and hamburger, although the correlation between meat doneness and concentration was greater for PhIP than for harmane. Evidence indicates that harmane was detectable in nanograms per gram quantities in cooked meat (especially chicken) and, moreover, was more abundant than other HCAs. There was some correlation between meat doneness and harmane concentration, although this correlation was less robust than that observed for PhIP. Data such as these may be used to improve estimation of human dietary exposure to this neurotoxin.     

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.     

Effects of (+)catechin and (−)epicatechin on heterocyclic amines‐induced oxidative DNA damage

The aim of the present study was to evaluate the protective effect of (+)‐catechin and (?)‐epicatechin against 2‐amino‐3,8‐ dimethylimidazo[4,5‐f]quinoxaline (8‐MeIQx), 2‐amino‐3,4,8‐trimethylimidazo[4,5‐f]‐quinoxaline (4,8‐diMeIQx) and 2‐amino‐1‐methyl‐6‐phenyl‐imidazo[4,5‐b]pyridine (PhIP)‐induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single‐cell gel electrophoresis or comet assay. Increasing concentrations of 8‐MeIQx, 4,8‐diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose‐dependent manner. Among those, PhIP (300 µm ) exerted the highest genotoxicity. (+)‐Catechin exerted protection against oxidized purines induced by 8‐MeIQx, 4,8‐diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)‐catechin. Otherwise, (?)‐epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8‐MeIQx and 4,8‐diMeIQx. One feasible mechanism by which (+)‐catechin and (?)‐epicatechin exert their protective effect towards heterocyclic amines‐induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O‐deethylation (CYP1A1) activity was moderately inhibited by (+)‐catechin, while little effect was observed by (?)‐epicatechin. However, (+)‐catechin showed the greatest increase in UDP‐glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)‐catechin was more efficient than (?)‐epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8‐MeIQx and 4,8‐diMeIQx. Copyright © 2010 John Wiley & Sons, Ltd.     

Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment.

Various methods of exposure assessment, such as questionnaires, sometimes combined with pictures of cooked meat, have been employed in investigations on the relationship between heterocyclic amines (HA) and health effects. However, as the content of heterocyclic amines vary greatly with cooking conditions, it is difficult to obtain an accurate estimate of the exposure. To improve the exposure assessment, the use of biomarkers has been investigated. The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is well characterised. In humans, the major part of the dose is excreted in urine within 24-48 h following a meal. A few percent is excreted as parent compounds, whereas the major part is metabolites. Urinary level of parent HA reflects only recent exposure. However, the pattern of excreted metabolites might indicate the capacity to activate or detoxify HAs. The excretion of glucuronide conjugates of N-hydroxy-PhIP and N-hydroxy-MeIQx could be a marker for the N-hydroxylation capacity and the dose of the proximate metabolites. Recently, we proposed 5-OH-PhIP as a marker for the ultimate reactive metabolite of PhIP, since it is formed from this compound as a by-product along with the formation of PhIP-DNA adducts. In a search for biomarkers reflecting exposure over some time, blood protein adducts with a longer lifespan have been investigated, and PhIP adducts of serum albumin and haemoglobin from meat-consuming humans were recently reported. Many compounds, like drugs, nicotine and narcotics, bind to melanin in hair and give information on exposure for longer time periods. In mice, PhIP is irreversibly incorporated in a dose-dependent manner into hair, and in humans exposed to an ordinary diet, it was found to vary from <50 to 5000 pg PhIP/g hair. The incorporation is also dependent on the content of eumelanin. The use of PhIP in hair as a biomarker of exposure is promising, but needs validation, using other methods of exposure assessment.     

Problems associated with the determination of heterocyclic amines in cooked foods and human exposure.

Epidemiological studies have shown diet to be an important factor in the global variation of human cancer rates. The presence of mutagenic/carcinogenic heterocyclic aromatic amines (HAs) in cooked foods has attracted a great deal of interest for more than 20 years. Accurate assessment of the human exposure to HAs requires food questionnaires that address cooking methods and reliable methods for the analysis of HAs in cooked foods, and of biomarkers of exposure. The complex food matrix, the low amounts of HAs present (ng/g), and the need for several isolation steps make accurate quantification difficult. Food composition, for example the concentrations and relative amounts of naturally occurring precursors, such as creatine, free amino acids and sugars and also the presence of enhancing or inhibiting compounds are known to greatly influence the formation of HAs. Cooking temperature and time are other important factors that affect the yield of HAs. One of the most abundant HAs, PhIP (2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine), is found typically in amounts up to around 35 ng/g, but there are some reports on much higher levels of PhIP. The levels of other HAs such as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and IFP (2-amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-b]pyridine) generally range from not detectable up to 10 ng/g, and AalphaC (2-amino-9H-pyrido[2,3-b]indole) up to 20 ng/g. Among the factors that influence human exposure to HAs are the type of food, cooking method, portion size and intake frequency. The estimated daily intake of HAs in different studies ranges from 0 to around 15 microg per person per day.     

Heterocyclic aromatic amines in domestically prepared chicken and fish from Singapore Chinese households.

Chicken and fish samples prepared by 42 Singapore Chinese in their homes were obtained. Researchers were present to collect data on raw sample weight, cooking time, maximum cooking surface temperature, and cooked sample weight. Each participant prepared one pan-fried fish sample and two pan-fried chicken samples, one marinated, one not marinated. The cooked samples were analyzed for five heterocyclic aromatic amine (HAA) mutagens, including MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline); 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline); 7,8-DiMeIQx (2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline); PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), and IFP (2-amino-(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine). A paired Student's t-test showed that marinated chicken had lower concentrations of PhIP (p<0.05), but higher concentrations of MeIQx (p<0.05) and 4,8-DiMeIQx (p<0.001) than non-marinated chicken, and also that weight loss due to cooking was less in marinated chicken than in non-marinated chicken (p<0.001). Interestingly, the maximum cooking surface temperature was higher for fish than for either marinated or non-marinated chicken (p<0.001), yet fish was lower in 4,8-DiMeIQx per gram than marinated or non-marinated chicken (p<0.001), lower in PhIP than non-marinated chicken (p<0.05), and lost less weight due to cooking than either marinated or non-marinated chicken (p<0.001). Fish was also lower in MeIQx and 7,8-DiMeIQx than marinated chicken (p<0.05). This study provides new information on HAA content in the Singapore Chinese diet.     

A mechanistic basis for the role of cycle arrest in the genetic toxicology of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of meat, induces tumors of the prostate, colon, and mammary gland when fed to rats. PhIP is readily absorbed and efficiently metabolized to a genotoxic derivative by CYP1 enzymes. Although metabolism and mutational potential of PhIP have previously been well characterized, the intervening cellular and genomic responses to the chemical are not fully understood. We have examined the cellular response to PhIP exposure in human mammary epithelial MCF10A cells, which retain characteristics of normal breast epithelial cells. Because these cells fail to activate PhIP, they were cocultured with a human lymphoblastoid cell line MCL-5, which constitutively expresses CYP1A1, and have been transfected to express human CYPs1A2, 2A6, 3A4, and 2E1. The MCL-5 cells were irradiated (2,000 rads) prior to coculture, rendering them unable to replicate yet still retaining metabolic competency. MCF10A cells were treated (in the presence of MCL-5 cells) with PhIP (1-100 microM) and harvested at various time-points. Compared to DMSO control, treatment (24 or 48 h) with PhIP resulted in a significant dose-dependent fall in cell number. Cells treated for 48 h then cultured in the absence of PhIP (and MCL-5 cells) for a further 6 days showed a much greater dose-dependent reduction in cell number. Flow cytometric analysis indicated that PhIP treatment (48 h) resulted in a dose-dependent accumulation of cells in the G1 population. Western blotting revealed elevated expression of p53 and the cyclin dependent kinase inhibitor p21WAF1/CIP1 after PhIP treatment. Levels of MDM2, a negative regulator of p53, and the hypophosphorylated form of RB were also elevated, consistent with the triggering of G1 cell cycle checkpoint. These cell cycle effects are critical, as they enable cells to effect genome repair, accept mutation, or eliminate excessively damaged cells.     

Insights into the o-acetylation reaction of hydroxylated heterocyclic amines by human arylamine N-acetyltransferases: a computational study

A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures, and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common polymorphisms may affect the structure of this protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclic amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the phosphate-binding loop was found using our models and will be discussed. Quantum mechanical calculations on the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP show that the reaction coordinates differ for these two compounds, but the activation barrier separating the reactant from the product are both low. The results of this study suggest that common polymorphisms in human NAT2 are distant from the active site and are more likely to destabilize the enzyme than affect catalysis. Additionally, the quantum mechanical calculations show that the observed differences in mutagenic activity between N-OH MeIQx and N-OH PhIP are not related to their acetylation reaction with NAT.     

Mechanisms of action of the carcinogenic heterocyclic amine PhIP

Formed during the cooking of meat, the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP) is mutagenic and carcinogenic. Although the metabolism and mutational effects of PhIP are well defined, the early cellular and genomic events by which it can induce neoplastic transformation are not yet fully characterised. These early cellular responses to genotoxic doses of PhIP were examined in a human mammary epithelial cell, MCF10A. Using Western blotting, PhIP was shown to induce expression of the DNA damage response proteins p53 and p21(WAF1/CIP1), and to inhibit cell growth while activating G1 cell cycle checkpoint, a consequence of PhIP-induced DNA damage. Using low doses of PhIP (previously shown to activate oestrogenic signalling), PhIP increased proliferation in the oestrogen receptor (ER)-negative MCF10A cell line and to activate the mitogen-activated protein kinase (MAPK) pathway. Inhibition of this pathway significantly reduced the PhIP-induced cell growth of MCF10A cells. The work presented here suggests that, further to its genotoxic properties, at levels close to human exposure PhIP stimulates cellular signalling pathways that are linked to the promotion and progression of neoplastic disease. It is possible that a combination of these DNA damaging and growth promoting properties provide a mechanism for the tumourigenicity of PhIP, and may be key determinants for the tissue specificity of PhIP-induced carcinogenesis.     

Formation and inhibition of heterocyclic aromatic amines in fried ground beef patties.

The effect of vitamin E and oleoresin rosemary on heterocyclic aromatic amine (HAA) formation in fried ground beef patties was studied. Patties were fried at three temperatures (175 degrees C, 200 degrees C, 225 degrees C) for 6 and 10 min/side to determine the conditions for optimum HAA formation. HAAs were isolated by solid phase extraction and quantitated by HPLC. Greatest concentrations were generated when patties were fried at 225 degrees C for 10 min/side, 31.4 ng/g 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 5.8 ng/g 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Vitamin E, when used at two concentrations (1% and 10% based on fat content) and added directly to the ground beef patties, reduced PhIP concentrations in the cooked patties by 69% and 72%, respectively. Smaller but more variable reductions were achieved for MeIQx. Comparable inhibition of HAA formation was achieved by the direct addition of vitamin E (1% based on fat content) to the surface of the patties before frying. Concentrations of five HAAs studied were all significantly reduced (P<0.006), with average reductions ranging from 45% to 75%. Oleoresin rosemary, when used at two concentrations (1% and 10% based on fat content), reduced PhIP formation by 44%.     

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.     

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.