Intestinal bacteria metabolize the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine following consumption of a single cooked chicken meal in humans.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic amine formed in meats during cooking. Although the formation of PhIP metabolites by mammalian enzymes has been extensively reported, the involvement of the intestinal bacteria remains unclear. This study examined the urinary and fecal excretion of a newly identified microbial PhIP metabolite 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) in humans. The subjects were fed 150 g of cooked chicken containing 0.88-4.7 microg PhIP, and urine and feces collections were obtained during 72 h after the meal. PhIP-M1 and its trideuterated derivate were synthesized and a LC/MS/MS method was developed for their quantification. The mutagenic activity of PhIP-M1, as analyzed using the Salmonella strains TA98, TA100 and TA102, yielded no significant response. Of the ingested PhIP dose, volunteers excreted 12-21% as PhIP and 1.2-15% as PhIP-M1 in urine, and 26-42% as PhIP and 0.9-11% as PhIP-M1 in feces. The rate of PhIP-M1 excretion varied among the subjects. Yet, an increase in urinary excretion was observed for successive time increments, whereas for PhIP the majority was excreted in the first 24h. These findings suggest that besides differences in digestion, metabolism and diet, the microbial composition of the gastrointestinal tract also strongly influences individual disposition and carcinogenic risk from PhIP.

A QSAR for the mutagenic potencies of twelve 2-amino-trimethylimidazopyridine isomers: structural, quantum chemical, and hydropathic factors.

An isomeric series of heterocyclic amines related to one found in heated muscle meats was investigated for properties that predict their measured mutagenic potency. Eleven of the 12 possible 2-amino-trimethylimidazopyridine (TMIP) isomers were tested for mutagenic potency in the Ames/Salmonella test with bacterial strain TA98, and resulted in a 600-fold range in potency. Structural, quantum chemical, and hydropathic data were calculated on the parent molecules and the corresponding nitrenium ions of all of the tested isomers to establish models for predicting the potency of the unknown isomer. The principal determinants of higher mutagenic potency in these amines are: (1) a small dipole moment, (2) the combination of b-face ring fusion and N3-methyl group, (3) a lower calculated energy of the pi electron system, (4) a smaller energy gap between the amine HOMO and LUMO orbitals (Pearson "softness"), and (5) a more stable nitrenium ion. Based on predicted potency from the average of six regression models, the isomer not yet synthesized and tested is expected to have a mutagenic potency of 0.77 revertants/microg in tester strain TA98, which is near the low end of the potency range of the isomers.

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.

Minimization of heterocyclic amines and thermal inactivation of Escherichia coli in fried ground beef.

BACKGROUND: Heterocyclic amine carcinogens are formed during the cooking of a number of foods, especially well-done meats. Lower temperatures and shorter cooking times can minimize the formation of these carcinogens, yet a major food safety concern is that pathogens in the meat must be thermally inactivated. This study investigated cooking techniques that minimize heterocyclic amine formation while simultaneously destroying contaminating bacteria. METHODS: Ground beef patties were inoculated with Escherichia coli K12 bacteria and fried to internal temperatures ranging from 35 degrees C to 70 degrees C in a skillet preheated to 160 degrees C, 180 degrees C, or 200 degrees C. Each patty was then analyzed for four common heterocyclic amines and for surviving bacteria. Additionally, the frequency of turning of the beef patty during cooking was varied (a single turn or multiple turns), length of time required for each patty to reach 70 degrees C was recorded, and heterocyclic amine levels were determined. An additional pan temperature of 250 degrees C was tested for its effect on heterocyclic amine formation but not on bacterial killing. Statistical tests were two-sided. RESULTS: Colony-forming bacteria were reduced by five orders of magnitude at internal temperatures greater than 60 degrees C, regardless of cooking method, and were completely inactivated at 70 degrees C. For patties turned just once, heterocyclic amine levels increased as the cooking temperatures increased. However, levels of heterocyclic amines were statistically significantly lower with turning every minute. For each pan temperature, patties reached 70 degrees C internal temperature sooner when they were turned every minute than when they were turned just once during cooking. CONCLUSION: Lowering the pan temperature and turning the patties frequently can greatly reduce the formation of heterocyclic amines and can simultaneously achieve bacterial inactivation with little or no increase in cooking time, ensuring a product that is safe for human consumption.

Heterocyclic amine content of cooked meat and risk of prostate cancer.

BACKGROUND: Some epidemiologic studies have described positive associations between prostate cancer risk and meat consumption, but underlying mechanisms have not been identified. Heterocyclic amines are mutagens formed during the cooking of meat. Well-done meat has been associated with increased risks of colorectal and breast cancers in humans. This study examined associations between prostate cancer risk and 1) estimated daily intake of heterocyclic amines from cooked meat and 2) level of cooked-meat doneness. METHODS: A population-based, case-control study involving 317 case patients with prostate cancer and 480 age-matched control subjects was carried out in Auckland, New Zealand. Levels of meat doneness and daily intake of heterocyclic amines were determined from self-reported dietary data and experimentally measured heterocyclic amine levels in locally sourced meat samples cooked under controlled conditions to varying degrees of doneness. RESULTS: The heterocyclic amines found in the highest concentrations in meat samples were 2-amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-b]pyridine (IFP) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) from well-done chicken and pork and very well-done beefsteak. Meat doneness was weakly and inconsistently associated with prostate cancer risk for individual types of meat, but increased risk was observed for well-done beefsteak (relative risk = 1.68; 95% confidence interval = 1.02-2.77; two-sided P for trend =.03). A weak positive gradient of increased risk was associated with estimated daily exposure to IFP but not with the other major heterocyclic amines. CONCLUSIONS: Meat doneness and estimated intake of heterocyclic amines from cooked meat were not clearly associated with prostate cancer risk.

Chemical analysis, prevention, and low-level dosimetry of heterocyclic amines from cooked food.

Potent mutagenic and carcinogenic heterocyclic amines are produced from heated food derived from muscle. These compounds are present at part-per-billion levels and consist primarily of the amino-imidazoazaarene class of chemicals. Additional mutagens present in the meat are not as clearly characterized. Commercial fried-beef patties (hamburgers) have low levels of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 0.1-0.68 ng/g meat for MeIQx and slightly lower for 4,8-DiMeIQx. The formation of these heterocyclic amines can be reduced by microwave pretreatment of meat, with the resulting liquid being poured off before frying. The Ames/Salmonella mutagenic activity was reduced to 5-10% of that of non-microwave-treated samples. MeIQx and DiMeIQx concentrations were reduced to 12% and 50% of levels in the non-microwave-treated samples, respectively. MeIQx adducts, as measured by accelerator mass spectrometry, were found to be linear with doses from 5 mg/kg to 500 ng/kg. Linear DNA binding at low doses is important for assuming linear risk estimation from the high animal-feeding doses causing cancer to the low human-dietary exposures. Extrapolating from the rodent TD50 dose to humans gives a maximum credible risk from consumption of heterocyclic amines of approximately 1/1000 exposed individuals.

Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef.

During the cooking of beef, the genotoxic heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed. Little is known about the fate of these compounds in humans or the factors affecting it. We have developed assays based on capillary column gas chromatography-negative ion mass spectrometry capable of the simultaneous measurement of MeIQx, DiMeIQx, and PhIP in cooked meat and in human urine using stable isotope labeled analogues. Ten normal, healthy male volunteers were invited to consume a standard cooked meat meal (400-450 g lean beef, cooked as patties on a griddle hotplate) on four separate occasions over a period of 14 months. Following consumption of the test meals, urine was collected from 0 to 8 h, during which time all free amines were excreted and analyzed for MeIQx, DiMeIQx, and PhIP. Subjects ingested 240 +/- 9 (SEM) g cooked meat, which contained 2.2 +/- 0.2 ng MeIQx/g meat, 0.7 +/- 0.1 ng DiMeIQx/g meat, and 16.4 +/- 2.1 ng PhIP/g meat. The variability in relative systemic bioavailability was assessed from the percentage of ingested amine excreted unchanged in the urine. Subjects excreted 2.1 +/- 1.1% of MeIQx and 1.1 +/- 0.5% of PhIP ingested as unchanged amine in the urine. Levels of DiMeIQx in urine, if present, were below the sensitivity of our assay (20 pg/ml) and could not be detected in any of the samples analyzed. Irrespective of dose, urinary excretion of unchanged MeIQx or PhIP (expressed as a percentage of the ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low and the pooled interday (intrasubject) coefficients of variation for both compounds were only 19 and 3.4%, respectively. In contrast, inter-subject (intraday) variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Based on these studies, it should be possible to use the percentage excretion of MeIQx and PhIP to assess the relative bioavailability of these compounds in humans.

Pan-fried meat containing high levels of heterocyclic aromatic amines but low levels of polycyclic aromatic hydrocarbons induces cytochrome P4501A2 activity in humans.

Heterocyclic aromatic amines (HAAs) are formed when meat juices are pyrolyzed. In humans HAAs are activated in vivo by cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2) to mutagens or carcinogens. While activity of NAT2 is noninducible, exposure to cigarettes, polycyclic aromatic hydrocarbons, and cruciferous vegetables has been shown to induce CYP1A2 activity in humans. To date, it is unknown if pan-fried meat, which is consumed at high levels in the United States, is capable of inducing CYP1A2. In order to address this issue, we measured CYP1A2 and NAT2 activities in 66 healthy nonsmokers (33 males and 33 females) in a controlled metabolic feeding study. The study was designed to minimize the influence of known inducers of CYP1A2. Subjects consumed meat pan-fried at a low temperature (100 degrees C) for 7 days followed by 7 days of meat pan-fried at a high temperature (250 degrees C). The low temperature-cooked meat had undetectable levels of HAAs while the high temperature-cooked meat contained high amounts of HAAs [9.0 ng/g of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2.1 ng/g of 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 32.8 ng/g of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)]. In contrast, total polycyclic aromatic hydrocarbon content was similar in both meat samples (10.7 ng/g in low temperature-cooked meat and 10.1 ng/g in high temperature-cooked meat). At the end of each period, subjects were tested for CYP1A2 and NAT2 enzyme activity by caffeine metabolism phenotyping. NAT2 activity remained unchanged throughout the study while CYP1A2 activity increased in 47 of 65 (72%) of the subjects after consuming high temperature-cooked meat (P < 0.0002), suggesting induction by some compound(s) formed during high temperature cooking. If HAAs are shown to be human carcinogens in epidemiological studies, then meat cooked at high temperatures may pose an increased cancer risk because it contains both inducers of CYP1A2 and procarcinogens MeIQx, DiMeIQx, and PhIP known to be activated by this enzyme.

High concentrations of the carcinogen 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine (PhIP) occur in chicken but are dependent on the cooking method.

Heterocyclic aromatic amines (HAAs) are mutagenic and carcinogenic compounds found in meats cooked at high temperatures. Although chicken is consumed in large quantities in the United States, there is little information on its HAA content. The objective of this study was to measure the five predominant HAAs (IQ, MeIQ, MeIQx, DiMeIQx, and PhIP) in chicken cooked by various methods to different degrees of doneness. Chicken breasts were panfried, oven-broiled, or grilled/barbecued. Whole chickens were roasted or stewed. Skinless, boneless chicken breasts were cooked to three degrees of doneness: just until done, well done, or very well done. High levels of PhIP (ranging from 12 to 480 ng/g cooked meat) were found in chicken breasts when panfried, oven-broiled, and grilled/barbecued but not in while roasted or stewed chicken. PhIP concentration increased in skinless, boneless chicken breast with longer cooking time, higher internal temperature, and greater degree of surface browning. PhIP concentration was also high in chicken breasts cooked with skin and bones. MeIQx and DiMeIQx levels increased with the degree of doneness, whereas IQ and MeIQ were not detectable in any of these chicken samples. Certain cooking methods produce PhIP, a known colon and breast carcinogen in rodents and possibly a human carcinogen, at substantially higher levels in chicken than has been reported previously in red meat.

Heterocyclic amine formation and the impact of structure on their mutagenicity.

The occurrence and formation of heterocyclic amines in foods is discussed in light of the consistent finding of a new class of imidazopyridines. In addition, a quantitative structure-activity relationship will be presented correlating the potency of these imidazopyridines to predicted chemical properties. Although no strong linear correlation is found between the potency and the chemical properties, a low dipole moment is found to be a qualitative predictor of high mutagenic potency.

In vivo human metabolism of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

To better understand the interactions of the pathways of activation and detoxification on the metabolism of the putative carcinogen, PhIP, we administered a dose of 70-84 microg [2-14C] PhIP (17.5 [microCi 14C) 48-72 h before scheduled colon surgery. Blood and urine collected for the next 48-72 h was evaluated by linear accelerator mass spectroscopy (AMS) and scintillation counting LC-MS to identify specific PhIP metabolites. The thermostable phenol sulfotransferase (SULT1A1) phenotype was correlated with the 4'-PhIP-SO4 levels in the urine at 0-4 h (R = 0.86, P = 0.059). The CYP1A2 activity had a negative correlation with PhIP serum levels at 1 h (R = 0.94, P = 0.06) and a positive correlation with urine N-OH-PhIP levels at 0-4 h (R = 0.85, P = 0.15). This low level radioisotope method of determining the influence of phenotype on metabolism will significantly improve our understanding of the interrelationships of these pathways and provide a critical foundation for the development of individual risk assessment.

Mutagenic activity of heterocyclic amines in cooked foods.

Mutagenic heterocyclic amines are generated in foods when they are cooked at temperatures over 150 degrees C. These compounds are present from 0.1 to 50 ppb, depending on the food and cooking conditions. These heterocyclic amines are not only present in cooked red meat, fish, and chicken, but are also present at lower levels in baked and fried foods derived from grain. Mutagenicity of fried beef hamburgers cooked at 230 degrees C is 800 +/- 37 TA98 revertants per gram cooked weight. We measured 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMelQx), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) formation at this temperature and found 3.0 +/- 2.0, 1.0 +/- 0.18, and 0.06 +/- 0.03 ng/g, respectively. 2-amino-1-methyl-6-phenylimidaz[4,5-b]pyridine (PhIP) was found at a higher concentration of 9.6 ng/g. In our laboratory we have shown these heterocyclic amines are capable of producing both reverse and forward mutations in Salmonella bacteria and forward mutations in Chinese hamster ovary cells (CHO). We have also been able to show a statistically significant increase in mutations in the pancreas of the "mutamouse" following PhIP exposure. The pancreas also shows relatively high DNA binding compared to other organs in the mouse. The number and type of mutations depend on the repair capacity of the cells for both Salmonella and CHO. In Salmonella the mutations are primarily 2-base deletions when the cells lack uvrB repair, but mutations are more complex (larger deletions and insertions) but lower in frequency when repair is functional.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the mutagens in cooked beef.

The purification of cooking mutagens depends on the extraordinary sensitivity of the Ames/Salmonella mutagenicity test and its usefulness for tracking the mutagens during the purification steps. Following aqueous/acid (pH 2) extraction of fried ground beef (cooked at 200, 250, or 300 degrees C), XAD-2 column adsorption and elution with acetone, and acidic and basic liquid/liquid extractions, the samples are separated into six distinct peaks with preparative reverse-phase HPLC. A total of nine distinct mutagens can be separated after two additional HPLC steps. These compounds fall into a class of compounds called aminoimidazoazaarenes (AIAs). The majority of the mutagenic activity is made up of MeIQx1 (m/z 213, C11H11N5), DiMeIQx (m/z 227, C12H13N5), trimethylimidazopyridine (TMIP) (m/z 176, C9H12N4) and phenylimidazopyridine (PhIP) (m/z 224, C13H12N4). Smaller contributions are from IQ (m/z 198, C11H10N4), MeIQ (m/z 213, C12H12N4), a nonpolar peak containing oxygen and two unidentified trace polar mutagens. Mass estimates (per kilogram uncooked beef) include: 15 micrograms for PhIP, 1.0 micrograms for MeIQx, 0.5 microgram for DiMeIQx, and 0.02 microgram for IQ. Because of the uncoupling of mutagenic and carcinogenic potencies of these aromatic amines, the PhIP, which contributes the highest mass content to the cooked meat, but has the lowest mutagenic potency, might ultimately make a significant contribution to the carcinogenicity.

Extended quantitative structure-activity relationships for 80 aromatic and heterocyclic amines: structural, electronic, and hydropathic factors affecting mutagenic potency.

The mutagenic/carcinogenic heterocyclic amines formed during the cooking of protein foods have been determined to be probable or possible human carcinogens. As part of a comprehensive study of the food mutagens, our laboratory has produced a series of quantitative structure-activity relationships (QSARs) of aromatic and heterocyclic amines, to attempt to elucidate the mechanisms of mutagenesis/carcinogenesis. Amines are genotoxically active only after activation by a series of reactions converting the parent compound to an electrophilic derivative, which is postulated to be a nitrenium ion that covalently binds to and damages DNA. An important agent in this conversion is cytochrome P450. In this report we develop a QSAR for 80 amines of diverse structure and a range of 10 orders of magnitude in mutagenic potency. New structural factors and quantum chemical ab initio and Hückel calculations are included. The results are interpreted to show that a main determinant of mutagenic potency is the extent of the aromatic pi-electron system. Small contributions are made by both the dipole moment and the calculated stability of the nitrenium ion. Multiple linear regression models account for nearly two-thirds of the variance in potency, leaving room for additional unknown factors. The role of cytochrome P450 1A in amine toxification is supported, and further theoretical and experimental research on its reaction mechanisms and modeling of its active site are proposed.

Quantitative structure-activity relationships of heterocyclic amine mutagens formed during the cooking of food.

The major protein-rich foods, particularly muscle meats, contain part-per-billion quantities of potent mutagens formed by frying or broiling to a well-done state. Related mutagens are formed by pyrolysis of amino acids or proteins and in heated model systems. The thermic mutagens so far identified are heterocyclic aromatic amines of aminoimidazo-azaarene (AIA) and aminocarboline classes. The chemicals require activation by enzymes to form metabolites reactive with nucleic acids. These thermic mutagens, and numerous synthetic congeners, exhibit an enormous range of potency as frameshift mutagens in the Ames/Salmonella assay. However, structural variations are nominal within the two classes. Structural parameters that appeared relevant to determining potency were selected for 38 AIAs and 23 amino-carbolines. For the AIA class these were: the number of fused rings, the number of heteroatoms in Rings 2 and 3, methyl substitution on imidazo ring nitrogen atoms, and methyl substitution on ring carbon atoms. For the amino-carboline class the structural parameters were: the position of the pyridine-type nitrogen atom in Ring 1, the substitution position of the exocyclic amino group on Ring 1, and methyl substitution on ring carbon atoms. These structural parameters may influence mutagenic potency in the following ways. 1) Electronic or steric effects may determine the reactivity and stability of the ultimate mutagenic metabolite. Optimal balance of reactivity and lifetime of this transient intermediate may be required for access to and reaction with nuclear DNA to cause mutations. 2) Substitution on the rings may block detoxication reactions. The structural parameters identified should prove useful in predicting the mutagenicity of untested compounds of these types.

Characterization of mutagenic activity in instant hot beverage powders.

Extracts of several grain-based coffee-substitute blends and instant coffees were mutagenic in the Ames/Salmonella test using TA98, YG1024, and YG1029 with metabolic activation. The beverage powders induced 150 to 500 TA98 and 1,150 to 4,050 YG1024 revertant colonies/g, respectively. Increased sensitivity was achieved using strain YG1024. No mutagenic activity was found in instant hot cocoa products. The mutagenic activity in the beverage powders was shown to be stable to heat and the products varied in resistance to acid nitrite treatment. Differential bacterial strain specificity, and a requirement for metabolic activation suggest that aromatic amines are present. Characterization of the mutagenic activity, using HPLC and the Ames test of the collected fractions, showed the coffee-substitute blends and instant coffees contain several mutagenic compounds. Known heterocyclic amines are not responsible for the major part of the mutagenic activity. The main mutagenic activity in grain-based coffee-substitute blends and instant coffees is due to several unidentified compounds, which are most likely aromatic amines.

Structure-mutagenicity relationships of four amino-imidazonaphthyridines and imidazoquinolines.

We tested four isomeric imidazonaphthyridines and one imidazoquinoline compound for mutagenic activity in the Ames/Salmonella mutagenicity assay, using strain TA98 and strain YG1024, an analogue of strain TA98 with elevated O-acetyltransferase levels. Their potency was related to calculated electronic parameters. Five compounds with a linear arrangement of 3 rings showed a positive response in strain YG1024. Compound 2 (1-methylimidazo[4,5-b][1,7]naphthyridin-2-amine) is the most mutagenic in both strains, giving specific activities of about 200 and 30 revertants per microgram in strains YG1024 and TA98, respectively. Three of the compounds were weak mutagens, giving a positive dose-response only in strain YG1024, with 3-5 revertants per microgram. A higher response of all five compounds in strain YG1024 as opposed to TA98 indicates that they require O-acetyltransferase activity for their metabolism. Mutagenic potencies in strain YG1024 were positively correlated to the energy of the LUMO (lowest unoccupied molecular orbital) of the nitrenium ion.

Liquid chromatography-tandem mass spectrometry method of urine analysis for determining human variation in carcinogen metabolism.

We developed a solid-phase extraction LC-MS-MS method for the analysis of the four major metabolites of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in human urine after a meal of well-done chicken. Ten volunteers each ate either 150 or 200 g of well-done chicken breast containing 9-21 microg of PhIP. Among the individual volunteers there is 8-fold variation in the total amount of metabolites and 20-fold variation in the relative amounts of individual metabolites, showing individual differences in carcinogen metabolism. PhIP metabolites were also detected in urine from a subject consuming chicken in a restaurant meal, demonstrating the method's sensitivity after real-life exposures.

Analysis of foods for heterocyclic aromatic amine carcinogens by solid-phase extraction and high-performance liquid chromatography.

Carcinogenic and mutagenic heterocyclic aromatic amines (HAA) are natural products often present at ng/g levels in muscle meats when they are cooked at temperatures over 150 degrees C. Using solid-phase extraction and high performance liquid chromatography (HPLC) with photodiode array UV detection, samples were analyzed for the following heterocyclic amines: DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline); IQ (2-amino-3-methylimidazo[4,5-f]quinoline); MelQx (2- amino-3,8-dimethylimidazo[4,5-f]quinoxaline); and PhIP (2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine). Quality control samples, analyzed periodically over two years in a blind study, show relative standard deviations ranging from 22 to 38% for the compounds found, variations typical for analysis at ng/g levels. Amounts range from undetectable levels (less than 0.1 ng/g) to hundreds of ng/g of PhIP for frying or grilling at high meat surface temperatures. Beef, chicken, pork and lamb can all have greater than 10 ng/g of PhIP. Ground chicken breast meat has lower amounts of heterocyclic amines than intact muscle pieces of the same size cooked identically. Restaurant prepared samples that we analyzed contained undetectable levels up to 14 ng/g total heterocyclic amines for a beef steak sample. Not extracted with the above method are related mutagenic heterocyclic amines, which have been reported in cooked foods in our laboratory and others. Method development using ion exchange on an SCX solid-phase extraction cartridge shows promise in providing a method for the quantitation of these mutagenic dimethyl-, trimethyl- and furo-imidazopyridines where a practical analysis method is needed.

PhIP metabolites in human urine after consumption of well-cooked chicken.

We devised an assay to quantify the metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human urine following a single exposure to well-cooked meat. Our method uses LC/MS/MS to detect four metabolites and four deuterated internal standard peaks in a single chromatographic run. N2-OH-PhIP-N2-glucuronide was the most abundant urinary metabolite excreted by the 12 individuals who participated in our study. N2-PhIP glucuronide was the second most abundant metabolite for 8 of the 12 volunteers. The stability of PhIP metabolism over time was studied in three of the volunteers who repeated the assay eight times over a 2.5 year-period. PhIP metabolite excretion varied in each subject over time, although the rate of excretion was more constant. Our results suggest that quantifying PhIP metabolites should make future studies of individual susceptibility and dietary interventions possible.