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.

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.

N-glucuronidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and N_hydroxy-PhIP by specific human UDP-glucuronosyltransferases.

Glucuronidation is a major metabolic pathway in the biotransformation of many xenobiotics. Recent studies have shown that in humans, UDP-glucuronosyltransferase (UGT)-mediated glucuronidation plays a critical role in the detoxification of food-borne carcinogenic heterocyclic amines. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant carcinogenic heterocyclic amine found in well-cooked meats, has been shown to be extensively glucuronidated in humans. To determine which UGT isozymes are involved in the biotransformation of PhIP and the cytochrome P4501A2-mediated reactive intermediate N-hydroxy-PhIP, microsomes expressing human UGT1A1, -1A4, -1A6 or -1A9 were incubated with PhIP and N-hydroxy-PhIP and the reaction products analyzed by HPLC and ESI-MS. Incubations containing N-hydroxy-PhIP and UGT1A1 expressing microsomes, with an apparent Km of 4.58 microM and a Vmax of 4.18 pmol/min/mg protein, had the highest capacity to convert N-hydroxy-PhIP to N-hydroxy-PhIP-N2-glucuronide. Microsomes expressing UGT1A9 produced N-hydroxy-PhIP-N3-glucuronide at the highest rate with an apparent Km and Vmax of 3.73 microM and 4.07 pmol/min/mg, respectively. A third previously undefined glucuronide accounted for 31% of the total glucuronides formed from the UGT1A4 expressing microsomes. No glucuronide conjugates were detected from microsomes expressing UGT1A6. Incubations containing PhIP as substrate formed direct PhIP-glucuronides in microsomes expressing UGT1A1, UGT1A4 and UGT1A9 but at levels averaging 53-fold lower than when N-hydroxy-PhIP was used as the substrate. Knowing the glucuronidation capacity of the specific UGT isozymes involved in PhIP and N-hydroxy-PhIP glucuronidation should help in determining the individual susceptibility to the potential cancer risk from exposure to PhIP.

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.

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.

Identification of urine metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine following consumption of a single cooked chicken meal in humans.

Many studies suggest that mutagenic/carcinogenic chemicals in the diet, like 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), may play a role in human cancer initiation. We have developed a method to quantify PhIP metabolites in human urine and have applied it to samples from female volunteers who had eaten a meal of cooked chicken. For this analysis, urine samples (5 ml) were spiked with a deuterium-labeled internal standard, adsorbed to a macroporous polymeric column and then eluted with methanol. After a solvent exchange to 0.01 M HCl, the urine extracts were passed through a filter, applied to a benzenesulfonic acid column, washed with methanol/acid and eluted with ammonium acetate and concentrated on a C(18) column. The metabolites were eluted from the C(18) column and quantified by LC/MS/MS. In our studies of human PhIP metabolism, eight volunteers were fed 200 g of cooked chicken containing a total of 27 microg PhIP. Urine samples were collected for 24 h after the meal, in 6 h aliquots. Although no metabolites could be found in urine collected from volunteers before eating the chicken, four major human PhIP metabolites, N:(2)-OH-PhIP-N:(2)-glucuronide, PhIP-N:(2)-glucuronide, 4'-PhIP-sulfate and N:(2)-OH-PhIP-N:3-glucuronide, were found in the urine after the chicken meal. The volunteers in the study excreted 4-53% of the ingested PhIP dose in the urine. The rate of metabolite excretion varied among the subjects, however, in all of the subjects the majority of the metabolites were excreted in the first 12 h. Very little metabolite was detected in the urine after 18 h. In humans, N:(2)-OH-PhIP-N:(2) glucuronide is the most abundant urinary metabolite, followed by PhIP-N:(2)-glucuronide. The variation seen in the total amount, excretion time and metabolite ratios with our method suggests that individual digestion, metabolism and/or other components of the diet may influence the absorption and amounts of metabolic products produced from PhIP.

Genetically modified Chinese hamster ovary cells for investigating sulfotransferase-mediated cytotoxicity and mutation by 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine.

To test the hypothesis that the sulfotransferase gene plays a role in the phase II bioactivation of PhIP, a heterocyclic amine found in cooked meats, we transfected the UV5P3 cell line with cDNA plasmids of human aryl sulfotransferases (HAST1 and HAST3). UV5P3 is a nucleotide excision repair-deficient and P4501A2-expressing CHO cell line that we have previously developed. Functionally transformed clones were identified by the differential cytotoxicity (DC) assay that used PhIP as the cytotoxic agent. Two clones designated 5P3H1 and 5P3H3, expressing HAST1 and HAST3, respectively, were chosen for further characterization. Correct fragment sizes of the sulfotransferase cDNAs were identified in both cell lines by polymerase chain reaction. Immunoblot analysis confirmed the expression of the sulfotransferase proteins. The addition of the sulfotransferase inhibitor DCNP decreased the cytotoxic effects of PhIP in a dose-dependent manner. The increase in cell growth was 6. 5-fold for 5P3H1 and 2.4-fold for 5P3H3, relative to values obtained without DCNP. Based on D(50) values, the dose that reduced the survival to 50% relative to untreated controls, the cytotoxic effect of PhIP was increased threefold for 5P3H1 and 1.87-fold for 5P3H3 cell lines over the parental UV5P3 line. There was also a small increase in the mutation response at the aprt locus. These newly established 5P3H1 and 5P3H3 sulfotransferase-expressing cells provide valuable mechanistic information of the bioactivation of PhIP and related compounds. Environ. Mol. Mutagen. 35:57-65, 2000. Published 2000 Wiley-Liss, Inc.

Occupational health in the USA in the 21st century.

Comparable to the confusion encountered in the birth of the machine age is the perplexing reconfiguration of the United States' health care system. Paralleling the advances in medicine have been the divesting mergers and downsizing of industry, coupled with globalization, which have released millions of long-time workers. The labour contingent is changing, with the addition of great numbers of women and immigrant workers, and the manufacturing economy has become one of service and information. Serving the occupational health (OH) needs of such a force have been the professional societies of physicians, nurses, and industrial hygienists, with their members providing care in a broad variety of facilities. It is possible that a national organization, including all these disciplines, would have a greater voice in the protection of workers' health. Immediate leadership of an occupational health service (OHS) can be rotated among the disciplines, so that competition for primacy among the professionals would end. The new workforce demands culture sensitivity among OH personnel and polylingual capabilities may be demanded in the future. Management skills will be required of all in OH, and greater participation of employees in OH policy will characterize the decades ahead. Nearly neglected up to now, occupational mental health programming will be required to meet the real needs of workers, and to counter the move to outsource OH services, where little patient contact results. Behavioural safety, total quality management, and application of the rapidly developing technologies in health care will define the 21st century efforts in OH. Remaining issues, such as violence, telecommuting injuries, the inclusion of alternative medicine, and women's health, among others, will see carry-over for resolution into the year 2000.

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.

Formation of mutagenic/carcinogenic heterocyclic amines in dry-heated model systems, meats, and meat drippings.

Mixtures of amino acids, creatine, and glucose simulating the composition of six different kinds of meats (beef, chicken breast, chicken thigh, turkey breast, pork, and fish) were dry-heated to simulate the formation of heterocyclic amines in meats. The presence of 16 heterocyclic amines was investigated in the model systems and in the six meats and their corresponding meat drippings to determine the importance of meat composition to heterocyclic amine formation. Nine mutagenic amines (IQ, MeIQ, 8-MeIQx, 4,8-DiMeIQx, PhIP, IQx, IFP, DMIP, and TMIP) were found to be present at concentrations >0.1 ng/g in some of the model systems and in some of the meats or pan residues. Heterocyclic amine concentrations clearly are affected by precursor composition in this model system, and the same nine heterocyclic amines formed in the meat and in the model system show that this is a well-controlled surrogate for the reaction conditions that occur in meats during cooking.

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.

Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.

Factors determining dietary intakes of heterocyclic amines in cooked foods.

The identification of heterocyclic amines (HCAs) in cooked foods has focused attention on the potential health effects from their consumption in the diet. Recent studies have estimated daily dietary intakes of HCAs that vary 10-fold and implicated different cooked meats as the prime source of HCAs in the diet. These varied estimates can be attributed to the different dietary assessment methods used in these studies, as well as the different levels of HCAs ascribed to the most commonly consumed cooked meats. Epidemiological studies utilizing information on dietary practice and food intake have found higher risks for several cancers among individuals consuming the highest levels of HCAs. These studies have highlighted the importance of using information on cooking methods in addition to food intake to accurately estimate dietary exposure to HCAs.

DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Epidemiology studies have indicated that certain dietary components, including well-cooked meat, are risk determinants for colon cancer. Cooked meat can contain significant quantities of heterocyclic aromatic amines (HCAs), which have been established as carcinogens in laboratory animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is usually the most mass-abundant HCA, with concentrations up to 480 ppb. We used accelerator mass spectrometry to establish whether DNA and protein adducts can be detected in humans exposed to a quantity of PhIP comparable with levels of exposure that occur in the diet. Five human volunteers were administered a dietary-relevant dose of [14C]PhIP (70-84 microg) 48-72 h before surgery for removal of colon tumors. Blood samples were collected at various time points, and albumin, hemoglobin, and WBC DNA were extracted for analysis by accelerator mass spectrometry. Tissue samples were collected during surgery and used to assess either tissue available doses of [14C]PhIP or adduct levels. The results of this study show: (a) PhIP is activated to a form that will bind to albumin, hemoglobin, and WBC DNA in peripheral blood. WBC DNA adducts were unstable and declined substantially over 24 h; (b) PhIP is bioavailable to the colon, with levels in normal tissue in the range 42-122 pg PhIP/g tissue; and (c) PhIP binds to both protein and DNA in the colon. DNA adduct levels in the normal tissue were 35-135 adducts/10(12) nucleotides, which was significantly lower than tumor tissue. The results of this study demonstrate that PhIP is bioavailable to the human colon following defined dietary-relevant doses and forms DNA and protein adducts.

Effects of a chargrilled meat diet on expression of CYP3A, CYP1A, and P-glycoprotein levels in healthy volunteers.

BACKGROUND & AIMS: Carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons present in chargrilled meat are substrates for inducible CYP1A and CYP3A enzymes and for P-glycoprotein. We examined whether consumption of a chargrilled meat diet results in induction of these proteins. METHODS: Ten healthy adults were fed a diet enriched with chargrilled meat for 7 days. Duodenal biopsy specimens were obtained on days 1, 5, and 12 and analyzed for CYP1A, CYP3A, and P-glycoprotein messenger RNA (mRNA) and protein. On days 5 and 12, hepatic CYP3A4 and CYP1A2 activities were measured and colon biopsies were performed. The levels of polycyclic aromatic hydrocarbon DNA adducts in peripheral blood mononuclear cells were measured on days 1, 4, 11, and 26. RESULTS: There was no detectable induction of CYP3A4, CYP3A5, or P-glycoprotein mRNAs or protein in small intestine or colon and no induction of hepatic CYP3A4 enzyme activity. In contrast, the chargrilled meat diet resulted in unequivocal induction of CYP1A enzymes in the liver and small intestine of each subject. There was an inverse correlation between the level of peripheral polycyclic aromatic hydrocarbon DNA adducts measured on day 11 and both liver CYP1A2 activity (P = 0.027) and enterocyte CYP1A1 protein concentration (P = 0.046). CONCLUSIONS: Ingestion of chargrilled meat results in induction of CYP1A enzymes but not CYP3A4 or P-glycoprotein. This observation, combined with the correlation between adduct levels and CYP1A expression, supports an adaptive role for CYP1A but not CYP3A4 or P-glycoprotein.

Food heating and the formation of heterocyclic aromatic amine and polycyclic aromatic hydrocarbon mutagens/carcinogens.

Heterocyclic aromatic amines (HAA) and polycyclic aromatic hydrocarbons (PAH) are mutagens and animal carcinogens sometimes formed when foods are heated or processed. Determining their role in cancer etiology depends on comparing human exposures and determining any significant dose-related effects. Chemical analysis of foods shows that flame-grilling can form both PAH and HAA, and that frying forms predominantly HAA. With detection limits of about 0.1 ng/g, amounts found in commercially processed or restaurant foods range from 0.1 to 14 ng/g for HAA, and levels of PAH up to 1 ng/g in a liquid smoke flavoring. Laboratory fried samples have greater amounts of PAH, up to 38 ng/g in hamburgers, and high levels of HAA, over 300 ng/g, are measured in grilled chicken breast. Understanding the processing conditions that form PAH and HAA can lead to methods to greatly reduce their occurrence in processed foods.