Journal of Chromatography B (v.802, #1)
FMiii: Full-title Page (iii).
Heterocyclic aromatic amines—still a challenge for scientists by S. Knasmüller; M. Murkovic; W. Pfau; G. Sontag (1-2).
Formation of heterocyclic aromatic amines in model systems by M. Murkovic (3-10).
Heterocyclic aromatic amines (HAs) are mutagenic and carcinogenic substances that are formed in significant amounts during heating of meat or fish at temperatures of at least 150 °C. To investigate the chemistry lying behind the formation of these harmful substances model systems were established. The first aim was to identify the naturally occurring precursors, namely creatinine, amino acids and carbohydrates. Later these model systems were used to develop strategies for a reduction of the content of the heterocyclic aromatic amines and for the evaluation of the reaction mechanisms that lead to the formation of these substances. All these aspects are discussed in this review.
Keywords: Model systems; Heterocyclic aromatic amines;
Formation and stability of heterocyclic amines in a meat flavour model system by M. Bordas; E. Moyano; L. Puignou; M.T. Galceran (11-17).
A model system based on a commercial meat flavour was used to evaluate the formation of heterocyclic amines, simulating the application of this seasoning in household cooking. The effects of different treatments in both dry and aqueous conditions were studied. The lyophilized meat flavour extract was heated at temperatures ranging between 100 and 200 °C for times ranging from 10 min to 2 h. Similarly, an aqueous suspension of the extract was heated at 175 °C for 1, 2 and 3 h. Precursors of HAs, such as creatinine, glucose, and the amino acids glycine, alanine and phenylalanine were added to the meat extract and their effect was tested by heating the mixture at 200 °C for 30 min, when dry conditions were used, and at 175 °C for 2 h in wet systems. All conditions led to the formation of HAs, PhIP being the amine that was detected at the highest level of concentration in most model systems (i.e. 173 ng g−1 at 200 °C, 30 min). Moreover, the addition of creatinine and amino acids to the meat extract flavour produced an important increase in IQ and MeIQx content.
Keywords: Food analysis; Heating temperature; Heating time; Precursors; Heterocyclic aromatic amines;
Evaluation of a new model system for studying the formation of heterocyclic amines by C. Messner; M. Murkovic (19-26).
Heterocyclic amines (HAs) are an important class of food mutagens and carcinogens, which can be found in cooked meat and fish. Increasing heating temperatures and times usually increase mutagenic activity in meat and meat extracts during cooking. We developed a model system, which allows to examine the effects of precursor composition and heating conditions (time and temperature) on the formation of HAs in meat. Homogenized and freeze dried meat samples (beef, pork chops, chicken breast and turkey breast) are heated with diethylene glycol in closed vials under stirring in a thermostated heating block. After an appropriate sample preparation (extraction and clean-up) ten different HAs were measured by HPLC analyses with gradient elution and mass selective detection. The time courses of HA-formation in the different kinds of meat at varying heating temperatures were determined up to heating times of 30 min. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was the most abundant HA in these experiments and reached the highest concentrations in the beef meat samples, as did the other HAs (MeIQ, AαC) at 220 °C in the heating block under stirred conditions. Additionally the influence of the antioxidant TBHQ (t-butylhydroquinone) on the formation of HAs in the model system was tested. However TBHQ effected only slight reductions of HA formation in all kinds of meat.
Keywords: Model system; Heterocyclic aromatic amines; 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; Food mutagens; Antioxidants;
Effects of heating time and antioxidants on the formation of heterocyclic amines in marinated foods by C.M Lan; T.H Kao; B.H Chen (27-37).
The effect of heating time and antioxidants on the heterocyclic amine (HAs) formation in marinated foods were studied. Food samples were cooked at 98±2 °C for 1, 2, 4, 8, 16 and 32 h in a closed pan in the presence of water, soy sauce and rock candy with or without antioxidants. The various HAs in marinated food samples and juice were analyzed by HPLC with photodiode-array detection. Results showed that the amount of HAs formed during heating followed an increased order for each increasing heating time. A larger variety and higher amount of HAs were generated in marinated pork when compared to marinated eggs and bean cake. In marinated juice, the levels of HAs were present in greater amount than in marinated foods. The incorporation of antioxidants Vitamin C, Vitamin E and BHT were found to be effective towards HAs inhibition, however, the effect was minor.
Keywords: Heating time; Food analysis; Antioxidants; Heterocyclic aromatic amines;
Blue cotton, Blue Rayon and Blue Chitin in the analysis of heterocyclic aromatic amines—a review by Kerstin Skog (39-44).
Heterocyclic amines (HCAs) are a group of compounds formed when protein-rich foods, such as meat or fish, are prepared under normal cooking conditions, such as frying, grilling, or broiling. To evaluate and estimate the risks associated with HCAs contained in the diet, it is important to determine the levels in cooked foods, and the levels of HCAs and metabolites in the body. HCAs are normally found at low amounts in a complex matrix, which necessitates a good purification method and a sensitive detection system. The objective of this review was to briefly present the current knowledge on the use of Blue Cotton, Blue Rayon and Blue Chitin in the analysis of HCAs.
Keywords: Blue cotton; Blue rayon; Blue chitin; Heterocyclic aromatic amines;
Evaluation of reversed-phase columns for the analysis of heterocyclic aromatic amines by liquid chromatography—electrospray mass spectrometry by E Barceló-Barrachina; E Moyano; L Puignou; M.T Galceran (45-59).
Liquid chromatography coupled to mass spectrometry (LC–MS), especially by the use of electrospray ionisation source (ESI), is currently used for the analysis of heterocyclic aromatic amines (HAs) in complex samples. The present paper describes the study of the performance of different narrow-bore reversed-phase columns to achieve the best chromatographic separation for the determination of 16 HAs by LC–ESI-MS in food samples. Different parameters such as peak symmetry, resolution and number of theoretical plates have been evaluated for each column, using different chromatographic conditions. The column that provided the best results was TSK Gel® Semi-Micro ODS-80TS of Tosohaas. Quality parameters have been established, obtaining good short-term precision in all cases (relative standard deviation (R.S.D.) lower than 7.7%) and low limits of detection (<13 pg injected in MS and <16 pg injected in MS/MS). The content of HAs in two beef extracts have been determined.
Keywords: Food analysis; Heterocyclic aromatic amines;
Preparation of a beef-extract as a laboratory reference material for the determination of heterocyclic amines by E Bermudo; R Busquets; E Barceló-Barrachina; L Puignou; F.J Santos; M.T Galceran (61-68).
The present paper describes the preparation of a suitable laboratory reference material (LRM) to validate analytical methods for the determination of heterocyclic amines (HAs) in foods. Three different lots of reference material were prepared using a beef extract which was contaminated with a well-known quantity of amines at different levels ranging from 10 to 75 ng/g. These materials were then lyophilised under determined conditions and, after grinding and sieving, homogenised and, finally, bottled and labelled. Homogeneity and stability studies were performed and no statistical differences were observed in the analysis of variances for within- and between-bottle results, thus demonstrating the homogeneity of the material. Stability at different storage temperatures (−18, +4, +25 and +40 °C) and times (1, 2, 3 and 6 months) was also tested. Therefore, the material can be considered homogeneous and stable and can be proposed for use in inter-comparison exercises for the determination of HAs.
Keywords: Food analysis; Laboratory reference material; Heterocyclic aromatic amines;
Analysis of heterocyclic amines in food products: interlaboratory studies by F.J Santos; E Barceló-Barrachina; F Toribio; L Puignou; M.T Galceran; E Persson; K Skog; C Messner; M Murkovic; U Nabinger; A Ristic (69-78).
A feasibility study and two interlaboratory exercises on the determination of selected heterocyclic amines (HAs) in beef extract, organised in the framework of a European project, are presented. The aim of these exercises was to improve the quality of the laboratories and to evaluate the performance of a standardised analytical method and also the methods currently used by each of the participants for the analysis of these compounds. Three lyophilised portions of a commercial beef material previously spiked with HAs at different concentration levels ranging from 10 to 75 ng g−1 were used as laboratory reference materials (lot A, B and C). Firstly, a feasibility study was carried out using a test standard solution and the beef extract (lot A), which contained only five HAs. Then, two interlaboratory exercises were carried out using the laboratory reference materials lot B and lot C, containing 10 selected HAs at two different concentration levels, 75 and 10 ng/g, respectively. The results obtained by all participant laboratories using the proposed method showed satisfactory agreement and the CV(%) between-laboratories obtained were from 8.3 to 24.1% for lot B and from 8.7 to 44.5% for lot C. The standardised method evaluated in these collaborative studies is therefore proposed for the analysis of HAs in food material. Moreover, LC–MS is recommended as the most suitable technique for the analysis of a large number of HAs in food samples.
Keywords: Food analysis; Inter-laboratory studies; Heterocyclic aromatic amines;
Occurrence of heterocyclic amines in several home-cooked meat dishes of the Spanish diet by R Busquets; M Bordas; F Toribio; L Puignou; M.T Galceran (79-86).
Heterocyclic amines (HAs) were determined in several of the most frequently eaten meat dishes in Spain such as fried beef hamburger, fried pork loin, fried chicken breast, fried pork sausages, griddled chicken breast, griddled lamb steak and griddled beef steak. All of the products tested were household cooked. The HAs were analysed in the selected meat dishes using an analytical method based on solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry. DMIP, MeIQx, 4,8-DiMeIQx, Norharman, Harman, PhIP, Trp-P-1, AαC and MeAαC were the amines most frequently found at concentrations of up to 47 ng g−1 of cooked meat. Glu-P-2, IQ, MeIQ, Glu-P-1, 7,8-DiMeIQx and Trp-P-2 were only found in a few of the meat dishes and their concentrations were lower than 1 ng g−1 of cooked meat. The highest amounts of HAs, especially PhIP and DMIP, were formed in fried chicken breast and the lowest were formed in fried beef hamburger and in fried pork sausages. Daily intake of HAs in Spain was estimated at 606 ng of mutagenic HAs per capita and day, DMIP and PhIP being the main contributors.
Keywords: Food analysis; Heterocyclic aromatic amines;
Determination of less polar heterocyclic aromatic amines in standardised beef extracts and cooked meat consumed in Austria by liquid chromatography and fluorescence detection by A Ristic; M Cichna; G Sontag (87-94).
An analysis method was developed for the determination of trace levels of less polar heterocyclic aromatic amines (HAs) in food samples. The development started from a frequently used sample pre-treatment scheme which was slightly improved to make it applicable with high-performance liquid chromatography (HPLC) with fluorescence detection. The method was applied for the analysis of a standardised beef extract containing 5–15 ng/g of HAs and the results are compared with those of the other participants in the same European project. In addition, the method was used for the analysis of less polar HAs in cooked meat consumed in Austria.
Keywords: Food analysis; Heterocyclic aromatic amines;
Determination of heterocyclic aromatic amines (HAs) content in samples of household-prepared meat dishes by L. Warzecha; B. Janoszka; U. Błaszczyk; M. Stróżyk; D. Bodzek; C. Dobosz (95-106).
Aminoazaarene content was investigated in 10 meat samples (including pork, beef, turey and chicken) thermally processed at home according to common recipes used by residents of Upper Silesia region in Poland. The clean-up procedure included tandem solid-phase extraction (SPE) using Extrelut-type columns filled with diatomaceous earth, propylsulphonic acid and chemically bounded phase-C18. Identification and quantitative analysis of HAs fraction was carried out using a HPLC system with DAD-type detector. Separation was achieved using TSK-gel ODS 80-TM column and a mixture of 5% acetonitrile and 95% triethylamine phosphate buffer (pH 3.3) as a mobile phase. The results of qualitative determinations were confirmed by GC–MS method. To achieve this, HAs fractions were derivatized to pentafluoropropionic acid (PFPA) amide derivatives. The summary content of five aminoazaarenes determined in investigated meat samples, i.e. 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), 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) falls within the range of 1.9–77.4 ng/g of sample. The calculated values of theoretically daily human exposure to five determined HAs were in the range of 0.2–7.7 μg per day per person.
Keywords: Food analysis; Heterocyclic aromatic amines; Aminoazaarenes;
Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection by U Gerbl; M Cichna; M Zsivkovits; S Knasmüller; G Sontag (107-113).
This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AαC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm×2 mm, 4 μm and LiChrospher 60 RP-select B, 250 mm×4 mm, 5 μm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and—after isolation of the analytes by several clean-up steps—for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.
Keywords: Heterocyclic aromatic amines; Coulometric electrode array detection; Food analysis;
Exposure to β-carbolines norharman and harman by W Pfau; K Skog (115-126).
The aromatic β-carbolines norharman and harman have been implicated in a number of human diseases including Parkinson’s disease, tremor, addiction and cancer. It has been shown that these compounds are normal body constituents formed endogenously but external sources have been identified. Here, we summarise literature data on levels of norharman and harman in fried meat and fish, meat extracts, alcoholic drinks, and coffee brews. Other sources include edible and medicinal plants but tobacco smoke has been identified as a major source. Exposure levels from these different dietary sources are estimated to a maximum of 4 μg norharman per kg body weight (bw) per day and 1 μg harman per kg bw per day. Exposure via tobacco smoke depends on smoking habits and type of cigarettes but can be estimated to 1.1 μg/kg bw for norharman and 0.6 μg/kg bw for harman per package of cigarettes smoked. Studies on toxicokinetics indicate that inhalative exposure leads to a rapid increase in plasma levels and high bioavailability of norharman and harman. Oral bioavailability is lower but there are indications that sublingual absorption may increase dietary uptake of β-carbolines. Endogenous formation can be estimated to be 50–100 ng/kg bw per day for norharman and about 20 ng/kg bw per day for harman but these rates may increase with high intake of precursors. Biomarker studies on plasma levels of β-carbolines reported on elevated levels of norharman, harman or both in diseased patients, alcoholics and following tobacco smoking or consumption of β-carboline-containing food. Cigarette smoking has been identified as major influence but dietary exposure may contribute to exposure.
Keywords: Reviews; β-Carbolines; Norharman; Harman;
Estimates of heterocyclic amine intake in the US population by G.A Keating; K.T Bogen (127-133).
HA-specific meat concentration estimates using a method that combines laboratory data to predict HA concentrations from meat type, cooking method and meat doneness were used with national dietary data to estimate daily HA intake for segments of the US population. PhIP was found to comprise ∼70% of US mean dietary intake of total HAs, with pan-frying and chicken being the single cooking method and meat type contributing the greatest to total estimated HA exposures. This analysis demonstrated significantly higher concentrations in grilled/barbecued meats than in other cooked meats. African-American males were estimated to consume nearly twofold and ∼35 to 40% more PhIP (and total HAs) than white males at ages <16 and >30 years, respectively.
Keywords: Heterocyclic amine intake;
Mutagens formed from β-carbolines with aromatic amines by Y Totsuka; T Takamura-Enya; R Nishigaki; T Sugimura; K Wakabayashi (135-141).
Norharman, widely distributed in our environment such as cigarette smoke and cooked foods, is not mutagenic to Salmonella strains, but becomes mutagenic to Salmonella typhimurium TA98 and YG1024 with S9 mix in the presence of aromatic amines, including aniline and o-toluidine. Therefore, we have designated norharman as a “co-mutagen”. Since, humans are simultaneously exposed to norharman and aromatic amines in daily life, it is important to clarify the mechanisms of its co-mutagenic action to further understanding of the potential genotoxic effects in humans. Regarding the mechanisms of this action of norharman with aniline, a mutagenic compound, 9-(4′-aminophenyl)-9H-pyrido[3,4-b]indole[aminophenylnorharman (APNH)] is produced by their interaction, and converted to the hydroxyamino derivative which eventually forms the DNA adduct, dG-C8-APNH through possible ultimate reactive forms with esterification, and this induces mutations. Also other aminophenyl-β-carboline compounds, such as 9-(4′-amino-3′-methylphenyl)-9H-pyrido[3,4-b]indole[amino-3′-methylphenylnorharman (3′-AMPNH)], 9-(4′-amino-2′-methylphenyl)-9H-pyrido[3,4-b]indole [amino-2′-methylphenylnorharman (2′-AMPNH)], 9-(4′-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole[aminophenylharman (APH)] and 9-(4′-amino-3′-methylphenyl)-1-methyl-9H-pyrido[3,4-b]indole[amino-3′-methylphenylharman (AMPH)], have been found on reaction of norharman or harman with aniline or toluidine isomers. These compounds showed mutagenic and clastogenic actions in bacterial and mammalian cells. Among them, APNH demonstrated the most potent activity, and it was most extensively studied. When APNH was administered as a single dose to F344 rats, severe testicular toxicity was observed after 6 days. Moreover, liver preneoplastic lesions (GST-P-positive foci) in the liver clearly developed in animals fed 10–50 ppm of APNH in the diet for 4 weeks. Since, APNH was detected in 24 h urine of rats upon simultaneous administration with norharman and aniline by gavage, it is likely to be also produced from norharman and aniline in the human body. From these findings, it is suggested that aminophenyl-β-carboline derivatives may be classified as one of the novel types of endogenous mutagens and carcinogens.
Keywords: Reviews; Mutagens; β-Carbolines; Norharman; Aniline;
PhIP metabolites in human urine after consumption of well-cooked chicken by K.S. Kulp; M.G. Knize; N.D. Fowler; C.P. Salmon; J.S. Felton (143-153).
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. N 2-OH–PhIP–N 2-glucuronide was the most abundant urinary metabolite excreted by the 12 individuals who participated in our study. N 2-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.
Keywords: Food analysis; Metabolism; 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; Heterocyclic aromatic amine;
Formation and analysis of heterocyclic aromatic amine–DNA adducts in vitro and in vivo by Robert J. Turesky; Paul Vouros (155-166).
The detection and quantification of heterocyclic aromatic amine (HAA)–DNA adducts, critical biomarkers in interspecies extrapolation of toxicity data for human risk assessment, remains a challenging analytical problem. The two main analytical methods currently in use to screen for HAA–DNA adducts are the 32 P -postlabeling assay and mass spectrometry, using either accelerated mass spectrometry (AMS) or liquid chromatography and electrospray ionization mass spectrometry (LC–ESI–MS). In this review, the principal methods to synthesize and characterize DNA adducts, and the methods applied to measure HAA–DNA adduct in vitro and vivo are discussed.
Keywords: Reviews; Heterocyclic aromatic amine–DNA adducts; DNA;
Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells by B.J Majer; F Kassie; Y Sasaki; W Pfau; H Glatt; W Meinl; F Darroudi; S Knasmüller (167-173).
Aim of the present study was the investigation of the genotoxicity of amino-α-carboline (AαC) in human derived cells and of its organ-specific effects in laboratory rodents. This heterocyclic amine (HA) is contained in fried meat and fish in higher concentrations than most other cooked food mutagens. In the present experiments, AαC caused dose-dependent induction of micronuclei in the human derived hepatoma cell line HepG2 at concentrations ≥50 μM. In contrast, no significant effects were seen in Hep3B, another human hepatoma cell line, which may be explained by the concurrent lower activity of sulfotransferase (SULT), an enzyme playing a key role in the activation of AαC. A positive result was also obtained in the single cell gel electrophoresis (SCGE) assay in peripheral human lymphocytes, but the effect was only significant at the highest concentration (1000 μM). In Fischer F344 rats and ICR mice, the liver was the main target organ for the formation of DNA adducts (at ≥50 mg/kg bw), and in lungs and colon substantially lower levels were detected. Identical organ specificity as in the DNA adduct measurements was seen in SCGE assays with rats, whereas in mice the most pronounced induction of DNA migration was observed in the colon. Comparison of our results with data from earlier experiments indicate that the genotoxic potency of AαC is equal to that of other HAs, which are contained in human foods in much smaller amounts. Therefore, our findings can be taken as an indication that the human health risk caused by exposure to AαC is higher than that of other HAs that are formed during the cooking of meat and fish.
Keywords: Genotoxicity; 2-Amino-9H-pyrido[2,3-b]indole; DNA;
Enzyme polymorphisms influencing the metabolism of heterocyclic aromatic amines by Luisa Airoldi; Cinzia Magagnotti; Roberta Pastorelli; Roberto Fanelli (175-181).
Heterocyclic aromatic amines are dietary carcinogens possibly involved in human carcinogenesis, DNA-adduct formation being an obligatory step in this multistage process. Heterocyclic amine binding to DNA largely depends on the balance between metabolic activation and detoxification pathways and DNA repair efficiency. Several genes coding for metabolic enzymes are polymorphic, which affects gene expression and/or enzyme activity. This paper briefly reviews the effect of polymorphisms of activating/detoxifying enzymes on the metabolism of heterocyclic amines. Despite some epidemiological evidence of an association between genetic polymorphisms and susceptibility to cancer possibly resulting from dietary exposure to heterocyclic aromatic amines (HA), the genetic polymorphisms had only slight effects on biomarker levels, suggesting the existence of further unknown factors.
Keywords: Heterocyclic aromatic amines metabolism; Enzyme polymorphisms;
Role of p53 and mismatch repair in PhIP-induced perturbations of the cell cycle by Romain Duc; Phaik-Mooi Leong-Morgenthaler (183-187).
Heterocyclic amines, found ubiquitously in our diet, are carcinogenic and mutagenic. Among this class of compounds, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is the most abundant. To further understand the carcinogenesis of this compound, we studied the effects of PhIP on the progression of human lymphoblastoid cells through the cell-cycle. Cells differing in p53 or mismatch repair status were used to evaluate the role of those proteins. Following PhIP-treatment, a dose and time-dependent accumulation of p53 was found in cells containing functional p53. The augmentation of the p53 protein, accompanied by increases in p21-WAF1, confirms that the p53 is activated. The increase in p53 was independent of the mismatch repair status of the cells. Perturbations in the cell-cycle were also observed. Twenty-four hours after PhIP treatment, the activation of the G2-M checkpoint was evident. Functional p53 and mismatch repair were not required for the PhIP-induced G2-M arrest. The G2-M arrests were reversible and are interpreted as necessary for the repair of the PhIP-DNA lesions. Under treatment conditions where less than 5% of the cells survived, the G2-M arrests were absent.
Keywords: p53; 2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine;
Use of antioxidants to minimize the human health risk associated to mutagenic/carcinogenic heterocyclic amines in food by P Vitaglione; V Fogliano (189-199).
Heterocyclic amines (HAs) are mutagenic/carcinogenic compounds formed in meat during cooking. Several efforts have been made to minimize the risk associated to HA human exposure. Supplementation with antioxidants is considered a promising measure to reduce HA exposure because of their ability as inhibitors of HA formation or as blocking/suppressing agents on HA biotransformation/metabolism. The aim of this review is to present the current knowledge on the capability of synthetic and natural antioxidants to modulate HA-induced mutagenicity/carcinogenicity. Data show a general trend towards a reduction of HA formation both in model systems and in real foods as well as an effective modulation of biotransformation and metabolism. Phenolic compounds, particularly those from tea and olive oil, seem to be the most effective, although a great variability is observed because of the concentration-dependent pro- and antioxidant effects.
Keywords: Reviews; Food analysis; Heterocyclic aromatic amines; Antioxidants;
Mechanisms by which resistant starches and non-starch polysaccharide sources affect the metabolism and disposition of the food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline by P. Kestell; S. Zhu; L.R. Ferguson (201-210).
Although both non-starch polysaccharides (NSP) and resistant starches (RS) are included in current definitions of dietary fibre, our previous work has suggested fundamental differences in the way in which these two classes of material affect the disposition and absorption of a dietary carcinogen. The present studies explore whether different effects on carcinogen metabolism could play a role in the contrasting patterns seen previously. Groups of female Wistar rats were pre-fed for 4 weeks one of five types of defined diet (AIN-76). The control diet contained 35% maize starch and no dietary fibre. The RS-containing diets had all the maize starch substituted with either Hi-maize™ or potato starch. In the NSP-containing diets, 10% of the maize starch was substituted with dietary fibre in the form of either lignified plant cell walls (wheat straw) or soluble dietary fibre (apple pectin). Pre-fed rats were gavaged with the food carcinogen, [2- 14 C ] 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and plasma and urinary metabolites characterized using HPLC at various time intervals after administration. After 4 h gavage, plasma from rats on both RS-containing diets contained significantly higher levels of intact IQ and lower levels of the major metabolites, IQ-5-O-glucuronide and IQ-5- sulfate, as compared with plasma from the negative control group at this time. In contrast, plasma from animals on the NSP-containing wheat straw diet (and to a lesser extent the apple pectin diet) showed significantly lower levels of intact IQ, and significantly higher levels of the two major metabolites, as compared with those from the control rats. These different metabolite profiles were also reflected in different urinary excretion profiles. Urine from rats pre-fed RS-containing diets revealed significantly slower metabolite excretion as compared with urine from rats that had been given the NSP-containing diets. Western blotting methodologies also profiled differences between the effects of these two types of dietary fibre in the expression of xenobiotic metabolizing enzymes. We conclude that changes in activity and expression of xenobiotic metabolising enzymes could play a role in the contrasting effects of these two types of dietary fibre on carcinogen uptake and disposition.
Keywords: Carcinogen metabolism; 2-Amino-3-methylimidazo[4,5-f]quinoline; Resistant starches; Non-starch polysaccharides;
Effect of intestinal microfloras from vegetarians and meat eaters on the genotoxicity of 2-amino-3-methylimidazo[4,5-f]quinoline, a carcinogenic heterocyclic amine by F Kassie; E.F Lhoste; A Bruneau; M Zsivkovits; F Ferk; M Uhl; T Zidek; S Knasmüller (211-215).
Aim of this study was to investigate the impact of intestinal microfloras from vegetarians and non-vegetarians on the DNA-damaging activity of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), a carcinogenic heterocyclic amine that is found in fried meats. Floras from four vegetarians (Seventh Day Adventists) and from four individuals who consumed high amounts of meats were collected and inoculated into germfree F344 rats. The rats were kept on isocaloric diets that either contained animal derived protein and fat (meat consumers group) or proteins and fat of plant origin (vegetarian groups). IQ (90 mg/kg bw) was administered orally, after 4 h the extent of DNA-damage in colon and liver cells was determined in single cell gel electrophoresis assays. In all groups, the IQ induced DNA-migration was in the liver substantially higher than in the colon. In animals harbouring floras of vegetarians, the extent of damage was in both organs significantly (69.2% in the liver, P<0.016 and 64.7%, P<0.042 in the colon, respectively) lower than in the meat consumer groups. Our findings show that diet related differences in the microfloras have a strong impact on the genotoxic effects of IQ and suggest that heterocyclic amines are less genotoxic and carcinogenic in individuals that consume mainly plant derived foods.
Keywords: Microflora; Genotoxicity; 2-Amino-3-methylimidazo[4,5-f]quinoline;
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 by Antony Boyce; Johannes Doehmer; Nigel J. Gooderham (217-223).
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.
Keywords: Heterocyclic aromatic amines mutagenicity; Resveratrol; 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline;
Effect of common Brassica vegetables (Brussels sprouts and red cabbage) on the development of preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in liver and colon of Fischer 344 rats by Maria Uhl; Fekadu Kassie; Sylvie Rabot; Bettina Grasl-Kraupp; Asima Chakraborty; Brenda Laky; Michael Kundi; Siegfried Knasmüller (225-230).
Aim of the present study was the investigation of effects of juices from commonly consumed Brassica vegetables (two cultivars of Brussels sprouts and two cultivars of red cabbage) on formation and development of preneoplastic lesions in colons (aberrant crypt foci, ACF) and livers (glutathione-S-transferase placental form, GST-P+) in male F344 rats. The foci were induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a widespread carcinogenic heterocyclic aromatic amine which is found in fried meats. Recently, we reported on pronounced protective effects in the two-organ foci model when the vegetable juices were given during the carcinogen treatment but several findings by other groups indicated that breakdown products of glucosinolates contained in Brassica vegetables cause tumour promotion in various organs of laboratory rodents. In the present study, the animals received the juices in the drinking water (5%) over a period of 20 days after treatment with IQ (100 mg/kg bw on 10 alternate days). To increase the foci yield (which facilitates the detection of modifying effects), the animals were fed with a modified (high fat, fibre free) AIN-76 diet. With exception of the sprout variety “Cyrus”, all juices lowered the number of GST-P+ foci as well as the foci area in the liver, but none of these effects was statistically significant. In the colon, none of the juices had an impact on crypt multiplicity (number of crypts/focus), whereas the number of ACF was decreased; only with the sprout variety Maximus the protective effect was significant (reduction 49%). The present findings show that administration of vegetable juices to the animals after the carcinogen does not increase the number and size of IQ-induced preneoplastic lesions in liver and colon.
Keywords: Brassica; 2-Amino-3-methylimidazo[4,5-f]quinoline; Heterocyclic aromatic amines;
Protective effects of Brussels sprouts, oligosaccharides and fermented milk towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced genotoxicity in the human flora associated F344 rat: role of xenobiotic metabolising enzymes and intestinal microflora by Christèle Humblot; Evelyne Lhoste; Siegfried Knasmüller; Karine Gloux; Aurélia Bruneau; Martine Bensaada; José Durao; Sylvie Rabot; Claude Andrieux; Fekadu Kassie (231-237).
We investigated the chemoprotective effects of four common constituents of the human diet, i.e. a fermented milk, inulin, oligofructose and Brussels sprouts, towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced genotoxicity in male Fischer 344 rats harbouring a human intestinal microflora. We found that the four dietary components significantly reduced IQ-induced DNA damage in hepatocytes (reduction ranged from 74% with inulin to 39% with Brussels sprouts) and colonocytes (reduction ranged from 68% with inulin to 56% with Brussels sprouts). This chemoprotective effect correlated with the induction of hepatic UDP-glucuronosyl transferase following Brussels sprouts consumption, and with alterations of bacterial metabolism in the distal gut (acidification, increase of butyrate proportion, decrease of β-glucuronidase activity) following inulin consumption.
Keywords: Brassica; Fermented milk; Genotoxicity; 2-Amino-3-methylimidazo[4,5-f]quinoline; Oligosaccharides;