harmine and 2-amino-3-8-dimethylimidazo(4-5-f)quinoxaline

Heterocyclic aromatic amines (HAAs) are process-induced food contaminants with high mutagenic and/or carcinogenic potential. Although the human gut microbiota is known to affect the metabolism of dietary constituents, its impact on HAA metabolism and toxicity has been little studied. Here, the glycerol-dependent metabolism of seven foodborne HAAs (AαC, Trp-P-1, harman, norharman, PhIP, MeIQx, and MeIQ) by the human fecal microbiota is investigated.. As analyzed by HPLC-DAD/FLD, the extent of conversion is strongly dependent on glycerol supplementation and HAA structure. AαC (60-100%) and the 2-aminoimidazoazarenes (up to 58%) are especially prone to microbial conversion. Based on high-resolution MS and/or NMR spectroscopy data, 70 fecal metabolites are identified in total, mainly formed by chemical reactions with one or two molecules of microbially derived reuterin. Moreover, it has been demonstrated that the human fecal microbiota can further transform reuterin adducts by reduction and/or hydroxylation reactions. Upon isolation, some reuterin-induced HAA metabolites appear to be partially unstable, complicating structural identification.. The formation of microbial metabolites needs to be incorporated into risk assessment considerations for HAAs in human health. In this study, several HAA metabolites, mainly reuterin-dependent, are identified in vitro, providing the basis for future human studies investigating microbial HAA metabolism.

Topics: Adult; Amines; Animals; Carbolines; Feces; Female; Food Contamination; Gastrointestinal Microbiome; Glyceraldehyde; Harmine; Heterocyclic Compounds, Fused-Ring; Humans; Male; Microsomes, Liver; Middle Aged; Propane; Quinolines; Quinoxalines; Rats, Wistar

Heterocyclic amines (HCAs) are primarily produced during high temperature meat cooking. These compounds have been intensively investigated as mutagens and carcinogens. However, converging data suggest that HCAs may also be neurotoxic and potentially relevant to neurodegenerative diseases such as Parkinson's disease (PD). The identification of new potential etiological factors is important because most PD cases are sporadic. Our group previously showed that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was selectively neurotoxic to dopaminergic neurons. However, PhIP is one of many HCAs, a class of compounds that exhibits wide structural variability. The goal of this study was to determine the neurotoxicity of the most prevalent and best studied HCAs from three subclasses: aminoimidazoaazarenes (AIA), α-carbolines, and β-carbolines. Using E17 rat primary midbrain cultures, we tested dopaminergic and non-dopaminergic neurotoxicity elicited by the following compounds: 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), PhIP, 1-methyl-9H-pyrido[3,4-b]indole (harmane), 9H-pyrido[3,4-b]indole (norharmane) and 2-amino-9H-pyrido[2,3-b]indole (AαC) at concentrations ranging from 100 nM-5 μM. All tested HCAs were selectively neurotoxic, though the dose required to elicit selective loss of dopaminergic neurons or decreases in dopaminergic neurite length was compound specific. Non-dopaminergic neurons were unaffected at all tested doses. The sensitivity (determined by threshold dose required to elicit selective neurotoxicity) appears to be unrelated to published mutagenic potency. Both AIA and α/β-carbolines produced oxidative damage, which was magnified in dopaminergic neurons vs. non-dopaminergic neurons as further evidence of selective neurotoxicity. These studies are expected to prompt clinical and mechanistic studies on the potential role of HCA exposure in PD.

Topics: Amines; Animals; Carbolines; Dopaminergic Neurons; Dose-Response Relationship, Drug; Harmine; Heterocyclic Compounds, 3-Ring; Mesencephalon; Molecular Structure; Nerve Degeneration; Neurites; Neurons; Primary Cell Culture; Quinolines; Quinoxalines; Rats

The effects of tissue antioxidant levels on formation of heterocyclic amines (HAs) and their mutagenicity in grilled lean beef were studied. Meat from 54 feedlot steers fed different levels of vitamin E (340, 690, 1040 and 1740 IU/animal/day) for 120-days was used to provide beef with different levels of antioxidants (α-tocopherol). Prevalent HAs were then analyzed by HPLC using UV/Fluorescence detection. Five major HAs were found: 2-amino-3,8-dimethyl-imidazo(4,5-F)Quinoxaline (MeIQx), 2-amino-3,4,7,8-tetramethyl-imidazo(4,5-F)Quinoxaline (TriMeIQx), ß-Carboline-9H-Pyrido[3,4-b]indole (Norharmane), 1-Methyl-9H-pyrido[3,4-b]indole (Harmane) and 2-amino-1-methyl-6-phenylimidaza(4,5-B)pyridine (PhIP). Total content of HAs in grilled lean beef ranged from 9.57 ng/g to 11.59 ng/g. There was, however, a trend (P=0.097) found for reduced mutagenicity with increasing tissue levels of α-tocopherol. The increasing dietary vitamin E significantly increased the α-tocopherol level in lean beef (P<0.001), but it had no significant (P>0.05) inhibitory effects on the content of individual and total HAs.

Topics: Amines; Animals; Antioxidants; Cattle; Chromatography, High Pressure Liquid; Cooking; Diet; Harmine; Imidazoles; Meat; Muscle, Skeletal; Mutagens; Quinoxalines; Salmonella; Vitamin E

The effects of Chinese spices on the profiles of 17 heterocyclic amines (HAs) from seven HA categories were investigated in roast beef patties using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) and principal component analysis. Three groups of HAs, imidazopyridines (PhIP, DMIP, and 1,5,6-TMIP), imidazoquinoxalines (MeIQx and 4,8-DiMeIQx), and β-carbolines (harman and norharman), were detected and quantified in all of the samples. The results demonstrated that the total HA and imidazopyridine profiles could clearly be affected by 1% pricklyash peel (14.1 ± 0.76 and 6.06 ± 0.32 ng/g), chilli (41.0 ± 0.01 and 23.0 ± 0.52 ng/g), and cumin (59.9 ± 2.44 and 31.1 ± 3.06 ng/g), in comparison with control values of 21.8 ± 2.40 and 14.3 ± 2.04 ng/g, respectively. The difference was only significant (p < 0.05) for cumin. The imidazoquinoxaline profile was significantly (p < 0.05) affected by 1% pricklyash peel (0.57 ± 0.05 ng/g) and cumin (2.36 ± 0.20 ng/g) compared to the control (1.16 ± 0.11 ng/g). The β-carboline profile was only markedly (p < 0.05) affected by 1% cumin (26.4 ± 0.82 ng/g) compared to the control (6.26 ± 0.26 ng/g). In general, pricklyash peel inhibited HA formation, whereas star anise, fennel, cumin, chilli, and black pepper promoted HA formation. The findings could facilitate the selection of spices in meat processing to minimize HA formation.

Topics: Amines; Animals; Capsicum; Cattle; Chromatography, High Pressure Liquid; Cooking; Cuminum; Foeniculum; Food Analysis; Harmine; Heterocyclic Compounds; Imidazoles; Meat; Piper nigrum; Principal Component Analysis; Pyridines; Quinolines; Quinoxalines; Spices; Tandem Mass Spectrometry

Heterocyclic amines (HAs) are formed as Maillard reaction products in the crust of meat products during heating processes. Two typical pizza toppings--salami and cooked ham--were analyzed for the presence of HAs after baking frozen pizzas at top and bottom temperatures of 250 and 230 °C, respectively. After baking pizza slices for 12 min, MeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline; 0.2 ng/g), 4,8-DiMeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; 0.5 ng/g), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 0.2 ng/g), norharman (4.5 ng/g), and harman (2.5 ng/g) were found in the ham toppings, whereas only the comutagenic norharman (107.4 ng/g) and harman (11.4 ng/g) were found in the salami toppings. The content of MeIQx and 4,8-DiMeIQx in ham increased from 0.3 to 1.8 ng/g and 0.8 to 1.6 ng/g, respectively, when the recommended baking time was increased from 15 min (manufacturer's specification) to 18 min at 230 °C. MeIQx was formed in salami when the heating time was extended to 18 min. Moreover, higher concentrations of PhIP in salami or ham slices were found when baking temperatures were 250 °C rather than 230 °C (baking time of 12 min). However, sensory tests showed that panelists preferred longer-baked pizzas due to an increased crispiness. Thus, results show that a substantial formation of HAs may occur in pizza toppings such as ham and salami, with ham being particularly susceptible when compared to salami. Formation of HAs increases with increasing baking time and temperature. The occurrence of the cupping of ham or salami slices during baking may also increase the formation of HAs.

Topics: Amines; Animals; Carbolines; Chromatography, High Pressure Liquid; Color; Consumer Behavior; Cooking; Freezing; Harmine; Hot Temperature; Humans; Imidazoles; Maillard Reaction; Meat Products; Quinoxalines; Swine; Taste

Heterocyclic amines (HAs) are an important class of food mutagens and carcinogens produced in meat cooked at high temperature. In the present study, the effects of various cooking methods: boiling, microwave cooking, charcoal-grilling, roasting, deep-frying and pan-frying on the formation of HAs in duck breast were studied. The various HAs formed during cooking were isolated by solid-phase extraction and analysed by HPLC. Results showed that both the varieties and contents of HAs and the cooking loss of duck breast increase along with increasing cooking temperature and time. Pan-fried duck breasts contained the highest amount of total HAs, followed by charcoal-grilling, deep-frying, roasting, microwave cooking and boiling. 9H-pyrido[3,4-b]indole (norharman) and 1-methyl-9H-pyrido[3,4-b]indole (harman) were detected in all of the cooked duck meat, with levels in the range of 0.1-33 ng g⁻¹. 2-Amino-1-methyl-6-phenylimidazo[4,5-f]pyridine (PhIP) was formed easily in duck meat cooked by pan-frying and charcoal-grilling in the range of 0.9-17.8 ng g⁻¹. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) was identified in duck meat cooked by charcoal-grilling and pan-frying, in the range of 0.4-4.2 ng g⁻¹. 2-Amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) was detected in amounts below 4.5 ng g⁻¹ in duck meat cooked by charcoal-grilling, roasting, deep-frying and pan-frying. The other HAs were detected in amounts below 10 ng g⁻¹. Colour development increased with cooking temperature, but no correlation with HAs' content was observed.

Topics: Amines; Animals; Carbolines; China; Chromatography, High Pressure Liquid; Cooking; Ducks; Food Contamination; Harmine; Heterocyclic Compounds; Hot Temperature; Imidazoles; Maillard Reaction; Meat; Microwaves; Mutagens; Quinolines; Quinoxalines; Solid Phase Extraction; Time Factors

In the present paper, we have developed a capillary liquid chromatography with MS detection for the determination at ngg⁻¹ levels of four heterocyclic aromatic amines (MeIQx, norharman, harman and harmine), a group of mutagenic and carcinogenic compounds that can potentially be produced in protein-rich food during processing operations. They have been determined in commercial ready-to-eat (RTE) smoked salmon and soft cheese treated with E-beam irradiation. On the basis of experimental design studies and operating conditions of MS detector, best chromatographic conditions were obtained using a Luna® C¹⁸ capillary column (150 mm × 0.3 mm I.D.) with a mixture of acetonitrile-ammonium formate 5 mM pH 3.6 buffer (13:87, v/v) as mobile phase. To improve sensitivity, large injection volumes (20 μL) and injection solutions of low elution strength were employed. Sample preparation procedure included a previous treatment with 1M NaOH, followed by two solid-phase extraction steps; firstly on diatomaceous earth and then on mixed-mode cartridges. Heterocyclic amines were detected neither in irradiated and in non-irradiated samples, indicating that they were not formed by the radiation effect even at doses higher than those indicated in the Food Safety Objective established by regulatory agencies. RTE food samples were spiked at concentration levels in the range 10-30 ngg⁻¹. Recoveries higher than 85% (n=3 for each spiked level) were obtained, showing the effectiveness of the proposed methodology.

Topics: Acetonitriles; Animals; Cheese; Chromatography, Liquid; Food Analysis; Food Irradiation; Harmine; Linear Models; Mass Spectrometry; Quinoxalines; Reproducibility of Results; Salmon; Seafood; Sensitivity and Specificity

Heterocyclic amines (HCAs) are potent mutagens/carcinogens to which humans are frequently exposed through the consumption of cooked meat and fish food. The effect of normal intake of HCAs and their role in the aetiology of human cancer is unknown. To some extent, limitations of the existing analytical methods in monitoring the low levels of HCAs in biological samples have hindered obtaining conclusive results. In this study, a method for the analysis of HCAs in human urine has been studied to detect HCAs and metabolites at levels resulting from consumption of food cooked at ordinary conditions. The analytical method consisted of extraction and clean-up by the novel technique liquid-phase microextraction combined with LC-MS/MS. The effect of pH during the extraction and hydrolysis step was examined. High sensitivity was achieved when the extraction was performed in raw urine adjusted to pH 5.5, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine being detected from 2 pg/g urine, levels comparable with a normal exposure. Good reproducibility and repeatability was obtained for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, below 9% using isotopic dilution. The performance of the method on 9H-pyrido[3,4-b]indole, 2-amino-1-methyl-6-(4'-hydroxyphenyl)imidazo[4,5-b]pyridine and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine was also studied.

Topics: Analytic Sample Preparation Methods; Carbolines; Carcinogens; Chromatography, High Pressure Liquid; Harmine; Humans; Hydrogen-Ion Concentration; Hydrolysis; Imidazoles; Limit of Detection; Microchemistry; Mutagens; Pyridines; Quinoxalines; Reproducibility of Results; Tandem Mass Spectrometry

A recently developed HPTLC/UV-FLD method was compared to the routinely used HPLC/UV-FLD method for the quantification of heterocyclic aromatic amines (HAA) formed at trace levels during the heating process of meat. For formation of these process contaminants under normal cooking conditions, beef patties were fried in a double-contact grill at 230 degrees C for five different frying times and extracted by solid-phase extraction. The HAAs most frequently found, that is, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline (4,8-DiMeIQx), 9 H-pyrido[3,4- b]indole (norharman), and 1-methyl-9 H-pyrido[3,4- b]indole (harman), were quantified by two chromatographic methods, which were orthogonal to each other (normal versus reversed phase system). Both methods showed a similar performance and good correlation of the results ( R (2) between 0.8875 and 0.9751). The comparison of running costs and run time in routine analysis proved HPTLC/UV-FLD to be more economical (factor of 3) and faster (factor of 4) due to its capability of parallel chromatography. The HAA findings calculated by standard addition increased with the heating time from <1 to 33 microg/kg related to 3-6 min of frying time. The precision (RSD) was between 7 and 49% (HPTLC) and between 5 and 38% (HPLC) at these very low HAA levels formed.

Topics: Amines; Animals; Carbolines; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Harmine; Heterocyclic Compounds; Hot Temperature; Imidazoles; Meat; Mutagens; Quinoxalines

Reversed phase ion-pair chromatography (RP-IPC) of seven heterocyclic aromatic amines encompassing quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP) and carboline derivatives (AalphaC, Harman, Norharman) was carried out with formate as counter ion in an aqueous eluent with acetonitrile as organic modifier. TSKgel ODS-80TS was used as the stationary phase. With the aim of acquiring a better insight into the mutual influence of ion-pair reagent and the organic modifier upon solute retention, the study was performed by using an experimental design approach able to evidencing the effect of the simultaneous variation of the two factors. A model for the chromatographic behavior of the amines is proposed that includes classical ion-pair mechanism involving formate in the case of MeIQx, PhIP, Harman and Norharman. A competitive ion-exchange mechanism was hypothesized to govern retention of quinoline compounds, whereas electrostatic interactions and hydrogen bond formation with the silanols of the stationary phase were judged to be responsible for the retention of AalphaC. Further, the chromatographic behavior of the analytes using the formic acid-ammonium formate buffer in the mobile phase was compared with that observed using acetic acid-ammonium acetate buffer. The method based on the use of RP IPC with tandem mass spectrometry when the eluent contained formate buffer at pH 2.8 exhibited higher detectability with respect to that achieved using the acetate buffer.

Topics: Acetonitriles; Amines; Buffers; Carbolines; Chromatography, Liquid; Formates; Harmine; Heterocyclic Compounds; Hydrogen-Ion Concentration; Imidazoles; Quinolines; Quinoxalines; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

A short-term effect of a meal of fried meat is a postprandial induction of hepatic and intestinal cytochrome P450 activity. In order to identify the components responsible for this effect we investigated the potency of food derived genotoxic heterocyclic aromatic amines (HA) to induce CYP1A1 in vitro. In two cell lines, the rat hepatoma cell line H4IIE and the human breast cancer cell line MCF-7, we investigated 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and Harman representing the different classes of HA at concentrations from 10(-8) to 10(-4) M. Induction of CYP1A1 was analysed on the mRNA level by semi-quantitative RT-PCR and the protein level (western blot using specific antibodies). The relative order of enzyme induction was Trp-P-1 with 1.4 x 10(-6) M (EC50 compared to TCDD 10(-9) M), MeAalphaC (1.4 x 10(-5)), Harman (2.1 x 10(-4)) and MeIQx (1.0 x 10(-3)). Furthermore, CYP1A1 enzyme activity was analysed as ethoxyresorufin-O-deethylase. While protein and mRNA analyses gave similar results, competitive inhibition impaired the enzyme activity assay. Inhibition of CYP1A1 activity was determined using microsomes of heterologous expressed CYP1A1. This dose-dependent inhibitory activity paralleled the induction potency. These results compare well with earlier data published for hepatic enzyme induction by HA observed in animal experiments. However, since the observed activities are rather weak and the amounts of HA ingested with a meal are low, there may be other factors involved in the observed postprandial enzyme induction in humans. On the other hand, concentrations in the micromolar range that are reached in high dosage animal experiments with HA may well influence cytochrome activity and, thus, influence the experimental outcome of these studies.

Topics: Amines; Animals; Carbolines; Carcinogens; Cooking; Cytochrome P-450 CYP1A1; Dose-Response Relationship, Drug; Enzyme Induction; Female; Harmine; Heterocyclic Compounds; Humans; Meat; Mutagens; Quinoxalines; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Pig meat shows natural variations in the concentrations of precursors of heterocyclic amines (HCAs), which may affect formation of HCAs in cooked pig meat. To study this, 26 pigs with an inherent genetic variation (carriers and noncarriers of the RN(-) allele) were subjected to different feeding regimes (conventional feed compared with feed composed according to organic standards). In addition, the effect of sex (castrated males or females) was considered when assessing chemical and technological meat quality parameters. Concentrations of precursors of HCAs, i.e., creatine, residual glycogen, dipeptides, and free amino acids, were analyzed in the raw meat, and the levels of some HCAs (4,8-DiMeIQx, MeIQx, PhIP, harman, and norharman) were then determined in fried meat patties prepared from these pigs. The RN genotype most affected technological meat quality parameters and the level of precursors of HCAs, especially the level of residual glycogen, where carriers of the RN(-) allele showed levels four times as high as those of noncarriers (75.3 +/- 2.6 compared with 17.2 +/- 2.4 micromol/g meat, least-squares means +/- SE). The increased level of residual glycogen resulted in about 50% lower amounts of total mutagenic HCAs in cooked meat compared with cooked meat from normal pigs. Fried meat from carriers of the RN(-) allele obtained darker crust color than meat from noncarriers. Feeding regime and sex did not significantly affect the chemical composition of the meat or the formation of HCAs.

Topics: Animals; Carcinogens; Eating; Female; Genotype; Harmine; Hot Temperature; Imidazoles; Male; Meat; Mutagens; Quinoxalines; Sex Characteristics; Swine