3-amino-1-methyl-5h-pyrido(4-3-b)indole and 2-amino-1-methyl-6-phenylimidazo(4-5-b)pyridine

During grilling of the roast beef the following heterocyclic aromatic amines were found: IQ=200.6 ng 100g(-1), MeIQx=719.8 ng 100 g(-1), MeIQ=532.9 ng 100g(-1), 4.8-diMeIQx=755.4 ng 100 g(-1), norharmane=507.0 ng 100 g(-1), harmane=1952.6 ng 100 g(-1), Phe-P 1=263.7 ng 100 g(-1), Trp-P 2=559.2 ng 100 g(-1), PhIP=1179.8 ng 100 g(-1) and AαC=51.7 ng 100g(-1). Their content was tested by using the method based on alkaline hydrolysis of the sample and the method based on solvent extraction of the grilled meat samples at different temperatures (without hydrolysis). The study showed that the heterocyclic aromatic amines produced during the grilling of beef are in a free form and chemically or physico-chemically bonded. The chemical forms of HAA formed in food have never been studied. For the purpose of the partial confirmation that HAA may be chemically or physico-chemically bonded, grilled beef samples were digested in vitro in model segments of the human digestive tract. Digestive enzymes, particularly proteolytic enzymes caused a statistically significant increase of free HAA determined by using solvent extraction without prior chemical hydrolysis of the sample.

Topics: Amines; Animals; Carbolines; Cattle; Cooking; Digestion; Humans; Hydrolysis; Imidazoles; Male; Meat; Mutagens; Quinolines; Quinoxalines; Solvents

Transgenic mouse assays have revealed that the mouse intestine, despite its resistance to carcinogenesis, is sensitive to the mutagenicity of some heterocyclic amines (HCAs). Little is known, however, about the level and localization of that sensitivity. We assessed the mutagenicity of four orally administered (20 mg/kg per day for 5 days) HCAs-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) hydrochloride, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate-in the intestine of male MutaMice. Two weeks after the last administration, we isolated epithelium from the small intestine, cecum, and colon and analyzed lacZ and cII transgene mutations. PhIP increased the lacZ mutant frequency (MF) in all the samples, and in the small intestine, cII and lacZ MFs were comparable. In the cII gene, G:C to T:A and G:C to C:G transversions were characteristic PhIP-induced mutations (which has also been reported for the rat colon, where PhIP is carcinogenic). In the small intestine, PhIP increased the cII MF to four-fold that of the control, but IQ, MeIQ, and Trp-P-2 did not have a significant mutagenic effect. In the cecum, cII MFs induced by IQ and MeIQ were 1.9 and 2.7 times those in the control, respectively. The MF induced by MeIQ in the colon was 3.1 times the control value. Mutagenic potency was in the order PhIP>MeIQ>IQ; Trp-P-2 did not significantly increase the MF in any tissue. The cecum was the most susceptible organ to HCA mutagenicity.

Topics: Amines; Animals; Bacteriophage lambda; Carbolines; Colon; Heterocyclic Compounds; Imidazoles; Intestine, Small; Intestines; Lac Operon; Male; Mice; Mice, Transgenic; Mutagenicity Tests; Mutagens; Quinolines; Transcription Factors; Viral Proteins

The in vivo genotoxicity of five heterocyclic amines-Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg), and PhIP (40 mg/kg)-in the mucosa of gastrointestinal and urinary tract organs (stomach, duodenum, jejunum, ileum, colon, and bladder) was studied by the alkaline single cell gel electrophoresis (SCG) (Comet) assay. Male CD-1 mice were sacrificed 1, 3, and 8 h after intraperitoneal injection. All the heterocyclic amines studied yielded statistically significant DNA damage in the colon but not the small intestine (duodenum, jejunum, and ileum) or urinary bladder. In this study, five heterocyclic amines were injected intraperitoneally to avoid the consequences of ingestion. Thus, the extensive damage to colon DNA was concluded to be due, at least in part, to a systemic effect.

Topics: Amines; Animals; Carbolines; Colon; Electrophoresis, Agar Gel; Gastric Mucosa; Imidazoles; Intestinal Mucosa; Male; Mice; Mutagenicity Tests; Mutagens; Quinolines; Urinary Bladder

Various kinds of mutagenic and carcinogenic heterocyclic amines (HCAs) are produced by heating protein-rich foods, such as meat and fish. To evaluate the risk of these HCAs in terms of human cancer development, exposure levels must be measured. We therefore analyzed their amounts in various kinds of cooked foods and in urine samples of healthy volunteers living in Tokyo. Based on the obtained quantitative data, daily exposure levels to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were calculated to be 0.3-3.9 and 0.005-0.3 microgram per person, respectively. Moreover, human DNA samples were analyzed with the 32P-postlabeling method, and colon, rectum and kidney tissues were found to contain an adduct spot corresponding to the standard 5'-pdG-C8-MeIQx by TLC and HPLC, at levels of 14, 18 and 1.8 per 10(10) nucleotides, respectively. The beta-carboline compound, norharman, is produced by heating L-tryptophan, and is known to be present in cooked foods and in cigarette smoke at higher levels than mutagenic and carcinogenic HCAs. While norharman is not itself mutagenic to Salmonella, it does become mutagenic to S. typhimurium TA98 with S9 mix in the presence of non-mutagenic aromatic amines like aniline and o-toluidine. When we examined whether DNA adducts are formed in the DNA of S. typhimurium TA98 by treatment with norharman and aromatic amines using 32P-postlabeling analysis, DNA adduct formation by norharman with aromatic amines was found to be related to the appearance of mutagenicity by norharman with aromatic amines.

Topics: Aniline Compounds; Carbolines; Colon; Diet; DNA Adducts; Environmental Exposure; Female; Harmine; Humans; Imidazoles; Male; Meat; Mutagenicity Tests; Quinoxalines; Salmonella typhimurium; Toluidines

Four mutagenic/carcinogenic heterocyclic amines (HCAs), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in organic extracts obtained by blue rayon hanging method from the Yodo River water were quantified. Blue rayon extracts obtained were separated in two stages of fractionation by reversed-phase high performance liquid chromatography (HPLC), and the quantification of corresponding fractions was performed by HPLC with an electrochemical detector for MeIQx and a fluorometric detector for Trp-P-1, Trp-P-2 and PhIP. The geometrical mean values of MeIQx, Trp-P-1, Trp-P-2 and PhIP in extracts collected at 11 locations from the Yodo River systems were 4.8, 26.9, 37.3, and 11.9 ng/g blue rayon equivalent, respectively. The total amounts of four HCAs accounted for mean 24% of the genotoxicity of blue rayon extracts evaluated by the umu test using an O-acetyltransferase-overproducing strain NM2009.

Topics: Amines; Animals; Carbolines; Carcinogens; Cellulose; Chromatography, High Pressure Liquid; Fresh Water; Imidazoles; In Vitro Techniques; Japan; Mutagenicity Tests; Mutagens; Quinoxalines; Rats; Salmonella typhimurium; Water Pollutants, Chemical

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.

Topics: Amines; Animals; Brain; Carbolines; DNA Damage; Electrophoresis; Imidazoles; Kidney; Liver; Male; Mice; Mice, Inbred Strains; Mutagens; Quinolines; Quinoxalines; Stomach

Lactic acid bacteria have been reported to have antimutagenic/anticarcinogenic properties in vitro and in vivo. One possible mechanism for this effect involves a physical binding of the mutagenic compounds to the bacteria. The purpose of the present investigation was to study the binding capacity of eight human intestinal or lactic acid bacterial strains for mutagenic heterocyclic amines formed during cooking of protein-rich food. Binding of the mutagens Trp-P-2, PhIP, IQ and MeIQx by the bacterial strains was analyzed by HPLC. There were only minor differences in the binding capacities of the tested strains but the mutagenic compounds were bound with markedly different efficiencies. Trp-P-2 was almost completely bound and the binding tended not to be of a reversible nature. The binding of PhIP, which reached about 50%, was important as PhIP is a major mutagen in the western diet. IQ and MeIQx were slightly less well bound. pH appeared to be of importance for the binding efficacy. Binding correlated well with the reduction in mutagenicity observed after exposure of the heterocyclic amines to the bacterial strains. The results indicate that cooked food mutagenic compounds, commonly found in the western meat-rich diet, can be bound to bacteria from the normal intestinal microflora in vitro.

Topics: Amines; Bifidobacterium; Biotransformation; Carbolines; Chromatography, High Pressure Liquid; Cooking; Food Contamination; Heterocyclic Compounds; Hot Temperature; Hydrogen-Ion Concentration; Imidazoles; Intestines; Lactobacillus; Meat; Mutagenicity Tests; Mutagens; Quinolines; Regression Analysis

The heterocyclic amines (HA) 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were mutagenic in V79 cells (Chinese hamster lung fibroblasts) using 6-thioguanine resistance as the marker of mutagenicity. Pancreas duct epithelial cells (DEC) from untreated hamsters, homogenates of pancreas ducts from untreated hamsters and those fed a high fat diet and human DEC were used to activate the heterocyclic amines. When hamster cells and tissues were used the optimum mutation frequencies (mutants/10(6) survivors) measured were: Glu-P-2, 10 +/- 1; MeIQ, 28 +/- 2 (DEC), 12 +/- 2 (control, duct homogenate), and 21 +/- 2 (high fat diet fed, duct homogenate); PhIP, 61 +/- 5. When human DEC were used the optimum mutation frequencies were: MeIQ, 32 +/- 4; PhIP, 35 +/- 3. 3,8-Dimethylimidazo[4,5-f]quinoxaline, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were not mutagenic in this assay.

Topics: Adolescent; Adult; Amines; Animals; Carbolines; Cells, Cultured; Cricetinae; Heterocyclic Compounds; Humans; Imidazoles; Lung; Male; Mutagens; Pancreatic Ducts; Quinolines

Two slightly different protocols, the plate incorporation method and the preincubation method, are used in the Ames Salmonella mutagen test. Using a preincubation method, we recently demonstrated efficient activation of a number of food-derived promutagens by extracts of mammalian cells expressing cDNAs of rat-liver cytochrome P450IA2 and of a P450IA2-IA1 hybrid. We report here that, for 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 1-aminoanthracene and several other promutagens, preincubation dramatically increased the number of revertant colonies in the Ames test when extracts of cytochrome P450IA2-containing transfected cells or low concentrations of rat-liver extracts were used as the source of activating enzymes. At higher concentrations of rat-liver extract protein, the effect of preincubation was less pronounced. The effect of preincubation was not due to the low protein concentrations in the assays since increasing the total protein concentration did not abolish the requirement for preincubation for the detection of MeIQ activation at low concentrations of rat-liver extract. In experiments where P450IA2 synthesized in transfected cells in culture is used to study promutagen activation, the plate incorporation protocol may seriously underestimate the capacity of cell extracts to activate promutagens. Thus, interlaboratory comparisons become difficult and unnecessarily large quantities of cell extract protein may be needed to detect promutagen activation. Whenever Ames test assays are carried out under conditions where P450 concentration limits revertant yield, it would be prudent to examine both the preincubation and plate incorporation protocol.

Topics: Aflatoxin B1; Anthracenes; Carbolines; Cytochrome P-450 CYP1A2; Cytochrome P-450 Enzyme System; Dihydroxydihydrobenzopyrenes; Dose-Response Relationship, Drug; Fluorenes; Humans; Imidazoles; Liver Extracts; Mutagenesis; Mutagenicity Tests; Mutagens; Oxidoreductases; Quinolines; Salmonella typhimurium; Transfection

For estimation of human exposures to carcinogenic heterocyclic amines, the amounts of four compounds, 3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), in human urine were measured. Twenty-four hour urine specimens were collected from ten healthy volunteers eating normal diet (five males and five females) and three inpatients (two males and a female) receiving parenteral alimentation, and the levels of the four heterocyclic amines were measured by HPLC after partial purification by treatment with blue cotton and ion exchange column chromatography. Trp-P-1, Trp-P-2, PhIP and MeIQx were detected in the 24 h urine samples of all healthy volunteers at levels of 0.04-1.43 ng, 0.03-0.68 ng, 0.12-1.97 ng and 11-47 ng respectively. As 1.8-4.9% of an oral dose of MeIQx is reported to be excreted unchanged in the urine, the daily exposure of humans to MeIQx was estimated to be 0.2-2.6 micrograms/person. The four heterocyclic amines were not detected in the urine of parenterally fed inpatients. These results indicate that humans are continually exposed to carcinogenic heterocyclic amines in food, and these compounds may not be formed endogenously.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carbolines; Diet; Female; Humans; Imidazoles; Male; Middle Aged; Parenteral Nutrition, Total; Quinoxalines