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

Heterocyclic amines (HCAs), such as Trp-P-1, PhIP, and IQ, are notorious food-born carcinogens. This study examined the inhibitory effects of HCAs on the expression of interleukin-8 (IL-8), which is an important chemokine for the initiation of innate immune responses that function by recruiting immune cells to inflamed sites. Among HCA types tested, only Trp-P-1 showed a robust inhibition of the lipopolysaccharide (LPS)-induced IL-8 expression at both mRNA and protein levels in a human monocytic cell-line, THP-1. However, Trp-P-1 did not inhibit the DNA-binding abilities of the transcription factors NF-kappaB, AP-1, and NF-IL6, all of which are known to regulate IL-8 transcription. Meanwhile, treatment with actinomycin D facilitated the Trp-P-1-induced down-regulation of IL-8 expression in LPS-stimulated THP-1 cells, implying that the inhibition might be due to a decrease in the stability of IL-8 mRNA rather than an attenuation of IL-8 transcription. Furthermore, Trp-P-1 also inhibited phosphorylation of p38 MAP kinase, which is involved in the regulation of IL-8 mRNA stability. Exogenous addition of ionomycin rescued both the IL-8 protein levels and phosphorylation of p38 MAP kinase inhibited by Trp-P-1. Collectively, these results suggest that Trp-P-1 suppresses LPS-induced IL-8 production in human monocytic cells through down-regulation of an intracellular calcium/p38 MAP kinase-dependent pathway, leading to the reduction of IL-8 mRNA stability.

Topics: Calcium; Carbolines; Carcinogens; CCAAT-Enhancer-Binding Protein-beta; Cell Line; DNA Adducts; Down-Regulation; Humans; Imidazoles; Immunity; Interleukin-8; Ionomycin; Lipopolysaccharides; Monocytes; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Quinolines; RNA Stability; RNA, Messenger; Transcription Factor AP-1

In order to study the mutagenic effects of heterocyclic aromatic amines (HAAs) in cells of human origin, five compounds, namely 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-3, 4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), the pyridoimidazo derivative 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), were tested in micronucleus (MN) assays with a human derived hepatoma (HepG2) cell line. All HAAs caused significant, dose-dependent effects. The activities of IQ, MeIQ, MeIQx and PhIP were similar (lowest effective concentrations 25-50 microM), whereas Trp-P-1 was effective at a dose of >/=2.1 microM. In addition, the HAAs were tested in MN assays with Chinese hamster ovary (CHO) cells and in Salmonella strain YG1024 using HepG2 cell homogenates as an activation mix. In the CHO experiments, positive results were obtained with Trp-P-1 and PhIP, whereas the other compounds were devoid of activity under all experimental conditions. The discrepancy in the responsivity of the two cell lines is probably due to differences in their acetylation capacity: enzyme measurements with 2-aminofluorene as a substrate revealed that the cytosolic acetyltransferase activity in the HepG2 cells is approximately 40-fold higher than that of the CHO cells. In the bacterial assays all five HAAs gave positive results but the ranking order was completely different from that seen in the HepG2/MN experiments (IQ > MeIQ > Trp-P-1 >/= MeIQx >> PhIP) and the mutagenic potencies of the various compounds varied over several orders of magnitude. The order obtained in bacterial tests with rat liver S9 mix was more or less identical to that seen in the tests with HepG2 cell homogenates but the concentrations of the amines required to give positive results were in general substantially lower (10(-5)-10(-1) microM). Overall, the results of the present study indicate that MN/HepG2 tests might reflect the mutagenic effects of HAAs more adequately than other in vitro mammalian cell systems due to the presence of enzymes involved in the metabolic conversion of the amines.

Topics: Amines; Animals; Arylamine N-Acetyltransferase; Carbolines; Carcinogens; Carcinoma, Hepatocellular; Cell Line; Cell Survival; CHO Cells; Cricetinae; Heterocyclic Compounds; Humans; Imidazoles; Liver Neoplasms; Mutagenicity Tests; Mutagens; Quinolines; Quinoxalines; Rats; Salmonella; Tumor Cells, Cultured

Precision-cut liver slices were prepared from male Fischer 344 rats, female CDF1 mice and humans (both male and female subjects). Liver slices were cultured for 24 hr in medium containing [3H]thymidine and either PhIP, IQ, MeIQ, MeIQx, Glu-P-1 or Trp-P-1, and then processed for auto-radiographic evaluation of unscheduled DNA synthesis (UDS). All six cooked food mutagens examined produced concentration-dependent increases in UDS in human liver slices. PhIP was the most potent compound examined, followed by MeIQx, IQ and then MeIQ, Glu-P-1 and Trp-P-1. Significant increases in UDS were observed with PhIP, IQ and MeIQx at concentrations as low as 5 microM in the culture medium. The same rank order of potency was not apparent in either rat or mouse liver slices. In rat liver slices only MeIQ significantly induced UDS, although positive results were obtained with two other genotoxins, namely 2-acetylaminofluorene and aflatoxin B1. Apart from MeIQx, all the cooked food mutagens produced significant increases in UDS in mouse liver slices. This study demonstrates the usefulness of precision-cut liver slices to evaluate species differences in xenobiotic-induced genotoxicity. Both marked compound and species differences in induction of UDS were observed. The data provide further evidence that dietary cooked food mutagens are potential human carcinogens.

Topics: 2-Acetylaminofluorene; Aflatoxin B1; Animals; Autoradiography; Carbolines; Cooking; Culture Techniques; DNA; Dose-Response Relationship, Drug; Female; Humans; Imidazoles; Liver; Male; Mice; Mutagens; Quinolines; Quinoxalines; Rats; Rats, Inbred F344; Structure-Activity Relationship

The effects of dietary bioantimutagens (compounds which have been shown to inhibit mutagenesis via interaction with DNA repair processes) on spontaneous and heterocyclic amine (HCA)-induced micronucleus (MN) frequencies were studied in metabolically competent human hepatoma (Hep-G2) cells. All the compounds tested (coumarin, vanillin, caffeine, tannic acid and cinnamaldehyde) caused a moderate increase of MN numbers in Hep-G2 cells at high concentrations (500 microg/ml); only tannic acid was also active at lower dose levels. In combination experiments with the HCA 2-amino-3-methylimidazo-[3,4-f]quinoline (IQ), post-treatment of the cells with bioantimutagens resulted in a pronounced (75-90%) decrease in MN. The most drastic effects were seen with vanillin, coumarin and caffeine which were active at concentrations < or = 5 microg/ml. Further experiments indicated that these compounds also attenuate the mutagenic effects of other HCAs (PhIP, MeIQ, MeIQx, Trp-P-1).

Topics: Acrolein; Amines; Antimutagenic Agents; Benzaldehydes; Caffeine; Carbolines; Carcinoma, Hepatocellular; Coumarins; DNA Repair; Heterocyclic Compounds; Humans; Hydrolyzable Tannins; Imidazoles; Micronucleus Tests; Mutagens; Quinolines; Tumor Cells, Cultured

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

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a mutagen/carcinogen derived from cooked foods which enhances the induction of mutations and chromosome aberrations by UV without microsomal activation. These co-mutagenic effects are considered to arise from inhibition of DNA excision repair at the incision step. However, the inhibition mechanism has not been clarified. In this study we show, using agarose gel electrophoresis, that Trp-P-1 inhibits incision by T4 endonuclease V, which cleaves DNA at the site of cyclobutane dimers. Trp-P-1 also inhibits the binding of this enzyme to UV-damaged DNA in a gel shift assay. In addition, the results of DNA unwinding assay with topoisomerase I suggest that Trp-P-1 intercalates into DNA molecules. The known intercalators ethidium bromide and acriflavine demonstrate similar effects in these experiments. However, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which showed no co-mutagenic effects in our previous study, does not demonstrate such effects. These results suggest that Trp-P-1 changes DNA conformation by intercalation, causing inhibition of binding of repair enzymes to UV-damaged DNA, and this in turn leads to inhibition of DNA excision repair and to co-mutagenic effects.

Topics: Carbolines; Deoxyribonuclease (Pyrimidine Dimer); DNA; DNA Damage; DNA Topoisomerases, Type I; DNA, Superhelical; Endodeoxyribonucleases; Ethidium; Imidazoles; Intercalating Agents; Mutagens; Nucleic Acid Conformation; Pyrimidine Dimers; Ultraviolet Rays; Viral Proteins

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

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

Most heterocyclic amines formed during the cooking of meat and fish have been shown to form adducts in the livers of rats. Recently, however, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), administered in the diet to Fischer 344 (F344) rats for 4 weeks, was shown to produce the highest levels of adducts in the heart. In the present study 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a:1',2'-d]imidazole (Glu-P-1) were given to F344 rats at carcinogenic dose levels (IQ 0.03%, MeIQx 0.04%, Trp-P-1 0.015%, Glu-P-1 0.05%) in the diet for 4 weeks. DNA adducts in the liver and heart were analyzed by 32P-postlabeling. DNA adducts were demonstrated to appear in the hearts of all animals exposed to heterocyclic amines at the following levels: IQ, 1.8 adducts/10(7) nucleotides, MeIQx, 3.8/10(7) ntd, Trp-P-1, 20/10(7) ntd and Glu-P-1, 7.2/10(7) ntd. Values for the heart were 10-20% of the respective liver adduct levels. Heart adducts increased linearly throughout the observed period when MeIQx was administered for up to 40 weeks. When MeIQx feeding was discontinued after 20 weeks and the animals subsequently given the basal diet, the adduct level at 20 weeks did not change during the following 20 weeks. A possible role for heart DNA alterations caused by food-borne heterocyclic amines in the development of age-related myopathies and cardiovascular disease is not inconceivable.

Topics: Administration, Oral; Aging; Amines; Animals; Carbolines; Diet; DNA; DNA Damage; Heart; Imidazoles; Kinetics; Liver; Male; Myocardium; Quinolines; Quinoxalines; Rats; Rats, Inbred F344