Antimutagens in gaiyou (Artemisia argyi levl. et vant.)

Antimutagens from gaiyou (Artemisia argyi Levl. et Vant., Compositae) were examined. The methanol extract prepared from aerial parts of this plant strongly reduced the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), when Salmonella typhimurium TA98 was used in the presence of the rat liver microsomal fraction. The antimutagens were purified chromatographically while monitoring the antimutagenic activity against Trp-P-2 with a modified Ames test employing a plate method. This purification resulted in the isolation of four strong antimutagens, 5,7-dihydroxy-6,3',4'-trimethoxyflavone (eupatilin), 5, 7,4'-trihydroxy-6,3'-dimethoxyflavone (jaceosidin), 5,7, 4'-trihydroxyflavone (apigenin) and 5,7, 4'-trihydroxy-3'-methoxyflavone (chrysoeriol) from the methanol extract. These antimutagenic flavones exhibited strong antimutagenic activity against not only Trp-P-2 but also against other heterocyclic amines, such as 3-amino-1,4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA(alpha)C) in S. typhimurium TA98. In contrast, they did not exhibit antimutagenic activity against benzo[a]pyrene (B[a]P), 4-nitroquinoline-1-oxide (4-NQO), 2-aminofluorene (2-AF), 2-nitrofluorene (2-NF) or furylfuramide (AF-2) in S. typhimurium TA98, or B[a]P, 4-NQO, 2-NF, AF-2, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium azide (SA) in Salmonella typhimurium TA100, whereas they decreased the mutagenicity caused by aflatoxin B(1) (AFB(1)) and 2-aminoanthracene (2-AA) in both of these tester strains. Regarding the structure-activity relationship, the tested flavones had distinct differences in the intensities of their antimutagenic activities according to the differences of their substitution patterns. Namely, the intensity of antimutagenic activities against Trp-P-2 decreased in the order of: 5,7,3',4'-tetrasubstituted flavones (IC(50): <0.1 mmol/plate), 5,7,4'-trisubstituted flavones (IC(50): 0.120-0.260 mmol/plate), 5,6,7,3',4'-pentasubstituted flavones (IC(50): 0.440-0. 772 mmol/plate). The four isolated flavones were also studied regarding their antimutagenic mechanisms with preincubation methods of the modified Ames test and emission spectroscopic analysis. The results suggested that all isolated flavones were desmutagens which directly inactivated Trp-P-2 or inhibited its metabolic activation.     

Structural analysis of a novel antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.

Six compounds were isolated from fresh rhizomes of fingerroot (Boesenbergia pandurata Schult.) as strong antimutagens toward 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in Salmonella typhimurium TA98. These compounds were 2',4',6'-trihydroxychalcone (pinocembrin chalcone; 1), 2',4'-dihydroxy-6'-methoxychalcone (cardamonin; 2), 5,7-dihydroxyflavanone (pinocembrin; 3), 5-hydroxy-7-methoxyflavanone (pinostrobin; 4), (2,4,6-trihydroxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (5), and (2,6-dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (panduratin A; 6). Compound 5 was a novel compound (tentatively termed 4-hydroxypanduratin A), and 1 was not previously reported in this plant, whereas 2-4 and 6 were known compounds. The antimutagenic IC(50) values of compounds 1-6 were 5.2 +/- 0.4, 5.9 +/- 0.7, 6.9 +/- 0.8, 5.3 +/- 1.0, 12.7 +/- 0.7, and 12.1 +/- 0.8 microM in the preincubation mixture, respectively. They also similarly inhibited the mutagenicity of 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). All of them strongly inhibited the N-hydroxylation of Trp-P-2. Thus, the antimutagenic effect of compounds 1-6 was mainly due to the inhibition of the first step of enzymatic activation of heterocyclic amines.     

Quantitation of carcinogenic heterocyclic aromatic amines and detection of novel heterocyclic aromatic amines in cooked meats and grill scrapings by HPLC/ESI-MS

A tandem solid-phase extraction method was used to isolate carcinogenic heterocyclic aromatic amines (HAAs) from cooked meats. The following 10 HAAs were identified by HPLC/ESI-MS/MS: 2-amino-9H-pyrido[2,3-b]indole (2-AalphaC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), and 2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]); the latter HAA has not previously been reported in cooked meats. The concentrations of these HAAs ranged from <0.03 to 15 ppb in cooked meats and poultry, to 75 ppb in cooked beef extract, and to 85 ppb in grill scrapings. The product ion scan mode was used to confirm the identities of these HAAs. Six other compounds were detected that appear to contain the N-methylimidazoquinoxaline skeleton on the basis of their product ion spectra, and these compounds are probable isomers of IQx, 8-MeIQx, and DiMeIQx. A number of known HAAs and novel HAAs of unknown genotoxic potential are formed at appreciable levels in cooked meats.     

Influence of lignification and feruloylation of maize cell walls on the adsorption of heterocyclic aromatic amines

Both epidemiological and experimental data indicate that a diet rich in fiber may reduce cancer risk. One possible mechanism is by adsorbing carcinogens and transporting them out of the body without metabolic activation. We investigated the role of fiber lignification and feruloylation on the adsorption of four of the most relevant heterocyclic aromatic amines in food: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 2-amino-9H-pyrido[2,3-b]indole (AalphaC). Adsorption experiments, under conditions mimicking the small intestine, were carried out using nonlignified and artificially lignified primary maize walls with defined lignin and ferulate/diferulate concentrations and defined lignin compositions. Lignin concentration and composition both influenced the adsorption of heterocyclic aromatic amines, especially the more hydrophobic types. Heterocyclic aromatic amine adsorption increased with lignin concentration. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-9H-pyrido[2,3-b]indole were better adsorbed by guaiacyl-rich lignins, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by syringyl-rich lignins, whereas the adsorption of 2-amino-3-methylimidazo[4,5-f]quinoline was not clearly influenced by lignin composition. Nonlignified cell walls adsorbed lesser amounts of heterocyclic aromatic amines. Variations in cell wall feruloylation had no effect on heterocyclic aromatic amine adsorption.     

Inhibitory effects of heterocyclic amine-induced DNA adduct formation in mouse liver and lungs by beer

An evaluation of the in vivo antigenotoxic potential of beer components on heterocyclic amines including 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 3-amino-1-methyl-5H-pyrido[4.3-b]indole (Trp-P-2) was determined with particular focus on the target organs of tumorigenesis, and the protective mechanisms involved were investigated. Beer-solution, consisting of a freeze-dried and dissolved sample, given as drinking water, reduced the formation of MeIQx-DNA adducts in mouse liver and lungs. Beer-solution added in the diet as a mimic of food additives also significantly reduced the amount of DNA adducts present in the liver, lung, and kidney DNA of mice fed with MeIQx compared to control mice fed with MeIQx in the absence of beer-solution. The amount of adducts present in the liver of mice with single or continuous administration of Trp-P-2 was significantly reduced when beer-solution was given as part of the diet compared to control mice given Trp-P-2 without beer-solution. Protective effects were observed both with lager- and stout-type samples. In an effort to investigate the mechanisms responsible for the observed protective effects, the effects of beer-solution on metabolizing enzymes for heterocyclic amines were examined. Beer-solutions inhibited the metabolic activation of Trp-P-2 to Trp-P-2(NHOH), as demonstrated by HPLC analysis. Considering the overall suppression of the genotoxicity of MeIQx and Trp-P-2 by beer, we have shown that beer components can inhibit the metabolic activation of heterocyclic amines and subsequently suppress the observed genotoxicity. The results of this study show that beer components are protective against the genotoxic effects of heterocyclic amines on target organs associated with tumorigenesis in vivo.     

Antimutagenic activity of flavonoids from Pogostemon cablin

A methanol extract from Pogostemon cablin showed a suppressive effect on umu gene expression of SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract was re-extracted with hexane, dichloromethane, butanol, and water. A dichloromethane fraction showed a suppressive effect. Suppressive compounds against furylfuramide in the dichloromethane fraction were isolated by SiO(2) column chromatography and identified as 7,4'-di-O-methyleriodictyol (1), 7, 3',4'-tri-O-methyleriodictyol (2), and 3,7,4'-tri-O-methylkaempferol (3). In addition, three flavonoids, ombuine (4), pachypodol (5), and kumatakenin (6), were isolated and identified from the dichrolomethane fraction. Compounds 1 and 3 suppressed >50% of the SOS-inducing activity at <0.6 micromol/mL, and the ID(50) values of both compounds were 0.25 micromol/mL. Compound 2 showed a weakly suppressive effect (17%) at a concentration of 0.6 micromol/mL, and compounds 4-6 did not. These compounds were also assayed with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver metabolizing enzymes. Compounds 3-6 suppressed >80% of the SOS-inducing activity of Trp-P-1 at <0.06 micromol/mL, and compounds 1 and 2 suppressed 87 and 63% at a concentration of 0.3 micromol/mL. In addition, these compounds were assayed with activated Trp-P-1, and the suppressed effects of these compounds were further decreased when compared to Trp-P-1. The antimutagenic activities of these compounds against furylfuramide, Trp-P-1, and activated Trp-P-1 were assayed by the Ames test using S. typhimurium TA100.     

Antimutagenic activity of polymethoxyflavonoids from Citrus aurantium

The methanol extract from Citrus aurantium showed a suppressive effect on umu gene expression of SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract from C. aurantium was successively re-extracted with hexane, dichloromethane, butanol, and water. A dichloromethane fraction showed a suppressive effect. The suppressive compounds in the dichloromethane fraction were isolated by SiO(2) column chromatography and identified as tetra-O-methylscutellarein (1), sinensetin (2), and nobiletin (3) by EI-MS and (1)H- and (13)C NMR spectroscopy. These compounds suppressed the furylfuramide-induced SOS response in the umu test. Gene expression was suppressed 67%, 45%, and 25% at a concentration of 0.6 micromol/mL, respectively. The ID(50) value (50% inhibition dose) of compound 1 was 0. 19 micromol/mL. These compounds were assayed with other mutagens, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver metabolizing enzymes, activated Trp-P-1, and UV irradiation. These compounds showed of all mutagen-induced SOS response in the umu test. In addition, compounds 1-3 exhibited antimutagenic activity in the S. typhimurium TA100 Ames test.     

Antimutagenic activity of isoflavones from soybean seeds (Glycine max merrill)

Two isoflavones, daidzein (1) and genistein (2), were isolated from soybean hypocotyls. Daidzein and genistein showed a suppressive effect on umu gene expression of the SOS response in Salmonellatyphimurium TA1535/pSK1002 against the mutagen 3-amino-1, 4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), which requires liver metabolizing enzymes. Compound 1 suppressed 73% of the SOS-inducing activity at concentrations <0.74 micromol/mL, and the ID(50) value was 0.37 micromol/mL. Compound 2 suppressed 95% of the SOS-inducing activity at concentrations <0.74 micromol/mL, and the ID(50) value was 0.17 micromol/mL. Compounds 1 and 2 were also assayed with the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) and activated Trp-P-1. In addition to the antimutagenic activities of daidzein and genistein against Trp-P-1, frylfuramide and activated Trp-P-1 were assayed by an Ames test using S. typhimurium TA100.     

Antimutagenic activity of phenylpropanoids from clove (Syzygium aromaticum)

Phenylpropanoids that possess antimutagenic activity were isolated from the buds of clove (Syzygium aromaticum). The isolated compounds suppressed the expression of the umu gene following the induction of SOS response in the Salmonella typhimurium TA1535/pSK1002 that have been treated with various mutagens. The suppressive compounds were mainly localized in the ethyl acetate extract fraction of the processed clove. This ethyl acetate fraction was further fractionated by silica gel column chromatography, which resulted in the purification and subsequent identification of the suppressive compounds. Electron impact mass spectrometry, IR, and (1)H and (13)C NMR spectroscopy were then used to delineate the structures of the compounds that confer the observed antimutagenic activity. The secondary suppressive compounds were identified as dehydrodieugenol (1) and trans-coniferyl aldehyde (2). When using 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) as the mutagen, compound 1 suppressed 58% of the umu gene expression as compared to the controls at a concentration of 0.60 micromol/mL, with an ID(50) (50% inhibitory dose) value of 0.48 micromol/mL, and compound 2 suppressed 63% of the umu gene expression as compared to the controls at a concentration of 1.20 micromol/mL, with an ID(50) value of 0.76 micromol/mL. Additionally, compounds 1 and 2 were tested for their ability to suppress the mutagenic activity of other well-known mutagens such as 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver metabolizing enzymes, and aflatoxin B(1) (AfB(1)) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver metabolizing enzymes and activated Trp-P-1 and UV irradiation. Compounds 1 and 2 showed dramatic reductions in their mutagenic potential of all of the aforementioned chemicals or treatment. For the search of the structure-activity relationship, the derivatives of 1 and 2 (1a and 2a-c) were also assayed with all mutagens. Finally, the antimutagenic activities of compounds 1, 1a, 2, and 2a-c against furylfuramide, Trp-P-1, and activated Trp-P-1 were assayed by the Ames test using the S. typhimurium TA100 strain.     

Quantitation of 13 heterocyclic aromatic amines in cooked beef, pork, and chicken by liquid chromatography-electrospray ionization/tandem mass spectrometry

The concentrations of heterocyclic aromatic amines (HAAs) were determined, by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS), in 26 samples of beef, pork, and chicken cooked to various levels of doneness. The HAAs identified were 2-amino-3-methylimidazo[4,5- f]quinoline, 2-amino-1-methylimidazo[4,5- b]quinoline, 2-amino-1-methylimidazo[4,5- g]quinoxaline (I gQx), 2-amino-3-methylimidazo[4,5- f]quinoxaline, 2-amino-1,7-dimethylimidazo[4,5- g]quinoxaline (7-MeI gQx), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline, 2-amino-1,6-dimethyl-furo[3,2- e]imidazo[4,5- b]pyridine, 2-amino-1,6,7-trimethylimidazo[4,5- g]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline, 2-amino-1,7,9-trimethylimidazo[4,5- g]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-9 H-pyrido[2,3- b]indole, and 2-amino-3-methyl-9 H-pyrido[2,3- b]indole. The concentrations of these compounds ranged from <0.03 to 305 parts per billion (micrograms per kilogram). PhIP was the most abundant HAA formed in very well done barbecued chicken (up to 305 microg/kg), broiled bacon (16 microg/kg), and pan-fried bacon (4.9 microg/kg). 7-MeI gQx was the most abundant HAA formed in very well done pan-fried beef and steak, and in beef gravy, at concentrations up to 30 microg/kg. Several other linear tricyclic ring HAAs containing the I gQx skeleton are formed at concentrations in cooked meats that are relatively high in comparison to the concentrations of their angular tricyclic ring isomers, the latter of which are known experimental animal carcinogens and potential human carcinogens. The toxicological properties of these recently discovered I gQx derivatives warrant further investigation and assessment.     

Quantification of heterocyclic aromatic amines in fried meat by HPTLC/UV-FLD and HPLC/UV-FLD: a comparison of two methods

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.     

Effect of pH on binding of mutagenic heterocyclic amines by the natural biopolymer poly(gamma-glutamic acid)

Poly(gamma-glutamic acid) (gamma-PGA), a nontoxic and biodegradable macropolymer, was evaluated for its efficiency in binding three mutagenic heterocyclic amines (HAs), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-p-2), as affected by pH in a batch mode. The maximum HA sorption was attained for pH 3-7 and decreased sharply for pH less than 3. Binding isotherms obtained at pH 2.5 and 5.5 showed different isotherm shapes that belong to S and L types, respectively. The isotherm data at pH 2.5 were well described by a linear form of the Langmuir equation, while at pH 5.5 it showed two distinct curves, which were precisely fitted as multiple Langmuir curves. The deviation of linearity in Scatchard plot proved the multisite HA sorption. The Brunauer-Emmett-Teller equation also fitted better to isotherm data at pH 5.5, suggesting a multisite sorption caused by multimolecular HA layers on gamma-PGA. High HA sorption levels of 1250, 667, and 1429 mg/g at pH 2.5 and 1429, 909, and 1667 mg/g at pH 5.5 were observed for MeIQ, 4,8-DiMeIQx, and Trp-p-2, respectively. Among the HAs studied, the sorption capacity correlated directly with hydrophobicity of HAs and inversely with the number of methyl groups in HA molecules. The plausible binding mechanism of HAs on gamma-PGA may include a combination of hydrophobic, hydrogen-bonding, ionic, and dipole-dipole interactions.     

Moscatilin from Dendrobium nobile, a naturally occurring bibenzyl compound with potential antimutagenic activity.

A bibenzyl compound that possesses antimutagenic activity was isolated from the storage stem of Dendrobium nobile. The isolated compound suppressed the expression of the umu gene following the induction of SOS response in Salmonella typhimurium TA1535/pSK1002 that have been treated with various mutagens. The suppressive compound was mainly localized in the n-hexane extract fraction of the processed D. nobile. This n-hexane fraction was further fractionated by silica gel column chromatography, which resulted in the purification and subsequent identification of the suppressive compound. EI-MS and (1)H and (13)C NMR spectroscopy were then used to delineate the structure of the compound that confers the observed antimutagenic activity. Comparison of the obtained spectrum with that found in the literature indicated that moscatilin is the secondary suppressive compound. When using 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) as the mutagen, moscatilin suppressed 85% of the umu gene expression compared to the controls at <0.73 micromol/mL, with an ID(50) value of 0.41 micromol/mL. Additionally, moscatilin was tested for its ability to suppress the mutagenic activity of other well-known mutagens such as 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), UV irradiation, 3-amino-1,4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), benzo[a]pyrene (B[a]P), and aflatoxin B(1) (AFB(1)). With all of the aforementioned chemicals or treatments, moscatilin showed a dramatic reduction in their mutagenic potential. Interestingly, moscatilin almost completely suppressed (97%) the AFB(1)-induced SOS response at concentrations <0.73 micromol/mL, with an ID(50) of 0.08 micromol/mL. Finally, the antimutagenic activities of moscatilin against furylfuramide and Trp-P-1 were assayed by the Ames test using the S. typhimurium TA100 strain. The results those experiments indicated that moscatilin demonstrated a dramatic suppression of the mutagenicity of only Trp-P-1 but not furylfuramide.     

Suppression of the SOS-inducing activity of mutagenic heterocyclic amine, Trp-P-1, by triterpenoid from Uncaria sinensis in the Salmonella typhimurium TA1535/pSK1002 Umu test

The methanol extract from Uncaria sinensis showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 3-amino-1,4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), which requires liver metabolizing enzymes. The methanol extract from U. sinensis was re-extracted with hexane, CH2Cl2, BuOH, and water, respectively. CH2Cl2 extract showed a suppressive effect. A suppressive compound 1 in CH2Cl2 extract was isolated by SiO2 column chromatography. Compound 1 was identified as ursolic acid by IR, electron ionization EI-MS, and NMR spectroscopy. Suppressive effects of ursolic acid (1) and its derivatives, methyl ursolate (1M), acetylursolic acid (1A), and methyl acetylursolate (1MA), were determined in the umu test. These compounds suppressed 61.3, 37.7, 71.5, and 37.8% of the Trp-P-1-induced SOS response at a concentration of 0.4 micromol/mL, respectively. The ID50 values of compounds 1 and 1A were 0.17 and 0.20 micromol/mL. In addition, these compounds were assayed with the activated Trp-P-1. Suppressive effects on activated Trp-P-1 were decreased as compared with those of Trp-P-1.     

Suppression of chemical mutagen-induced SOS response by alkylphenols from clove (Syzygium aromaticum) in the Salmonella typhimurium TA1535/pSK1002 umu test

A methanol extract from clove (Syzygium aromaticum) showed a suppressive effect of the SOS-inducing activity on the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) in the Salmonella typhimurium TA1535/pSK1002 umu test. The methanol extract was re-extracted with hexane, dichloromethane, ethyl acetate, butanol, and water. The hexane fraction showed a suppressive effect. Suppressive compounds in the hexane fraction were isolated by silica gel column chromatography and identified as trans-isoeugenol (1) and eugenol (2) by GC, GC-MS, IR, and (1)H and (13)C NMR spectroscopy. Compounds 1 and 2 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1 and 2 suppressed 42.3 and 29.9% of the SOS-inducing activity at a concentration of 0.60 micromol/mL. These compounds were assayed with other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In addition, compounds 1 and 2 were assayed with aflatoxin B(1) (AfB(1)) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver metabolizing enzymes. These compounds showed suppressive effects of the SOS-inducing activity against furylfuramide, 4NQO, AfB(1), and Trp-P-1. To research the structure-activity relationship, methyl esters of 1 and 2 (1Me and 2Me) and o-eugenol (3), as compounds similar to 2, were also assayed with all mutagens. Compounds 1Me, 2Me, and 3 showed weak suppressive effects of the SOS-inducing activity against furylfuramide.     

Addition of various carbohydrates to beef burgers affects the formation of heterocyclic amines during frying

The influence of the addition of carbohydrates with different physicochemical properties on weight loss and formation of heterocyclic amines (HAs) during the frying of beef burgers was examined. Furthermore, the capability of carbohydrates to bind HAs was tested. Beef burgers containing 1.5% NaCl and 0.3% tripolyphosphate (reference), with the addition of 1.5% carbohydrate, were fried for 5 min at 200 degrees C in a double-sided pan fryer. The beef burgers were analyzed for HAs with solid phase extraction and liquid chromatography/mass spectrometry. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 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), and 9H-pyrido[3,4-b]indole (Norharman) were detected in all of the beef burgers. The addition of carbohydrates affected both the weight loss and the formation of HAs during cooking. The formation of HAs could be correlated to depend on both the weight loss and the type of the added carbohydrate. Of the 11 different carbohydrates tested, raw potato starch was most capable of inhibiting the formation of HAs, while potato fiber gave the lowest weight loss and a comparably low amount of PhIP. Wheat bran and potato fiber were found to reversibly bind HAs. It is concluded that adding small amounts of certain carbohydrates may be a simple and effective way of reducing the amount of HAs and can easily be applied in households and commercial preparations of beef burgers.     

Reaction of tryptophan with carbohydrates: identification and quantitative determination of novel beta-carboline alkaloids in food

The formation of various carbohydrate-derived beta-carbolines was observed when model reactions of tryptophan with glucose were studied by means of HPLC with diode array detection, as well as by means of HPLC-MS. Isolation of these compounds and subsequent characterization by tandem mass spectrometry and NMR spectroscopy led to the identification of diastereomeric 1-(1,3,4,5-tetrahydroxypent-1-yl)-9H-pyrido[3,4-b]indoles (1a/b), 1-(1,4,5-trihydroxypent-1-yl)-9H-pyrido[3,4-b]indoles (2a/b), and E/Z isomers of 1-(1,5-dihydroxypent-3-en-1-yl)-9H-pyrido[3,4-b]indole (3a/b). HPLC-MS was used to prove the presence of these novel beta-carboline alkaloids in various food samples. In addition, quantitative determination of beta-carbolines 1a, 1b, and 2a/b in numerous products was achieved by means of HPLC with fluorometric detection. Concentrations ranged from 12 to 1922 microg/L for 1a and 1b and from 3 to 644 microg/L for 2a/b. The highest concentrations of all carbohydrate-derived beta-carbolines under study were found in ketchup, soy sauce, and fish sauce.     

Inhibitory effects of beer and other alcoholic beverages on mutagenesis and DNA adduct formation induced by several carcinogens

The possibility that beer and other alcoholic beverages could be antimutagenic against the heterocyclic amines (HAs), a group of carcinogens produced on cooking proteinaceous foods, has been explored. In the Salmonella mutation assays, beer showed inhibitory effects against several HAs [preactivated Trp-P-1, Trp-P-2(NHOH), and Glu-P-1(NHOH)] that are directly mutagenic in bacteria. Japanese sake, red and white wines, and brandy were also effective. However, ethyl alcohol alone did not show these effects. The formation of O(6)-methylguanine by N-methyl-N'-nitro-N-nitrosoguanidine in the DNA of Salmonella YG7108 was also inhibited by beer. Nonvolatile beer components were administered orally to CDF(1) mice together with Trp-P-2. Adducts in the liver DNA were significantly decreased by the beer, as compared to those in controls fed Trp-P-2 only. Although several phenolic compounds known to be present in beer were antimutagenic toward these mutagens, their effects were very small. It was concluded that some yet to be identified component(s) of beer is (are) responsible for this antimutagenicity.     

Antimutagenicity of some edible Thai plants,and a bioactive carbazole alkaloid,mahanine, isolated from Micromelum minutum

The antimutagenic activity against Trp-P-1 of methanolic extracts of 118 samples (108 species) of edible Thai plants was examined by the Ames Test. The activity was evaluated by the amount of plant extracts which suppressed 90% of the mutagenesis (ED90). Five plants, Micromelum minutum, Oroxylum indicum, Cuscuta chinensis, Azadirachta indica, and Litsea petiolata, exhibited significant activity with antimutagenic ED90 values lower than 5 microL/plate (0.1 mg of dry plant material equivalent). The activity-guided fractionation of the extract of M. minutum, which exhibited the highest antimutagenic activity in the screening, resulted in the isolation of an active principle, (+)-mahanine (1) as confirmed by its physicochemical properties. Compound 1 showed a wide variety of biological activity, including antimutagenicity against heterocyclic amines such as Trp-P-1 with an IC50 of 5.2 microM, cytotoxicity against a tumor cell line HL60 with a MIC100 of 4.0 microg/mL, and antimicrobial activity against Bacillus cereus and Staphylococcus aureus with MIC100 values of 6.25 and 12.5 microg/mL, respectively.     

Effect of high water-holding capacity on the formation of heterocyclic amines in fried beefburgers

Mutagenic/carcinogenic heterocyclic amines (HAs) are formed in the crust during the cooking of meat. The influence of cooking loss, time, and temperature on the formation of HAs was investigated in fried beefburgers. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) were identified in all samples. The amounts of PhIP, MeIQx, and 4,8-DiMeIQx increased with increasing cooking time, and this effect was significant for all three HAs. The pan temperature had a significant effect on the formation of PhIP and 4,8-DiMeIQx. The addition of NaCl/sodium tripolyphosphate to the beefburgers reduced the cooking loss and decreased the formation of PhIP, MeIQx, and 4,8-DiMeIQx. This decrease was significant for MeIQx and 4,8-DiMeIQx. The results clearly show that it is possible to modify cooking practices to minimize the formation of HAs.