Antimutagenic Effect of Methanolic Extracts from Peanut Hulls

The antimutagenic effects of methanolic extracts of peanut hulls (MEPH) were evaluated by the Ames test. MEPH inhibited the mutagenicity of 4-nitroquinoline-N-oxide (NQNO), a direct-acting mutagen. MEPH also inhibited the mutagenicity of some indirect-acting mutagens and decreased in the order of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ)>aflatoxin B₁ (AFB₁)>2-amino-6-methyldipyrido(1,2-a : 3′, 2′-d)imidazole (Glu-P-1) > 3-amino-1,4-dimethyl-5H-pyridol(4,3-b)indole (Trp-P-1) > benzo(a)pyrene (B(a)P) for 5. typhimurium TA98, and IQ > Trp-P-1 > Glu-P-1 > AFB₁ > B(a)P for S. typhimurium TA100.     

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

Structural Elucidation of Rotihibin B by Tandem Mass Spectrometry

The mutagenicity and desmutagenicity of extracts of soybeans heated at 225 ± 5°C were investigated by the Ames test. The soybeans were refluxed in water, methanol, or diethylether for 2h. The aqueous and methanol extracts (2–4 mg/plate) of the heated soybeans exhibited strong desmutagenic activity of 43–92% against heterocyclic amines (Trp-P-1, Glu-P-2, IQ, MeIQx, PhIP), while no mutagenicity was observed. The desmutagenicity of the heated soybean extracts remained even after denaturation by 0.1 N HCI in vitro and absorption by the rat small intestine. The desmutagenic mechanism for heated soybeans was evaluated, and it was verified that the soybean extract exhibited its desmutagenicity by blocking the mutagenicity of activated Trp-P-1, and not by inhibiting the S9 enzyme system.     

Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative

Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH)) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.     

Antimutagenic Acitivity against Trp-P-1 of the Edible Thai Plant,Oroxylum indicum Vent.

A methanolic extract of Oroxylum indicum strongly inhibited the mutagenicity of Trp-P-1 in an Ames test. The major antimutagenic constituent was identified as baicalein with an IC₅₀ value of 2.78±0.15 μM. The potent antimutagenicity of the extract was correlated with the high content (3.95±0.43%, dry weight) of baicalein. Baicalein acted as a desmutagen since it inhibited the N-hydroxylation of Trp-P-2.     

Coenzymatic Activity of Epinephrine for Vitamin B2-Aldehyde Forming Enzyme and Its Physiological Significance

The effects of artificial food dyes on the mutagenicity of carcinogenic mutagens were examined using the Salmonella/microsome system. Indigocarmine (IC), an indigoid dye widely used for coloring foods and for clinical tests, enhanced the mutagenic activity of Trp-P-1, a carcinogenic pyrolysate of tryptophan, depending on the dose of IC. His⁺ revertants of TA 98 induced by Trp-P-1 were two to four times greater in the presence of 10 to 50 μg/plate of IC than those in the absence of IC.

IC also enhanced the mutagenicity of Trp-P-2, another carcinogenic pyrolysate of tryptophan, while the activities of other mutagens such as MNNG, 4-NQO, AF-2, BP, Glu-P-1 were not affected.     

The Piperaceae Amides I: Structure of Pipercide,A New Insecticidal Amide from Piper nigrum L.

It was evidenced that mutagenic principles in tryptophan pyrolysate, 3-amino-1,4-dimethyl-5H pyrido(4,3-b) indole and 3-amino-1-methyl-5H pyrido(4,3-b) indole (abbreviated as Trp-P-1 and Trp-P-2, respectively) bind to DNA without activation by rat liver microsomes. The bindings of Trp-P-1 and Trp-P-2 were not random and did not introduce strand scissions into DNA. Trp-P-1 bound more easily than Trp-P-2. The bindings of these mutagenic principles to DNA were concluded by using negatively superhelical simian virus 40 (SV40) DNA from following experimental data. (1) The intensity of ethidium bromide (EtBr)-DNA fluorescence by illumination with UV light and the electrophoretic mobility of superhelical DNA in agarose gel decreased as a function of the amounts of Trp-P-1 and Trp-P-2. (2) In vitro RNA synthesis catalyzed by Escherichia coli DNA-dependent RNA polymerase and nick-translation catalyzed by Escherichia coli DNA polymerase I (Kornberg enzyme) were inhibited significantly on DNA treated with Trp-P-1 and Trp-P-2. (3) The negative superhelicity of SV40 DNA introduces unpaired regions into DNA. These regions can be cleaved by single-strand-specific S1 endonuclease to generate unit length linear duplex molecules. It was found that this S1-sensitivity of DNA decreased by treatment with Trp-P-1. (4) The cleavage patterns of Trp-P-1 treated DNA with five restriction endonucleases were investigated. The protection of the cleavage site by the drug was observed against HincII, HindIII and EcoRII, whereas not against HaeIII and HinfI. These results show that the binding of Trp-P-1 to DNA is not random. Identical results were also obtained in Trp-P-2.

However, the bindings of Trp-P-1 and Trp-P-2 were not so tight, and phenol extraction of the complex dissociated these drugs from DNA.     

Mutagenicity of Intermediates Produced in the Early Stage of the Browning Reaction of Triose Reductone with Nucleic Acid Related Compounds on Bacterial Tests

Two types of reductive intermediates, linear and tricyclic forms, isolated from browning mixtures of triose reductone (TR) with guanine and its derivatives showed evident mutagenicity on Salmonella typhimurium TA 100 without S-9 mixture. The linear intermediates, N²-(3-oxo-2-hydroxypropenyl) compounds of guanine, guanosine, 2′(3′)-guanylic acid and 5′-guanylic acid were more effective than the tricyclic one, l, N²-(2-hydroxypropenylidene)guanine, though they were far less active than 4-nitroquinoline-N-oxide. No acceleration in mutagenicity was observed with Cu^{2 +} and other metal ions. The reaction mixtures of TR and nucleic acid bases were also mutagenic on TA 100. Intermediates of TR with guanine and its derivatives did not have a lethal effect in Recassays with Bacillus subtilis.     

Antimutagenic activity against trp-P-1 of the edible Thai plant, Oroxylum indicum vent.

A methanolic extract of Oroxylum indicum strongly inhibited the mutagenicity of Trp-P-1 in an Ames test. The major antimutagenic constituent was identified as baicalein with an IC50 value of 2.78+/-0.15 microM. The potent antimutagenicity of the extract was correlated with the high content (3.95+/-0.43%, dry weight) of baicalein. Baicalein acted as a desmutagen since it inhibited the N-hydroxylation of Trp-P-2.     

N-Terminal Amino Acid Sequence of the Chlorophyll-binding Protein CP-47 of Photosystem 2 in the Thermophilic Cyanobacterium Synechococcus elongatus

Effects of caffeic acid and chlorogenic acid on mutagenicity were studied using the Salmonella typhimurium system. These compounds had inhibitory effects on the mutagenicity of Trp-P-1 and Glu-P-2. Caffeic acid completely eliminated the mutagenicity induced by activated Glu-P-2. Some compounds analogous to caffeic acid, such as cinnamic acid, coumaric acid, and ferulic acid, also significantly decreased the mutagenicity of Glu-P-2.     

Antimutagenic Effect of Amino Acids on the Mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

The antimutagenic activity of protein-constituting amino acids except histidine on the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was investigated in vitro using Salmonella typhinurium TA-100 as an indicator bacterium (Ames test), and concentrations (IC₅₀) of amino acids that inhibit 50% of the mutagenecity were measured. Cysteine was found to be most active and glycine, tryptophan, lysine, and arginine were strong antimutagenic amino acids. Other amino acids showed moderate or weak antimutagenic activities, depending on the amino acids. The results indicate that amino acids play a substantial role in chemoprevention of N-nitroso amine-induced mutagenicity.     

Facile Synthesis of 4-O-β-N-Acetylchitooligosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone Based on the Transformation of Chitooligosaccharide and Its Suppressive Effects against the Furylfuramide-Induced SOS Response

A facile synthesis method is described for transforming the reducing-end residue of chitooligosaccharides (DP 2–4) into lactone. The desired 4-O-β-N-acetylchitooligosyl lactones (GN_nL) were conveniently prepared from chitooligosaccharides by consecutive dehydration and oxidation reactions to afford 4-O-β-tri-N-acetylchitotriosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN₃L), 4-O-β-di-N-acetylchitobiosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN₂L), and 4-O-β-2-acetamido-2-deoxy-D-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GNL). The resulting lactone derivatives exhibited considerable suppression (42.6–54.3% at a concentration of 400 µM) in umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2). Lactonization of the chitooligosaccharides was found to be essential for their suppression of the SOS-inducing activity.     

Cleavage Maps of Dehalogenation Plasmid pUOl and Its Deletion Derivative Harbored in Moraxella sp.

Inhibitory effects of lactic acid bacteria for dairy use on the mutagenicities of some volatile nitrosamines were investigated in vitro using a Salmonella typhimurium TA 98 streptomycin-dependent strain (SD 510) as an indicator bacterium. Among 40 strains examined, Leuconostoc paramesenteroides R-62, R-8, Streptococcus lactis subsp. diacetylactis R-63, and St. cremoris R-48 strongly inhibited the mutagenicity of N-nitroso-diethylamine (NDEA) and moderately N-nitroso-dimethylamine (NDMA), but not N-nitroso-piperidine (NPIP) or N-nitroso-pyrrolidine (NPYR). In addition, the filtrates obtained from cell suspensions of the lactic acid bacteria examined inhibited the mutagenicity of NDEA.     

Peptization of the Gel of Konjac Mannan

The addition of 1,8-pyrenequinone into the assay system containing rat liver homogenates (S-9) caused an approximately 10-fold increase in the mutagenicity of 2-acetylaminofluorene (AAF) in the current Salmonella reversion assay system. Since no chemical reaction between 1,8-pyrenequinone and AAF was observed, the in vitro effects of 1,8-pyrenequinone on the metabolisms of AAF with S-9 mix were studied. The enhancement of mutagenicity by 1,8-pyrenequinone was not dependent on the dose of NADPH under the present assay condition. The mutagenicity of AAF was increased approximately 4-fold by the addition of 1,8-pyrenequinone into microsomes, whereas it remained at the spontaneous level in the presence of cytosol. However, by reconstituting microsomes with cytosol, the mutagenicity enhancing activity was recovered to the original level. Since 1,8-pyrenequinone inhibited the AAF hydroxylase activity, chemical analysis of the incubation mixture of AAF was tried. This indicated that a higher amount of unmetabolized AAF remained and higher amounts of 2-aminofluorene and N-hydroxy-2-acetylaminofluorene were accumulated in the presence of 1,8-pyrenequinone compared with those in the absence of 1,8-pyrenequinone. From these results, it seems probable that 1,8-pyrenequinone inhibits C-hydroxylation (the detoxifying pathway) and promotes N-hydroxylation (the activating pathway) as well as deacetylation in the AAF metabolism.     

Study on mutagenicity and antimutagenicity of BHT and its derivatives in a bacterial assay

The mutagenicity of butylated hydroxytoluene (BHT) and its derivatives was investigated by the Ames method using Salmonella typhimurium TA98 and TA100 with or without S9 mix. The compounds were not mutagenic in either indicator strain at concentrations ranging from 50 to 330 micrograms/plate (SQ: 3,5,3',5'-tetra-tert-butylstilbenequinone; VI-III: unidentified), 500 micrograms/plate (BE: 3,5,3',5'-tetra-tert-4,4'-dihydroxy-1,2-diphenylethylene; VI: 2,6-di-tert-butyl-4-methyl-4-tert-butylperoxy-2,5-cyclohexadienone ; VI-I: unidentified; VI-II: 3-acetyl-2,5-di-tert-cyclopenta-2,4-dienone) and 1000 micrograms/plate (BHT). The antimutagenic effects of BHT and its derivatives on mutagenesis by chemical agents were investigated in Salmonella typhimurium TA98 and TA100 and Escherichia coli WP-2 hcr-. VI-II suppressed the mutagenesis induced in TA98 and TA100 by 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and that induced in WP-2 hcr- by 4-nitroquinoline-1-oxide (4NQO) without decreasing cell viability. In WP-2 hcr-, the mutagenesis induced by AF-2 and ethyl methanesulfonate was also suppressed significantly. Mutations induced by methyl methanesulfonate were slightly inhibited. However, VI-II had no effect on the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Effect of glutathione and uridine-5'-diphosphoglucuronic acid on the mutagenicity of tryptophan pyrolysis products (Trp-P-1 and Trp-P-2) by rat-liver and -intestine S9 fraction

In the Ames test, after the addition of glutathione (GSH) or uridine-5'-diphosphoglucuronic acid (UD-PGA), we observed for Trp-P-1 an unchanged or a reduced mutagenicity by both the liver and intestine S9 fraction. For Trp-P-2, the same was true when we used the intestine S9 fraction. In the presence of liver S9 fraction, Trp-P-2 mutagenicity was also decreased by the addition of UDPGA but was increased by the addition of GSH. These results show that cofactors for glucuronide and GSH conjugation may alter the metabolic activation of Trp-P-1 and Trp-P-2 and consequently their mutagenicity.     

Inhibition of mutagenicity of N-nitrosamines by tobacco smoke and its constituents

Tobacco smoke is a complex chemical mixture including pyridine alkaloids and N-nitrosamines, with the concentration of the former several orders of magnitude higher than that of the N-nitrosamines. The major biologically important N-nitrosamines present in tobacco smoke are N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosonornicotine (NNN). These nitrosamines require metabolic activation by cytochrome P-450s for the expression of mutagenicity. Although nicotine, the major pyridine alkaloid in tobacco, has been shown to inhibit the metabolic activation of NNK, its effect on the mutagenicity of NNK and other N-nitrosamines has not been reported. In the present study, the ability of three pyridine alkaloids (nicotine, cotinine, nornicotine) and aqueous cigarette smoke condensate extract (ACE) to inhibit the mutagenicity of tobacco-related N-nitrosamines was tested on Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9). All three of the pyridine alkaloids tested, as well as ACE, inhibited the mutagenicity of NDMA and NNK, but not NNN, in a concentration-dependent manner. The induction of SCEs in mammalian cells (CHO) by NNK in the presence of metabolic activation was also significantly reduced by nicotine and cotinine. None of the observed reductions in mutagenicity could be explained by cytotoxicity. These results demonstrate that tobacco smoke contains chemicals, pyridine alkaloids and other unidentified constituent(s), which inhibit the mutagenicity of N-nitrosamines.     

Protection against the bacterial mutagenicity of heterocyclic amines by purpurin, a natural anthraquinone pigment.

Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a naturally occurring anthraquinone pigment found in species of madder root. We have found that the presence of purpurin in bacterial mutagenicity assays is responsible for a marked inhibition of mutagenicity induced by food-derived heterocyclic amines. Purpurin was found to be a better inhibitor of Trp-P-2-dependent mutagenicity than either epigallocatechin gallate or chlorophyllin both of which are well-established anti-mutagenic components of diet. Inhibition of Trp-P-2(NHOH) mutagenicity by purpurin was dependent upon pH. It was a better inhibitor in neutral than acidic conditions. Purpurin was protective against the direct mutagen Trp-P-2(NHOH) in both the presence and the absence of hepatic S9 but required pre-incubation. Finally, purpurin was responsible for the inhibition of human CYP1A2 and human NADPH-cytochrome P450 reductase and a decrease in the bioactivation of Trp-P-2 by these enzymes when they were expressed in Salmonella typhimurium TA1538ARO. However, inhibition of Trp-P-2(NHOH)-dependent mutations suggests purpurin also has a direct effect on this mutagen in addition to inhibiting its formation by CYP1A2.     

Deacylation of carcinogenic 5-nitrofuran derivatives by mammalian tissues

The deacylations of N-[4-(5-nitro-2-furyl)-2-thiazolyl] fonnamide (FANFT), N-[4-(5-nitro-2-furyl)-2-thiazolyl] acetarnide (NFTA) and formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl] hydrazide (FNT) by liver, kidney, small intestines and stomach of mouse, rat, hamster and guinea pig were investigated. FANFT was deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT). FANFT formamidase activity was higher in the liver and small intestines of mouse, hamster and guinea pig, and small intestines and stomach of rat. There was no detectable FANFT formamidase activity in the stomach of the mouse and hamster. Neither NFTA nor FNT was deacylated by the rodent tissue homogenates studied. It is suggested that (1)4 ANFT is a metabolite of FANFT but not NFTA; (2) 2-hydrazino-4-(5-nitro-2-furyl)thiazole (HNFT) may not be a metabolite of FNT; and (3) the induction of tumors by FANFT, NFTA and FNT may not be due to a common carcinogenic metabolite, although these chemicals demonstrate similar organ specificities in some of these rodents.     

Antimutagenic activity of flavonoids from Chrysanthemum morifolium

A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the 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, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction showed a suppressive effect. Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and (1)H and 13C NMR spectroscopy. Compounds 1-4 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1-4 suppressed 60.2, 75.7, 90.0, and 66.6% of the SOS-inducing activity at a concentration of 0.70 micromol/ml. The ID50 (50% inhibitory dose) values of 1-4 were 0.62, 0.55, 0.44, and 0.59 micromol/ml. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100.