Inhibitory effect of curcumin and its natural analogues on genotoxicity of heterocyclic amines from cooked food

Curcumin (C) and its natural analogues demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC), known for their potent anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic effects, were tested for their possible inhibitory effects against seven cooked food mutagens (heterocyclic amines): 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), 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-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor induced rat liver S9 homogenate. In the present investigations, curcumin as well as its two natural analogues i.e., dmC and bdmC were found to be highly effective in suppressing genotoxicity of all the tested cooked food mutagens in a dose-dependent manner, in both the frame shift (TA98) as well as base pair mutation sensitive (TA100) strains of S. typhimurium. However, bdmC appeared to be a relatively less active antimutagen compared to C and dmC. More than 80% inhibition of mutagenicity was observed at 200 microg/plate in case of C and dmC in both TA98 and TA100 against all tested cooked food mutagens. Where as, bdmC showed 39-79% inhibition in TA100 and 60-80% inhibition in TA98, at a dose of 200 microg/plate. These findings warrant further biochemical, enzymatic and in vivo investigations in animal models as well as in humans to establish the chemoprotective effect of these agents against mutagenic heterocyclic amines found in cooked food.     

Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane--a constituent of broccoli

Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines). These include imidazoazaarenes such as 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-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9. Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.     

Mutagenicity of some heterocyclic amines in Salmonella typhimurium with metabolic activation by human red blood cell cytosol.

Purified human red blood cell cytosol was used to activate the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) into mutagenic intermediate(s) in the Salmonella test. The liquid preincubation method in the presence of strain TA98 was utilized. In order to understand the mechanism involved in this metabolic activation, some modulators were incorporated in the medium. The results suggest that an oxygenated hemoprotein, probably oxyhemoglobin, is involved in the activation into genotoxic intermediate(s).     

Inactivation of potent pyrolysate mutagens by chlorinated tap water

The potent mutagens 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2, 62450-07-1), 2-amino-6-methyldipyrido-[1,2-a:3', 2'-d]imidazole (Glu-P-1, 67730-11-4) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 76180-96-6), isolated from pyrolysates of tryptophan and glutamic acid and from broiled sardines, respectively, were effectively degraded by chlorinated tap water with a concomitant loss of mutagenicity toward Salmonella typhimurium TA98 and TA100. The half-life of 10 microM IQ in the presence of 1.5 ppm of residual chlorine was less than 10 sec; those of Glu-P-1 and Trp-P-2 were 0.5-1 and 2-3 min, respectively. This means that a glass of chlorinated tap water (150 ml) containing 1.5 ppm of residual chlorine can break down about 200 micrograms of these pyrolysate mutagens within a couple of minutes.     

Binding of mutagenic pyrolyzates to fractions of intestinal bacterial cells.

The binding of mutagenic pyrolyzates to cell fractions from some gram-negative intestinal bacteria and to thermally treated bacterial cells was investigated. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were effectively bound by several of the bacterial cells. The cell wall skeletons of all bacteria effectively bound Trp-P-1 and Trp-P-2. Their cytoplasmic fractions retained Trp-P-1 and Trp-P-2, but to a lesser extent than the cell wall skeletons. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) was not found in their cytoplasmic fractions. These cell wall skeletons also bound 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-amino-5-phenylpyridine (Phe-P-1), IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQX). The amount of each mutagen bound differed with the type of mutagen and the bacterial strain used. The outer membrane of Escherichia coli IFO 14249 showed binding of about 123.7 micrograms/mg of Trp-P-2, and its cytoplasmic membrane bound 57.14 micrograms/mg. Trp-P-2 bound to the bacterial cells was extracted with ammonia (5%), methanol, and ethanol but not with water.     

Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods

Samples of 7 foods commonly eaten in the Northeast of China (i.e. fried and broiled fishes and broiled meat) were tested for mutagenicity on Salmonella typhimurium TA98 with S9 mix. The basic fractions of the samples were mutagenic, inducing 33-2930 revertants/g of cooked food. Fried walleye pollack (a kind of cod fish heated on a stainless steel pan) showed the highest mutagenicity, so attempts were made to isolate mutagens from the basic fraction of this food. The mutagens were purified by treatment with blue cotton and HPLC on a semi-preparative ODS column and analytical cation exchange and ODS columns. 5 mutagens were isolated and identified as 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), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). 1 g of fried fish was estimated to contain 0.16 ng of IQ, 0.03 ng of MeIQ, 6.44 ng of MeIQx, 0.10 ng of 4,8-DiMeIQx and 69.2 ng of PhIP. MeIQx and PhIP accounted for 24% and 4.7%, respectively, of the total mutagenicity. The other 3 heterocyclic amines were each responsible for only 0.3-1.2% of the total mutagenicity.     

Binding activity of natto (a fermented food) and Bacillus natto isolates to mutagenic-carcinogenic heterocyclic amines.

The fermented food, whole meal Natto, viscous polymeric material from Natto, Natto bean, cooked soya bean, and 28 bacterial isolates from Natto were studied for their binding capacity to foodborne mutagenic-carcinogenic heterocyclic amines. The mutagenic heterocyclic amines used were Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole); Trp-P-2 (3-amino-1-methyl-5H-pyrido(4,3-b)indole); Glu-P-1 (2-amino-6-methyldipyrido(1,2-a:3'2'-d)imidazole); PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine); IQ (2-amino-3-methylimidazo(4,5-f)quinoline); MeIQ (2-amino-3,4-dimethylimidazo(4,5-f)quinoxaline); MeIQx (2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline); and MeAalphaC (2-amino-3-methyl-9H-pyrido(2,3)indole). The lyophilized Natto and other fractions of Natto exhibited high binding activity towards Trp-P-1, Trp-P-2, PhIP, and MeAalphaC, while Glu-P-1, IQ, and MeIQ were not effectively bound. The binding capacity of bacterial isolates (Bacillus natto) were isolate-mutagen dependent. Heat treated lyophilized cells, cell wall, and cytoplasmic contents of the bacterial isolate with the highest binding capacity were analyzed for their ability to bind different heterocyclic amines. The results indicate the importance of the cell wall in binding to heterocyclic amines, whereas the cytoplasmic contents were less effective. Heat-treated cells were not much different from that of viable cells in their binding. The impact of different factors, such as pH, incubation time, metal ions, different concentrations of sodium chloride and alcohol, various enzymes, and acetylation of mutagens on binding of Trp-P-1 and IQ, were discussed. The significance of the present results is also discussed from the viewpoint that Natto, a fermented food, is able to scavenge dietary mutagenic heterocyclic amines through binding.     

Developmental changes in hepatic activation of dietary mutagens by mice

Metabolic activation of the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and aflatoxin B1 by female BALB/c mice of different ages (2-24 weeks) was investigated in vivo and in vitro using Salmonella typhimurium TA98 as the indicator organism. The in vivo activation of the three mutagens was investigated in 4- and 24-week-old mice using an intrasanguineous host-mediated assay. All three compounds showed reduced levels of activation with the older hosts. Hepatic S9 fractions from female mice of varying ages between 2 and 24 weeks were used in the in vitro mutagenicity assay. To achieve optimal activation to bacterial mutagens, 5% S9 was required for aflatoxin B1 and Trp-P-2 and 10% S9 for MeIQ; age of donor generally had little effect on the profile of these protein activation curves. Under these optimal conditions MeIQ and Trp-P-2 both exhibited, as before, age-dependent decreases in activation over a wide range of mutagen concentrations, however the in vitro activation of aflatoxin showed no consistent change with age. Spectrophotometric measurements of S9 cytochrome P-450 content showed a decrease in concentration with increasing age, but this was not sufficient to account for changes observed in hepatic mutagen activation. However, changes in the activities of certain cytochrome P-450 isoenzymes and cytosolic GSH-transferases, which in turn result in changes in the activation and detoxification capacity of the liver, would appear to explain age-dependent changes in the activity of mutagens in vivo.     

Potent antimutagenic activity of white tea in comparison with green tea in the Salmonella assay.

There is growing interest in the potential health benefits of tea, including the antimutagenic properties. Four varieties of white tea, which represent the least processed form of tea, were shown to have marked antimutagenic activity in the Salmonella assay, particularly in the presence of S9. The most active of these teas, Exotica China white tea, was significantly more effective than Premium green tea (Dragonwell special grade) against 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and four other heterocyclic amine mutagens, namely 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Mechanism studies were performed using rat liver S9 in assays for methoxyresorufin O-demethylase (MROD), a marker for the enzyme cytochrome P4501A2 that activates heterocyclic amines, as well as Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ). White tea at low concentrations in the assay inhibited MROD activity, and attenuated the mutagenic activity of N-hydroxy-IQ in the absence of S9. Nine of the major constituents found in green tea also were detected in white tea, including high levels of epigallocatechin-3-gallate (EGCG) and several other polyphenols. When these major constituents were mixed to produce "artificial" teas, according to their relative levels in white and green teas, the complete tea exhibited higher antimutagenic potency compared with the corresponding artificial tea. The results suggest that the greater inhibitory potency of white versus green tea in the Salmonella assay might be related to the relative levels of the nine major constituents, perhaps acting synergistically with other (minor) constituents, to inhibit mutagen activation as well as "scavenging" the reactive intermediate(s).     

Dietary fat modifies the in vivo mutagenicity of some food-borne carcinogens

Female BALB/c mice were fed a low fat diet (1% safflower oil, by weight) or one supplemented with 25% (by weight) of beef fat or olive oil. The abilities of these diets to modify the in vitro and in vivo hepatic conversion of the dietary carcinogens aflatoxin B1, 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) to bacterial mutagens was evaluated. Dietary olive oil appeared to increase the metabolism of both MeIQ and Trp-P-2 to bacterial mutagens in vivo using the intrasanguineous host-mediated assay. Feeding mice either of the high-fat diets increased hepatic conversion of these two compounds to bacterial mutagens in vitro. Dietary fat had no effect on the metabolism of aflatoxin B1. Subsequent experiments suggested that the in vivo effects of dietary olive oil on MeIQ and Trp-P-2 mutagenesis were due to the induction of hepatic enzyme activities rather than to increased rates of uptake of the carcinogen from the gut-lumen.     

Identification of mutagenic heterocyclic amines (IQ, Trp-P-1 and AalphaC) in the water of the Danube river

Three mutagenic heterocyclic amines, 2-amino-3-methylimidazo-[4, 5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), were isolated and identified in water from the Danube River in Vienna. Heterocyclic amines were extracted from river water by the blue rayon hanging method, and analyzed by gas chromatography with a nitrogen-phosphorous detector (GC-NPD) and GC-mass spectrometry (GC-MS) after conversion into their N-dimethylaminomethylene derivatives. Identity of IQ, Trp-P-1 and AalphaC in the river water was confirmed by GC-MS. The contents of IQ, Trp-P-1 and AalphaC were estimated by GC-NPD at 1.78+/-0.17, 0.14+/-0.02 and 0.44+/-0.02 ng/g blue rayon equivalent (n=3), respectively. The total amounts of these amines accounted for 26% of the mutagenicity of blue rayon extracts evaluated by the Ames test using TA98 with metabolic activation.     

Regional mutagenicity of heterocyclic amines in the intestine: mutation analysis of the cII gene in lambda/lacZ transgenic mice

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.     

Antimutagenic effects and possible mechanisms of action of vitamins and related compounds against genotoxic heterocyclic amines from cooked food.

Possible antimutagenic activity of 26 vitamins and related compounds - ascorbic acid, beta-carotene, cyanocobalamin, folic acid, nicotinic acid, nicotinamide, pantothenic acid, pyridoxale, pyridoxamine, pyridoxine, retinal, retinol, retinoic acid, retinyl acetate, retinyl palmitate, riboflavin, riboflavin 5'-phosphate, flavin adenine dinucleotide (FAD), alpha-tocopherol, alpha-tocopherol acetate, vitamins K(1), K(3), K(4), 1, 4-naphthoquinone, and coenzyme Q(10) - was tested against six heterocyclic amine (HCA) mutagens, i.e., 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), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in the Salmonella/reversion assay using tester strains Salmonella typhimurium TA 98 and TA 100. Retinol, retinal, riboflavin, riboflavin 5'-phosphate, FAD, vitamins K(1), K(3), K(4), 1, 4-naphthoquinone, and coenzyme Q(10) caused a concentration-dependent decrease in the mutagenicity of all six mutagens in both tester strains. Quantification of antimutagenic potencies by calculating ID(50)1000; vitamin K(1): 401-740; vitamin K(3) (menadione): 85-590; vitamin K(4): 45-313; 1,4-naphthoquinone: 170-290; coenzyme Q(10): 490-860. In general, there were no major differences between HCAs tested except in part with Trp-P-2 nor between the two tester strains. In enzyme kinetic experiments with Salmonella, retinol, vitamins K(3), and K(4) behaved as competitive inhibitors of IQ induced mutagenesis. However, at the highest concentration of menadione (200 nmol/plate) and of riboflavin 5'-phosphate (2000 nmol/plate), non-competitive inhibition was observed. At other concentrations of riboflavin 5'-phosphate and at all concentrations of FAD, meaningful interpretation of enzyme kinetics were not possible. Reduction of the activity of 7-ethoxy- and 7-methoxyresorufin-O-dealkylases with IC(50) values of 2.03-30.8 microM indicated strong inhibition of 1A1 and 1A2 dependent monooxygenases by menadione and retinol. Riboflavin 5'-phosphate and FAD were less effective (IC(50): 110-803.7 microM). Nicotinamide-adenine-dinucleotidephosphate (NADPH) cytochrome P-450 reductase was not affected by retinoids but stimulated by naphthoquinones and both riboflavin derivatives up to about 50 and 80%, respectively. Again, the mutagenic activity of N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoline (N-OH-IQ) in Salmonella was not suppressed by K-vitamins but marginally reduced by retinol, retinal, and FAD but distinctly by riboflavin 5'-phosphate. In various experiments designed for modulation of the mutagenic response, inhibition of metabolic activation of IQ to N-OH-IQ was found to be the only relevant mechanism of antimutagenesis of menadione while a weak contribution of an other way seemed possible for retinol and FAD.     

Activation of the food mutagens IQ and MeIQ by hepatic S9 fractions derived from various species

The ability of hepatic S9 mixes derived from different rodent species (rat, mouse, Syrian and Chinese hamster) to activate the mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was investigated using Salmonella typhimurium strain TA98. In general, the mutagenicity of IQ and MeIQ was greatest in the presence of S9 fractions from Swiss albino mice and least from fractions derived from Chinese hamsters. However, treatment of rats or hamsters with Aroclor 1254 had little or no effect on the activation of IQ or MeIQ to mutagens.     

Metabolic activation of food mutagens by liver and lung enzymes from rats, pigs and oxen

Mutagenic activation of the 3 cooked food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was compared in liver and lung enzyme preparations from oxen, pigs and rats. Liver preparations from oxen were the most efficient in activating the mutagens, while the rat enzymes were more active than those from pigs. The different cooking mutagens showed different mutagenic potential. MeIQ was the most potent mutagen, followed by IQ and MeIQx in descending order. In oxen, MeIQx was as potent as IQ. The activation with the lung enzymes was 2-3 orders of magnitude lower than with liver. Furthermore, species differences in mutagenic activation with lung enzymes were small compared with liver enzymes. In lung preparations the differences between IQ and MeIQ were small, but in all 3 animal species the mutagenicity of MeIQx was 1 order of magnitude lower than that of the other 2 mutagens.     

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.     

The antimutagenic properties of a polysaccharide produced by Bifidobacterium longum and its cultured milk against some heterocyclic amines

The antimutagenicity and fermentation pattern of three Bifidobacterium longum strains (B. longum, B. longum PS+, and B. longum PS-) in skim milk were studied. The increase in fermentation time significantly increased antimutagenicity with all strains tested against the mutagenicity of both 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in an Ames-like test using streptomycin-dependent strain SD510 of Salmonella typhimurium TA98. Bifidobacterium longum PS+, a polysaccharide-producing strain, had a longer lag phase but showed the highest inhibition percentage against both mutagens tested. The viability of B. longum PS+ cells was not affected by the low pH of 4.1, probably owing to the protection offered by the polysaccharide produced. The antimutagenicity of the fermented milk against Trp-P-1 was dose dependent. The strains were also able to bind with different amino acid pyrolysates, and B. longum showed the highest binding. Acetone extracts of fermented skim milk dissolved in water showed less antimutagenicity than extracts dissolved in dimethylsulfoxide. The isolated crude polysaccharide from B. longum PS+ showed a dose-dependent inhibition of the mutagenicity of Trp-P-1. Thus, we conclude that the polysaccharide of B. longum PS+ can be used as an antimutagen.     

Inhibition of the metabolism of mutagens occurring in food by arachidonic acid.

Hepatic microsomal fractions (microsomes) were prepared from male Sprague-Dawley rats. The effect of arachidonic acid on the conversion of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay (indicator: Salmonella typhimurium TA98). Arachidonic acid inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA-repair processes within the bacterial cell. The activation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and aflatoxin B1 (AFB1) was also inhibited by this polyunsaturated fatty acid.     

Modulating effect of tanshinones on mutagenic activity of Trp-P-1 and benzo[a]pyrene in Salmonella typhimurium.

The modulating effects of the Chinese medicinal plant 'Tan-shen', the radix of Salvia miltiorrhiza Bunge, on the mutagenic activities of Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) and B(a)P (benzo[a]pyrene) were investigated using Salmonella typhimurium TA98. Ether- and hot water-extracted 'Tan-shen' enhanced both mutagens at low concentrations, but suppressed them at high concentrations. Extracts by ether treatment were more effective than those extracted by hot water. Dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA were isolated from the ether extract by high performance liquid chromatography (HPLC) and were recognized to be the mutagenic modulators. 4 tanshinones enhanced the mutagenicity of Trp-P-1 by 8-24-fold at 20 micrograms/plate and the enhancement was reduced at the higher concentration. Dihydrotanshinone I suppressed Trp-P-1 activity completely at 100 micrograms/plate.     

Subsequent products after antioxidant actions of beta-carotene and alpha-tocopherol have no Salmonella mutagenicity

Beta-carotene and alpha-tocopherol are important antioxidants biologically, but whether their oxidized products are toxic or not remains to be discovered. Here, we chromatographically separated 5 pure products or isomeric mixtures from reaction mixtures of beta-carotene and reactive oxygens, and 17 lipid-radical scavenging products of alpha-tocopherol. The products were tested for mutagenicity using Salmonella typhimurium TA98, TA100, TA102, and TA104, in the presence and absence of S9. None showed mutagenicity against any of the four strains, or cytotoxicity that influenced the survival of the bacteria. Lipid-peroxides have been known to increase the formation of mutagens from dietary procarcinogens such as heterocyclic amines. So, we also measured the activity to increase 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) mutagenicity. The products from beta-carotene and alpha-tocopherol did not increase, but rather several of them suppressed, the mutagenicity of Trp-P-2. Thus, the products of beta-carotene and alpha-tocopherol formed after the antioxidant actions were not genotoxic.