The possible role of probiotics as dietary antimutagens

Possible antimutagenic actions of probiotics - mainly lactic acid bacteria - were examined using in vitro and in vivo test systems.In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli and its cellfree culture broth exhibited antimutagenic action only on beef extract.The actions of probiotics were more homogeneous when living animais were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli.     

Antimutagenic and antioxidant/prooxidant activity of quercetin

The present study has been performed to evaluate the antimutagenic activity of quercetin, ascorbic acid and their combination against an oxidative mutagen. An effort was also made to correlate this activity to the in vitro antioxidant activity of these agents. Antimutagenicity testing was done in Ames Salmonella Assay system using Salmonella typhimurium TA102 against t-butylhydroperoxide as an oxidative mutagen. In vitro antioxidant scavenging activity was tested for DPPH free radical, superoxide anion, hydrogen peroxide and hydroxyl radical in their specific test systems. Quercetin (0.5-8 nmole/plate) and ascorbic acid (0.1-100 micromole/plate) showed significant effect. Quercetin (4 and 8 nmole/plate) when combined with ascorbic acid (500 nmole/plate) showed an increase in the antimutagenic activity. In vitro antioxidant activity of quercetin was better than ascorbic acid in all the test systems used. The study indicated that the antimutagenic activity of quercetin was not solely accountable by its antioxidant nature. However, in vitro free radical scavenging activity of quercetin correlated well with the antimutagenic activity.     

The metabolic activation of 4-nitro-o-phenylenediamine by chlorophyll-containing plant extracts: The relationship between mutagenicity and antimutagenicity

Chlorophyllin, the sodium and copper salt of chloropyll, and chlorophyll a, and chlorophyll b were tested for their ability to inhibit the mutagenic activity of the direct-acting mutagen 4-nitro-o-phenylenediamine (NOP) and its plant-activated mutagenic enhancement. All three forms of chlorophyll were antimutagenic against both NOP and its plant-activated product, with chlorophyllin proving most effective. Chlorophyll-containing plant extracts, however, proved very efficient at activating NOP into a mutagen of greater potency. When these extracts were assayed for total chlorophyll content it was found that they contained far less chlorophyll than was required for an antimutagenic effect to occur. Thus, the balance between chemical mutagen activation and/or enhancement by chlorophyll-containing plant extracts and the potential antimutagenicity of these plant extracts is a function of chlorophyll concentration. The data presented here indicate that this balance must be taken into consideration in future studies investigating the efficacy of complex natural plant extracts as antimutagenic substances.     

The possible role of probiotics as dietary antimutagen.

Possible antimutagenic actions of probiotics--mainly lactic acid bacteria--were examined using in vitro and in vivo test systems. In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli) and its cell-free culture broth exhibited antimutagenic action only on beef extract. The actions of probiotics were more homogeneous when living animals were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli.     

Chemical investigation of different extracts and essential oil from the tubers of (Tunisian)Cyperus rotundus. Correlation with their antiradical and antimutagenic properties

The mutagenic potential of aqueous, Total Oligomers Flavonoids (TOF), ethyl acetate, and methanol extracts as well as essential oil (EO) obtained from tubers ofCyperus rotundus L. was assessed by “Ames assay”, usingSalmonella tester strains TA98 and TA100, and “SOS chromotest” usingEscherichia coli PQ37 strain with and without an exogenous metabolic activation system (S9). None of the different extracts showed a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test” and the “SOS chromotest”. Our results showed thatC. rotundus extracts have antimutagenic effects withSalmonella typhimurium TA98 and TA100 strains towards the mutagen Aflatoxin B1 (AFB1), as well as withE. coli PQ37 strain against AFB1 and nifuroxazide mutagens. A free radical scavenging test was used in order to explore the antioxidant capacity of the extracts obtained from the tubers ofC. rotundus. TOF, ethyl acetate and methanol extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. These extracts showed IC50 values of respectively 5, 20 and 65 μg/ml. The beneficial effects of TOF, ethyl acetate, methanol and essential oil extracts ofC. rotundus have been assessed by antioxidant and antimutagenic activities.     

Antimutagenic properties of fresh-water blue-green algae

The antimutagenic properties of whole fresh-water blue-green algaeAphanisomenon flos-aquae, marketed under the commercial name “Alpha Sun” were tested using the Ames test. Simultaneous addition of both algae and Nitrovin (a mutagen) to the test medium did not reduce the mutagenic activity. On the other hand, addition of freeze-dried blue-green algae to the test medium 2–24 h before the application of mutagen reduced its mutagenic activity.     

Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents

In the present study, the chemical composition of Origanum compactum essential oil was determined by gas chromatography and mass spectrometry, and its mutagenic and antimutagenic activities were investigated by the somatic mutation and recombination test (SMART) in Drosophila melanogaster. No significant increase in the number of somatic mutations was observed with the essential oil tested using both the standard (ST) and high bio-activation (HB) cross. In order to investigate the antimutagenic effect of the essential oil, we have tested the effect on the indirect-acting mutagen urethane (URE), as well as the direct-acting mutagen methyl methanesulfonate (MMS). O. compactum essential oil showed a strong inhibitory effect against URE-induced mutagenicity, especially with the HB cross. However, only a weak inhibitory effect on the mutagenicity induced by MMS was observed. These results suggest that the detected antimutagenicity could be mediated by an inhibitory effect on metabolic activation. The essential oil was fractionated to identify the components responsible of the suppressing effect detected. Seven fractions were obtained: two of them showed the most potent inhibitory effect against URE-induced mutagenicity and were further fractionated. The sub-fractions obtained from the second chromatographic fractionation were tested for their antimutagenic activity, together with carvacrol and thymol. The highest antimutagenic effect obtained with the sub-fractions was similar to the effect of the crude essential oil, as well as to the effect of carvacrol alone. These results suggest the absence of a synergic antimutagenic effect between the components of O. compactum essential oil and indicate that carvacrol was the most active oil component.     

The metabolic activation of 4-nitro-o-phenylenediamine by chlorophyll-containing plant extracts: the relationship between mutagenicity and antimutagenicity

Chlorophyllin, the sodium and copper salt of chloropyll, and chlorophyll a, and chlorophyll b were tested for their ability to inhibit the mutagenic activity of the direct-acting mutagen 4-nitro-o-phenylenediamine (NOP) and its plant-activated mutagenic enhancement. All three forms of chlorophyll were antimutagenic against both NOP and its plant-activated product, with chlorophyllin proving most effective. Chlorophyll-containing plant extracts, however, proved very efficient at activating NOP into a mutagen of greater potency. When these extracts were assayed for total chlorophyll content it was found that they contained far less chlorophyll than was required for an antimutagenic effect to occur. Thus, the balance between chemical mutagen activation and/or enhancement by chlorophyll-containing plant extracts and the potential antimutagenicity of these plant extracts is a function of chlorophyll concentration. The data presented here indicate that this balance must be taken into consideration in future studies investigating the efficacy of complex natural plant extracts as antimutagenic substances.     

Antimutagenicity of hops (Humulus lupulus L.): bioassay-directed fractionation and isolation of xanthohumol

Bioassay-directed fractionation with a Salmonella/microsomal assay against the food borne mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used to identify antimutagenic components of hops. Hops pellets extracted with diethylether showed antimutagenic activity against mutations induced by IQ. Fractionation of the diethylether extract (DE) by column chromatography, followed by semi-preparative HPLC yielded two fractions (E4b and E4d) with strong antimutagenic activity against IQ induced mutations. Separation of fraction E4b resulted in inactive fractions, while fraction E4d has been identified to be xanthohumol. In mammalian test system with human hepatoma HepG2 cells fraction E4d at 10 μg/ml completely prevented formation of IQ induced DNA damage. These results indicate that xanthohumol is a very promising potential protective agent against genotoxicity of food borne carcinogens, which warrants further investigation.     

Antimutagenicity of an acetone extract of yogurt

Reconstituted non-fat dry milk powder, fermented by a mixture of Streptococcus thermophilus CH3 and Lactobacillus bulgaricus 191R to produce yogurt, was freeze-dried and extracted in acetone. After evaporation of the acetone, the extract was dissolved in dimethyl sulfoxide (DMSO) and tested for antimutagenicity. In the Ames test, significant dose-dependent activity was observed against N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-N-oxide (4NQO), 3,2′-dimethyl-4-aminobiphenyl (DMAB), 9,10-dimethyl-1,2-benz[a]anthracene (DMBA), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2). Weak activity was observed against 1,2,7,8-diepoxyoctane (DEO), and no activity was observed against methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), or aflatoxin B1 (AFB1). In a related assay (Saccharomyces cerevisiae D7), significant antimutagenic activity was detected against MNNG and 4NQO.Activity against the experimental colon carcinogens MNNG and DMAB was examined further, as assayed in the Ames test (Salmonella typhimurium TA100). Compounds responsible for both activities were less soluble in aqueous solutions than in DMSO. Adjustment of yogurt pH to 3, 7.6, or 13 prior to freeze-drying and acetone extraction did not significantly alter the amount of anti-MNNG activity recovered. In contrast, extractability of anti-DMAB activity was significantly greater at acidic pH. Conjugated linoleic acid, a known dairy anticarcinogen, failed to inhibit mutagenesis caused by either mutagen, suggesting that other yogurt-derived compound(s) are responsible. Unfermented milk was treated with lactic acid, yogurt bacteria without subsequent growth, or both, to determine if formation of antimutagenic activity required bacterial growth. Extracts of the milk treatments exhibited the same weak antimutagenicity observed in unfermented milk, approximately 2.5-fold less than in the yogurt extracts, suggesting that antimutagenic activity is associated with bacterial growth.     

Antimutagenicity of propionic acid bacteria.

The antimutagenic effect of dialysed cell extracts of 4 strains of propionic acid bacteria was examined against the mutagenicity of sodium azide in the TA1535 tester strain of Salmonella typhimurium using the Ames test. It was noted that dialysates of 2 strains of Propionibacterium shermanii, P. pentosaceum and P. acnes, significantly reduced sodium azide-induced revertants. The dialysate of propionic acid cocci did not show an antimutagenic effect. The inhibitory activity was enhanced if the mutagen and extract were coincubated for 20 min prior to performing the mutagenicity assay. Antimutagenicity of dialysates from P. shermanii VKM-103 against MNNG and 9-aminoacridine was shown in S. typhimurium strains TA1535 and TA97. The antimutagenic activity was found in the protein fraction of the cell extract of P. shermanii. The proteins of the dialysate of P. shermanii were separated using a Toyopearl gel column into 3 main peaks according to their molecular weights. The antimutagenic activity towards sodium azide was found in the second and the third peaks. We suggest that dialysates of the cells of propionic acid bacteria contain several kinds of antimutagenic substances with different molecular weights.     

Humic acids reduce the genotoxicity of mitomycin C in the human lymphoblastoid cell line TK6

The antimutagenic/desmutagenic activity of a leonardite humic acid (LHA) and a soil humic acid (SHA) was studied in the cultured human lymphoblastoid cell line TK6 treated with mitomycin C (MMC) as reference mutagen by evaluating the induction of micronuclei (MN). Two different concentrations of HA were used, 2.5 and 10 microg/ml, in three different treatments: (1) HA alone (genotoxic test); (2) HA after 2-h pre-incubation with 0.3 microM of MMC (desmutagenic test) and (3) combinations of HA and MMC at 0.3 microM without pre-incubation (antimutagenic test). Neither of the HA used alone did produce genotoxic effects, but both HAs reduced significantly the frequencies of MN induced by MMC, especially in the desmutagenic test. A slight cell-protective effect against the cytotoxicity of MMC was also exhibited by the two HAs in the desmutagenic test. The LHA showed a desmutagenic/antimutagenic activity that was more pronounced than that of SHA, which is possibly related to the higher carboxylic group content and lower phenolic group content of LHA. These results confirm the antigenotoxic action exerted by HAs in human cells, similarly to what has been previously observed in various plant species.     

Ginseng extract exhibits antimutagenic activity against induced mutagenesis in various strains of Salmonella typhimurium

Ginseng has been reported to exhibit antioxidant and antimutagenic activity. The present study was undertaken with a view to confirm whether the antioxidant activity of Ginseng is responsible for its antimutagenic action. The concentrated root extract of Panax ginseng (Ginseng extract I) and its lyophilized powder (Ginseng extract II) obtained from two different manufacturing houses, were tested against mutagenesis using the well-standardized Ames microsomal test system. The extracts exhibited antimutagenic effect against hydrogen peroxide induced mutagenesis in TA100 strain, and against mutagenesis produced by 4-nitroquinoline-N-oxide in both TA98 and TA100 strains of Salmonella typhimurium. Both the extracts failed to show any antimutagenic potential against tert-butyl hydroperoxide (an oxidative mutagen) in TA102 strain, a strain highly sensitive to active oxygen species. The extracts also indicated a weak antioxidant activity in a series of in vitro test systems viz., 1,1-diphenyl picryl hydrazyl (DPPH) assay, hydrogen peroxide scavenging and superoxide anion scavenging. The results indicate that the protective effects shown by ginseng extract(s) against 4-nitroquinoline-n-oxide and hydrogen peroxide induced mutagenesis in TA98 and TA100 could mainly be due to its property to initiate and promote DNA repair rather than free radical scavenging action.     

Cytotoxic, mutagenic and antimutagenic screening of Arenosclera brasiliensis acetone and ethanol extracts

The marine environment is a rich source of biologically active compounds with pharmacological properties. Marine organisms often produce secondary metabolites with structural features different from those produced by terrestrial ones, and the Phylum Porifera seems to be one of the most productive in this sense. This study was undertaken to provide data on mutagenic and antimutagenic activities from an acetone (Areac) and an ethanol (Areet) extract obtained from Arenosclera brasiliensis, an endemic Brazilian sponge. A qualitative Salmonella reverse mutation test was performed with the TA97, TA98, TA100, and TA102 strains by incubating cells with Areac and Areet in the presence and absence of a known mutagen. A cytotoxic evaluation of the extracts was also performed. A. brasiliensis did not display any mutagenic activity, but Areac showed significant toxicity against test strains. In the antimutagenic assay, a reduction in the number of his+ revertants was observed for the TA97, TA100 and TA102 strains treated with Areac when compared to the positive controls. Areet treatment showed protective activity against DNA lesions only for the TA100. These results are in agreement with those obtained previously with other A. brasiliensis extracts, suggesting an antimutagenic activity.     

Antimutagenicity of some citrus fruits in Salmonella typhimurium

The antimutagenic effect of 10 citrus fruit juices was observed against the mutagenicity of N-nitro-o-phenylenediamine (NPD) in TA97a and sodium azide in TA100 tester strains of Salmonella typhimurium using the Ames test. It was noticed that the juices of all these fruits reduced significantly the NPD and sodium azide induced revertant colonies. The inhibitory activity was enhanced if the mutagen and juice were co-incubated for about 30 min at 37 degrees C prior to performing the mutagenicity assay. Dilution with distilled water led to the reduction in the inhibitory activity. The antimutagenic activity of synthetic ascorbic acid or citric acid or combined ascorbic acid and citric acid was also seen. But the results with fruit juices tempted us to believe that in addition to ascorbic acid and citric acid, the presence of other factor(s) possessing antimutagenic properties cannot be ruled out.     

A monitoring technique of piezoelectric impedance analysis used in tea polyphenols (TP) antimutagenic characteristics

A new method was proposed for studying antimutagenic characteristics of Tea Polyphenols (TP) using piezoelectric impedance analysis during the Salmonella typhimurium strain TA100 growth process in the presence of mutagen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (abbreviated NNK). Compared with the general method of antimutagenic investigation, the proposed method provided real-time, multidimensional response information about the antimutagenic characteristics during the bacterial growth process. The results showed that TP was allowed to be antimutagenic at the lowest concentration of 0.25 μg/ml, and the inhibitory effect of TP presented a linear relationship with its dosage in the range of 0.25–2.5 μg/ml. And the relationship between the bacterial growth kinetic parameters and the TP dosage was also obtained. The present method provided a new approach for studying microbial antimutagenic characteristics.     

Evaluation of antimutagenic and protective effects of <Emphasis Type="Italic">Parkinsonia aculeata</Emphasis> L. leaves against H<Subscript>2</Subscript>O<Subscript>2</Subscript> induced damage in pBR322 DNA

The in vitro antimutagenic and DNA protecting potential of organic (methanol, hexane, n-butanol) and aqueous extract/fractions of Parkinsonia aculeata L. (Fabaceae) was investigated by employing Ames assay and DNA nicking assay. DNA damage by hydroxyl radicals was effectively inhibited by all the extract/fractions. A marked antimutagenic effect was observed against 4-Nitro-o-phenylenediamine and sodium azide (direct acting mutagens) and 2-Aminofluorene (indirect acting mutagen) in TA98 and TA100 strains of Salmonella typhimurium. In Ames assay, two different modes of experiments i.e. pre-incubation and co-incubation were performed and it was observed that all the extract/fractions showed better results in the pre-incubation as compared to co- incubation mode. Out of all the extract/fractions tested, n-butanol fraction was found to be the most effective in preventing DNA damage and inhibiting mutagenesis. UHPLC analysis of extract/fractions revealed presence of polyphenols such as gallic acid, catechin, chlorogenic acid, caffeic acid, umbelliferone, coumaric acid, rutin, and ellagic acid etc. DNA protecting and antimutagenic activity of this plant could be attributed to presence of these polyphenols. The results of this study indicate the presence of potent antioxidant factors in Parkinsonia aculeata L, which are being explored further for their mechanism of action.     

Antimutagenic activity of green tea and black tea extracts studied in a dynamic in vitro gastrointestinal model

An in vitro gastrointestinal model, which simulates the conditions in the human digestive tract, was used to determine potential antimutagenic activity of extracts of black tea and green tea. In this paper, results are presented on the availability for absorption of potential antimutagenic compounds present in tea and on the influence of the food matrix on this activity. Between 60 and 180min after the tea was introduced into the model, antimutagenic activity was recovered from the jejunal compartment by means of dialysis: the dialysate appeared to inhibit the mutagenicity of the food mutagen MeIQx in the direct plate assay with Salmonella typhimurium (Ames test). The maximum inhibition was measured at 2h after the start of the experiment and was comparable for black tea and green tea extract. To determine the influence of food matrices on the antimutagenic activity of tea, the model was loaded with black tea together with milk or a homogenized standard breakfast. The maximum inhibition observed with black tea was reduced by 22, 42 and 78% in the presence of whole milk, semi-skimmed milk, and skimmed milk, respectively. Whole milk and skimmed milk abolished the antimutagenic activity of green tea by more than 90%; for semi-skimmed milk the inhibition was more than 60%. When a homogenized breakfast was added into the model together with the black tea extract, the antimutagenic activity was completely eliminated. When tea and MeIQx were added together into the digestion model, MeIQx mutagenicity was efficiently inhibited, with green tea showing a slightly stronger antimutagenic activity than black tea. In this case, the addition of milk had only a small inhibiting effect on the antimutagenicity.Antioxidant capacity and the concentration of catechins were also measured in the jejunal dialysates. The reduction in antimutagenic activity corresponded with reduction in antioxidant capacity and with a decrease of concentration of three catechins, viz. catechin, epigallocatechin gallate and epigallocatechin. The in vitro gastrointestinal model appears to be a useful tool to study the antimutagenicity of food components.     

Glycine betaine in beer as an antimutagenic substance against 2-chloro-4-methylthiobutanoic acid, the sanma-fish mutagen

Beer can inhibit the mutagenicity of the sanma-fish mutagen, 2-chloro-4-methylthiobutanoic acid (CMBA) in Salmonella typhimurium TA100 and TA1535. The antimutagenic component was isolated from beer and identified as glycine betaine, a compound known to be distributed widely in plants and animals including humans. Beer also contains components that interfere the antimutagenic action of glycine betaine. Glycine betaine seems to antagonize CMBA in a specific manner, since several other direct-acting mutagens tested were not subject to inhibition by glycine betaine. CMBA was stable in the presence of glycine betaine under neutral conditions. Since a treatment of Salmonella with glycine betaine before the bacteria was exposed to CMBA resulted in inhibition of the mutagenesis, the antimutagenic action of glycine betaine may be taking place inside the cells. These observations suggest that the mutagenic action of CMBA may be modified by the presence of both extracellular and intracellular glycine betaine.     

Antimutagenic effects of the mushroom Agaricus blazei Murrill extracts on V79 cells

Agaricus blazei Murrill, a native mushroom in Brazil, has been widely consumed in different parts of the world due to its medicinal power. Its anticarcinogenic activity has been shown in experimental animals, and antimutagenic activity has been demonstrated only in Salmonella. In this work, the mutagenic and antimutagenic activities of mushroom teas of strains AB96/07, AB96/09 and AB97/11 were evaluated in Chinese hamster V79 cells, using the comet assay and the micronucleus test. The cells were treated with three different concentrations (0.05, 0.1 and 0.15) of teas prepared from a 2.5% aqueous solution, under three different temperatures: (1) room (20-25 degrees C); (2) ice-cold (2-8 degrees C); and (3) warm (60 degrees C). The teas were applied in co-, pre- and post-treatments in combination with the mutagen methyl methanesulfonate (MMS; 1.6x10(-4) and 4x10(-4)M). The duration of the treatment was 1h in the comet assay and 2h in the micronucleus test. The results showed that the mushroom was not mutagenic itself. Nevertheless, the mushroom is an efficient antimutagen against the induction of micronuclei by MMS in all concentrations and preparations tested. The observed reductions in the frequencies of micronuclei ranged from 61.5 (room temperature 0.1% tea in post-treatment) to 110.3% (co-treatment with warm and ice-cold 0.15% tea). In the comet assay, the antimutagenic activity was detected only when the cells were pre-treated with the following teas: warm 0.1 and 0.15%, room temperature 0.05% and ice-cold 0.1%. The results indicate that the mushroom A. blazei extracts are antimutagenic when tested in V79 cells.