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.     

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.     

Bone marrow and lymphocyte cytogenetics of rhesus monkeys (Macaca mulatta) treated with the clastogen mitomycin C

Rhesus monkeys (Macaca mulatta) were used to determine their effectiveness as experimental animals for different cytogenetic tests with mitomycin C (MC). The micronucleus test (MNT) and/or chromosome analysis of blood and bone marrow were made before and/or after the treatment with mitomycin C. Thus, the controls data and treated data were obtained from the same animals. With the employed methology, the micronucleus test could not be performed on living animals. Less chromosomal damage was detected in the micronucleus test of post-mortem samples than in the chromosome analysis of bone marrow. No influence by the mutagen could be observed in lymphocyte chromosomes at any of the different times of analysis. In contrast to this, bone-marrow chromosomes seemed to be highly affected by mitomycin C at day 1, 2 and 3 after injection. However, before treatment and at day 14, 16 and 17 after treatment there was no visible increase in chromosomal aberration in bone marrow.     

Mutagenic activity of the pesticide hexachlorobutadiene

Pesticide hexachlorobutadiene (HCBD) has been studied for its mutagenic activity in two test systems: in vivo and in vitro. The above pesticide manifested a mutagenic activity in the bone marrow cells under peroral and inhalation effect. No clastogenic action in the human peripheral lymphocyte culture was observed. Results of this study and data available in literature permit concluding that HCBD is an indirect mutagen.     

Antimutagenic effect of Agaricus blazei Murrill mushroom on the genotoxicity induced by cyclophosphamide

Agaricus blazei Murrill extracts have previously been shown to have anticarcinogenic and antimutagenic properties. These results suggest that antimutagenic activity, besides the modulation of the immune system, might be involved in the anticarcinogenic action of A. blazei. To investigate the possible antimutagenic effect of A. blazei in vivo, we evaluated its effect on clastogenicity induced by cyclophosphamide (CP) in mice, using the micronucleus test in bone marrow (MNPCE) and in peripheral blood (MNRET). Male Swiss mice were treated with CP (25 or 50mg/kg i.p.) or with CP plus mushroom solution at three different temperatures: 4, 21, and 60 degrees C. Aqueous solution of a mixture from various lineages of the mushroom inhibited induction of micronuclei by CP in bone marrow and in peripheral blood of mice. In contrast to the mixture of lineages, a single isolated lineage did not lead to a reduction of CP-induced MN frequencies in either bone marrow or blood cells of mice. The results suggest that under certain circumstances these mushrooms exhibit antimutagenic activities that might contribute to an anticarcinogenic effect.     

Quantitative characterization of antagonistic activity of lactobacilli

In this work the method of serial dilutions of lactobacilli in two-layer agar was used. On the agar surface bacterial or fungal cultures were applied at different time intervals. A special quantitative characteristic was introduced. L. plantarum strain 8P-A3 was shown to have the maximum antagonistic activity. In great amounts L. casei and L. reuteri are capable to suppress the growth of bacteria and fungi. All lactobacilli under study produced a pronounced bactericidal effect on Pseudomonas, had different influence on the viability of Escherichia and staphylococci and exhibited fungistatic and fungicidal action only when inoculated at high concentrations.     

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.     

The anticlastogenic potential of fatty acid methyl esters

To test for possible anticlastogenic effects of fatty acids, the methyl esters of fatty acids--short-chain to long-chain--were examined on busulfan in Chinese hamster bone-marrow cells using the chromosome aberration test. When the experimental animals were treated with fatty acid esters and the mutagen, the chromosome-breaking actions of busulfan were not modulated by the short-chain fatty acids, but the fatty acids from lauric acid (C12) up to nonadecanoic acid (C19) reduced the rate of aberrant metaphases from 9.4 to about 3% at doses of 100 mg/kg and less. Other chemical properties of the fatty acids (saturated or not, number of double bonds, even- or odd-numbered) had no influence on the anticlastogenic effects. The only exceptions to this rule were arachidonic acid, which had no effect, and gamma-linolenic acid, which had no consistent effect on the action of busulfan.     

Evidence for extrachromosomal elements in Lactobacillus.

Three strains of lactobacilli, Lactobacillus casei subsp. casei 64H, L. casei subsp. rhamnosus OC91, and L. coryniformis M34, were examined for the presence of plasmids. Plasmids of molecular weights of 23 x 10(6) and 16 x 10(6) were found in the first two strains respectively. This represents the first evidence for plasmids in lactobacilli; their function is not presently known.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Culture media for differential isolation of Lactobacillus casei Shirota from oral samples

This study aimed to develop a solid culture medium for differential isolation of the probiotic strain Lactobacillus casei Shirota (LcS) and for selective cultivation of lactobacilli present in oral samples. Type strains of lactobacilli and isolates from commercial probiotic products were inoculated onto modified de Man Rogosa Sharpe agar (termed 'LcS Select'), containing bromophenol blue pH indicator, vancomycin and reducing agent L-cysteine hydrochloride for differential colony morphology development. L. casei Shirota cultured on the novel medium produced distinctive colony morphologies, different from other lactobacilli tested. LcS-characteristic colonies were recovered on LcS Select medium from samples of saliva and tongue plaque following a four-week probiotic intervention study. The viable count of presumptive LcS colonies correlated with those isolated on a non-commercial lactitol-LBS-vancomycin agar (LLV) developed for a selective isolation of LcS from faeces. The novel LcS Select medium proved suitable for differential isolation of the probiotic strain L. casei Shirota from oral samples containing mixed microbial populations. It can also be used for selective growth of vancomycin-resistant lactobacilli. There are few available culture media that are sufficiently selective to enable isolation of probiotic strains from mixed populations. LcS Select medium provides a cheaper, yet effective tool in this context.     

THE NORMAL FLORA OF THE BOVINE RUMEN. IV. QUALITATIVE STUDIES OF LACTOBACILLI FROM COWS AND CALVES

SUMMARY: Two hundred and four samples of rumen contents were taken from eight fistulated cows receiving various diets, and seventy-one samples from twelve calves. From dilution counts of these samples, 91 isolates of Gram-positive catalase-negative rods were obtained, and by means of physiological and serological tests, 78 (95%) out of 82 lactobacilli were characterized as follows: Lactobacillus fermenti (33 strains), L. acidophilus (21), L. casei (17) and L. plantarum (7). Indications were obtained of progressive establishment of lactobacilli in the rumen of the growing calf, L. fermenti predominating. In the cow, L. acidophilus was generally associated with hay plus concentrates diets and L. casei with silage, whereas with grass small numbers of several species of lactobacilli were found.     

Studies on the genotoxicity of asataf (acephate), an organophosphate insecticide, in a mammalian in vivo system

The genotoxic potential of asataf (acephate) was evaluated by a battery of in vivo tests: bone marrow chromosome aberrations, micronucleus, sperm-shape abnormality and dominant lethal tests in mice. A significant enhancement in the percentage of chromosome aberrations was noticed in 3 doses, 3 routes and 3 h after asataf treatment of groups of mice as well as in chronic (sub-acute) treatment. A significant difference in the occurrence of micronuclei was found only at the highest dose whereas all the results of the sperm-shape abnormality test were highly significant. In the dominant lethal mutagenicity assay only the result (dead implants) of a single week (3rd) with the higher dose differed significantly from control. On the basis of the present in vivo results in mouse test systems asataf may be considered to be a potential mutagen.     

Antimutagenic effect of an antioxidant in mammals

To test a possible antimutagenic activity of ethoxyquin (EQ) in bone-marrow cells, 3 cytogenetic tests with distinct genetic end-points were applied. Cyclophosphamide (CPA), serving as test mutagen, and the antioxidant EQ were administered immediately following each other to Chinese hamsters by stomach tube. Whereas the CPA dose was the same in each test, the EQ doses were increased up to a ratio of CPA/EQ = 1:25, in some cases up to 1:50. The formation of SCEs induced by CPA was not influenced by EQ--even at the highest dose. In the micronucleus test, however, EQ drastically reduced the micronucleus rate even at the lowest dose applied (20 mg/kg) and abolished the CPA effects at a dose of 100 mg/kg. This action of EQ against CPA was also found in the rat and mouse in the micronucleus test system. Two inbred strains of mouse showed similar reactions, but the induced micronucleus rate was higher and its decrease in response to increasing doses of EQ was more delayed. In the chromosome aberration test, EQ also showed a distinct anticlastogenic response. At higher EQ doses, all CPA-induced chromosomal damage was reduced down to the level of spontaneous rates. The anticlastogenic effect of EQ was quantitatively similar in the micronucleus and chromosome aberration tests. Only minor qualitative differences were recognizable.     

Biological effect of extracellular peptide factor from Luteococcus japonicus subsp. casei on probiotic bacteria

The biological effect of the extracellular peptide reactivating factor (RF) from Luteococcus casei on cells of probiotic cultures was studied. The RF showed the protective and reactivating effects on the Bifidobacterium bifidum cells under the action of bile salts and an acidic stress. Also, it acted as a cryoprotector during lyophilisation and long-term culture storage. The RF and the L. casei culture liquid (CL) were shown to have bifidogenic properties. The degree of protection and reactivation of lactic-acid bacteria under the action of bile salts depended on the particular strain properties. The maximum degree of protection (more than thirteen-fold) and reactivation (close to three-fold) was found in Lactobacillus casei, while the minimum values were characteristic of Lactobacillus reuterii. The resistance of lactobacilli to bile was increased in the row of L. acidophilus, L. casei, L. plantarum, L. rhamnosus, and L. reuterii correlating with the RF protection degree.     

Murine bone marrow culture system for cytogenetic analysis

A mouse bone marrow culture system for examining genotoxicity of agents by first exposing animals in vivo then growing cells in vitro is presented. This assay can also be used for in vitro and/or for the in vivo and in vitro comparative cytogenetic studies. The protocol involves culturing of approximately 1,000,000 nucleated cells obtained from mice tibia and femora in 5 ml of Ham's F-12 medium containing 20% fetal bovine serum, 10% whole uterus extract from pregnant mice and 1% penicillin-streptomycin. The use of flasks and mouse uterus extract for culturing are important steps for higher mitotic yield. The addition of 20 microM BrdU for 24 h helps in the differentiation of sister chromatids for sister-chromatid exchange (SCE) analysis. Cyclophosphamide, given to mice through intraperitoneal injection, induced significant dose-related SCEs in culture. Trinitrofluorenone, a direct-acting mutagen, caused dose-related SCEs in in vitro bone marrow cell culture.     

Experimental study of the antimutagenic properties of 5-methylresorcinol

A study was made of the effect of 5-methylresorcinol (5-MR) on mutagenic activity of benzo(a)pyrene (BP) and of the action of gamma-radiation in in-vitro and in-vivo systems. The induction of direct gene mutations in Chinese hamster cells V-79 and micronuclei in mouse bone marrow reticulocytes was efficiently suppressed by 5-MR treatment. The antimutagenic activity of 5-MR can be explained by inhibition of free-radical processes.     

Cytogenetic effects of 1,4-dihydropyridine in various test systems

Cytogenetic effect of 1,4-dihydropyridine was studied in different test-systems. The preparation is shown to decrease the level of complete sex-chromosome losses in Drosophila and chromosome aberration frequency in Allium fistulosum seedlings. The preparation does not affect spontaneous mutability of bone marrow cells in mice, high doses of the preparation have no mutagenic potential. Thus, 1,4-dihydropyridine shows antimutagenic activity reducing the chromosome mutation level in sex and somatic cells of eucaryotic organisms. Absence of the effect on mice chromosomes may testify to the specificity of 1,4-dihydropyridine action.     

Screening substances derived from cultures of medicinal plants for antimutagenic activity in the Escherichia coli-bacteriophage lambda system

In Escherichia coli--bacteriophage lambda system protective properties of the extracts derived from the biomass of cultured Panax ginseng, Polyscias filicifolia, Rhodiola rosea, Ungernia victoris cells, and those from intact Rhodiola roots have been studied. Escherichia coli--bacteriophage lambda system responsiveness was found to vary with the test-object state, namely: the deleted bacteriophage form (lambda-4) as well as previously mutagenized bacteriophage were more sensitive to the mutagenic and antimutagenic influence versus the native bacteriophage lambda +. The contribution of extracts in the induction and realization of the lethal injuries in phages caused by nitrite acid in extracellular phage (conditions in vitro) was estimated thus enabling to discriminate between the protective and antimutagenic extract activities. Protective extract effect in the given test-system appeared to be higher their antimutagenic action. With the most responsive bacteriophage variant the extracts from the biomass of cultured Rh. rosea and P. filicifolia cells showed high protective and somewhat lower antimutagenic activities. With other phages significant antimutagenic potential of extracts was demonstrated, which by their protective effect could be arranged in a raw as follows U. victoris > P. ginseng > P. filicifolia. The primary screening for the antimutagenic effect of preparations in the prokaryotic systems could be reduced to the investigation of their effects on the object inactivation exposed to the mutagen in vitro.     

Lactobacilli Causing Spoilage of Acetic Acid Preserves

Twentyseven cultures of lactobacilli, isolated from 27 packs of spoiled vinegar preserves, which originated from 21 manufacturers and included 12 varieties of product, were examined according to the system of Rogosa & Sharpe and by other tests. One culture conformed to the characteristics of Lactobacillus casei var. casei ; 2 cultures to those of L. brevis ; 4 cultures to those of L. buchneri ; and 20 to those of L. fructivorans. On the basis of the results and an examination of the literature it is suggested that L. trichodes is a synonym of L. fructivorans.