Food borne yeasts as DNA-bioprotective agents against model genotoxins.

Yeasts isolated from Italian beverages and foods (wine and cheeses) were identified as Saccharomyces cerevisiae and Debaryomyces hansenii by sequencing the D1/D2 domain of the 26S rRNA gene and differentiated, at strain level, by microsatellite PCR fingerprinting and RAPD-PCR. All the strains showed antioxidant activity, as demonstrated by their ability to scavenge the free radical diphenyl-1-picrylhydrazyl (DPPH). Furthermore, tested strains revealed high in vitro inhibitory activity against two model genotoxins, 4-nitroquinoline-1-oxide (4-NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as showed by short-term methods with different target cells: SOS-Chromotest with Escherichia coli PQ37 and Comet assay with HT-29 enterocytes. High inhibitory activity towards 4-NQO was associated with cell viability, while heat-inactivated cells showed a reduced antigenotoxic capability. Surprisingly, high inhibition of MNNG genotoxicity was observed even with heat-treated cells. Moreover, the strains able to inhibit the genotoxins induced some changes in the spectroscopic properties of the original compound. This result perfectly agrees with the information obtained by the two bioassays. Interestingly, strains characterized for antioxidant and antigenotoxic properties, also presented acid-bile tolerance, indicating that food autochthonous yeasts could be expected to reach gut in viable form and thus prevent genotoxin DNA damage in situ.

Raw milk traditional Italian ewe cheeses as a source of Lactobacillus casei strains with acid-bile resistance and antigenotoxic properties.

Twenty-five Lactobacillus casei group strains isolated from ewe cheeses from Abruzzo region, central Italy, were identified by 16S rRNA gene sequencing, differentiated by RAPD-PCR analysis and characterized as in vitro for acid-bile tolerance and antigenotoxic properties. All the strains were very susceptible to simulated gastric fluid (pH 2.0) but most of them recovered viability (ca. 2-3 log-units) when transferred and maintained in simulated intestinal fluid (0.5% w/v bovine bile) for 3 h. Some strains showed potential for deactivating representative genotoxins as highlighted by the SOS-Chromotest. Twelve were active and nine moderately active against 4-nitroquinoline-1-oxide, and one active and only one moderately active against N-methyl-N'-nitro-N-nitrosoguanidine. The active strains produced evident spectroscopic modification of genotoxins after co-incubation. Most isolates with antigenotoxic activity resulted as acid-bile tolerant demonstrating that cheese autochthonous lactobacilli may reach the gut as a viable form and prevent genotoxin DNA damage.

Modulatory activity of a Lactobacillus casei strain on 1,2-dimethylhydrazine-induced genotoxicity in rats.

The present study was designed to investigate the putative antigenotoxic effects of supplementing the diet of rats treated with the colon carcinogen 1,2-dimethylhydrazine hydrochloride (DMH) with a Lactobacillus casei strain using an in vivo approach. The antigenotoxic response was evaluated in colon and liver cells using the alkaline comet assay. Since the balance between the bioactivation and detoxification metabolic pathways is crucial for the formation of toxic and genotoxic metabolites, alterations in the level of some xenobiotic metabolizing enzymes (XME) were studied in liver preparations. In the challenge group (L. casei + DMH), lactobacilli-supplemented diet, there was a decrease in the extent of DMH-induced DNA damage, especially in colon cells. Compared with control rats, there was less basal DNA damage in colon cells of rats fed on a lactobacilli-supplemented diet. These findings are the first to give clear evidence of DNA-protective effects of lactobacilli against basal DNA damage. Moreover, the chemopreventive effects were accompanied by changes in the activities of several XME. The observed decrease in the concentration of nonenzymatic antioxidants (i.e. GSH) and the reduced activity of enzymatic antioxidants (i.e., GST, GPx, and SOD) in liver could reflect an overall reduction in the level of oxidative stress in rats on a diet supplemented with the L. casei suspension compared with control rats (basal state). Thus, the concentrations of GSH and the activities of GST, GPx, and SOD could be downregulated by supplementing the diet with L. casei as a response to an improved antioxidant status.

Evidence for in vitro anti-genotoxicity of cheese non-starter lactobacilli.

The inhibition of direct acting DNA reactive agents by 63 non-starter lactobacilli isolated from raw ewes milk cheeses was examined by short-term assay (SOS-Chromotest) and compared with already characterized starter lactobacilli. The screening revealed strains active against the nitroarene 4-nitroquinoline-1-oxide (NQO) and the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in different species of the genus Lactobacillus (L. rhamnosus, L. casei, L. plantarum, L. brevis, Lactobacillus spp.). It was proved that the anti-genotoxicity was strain-dependent, and always associated with spectroscopic modification of genotoxins. The frequency of strains inhibiting nitroarene genotoxicity was comparable for non-starter and starter lactobacilli, whereas inhibition of the alkylating agent was largely predominant in non-starter isolates. Seventeen strains presented inhibitory activity against both genotoxins. DNA RAPD-PCR performed with M13, Pro-Up and RPO2 primers on the lactobacilli under examination showed genetic diversity in these strains. The non-starter isolates clustered in seven groups and the strains presenting a high degree of activity against 4-nitroquinoline-1-oxide clustered in a single group with a similarity around 75%. Interestingly, the strains with anti-genotoxic properties also showed acid-bile tolerance, indicating that the autochthonous lactobacilli which survive cheese ripening may also reach the gut as viable cells and could prevent genotoxin DNA damage to enterocytes, as is desirable for probiotic bacteria.

Inhibition of 4-nitroquinoline-1-oxide genotoxicity by Bacillus strains.

The effect of 16 Bacillus strains from pharmaceutical probiotic preparations (Bacillus spp.) and collection (B. subtilis, B. firmus, B. megaterium, B. pumilus) on genotoxicity induced by the known mutagen 4-nitroquinoline-1-oxide (4-NQO) was studied using the short-term bacterial assay SOS-chromotest. with Escherichia coli PQ37 as the tester organism. It was found that the activity of 0.1 mM 4-NQO was reduced (P < 0.01) after coincubation with Bacillus suspensions (10(8) CFU/ml for 150 min at 37 degrees C). All isolates showed potential for deactivating 4-NQO, and genotoxicity inhibition ranged from 92.9 to 100%. There were no appreciable differences in behaviour observed among probiotic and collection strains or in relation to species. The observed antigenotoxicity was associated with a clear-cut modification of 4-NQO molecular characteristics.

Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest.

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.