Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

The role of fungi in cheese ripening

Fungi are important in the manufacture of two types of cheese—blue-veined cheeses, and Camembert and Brie. Among the former are Roquefort, Gorgonzola and Stilton, dependent on the mold Penicillium roqueforti and the bacterium Streptococcus lactis. Camembert and Brie require Penicillium camemberti and lactic acid- producing streptococci; the mold Oospora lactis and the organism Bacterium linens may also play roles in their manufacture.     

Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms

An aqueous two-phase system, consisting of poly(ethylene glycol) (PEG) and dextran, was employed to separate polymerase chain reaction (PCR)-inhibitory substances from bacterial cells. The PCR inhibition of four soft cheeses was examined and three of them were found to be strongly PCR-inhibitory. Extraction of the PCR-inhibitory soft cheeses inoculated with Listeria monocytogenes in an aqueous two-phase system containing 8% (w/w) PEG 400 and 8% (w/w) dextran 500, was found to lower the PCR detection level of L. monocytogenes by more than four orders of magnitude in two of the cheeses compared to the case where no such sample pretreatment was performed. Depending on the type of cheese used, the PCR-inhibitory factors were found to be enriched in either the top or botton phase in the aqueous two-phase system. These results show that different soft cheeses contain different types and amounts of PCR-inhibitory substances.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Synthesis of Rat Casein Antigen in Escherichia coli

Fumes from phospholipids pyrolyzed at 500~700°C did not themselves show any mutagenicity on Salmonella strains, but when the pyrolyzates were treated with a sodium chloride precipitate, active carbon, or an anionic exchange resin, the filtrates were found to be mutagenic on Salmonella TA 100. Tests confirmed that the phospholipid pyrolyzates contained both mutagenic and inactivating substances of this mutagenicity. Low level mutagenicity was produced on Salmonella TA 98, but there was no such activity on the other strains. Preincubation of the pyrolyzates with S-9 mix had no activating effect on mutagenicity. The inactivating substances of the mutagenicity were isolated and identified as long chain fatty acids.     

Absence of mutagenic action of 5 beta-cholan-24-oic acid derivatives in the bacterial fluctuation and standard Ames tests

Published data on the mutagenicity of 3 bile acids in the bacterial fluctuation test are conflicting. Eleven 5 beta-cholanoic acids including 2 of the bile acids were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in the fluctuation tests. In any of these bile acids at the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. Similarly, none of these compounds showed positive mutagenicity in both strains in the standard Ames test either with or without hepatic metabolic activation. Our results support the claim that 3 bile acids are not mutagenic, and indicate that the initiation activity of 5 beta-cholanoic acids is not demonstrable with a short-term assay using Salmonella strains.     

Microbial Activation of Wooden Vats Used for Traditional Cheese Production and Evolution of Neoformed Biofilms

Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differences in terms of microbial levels and composition of the neoformed biofilms. The levels of the microbial groups investigated during cheese production showed significant differences between the control trials and between the control and experimental trials, but the differences were not particularly marked between the TA2 and TZ2 productions, which showed the largest numbers of mesophilic lactic acid bacterium (LAB) cocci. LAB populations were characterized phenotypically and genotypically, and 44 dominant strains belonging to 10 species were identified. Direct comparison of the polymorphic profiles of the LAB collected during cheese making showed that the addition of the NWSC reduced their biodiversity. Sensory evaluation showed that the microbial activation of the wooden vats with the multistrain Lactococcus culture generated cheeses with sensory attributes comparable to those of commercial cheese. Thus, neoformed biofilms enable a reduction of microbial variability and stabilize the sensorial attributes of Vastedda cheese.     

Mutagenicity of and Formation of Oxygen Radicals by Trioses and Glyoxal Derivatives

Dihydroxyacetone, glyceraldehyde, glyoxal, methyl glyoxal, and glyoxylic acid were found to show mutagenicity on Salmonella typhimurium TA 100. The mutagenicities of these substances were inhibited by the addition of S-9 or some free radical scavengers. The alkaline buffered solutions of these mutagenic substances were found to reduce Nitro Blue tetrazolium chloride. DNA was degraded by the addition of these mutagenic substances. It has also been confirmed that free radicals derived from autoxidation of these substances are responsible for their mutagenicity.     

Occurrence of Streptococcus macedonicus in Italian cheeses

A new approach for the detection and enumeration of Streptococcus macedonicus in cheese was developed. The method which is based on a first screening of cheeses by a PCR assay specific for S. macedonicus followed by plating positive samples on a differential medium (SM medium) was applied to 51 samples derived from PDO and traditional Italian cheeses. Streptococcus macedonicus was found in 16 of the 51 samples examined in the present work. With the exclusion of an Asiago cheese sample in which very high numbers of S. macedonicus (7.13 log CFU g(-1)) were found, the counts of S. macedonicus in SM medium ranged from 2.48 to 4.70 log CFU g(-1). In the same cheeses, total streptococci enumerated onto M17 agar were found at higher concentrations with values up to 7.88 log CFU g(-1). The system developed was particularly useful for the differential count of S. macedonicus in cheese and allowed to evaluate the occurrence of this species within the complex microbial lactic acid bacteria (LAB) population, which is typical of traditional cheeses. Results showed that in the examined cheeses S. macedonicus cannot be considered as a dominant LAB species.     

Human saliva inactivates mutagenicity of carcinogens

Mutagenicities of AF-2, MNNG, 4NQO, aflatoxin B₁, benzo[a]pyrene P-1 with or without metabolic activation, were inactivated by treatment with human saliva to a great extent in the Ames test with salmonella typhimurium test strains TA98 and TA100. Mutagenic activities of quercetin, pyrolsates of beef, salmon and sodium glutamate, and condensate of cigarette smoke were also decreased to some extent by saliva treatment, but no significant effect was found on the activity of MMS and pyrolysate of polypeptone. These effects showed individual variations.The inhibition of AF-2 mutagenicity by saliva varied with temperature in TA100 but not in TA98 cultures. Boiled saliva inactivated AF-2 mutagenicity in TA98 to some extent but not in TA100 cultures. Inactivation of AF-2 mutagenicity by saliva treatment was completed within 30 sec.Complex mechanisms may be involed in the inactivation of mutagenicity of carcinogens by saliva, including biochemical reactions with enzymes, vitamins, etc. and/or adsorption with high molecular weight substances in saliva such as proteins, bacterial cells, mucous materials, etc.     

Urine mutagenicity of petroleum plant workers

The mutagenicity of urinary extracts from workers employed in a petroleum plant was analyzed by means of the plate test using S. typhimurium strains TA98 and TA100. Mean urinary mutagenic activities in TA98 were 2-14 times higher in the petroleum plant workers than in the control group. In TA100 these differences were even bigger, the mutagenicity in petroleum plant workers' urine being 3-42 times higher than in the control group. These results suggest that the environmental exposure of people to mutagenic substances is markedly increased in a petrochemical plant.     

Assessing the Mutagenic Potential of Surface Sediments from Beijing Guanting Reservoir to Salmonella typhimurium

Mutagenicity of organic extracts from Beijing Guanting Reservoir sediments was investigated with TA98 and TA100 of Salmonella typhimurium. TA98 and TA100 were employed to detect frameshift mutation and base-pair substitution mutation, respectively. For TA100, no positive result was found, while TA98 was more sensitive and pro-mutagenic frameshift mutagens were mainly detected in sediments. Sediments from northern and southern Guanting Reservoir were at potential mutagenic risk. No mutagenicity was found in the sediments from the entrance of the tributaries, but strong mutagenicity was observed in the sediments from the outlet of the reservoir. Chemical analysis was also performed, and poor correlation was found between mutagenicity and organochlorine pesticides (OCPs). However, significant positive correlation was found for polycyclic aromatic hydrocarbons (PAHs) (r = 0.603–0.946), which showed that PAHs were dominated for the tested mutagenicity in the sediments. Polychlorinated biphenyls (PCBs) might induce mutagenicity or promote the mutagenicity of other substances.     

Absence of mutagenic action of 5β-cholan-24-oic acid derivatives in the bacterial fluctuation and standard Ames tests

Published data on the mutagenicity of 3 bile acids in the bacterial fluctuation test are conflicting. Eleven 5β-cholanoic acids including 2 of the biie acids were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in the fluctuation tests. In any of these bile acids at the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. Similarly, none of these compounds showed positive mutagenicity in both strains in the standard Ames test either with or without hepatic metabolic activation. Our results support the claim that 3 bile acids are not mutagenic, and indicate that the initiation activity of 5β-cholanoic acids is not demonstrable with a short-term assay using Salmonella strains.     

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.     

Characterization of organic extracts from standard reference materials 1649, 'urban dust/organics,' and 1650, 'diesel particulate matter', using a microsuspension assay. A WHO/IPCS/CSCM study.

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 'urban dust/organics' (air particles), and 1650, 'diesel particulate matter' (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation assay. In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98-S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/micrograms, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/microgram, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 -S9 but the TA98-1,8-DNP6 levels were.     

The effect of gamma-radiation on smoked fish using short-term mutagenicity assays

The effect of gamma-radiation on the mutagenicity potential of wood-smoked fish (Rastrelliger sp.) was investigated. Smoked fish were irradiated with radiation doses of 2.0, 4.0, 6.0 and 8.0 kGy. The DMSO extracts of non-radiated and irradiated smoked fish were tested for mutagenicity using the Ames plate incorporation assay, host-mediated assay, and the micronucleus test. It was observed that gamma-irradiation did not induce any significant increase in the number of revertants of TA98, TA100 and TA104 as compared with the non-radiated smoked fish. Results of the host-mediated assay and the micronucleus test showed no difference in the mutagenic response of non-radiated and irradiated smoked fish. The results indicate that gamma-radiation does not introduce mutagens in smoked fish.     

Quantitative detection of Corynebacterium casei in cheese by real-time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10(5) to 10(10) CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10(5) CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.     

Mutagenic effect of amniotic fluid from smoking women at term

Concentrated term amniotic fluid samples from 44 women smokers and 44 controls were investigated with respect to mutagenic effect in the Salmonella/mammalian-microsome mutagenicity test, using tester strains TA98 and TA100. Tests with freeze-dried specimens of term amniotic fluid showed increases in the number of revertant colonies over background values, regardless of smoking status. However, samples from heavy smokers produced a higher number of revertants than did samples from nonsmokers in several experiments with tester strain TA98. The increase was statistically significant, using either total tar content or number of cigarettes smoked to identify heavy smokers. Experimental series with tester strain TA100 also resulted in higher group means for heavy smokers than for nonsmokers, but the difference was not statistically significant with the concentrations used in this assay. We conclude that heavy smokers may expose their unborn children to mutagenic substances.     

Mutagenicity of stemphyltoxin III, a metabolite of Alternaria alternata

Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.     

Mutagenicity of stemphyltoxin III, a metabolite of Alternaria alternata.

Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.