Detection of mutagens produced by fungi with the Salmonella typhimurium assay.

Forty-one fungal isolates (one isolate per species) representing common plant pathogens and food crop contaminants were grown on sterile, polished rice and assayed for mutagenic activity in the Salmonella typhimurium-microsome system. Initially, single doses of aqueous and chloroform extracts of the moldy rice were assayed against the TA100 tester strain by incorporating extracts into the growth medium and by applying small quantities on disks placed on the agar surface. Suspected activity was examined further by analysis of several doses in the plate incorporation assay. Extracts of two aflatoxin-producing isolates (Aspergillus flavus and A. parasiticus) showed pronounced mutagenic activity, as did extracts of five other isolates (A. heterothallicus, A. nidulans, A. terricola, Alternaria tenuis, and Fusarium moniliforme) which did not contain detectable aflatoxins. Seven additional isolates (Botrytis cineria, Ceratocystis fimbriata, Cladosporium herbarum, Fusarium solani f. sp. pisi, Penicillium oxalicum, Thermomyces lanuginosus, and Verticilium albo-atrum) revealed activity which was possibly mutagenic; i.e., mutagenic responses were not observed in both the disk and incorporation assays, and clear dose-related activity was not observed in the incorporation assay. Extracts of the remaining fungi were not mutagenic in the bacterial assay.     

Mutagenic activity of Fusarium moniliforme isolates in the Salmonella typhimurium assay.

A total of 33 isolates of Fusarium moniliforme from several food or feed crops were grown on sterile cracked corn, and chloroform-isopropanol extracts were assayed for mutagenic activity in the Salmonella typhimurium-microsome system by using tester strain TA98 or TA100 or both. Extracts of 21 (64%) of the isolates assayed against TA100 were mutagenic. Activities of seven of these extracts were increased markedly with incorporation of the liver homogenate (S-9) into the assay. Seven (33%) of the isolates assayed against TA98 were weakly active, with the liver homogenate having little effect on reversion rates.     

Mutagenic activity of Fusarium moniliforme isolates in the Salmonella typhimurium assay.

A total of 33 isolates of Fusarium moniliforme from several food or feed crops were grown on sterile cracked corn, and chloroform-isopropanol extracts were assayed for mutagenic activity in the Salmonella typhimurium-microsome system by using tester strain TA98 or TA100 or both. Extracts of 21 (64%) of the isolates assayed against TA100 were mutagenic. Activities of seven of these extracts were increased markedly with incorporation of the liver homogenate (S-9) into the assay. Seven (33%) of the isolates assayed against TA98 were weakly active, with the liver homogenate having little effect on reversion rates.     

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.     

Release of mutagens from finished leather

Extracts of a leather widely used in the furniture and dress-making industries were tested for their mutagenic activity in the Salmonella/microsome assay. Extracts obtained after vigorous treatment of leather samples in a Soxhlet apparatus with toluene or ethanol were mutagenic in strain TA98 of S. typhimurium in the absence of S9 mix. The analysis of extracts of leather at various intermediate stages of processing showed that the mutagenic activity appeared after the coloration process. The responsible compound was identified to be an azo dye (Color Index: Acid Brown 83) whose mutagenic potency was about 4 revertants/micrograms.     

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 asssay.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/μg, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/μg, 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.     

The bactericidal activity of tea against Helicobacter pylori

Extracts of black and green tea inhibited in-vitro growth of six clinical isolates of Helicobacter pylori in an agar diffusion assay. Tea extracts killed H. pylori (10⁶ cfu ml^-1) within 5 h. Heat treatment of extracts did not affect the inhibitory or bactericidal activity.     

Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China

The aim of this research is to investigate the impact of air pollution on the population in Shanghai. The genotoxicity of extractable organic matter (EOM) from the air particles was investigated by the means of the Salmonella plate incorporation assay, rat hepatocyte unscheduled DNA repair assay, and mice micronuclei test. The airborne particles were collected in 13 locations during the summer of 1992 and winter of 1993. The crude extracts were fractionated by acid-base partitioning into acid, base and neutral fractions. The neutral fractions were further fractionated by resin-silica gel column chromatography into three subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The mutagenicity was detected with TA98, but was not detected with TA100. The mutagenic activity was the greatest in the acid, aromatic and polar fractions from summer samples. The fractions from the winter samples did not show clear differences. There was no substantial location-related variance in the mutagenic potencies of EOM, but substantial location- or time-related variances in the mutagenic potencies of the airborne particles per cubic meter air were found. While rat hepatocyte unscheduled DNA synthesis (UDS) assay revealed genotoxicity for all the samples, there was no big variance in the genotoxicity of the fractions. The mouse micronuclei test showed results similar to the UDS assay. The difference of locality did not have statistical significance.     

Antagonistic activity of Penicillium oxalicum Corrie and Thom,Penicillium decumbens Thom and Trichoderma harzianum Rifai isolates against fungi,bacteria and insects in vitro

The antibiotic activity of 70 isolates belonging to the genera Aspergillus, Penicillium, Fusarium, Alternaria and Trichoderma was tested as preliminary screening. The highest activity was obtained with three Penicillium oxalicum isolates, one Penicillium decumbens isolate and the Trichoderma harzianum isolate. After that, we chose these five isolates in order to carry out other studies with bacteria, fungi and insects. Extracts from these isolates were obtained. The extracts were tested for antibiotic activity with positive results, which implies that metabolite production is involved in this antagonistic effect. The highest activity was shown by T. harzianum and P. oxalicum extracts, but there was high variability among P. oxalicum isolates.     

Mutagenicity of extracts from typewriter ribbons and related items

Extracts of typewriter ribbons and carbon papers were found to be mutagenic in the Salmonella/microsome assay with strain TA98. Fractionation of ribbon extracts indicates that at least 2-3 different classes of mutagenic component are present in these extracts. Nitro-containing compounds may be responsible for the high mutagenicity observed for some of the ribbon extracts in the absence of S9. The results indicate that impurities in the products may be causing part of the mutagenic effect.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Mechanism of cellulose synthesis in Agrobacterium tumefaciens.

Extracts of Agrobacterium tumefaciens incorporated UDP-[14C]glucose into cellulose. When the extracts were fractionated into membrane and soluble components, neither fraction was able to synthesize cellulose. A combination of the membrane and soluble fractions restored the activity found in the original extracts. Extracts of cellulose-minus mutants showed no significant incorporation of UDP-glucose into cellulose. When mixtures of the extracts were made, the mutants were found to fall into two groups: extracts of mutants from the first group could be combined with extracts of the second group to obtain cellulose synthesis. No synthesis was observed when extracts of mutants from the same group were mixed. The groups of mutants corresponded to the two operons identified in sequencing the cel genes (A. G. Matthysse, S. White, and R. Lightfoot. J. Bacteriol. 177:1069-1075, 1995). Extracts of mutants were fractionated into membrane and soluble components, and the fractions were mixed and assayed for the ability to synthesize cellulose. When the membrane fraction from mutants in the celDE operon was combined with the soluble fraction from mutants in the celABC operon, incorporation of UDP-glucose into cellulose was observed. In order to determine whether lipid-linked intermediates were involved in cellulose synthesis, permeablized cells were examined for the incorporation of UDP-[14C]glucose into material extractable with organic solvents. No radioactivity was found in the chloroform-methanol extract of mutants in the celDE operon, but radioactive material was recovered in the chloroform-methanol extract of mutants in the celABC operon. The saccharide component of these compounds was released after mild acid hydrolysis and was found to be mainly glucose for the celA insertion mutant and a mixture of cellobiose, cellotriose, and cellotetrose for the celB and celC insertion mutants. The radioactive compound extracted with chloroform-methanol form the celC insertion mutant was incorporated into cellulose by membrane preparations from celE mutants, which suggests that this compound is a lipid-linked intermediate in cellulose synthesis.     

Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents

1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s(20,w) values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen-detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).     

Characterization of the Late Steps of Microbial Heme Synthesis: Conversion of Coproporphyrinogen to Protoporphyrin

Cell-free extracts of various cytochrome-containing, heterotrophic microorganisms were examined for ability to convert coproporphyrinogen to protoporphyrin. Extracts of Escherichia coli and Pseudomonas denitrificans readily accumulated large amounts of protoporphyrin when assayed under aerobic conditions. However, protoporphyrin did not accumulate under either aerobic or anaerobic conditions of assay or in the presence of various supplements in extracts of the aerobe Micrococcus lysodeikticus, the facultative anaerobe Staphylococcus aureus, or the anaerobe Vibrio succinogenes. Protoporphyrin also accumulated when extracts of E. coli and P. denitrificans were incubated aerobically with the early heme precursor, delta-amino levulinic acid (ALA). This protoporphyrin accumulation was markedly stimulated by the iron chelator, o-phenanthroline. Extracts of S. aureus and M. lysodeikticus accumulated coproporphyrin, but not protoporphyrin when incubated with ALA. The enzyme system in extracts of E. coli which converts coproporphyrinogen to protoporphyrin under aerobic conditions of assay was also partially characterized. This conversion was stimulated by the iron chelator, o-phenanthroline, the respiratory inhibitor, cyanide, and the reducing agent, thioglycolate. Dialysis of the extract did not diminish enzyme activity. Certain alternate electron acceptors and nitrite caused a marked inhibition of the conversion. These results indicate that this late step in heme synthesis, the conversion of coproporphyrinogen to protoporphyrin, can be readily demonstrated in extracts of some, but not all, cytochrome-containing bacteria and that the aerobic conversion in E. coli exhibits many characteristics similar to those demonstrated for the aerobic conversion previously studied in liver mitochondria.     

Influence of the interactions among ecological variables in the characterization of zearalenone producing isolates of Fusarium spp

To carry out the physiological characterization of Fusarium graminearum and F. culmorum isolates with regard to its zearalenone producing ability, an in-depth experiment with a full factorial design was conducted. The effects and mutual interactions of temperature, moisture, substrate and isolate on the production of the toxin were studied. The study was done with twelve isolates of Fusarium (7 of F. graminearum and 5 of F. culmorum). The analysis of variance shows that there is a complex interaction of all of these factors, which can influence the relative concentrations of the mycotoxin produced, and hence, the correct physiological characterization of the strain. All the tested cultures were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.     

Butylated hydroxyanisole, butylated hydroxytoluene and tert.-butylhydroquinone are not mutagenic in the Salmonella/microsome assay using new tester strains

The phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert.-butylhydroquinone (TBHQ) were reassessed for mutagenic activity using the recently developed Salmonella tester strains TA97, TA102 and TA104, and in addition TA100. None of the phenolic antioxidants showed mutagenic activity, either with or without metabolic activation. At doses of 100 micrograms/plate and higher all 3 phenolic antioxidants exhibited toxic effects. A modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Combinations of BHA and BHT, tested to detect possible synergistic effects, did not exert mutagenic activity.     

Genotoxic and mutagenic activity of environmental air samples from different rural, urban and industrial sites in Flanders, Belgium

The present study reports mutagenic and genotoxic activities associated with ambient air collected at 15 sites characteristic for urban, industrial or rural conditions in Flanders. Airborne particulates (PM10) and semi-volatile compounds were collected on quartz filters (QF) and polyurethane foam (PUF) cartridges using a high-volume sampling device. The mutagenic and genotoxic potency of the organic extracts--Soxhlet extraction with acetone--was determined by use of the Salmonella mutagenicity standard plate-incorporation assay and the Vitotox assay, respectively. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). Ambient air samples contained significant PAH levels and mutagenic activities at all 15 sites: direct mutagenicity of up to 47 revertants per cubic meter was found in the QF extracts and more limited activity of up to 11 rev m(-3) in the PUF extracts. Metabolic activation of PUF extracts resulted in an important increase in mutagenic activity, up to 30 rev m(-3), but no such increase was observed for QF extracts. The highest values were observed outside large cities at industrial sites and at a rural site contaminated by pollution from a chemical plant at a distance of 4 km. Also at the background location near the North Sea a significant mutagenic activity was measured in the QF extracts (+S9: 9 rev m(-3); -S9: 7 rev m(-3)). Apparently, there is in Flanders a significant background exposure level to airborne mutagenicity, even in areas with limited or no nearby pollution sources. Based on the concentrations of 10 mutagenic PAHs and supposing additivity of their specific mutagenicities, only a few percent (mean 3%) of the observed indirect mutagenic activity could be explained. This implies that most mutagenic activity originated from other substances that were not identified or measured in our chemical analysis. This underscores the importance of bio-monitoring measurements.     

Mutagens in rat urine after dermal application of 1,3-diaminobenzene

The mutagenicity of urine from rats treated topically on the skin with 1,3-diaminobenzene was studied by the Salmonella/mammalian-microsome assay. Urine samples were either passed directly through micropore filters or extracts were prepared using XAD-2 resin before testing in the frameshift strain TA98. Significant mutagenic activity was found only after metabolic activation with rat-liver microsomes. The activity was higher in extracts from rats treated with a mixture of hydrogen peroxide and 1,3-diaminobenzene than from rats which were exposed to 1,3-diaminobenzene only. After fractionation of the urine by HPLC it could be demonstrated that the mutagenic activity was not due to the parent amine but related to metabolites in two of the fractions. To a lesser extent these two partially purified fractions were also mutagenic without S9 activation even though it was not possible to demonstrate this effect in unfractionated urine extracts. A third fraction containing two metabolites did not exert demonstrable mutagenic activity. The implications for the assessment of hazard to man are discussed.     

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.