Chemical characterization of direct-acting airborne mutagens: The functional group

Durham NC air was sampled, extracted, and bioassayed for mutagenic activity in Salmonella typhimurium TA98. Portions of the extracts were treated with sodium borohydride over copper(II) acetylacetonate to reduce any nitroaromatic substances to their corresponding amines. All of the reduced extracts were not directly mutagenic, but the 2 that were derived from cold-weather air samplings did contain substances that could be activated oxidatively. These “indirect-acting” mutagens were present in the same 2 reduced extracts that contained detectable concentrations of aromatic amines. These results suggest that a major portion of the total mutagenic activity in air-pollution particles is contributed by nitro-substituted compounds that are detectable as their corresponding amines. They also suggest that the atmospheric concentrations of these substances may be high in the colder months of Durham's year.     

Mutagenicity in Salmonella typhimurium mutants of serum extracts from airborne particulates

Airborne particulates collected from urban and non-urban air were extracted with calf serum or benzene, and their mutagenic potencies were evaluated in the Salmonella reversion assay. The serum extracts were mutagenic to strains TA98 and TA100 and contained both direct- and indirect-acting mutagens. Mutagenic activities for TA98 recovered from the particulates by serum or benzene extraction were much less in the serum extracts than in the benzene extracts. There was no significant difference in mutagenic potencies of the extracts between the urban and non-urban particulates, irrespective of the presence of S9 mix. The calculated mutagenic activities per m3 of air, however, were greater for urban air than for non-urban air, because of higher concentration of particulates in urban air than in non-urban air. Serum effectively reduced both direct and indirect mutagenic activities of the benzene extracts except for an insufficient reduction in direct mutagenicity at a high dose of benzene extracts. These findings suggest that serum could contribute greatly to decrease the mutagenicity of airborne particulates by mechanisms such as less efficient solubilization of mutagenic components and inactivation by protein binding. Biological availability of mutagens, therefore, should be considered for evaluation of actual mutagenic hazard by airborne particulates.     

Ames test: mutagenic activity of airborne particles collected on airconditioner filters during the fire at a Sandoz storehouse in Schweizerhalle on November 1, 1986

The mutagenic activity of methanol extracts from airconditioner filters was tested using the standard plate-incorporation procedure for the Ames test and the strains Salmonella typhimurium TA97, TA98 and TA100 as test organisms. In a first set of experiments filters from 4 buildings were investigated. For each building the mutagenic activity of a filter which was in use during the fire (fire-exposed) was compared with the mutagenic activity of a filter which was exposed for a similar time span to normal urban air (non-exposed). While for 1 pair of filters the non-exposed extract was more mutagenic than the fire-exposed material, the opposite was found for 2 other filter pairs, and in 1 case there was hardly any difference in the mutagenic activities of the fire-exposed and the non-exposed filter extracts. Overall differences by factors up to 100 were observed in the mutants/m3 air values of the most and the least mutagenic filter extracts. The second group of experiments was performed to investigate the variations in mutagenic activity of filter extracts occurring due to changes in the weather conditions. Airborne particles were collected for 3 consecutive periods of 10-11 days at the 2 buildings where the extracts of the fire-exposed filters had been found to be more mutagenic than the corresponding control materials. The differences between the strongest and the weakest mutagenic filter extracts of these series were similar to the differences observed previously between the fire-exposed filters and the non-exposed filter materials. The highest mutagenic activities found in the second group of experiments was similar, at both sites, to the mutagenic activities measured in the fire-exposed extracts from these 2 buildings. Since the differences in the mutagenic activities of filters exposed to urban air during the different meteorological conditions were similar to the differences observed between fire-exposed and non-exposed materials, it is not possible to state whether the fire led to the distribution of mutagenic chemicals, although it is theoretically possible. However, based on the observation that the maximal mutagenic activities of the fire-exposed and the non-exposed extracts were similar, the present study allows the conclusion that the mutagenic burden for the population of the area of Basel was not significantly increased by the fire in Schweizerhalle on November 1, 1986.     

Mutagenicity studies on complex environmental mixtures: selection of solvent system for extraction.

The mutagenic activities in the Salmonella/microsome assay of dichloromethane (DCM) and acetone extracts of complex environmental mixtures were compared. The particulate samples used in the IPCS collaborative study were Soxhlet-extracted twice with DCM followed by a third extraction with acetone. Compared with the mutagenic activity of the first extract, the third (acetone) extract of the urban particulate matter showed a relatively high mutagenic activity. In contrast to this the third extract of the diesel particulate matter contributed very little additional mutagenic activity. Furthermore, 10 filter samples of air particulates from a suburban airport area were collected for comparison of the extraction efficiency of DCM and acetone. Each sample was divided into two samples of identical size followed by extraction with acetone and DCM, respectively. No clear difference in the mutagenic activity of these extracts was observed in strains TA98 and TA98NR. It is concluded that for ambient air particulates (but not emission samples) acetone may extract some mutagenic compounds which are not extracted by DCM. The amount of these additional extractable compounds seems to depend on the composition of the sample. As DCM extracts are better suited for further fractionation and chemical analysis DCM is considered to be the best choice for a general solvent system for extraction of complex environmental mixtures.     

Sodium azide mutagenesis in mammals: inability of mammalian cells to convert azide to a mutagenic intermediate

Sodium azide is unique among mutagens. It is highly mutagenic in many plant and bacterial species but marginally mutagenic in mammalian cells. A possible explanation for this difference in mutagenic efficiency may lie in the inability of mammalian cells to convert azide to the putative ultimate mutagen. Normal human fibroblasts and Chinese hamster cells or cell-free extracts from these cell lines were treated with azide and the sonicates tested for mutagenicity in Salmonella strain TA1530. The data suggest that neither cell line was capable of converting azide to a mutagenic intermediate. In addition, both cell lines expressed the enzyme O-acetylserine(thio)-lyase which is responsible for the conversion of azide to azidoalanine, the putative mutagenic intermediate. Although mammalian cells possess the enzyme responsible for the conversion of azide to azidoalanine, they appear incapable of converting azide into a mutagenic intermediate in appreciable quantities. Further, the data support the conclusion that azide may be further modified in mammalian cells to an intermediate that is not genotoxic.     

Vinyl chloride dependent mutagensis: effects of liver extracts and free radicals.

The mutagenic effects of vinyl chloride (VC) on Salmonella typhimurium strain TA1530 are enhanced by mouse or rat liver extracts. The extracts prepared from mice pretreated either with vinyl chloride or the microsomal enzyme inducer, Aroclor 1254, did not produce any greater stimulation of VC-dependent mutagenesis than extracts from untreated animals. These same extracts, however, differed markedly in their capacity to stimulate the mutagenicity of dimethylnitrosamine (DMN), a compound which is converted to a mutagen by an NADPH dependent microsomal mixed function oxidase. The order of activity of the extracts with DMN was Aroclor pretreated is greater than untreated is greater than VC pretreated. Furthermore, the stimulatory effect of the liver extracts on VC mediated mutagenesis did not require NADPH and was still evident in liver extracts in which the microsomal mixed function oxidase system had been heat inactivated. The mutagenic activity of VC also was found to be stimulated by riboflavin in the presence of light suggesting that free radicals may be involved in VC dependent mutagenesis.     

Crude tea extracts decrease the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine in vitro and in intragastric tract of rats

The effects of tea extracts and their ingredients, catechins and L-ascorbic acid (AsA), on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in vitro and in the stomachs of rats using E. coli WP2 and S. typhimurium TA100. The extracts of green tea and black tea leaves decreased the mutagenic activity of MNNG to E. coli WP2 in vitro in a desmutagenic manner. Catechins such as (-)-epigallocatechin from green tea leaves and the low-molecular-weight tannin fraction isolated from black tea extract with HP-20 resin also exhibited inhibitory effects against the mutagenic activity of MNNG. A desmutagenic effect of AsA on MNNG-induced mutagenicity was observed depending on the dose, though it was complicated. The effects were also demonstrated in the stomachs of rats by assaying the bacterial mutagenic in vitro; the tea extracts previously given orally to rats reduced the mutagenic activity of MNNG remarkably, though simultaneous administration showed less effect. The effectiveness of tea extracts for the decrease of MNNG-induced mutagenesis in vitro and in vivo suggests that the habitual drinking of tea may reduce the tumor-initiating potency of MNNG-type nitrosoureido compounds if they are formed in the stomach.     

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.     

Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

Several known indole compounds are not important precursors of direct mutagenic N-nitroso compounds in green cabbage

In this study we investigated the role of indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan in the formation of N-nitroso compounds in green cabbage extracts. Green cabbage extracts were separated by gel permeation chromatography. Fractions were treated with nitrite, tested for mutagenicity and analysed for total N-nitroso content. Fractions in which spiked indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan eluted appeared to be low in mutagenic activity and contained relatively small amounts of N-nitroso compounds. To detect indole compounds other than the ones used in the gel permeation chromatography experiments, high-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed of green cabbage extracts. Indole-3-carboxaldehyde was found to be the most commonly occurring indole compound, but it did not show direct mutagenic activity upon nitrite treatment. Indole-3-acetonitrile was the second most common compound; although it was mutagenic after nitrite treatment, its contribution to the mutagenicity of nitrite-treated green cabbage was roughly estimated to be only 2%. No other indole compounds were detected. From this study we conclude that neither the tested indole compounds nor indole-3-carboxaldehyde play a significant role in the formation of direct mutagenic N-nitroso compounds in nitrite-treated green cabbage extracts.     

Conversion of Congo red and 2-azoxyfluorene to mutagens following in vitro reduction by whole-cell rat cecal bacteria

Congo red, an azo dye derived from benzidine, and 2-azoxyfluorene, a derivative of 2-aminofluorene, were reduced during overnight incubation with a suspension of rat intestinal bacteria. High performance liquid chromatography and ultraviolet spectral analysis verified the presence of benzidine in extracts of the Congo red incubations and 2-aminofluorene in extracts of the 2-azoxyfluorene incubations. Extracts of the Congo red incubations were mutagenic toward Salmonella typhimurium TA1538 in the presence of a post-mitochondrial activating system, but Congo red was not mutagenic without this reductive pretreatment. Thus, the utility of the Ames test in screening for potential mutagens may be expanded by a reductive pretreatment utilizing cecal bacteria.     

Mutagenicity in salmonella of hazardous wastes and urine from rats fed these wastes

15 hazardous industrial waste samples were evaluated for mutagenicity in the Salmonella plate-incorporation assay using strains TA98 and TA100 in the presence and absence of Aroclor 1254-induced rat liver S9. Dichloromethane/methanol extracts of the crude wastes were also evaluated. 7 of the crude wastes were mutagenic, but only 2 of the extracts of these 7 wastes were mutagenic; extracts of 2 additional wastes also were mutagenic. In addition, 10 of the crude wastes were administered by gavage to F-344 rats, and 24-h urine samples were collected. Of the 10 raw urines evaluated, 3 were mutagenic in strain TA98 in the presence of S9 and beta-glucuronidase. The 3 crude wastes that produced these 3 mutagenic urines were, themselves, mutagenic. Adequate volumes of 6 of the 10 raw urines were available for extraction/concentration. These 6 urines were incubated with beta-glucuronidase and eluted through Sep-Pak C18 columns; the methanol eluates of 3 of the urines were mutagenic, and these were the same 3 whose raw urines also were mutagenic. In general, the C18/methanol extraction procedure reduced the cytotoxicity and increased the mutagenic potency of the urines. To our knowledge, this is the first report of the mutagenicity of urine from rodents exposed to hazardous wastes. Based on the present results, the use of only strain TA98 in the presence of S9 might be adequate for general screening of hazardous wastes or waste extracts for genotoxicity. The urinary mutagenesis assay does not appear to be a useful adjunct to the Salmonella assay for screening hazardous wastes. The problems associated with chemically fractionating diverse types of hazardous wastes for bioassay are also discussed.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay

This paper describes the screening studies of 104 commercial crude drugs for mutagenicity by the rec-assay with Bacillus subtilis as well as the reversion assay with Ames strains TA98 and TA100 of Salmonella typhimurium. The rec-assays showed that 13 water extracts and 27 methanol extracts of the crude drugs were positive. The Ames assays with or without metabolic activation showed that 24 water extracts and 16 methanol extracts were mutagenic. In total, mutagenic activities were found in 45 samples among the 104 crude drugs tested.     

The mutagenic potential of madder root in dyeing processes in the textile industry

The roots of Rubia tinctorum L. have a long tradition of being used in dyeing processes of textiles from centuries ago until the present time. The colouring principles belong to the class of hydroxyanthraquinones. Concern arose because several of these compounds were recognised as mutagenic in vitro and even carcinogenic in rodents. To assess the possible risk to humans caused by coloured textiles, mutagenicity was investigated with two madder root samples of different origin (Iran and Bhutan) along the entire dyeing process from root extracts to the dyed wool. The Salmonella/microsome test (Ames assay) with the strains TA98, TA100 and TA1537 was used. Significant mutagenic effects could be detected in madder root extracts and also in the final product, the dyed wool. Madder root from Iran showed considerably higher mutagenic responses than samples from Bhutan. Analytical investigations of the extracts by HPLC showed the presence of a spectrum of anthraquinones typical for madder root. Three mutagenic compounds, lucidine, rubiadine and purpuroxanthine, together with the non-mutagenic alizarine could be detected. The mutagenic response of the different samples was positively correlated with the concentration of the mutagenic anthraquinones, and with lucidine in particular. Based on these investigations a risk to dye-house workers and users of textiles dyed with R. tinctorum must be anticipated.     

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.     

Heterocyclic aromatic amine content of selected beef flavors

Previous work has shown that meat extracts contain potent mutagenic and/or carcinogenic heterocyclic aromatic amines (HAAs). Because meat extracts and some beef flavors are produced from similar precursors and processing steps, the beef flavors may also contain HAAs. This study analyzed 24 commercial beef flavors and 2 food-grade beef extracts for creatine and creatinine concentrations, mutagenic activity and HAA concentrations (IQ, MeIQ, MeIQ_x, DiMeIQ_x, Glu-P-1, Glu-P-2 and PhIP). The creatine and creatinine levels of the flavors ranged from 0 to 73 and from 0 to 21 mg/g (dry wt.), respectively. The mutagenic activities of the flavors ranged from 0 to 3200 Salmonella typhimurium TA98 revertants/g (dry wt.). No direct relationship was found between creatine and/or creatinine concentrations and mutagenic activities. However, flavors with high creatine (> 1.5 mg/g) or creatinine (> 2 mg/g) levels exhibited higher mutagenic activities than did flavors with low levels of these compounds. Flavors with high mutagenic activities (> 1500 revertants/g) contained measurable amounts of HAAs. Three flavors contained MeIQ_x (7.2–21.2 ng/g [dry wt.]) and one contained DiMeIQ_x (4.2 ng/g [dry wt.]).     

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.     

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.     

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.