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.     

Mutagenicity of native cigarette mainstream smoke and its gas/vapour phase by use of different tester strains and cigarettes in a modified Ames assay

The "Bacterial Reverse Mutation Assay" is generally accepted to analyse the genotoxic capacity of single compounds or complex mixtures such as cigarette-smoke condensates. With an adapted and modified Ames assay, the mutagenicity of native cigarette mainstream whole smoke (WS) and its gas/vapour phase (GVP) was studied. The bacteria were directly exposed to the smoke in a CULTEX1 system closely connected to a smoking robot (VC10). A variety of standard tester strains (TA98, TA100, TA1535, TA1537, TA1538, TA102, WP2uvrApKM101) and descendants of TA98 (YG1021, YG1024, YG1041) and TA100 (YG1026, YG1029 and YG1042) were exposed to whole and filtered smoke of the research cigarette K2R4F to find the most sensitive strains for analysing the mutagenic activity of these test atmospheres. Mutagenicity of WS was detected by TA98, TA100 and their YG descendant strains as well as by WP2uvrApKM101 in the presence of S9 mix. The GVP induced a mutagenic signal in TA100, YG1029 and YG1042 and WP2uvrApKM101 only in the absence of S9 mix. To detect mutagenicity in WS the presence of the plasmid pKM101 is required and a frame-shift mutation is more effective than a missense mutation. To detect mutagenicity in GVP, the presence of the plasmid pKM101 and a missense mutation are required. The differentiating capacity of this modified Ames assay was demonstrated by exposing strain TA98 to WS and TA100 to the GVP of cigarettes with different tar content. The mutagenic activity of WS and the GVP increased with rising tar content of the cigarettes with two exceptions in WS. Thus, the concept of tar content alone is misleading and does not reflect the mutagenic activity of a cigarette.     

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.     

Application of Salmonella strains with altered nitroreductase and O-acetyltransferase activities to the evaluation of the mutagenicity of airborne particles

The Ames test was applied to evaluation of the mutagenicity of month's samples of airborne particles from the center of Wroc?aw (SW Poland) collected in August and December 1997. The strains used for the study were TA 98, TA 100 and their derivatives: TA 98 NR, YG 1021, YG 1024, YG 1026, YG 1029, YG 1041, YG 1042. Both studied samples were mutagenic for almost all tested strains, with the exception of the August sample which did not influence the strain TA 100 without the metabolic activation with the S9 fraction. The December sample exhibited higher genotoxic activity than the August sample. Mutagenicity ratios of the strains with reduced nitroreductase and O-acetyltransferase activities were higher, and of the strain without the nitroreductase--lower than those of the parent strains. This indicates that nitro and amino derivatives of PAHs are responsible for the significant proportion of total mutagenicity of the studied samples of particulates. Metabolic activation with the S9 fraction caused the increase of the mutagenic activity of the samples, which indicates the presence of promutagens. The GC-MS analysis revealed the presence of known indirect mutagens from the PAHs group.     

Comparison of two extraction procedures for the assessment of sediment genotoxicity: implication of polar organic compounds

Four sediment samples (Va?ne Airport VA, Va?ne Center VC, Va?ne North VN and Reference North RN) were collected in the Berre lagoon (France). Sediments were analyzed for polycyclic aromatic hydrocarbons (PAHs) by use of pressurized fluid extraction with a mixture of hexane/dichloromethane followed by HPLC with fluorescence detection analysis. Organic pollutants were also extracted with two solvents for subsequent evaluation of their genotoxicity: a hexane/dichloromethane mixture intended to select non-polar compounds such as PAHs, and 2-propanol intended to select polar contaminants. Sediment extracts were assessed by the Salmonella/microsome mutagenicity test with Salmonella typhimurium TA98+S9 mix and YG1041±S9 mix. Extracts were also assessed for their DNA-damaging activity and their clastogenic/aneugenic properties by the comet assay and the micronucleus test with Chinese Hamster ovary (CHO) cells. The PAH concentrations were 611ngg(-1)dw, 1341ngg(-1) dw, 613ngg(-1)dw and 482ngg(-1)dw for VA, VC, VN and RN, respectively. Two genotoxic profiles were observed, depending on the extraction procedure. All the non-polar extracts were mutagenic for TA98+S9 mix, and VA, VC, VN sediment samples exerted a significant DNA-damaging and clastogenic activity in the presence of S9 mix. All the polar extracts appeared mutagenic for TA98+S9 mix and YG104±S9 mix, and VA, VC, VN were genotoxic and clastogenic both with and without S9 mix. These results indicate that the genotoxic and mutagenic activities mainly originated from PAHs in the non-polar extracts, while these activities came from other genotoxic contaminants, such as aromatic amines and nitroarenes, in the polar extracts. This study focused on the important role of uncharacterized polar contaminants such as nitro-PAHs or aromatic amines in the global mutagenicity of sediments. The necessity to use appropriate extraction solvents to accurately evaluate the genotoxic hazard of aquatic sediments is also highlighted.     

Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity

N-Nitroso compounds, such as N-nitrosodiethylamine (NDEA), are a versatile group of chemical carcinogens, being suspected to be involved in gastrointestinal tumors in humans. The intestinal microflora can modify a wide range of environmental chemicals either directly or in the course of enterohepatic circulation. Nitroreductases from bacteria seem to have a wide spectrum of substrates, as observed by the reduction of several nitroaromatic compounds, but their capacity to metabolize N-nitroso compounds has not been described. To elucidate the participation of nitroreductase or acetyltransferase enzymes in the mutagenic activity of NDEA, the bacterial (reverse) mutation test was carried out with the strains YG1021 (nitroreductase overexpression), YG1024 (acetyltransferase overexpression), TA98NR (nitroreductase deficient), and TA98DNP6 (acetyltrasferase deficient), and YG1041, which overexpresses both enzymes. The presence of high levels of acetyltransferase may generate toxic compounds that must be eliminated by cellular processes or can lead to cell death, and consequently decrease the mutagenic effect, as can be observed by the comparison of strain TA98DNP6 with the strains TA98 and YG1024. The slope curves for TA98 strain were 0.66 rev/microM (R(2) = 0.51) and 52.8 rev/microM (R(2) = 0.88), in the absence and presence of S9 mix, respectively. For YG1024 strain, the slope curve, in the presence of S9 mix was 6897 rev/microM (R(2) = 0.78). Our data suggest that N-nitroso compounds need to be initially metabolized by enzymes such as cytochromes P450 to induce mutagenicity. Nitroreductase stimulates toxicity, while acetyltransferase stimulates mutagenicity, and nitroreductase can neutralize the mechanism of mutagenicity generating innoccuos compounds, probably by acting on the product generated after NDEA activation.     

Genotoxicity of urban air pollutants in the Czech Republic. Part I. Bacterial mutagenic potencies of organic compounds adsorbed on PM10 particulates

As part of a long-term program to investigate the impact of air pollution on the health of a population in a polluted region in Northern Bohemia, mutagenicity of extractable organic matter (EOM) from air particles PM10 was investigated by the means of Salmonella typhimurium indicator strains TA98 and YG1041 using the Ames plate incorporation assay. The air samples were collected in both the polluted and the control districts during the summers and winters of 1993-1994. In the polluted district, the collection was repeated during the winter of 1996-1997. The crude extracts from filters pooled according to the locality and the season were fractionated by acid-base partitioning into acid, base, and neutral fractions. The neutral fractions were further fractionated by silica gel column chromatography into five 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 indirect mutagenic potency of aromatic subfractions containing polycyclic aromatic hydrocarbons (PAHs) was generally low. The mutagenic potency detected with TA98 was more distinct only in the winter sample 1993-1994 from the polluted area, where the aromatic subfraction accounted for 23% of total mutagenicity. In both strains, the highest direct-acting mutagenicity was found in slightly polar fractions containing nitro-PAHs. The mutagenic potency detected with YG1041 was about two orders of magnitude higher than that detected with TA98. No substantial locational- or time-related variances in the mutagenic potencies of EOM, or in the spectrum of chemical components identified in individual fractions were found. The polluted district, in comparison to the control district, was found to have higher amounts of EOM, carcinogenic PAHs and mutagenicity of air particles (rev/m(3)). The fractionating process, combined with the bacterial mutagenicity test, confirmed that nitro-derivatives are the most important contributors to the bacterial mutagenicity of air particles. However, this study did not fulfill the expectancy to bring substantially new, clear-cut information on the composition and the biological activity of air pollution in both districts.     

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.     

Mutagenic characteristics of river waters flowing through large metropolitan areas in North America

The hanging technique using blue rayon, which specifically adsorbs mutagens with multicyclic planar structures, has the advantages over most conventional methods of not having to bring large volumes of water back to the laboratory for extraction of organic materials. Therefore, for the same effort the hanging blue rayon technique allows for the analysis of more samples from remote sites, although it has a disadvantage of not allowing quantitative analysis. In this study, the blue rayon hanging technique was used to collect organic mutagens in river waters that flow through metropolitan areas in northeastern North America. Monitoring was performed at a total of 21 sites: the Providence River system (4 sites), the Charles River (2 sites), the Potomac River (6 sites), the St. Lawrence River (5 sites), the Hudson River (3 sites), and the East River (1 site). Mutagenicity was evaluated using the Salmonella assay with strains TA98, TA100, YG1024, YG1041, and YG1042 with and without metabolic activation. The results demonstrated that strains YG1041 and YG1024 were much more sensitive than TA98 with S9 mix. Fifteen samples out of 21 were positive in YG1041 with S9 mix. Six samples gave 5000-18,400 revertants/g blue rayon equivalent. YG1042 was also much more sensitive than TA100. Eight samples were positive in YG1042 with S9 mix. The highest activity was 10,200 revertants/g blue rayon equivalent. The overall results showed that rivers flowing through major cities in North America contain frameshift-type, aromatic amine-like mutagenic activity. However, the levels of mutagenic activity in these rivers were much lower than expected based on prior analyses and calculated population-to-discharge ratios. Further research, such as detailed chemical analyses and/or simultaneous comparisons of several different adsorbents (e.g. XAD and blue rayon), will be needed to clarify the observed differences between North American blue rayon values and published values for European and Asian river systems.     

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.     

Mutagenicity of polycyclic aromatic hydrocarbon fractions extracted from urban air particulates

Polycyclic aromatic hydrocarbon (PAH) fractions, purified from extracts of airborne particles collected in the area of Genoa municipality, were assayed for mutagenicity in the Salmonella/microsome test. PAH fractions accounted for only a portion of the total mutagenic activity and also displayed a different specificity of genetic activity, as compared to unfractionated material. The analysis of 224 samples collected from January 1986 to November 1987 in 10 different localities led to a large number of positive results in strain TA100 with S9 mix and, less frequently, also in TA98 without metabolic activation. Mutagenicity was related to the intensity of anthropogenic atmospheric pollution, and showed some seasonal variations, although it was not possible to discriminate particular sources of pollution on the basis of mutagenicity patterns. The mutagenic potency in TA100 (S9+) of airborne PAH fractions was significantly correlated with the concentration of individual PAHs in most of the monitored localities. The spectrum of mutagenicity of monthly samples pooled from several localities in S. typhimurium strains, with and without S9 mix, provided evidence for some contribution of nitro derivatives of PAHs or possibly also of other compounds present in the same fractions. The results obtained are discussed in view of their predictive value as indicators of potential health hazards, and of the reliability of this biological tool as a complement to chemical analyses in the evaluation of ambient air pollution.     

Mutagenicity of urine from rats after 1-nitropyrene and 2-nitrofluorene administration using new sensitive Salmonella typhimurium strains YG1012 and YG1024

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2-30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without beta-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of beta-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N,O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Urinary mutagenicity tests in lead-exposed workers

Urinary mutagenic activity detected by the bacterial fluctuation assay, using Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA with and without metabolic activation (S9 mix), was studied in a group of 21 workers exposed to inorganic lead and a control group of 22 non-occupationally exposed subjects. Occupational exposure to inorganic lead had no effect on urinary mutagenicity in the strains considered, with or without metabolic activation. In smokers (exposed and non-exposed), urinary mutagenic activity appeared to increase compared to non-smokers (exposed and non-exposed), only with Salmonella typhimurium TA98 in the presence of S9 mix.     

Photomutagenicity of 16 polycyclic aromatic hydrocarbons from the US EPA priority pollutant list

The photomutagenicity of 16 polycyclic aromatic hydrocarbons (PAHs), all on the United States Environmental Protection Agency (US EPA) priority pollutant list, was studied. Concomitant exposing the Salmonella typhimurium bacteria strain TA102 to one of the PAHs and light (1.1 J/cm2 UVA+2.1 J/cm2 visible) without the activation enzyme S9, strong photomutagenic response is observed for anthracene, benz[a]anthracene, benzo[ghi]perylene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, and pyrene. Under the same conditions, acenaphthene, acenaphthylene, benzo[k]fluoranthene, chrysene, and fluorene are weakly photomutagenic. Benzo[b]fluoranthene, fluoranthene, naphthalene, phenanthrene, and dibenz[a,h]anthracene are not photomutagenic. These results indicate that PAHs can be activated by light and become mutagenic in Salmonella TA102 bacteria. At the same time, the mutagenicity for all the 16 PAHs was examined with the standard mutagenicity test with 10% S9 as the activation system. Benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene, acenaphthylene, and fluorene are weakly mutagenic, while the rest of the PAHs are not. In general, the photomutagenicity of PAHs in TA102 does not correlate with their S9-activated mutagenicity in either TA102 or TA98/TA100 since they involve different activation mechanisms.     

Structural activity relationship between Salmonella-mutagenicity and nitro-orientation of nitroazaphenanthrenes

Nitroazaphenanthrenes (NAphs) and their N-oxides (NAphOs) were synthesized as derivatives with nitrogen atoms in the 1, 4, and 9 positions of phenanthrene rings, and as nitrated derivatives substituted at the 1, 2, 3, 4, 5, 6, 7, and 8 positions of phenanthrene rings. To determine the structure activity relationship of these derivatives, all 19 isomers were bioassayed with Salmonella tester strains. NAphs substituted at the 4, 6, 7 and 8 positions were mutagenic for TA98, and 1-, 2-, and 3-N-9-AphOs, 6-N-1-AphO and 6-N-4-AphO were mutagenic for TA98 and TA100 without the S9 mix, while 5-N-1-AphO and 5-N-9-AphO were non- or weakly mutagenic. Nitrated derivatives, 6-N-4-Aph, 6-N-9-Aph, 6-N-1-AphO, and 6-N-4-AphO, were powerful mutagens for TA98 and TA100. Mutagenicity was enhanced by mutant strains producing nitroreductase, such as YG1021 and 1026, and by those producing O-acetyltransferase, such as YG1024 and 1029. Nitro derivatives substituted at positions 4 and 5 in the phenanthrene rings were perpendicular, while those at positions 2, 3, 6 and 7 were coplanar to the phenanthrene rings. NAphs substituted at the 1 and 8 positions were noncoplanar due to steric hindrance of the aromatic proton at the peri position. On the other hand, 1,5- and 1,8-dinitro-4-azaphenanthrenes showed high mutagenicity for strains TA98 and TA100 in the absence of the S9 mix, and were strongly enhanced by nitroreductase and O-acetyltransferase, over-producing mutants. Therefore, it was found that the mutagenic potency of NAphs and NAphOs was closely associated with the chemical properties and orientation of nitro substitution of aromatic rings.     

Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium

The nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 tested nitrophenazines, 4 were mutagenic in the absence of a microsomal metabolic activation system (S9 mix) and were more mutagenic in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1.7- less than 2- less than 2.8- less than 2.7-substituted phenazine. Of 7 tested amino derivatives, 4 exhibited mutagenic activity with S9 mix in TA98. 1-Nitro-, 1-amino, 1.6-dinitro-, 1.9-dinitro-, 1.6-diamino- and 1.9-diamino-phenazine were not mutagenic. As regards the relationship between mutagenic potency and chemical structure of the phenazines, the results suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 and/or 7 position. Furthermore, 2.7-disubstituted phenazines were extremely mutagenic, 2.7-dinitrophenazine and 2.7-diaminophenazine induced 36,450 and 12,110 rev./nmole, respectively. In the preliminary study, 2.7-diaminophenazine was identified by gas chromatography/mass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide.     

Biological activities of organic compounds adsorbed onto ambient air particles: comparison between the cities of Teplice and Prague during the summer and winter seasons 2000-2001

The capital of the Czech Republic, Prague, appears today to be one of the most polluted residential areas in the country, whereas air pollution in the Northern Bohemia region (the former "Black Triangle Region") has substantially decreased during the last decade, especially with respect to the gaseous pollutant SO(2). This study evaluated the biological activities of complex mixtures of organic compounds adsorbed onto ambient air particles (PM10) collected during the summer and winter seasons of 2000-2001 at three monitoring sites--Teplice (TP), Prague-Smíchov (PRG-SM) (city centre) and Prague-Libus (PRG-LB) (suburban area). The following short-term in vitro assays with strikingly different endpoints were used: a bacterial mutagenicity test using the Salmonella typhimurium tester strain TA98 and YG1041, an acellular assay (CT DNA) combined with 32P-postlabelling to evaluate DNA adduct-forming potency and the chick embryotoxicity screening test (CHEST). The results of the mutagenicity test with the YG1041 strain, the acellular genotoxicity (DNA adducts) and the embryotoxicity tests responded to the amount of eight carcinogenic polycyclic aromatic hydrocarbons (PAHs) analysed in the EOM (dichloromethane extractable organic matter) samples tested. Nevertheless, the biological effects of the EOM did not differ between locations. The highest biological activity of the ambient air in terms of organic compounds associated with particles (per unit volume of air) was seen in the Prague city centre during both summer and winter seasons. At this location, B[a]P concentration ranged from 0.1 to 8.9 ng/m(3) (mean 0.3 and 3.6 ng/m(3) for summer and winter seasons, respectively), 13 PAHs ranged from 11 to 343 ng/m(3) (mean 52 and 160 ng/m(3) for summer and winter seasons, respectively). Generally, using in vitro tests, higher ambient air activity was found in the winter season as compared with the summer season at all three monitoring sites (TA98 +S9, approximately 4-fold; YG1041 -S9, approximately 5-fold; YG1041 +S9, approximately 8-fold; CT DNA +S9, approximately 10-fold; CHEST, approximately 10-fold; B[a]P, carcinogenic PAHs and total PAHs analysed, more than 10-fold). The different proportions of individual PAHs found in the summer and winter samples suggested traffic as a major emission source in the summer and, additionally, residential heating in the winter season at all three monitoring sites. The DNA adduct patterns resulting from the in vitro acellular assay also demonstrated similar major emission sources at all three locations. The study shows that particle-bound carcinogenic-PAH concentrations may be taken as an index for the biologically active (mutagenic, genotoxic, embryotoxic) components in air particulate samples. Therefore, high-quality monitoring data of carcinogenic PAHs may be useful for epidemiological studies of the impact of air pollution on the health of the population and for helping decision makers to improve our environment.     

The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay

To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks. Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100). In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity. No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected. In another series, soils sampled at the roadside and at distances of 10 and 50m of five roads near Mainz expressed 10-20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils. Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated. Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides. The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, approximately 4% to fractions designed polar neutrals, approximately 8% to polar aromatics, approximately 7% to dichloromethane solubles, and approximately 79% to cylohexane solubles, among them approximately 63% acetone soluble compounds. The major part of mutagenicity (55-65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles ( approximately 10% each) summarizing the results obtained with S. typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9. The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity. With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz[a,h]anthracene (4.18mg/kg soil), benzo[a]pyrene (1.96mg), benzofluoranthenes (0.14mg), and benz[a]anthracene (0. 18mg) were present in the acetone subfraction of cyclohexane solubles. Concentrations and mutagenic activities, however, did not correlate. Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene. Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5-15cm depth and a subsequent decrease with very little activity remaining deeper than 35cm. In human lymphocyte cultures, the fraction of polar aromatics, 0.01-0. 3microg/ml, induced 11.27+/-4.76-20.70+/-6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10. 16+/-4.83 SCE per cell) and 12.77+/-6.53-17.87+/-4.93 SCE per cell in the presence of S9 (solvent control: 8.37+/-3.92 SCE per cell). However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals. Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000mg/kg p.o. with all fractions.     

Role of bacterial nitroreductase and O-acetyltransferase in urine mutagenicity assay of rats exposed to 2,4,6-trinitrotoluene (TNT)

The urine mutagenicity of rats exposed to 2,4,6-trinitrotoluene (TNT) by i.p. injection was studied in the Salmonella assay using indicator strains with various levels of 'classical' nitroreductase or acetyl-CoA:N-hydroxylarylamine O-acetyltransferase activity. The strains used were the conventional Salmonella typhimurium TA98, nitroreductase-deficient TA98NR and -overproducing YG1021, and O-acetyltransferase-deficient TA98/1,8-DNP6 and -overproducing YG1024. TA98, YG1021 and YG1024 clearly detected the increase of direct urine mutagenicity. A slight increase of mutagenicity was also detected with metabolic activation in YG1021 and YG1024. High levels of both nitroreductase and O-acetyltransferase significantly increased the sensitivity of the indicator strain to the mutagenicity of urine caused by TNT exposure, while the nitroreductase- or O-acetyltransferase-deficient strains gave negative responses.