The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity

Mutagens in urban air pollution come from anthropogenic sources (especially combustion sources) and are products of airborne chemical reactions. Bacterial mutation tests have been used for large, multi-site, and/or time series studies, for bioassay-directed fractionation studies, for identifying the presence of specific classes of mutagens, and for doing site- or source-comparisons for relative levels of airborne mutagens. Early research recognized that although carcinogenic PAHs were present in air samples they could not account for the majority of the mutagenic activity detected. The mutagenicity of airborne particulate organics is due to at least 500 identified compounds from varying chemical classes. Bioassay-directed fractionation studies for identifying toxicants are difficult to compare because they do not identify all of the mutagens present, and both the analytical and bioassay protocols vary from study to study. However, these studies show that the majority of mutagenicity is usually associated with moderately polar/highly polar classes of compounds that tend to contain nitroaromatic compounds, aromatic amines, and aromatic ketones. Smog chamber studies have shown that mutagenic aliphatic and aromatic nitrogen-containing compounds are produced in the atmosphere when organic compounds (even non-mutagenic compounds) are exposed to nitrogen oxides and sunlight. Reactions that occur in the atmosphere, therefore, can have a profound effect on the genotoxic burden of ambient air. This review illustrates that the mutagenesis protocol and tester strains should be selected based on the design and purpose of the study and that the correlation with animal cancer bioassay results depends upon chemical class. Future emphasis needs to be placed on volatile and semi-volatile genotoxicants, and on multi-national studies that identify, quantify, and apportion mutagenicity. Initial efforts at replacing the Salmonella assay for ambient air studies with some emerging technology should be initiated.     

Mutagenicity of airborne particles from four cities in Taiwan.

The mutagenicity of airborne particles from 8 urban and suburban locations in each of four cities, Taipei, Hsinchu, Taichung, and Kaohsiung, in Taiwan area were investigated with S. typhimurium strain TA98 by Ames Salmonella/microsomal test. The average mutagenic activity of airborne particulate samples from Taipei and Kaohsiung was higher than that from Hsinchu and Taichung with or without metabolic activation. The major direct-acting mutagenic compounds of airborne particulate samples from Taipei and Kaohsiung was similar to that of standard dinitropyrenes mixture (DNPs) in the retention time of HPLC. Moreover, the contents of DNPs of airborne particulate samples from Taipei and automobile exhaust partially purified through Sephadex LH-20 gel filtration and semipreparative HPLC were determined by HPLC. DNPs was major direct-acting mutagens of the urban air samples from Taipei and their major pollutants might be from automobile exhaust. However, the major mutagenic compounds of airborne particulate samples from Hsinchu and Taichung did not correspond to any of the standard compounds tested. The content of benzo[a]pyrene (B[a]P) of airborne particulate samples was also determined by HPLC. The concentration of B[a] P was 0.05-0.62 ng/m3 air sample. The B[a] P contents of airborne particulate samples from four cities in Taiwan did not show good correlation with their mutagenic activity. Thus, we concluded that B[a] P was not a major indirect-acting mutagenic compound in the tested air samples.     

Mutagenicity of 43 structurally related heterocyclic compounds and its relationship to their carcinogenicity.

43 heteropolycyclic compounds belonging to a homologous series were investigated for mutagenicity. The results are compared with carcinogenicity data obtained with the same batches of compounds under conditions identical for all of them. Mutagenicity was tested in the Ames test with Salmonella typhimurium strains TA1535, TA1537 and TA100 in the presence and absence of liver 10 000 g supernatant from rats treated with Aroclor 1254. Carcinogenicity was tested by injection of the compounds into subcutaneous tissue of XVIInc/Z mice. 18 test compounds showed carcinogenic activity, some strongly, others only weakly. Of these, 17 were detected as mutagens: one weak carcinogen did not revert the Salmonella strains. No quantitative correlation was observed between the extents of the mutagenic and the carcinogenic effects. Of the 25 substances that did not produce tumours, 13 showed mutagenicity (12 in the presence, 2 in the absence, of the liver homogenate). The mutagenic effects of these compounds were quantitatively similar to those of the compounds that produced tumours. The most sensitive strain of Salmonella typhimurium was TA100. It detected all 30 mutagens. TA98 was mutated by 25 compounds, TA1537 by 16 compounds. No mutagenic effects were seen with TA1535. Possible reasons for the high percentage of apparently "false positives" in the Ames test and the lack of a quantitative correlation between the potency of the mutagenic and carcinogenic effects are discussed. It is suggested that the complexity of the metabolism of these heterocyclic compounds may lead to critical differences in metabolism in mouse subcutaneous tissue in vivo and in liver homogenates from rats treated with Aroclor. Therefore the present study will be extended to life-long oral and intrahepatic carcinogenicity tests leading to a higher proportion of metabolism in the liver.     

Relationships between structures and mutagenic potencies of 16 heterocyclic nitrogen mustards (ICR compounds) in Salmonella typhimurium

16 heterocyclic nitrogen mustards (ICR compounds), which were synthesized for use as possible antitumor agents by Creech and coworkers, were tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1536, TA1537, TA1538, TA98 and TA100. The compounds were incorporated into the top agar at 5 doses: 0.5, 1, 2.5, 5 and 10 micrograms/plate. All of the compounds were negative in TA1535 except ICR 449, which was positive in all 6 strains. The other 15 compounds were positive in the remaining strains with the following exceptions: ICR 371 and 355 were negative in TA100; ICR 445 was negative in TA98 and TA100; and ICR 360 was negative in TA1537, TA1538, TA98 and TA100. Good qualitative agreement was observed between the mutagenic and antitumor activities of the 16 compounds, and between the mutagenic and carcinogenic activities of the 5 compounds that have been tested for carcinogenicity by Peck and coworkers. However, no significant correlation was found between mutagenic potency in Salmonella and antitumor potency in mice for the 16 compounds. Also, for the 5 compounds that have been tested for carcinogenicity, no significant correlation was found between their mutagenic potency in Salmonella and their carcinogenic potency in mice. In Salmonella, the secondary (2 degrees) amines generally were more mutagenic than their tertiary (3 degrees) amine homologs, although the opposite result has been reported in certain eukaryotes. Relationships between structures and potencies for the different nuclei of the 16 ICR compounds are discussed, as are similarities and differences in strain sensitivities. We conclude that the Salmonella his reversion test is not a good predictor of the antitumor and carcinogenic potencies of these ICR compounds.     

Mutagenic and carcinogenic potential of a textile azo dye processing plant effluent that impacts a drinking water source

Recently a textile azo dye processing plant effluent was identified as one of the sources of mutagenic activity detected in the Cristais River, a drinking water source in Brazil [G.A. Umbuzeiro, D.A. Roubicek, C.M. Rech, M.I.Z. Sato, L.D. Claxton, Investigating the sources of the mutagenic activity found in a river using the Salmonella assay and different water extraction procedures, Chemosphere 54 (2004) 1589-1597]. Besides presenting high mutagenic activity in the Salmonella/microsome assay, the mutagenic nitro-aminoazobenzenes dyes CI Disperse Blue 373, CI Disperse Violet 93, and CI Disperse Orange 37 [G.A. Umbuzeiro, H.S. Freeman, S.H. Warren, D.P. Oliveira, Y. Terao, T. Watanabe, L.D. Claxton, The contribution of azo dyes in the mutagenic activity of the Cristais river, Chemosphere 60 (2005) 55-64] as well as benzidine, a known carcinogenic compound [T.M. Mazzo, A.A. Saczk, G.A. Umbuzeiro, M.V.B. Zanoni, Analysis of aromatic amines in surface waters receiving wastewater from textile industry by liquid chromatographic with eletrochemical detection, Anal. Lett., in press] were found in this effluent. After approximately 6 km from the discharge of this effluent, a drinking water treatment plant treats and distributes the water to a population of approximate 60,000. As shown previously, the mutagens in the DWTP intake water are not completely removed by the treatment. The water used for human consumption presented mutagenic activity related to nitro-aromatics and aromatic amines compounds probably derived from the cited textile processing plant effluent discharge [G.A. Umbuzeiro, D.A. Roubicek, C.M. Rech, M.I.Z. Sato, L.D. Claxton, Investigating the sources of the mutagenic activity found in a river using the Salmonella assay and different water extraction procedures, Chemosphere 54 (2004) 1589-1597; G.A. Umbuzeiro, H.S. Freeman, S.H. Warren, D.P. Oliveira, Y. Terao, T. Watanabe, L.D. Claxton, The contribution of azo dyes in the mutagenic activity of the Cristais river, Chemosphere 60 (2005) 55-64]. Therefore, it is important to evaluate the possible risks involved in the human consumption of this contaminated water. With that objective, one sample of the cited industrial effluent was tested for carcinogenicity in the aberrant crypt foci medium-term assay in colon of Wistar rats. The rats received the effluent in natura through drinking water at concentrations of 0.1%, 1%, and 10%. The effluent mutagenicity was also confirmed in the Salmonella/microsome assay with the strains TA98 and YG1041. There was an increased number of preneoplastic lesions in the colon of rats exposed to concentrations of 1% and 10% of the effluent, and a positive response for both Salmonella strains tested. These results indicate that the discharge of the effluent should be avoided in waters used for human consumption and show the sensitivity of the ACF crypt foci assay as an important tool to evaluate the carcinogenic potential of environmental complex mixtures.     

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.     

Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment

Aromatic amines represent one of the most important classes of industrial and environmental chemicals: many of them have been reported to be powerful carcinogens and mutagens, and/or hemotoxicants. Their toxicity has been studied also with quantitative structure-activity relationship (QSAR) methods: these studies are potentially suitable for investigating mechanisms of action and for estimating the toxicity of compounds lacking experimental determinations. In this paper, we first summarized the QSAR models for the rodent carcinogenicity of the aromatic amines. The gradation of potency of the carcinogenic amines depended firstly on their hydrophobicity, and secondly on electronic (reactivity, propensity to be metabolically transformed) and steric properties. On the contrary, the difference between carcinogenic and non-carcinogenic aromatic amines depended mainly on electronic and steric properties. These QSARs can be used directly for estimating the carcinogenicity of aromatic amines. A two-step prediction is possible: (1) estimation of yes/no activity; (2) if the answer from step 1 is yes, then prediction of the degree of potency. The QSARs for rodent carcinogenicity were put in a wider context by comparing them with those for: (a) Salmonella mutagenicity; (b) general toxicity; (c) enzymatic reactions; (d) physical-chemical reactions. This comparative QSAR exercise generated a coherent global picture of the action mechanisms of the aromatic amines. The QSARs for carcinogenicity were similar to those for Salmonella mutagenicity, thus pointing to a similar mechanism of action. On the contrary, the general toxicity QSARs (both in vitro and in vivo systems) were mostly based on hydrophobicity, pointing to an aspecific mechanism of action much simpler than that for carcinogenicity and mutagenicity. The oxidation of the amines (first step in the main metabolic pathway leading to carcinogenic and mutagenic species) had identical QSARs in both enzymatic and physical-chemical systems, thus providing evidence for the link between simple chemical reactions and those in biological systems. The results show that it is possible to generate mechanistically and statistically sound QSAR models for rodent carcinogenicity, and indirectly that the rodent bioassay is a reliable source of good quality data.     

In vitro and in vivo mutagenicity studies with airborne particulate extracts

The contribution of nitro compounds to airborne particulate mutagenicity was studied with Salmonella typhimurium strains TA98, TA98NR, TA98/1,8DNP6. The results obtained indicate that nitropyrenes play a minor role in air particulate mutagenicity. Seasonal variations indicate a relatively greater contribution of nitro compounds to the mutagenicity of spring and summer samples. Fractionation of extracts into acidic, neutral and basic components shows that neutral compounds account for about two-thirds of the total mutagenic activity. Attempts to extract mutagens adsorbed onto particulate matter with aqueous media were almost completely negative. No significant mutagenicity was detected in urine and faecal extracts and in plasma samples of Sprague-Dawley rats treated with air particulate extracts at 80 mg/kg either per os or by i.p. injection. Negative results were obtained in the micronucleus test with Swiss mice treated at 200 and 400 mg/kg (twice by i.p. injection). A significant decrease in liver aminopyrine-N-demethylase was observed in Swiss mice injected with air particulate extracts or its basic and neutral fractions. In vitro experiments suggest a direct interaction of test materials with microsomal cytochrome P-450.     

Detection of oxidative mutagens in an urban air-particulate extract: a preliminary study.

The Ames assays strains TA98 and TA100 have been useful in characterizing complex mixtures from organic solvent extracts of particles from diesel-powered vehicles, ambient air, and other sources. In this paper we report preliminary experiments using TA102, a bacterial strain that detects compounds that can oxidize DNA, to characterize the mutagenicity of an ambient air sample collected in Ann Arbor, MI. Four sets of ambient air filters were collected in duplicate over a period of several days. The mutagenicities of methylene chloride extracts of these filters were compared using strains TA98, TA100 and TA102. The concentration-mutagenicity data for TA98 and TA100 were linear over the concentration range 0-200 micrograms extract/plate. The mutagenicity of the extracts using TA102 was much lower than the other two strains and was non-linear over the concentration range tested. These results suggest that it would be difficult to use TA102 to identify the oxidative mutagens present in an ambient air particulate extract.     

Evidence for the presence of mutagenic compounds other than chromium in particles from mild steel welding

A modified Salmonella/microsome liquid culture assay was used to investigate the mutagenicity of the particulate fraction from mild steel welding. Previous reports have implicated compounds of chromium VI as the mutagenic and toxic agents in welding fumes, since only the particles from welding on stainless steel, which contains 15-25% chromium, were mutagenic, whereas particles from welding on mild steel, which contain less than 0.1% chromium, were not mutagenic or toxic. In this investigation, mild steel particles were shown to contain direct-acting and promutagenic compounds that induced frameshift mutations. The mutagenic agents, which were insoluble in sodium phosphate buffer, did not include chromium VI or organic compounds. Further, the expression of mutation appears to require a cell-particle interaction for the release of the mutagenic species from the particles.     

Comparative evaluation of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains for rapid detection of chemical mutagens and carcinogens

The aim of the present study was to evaluate the usefulness of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains in a mutagenicity/carcinogenicity screen, possibly as complements to the Ames test. 70 carcinogenic and non-carcinogenic compounds, representing a variety of chemical structures, were tested for their DNA-damaging effects, using 6 different DNA-repair-deficient bacterial strains. 2 Bacillus subtilis systems, H17/M45 and HLL3g/HJ-15, were used. The susceptibility of Escherichia coli AB1157 was compared with the susceptibility of 4 recombination-deficient mutants, JC5547, JC2921, JC2926 and JC5519. The test compounds were applied onto paper disks (spot test, ST), or incorporated into a top agar layer (agar-incorporation test, AT). The 2 B. subtilis systems were generally found to be more sensitive and reliable than the assays using E coli. The incorporation of the test compounds in the agar increased the sensitivity of the test for polycyclic aromatic hydrocarbons and other poorly water-soluble compounds. Hydrazines and several other highly polar chemicals could be tested more efficiently when applied onto paper disks. About 30% of the test compounds did not induce any growth inhibition and so could not be tested properly. In order to evaluate the ability of these DNA-repair tests to complement the Ames Salmonella mutagenicity test in a genetic toxicology screening program, results from this study were compared with published data both on mutagenicity in the Ames test and on carcinogenicity. 8 carcinogens generally found to be non-mutagenic for Salmonella were tested: 2 showed DNA-damaging properties (mitomycin C, 1,2-dimethylhydrazine), 5 failed to do so (actinomycin D, griseofulvin, thioacetamide, diethylstilbestrol, safrole), and one (thiourea) was not toxic, so that no classification was possible. 2 non-carcinogenic bacterial mutagens were examined; one, sodium azide, was equitoxic for repair-proficient and -deficient strains, while the other, nitrofurantoin, primarily inhibited repair-deficient strains. The DNA-repair tests failed to indicate the mutagenic and carcinogenic properties of acridine orange. Nalidixic acid, a non-mutagenic DNA synthesis inhibitor, damaged bacterial DNA. Apart from the differences summarized above, carcinogenicity was indicated correctly by the Salmonella S9 assay and most sets of DNA-repair-deficient and DNA-repair-proficient tester strains evaluated in this study. Thus, several more carcinogens could be detected by performing the Ames test and the bacterial DNA-repair tests in tandem than by using either test alone. Nevertheless, the use of both bacterial in vitro systems in a battery of short-term tests for mutagenicity/carcinogenicity evaluation is not considered to be ideal, since the Ames test and the pairs of DNA-repair-deficient and DNA-repair-proficient tester strains used had several shortcomings in common under the conditions of this study.     

Mutagenic activity as a parameter to assess ambient air quality for protection of the environment and human health

Atmospheric pollution has significant effects on maintaining the integrity of ecosystems and on the population's quality of life. Epidemiological studies have clearly associated related health problems, especially respiratory diseases, with exposure to air pollution. Organic compounds adsorbed to the airborne particulate matter are mutagenic in the Salmonella/microsome assay, and a considerable number of them are known to be carcinogenic to rodents. Studies performed at four sites within the urban area of Porto Alegre, capital of the state of Rio Grande do Sul, identified higher mutagenic activity at the sites with heavier vehicle traffic in assays without and with metabolic activation. The responses varied at different seasons of the year, and the highest revertants per cubic meter (rev/m(3)) values were observed in spring for moderately polar compounds, and in summer for non-polar ones. A pilot study was also performed in the region under the influence of a industrial petrochemical area. Most of the sites studied within the industrial area, as compared to others sampled in the nearby environment, presented higher levels of mutagenic activity independent of total suspended particulates (TSP) concentration in the sample. In the urban and industrial regions, the observed mutagenic activities were strongly associated with the presence of polycyclic aromatic hydrocarbons (PAHs). The responses observed in the TA98NR and TA98/1,8-DNP(6) strains suggest the activity of nitrocompounds in both studies. The Salmonella/microsome assay is a sensitive method to define areas contaminated by these compounds, even in samples with TSP values that are consistent with the legal environmental quality standards.     

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.     

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.     

Air pollution combustion emissions: characterization of causative agents and mechanisms associated with cancer, reproductive, and cardiovascular effects

Combustion emissions account for over half of the fine particle (PM(2.5)) air pollution and most of the primary particulate organic matter. Human exposure to combustion emissions including the associated airborne fine particles and mutagenic and carcinogenic constituents (e.g., polycyclic aromatic compounds (PAC), nitro-PAC) have been studied in populations in Europe, America, Asia, and increasingly in third-world counties. Bioassay-directed fractionation studies of particulate organic air pollution have identified mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAH), nitrated PAH, nitro-lactones, and lower molecular weight compounds from cooking. A number of these components are significant sources of human exposure to mutagenic and carcinogenic chemicals that may also cause oxidative and DNA damage that can lead to reproductive and cardiovascular effects. Chemical and physical tracers have been used to apportion outdoor and indoor and personal exposures to airborne particles between various combustion emissions and other sources. These sources include vehicles (e.g., diesel and gasoline vehicles), heating and power sources (e.g., including coal, oil, and biomass), indoor sources (e.g., cooking, heating, and tobacco smoke), as well as secondary organic aerosols and pollutants derived from long-range transport. Biomarkers of exposure, dose and susceptibility have been measured in populations exposed to air pollution combustion emissions. Biomarkers have included metabolic genotype, DNA adducts, PAH metabolites, and urinary mutagenic activity. A number of studies have shown a significant correlation of exposure to PM(2.5) with these biomarkers. In addition, stratification by genotype increased this correlation. New multivariate receptor models, recently used to determine the sources of ambient particles, are now being explored in the analysis of human exposure and biomarker data. Human studies of both short- and long-term exposures to combustion emissions and ambient fine particulate air pollution have been associated with measures of genetic damage. Long-term epidemiologic studies have reported an increased risk of all causes of mortality, cardiopulmonary mortality, and lung cancer mortality associated with increasing exposures to air pollution. Adverse reproductive effects (e.g., risk for low birth weight) have also recently been reported in Eastern Europe and North America. Although there is substantial evidence that PAH or substituted PAH may be causative agents in cancer and reproductive effects, an increasing number of studies investigating cardiopulmonary and cardiovascular effects are investigating these and other potential causative agents from air pollution combustion sources.     

Mutagenic activity of airborne particulate matter as an indicative measure of atmospheric pollution

Mutagenic activity of organic extracts of airborne particulate matter at four different sites within the urban area of the city of Porto Alegre, Brazil, was investigated using the Salmonella/microsome assay, with the Kado microsuspension method. The extracts were obtained by sonication, sequentially extracted according to polarity, with cyclohexane (CX) and dichloromethane (DCM) solvents. The different fractions were tested for mutagenicity with the Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, without S9 mix metabolic activation. A positive frameshift mutagenic response was observed for non-polar (CX) and/or moderately polar (DCM) compounds at the different sites. The responses varied at different seasons of the year, and the highest revertants per m3 (rev/m3) values were observed at the site subject to the strongest influence of automotive vehicles (site 3) in spring (17.13 rev/m3) in DCM fractions, and in summer (13.01 rev/m3) in CX fractions. The responses observed for the TA98NR and TA98/1,8-DNP6 strains suggest the contribution of nitrocompounds to the mutagenic activity observed. Although there appears to be an indicative association between the increased mass per unit volume of air (TSP) and the mutagenicity of organic extracts of airborne particulate matter in the present study, the Salmonella/microsome assay was a sensitive method to define areas contaminated by genotoxic compounds, even in samples that present TPS values acceptable by the environmental quality standards established by law.     

Involvement of nitro-compounds in the mutagenicity of urban Pm2.5 and Pm10 in Turin

Fine particles can be active carriers of toxic compounds into the alveoli of the lungs. Among these compounds are numerous mutagens and carcinogens. The direct mutagenicity per unit mass of fine particulate matter (PM) is significantly higher than that of coarse particles, especially in urban areas. In this study, the mutagenic properties of urban PM2.5 and PM10 were evaluated, and the role of nitro-compounds was estimated. PM2.5 and PM10 samplings, and measurements of NOx and some PAHs were performed daily in 2007 in Turin, following a consolidated in vitro test - the Salmonella mutagenicity assay - conducted with organic extracts of PM2.5 and PM10. The mutagenic properties were assessed for each month of sampling with Salmonella typhimurium strain TA98 and TA98-derived strains: a nitroreductase-deficient mutant strain (TA98NR) and an additional nitroreductase-producing plasmid strain (YG1021). The annual measured mean levels of PM2.5 and PM10 were 34±20 and 48±18μg/m(3). The PM2.5/PM10 ratio ranged from 0.36 to 0.89. The Salmonella assay showed higher mutagenicity in autumn/winter (20±15 TA98NR; 54±39 TA98; 173±161 YG1021 net revertants/m(3)) compared with spring/summer (2±2 TA98NR; 7±8 TA98; 24±27 YG1021 net revertants/m(3)) (p<0.01). There are also statistically significant seasonal differences in the gravimetric analysis data. The number of TA98 net revertants per μg of PM2.5 is 6.5 times greater than per μg PM10. Moreover, the bioassay results showed an amplified response in the YG1021 strain and a reduced response in the TA98NR strain. The net revertant ratio TA98NR/YG1021 is 11±4 for organic extracts of PM2.5 and 13±6 for extracts of PM10 (p<0.01). There is a significant correlation between the NOx and PAH concentrations. These findings illustrate the relevant role of nitro compounds, and they underline the priority in improving preventive measures to reduce air pollution by nitrated molecules.     

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.     

Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents

Alkylating agents, because of their ability to react directly with DNA either in vitro or in vivo, or following metabolic activation as in the case of the dialkylnitrosamines, have been used extensively in studying the mechanisms of mutagenicity and carcinogenicity. Their occurrence is widespread in the environment and human exposure from natural and pollutant sources is universal. Since most of these chemicals show varying degrees of both carcinogenicity and mutagenicity, and exhibit compound-specific binding patterns, they provide an excellent model for studying molecular dosimetry. Molecular dosimetry defines dose as the number of adducts bound per macromolecule and relates the binding of these adducts to the human mutagenic or carcinogenic response. This review complies DNA alkylation data for both methylating and ethylating agents in a variety of systems and discusses the role these alkylation products plays in molecular mutagenesis.     

Oxidative stress-induced DNA damage by particulate air pollution

Exposure to ambient air particulate matter (PM) is associated with pulmonary and cardiovascular diseases and cancer. The mechanisms of PM-induced health effects are believed to involve inflammation and oxidative stress. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from the surface of particles, soluble compounds such as transition metals or organic compounds, altered function of mitochondria or NADPH-oxidase, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species. Resulting oxidative DNA damage may be implicated in cancer risk and may serve as marker for oxidative stress relevant for other ailments caused by particulate air pollution. There is overwhelming evidence from animal experimental models, cell culture experiments, and cell free systems that exposure to diesel exhaust and diesel exhaust particles causes oxidative DNA damage. Similarly, various preparations of ambient air PM induce oxidative DNA damage in in vitro systems, whereas in vivo studies are scarce. Studies with various model/surrogate particle preparations, such as carbon black, suggest that the surface area is the most important determinant of effect for ultrafine particles (diameter less than 100 nm), whereas chemical composition may be more important for larger particles. The knowledge concerning mechanisms of action of PM has prompted the use of markers of oxidative stress and DNA damage for human biomonitoring in relation to ambient air. By means of personal monitoring and biomarkers a few studies have attempted to characterize individual exposure, explore mechanisms and identify significant sources to size fractions of ambient air PM with respect to relevant biological effects. In these studies guanine oxidation in DNA has been correlated with exposure to PM(2.5) and ultrafine particles outdoor and indoor. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. Related biomarkers and personal monitoring may be useful tools for risk characterization.