Mutagenicity and chemical analysis of sequential organic extracts of airborne particulates

To obtain insight into the identity of chemicals associated with the mutagenicity of United States National Institute of Standards and Technology (NIST) Standard Reference Materials SRM 1649 (urban dust) and SRM 1650 (diesel particulate), parallel mutagenicity tests and chemical analyses were performed on dichloromethane and sequential organic extracts of these samples. SRM 1649 and 1650 were sequentially extracted with five organic solvents of increasing polarity, in order to partition mutagenic components into discrete fractions. The solvents (with associated polarity index) were as follows: (1) hexane (0.0); (2) hexane:diethyl ether 9:1 (0.29); (3) hexane:diethyl ether 1:1 (1.45); (4) diethyl ether (2.9); (5) methanol (6.6). 0.9270 g of SRM 1649, and 0.0510 g of SRM 1650 were each extracted three times with 8 ml of each of the solvents, the three aliquots were pooled, and analysed for target organics or solvent-exchanged into DMSO for mutagenicity testing in Salmonella typhimurium strains TA98 and TA100.The dichloromethane extracts of SRM 1649 and SRM 1650 contained direct-actin mutagens in Salmonella strains TA98 and TA100; SRM 1650 was significantly more potent than SRM 1649 in either strain. Addition of S9 caused a large decrease in mutagenicity of each extract, although SRM 1650 remained more potent. An interesting pattern of mutagenicity was observed for the sequential extracts of SRM 1649 and SRM 1650: the mutagenic potency of SRM 1649 extracts increased with increasing polarity of the extraction solvent while the response of the SRM 1650 extracts was the opposite. This suggests that the direct-acting mutagens in SRM 1650 are unlike those in SRM 1649. The response, though diminished, was largely unchanged when S9 was included in the test mixture.Chemical analyses on the various extracts were performed using a Hewlett-Packard model 5890 gas chromatograph equipped with a model 5970B mass selective detector (GC-MSD), and a 0.3 μm film thickness cross-linked methyl silicone capillary column (HP 1909A-101). Selected ion monitoring (SIM) methods were used to analyze for 105 target compounds including PAHs and nitro-PAHs. Chemical analysis of the dichloromethane extracts of SRM 1649 and SRM 1650 identified three main classes of compounds: polyaromatic hydrocarbons (PAH), vitro-polyaromatic hydrocarbons (NO₂-PAHs) and heterocyclics. The concentration of target compounds and the proportion of vitro-PAHs and heterocyclic compounds were considerably greater in SRM 1650 than in SRM 1649, consistent with the observed differences in their mutagenic potency. However, the different responses of the dichloromethane extracts in TA98 and TA100 suggest the presence of different (unidentified) compounds.Many of the target compounds were detected at least once in the sequential extracts from SRM 1649 and SRM 1650. There was no evident relationship between the occurrence of extracted organics, or classes of organics, and the polarity of solvents, except that, generally, the largest amount and variety of compounds were recovered in the first and second extracts (hexane; hexane:diethyl ether, 9:1). Preliminary examination of the chemical analysis results did not provide an explanation of the observed trends in mutagenic response. No single class of chemicals or individual compound was found to account for the observed pattern of mutagenicity. Compounds other than those identified must also contribute to the observed mutagenicity of any of the SRM 1649 and SRM 1650 extracts.     

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.     

Mutagenic and chemical assay of extracts of airborne particulates

The mutagenic activity of extracts of airborne particulates was evaluated in the Salmonella system. The mutagenicity of airborne particulates was not always correlated with the content of benzo[a]pyrene (B[a]P) in the complex mixtures, especially when the samples were collected at different sites.Large-scale fractionation of extracts of airborne particulates was used to determine the content of specific mutagenic chemicals. The neutral fraction of material soluble in cyclohexane and nitromethane contained the polycyclic aromatic hydrocarbon (PAH) compounds, which accounted for 27.9% of the mutagenic activity of the whole extracts. 9 kinds of PAH compound were identified quantitatively by thin-layer chromatography. They included, per 1000 m³ of air, 12.6 μg of benzo[e]pyrene (B[e]P), 10.7 μg of chrysene (CHRY), 10.0 μg of fluoranthene (FL), 6.43 μg of benzo[ghi]perylene (B[ghi]P), 5.75 μg of benz[a]anthracene (B[a]A), 5.33 μg of B[a]P, 3.38 μg of pyrene (PYR), 1.83 μg of coronene (COR), and 1.34 μg of perylene (PERY).Mutagenicity of the ether-soluble acidic, basic and methanol-soluble neutral fractions accounted for 10.9, 9.71 and 6.78% of the total mutagenic activity of crude extract, respectively, when assayed in strain TA98 with liver S9 fraction. The total recovery of mutagenic activity after fractionation was 58%.Two acidic fractions (weak and strong ether-soluble acids) and the methanol-soluble neutral fraction reverted strain TA98 dramatically to prototrophy in the presence of rat-lung S9 fraction more than liver. But the mutagenic chemicals in these fractions remain to be clarified. Direct mutagens were present in essentially all fractions.The particulates, which had diameters ranging from 0.3 to 1.0 μm and were able to penetrate alveoli, contained a high content of mutagens.     

Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts.

The mutagenicity spectra of the organic extracts of both airborne particulate matter and diesel and gasoline soot particles were determined using a battery of 9 bacterial strains of different genetic specificity. The assays with crude extracts and with fractionated acidic, neutral and basic components revealed striking differences in the patterns of mutagenic responses produced by each of the complex mixtures investigated. The mutagenicity of air particulate matter was shown to depend mainly on direct-acting acidic and neutral compounds, with a lesser contribution of basic promutagens which required exogenous metabolic activation by liver S9. The assays with a diesel soot extract indicated the prevailing contribution of direct-acting acidic and neutral compounds, and suggested an important role also for nitro derivatives other than nitropyrenes. The gasoline exhaust was characterized by powerful promutagenic compounds, belonging to either the acidic, neutral or basic fractions. The implications of these results are discussed with respect to the contribution of engine exhausts to air pollution, and the possible use of mutagenicity spectra in the analysis of environmental complex mixtures.     

Mutagenicity and antimutagenicity of air-borne particulates

A methanol extract of air-borne particulates collected in a suburban area of Okayama City showed not only mutagenicity but also antimutagenicity in the Ames test. Thus, when the mutagenicity of this preparation in Salmonella typhimurium TA98 (with metabolic activation) was measured, we observed that the dose response reached a plateau at 27 m3 air equivalent of the particulate, showing approximately equal numbers of revertants in the dose range 27-270 m3 equivalent. This plateau formation was not seen in the dose response of a blue-cotton extract of this preparation: the extract gave a linearly increasing dose response up to 270 m3 equivalent. This finding suggests that some factors that inhibit the mutagenicity were present in this methanol extract and that these inhibitors were not adsorbable to blue cotton, an adsorbent selective for compounds having 3 or more fused rings. From the portion unadsorbed to blue cotton, we isolated the antimutagenic factors and identified them as long-chain fatty acids: palmitic, stearic, oleic and linoleic acids. Analysis of several samples, including those from other parts of Japan, has suggested that these antimutagenic fatty acids are ubiquitous in air-borne particulates.     

Mutagenicity of particulates from the laboratory combustion of plastics

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (nitro-PAHs) have been identified in airborne particulate organic matter extracts. The pollutant sources were generally contributed by motor vehicles and industrial activity. Massive quantities of urban solid wastes, containing plastic materials such as PVC, PET, PS, and PE, burnt in the open air in local garbage dumps are frequently found in developing countries. In this study, the smog particulates from the combustion of these synthetic polymers were produced in a laboratory combustion chamber. The mutagenicity of acetone extracts from the smog particulates was evaluated with Salmonella typhimurium TA98 and TA100 in the presence and absence of S9 mix. Four samples in TA98 exhibited higher mutagenicity than those in TA100. The greatest mutagenicity was observed from the extracts of particulates from combustion of PVC followed by that of PS, PET, and PE. To determine the major mutagenic compounds in these samples, mutagens were partially purified through TLC and their mutagenicity was monitored with TA98. 1-NP and DNPs in the above samples were also determined by HPLC. The amounts of 1-NP and DNPs generally corresponded with their mutagenicity. Higher levels of 1-NP and DNPS from the combustion of PVC, PET, and PS. the combustion of synthetic polymer wastes might be responsible for the presence of high levels of 1-NP and DNPs in Taiwan urban air.     

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.     

Characterization of airborne particulates in bangkok urban area by neutron activation analysis

Samples of airborne particulates were collected in a residential area and in an area near a busy highway in Bangkok during the period from January 1997 to May 1998. A stacked filter system was used for the former site and a Partisol 2000 was used for the latter site. Both 2.5 Μm and 10-Μm particulates were collected every week. The total suspended particulate matters were also collected at the latter site. The samples were analyzed by neutron activation analysis utilizing neutron flux from a 2-MW TRIGA MARK III research reactor. The elements most frequently detected in the airborne particulates were Al, As, Br, Ca, Ce, Cl, Co, Cr, Cs, Fe, I, K, La, Mg, Mn, Na, Rb, Sb, Sc, Sm, Th, Ti, V, and Zn. The enrichment factor and factor analysis were used to investigate trends, sources, and origin of the atmospheric aerosols. Anthropogenic elements in road dust, construction dust, motor vehicles emission, and other combustion components were identified. A comparative study of data between both sites was performed and it was found that the mass concentration in the area close to the highway was about three times higher than in the residential area.     

Mutagenicity and polycyclic aromatic hydrocarbons analysis of airborne particulate matters from Taipei City

The mutagenic activity of dichloromethane extracts of 147 air particulate samples collected from 8 stations during December 1986-June 1987 in Taipei city was consistently higher in S. typhimurium strain TA 98 than in strain TA 100 in the presence of S9 mixture. Among the 8 stations, Nan Kang Police Station, Fu Hsing Elementary School, and Chung Hsing University which were located in the industrial district, downtown area, and heavy traffic zone, respectively, had significantly higher levels of PAHs than the other stations. In contrast, the levels of PAHs were much lower in the suburban station, near Pei Tou Elementary School. However, PAH contents of the air particulate samples collected from these stations did not show good correlation with their mutagenicity. The air particulates collected at some stations on Sunday when the traffic changed from heavy to light showed lower mutagenicity and PAH contents as compared with the other weekdays at the same stations. On the contrary, the samples collected at Pei Tou station in a suburban area where the traffic changed from light to heavy on Sunday showed higher mutagenicity and PAH contents. The monthly average of PAHs of air particulate samples collected over a 7-month period from 8 stations in Taipei city was lower than the average in 1980. Moreover, when compared with other countries, such as U.S.A., the Netherlands, West Germany, Italy, Norway, and Japan, the levels of PAHs and mutagenicity of air particulate matters in Taipei city were similar or slightly lower. The mutagenicity and contents of PAHs of air particulates collected from burnt ABS were significantly higher than those of burnt PVC. One sample PT-6-3 was collected while a nearby garbage collection area was on fire. The mutagenicity of that sample increased 3 to 16 fold and contained an 11 to 33 times higher content of the six PAHs (BaP, BeP, BbF, BaA, Chr, and DbA) as compared with the other samples collected at the same location at a different time. The higher mutagenicity and PAH contents of that sample might be due to the pollution of the air from combustion of the garbage containing products made of ABS.     

Mutagenicity studies of ambient airborne particles. II. Comparison of extraction methods

Organic materials were extracted with acetone from airborne particles by shaking, soxhletion and sonication for varying durations. 4-h, 1-h and 1/8-min extractions by shaking, soxhletion and sonication, respectively gave maximum his+ revertants with the Ames Salmonella/microsome assay. In a comparative study of extraction methods, sonication gave the highest and soxhletion the lowest mutagenic response. It appears that sonication with acetone is the best procedure for the extraction of mutagens from airborne particles as shown by Ames assay and Arar assay systems in Salmonella typhimurium.     

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.