Diesel particulate extracts in bacterial test systems

The Ames bacterial mutagenicity test system was used to evaluate parameters which may affect the mutagenic activity of diesel particulate extracts. The optimal extraction conditions, extractability of mutagens by simulated biological fluids and the effect of collection method were investigated. The role of solvent was examined by extracting diesel particles with methanol, acetone, cyclohexane, ethyl acetate, n-hexane, dichloromethane, benzene and a benzene-ethanol mixture. Of these, the dichloromethane extract exhibited the highest activity in the Ames test, although methanol yielded the largest extractable mass. Diesel particles were also extracted by dimethyl sulfoxide (DMSO) and four other simulated biological fluids for 48 h at 25, 37, and 45°C to study the effects of temperature. The mutagenic activity of the DMSO extract began to decline at temperatures higher than 37°C after 8 h of incubation. Fetal calf serum was the only simulated biological fluid which eluted mutagenic activity from the particles. No activity was detected in the 0.5% bovine serum albumin, simulated lung surfactant and saline extracts. Diesel particles collected by electrostatic precipitation (ESP) and filtration were studied. The mutagenic activities of both extracts were comparable when expressed as revertants per mg of particle. After the extracts were separated into nine fractions by a solvent partitioning scheme, the majority of the activity was found in the neutral-nonpolar II, neutral polar, strong acid and weak acid fractions. The acid salt fraction from the ESP sample was inactive. These results demonstrate that differences in the extraction conditions can result in differences in the mutagenic activity of diesel particulate extract. Since the mutagens in the extracts are not readily extractable by simulated biological fluids, the question of bioavailability of mutagens in diesel particles must be considered in the final assessment of their potential effects in biological systems and organisms.     

Genetic biomonitoring of an urban population exposed to mutagenic airborne pollutants

Biomonitoring studies have increased as a consequence of risks and effects to human health on exposure to environmental contaminants, mainly air pollutants. Genetic biomarkers are useful tools for the early assessment of exposure to occupational and environmental pollution. The objective of the present study was to investigate genotoxic effects on people residing and/or working downwind from an oil refinery in southern Brazil and the mutagenic activity of airborne particulate matter (PM10). Samples of peripheral blood and buccal mucosa cells were evaluated using the single-cell gel electrophoresis assay (comet assay) and the micronucleus (MN) assay, respectively. PM10 samples were collected in the target site and the organic matter extraced with dichloromethane was assessed for mutagenic activity in the Salmonella/microsome assay. The exposed group (n = 37) was compared to a reference group (n = 37) of subjects living in an urban area with limited traffic and industrial influence, located far from the main industrial areas. All PM10 organic extracts showed mutagenic positive responses and the effect decreased in the presence of S9 mix indicating that the predominant compounds present were direct-acting mutagens. The responses of YGs strains are consistent with aromatic amines and nitroarenes being present in the PM10 extracts. The group in the area under the influence of the oil refinery (exposed group) showed significantly higher DNA damage in lymphocytes than the reference group. The MN frequencies in buccal mucosa were very low for both groups and no difference between groups was observed. No association was found between age and tobacco smoking habit and level of DNA damages measured by the comet assay. The results indicate that the comet assay was a sensitive tool to detect DNA damage in subjects under the influence of an oil refinery, with marked genotoxic activity in the atmospheric environment.     

Salmonella/microsome mutagenicity assays of exhaust from diesel and gasoline powered motor vehicles

Motor vehicle exhaust from prechamber injection diesel and gasoline powered passenger cars, sampled during US FTP 1973 test cycles and comprising both particulate matter and compounds condensable at ambient temperature, has been assayed for mutagenicity in the Salmonella/microsome test. Mutagenic components were to a large extent active in the absence of the mammalian microsomal preparation. The mutagenicity of both particulate matter and condensate from diesel exhaust and condensate from gasoline exhaust was decreased in the presence of the microsomal preparation whereas the mutagenicity of particulate matter from gasoline exhaust was enhanced by microsomal activation. A comparison between the investigated diesel and gasoline exhaust samples shows that the mutagenic effect in the Salmonella test of the diesel exhaust is more than ten times higher than that of the gasoline exhaust. Fractionation with respect to polarity indicates that the mutagenic components mainly are distributed in neutral aliphatic, aromatic, and oxygenated fractions. Tests for mutagenic monofunctional nitroarenes by an anaerobic assay indicate that such compounds at most are marginally present in the exhaust samples as compared with their presence in airborne particulate matter collected in an urban environment.     

Evaluation of techniques used in the preparation of diesel extract samples for mutagenicity studies

Diesel exhaust particles were used to compare methods and techniques used in the preparation of particulate matter for microbial mutagenesis testing. Investigated in this study were extraction, concentration, and solvent exchange methodologies as they affected recovery of mutagenic material from diesel samples using a Salmonella typhimurium plate incorporation assay. Solvent removal methods applicable for use in determining the mass concentration of extracts were also evaluated. Results indicated that particle samples Soxhlet extracted with dichloromethane yielded higher levels of mutagenic activity than did comparative samples utilizing sonication. No difference was seen between rotary evaporation or Kuderna-Danish macro concentration of extracts to volumes > 50 mL. In comparison of micro concentration techniques to volumes < 10 mL, vortex evaporation was found to be more efficient than a modified micro Kuderna-Danish method in recovery of mass and mutagenicity. Solvent exchanged samples were found to yield higher recoveries of mutagenic activity than samples taken to dryness and then reconstituted in the bioassay solvent. A dry mass weighing procedure utilizing desiccation was found to be more acceptable than either the use of an infrared heat lamp or nitrogen blowdown for solvent removal.     

Diesel soot: Mutation measurements in bacterial and human cells

Both hot pipe and dilution chamber samples of the exhaust from a diesel (Oldsmobile 350) engine have been collected, extracted with methylene chloride and those extracts have been tested for mutagenicity in forward mutation assays in human lymphoblasts and S. typhimurium. In the absence of a metabolic activation system, the extract was significantly mutagenic to the bacteria in the range of 0 to 30 μg/ml, but introduced no mutations in human cells at concentrations up to 200 μg/ml under the same conditions of assay medium. However, when assayed in the presence of a postmitochondrial supernatant derived from rat liver, the the soot extracts were significantly mutagenic to both bacteria and human cells in the range of 50–100 μg/ml. Fractionation of the soot extract on the basis of polarity by sequential elution from a silicic acid column permitted concentration of the mutagenic activity in the alkane/toluene eluate, as determined by bacterial assays. Preliminary characterization of this fraction and preliminary studies of pure compounds leads us to suspect the alkyl substituted phenanthrenes as representing at least a significant fraction of the mutagenic activity of this alkane/toluene eluate.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: In vitro mutagenesis and oncogenic transformation

Extracts from emissions of four diesel engines, a gasoline engine and three related environmental samples were tested in four in vitro assay systems designed to detect carcinogenic or mutagenic activity of chemicals. Samples from three of four diesel extracts, the gasoline engine, and all three related samples were positive in an enhancement of viral transformation assay. Two diesel samples, the gasoline engine extract and extract from coke oven emissions were positive for mutation induction in Chinese hamster ovary cells. Only the gasoline engine extract and the coke oven sample were positive in a DNA fragmentation assay using alkaline sucrose gradients. Experiments using chemical transformation of Syrian hamster embryo cells as an assay method have not been completed.     

Mutagenic and carcinogenic potency of diesel and related environmental emissions: Salmonella bioassay

Due to the expected increase in the percentage of diesel vehicles in the United States, the Environmental Protection Agency must evaluate the health effects associated with exposure to diesel emissions. Respirable particles from a variety of combustion sources have the potential of being carcinogenic and mutagenic. The objective of these studies was to determine the relative biological activity of the organic material adsorbed on these particles in in vitro mutagenesis bioassays. The organic extracts from the following series of emission sources were bioassayed in the Salmonella assay for mutagenic activity: (1) a light-duty Oldsmobile diesel 350 engine; (2) a heavy-duty Caterpillar diesel engine; (3) a light-duty Nissan engine; (4) a Volkswagen Rabbit diesel engine; (5) cigarette smoke; (6) roofing tar; (7) coke oven; and (8) a gasoline catalyst Mustang. This paper provides a comparison of these sources within the Salmonella bioassay and also demonstrates how bacterial systems can be used as a quality assurance measure in in vivo testing.     

Detection and average content levels of carcinogenic and mutagenic compounds from the particulates on diesel and gasoline engine mufflers

Carcinogenic and mutagenic compounds, which were extracted from the particulates that adhered to inner surfaces of diesel and gasoline engine mufflers, were quantified by the series method of Soxhlet extraction, liquid-liquid partition, thin-layer chromatography, and spectrofluorometry. Mutagenic activity of their neutral and acidic fractions was tested in the improved Ames assay by the preincubation method with Salmonella typhimurium TA98 in the presence and absence of metabolic activation system (S-9 mix). The average content levels (μg/g tar) of polycyclic aromatic hydrocarbons from gasoline engine cars were greater than those from diesel engine vehicles. However, the levels of nitro derivatives of PAHs and polycyclic quinones from the diesel engines were greater than from the gasoline engines. Mutagenic activity of the diesel acidic fraction was the highest among the diesel and gasoline fractions, and was significantly higher in the absence of the S-9 mix. Furthermore, the relative value (R_{c = 0}) of infrared absorption of carbonyl stretching vibration to that of methylene asymmetric stretching vibration of the diesel acidic fraction was the highest among the diesel and gasoline fractions. These results strongly suggest that highly direct-acting mutagens in the acidic fraction are at higher levels in diesel emission particulates than those from gasoline, and that these mutagens are carboxylic acid, aldehyde, and alcohol derivatives of PAHs and NPAHs.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: In vitro mutagenesis and DNA damage

The Saccharomyces cerevisiae D3 recombinogenic assay, the assay for forward mutagenesis in L5178Y mouse lymphoma cells, and the sister chromatid exchange (SCE) assay using Chinese hamster ovary cells were used to evaluate the in vitro mutagenic and DNA-damaging effects of eight samples of diesel engine emissions and related environmental emissions. The recombinogenic assay was not sufficiently sensitive for this evaluation, but mutagenicity was detected in the L5178Y mutagenesis assay following exposures of the cells to all of the emission samples, and DNA damage in the SCE assay was induced by most of the emission samples in the presence and absence of metabolic activation. The observation of positive results in the absence of activation indicated that the samples contained substances that were direct-acting mutagens and DNA-damaging agents.     

Deposition and biological availability of diesel particles and their associated mutagenic chemicals

To estimate the human health risk of inhaled diesel particles, it is necessary to know their deposition and retention in the respiratory tract and the rate of dissociation of mutagenic compounds associated with the particles. The deposition of a chain aggregate aerosol of ⁶⁷Ga₂O₃ with size and shape characteristics similar to diesel exhaust particles has been evaluated using Beagle dogs. Approximately one-third of the inhaled activity is deposited in the respiratory tract with most of the particles deposited in the lung. The mutagenic activity present in dichloromethane, dog serum, dog lung lavage fluid, saline, dipalmitoyl lecithin (DPL) and albumin following incubation of these fluids with diesel exhaust particles was determined in the Ames Salmonella system. As observed by other investigators, large quantities of mutagenic activity were removed by dichloromethane. A very small amount of mutagenic activity was removed by the serum and lavage fluid over a 3-day incubation period. No activity was detected following elution with the other solvents. The finding that minimal mutagenic activity could be demonstrated in the biological media following incubation with diesel exhaust particles may be due to a lack of removal of mutagens from the particles or an inactivation of removed mutagens by protein binding or other processes.     

DNA-binding studies with diesel exhaust particle extract

This study examines whether chemical components from diesel exhaust particulates react with DNA to form covalently bound adducts. Experiments in this report describe the in vitro reaction of purified DNA with a dichloromethane extract of diesel exhaust particulates in the absence or presence of enzyme activation by rat liver microsomes. The reactivity of the particle extract was compared to that of benzo[a]pyrene metabolites using low temperature fluorescence techniques which detect small quantities of polycyclic aromatic compounds bound to DNA. Incubation of DNA with the particle extract in the presence of microsomal enzymes produced no detectable fluorescent adducts in contrast to model experiments using benzo[a]pyrene. However, addition of the particle extract to incubation mixtures containing benzo[a]pyrene markedly decreased formation of benzo[a]pyrene-DNA adducts because the particle extract inhibits microsomal enzymes which activate benzo[a]pyrene and other polycyclic aromatic hydrocarbons. In the absence of microsomal enzymes, fluorescent material was detected in DNA exposed to high concentrations of the particle extract, but probably not as a result of covalent binding because the mutagenic activity of the particle extract remained unchanged during prolonged incubation with DNA. This stability is in contrast to the rapid decrease in mutagenic activity of benzo[a]pyrene-4,5-oxide during incubation with DNA. Thus, direct mutation of bacteria by the particle extract may require activation by bacterial enzymes as is known to occur with nitroaromatic compounds.     

Mutagenic properties of PM2.5 urban pollution in the Northern Italy: The nitro-compounds contribution

PM2.5 is the breathable fraction of the particulate matter and some adverse health effects, such as respiratory functionality, cardiological diseases and cancer, can be in some measure attributable to this risk factor exposure. Some of the most carcinogen compounds transported by PM2.5 are nitro-compounds. In this study, a strengthened in vitro bioassay — able to predict the mutagenic/carcinogenic activity of the environmental mixtures — was conducted on PM2.5 organic extracts to define the nitro-compounds burden. PM2.5 air pollution was daily monitored, during 2006, in three cities located in the Northern part of Italy (Torino, Pavia and Verona) and the mutagenic properties of the PM2.5 organic extracts were assessed with the Ames test. The bacterial used in this study were three Salmonella typhimurium strains: TA98, nitroreductase-less mutant TA98NR and YG1021 carrying a nitroreductase-producing plasmid. The annual PM2.5 mean level measured in Torino was 46.5 (± 31.6) μg/m³, in Pavia 34.8 (± 25.1) μg/m³, and in Verona 37.3 (± 27.8) μg/m³, while the mutagenicity expressed as TA98 net reverants/m³ was 28.0 (± 22.1), 28.3 (± 24.9), and 34.2 (± 30.9) respectively. Monthly pool bioassays, conducted with the three different strains, showed a greater mutagenic response of the YG1021 in each city. The relationship among the mutagenic answers for YG1021:TA98:TA98NR was about 6:3:1 (p < 0.001). Over nitroreductase activity enhanced the response of 2.2, 2.0 and 1.7 times for Torino, Pavia, and Verona (ANOVA Torino p < 0.05) respectively. Without nitroreductase activity the genotoxicity was limited. These biological findings are able to describe a relevant role played by the nitro compounds in the mutagenic properties of the urban PM2.5 in the Padana plain; moreover the bacterial nitroreductase plays a predominant role in DNA interaction primarily for Torino PM2.5 extracts.     

Assessment of mutagenic risks of emissions from coal power plants

This study deals with atmospheres generated by coal burning, in order to verify whether aerosols collected in the environment of a thermal coal power plant produce a mutagenic effect. Biological tests were conducted (Ames' test) in order to study the mutagenic effects and to assess and compare risks. First, a chemical treatment was applied to the aerosols in order to separate mineral and organic fractions (hydrophile, aromatic, and aliphatic). Polyaromatic hydrocarbons were then evaluated and the mutagenic activity of every fraction was determined. The results obtained were compared by estimating the “mutagenic effect speed” parameters of these various fractions.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: Summary and discussion of the results

The proposed conversion from gasoline powered automobiles to diesel powered vehicels has prompted the Environmental Protection Agency to evaluate the potential health effects associated with exposure to diesel emissions. At present, there is no direct epidemiological link between this exposure and human health. Therefore, a research program was constructed to compare the health effects associated with diesel emissions with those from other emission sources for which epidemiological information was available. The emission sources chosen were cigarette smoke, roofing tar, and coke oven. An additional comparative emission source which was a gasoline catalyst engine. Respirable particles from a variety of combustion sources have the potential of being carcinogenic and mutagenic. The objective of these studies was to determine the relative biological activity of the organic material adsorbed on these particles in both in vitro mutagenesis and in vitro and in vivo bioassays. The organic extracts from the following series of emission sources were quantitatively bioassayed in a matrix of tests for their carcinogenic and mutagenic activity: (1) a light-duty Oldsmobile diesel 350 engine; (2) a heavy-duty Caterpillar diesel engine; (3) a light-duty Nissan engine; (4) a Volkswagen Rabbit diesel engine; (5) cigarette smoke; (6) roofing tar; (7) coke oven; and (8) a gasoline catalyst Mustang. The test matrix consisted of the following bioassay: reverse mutation in Salmonella typhimurium; mitotic recombination in Saccharomyces cerevisiae; DNA damage in Syrian hamster embryo cells (SHE); sister chromatid exchange in CHO cells; gene mutation in L5178Y mouse lymphoma cells, Balb/c 3T3 mouse embryo fibroblasts and CHO cells; viral enhancement of SHE cells; oncogenic transformation in Balb/c 3T3 cells; and skin tumor initiation in SENCAR and C57 black mice. The results of this test matrix are discussed.     

The DNA damage activity (DDA) assay and its application to river waters and diesel exhausts

An extremely sensitive assay has been developed for DNA-damaging chemicals using a DNA repair-deficient strain of Escherichia coli. Considerable DNA-damaging activity has been demonstrated by this assay in natural surface-water samples and in diesel-exhaust particulate extracts.     

Mutagenic properties of PM2.5 air pollution in the Padana Plain (Italy) before and in the course of XX Winter Olympic Games of "Torino 2006"

PM2.5 is one of the most important aspects of environmental health. This air pollutant is breathable and it is implicated in several chronic adverse health effects such as the decrease of respiratory functionality and cancer. Several in vitro bioassays are able to predict the mutagenic/carcinogenic activity of the environmental pollutants and mixtures of them. In this study PM2.5 air pollution was daily monitored in three cities located in the Northern part of Italy and the mutagenic properties of the PM2.5 organic extracts were also assessed. Samplings lasted 14 months and cover the period of the Winter Olympic Games of "Torino 2006". In this work, the levels of PM2.5, its mutagenic properties (detected with Salmonella typhimurium assay), the role of the Olympic Games as environmental factor and some meteorological data are discussed. The mean concentration of PM2.5 measured in Torino was 45.4 (+/-30.6) microg/m(3), in Pavia 37.6 (+/-25.6) microg/m(3), in Verona 43.1 (+/-28.5) microg/m(3). Findings of the monthly pool bioassay were in Torino 107 (+/-104) net revertans/m(3), in Pavia 108 (+/-89) net revertans/m(3), in Verona 128 (+/-109) net revertans/m(3). The Olympic Games period data show that PM2.5 pollution and its load of mutagenic potential are different and partially independent phenomena. The Olympic Games had not a great impact on the PM2.5 pollution. The exclusive PM2.5 gravimetric analysis shows a potential human risk if compared with the latest international guide values but it does not describe exhaustively the human health risk associated to the presence of this particular air pollutant. Moreover, the chemical and biological activity qualification of the PM organic extracts as a whole, can instead improve the knowledge.     

Study of total organic halogen as a means to detect groundwater contamination

Total organic halogen (TOX) was evaluated as a means to detect contamination of groundwater by halogenated organics. Groundwater samples from monitoring wells at 10 Canadian landfill sites were analyzed for carbon adsorbable TOX, total organic carbon and volatile organics. Low concentrations of TOX (range, ND-44; median 8 μg Cl L^?1) were found at nine sites, and elevated TOX levels (?2072 μg Cl L^?1) at the tenth site. This latter site was studied in greater detail, and TOX and GC-MS analyses were carried out for volatile, base neutral, and acid extractable fractions of the water in six monitoring wells at this site. The TOX and GC-MS techniques were found to provide complementary information. GC-MS analysis detected high concentrations of 1,2-dichloroethylene, trichloroethylene, toluene, and ethylbenzene in some of the wells. TOX measurements, however, indicated the presence of halogenated contaminants in the base-neutral and acid extractable fractions, which had not been detected by GC-MS.     

Mutagenic and carcinogenic potency of extracts from diesel related environmental emissions: Simultaneous morphological transformation and mutagenesis in BALB/c 3t3 cells

Particulate extracts from six different environmental emission sources were assayed for genotoxic activity in mouse BALB/c 3T3 clone A31-1 cells. All compounds were tested simultaneously for both transforming and mutagenic (induction of ouabain-resistance) potential with and without exogenous metabolic activation in the form of a 9000 × g postmitochondrial hepatic supernatant fraction from Aroclor-1254 induced Fischer 344 rats. Dichloromethane particulate extracts from the exhaust of two light duty diesel engines (Oldsmobile and Nissan), one heavy duty diesel engine (Caterpillar) and one late model gasoline engine (Mustang II) were assayed in an identical manner to particulate extracts from the emissions of a roofing tar pot and a coke oven. No clear dose-dependent responses were observed, but several of the samples showed significant transforming and mutagenic activity. A qualitative ranking system showed the activity of these particulate extracts for either mutagenesis or transformation was: coke oven = Mustang II gasoline engine > Nissan diesel engine > roofing tar. Particulate extracts from the Oldsmobile diesel engine and the Caterpillar diesel engine showed essentially no activity.     

Identification and quantitative confirmation of dinitropyrenes and 3-nitrobenzanthrone as major mutagens in contaminated sediments

Polar fractions of a sediment extract of the industrial area of Bitterfeld, Germany, have been subjected for effect-directed identification of mutagens using the Ames fluctuation assay with TA98. Mutagenicity could be well recovered in several secondary and tertiary fractions. Dinitropyrenes and 3-nitrobenzanthrone could be confirmed to contribute great shares of the observed mutagenicity. In addition, a multitude of polar polycyclic aromatic compounds has been tentatively identified in mutagenic fractions including nitro-PAHs, azaarenes, ketones, quinones, hydroxy-compounds, lactones and carboxylic acids although their contribution to mutagenicity could not be quantified due to a lack of standards. Diagnostic Salmonella strains YG1024 and YG1041 were applied to confirm the contribution of nitro-aromatic compounds. We suggest the inclusion of dinitropyrenes and 3-nitrobenzanthrone into sediment monitoring in order to minimize the mutagenic risk to aquatic organisms and to human health.     

Evaluation of the water genotoxicity from Santos Estuary (Brazil) in relation to the sediment contamination and effluent discharges

The genotoxic activity of water samples collected in 9 different sites within the area of the Santos estuary was preliminary evaluated, and related to previous data on the genotoxicity of sediments and the contents of PAHs in both water and sediment samples. The liquid discharge of a steel mill (coke plant), known to be mutagenic, was chemically analyzed to determine its PAH content. For the water evaluation we employed the Salmonella/microsome assay with the strains TA98 and TA100 with and without S9 mix in the plate incorporation method. The water was filtered with an AP20 membrane before being extracted with XAD4 at natural and acidic pH. The industrial effluent was filtered in 0.45 microm membranes before being extracted with the liquid/liquid method. Both membranes containing the particulate material were extracted using ultrasonication. PAHs were found associated with the suspended particles present in the industrial effluent in accordance with mutagenicity data previously reported. In relation to the estuarine waters, sites 1 and 5 presented low levels of mutagenic activity only in the filtered water (liquid fraction) extracts. At site 3, both the filtered water and particulate solids presented also low mutagenicity. Results show that the mutagenic activity observed in water could not be directly related to the genotoxic activity and PAHs contents of the bottom sediments.