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.     

Mutagenic activity of diesel emission particulate extracts and isolation of the mutagenic fractions

Mutagenic activity in diesel emission particulate extracts was detected by the Salmonella typhimurium/microsome assay. Direct-acting mutagens as well as promutagens requiring metabolic activation were detected. The extracts were fractionated into acidic, basic, and neutral fractions, and the neutral fraction was chromatographed into seven subfractions. Differences in the mutagenic potency of these fractions and subfractions were determined by the Salmonella assay. Fractions containing as yet unidentified compounds, but not polynuclear aromatic hydrocarbons, were found to make a major contribution to mutagenic activity of the extracts.     

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.     

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.     

Indoor air exposure to coal and wood combustion emissions associated with a high lung cancer rate in Xuan Wei, China

Residents of Xuan Wei County in China have unusually high lung cancer mortality that cannot be attributed to tobacco use or occupational exposure. They are exposed to smoke from unvented, open pit coal or wood fires (often used for cooking and heating). The variation in lung cancer rates among communes within the county suggests that indoor combustion of smoky coal may be the prime determinant of lung cancer. To characterize the air in Xuan Wei homes, samples of air particles and semivolatile organic compounds were collected from homes located in two communes; one commune has a high rate of lung cancer, and the other has a low rate. Samples collected in the commune where the lung cancer rate is high and where smoky coal is the predominant fuel contained high concentrations of small particles with high organic content; organic extracts of these samples were mutagenic. Samples from homes in the wood-burning commune, which has a low rate of lung cancer, consisted mostly of larger particles of lower organic content and mutagenicity. The smoky coal sample was a mouse skin carcinogen and was a more potent initiator of skin tumors in comparison to the wood or smokeless coal sample.     

Basic characterization of reference NOM from Central Europe — Similarities and differences

The FA and HA fractions represent 20 to 50% of the DOC of the original samples, depending on the origin, and serve as reference materials. The original aqueous samples and the aqueous solutions of the reference materials were characterized with methods suited for measuring samples at natural concentrations. The DOC normalized values of the spectral absorbances (λ = 254 nm; λ = 436 nm) in the original water samples were highest for the brown water and brown coal water and lowest for the secondary effluents. The FA fractions were similar in the UV-absorbance with the exception of the low-absorbing groundwater FA, reflecting a low density of double bonds. The DOC-normalized proton capacity of the FA fractions (10 to 20 μmol/mg) was highest for the brown coal, brown water, and soil-derived samples and again lowest for the groundwater FA. The high values are due to a higher contribution of the weak acids of phenolic type. Specific data for copper complexation capacity (0.5 to 2.5 μmol/mg) went parallel to the proton capacity. The characterization of the FAs by gel chromatography with organic carbon detection revealed three major fractions. The refractory part at short retention time dominated in the brown water and soil-derived samples, whereas the fractions for the lower molecular mass acids at long retention times were dominant in the secondary effluent and the brown coal isolate. Comparison with UV-detected chromatograms revealed a strong decrease in the specific absorbance of the secondary effluent. Up to 3% of the total mass of the FAs could be identified as hydrolyzable amino acids with the highest yield in the sample from secondary effluent and the lowest one in the groundwater sample. The nitrogen content was always higher in the FAs compared to the HAs of the same origin. The application of gel chromatography fluorescence and fluorescence decay time distribution analysis turned out to be a useful tool to differentiate between the different fractions of NOM, the isolates obtained by different methods (e.g., XAD-8, membrane separation) and the fractions of the samples from different origin and age. This is of special value, for the information can also be obtained for samples with natural NOM concentrations, even though further investigations required for full understanding of the data. This work is part of a multi-disciplinary research program (ROSIG: Refraktäre Organische Säuren in Gewässern), funded by the German Research Association (DFG).     

Preparation of size-selected fibers and fibrous aerosols for biological experiments

A size-selective procedure was developed for the preparation of fiber fractions of uniform size of chrysotile, crocidolite, amosite, and glass. The raw material was first ground to a suitable fineness, then suspended in liquids by ultrasonic agitation and separated by fiber diameter by means of slow sedimentation. The fractionation by fiber length was done by a vibrating sieve method. Fibrous aerosols of different size distributions could be prepared by using a vibrating bed aerosol generator.     

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.     

Evaluation of the mutagenic potential of some compounds using Salmonella typhimurium

The mutagenicity of sodium nitrite, potassium chromate, O-tolidine, and smoke condensate from tobacco was shown on Salmonella typhimurium TA 1535. Sodium nitrite was definitely mutagenic on TA 1535 without S-9 mix. Potassium chromate without S-9 mix was also mutagenic on TA 1535. However, addition of S-9 mix resulted in a complete loss of potassium chromate mutagenicity. O-tolidine with and without S-9 mix elicited a very weak and variable mutagenic effect for strain TA 1535. Smoke condensate from pipe tobacco without S-9 mix elicited a weak mutagenic effect, whereas with S-9 mix it showed no mutagenicity on TA 1535.     

Mineralogical,chemical and toxicological characterization of urban air particles

Systematic characterization of morphological, mineralogical, chemical and toxicological properties of various size fractions of the atmospheric particulate matter was a main focus of this study together with an assessment of the human health risks they pose. Even though near-ground atmospheric aerosols have been a subject of intensive research in recent years, data integrating chemical composition of particles and health risks are still scarce and the particle size aspect has not been properly addressed yet. Filling this gap, however, is necessary for reliable risk assessment. A high volume ambient air sampler equipped with a multi-stage cascade impactor was used for size specific particle collection, and all 6 fractions were a subject of detailed characterization of chemical (PAHs) and mineralogical composition of the particles, their mass size distribution and genotoxic potential of organic extracts. Finally, the risk level for inhalation exposure associated to the carcinogenic character of the studied PAHs has been assessed. The finest fraction (< 0.45 μm) exhibited the highest mass, highest active surface, highest amount of associated PAHs and also highest direct and indirect genotoxic potentials in our model air sample. Risk assessment of inhalation scenario indicates the significant cancer risk values in PM 1.5 size fraction. This presented new approach proved to be a useful tool for human health risk assessment in the areas with significant levels of air dust concentration.     

Instantaneous dust exposure monitors

This is a review of investigations conducted to evaluate portable, direct reading instruments employing the principles of light scattering and the attenuation of beta-rays to measure the respirable mass concentration of airborne particulates. Data are presented on comparative measurements obtained with several different models of beta-ray attenuation instruments manufactured by the GCA Corporation and light scattering instruments manufactured by GCA Corporation, USA, Ernst Leitz, GmbH, Germany, Rotheroe and Mitchell, England, and Sibata Chemical Apparatus Manufacturing Company, Ltd., Japan, using aerosols of coal, limestone, Arizona road dust, and silica. The effect of aerosol parameters (concentration, size distribution, index of refraction) on instrument calibration and precision are discussed.     

Lead isotopes in environmental sciences: a review

Lead (Pb) isotopic analyses proved to be a very efficient tool for tracing the sources of local and global Pb pollution. This review presents an overview of literature published on the use of Pb isotopic analyses of different environmental matrices (atmospheric aerosols, lichens, tree rings, peat deposits, lake, stream, marine sediments, soils, etc.). In order to gain more insight, the isotopic compositions of major sources of Pb in the environment as determined by several authors are described in detail. These include, above all, the former use of leaded gasoline, coal combustion, industrial activities (e.g., metallurgy) and waste incineration. Furthermore, this review summarises analytical techniques (especially ICP-MS) used for the determination of Pb isotopes in environmental samples.     

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.     

从气溶胶微物理属性分析冬季重庆地区霾的垂直分布特征

采用2007~2011年冬季(12月~次年2月)CALIPSO星载激光雷达L1监测数据,通过分析532 nm总后向散射系数、体积退偏比和色比,对重庆地区冬季霾期间气溶胶光学和微物理特性的垂直分布进行了研究。结果表明:重庆地区冬季霾期间,大气散射能力随高度减弱,在0~1 km高度最强;各高度层气溶胶粒子规则性与粒径大小的分布情况与全年总体分布情况基本相同,但变化趋势单调性更强,0~1 km高度层规则的、大颗粒气溶胶所占比例最大,3~4 km高度不规则的、小颗粒气溶胶所占比例最大;年际变化上,气溶胶散射强度逐年增强,其中2008~2010年变化较小,2011年明显增强;各年均以规则的、小颗粒气溶胶为主,2011年不规则气溶胶所占比例最大,而大颗粒气溶胶所占比例逐年增大。     

Contamination of terrestrial gastropods, Helix aspersa maxima, with 137Cs, 85Sr, 133Ba and 123mTe by direct, trophic and combined pathways

(137)Cs, (85)Sr, (133)Ba and (123m)Te contaminations of terrestrial gastropods, Helix aspersa maxima, by direct deposition, labelled food ingestion or combined (trophic and direct pathways) exposure were carried out under laboratory conditions. The aim of this study was to compare the three contamination pathways: direct, trophic and combined, in terms of individual mortality, radionuclide uptake, depuration and distribution in the tissues. An initial group of 30 snails (2 years old) was exposed to radioactive aerosols during a 20-h period. These aerosols were assumed to be representative of those that would be released during a nuclear accident occurring in a PWR. A second group of 50 snails (same age) was submitted to an ingestion of commercial food contaminated by the same aerosols, twice a week for 21 days (flour at a feeding rate of about 0.2g). A third group of 40 snails was submitted to a combined exposure: exposure to radioactive aerosols (20h), followed by ingestion of flour contaminated by the same aerosols, twice a week for 21 days. No significant difference between the three groups and a reference group of 10 snails was observed, neither in growth nor in mortality. Concerning the direct pathway, at the end of direct deposition (about 1 day after the beginning), cesium was the most bioavailable element, distributed rather homogeneously throughout the whole body (13% of the total Cs in all organs excepting the digestive system and 28% in the muscle). Strontium was measured in the shell (about 70%). Barium was found in the muscle (20%) and in the shell (65%). Tellurium was mainly present in the shell (70%) and in the digestive system (20%). After 21 days of depuration, the faeces eliminated 42% of the Te. As for contamination by ingestion, Te mainly accumulated in the digestive system (72% of Te present in the total body), Ba accumulated in the muscle (75%) and Sr in the shell (70%). Concerning contamination by combined pathways, at the end of the 21-day exposure, the 4 radionuclides had the same tendency as direct deposition. However, the effect of the trophic pathway was significant: it causes an 18% increase of Sr in the shell and an 7% increase of Cs in the digestive system in comparison to direct deposition, resulting in a final 86% in the shell and 27% in the digestive system.     

The effect of iron on the biological activities of erionite and mordenite

Epidemiological data has demonstrated that environmental and/or occupational exposure to mineral particulates may result in the development of pulmonary fibrosis, bronchogenic carcinoma and malignant mesothelioma many years following exposure. It has been suggested that the genotoxic effects of fibrous particulates, such as asbestos, is due in part to the generation of reactive oxygen species (ROS) from iron associated with the particulates. However, the molecular mechanisms by which mineral particulates induce ROS that results in genotoxic damage remains unclear. The naturally occurring zeolites, erionite and mordenite share several physiochemical properties but they elicit very different biological responses, with erionite, a fibrous particulate, being highly toxic, and mordenite, a nonfibrous particulate, being relatively benign. We are using these natural zeolites as a model system to determine what physicochemical properties of these zeolites are responsible for their biological response(s) and to evaluate the parameters that influence these responses. The purpose of the present study was to determine the mutagenic potential of erionite and mordenite and to determine whether this mutagenic potential was modulated by iron. The results of this study using the Chinese hamster ovary cell line AS52 demonstrated that erionite was more cytotoxic than mordenite. However, the cytotoxicity of both zeolites was increased in the presence of physiological concentrations of ferrous chloride. Ferrous ions (5-20 microM) significantly (p<0.001) increased the cytotoxicity of mordenite, but only at the highest concentration (16 microg/cm(2)) of mordenite tested. Conversely, only the highest concentration (20 microM) of ferrous ion significantly (p<0.001) increased the cytotoxicity of erionite, but this enhanced cytotoxicity occurred over a wider concentration range (6-16 microg/cm(2)) of erionite. Mordenite was not mutagenic at any of the concentrations tested, and the mutagenic potential of mordenite was not enhanced by the addition of ferrous ion. Conversely, erionite was mutagenic in a dose-response manner at concentrations greater than 6 microg/cm(2) and the mutagenic potential of erionite was significantly enhanced by the addition of ferrous ions. These results suggest that while the cytotoxicity of mordenite and erionite may be related to the ability of these fibers to transport iron into a cell, the different coordination state of iron associated with the two fiber surfaces is critical for inducing genotoxic damage.     

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.     

Microbial mutagenicity and toxicity of newly synthesized heterocyclic N-oxides

Newly synthesized heterocyclic N-oxides were tested for their mutagenicity using the Ames Test. DX1 was potentially mutagenic in Salmonella typhimurium TA 100 and TA 98 with and without the S-9 mixture. WO-25 and WO-20, which are structurally related to DX1 did not show genetic change in the strains used. The antibiotic activity of these chemicals was also tested using gram negative and gram positive bacteria. The mutagenic chemical DX1 had more killing effect in gram positive bacteria than WO-25 and WO-20.     

Determination of 129I/127I in aerosol samples in Seville (Spain)

In this work we present results of the (129)I/(127)I ratio in aerosols of Seville, Southwest of Spain (37.4 degrees N,6 degrees W). A radiochemical method is applied to extract the iodine from the aerosols and prepare samples to be measured by accelerator mass spectrometry (AMS) at the ETH facility in Zürich. We have found the possibility of monitoring the (129)I/(127)I isotopic ratio on a two-days basis with sensitivities in the order of 10(4)-10(5) atoms (129)I/m(3), and values of 10(-8)-10(-9) for the isotopic ratio.     

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.