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.     

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.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: Two-stage carcinogenesis in skin tumor sensitive mice (SENCAR)

Skin tumors can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation phase), followed by repetitive treatment with a noncarcinogenic tumor promoter. There is a very good dose-response relationship between the induction of the number of papillomas per mouse at early times (10 to 20 weeks) by either tumor initiators and promoters and the final carcinoma incidence after a longer latency (20 to 50 weeks) in SENCAR mice. This system not only can be used to determine the tumor initiating and promoting activities of a compound but if the agent is given repeatedly by itself one can also determine if it is a complete carcinogen, i.e., if it has both tumor initiating and promoting activity. In addition, if the agent is given concurrently with a known complete carcinogen or a tumor initiator one can also determine if the agent has cocarcinogenic or cotumor initiating activity or even possibly anticarcinogenic activity. Likewise, if the agent is given concurrently with a known tumor promoter one can determine if the agent has copromoting or antipromoting activity. Using the SENCAR skin carcinogenesis system we have undertaken the determination of the skin carcinogenic, cocarcinogenic, tumor initiating and promoting activities of various diesel emission particle extracts as well as for comparative purposes, standards such as benzo(a)pyrene and 12-0-tetradecanoylphorbol-13-acetate and extracts of emissions from a gasoline engine, roofing tar, coke oven and cigarette smoke condensate. Most of the studies are still in progress but some preliminary data is available on the comparative tumor initiating activities of the various samples at 14 weeks. Caterpillar diesel and cigarette smoke condensate were essentially without activity, whereas the Mustang gasoline catalyst and Olds diesel gave extremely low values although they were slightly above background. Roofing tar, coke oven and Nissan Diesel all gave moderate activity at high doses (1 to 10 mg) but when compared to benzo(a)pyrene were relatively low values.     

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 extracts of diesel and related environmental emissions: Study design, sample generation, collection, and preparation

A major diesel emissions research program has been initiated by the U.S. Environmental Protection Agency to assess the human health risk associated with increased use of diesel automobiles. This program is intended to establish the mutagenic and carcinogenic potency of complex organics associated with diesel particles as well as comparative particle-bound organics from other environmental emissions for which human epidemiological data are available. The mobile source samples selected for this study were collected from a heavy-duty diesel engine, a series of light-duty diesel passenger cars, and a gasoline catalyst automobile. The comparative source samples incorporated into the study were cigarette smoke condensate, coke oven emissions, roofing tar emissions, and benzo(a)pyrene. The samples were tested using three mutagenic assays and four carcinogenic assays as prescribed by a test matrix. This report describes the study design, particle generation, and sample collection and preparation. A brief summary of the bioassays is also included.     

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.     

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.     

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.     

Particle size measurements of automotive diesel emissions

The automative diesel engine has long been acknowledged as being “dirtier” than the spark ignition engine and its particulate emissions may be carcinogenic. Possible solutions to the diesel emission problem are combustion modification or aftertreatment devices. Selection of candidate aftertreatment devices requires knowledge of the physical and chemical properties of the particles, including particle morphology, size distribution, mass concentration and emission rates in the exhaust gas stream. The study reported here represents the first of a series of experiments designed to characterize the exhaust emissions and test various aftertreatment devices. This paper deals only with the particulate characterization phase of the program. Results of size distribution, particle concentration and mass emission rate measurements for a 5.71 displacement Oldsmobile diesel engine are given for a variety of engine operating conditions.     

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.     

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.     

Enhanced susceptibility to infection in mice after exposure to dilute exhaust from light duty diesel engines

A series of experiments was conducted in which groups of mice were first exposed for various durations to diluted exhaust from light duty diesel engines and then briefly to an infectious aerosol generated by nebulizing cultures of a bacterial pathogen (Streptococcus). Typically, postinfection mortality was significantly greater in groups exposed to exhaust than in their corresponding control groups exposed to purified air only. Data of recent diesel and of past diesel- and catalyst-treated gasoline engine exhaust experiments suggest a somewhat greater excess mortality from (enhanced susceptibility to) bacterial infection in mice exposed to diesel exhaust than in those exposed to catalytic gasoline exhaust. Limited data on acute tests of NO₂ and acrolein vapor alone suggest that the infectivity-enhancing effect of diesel exhaust could be accounted for in large part by these components. Exposures to diesel exhaust, NO₂, or acrolein did not enhance the mortality response to a viral pathogen (A/PR8-34).     

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.     

Preparation and characterization of diesel exhaust particles for biological experiments

Bioassays of diesel engine exhaust components are being conducted at IITRI to determine toxic and carcinogenic potentials of the exhaust. The bioassay method, intracheal instillation of saline suspensions of test materials in hamsters, requires preparation of stable suspensions of test materials. A method to prepare suspensions of whole particle diesel exhaust in saline has been developed. The diesel exhaust particle material was supplied to IITRI as a dry, loose powder by the U.S. EPA from a light duty diesel test engine. Preliminary characterizations of the powders indicated aggregation of exhaust particles had occurred both before and during capture on collection substrates. Flake-like sheets and hollow spheres of aggregated particles up to 150 μm in sie present in the powders. Therefore, the powder were ball-milled to geometric particle sizes more amenable to the animal administration technique to be employed. Grinding, suspension preparation and particle concentration assaying methods have been developed. Particle (geometric) size and morphological characterizations have also been performed on the as-received powders and prepared suspensions. A method to prepare emulsions (liquid-liquid suspensions) of the dichloromethane extracts of whole particle diesel exhaust has also been developed.     

Diesel particulate matter: Chemical and biological assays

This study involves the chemical characterization and biological assay of diesel particulate matter generated from a single-cylinder, direct-injected diesel engine. This engine was powered with a pure hydrocarbon fuel. The total extract obtained with this fuel had a positive biological activity by various biological assays, although the raw particulate matter was negative. The methodology of sampling and the assays are discussed together with the implications of the results to date.     

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.     

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.     

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.     

Lymphatic transport of inhaled diesel particles in the lungs of rats and guinea pigs exposed to diluted diesel exhaust

Factors influencing the disposition of the inhaled diesel particles were studied by analyzing the deposition of radioactively labelled diesel particles in the respiratory system, by determining the specific function of alveolar cellular mechanisms in the primary defense against inhaled particles and by identifying the important role of the lymphatic system in the lung clearance of experimental animals exposed to diluted emissions from a diesel engine. Radioactive ¹³¹Barium was used as a tracer of diesel particles and the deposition efficiency was determined to be 15%±6% of the inhaled dose in the Fischer 344 rat strain. The number of cells obtained by bronchial lavage increased significantly after a prolonged exposure to a concentration of 1500 μg/m³ of diesel particles. The increased cell number was more than twofold, contained two distinct cell populations (alveolar macrophages and neutrophils) and represented a reactive mobilization of the defense mechanisms in the organism. Light microscopy studies investigated the role of lymphatic transport of the particulate matter and revealed that the peribrochial and perivascular aggregates of lymphoid tissue contained diesel particles even after short exposure periods at low dose levels. With the increasing burden of particles in the respiratory system, the coloration of hilar and mediastinal lymph nodes continuously changed to gray and finally to dark black, depending upon the dose level and exposure. However, at all exposure levels, most of the diesel particles in the alveoli were phagocytized by an increased alveolar cellular defence and particle-containing macrophages were actively moving towards the mucociliary escalator or towards lymphatic channels leading to peribronchial lymphoid aggregates and bronchial or mediastinal lymph nodes. In the lymph nodes, alveolar macrophages containing diesel particles were found mostly in the afferent subcapsular lymphatic vessels and marginal sinuses. In the later stages, cellular structure disintegrated and large aggregates of particulate matter were dispersed throughout the medullary cords with increasing accumulation towards the hilus. It is concluded that the lymphoid aggregates and lymphatic nodes play an important role in sequestering diesel particles or particle-containing phagocytizing cells and provide a pathway, in addition to the mocociliary clearance for particulate removal from the deep pulmonary region.