Carcinogenicity of diesel exhaust as tested in strain a mice

Groups of strain A mice were exposed to diesel exhaust by inhalation and diesel particulate by intraperitoneal injection. The animals were exposed from seven to eight weeks and then sacrificed 26–30 weeks postexposure. Other animals were exposed for up to seven months by the inhalation route. Some animals were promoted using urethane at a dose below which tumors would occur. There was no increase in incidence of pulmonary adenomas in the animals exposed to either diesel exhaust or diesel particulate over the control animals. In the animals which were promoted using urethane at a low dose, there was a significant increase in pulmonary adenomas. Diesel particulate was found in the lungs and bronchial lymph nodes of animals exposed to diesel exhaust 26–30 weeks after cessation of exposure.     

Physical characterization of diesel exhaust particles in exposure chambers

Since the deposition of particulate in the respiratory system is strongly influenced by particle size, a correct assessment of this parameter is important for any inhalation experiment studying the potential health effects of air pollutants. Measuring the distribution of particles according to their aerodynamic diameter and mechanical mobility diameter is crucial in analyzing the deposition of submicron particles in the lower respiratory system. Cascade impactor measurements of diluted diesel exhaust in 12.6 m³ animal exposure chambers in the GMR Biomedical Science Department showed that the mass median aerodynamic diameter of the aerosol was 0.2 μm with 88% of the mass in particles smaller than 1 μm. Diffusion battery measurements showed that the mass median mechanical mobility diameter was about 0.11 μm. Transmission electron micrographs of particles deposited on chamber surfaces revealed both agglomerates and nearly spherical particles. The particles in these chambers are similar in size and shape to diesel particles described elsewhere. The flux of diesel particles to food surfaces was measured. Calculations of the expected daily dose by inhalation and by feeding showed that the “worst case” dose by feeding was only about one-tenth the dose by breathing.     

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.     

Introduction to and experimental conditions during the first year of the GMR chronic inhalation studies of diesel exhaust

A chronic exposure study was initiated to determine the effects of diesel exhaust on the health of experimental animals. For this purpose, test atmospheres of clean air (control) or freshly diluted diesel exhaust at concentrations of 250, 750, and 1500 μg/m³ were supplied to four 12.6 m³ inhalation chambers which housed rats and guinea pigs. Diesel aerosol size and concentration, as well as chamber temperature and relative humidity, were continually monitored and controlled to maintain the exposure dose levels and an environment of 22±2°C and 50%±20% relative humidity. The concentrations of CO and NO_x were found to be 5.8±1.0 mg/m³ and 7.9±1.0 mg/m³ above ambient in the chamber containing 1500 μg/m³ of particulate. Animals were supplied from the chambers, on a random basis, for both intramural and extramural studies throughout the exposure period. The experiment ran uninterrupted for over twelve months with mean diesel particle mass concentrations within 2% of the target values.     

Pulmonary function changes in Chinese hamsters exposed six months to diesel exhaust

Chinese hamsters were exposed for eight hours per day to automotive diesel exhaust emissions which were diluted with air (18 to 1) and had a particulate level of 6.4 mg/m³. Pulmonary function measurements were made after six months exposure. Body weight (BW), lung weight (LW), vital capacity (VC), residual volume by water displacement (RV_w) and by gas dilution (RV_D), alveolar volume (V_A), and carbon monoxide transfer factor (D_LCO) were measured. LW showed a significant increase in the diesel exposed animals (P < 0.01) while VC, RV_W, and D_LCO showed decreases (P < 0.01). Static deflation volume-pressure curves showed depressed deflation volumes for diesel exposed animals when volumes were corrected for body weight and even greater depressed volumes when volumes were corrected for lung weight. However, when volumes were expressed as percent vital capacity, the diesel exposed animals had higher lung volumes at 0 and 5 cm H₂O. Results of the pathological examination of the lung tissue will be necessary for final analysis of our findings. However, preliminary interpretation indicates possible emphysematous changes which are compatible with the observed decrease in D_LCO.     

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.     

Electron paramagnetic resonance spectrometry of diesel particulate matter

Electron paramagnetic resonance (EPR) spectrometry has been used to study diesel particulate matter. The EPR signals for a graphitized carbon black and particles collected from two different diesel engines have been compared. Variations in the line widths and signal intensities due to various chemical and physical pretreatments of the particles were observed. The EPR signals for the diesel particulates were shown to be sensitive to oxygen, nitric oxide, nitrogen dioxide, and ultraviolet and visible radiation. These results suggest that EPR may be a convenient means for characterizing particles from different sources or for demonstrating particle reactivities. The demonstrated photochemical reactivities of the airborne particles have significant environmental health implications.     

Pulmonary function testing of animals chronically exposed to diluted diesel exhaust for 267 days

Thw purpose of this work was to assess the potential effect that chronic inhalation of diesel exhaust may have on lung mechanics and lung volumes. Noninvasive pulmonary function tests have been conducted repeatedly on 25 rats exposed to diesel exhaust at a particulate concentration of 1500 μg/m³, for 20 h/day, days/week for 267 days. The same tests were conducted on 25 clean air control animals. When the data is normalized, there are no apparent functionally significant changes occurring in the lungs that may be attributed to the chronic inhalation of diesel exhaust.     

Seasonal distribution and modeling of diesel particulate matter in the Southeast US

The fine and ultra fine size of diesel particulate mater (DPM) are of great health concern and significantly contribute to the overall cancer risk. In addition, diesel particles may contribute a warming effect on the planet's climate. The composition of these particles is composed principally of elemental carbon (EC) with adsorbed organic compounds, sulfate, nitrate, ammonia, metals, and other trace elements. The purpose of this study was to depict the seasonality and modeling of particulate matter in the Southeastern US produced by the diesel fueled sources (DFSs). The modeling results came from four one-month cases including March, June, September, and December to represent different seasons in 2003 by linking Models-3/CMAQ and SMOKE. The 1999 National Emissions Inventory Version 3 (NEI99) was used in this analysis for point, area, and non-road sources, whereas the National Mobile Inventory Model (NMIM) was used to create the on-road emissions. Three urban areas, Atlanta, Birmingham, and Nashville were selected to analyze the DPM emissions and concentrations. Even though the model performance was not very strong, it could be considered satisfactory to conduct seasonal distribution analysis for DPM. Important hourly DPM seasonality was observed in each city, of which higher values occurred at the morning traffic rush hours. The EC contributions of primary DPM were similar for all three sites (~ 74%). The results showed that there is no significant daily seasonality of DPM contribution to PM_2.5 for any of these three cities in 2003. The annual DPM contribution to total PM_2.5 for Atlanta, Nashville, and Birmingham were 3.7%, 2.5%, and 2.2%, respectively.     

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.     

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 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.     

Control strategies for particulate emissions from vehicular diesel exhaust

The advantages and disadvantages of several possible control strategies for particulate emissions from vehicular diesel exhaust are discussed. The evaluation of the potential usefulness of the various control strategies is based on available data concerning the mass loading and particle size distribution and on anticipated control standards. Several studies have been made on devices for removing particulate emissions from vehicular diesel exhaust. These studies, which include the techniques of filtration, wet scrubbers, and electrostatic precipitation, are summarized. A comparison of the various control devices is made based on such factors as size, efficiency, and maintenance requirements.     

Test of diesel exhaust emissions in the rat liver foci assay

In the initiation/promotion assay in rat liver, partial hepatectomy is used to enhance initiation, and a choline-devoid diet as promoter. The induction of carcinogenesis is determined by the focal appearance of gamma glutamyl transpeptidase (GGT) positive hepatocytes. We adopted this assay to diesel exhaust emission by performing a partial hepatectomy, and exposing the rats to either clean air or diesel exhaust emission. The rats were fed either a choline-devoid or a choline-supplemented diet for three or six months. The animals were sacrificed and liver sections stained for GGT were examined for the presence of foci of GGT(+) hepatocytes. The results indicate that diesel exhaust exposure does not result in a systemic dose of carcinogens sufficient to be detected in the liver foci assay.     

Behavioral alterations due to diesel exhaust exposure

Several experiments examining the effects of diesel exhaust on the behavior of rats are reported. Animals were exposed either as adults or neonates. The spontaneous locomotor activity (SLA), measured in standard running wheel cages, of adult rats exposed for 8 h/day, 7 days/week was significantly less than that of controls. Experiments involving diesel exhaust exposure to neonatal rats indicated that adult rats, exposed to diesel exhaust during their neonatal lives, were significantly less active as measured by SLA. Adult rats, exposed to 20 h diesel per day as neonates, were placed in skinner boxes after the SLA experiment described above had been completed. The exhaust exposed animals showed significantly decreased acquisition of a food reinforced bar pressing task. All animals that learned this task extinguished at the same rate. The results of the neonatal diesel exhaust experiments support the hypothesis that diesel exhaust exposure during development of an organism can lead to behavioral differences in adulthood.     

A study of diesel emissions on Drosophila

A sex-linked recessive lethal test was performed on male fruit flies of the species Drosophila melanogaster, (Oregon-R strain), exposed to an approximate five-fold dilution of exhaust from a diesel engine. The eight hour exposure was achieved by drawing diluted diesel exhaust from a three cubic meter stainless steel exposure chamber housing laboratory animals through a two liter reaction flask modified for use with Drosophila. A preconditioned sampling bag was used to collect the emissions after passing through the exposure chamber containing the flies. Results of analyses performed on the diesel exhaust mixture showed the following: carbon dioxide—0.17%, carbon monoxide—12.2 ppm, hydrocarbons—11.6 ppm, nitrogen oxide—3.8 ppm, nitrogen dioxide—2.9 ppm, sulphur dioxide—1.0 ppm, and particulates—2.18 mg/m³. Two broods of the F₂ generation were investigated for the occurrence of recessive lethal events. These broods approximated the developing gametogenic stages of mature sperm (P₁ matings on days 2 and 3 postexposure) and spermatocytes (P₁ matings on days 8 and 9). Additionally, the F₃ generation was evaluated for the occurrence of mosaic recessive lethal events which might escape detection in the F₂ generation. An equal number of F₂ and F₃ flies for both broods served as concurrent controls. Results indicate that, under the conditions tested, the diesel exhaust did not increase the mutation frequency of the exposed flies (F₂ rate = 0.30%, F₃ rate = 0%) when compared to the concurrent controls (F₂ rate = 0.37%, F₃ rate = 0.15%).     

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.     

Facilities for diesel exhaust studies

It is the major intent of this report to examine the animal inhalation facilities at the EPA Health Effects Research Laboratory in Cincinnati, Ohio, associated with the exposure to the exhaust of diesel-powered equipment. The facility serves as a center for toxicologic evaluation, in relevant animal models, of potentially hazardous environmental pollutants from diesel engine exhaust. Briefly, the entire engine exhaust is mixed with filtered and conditioned air in a dilution tube. That diluted exhaust enters a large volume mixing chamber and a portion passes through dynamic flow irradiation chambers (to simulate sunlight) and is then conducted to animal exposure chambers. The system provides nonirradiated exhaust in the same concentration (directly from the mixing chamber) and also filtered, conditioned ambient air for control animal exposure. The major physical components of the diesel emission system include: air purifiers, animal exposure chambers, engine-dynamometers, irradiation chambers, monitoring instrumentation and data acquisition systems.     

Radiation measurements and radioecological aspects of fallout from the cherbonyl reactor accident

Fallout from the Chernobyl reactor accident has been monitored for about one year in Thessaloniki in Northern Greece. Fifteen different short-lived, three relatively long- and one long-lived fission products were identified in air, precipitation, soil, grass and milk samples. The iodine-131 and cesium-137 concentrations in air reached 6·5 and 3 Bq m⁻³ respectively, on 6 May, 1986. The external exposure dose rate rose to five times the normal background level. It was estimated that the accumulated dose equivalent to the adult thyroid from inhaled iodine-131 averaged 96 μSv, while the body burden from inhaled radiocesium nuclides averaged 2 μSv, 1000 times lower than that corresponding to the estimated dose equivalent from ingestion of foodstuff, which averaged 2 mSv for the first year after the accident.