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.     

Effects of EGR on performance and emissions of a diesel engine fuelled with balanites aegyptiaca/diesel blends

In this study, balanites Aegyptiaca (L.) Del biodiesel was blended in proportions of 10% and 20% on the volume basis with diesel fuel and tested in a single cylinder, VCR diesel engine under measured load conditions with varied EGR rates (0, 10 and 20%). The results showed that B10 and B20 blends shown a significant reduction rate in terms of NO_x emissions that were familiar with biodiesel blends. At peak load conditions, BTE increased slightly for test fuel blends compared with pure diesel fuel while the BSFC rate and EGT suffered from increasing and decreasing nature with respect to blending percentage. From the emissions point of view, with the increase in blends percentage, a significant reduction rate is observed in terms of CO and HC concentrations (up to 12.34 and 17.5%, respectively) while NOx emissions decreased at peak load conditions (up to 24.34%). HC and CO emissions decreased with increase in blends percentage. However, lower levels of NO_x and EGT (up to 21.37 and 8.47%, respectively) and the average increase in terms of BTE and BSFC (up to 2.83 and 2.9%, respectively) can be realised with B20 test fuel blend under 20% EGR rate.     

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: 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.     

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.     

A review of in vitro testing systems applicable to diesel health effects research

In vitro mutagenicity and carcinogenicity testing techniques are currently being used to assess the potential risk to man of exposure to diesel exhaust emissions. This paper examines general considerations of such systems, the types of in vitro tests currently available, the advantages and disadvantages of each cell line and type of test, the limitations of in vitro techniques, the alternative human cell lines that could be utilized for diesel health effects studies, and recommendations for future research employing in vitro methods.     

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.     

Measurement of unregulated emissions: Some heavy duty diesel engine results

Development of analytical capabilities to evaluate unregulated emissions are discussed. The sampling and analysis methods along with some problem areas are included. Results obtained on three heavy-duty diesel engines are reported. Emphasis was placed on analysis of the particulate fraction including the solvent extractable material. Preliminary experiments suggest appreciable quantities of organic extract and BaP pass through the primary filter. Other species analyzed to obtain a baseline for emission reduction research included aldehydes, sulfates, sulfur dioxide, ammonia, hydrogen sulfide, and hydrogen cyanide. Of primary concern is the proper assessment of the results.     

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.     

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.     

In vivo detection of mutagenic effects of diesel exhaust by short-term mammalian bioassays

Male Chinese hamsters were exposed to diesel exhaust and clean air for six months at the Center Hill Facility of the U.S. Environmental Protection Agency in Cincinnati, Ohio. The animals were kept in specially constructed inhalation chambers and exposed to clean air or diesel exhaust for eight hours daily. The animals were sacrificed and slides prepared to study the mutagenic effects of diesel exhaust by four in vivo short term mammalian bioassays. Sperm morphology bioassay revealed a 2.67-fold increase in sperm abnormalities in the animals exposed to diesel exhaust as compared to those exposed to fresh air. Micronucleus bioassay revealed a 50% increase in the number of micronuclei in polychromatic erythrocytes obtained from animals exposed to diesel exhuast. However, no increase in sister chromatid exchange or chromosomal abnormalities was observed in bone marrow cells of animals treated with diesel exhaust. During these studies a decrease in mitotic index was observed in animals treated with diesel exhaust.     

Genotoxicity of syrian hamster lung cells treated in vivo with diesel exhaust particulates

Polycyclic aromatic hydrocarbons extracted and concentrated from diesel exhaust particulates have been shown to be mutagenic and carcinogenic, but attempts to induce pulmonary tumors through chronic inhalation of diesel exhaust by experimental animals have failed. We have attempted to resolve this incongruity by measuring chromosomal damage in lung tissue of chronically exposed hamsters, using the highly sensitive test for genotoxic chemical agents, sister chromatid exchange (SCE) analysis. To determine the degree of responsiveness of the test system to both diesel exhaust particulates and benzo(a)pyrene (BaP), these agents were instilled intratracheally into anesthetized hamsters as suspensions in 0.25 ml volumes of Hank's balanced salt solution (HBSS). Lung tissues from these animals were subsequently cultured in vitro and chromosomes from the resulting cell divisions were scored for exchanges of chromatin between sister chromatids. Control animals, treated weekly with 0.25 ml of BSS for 10 weeks, showed an average value of 12 SCE's per cell, while animals treated weekly with 200 ng BaP over a 10-week period showed an average of 17 SCE's per cell. HBSS, given as a single treatment, also produced an average of 12 SCE's per cell in control animals, but animals treated with a single instillation of 12.5 μg BaP showed an average SCE value of 19. These data confirmed that the procarcinogen BaP can be metabolically activated by lung cells in vivo and also demonstrated the efficacy of using this technical approach to study the effect of chemical mutagens that enter the lungs. Diesel exhaust particulates, administered in a range from 0 to 20 mg per hamster over a 24 h exposure period, produced a linear SCE dose-response ranging from 12 to 26 SCE's per metaphase. This curve suggested that a concentration of 3 mg of diesel particulates per hamster would not produce a statistically significant increase in SCE's above control values. One group of 8 hamsters, chronically exposed to diesel exhaust particulates for 3 months showed an average of 12 SCE's per cell. This was equivalent to a set of 5 control animals which also showed an average of 12 SCE's per cell. Although the scope of this study was limited, the data demonstrated that diesel exhaust particulates can induce genotoxic damage but a 3-month exposure to 6 mg/m³ of diesel exhaust particulates was insufficient to produce measurable mutagenic changes in lung cells. This negative response is consistent with the results from other studies in which similar exposures failed to produce pulmonary tumors.     

Time and cost characterisation of emissions from non-road diesel equipment for infrastructure projects

The construction process contributes to pollutant emissions, particularly through the operation of diesel- and gasoline-powered equipment. In the past decade, a series of investigations were undertaken to quantify these emissions for a variety of non-road construction equipment performing different activities and undergoing different duty cycles, and a model to estimate quantities of six types of pollutant was developed. This paper uses that model to estimate emissions for four street and utility construction projects which no one has done previously. We combined information from company records with standard construction industry manuals to estimate total emissions for the projects and to examine the pollution patterns and magnitudes. The street construction projects all had similar emission profiles with a large peak at the beginning and a steady output of emissions throughout the duration of the project. For example, in two of the projects studied, half of all CO₂ emissions were produced before the projects were 40% completed. Results showed that demolition and earthwork are the activities with the largest contribution. The equipment types with the largest contribution are backhoes, front-end loaders, bulldozers and trenchers. Trenchers, for example, produced 30% of all emissions on the projects on which they were used.     

An industrial hygiene characterization of exposure to diesel emissions in an underground coal mine

Problems exist in the United States' effort to achieve energy self-sufficiency. Increasing coal production to assure energy self-sufficiency is a prime problem for the rest of the century and beyond. The use of diesels in underground coal mines has been suggested as a mining method to aid in this needed, increased production. Many questions exist about the effects on humans in such environments. NIOSH Division of Respiratory Disease Studies has undertaken a research effort to characterize the environments of existing diesel coal mines. The results of one of the studies will be presented. Preliminary assessments of carbon monoxide, nitrogen dioxide, C₁-C₅ aldehydes and organic acids, aliphatic hydrocarbons, sulfates, total and respirable dust, and polycyclic aromatic hydrocarbons (PAH) are presented. Nitrogen dioxide and total aldehydes are suggested as possible species to quantify diesel exposure.     

Interactions between diesel emissions and gaseous copollutants in photochemical air pollution: Some health implications

A complete assessment of the health effects of diesel emissions must take into account the possible chemical transformations (and associated biological impacts) of particulate organic matter (POM) due to reactions with the many gaseous copollutants which have now been unambiguously demonstrated to be present in atmospheres burdened by photochemical air pollution. These copollutants include the “trace” species, nitric (HNO₃) and nitrous (HONO) acids, the nitrate radical (NO₃), formaldehyde (H₂CO) and formic acid (HCOOH), as well as the criteria pollutants, ozone (O₃) and nitrogen dioxide (NO₂). Techniques for establishing the atmospheric concentrations of the trace pollutants (and their spatial and temporal variations) are briefly described, and we present results of investigations into the reactions of polycyclic aromatic hydrocarbons (PAH) coated on filters and exposed to ambient concentrations of O₃ and NO₂. Environmental health implications of these results are discussed and include the potential for sampling “artifacts” and their possible effects on the correlation (or lack thereof) between ambient PAH levels and urban lung cancer rates, as well as the problems associated with understanding the appropriate POM “dose” to be employed in animal testing and assessments of impacts on human health.     

Metaphase analysis, micronuclei assay, and urinary mutagenicity assay of mice exposed to diesel emissions

Female Swiss mice were exposed 8 h/day to diesel exhaust for 1, 3, and 7 weeks. Urine was collected overnight for 4 days prior to sacrifice while the mice continued to be exposed for eight hours during the day. The presence of mutagens was determined by the Ames Salmonella test. One hour prior to sacrifice each mouse received 1 mg/kg colcemide. After sacrifice, the marrow from each femur was obtained. The marrow from one femur was used to prepare slides for metaphase analysis and the other for micronuclei assay. Other mice received IP 50 mg/kg cyclophosphamide 24 h prior to sacrifice or 1 μmole/kg benzo(a)pyrene in each of four daily doses prior to sacrifice and served as positive controls. The Ames Salmonella assay of the unconcentrated urine after 1, 3, and 7 weeks and concentrated urine after 7 weeks exposure to diesel exhaust did not significantly vary from clean air controls. In the micronucleus test, and metaphase analysis, cyclophosphamide produced a strong positive response and the 7 week diesel exposure was not different from clean air controls.     

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.     

Benzo[a]pyrene metabolism in mice exposed to diesel exhaust: II. Metabolism and excretion

In this study we examined the effect of diesel exhaust (DE) exposure on in vivo metabolism of benzo[a]pyrene (BaP). DE-exposed and unexposed A/Jax mice of group B were instilled intratracheally with ³H-BaP. At each time point of 2, 24, and 168 h after instillation five mice were killed and the liver, lungs, and testes were removed and frozen. Aliquots of the organs were homogenized in 2 ml water and each received 3 volumes of cold ethanol. Radioactivity in supernatant and precipitate was measured. The supernatant extracts were subjected to HPLC analysis on ALOX-T and on Zorbax ODS. The ALOX-T method was a modification of Autrup's procedure for conjugate assay (Biochem. Pharmacol.28, 1727, 1979). Fractions were (a) free BaP; (b) nonconjugated primary metabolites; (c) sulfate conjugates; (d) glucuronides, glutathiones, and other conjugates. By 2 h after instillation primary metabolites were found in liver and lung, but very little was conjugated. The unconjugated BaP was mainly in the form of free BaP and phenolic metabolite(s). The lungs of DE-exposed mice had less capacity to dispose of “bound” BaP 1 week after instillation.     

Benzo[a]pyrene metabolism in mice exposed to diesel exhaust: I. Uptake and distribution

In this study we examined the effect of diesel exhaust (DE) exposure on the disposition of a typical polycyclic aromatic hydrocarbon. DE-exposed and nonexposed A/Jax mice were divided into three groups and each mouse instilled intratracheally with benzo[a]pyrene (BaP). One group (A) received ¹⁴C-BaP, and at intervals of 2, 24, and 168 h, three mice from the group were killed and quick frozen for whole body autoradiography. Sagittal sections were cut at 0.5 mm intervals and autoradiograms prepared. Adjacent sections were studied so that radioactive areas were matched to specific organs. The second group (B) received ³H-BaP and at 2, 24, and 168 h these mice were killed. Livers, lungs, and testes were weighed and frozen. From these tissues metabolites were analyzed; these data are reported in the next paper. Histofluorescent examination of tissues from mice instilled with nonradioactive BaP (group C) confirmed that BaP was present in the lung. The autoradiography data are the basis for elucidating the BaP distribution in the mouse. Within 2 h after instillation radioactivity was detected in the entire animal, with most in lungs, liver, and GI tract. By 24 h after instillation considerable radioactivity had redistributed to the GI tract. At 168 h after instillation only a trace of label was found in the GI mucosa.     

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.