A controlled inhalation diesel exhaust exposure facility with dynamic feedback control of PM concentration

A facility has been assembled that provides a controlled inhalation exposure to freshly diluted and mixed diesel exhaust using a diesel engine under load and a two-stage exhaust dilution system with dynamic feedback control. The concentrations of particulate matter less than 2.5 microm in diameter (PM(2.5)), particulate carbon, and gaseous pollutants including carbon monoxide and oxides of nitrogen (NOx) have been characterized and the exposure conditions have been found to be both stable and reproducible. Control of the PM(2.5) concentration at intended levels relies on the relatively linear relationship between particle light scattering and exhaust particle mass concentration. While the exposure system does not entirely replicate diesel exhaust conditions in the atmosphere due to the relatively low ratio of nitrogen dioxide to total NOx, the fine particulate matter size distributions are quite similar to those of aged diesel exhaust. The facility enables study of the relationship between diesel exhaust and cardiovascular and respiratory health effects in human and animal models.     

A Controlled Inhalation Diesel Exhaust Exposure Facility with Dynamic Feedback Control of PM Concentration

A facility has been assembled that provides a controlled inhalation exposure to freshly diluted and mixed diesel exhaust using a diesel engine under load and a two-stage exhaust dilution system with dynamic feedback control. The concentrations of particulate matter less than 2.5 μ m in diameter (PM_2.5), particulate carbon, and gaseous pollutants including carbon monoxide and oxides of nitrogen (NO_x) have been characterized and the exposure conditions have been found to be both stable and reproducible. Control of the PM_2.5 concentration at intended levels relies on the relatively linear relationship between particle light scattering and exhaust particle mass concentration. While the exposure system does not entirely replicate diesel exhaust conditions in the atmosphere due to the relatively low ratio of nitrogen dioxide to total NO_x, the fine particulate matter size distributions are quite similar to those of aged diesel exhaust. The facility enables study of the relationship between diesel exhaust and cardiovascular and respiratory health effects in human and animal models.     

Responses of cultured human airway epithelial cells treated with diesel exhaust extracts will vary with the engine load

Epidemiologic evidence suggests that increased morbidity and mortality are associated with the concentrations of ambient air particulate matter (PM). Many sources contribute to the particulate fraction of ambient air pollution, including diesel exhaust particulates (DEP). Diesel exhaust also contributes gas-phase pollutants to the atmosphere, and gaseous copollutants may influence the toxicity of PM. The composition of diesel exhaust varies greatly depending on the engine load conditions as well as other factors. To determine whether different diesel exhaust composition can affect lung cell resposes, the effects of of diesel exhaust extracts derived from different engine loads were examined on normal human bronchial epithelial cells (NHBE) in vitro. Diesel exhaust was collected into chilled impingers containing phosphate-buffered saline (PBS). Cultured NHBE cells were treated with 0 to 500 microg/well extract from approximately 0% engine load (termed low load or LL) or extract from approximately 75% engine load (termed high load or HL) for 24 h. The HL extract was cytotoxic at 500 microg compared to controls as measured by (51)Cr release. Production of the neutrophil chemotaxin interleukin 8 (IL-8) was decreased 4.7-fold in cells treated with 500 microg LL extract, whereas cells treated with 500 microg HL extract showed a 2.4-fold increase in IL-8 release. Production of the inflammatory and immune system mediator prostaglandin E(2) (PGE(2)) was increased up to 2.5-fold in cells treated with HL extract, but unchanged with other treatments. Melittin stimulation of cells showed that the LL extract had an inhibitory effect on PGE(2) release at 500 microg. Differences in carbonyl content of the extracts were found by high performance liquid chromatography/mass spectroscopy HPLC/MS, with the HL extract having more intermediate size carbonyls (i.e. with six to nine carbons). The data suggest that the response of NHBE cells to treatment with diesel exhaust will vary depending on the constituent components of the exhaust.     

A trivalent dimethylarsenic compound, dimethylarsine iodide, induces cellular transformation, aneuploidy, centrosome abnormality and multipolar spindle formation in Syrian hamster embryo cells

The abilities of dimethylarsine iodide (DMI), a model compound of trivalent dimethylarsenicals, to induce cellular transformation, aneuploidy, centrosome abnormality, and multipolar spindle formations were investigated using the Syrian hamster embryo (SHE) cell model. Cellular growth was decreased in a concentration-dependent manner by treatment with DMI at concentrations over 0.1 microM. Treatment with DMI at concentrations from 0.1 to 1.0 microM induced morphological transformation in SHE cells. The transforming activity of DMI, determined by the frequency of morphologically transformed colonies, was approximately 30 times higher than that induced by treatment with the same concentration of sodium arsenite. Flow cytometry suggested an increase in the aneuploid population caused by DMI, as shown by the appearance of hypo-2N, hypo-4N and hypo-8N. DMI also caused abnormal staining of gamma-tubulin, indicating loss of centrosome integrity and a resultant induction of multipolar spindles in mitotic cells. Mitotic cells with centrosomes that coalesced partly at the cell periphery, not the cell center, were detected as early changes that resulted in multipolar spindles. These findings indicate that DMI has transforming activity in SHE cells. Moreover, the results suggest the importance of centrosome abnormalities as a causal change of DMI-induced aneuploidy.     

Comparison of cytotoxicity and genotoxicity induced by the extracts of methanol and gasoline engine exhausts.

Gasoline engine exhaust has been considered a major source of air pollution in China, and methanol is considered as a potential substitute for gasoline fuel. In this study, the genotoxicity and cytotoxicity of organic extracts of condensate, particulate matters (PM) and semivolatile organic compounds (SVOC) of gasoline and absolute methanol engine exhaust were examined by using MTT assay, micronucleus assay, comet assay and Ames test. The results have showed that gasoline engine exhaust exhibited stronger cytotoxicity to human lung carcinoma cell lines (A549 cell) than methanol engine exhaust. Furthermore, gasoline engine exhaust increased micronucleus formation, induced DNA damage in A549 cells and increased TA98 revertants in the presence of metabolic activating enzymes in a concentration-dependent manner. In contrast, methanol engine exhaust failed to exhibit these adverse effects. The results suggest methanol may be used as a cleaner fuel for automobile.     

Identification of mammalian cell genotoxins in respirable diesel exhaust particles by bioassay-directed chemical analysis

A bioassay-directed chemical analysis which consists of mammalian cell bioassays (comet assay, CBMN assay and EROD-microbioassay) in conjunction with analytical measurements was performed to identify the most biologically active compounds of the diesel exhaust particulate matters (DEPs) on mutagenic activity. These bioassay systems were suitable to estimate the mammalian genotoxic potentials of pollutants present in low concentrations in limited environmental samples, as is the case with DEPEs. The results from mutagenic assay showed that the aromatic and slightly polar fraction of DEPs induced chromosomal damage and DNA breakage in a non-cytotoxic dose. It was also revealed that indirect-acting mutagens may mainly contribute to the mutagenic effect of aromatic fraction via the enzyme metabolism system. In the aromatic fraction, several indirect-acting mutagenic PAHs such as dibenzo(a,h)anthracene, chrysene, and 1,2-benzanthracene were detected by GC-MS and the complex mixture effect of this fraction was quantified in terms of its biological-TCDD equivalent concentration (bio-TEQ) which was 32.82 bio-TEQ ng/g-DEPs by EROD-microbioassay. Conclusively, we confirmed that indirect-acting mutagens contained in aromatic fraction may be important causatives of the genotoxicity of extracts of DEPs by integrating the results obtained from a mammalian cell bioassay-directed fractionation.     

Effects of environmental air pollutants on CFTR expression and function in human airway epithelial cells

The airway epithelium is exposed to a variety of air pollutants, which have been associated with the onset and worsening of respiratory diseases. These air pollutants can vary depending on their composition and associated chemicals, leading to different molecular interactions and biological effects. Mucociliary clearance is an important host defense mechanism against environmental air pollutants and this process is regulated by various ion transporters including the cystic fibrosis transmembrane conductance regulator (CFTR). With evidence suggesting that environmental air pollutants can lead to acquired CFTR dysfunction, it may be possible to leverage therapeutic approaches used in cystic fibrosis (CF) management. The aim of our study was to test whether environmental air pollutants tobacco smoke extract, urban particulate matter, and diesel exhaust particles lead to acquired CFTR dysfunction and whether it could be rescued with pharmacological interventions. Human airway epithelial cells (Calu-3) were exposed to air pollutant extracts for 24 h, with and without pharmacological interventions, with readouts of CFTR expression and function. We demonstrate that both tobacco smoke extract and diesel exhaust particles led to acquired CFTR dysfunction and that rescue of acquired CFTR dysfunction is possible with pharmacological interventions in diesel exhaust particle models. Our study emphasizes that CFTR function is not only important in the context of CF but may also play a role in other respiratory diseases impacted by environmental air pollutants. In addition, the pharmacological interventions approved for CF management may be more broadly leveraged for chronic respiratory disease management.     

ACTIVATION OF CELLULAR ONCOGENES IN HUMAN DIPLOID CELL STRAIN (KMB-13) CELLS BY SCOOTER EXHAUST PARTICULATE MATTER EMISSIONS

Scooter exhaust particulate matter emissions were found to be mutagenic and to induce potential carcinogenicity. To further explore the mechanisms of mutagenicity and potential carcinogenicity of scooter exhaust particulate matter emissions, immunohistochemistry assays were carried out to detect the expression of some oncogenes and tumor suppressor genes in human diploid cell strain (KMB-13) cells, which were morphologically transformed by the organic extracts of scooter exhaust particulate matter emissions. An ABC diagnostic kit was used to investigate the expression of c-myc, p21, p53, and p16 proteins in the transformed cells. The c-myc and p21 proteins showed marked positive staining compared to control. The data suggest that the mutagenicity and potential carcinogenicity of the scooter exhaust particulate matter emissions correlate with the activation of some cellular oncogenes.     

Chronic Diesel Exhaust Exposures of Rats Demonstrate Concentration and Time-Dependent Effects on Pulmonary Inflammation

Long-term, repeated exposure to particles in air pollution increases the risk for chronic respiratory diseases and cardiorespiratory mortality. The biological linkages remain poorly understood in chronic exposure to particle matter. To elucidate and verify these linkages, we investigated long-term exposure to diesel emission with respect to dose dependence and the effect of components without particles from diesel emission in rats. Wistar rats were exposed to filtered air (C group), diesel exhaust at low (L group), medium (M group), and high level (H group), or at a medium concentration diesel exhaust without particulate matter (MG group), for 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo. Anesthetized animals were sacrificed and bronchoalveolar lavage (BAL) fluid from the lung and blood from the right ventricle were collected. Various biomarkers of inflammation and components of mucus and surfactant were determined. Changes in total cell counts and cell differentiation, total protein, mucus and surfactant components, and prostaglandin E 2 in BAL fluid, but not biomarkers in plasma, showed statistical differences among the C, L, M, and H groups during the experimental period. The changes in these biomarkers in the H group were greater than those in the M group, whereas those in the L group showed no significant changes compared with those in the C group during the experimental period. The onset of significant changes in inflammatory cells and these biomarkers in BAL fluid for the M and H groups was at 6 to 12 mo of exposure. The maximum level was reached at 12 to 18 mo of exposure. Although BAL prostaglandin E 2 decreased significantly at 6 mo of exposure in the M and H groups, this trend was not observed in the C and L groups. Animals exposed to a medium level of diesel exhaust without particulate matter showed significantly less inflammatory cells and various biomarkers in BAL fluid than animals exposed to the same level of diesel exhaust with particulate matter during the experimental period. These findings suggest that biological response to inhaled particles is aggravated during chronic exposure to diesel exhaust dose-dependently. Inflammation and overproduction of mucus and surfactant components reached a plateau at 12 or 18 mo of exposure during a 24-mo experimental period. No adverse effect of particles (less than 1.0 mg particles/m 3 of diesel emission) was observed in these rats. However, our data suggest that particulate matter plays an important role during development of chronic lung injury induced by diesel emission exhaust.     

In vitro cytokine release from rat type II pneumocytes and alveolar macrophages following exposure to JP-8 jet fuel in co-culture

Exposure of Chinese hamster V79 cells to extracts of airborne pollutants induced formation of multipolar or incomplete mitotic spindles. To find out whether overexpression of the HSP70 chaperone protein could protect spindles against airborne toxins we constructed V79 cells stably transfected with an expression vector containing rat heat-inducible hsp70.1 gene under the control of a constitutive CMV promoter. When cells were incubated with extracts of airborne pollutants (5–20 μg/ml) no protective effect of the HSP70 protein against mitotic spindle damage was observed. Moreover, at 20 μg/ml of extracts of airborne toxins the frequency of mitotic malformations was even higher in HSP70-overexpressing cells than in control ones. Extracts of airborne pollutants of 50 μg/ml blocked the formation of mitotic figures both in control and HSP70-overexpressing cells and led to destruction of cell nuclei. However, the HSP70-overproducing cells exhibited higher survival rates when exposed to heat shock and airborne toxins than the control ones, as determined by MTT assay. This suggests that HSP70 overexpression—a frequent feature of cancer cells—should be considered as a factor facilitating survival of cells with damaged mitotic spindles and aberrantly segregated chromosomes.     

Chronic diesel exhaust exposures of rats demonstrate concentration and time-dependent effects on pulmonary inflammation

Long-term, repeated exposure to particles in air pollution increases the risk for chronic respiratory diseases and cardiorespiratory mortality. The biological linkages remain poorly understood in chronic exposure to particle matter. To elucidate and verify these linkages, we investigated long-term exposure to diesel emission with respect to dose dependence and the effect of components without particles from diesel emission in rats. Wistar rats were exposed to filtered air (C group), diesel exhaust at low (L group), medium (M group), and high level (H group), or at a medium concentration diesel exhaust without particulate matter (MG group), for 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo. Anesthetized animals were sacrificed and bronchoalveolar lavage (BAL) fluid from the lung and blood from the right ventricle were collected. Various biomarkers of inflammation and components of mucus and surfactant were determined. Changes in total cell counts and cell differentiation, total protein, mucus and surfactant components, and prostaglandin E(2) in BAL fluid, but not biomarkers in plasma, showed statistical differences among the C, L, M, and H groups during the experimental period. The changes in these biomarkers in the H group were greater than those in the M group, whereas those in the L group showed no significant changes compared with those in the C group during the experimental period. The onset of significant changes in inflammatory cells and these biomarkers in BAL fluid for the M and H groups was at 6 to 12 mo of exposure. The maximum level was reached at 12 to 18 mo of exposure. Although BAL prostaglandin E(2) decreased significantly at 6 mo of exposure in the M and H groups, this trend was not observed in the C and L groups. Animals exposed to a medium level of diesel exhaust without particulate matter showed significantly less inflammatory cells and various biomarkers in BAL fluid than animals exposed to the same level of diesel exhaust with particulate matter during the experimental period. These findings suggest that biological response to inhaled particles is aggravated during chronic exposure to diesel exhaust dose-dependently. Inflammation and overproduction of mucus and surfactant components reached a plateau at 12 or 18 mo of exposure during a 24-mo experimental period. No adverse effect of particles (less than 1.0 mg particles/m(3) of diesel emission) was observed in these rats. However, our data suggest that particulate matter plays an important role during development of chronic lung injury induced by diesel emission exhaust.     

Monitoring the inflammatory potential of exhaust particles from passenger cars in mice

This study presents different research techniques linked together to improve our understanding of the particulate matter (PM) impacts on health. PM samples from the exhaust of different vehicles were collected by a versatile aerosol concentration enrichment system (VACES). Waterborne PM samples were collected with this technique, thus retaining the original physicochemical characteristics of aerosol particles. PM samples originated from a gasoline Euro 3 car and two diesel cars complying with the Euro 2 and Euro 4 standards, respectively. The Euro 2 diesel car operated consecutively on fossil diesel and biodiesel. The Euro 4 car was also retrofitted with a diesel particle filter. In total, five vehicle configurations and an equal number of samples were examined. Each sample was intratracheally instilled to 10 mice at two different dose levels (50 and 100 μL). The mice were analyzed 24 h after instillation for acute lung inflammation by bronchoalveolar lavage and also for hematological changes. Results show that a moderate but still significant inflammatory response is induced by PM samples, depending on the vehicle. Several organic and inorganic species, including benz(a)anthracene, chrysene, Mn, Fe, Cu, and heavy polycyclic aromatic hydrocarbons (PAHs), as well as the reactive oxygen species content of the PM suspensions are correlated to the observed responses. The study develops conceptual dose-response functions for the different vehicle configurations. These demonstrate that inflammatory response is not directly proportional to the mass dose level of the administered PM and that the relative toxicity potency depends on the dosage level.     

Short-Term Exposure to Diesel Exhaust Induces Nasal Mucosal Hyperresponsiveness to Histamine in Guinea Pigs

The increasing prevalence of allergic rhinitis in many countriesis becoming a social problem. It is important to determine whetherair pollutants are related to the increase in the prevalencerate of allergic rhinitis or not. In this respect, it is necessaryto elucidate whether exposure to air pollutants affects thenasal mucosa and causes nasal mucosal hyperresponsiveness tochemical mediators released by antigen-antibody reactions. Aprevious study revealed that diesel exhaust participates arepotent in augmenting increases in nasal congestion and nasalsecretion induced by histamine (T. Kobayashi and T. Ito, 1995,Fundam. Appl. Toxicol. 27,195–202). In the present study,using a rhinitis model of guinea pigs, we investigated whethershort-term (3-hr) exposure to diesel exhaust induces nasal mucosalhyperresponsiveness to histamine. Guinea pigs of each groupwere exposed to filtered air or to a low or high concentrationof diesel exhaust (1 and 3.2 mg/m³ particulates in diluted dieselexhaust, respectively) for 3 hr. After diesel exhaust exposure,sneezing frequency, nasal secretion from the nostril, and intranasalairway resistance induced by histamine were measured as indicesof sneezing response, rhinorrhea, and nasal congestion, respectively.Short-term exposure to a low or high concentration of dieselexhaust itself did not induce sneezing, nasal secretion, ornasal congestion. However, short-term exposure to a high concentrationof diesel exhaust augmented sneezing and nasal secretion, butnot nasal congestion, induced by histamine. In conclusion, short-termexposure to diesel exhaust potently induces nasal mucosal hyperresponsiveness.     

Biodiesel versus diesel exposure: Enhanced pulmonary inflammation,oxidative stress,and differential morphological changes in the mouse lung

The use of biodiesel (BD) or its blends with petroleum diesel (D) is considered to be a viable approach to reduce occupational and environmental exposures to particulate matter (PM). Due to its lower particulate mass emissions compared to D, use of BD is thought to alleviate adverse health effects. Considering BD fuel is mainly composed of unsaturated fatty acids, we hypothesize that BD exhaust particles could induce pronounced adverse outcomes, due to their ability to readily oxidize. The main objective of this study was to compare the effects of particles generated by engine fueled with neat BD and neat petroleum-based D. Biomarkers of tissue damage and inflammation were significantly elevated in lungs of mice exposed to BD particulates. Additionally, BD particulates caused a significant accumulation of oxidatively modified proteins and an increase in 4-hydroxynonenal. The up-regulation of inflammatory cytokines/chemokines/growth factors was higher in lungs upon BD particulate exposure. Histological evaluation of lung sections indicated presence of lymphocytic infiltrate and impaired clearance with prolonged retention of BD particulate in pigment laden macrophages. Taken together, these results clearly indicate that BD exhaust particles could exert more toxic effects compared to D.     

Obese mice are resistant to eosinophilic airway inflammation induced by diesel exhaust particles

Particulate matter can exacerbate respiratory diseases such as asthma. Diesel exhaust particles are the substantial portion of ambient particulate matter with a <2.5 µm diameter in urban areas. Epidemiological data indicate increased respiratory health effects of particulate matter in obese individuals; however, the association between obesity and diesel exhaust particle‐induced airway inflammation remains unclear. We aimed to investigate the differences in susceptibility to airway inflammation induced by exposure to diesel exhaust particles between obese mice (db/db) and lean mice (db/+m). Female db/db and db/+m mice were intratracheally administered diesel exhaust particles or vehicle every 2 weeks for a total of seven times. The cellular profile of bronchoalveolar lavage fluid and histological changes in the lungs were assessed and the lungs and serum were analyzed for the generation of cytokines, chemokines and soluble intercellular adhesion molecule 1. Diesel exhaust particle exposure‐induced eosinophilic infiltration in db/+m mice accompanied by T‐helper 2 cytokine, chemokine and soluble intercellular adhesion molecule 1 expression in the lungs. In contrast, it induced mild neutrophilic airway inflammation accompanied by elevated cytokines and chemokines in db/db mice. The lungs of db/db mice exhibited decreased expression of eosinophil activators/chemoattractants such as interleukin‐5, interleukin‐13 and eotaxin compared with those of db/+m mice. In addition, serum eotaxin and monocyte chemotactic protein‐1 levels were significantly higher in db/db mice than in db/+m mice. In conclusion, obesity can affect susceptibility to diesel exhaust particle‐induced airway inflammation, which is possibly due to differences in local and systemic inflammatory responses between lean and obese individuals. Copyright © 2013 John Wiley & Sons, Ltd.     

Cytotoxicity of diesel exhaust particle extract--a comparison among five diesel passenger cars of different manufactures

The cytotoxicity of the dichloromethane extracts of diesel exhaust particles from passenger cars of different manufactures was studied in cultured chinese hamster ovary cells. While exhaust particles from diesel cars of the same make and model yielded extracts of similar cytotoxicity, those from cars of different manufactures yielded extracts with a 3-fold difference in cytotoxicity. Using data on the percentages of extractable organic chemicals and total exhaust particulate emission rates, the emission rate of cytotoxin into the environment from the different cars were calculated. Of the 3 factors that could affect the emission rate of cytotoxins (cytotoxicity of the extractable chemicals, amount of cytotoxins per particle, and particulate emission rate), the differences in particulate emission rates were found to be the predominant factors leading to the differences in the emission rate of cytotoxins. Our findings indicate the need to consider other chemical and physical data, not just the activities of the extracts, when the potential health risk due to the exhaust emissions of different automobiles are compared.     

Comparison of immunotoxic effects induced by the extracts from methanol and gasoline engine exhausts in vitro

Gasoline engine exhaust has been considered as a major source of air pollution in China. Due to lower cyto- and geno-toxicity effects of methanol engine exhaust, methanol is regarded as a potential substitute for gasoline. We have previously compared cyto- and geno-toxicities of gasoline engine exhaust with that of methanol engine exhaust in A549 cells (Zhang et al., 2007).To characterize the immunotoxic effects for gasoline and methanol engine exhausts in immune cell, in this study, we further compared effects of gasoline and methanol engine exhausts on immune function in RAW264.7 cell and rabbit alveolar macrophages. Results showed that both gasoline and methanol engine exhaust could evidently inhibit RAW264.7 cell proliferation, promote RAW264.7 cell apoptosis, decrease E-rosette formation rate and inhibit anti-tumor effects of alveolar macrophages, at the same time, these effects of gasoline engine exhaust were far stronger than those of methanol engine exhaust. In addition, gasoline engine exhaust could significantly inhibit activities of ADCC of alveolar macrophages, but methanol engine exhaust could not. These results suggested that both gasoline and methanol engine exhausts might be immunotoxic atmospheric pollutants, but some effects of gasoline engine exhaust on immunotoxicities may be far stronger than that of methanol engine exhaust.