Part 1. Assessment of carcinogenicity and biologic responses in rats after lifetime inhalation of new-technology diesel exhaust in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to 2007-compliant new-technology diesel exhaust (NTDE*). The a priori hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions "... will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used ... although some biological effects may occur." This hypothesis was tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay. Indicators of pulmonary toxicity in rats were measured after 1, 3, 12, 24, and 28-30 months of exposure. Similar indicators of pulmonary toxicity were measured in mice, as an interspecies comparison of the effects of subchronic exposure, after 1 and 3 months of exposure. A previous HEI report (Mauderly and McDonald 2012) described the operation of the engine and exposure systems and the characteristics of the exposure atmospheres during system commissioning. Another HEI report described the biologic responses in mice and rats after subchronic exposure to NTDE (McDonald et al. 2012). The primary motivation for the present chronic study was to evaluate the effects of NTDE in rats in the context of previous studies that had shown neoplastic lung lesions in rats exposed chronically to traditional technology diesel exhaust (TDE) (i.e., exhaust from diesel engines built before the 2007 U.S. requirements went into effect). The hypothesis was largely based on the marked reduction of diesel particulate matter (DPM) in NTDE compared with emissions from older diesel engine and fuel technologies, although other emissions were also reduced. The DPM component of TDE was considered the primary driver of lung tumorigenesis in rats exposed chronically to historical diesel emissions. Emissions from a 2007-compliant, 500-horsepower-class engine and after treatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors. components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hours/day (overnight, during the rats' most active period), 5 days/week. Responses to exposure were evaluated via hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, histopathology, and pulmonary function. The exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components from exhaust were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of DPM and volatile and semi-volatile organic compounds (VOCs and SVOCs). Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to NTDE. The major outcome of this study was the absence of pre-neoplastic lung lesions, primary lung neoplasia, or neoplasia of any type attributable to NTDE exposure. The lung lesions that did occur were minimal to mild, occurred only at the highest exposure level, and were characterized by an increased number and prominence of basophilic epithelial cells (considered reactive or regenerative) lining distal terminal bronchioles, alveolar ducts, and adjacent alveoli (termed in this report "Hyperplasia; Epithelial; Periacinar"), which often had a minimal increase in subjacent fibrous stroma (termed "Fibrosis; Interstitial; Periacinar"). Slight epithelial metaplastic change to a cuboidal morphology, often demonstrating cilia, was also noted in some animals (termed "Bronchiolization"). In addition to the epithelial proliferation, there was occasionally a subtle accumulation of pulmonary alveolar macrophages (termed "Accumulation; Macrophage") in affected areas. The findings in the lung progressed slightly from 3 to 12 months, without further progression between 12 months and the final sacrifice at 28 or 30 months. In addition to the histologic findings, there were biochemical changes in the lung tissue and lavage fluid that indicated mild inflammation and oxidative stress. Generally, these findings were observed only at the highest exposure level. There was also a mild progressive decrease in pulmonary function, which was more consistent in females than males. Limited nasal epithelial changes resulted from NTDE exposure, including increases in minor olfactory epithelial degeneration, hyperplasia, and/or metaplasia. Increases in these findings were present primarily at the highest exposure level, and their minor and variable nature renders their biologic significance uncertain. Overall, the findings of this study demonstrated markedly less severe biologic responses to NTDE than observed previously in rats exposed similarly to TDE. Further, the effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors.

The National Environmental Respiratory Center (NERC) experiment in multi-pollutant air quality health research: I. Background, experimental strategy and critique.

The National Environmental Respiratory Center Program was initiated as an experiment to explore strategies for identifying the components of complex air pollution mixtures that cause health effects associated statistically with air pollution. A strategy involving multivariate analysis of a composition-concentration-response database was adopted. A novel database was created by exposing rodents daily for up to six months to one of four combustion-related mixtures and measuring respiratory, cardiovascular and general toxicological responses after one week or six months of exposure. The mixtures included multiple concentrations of diesel and gasoline engine exhaust, hardwood smoke and simulated downwind coal combustion emissions. After reporting the biological effects of each mixture and comparing effects among them, 47 significant effects were selected for multiple additive regression tree analysis to identify putative causal components. Although the four mixtures provided a database marginally sufficient for the analysis, the results suggested the putative causes of 19 significant effects with acceptable confidence. This article describes and critiques the Program and its strategy. The integrated results are presented in two accompanying papers, and mixture-specific results were presented in preceding papers, which are cited. The experiment demonstrated the potential utility of the general approach and identified certain cause-effect relationships for confirmatory studies. A follow-up study provided support for causation by the components implicated for one of those relationships. The advantages and disadvantages of the Program's management and funding strategies are discussed.

The National Environmental Respiratory Center (NERC) experiment in multi-pollutant air quality health research: III. Components of diesel and gasoline engine exhausts, hardwood smoke and simulated downwind coal emissions driving non-cancer biological responses in rodents.

An approach to identify causal components of complex air pollution mixtures was explored. Rats and mice were exposed by inhalation 6 h daily for 1 week or 6 months to dilutions of simulated downwind coal emissions, diesel and gasoline exhausts and wood smoke. Organ weights, hematology, serum chemistry, bronchoalveolar lavage, central vascular and respiratory allergic responses were measured. Multiple additive regression tree (MART) analysis of the combined database ranked 45 exposure (predictor) variables for importance to models best fitting 47 significant responses. Single-predictor concentration-response data were examined for evidence of single response functions across all exposure groups. Replication of the responses by the combined influences of the two most important predictors was tested. Statistical power was limited by inclusion of only four mixtures, albeit in multiple concentrations each and with particles removed for some groups. Results gave suggestive or strong evidence of causation of 19 of the 47 responses. The top two predictors of the 19 responses included only 12 organic and 6 inorganic species or classes. An increase in red blood cell count of rats by ammonia and pro-atherosclerotic vascular responses of mice by inorganic gases yielded the strongest evidence for causation and the best opportunity for confirmation. The former was a novel finding; the latter was consistent with other results. The results demonstrated the plausibility of identifying putative causal components of highly complex mixtures, given a database in which the ratios of the components are varied sufficiently and exposures and response measurements are conducted using a consistent protocol.

Identification of chemical components of combustion emissions that affect pro-atherosclerotic vascular responses in mice.

Combustion emissions cause pro-atherosclerotic responses in apolipoprotein E-deficient (ApoE/⁻) mice, but the causal components of these complex mixtures are unresolved. In studies previously reported, ApoE⁻/⁻ mice were exposed by inhalation 6 h/day for 50 consecutive days to multiple dilutions of diesel or gasoline exhaust, wood smoke, or simulated "downwind" coal emissions. In this study, the analysis of the combined four-study database using the Multiple Additive Regression Trees (MART) data mining approach to determine putative causal exposure components regardless of combustion source is reported. Over 700 physical-chemical components were grouped into 45 predictor variables. Response variables measured in aorta included endothelin-1, vascular endothelin growth factor, three matrix metalloproteinases (3, 7, 9), metalloproteinase inhibitor 2, heme-oxygenase-1, and thiobarbituric acid reactive substances. Two or three predictors typically explained most of the variation in response among the experimental groups. Overall, sulfur dioxide, ammonia, nitrogen oxides, and carbon monoxide were most highly predictive of responses, although their rankings differed among the responses. Consistent with the earlier finding that filtration of particles had little effect on responses, particulate components ranked third to seventh in predictive importance for the eight response variables. MART proved useful for identifying putative causal components, although the small number of pollution mixtures (4) can provide only suggestive evidence of causality. The potential independent causal contributions of these gases to the vascular responses, as well as possible interactions among them and other components of complex pollutant mixtures, warrant further evaluation.

Part 1. Biologic responses in rats and mice to subchronic inhalation of diesel exhaust from U.S. 2007-compliant engines: report on 1-, 3-, and 12-month exposures in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to diesel exhaust (DE*) from 2007-compliant engines. The preliminary hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions ". . . will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used . . . although some biological effects may occur." This hypothesis is being tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay, measuring indicators of pulmonary toxicity in rats after 1, 3, 12, and 24-30 months of exposure (final time point depends on the survival of animals), and measuring similar indicators of pulmonary toxicity in mice after 1 and 3 months of exposure. This report provides results of exposures through 3 months in rats and mice. Emissions from a 2007-compliant, 500-horsepower-class engine and aftertreatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four treatment groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hr/dy (overnight), 5 dy/wk. Rats were evaluated for hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, and histopathology after 1 month of exposure, and the same indicators plus pulmonary function after 3 months. Mice were evaluated for BAL, lung cell proliferation, and respiratory tract histopathology after 1 month of exposure, and the same indicators plus hematology and serum chemistry after 3 months. Samples from both species were collected for ancillary studies performed by investigators who were not at LRRI and were funded separately. Exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of diesel particulate matter (DPM) and volatile and semivolatile organic compounds (VOCs and SVOCs). There were no exposure-related differences in mortality or clinically evident morbidity. Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to DE. There was evidence of early lung changes in the rats, accompanied by a number of statistically significant increases in inflammatory and oxidative stress indicators, and some evidence of subtle changes in pulmonary function. In general, statistically significant effects were observed only at the highest exposure level. The mice did not have the same responses as the rats, but did have small but statistically significant increases in lavage neutrophils and the cytokine IL-6 at 1 month (but not at 3 months). These findings suggest that the rats were more sensitive than mice to the subchronic exposures.

Effects of simulated downwind coal combustion emissions on pre-existing allergic airway responses in mice.

CONTEXT: Coal-fired power plant emissions can contribute a significant portion of the ambient air pollution in many parts of the world. OBJECTIVE: We hypothesized that exposure to simulated downwind coal combustion emissions (SDCCE) may exacerbate pre-existing allergic airway responses. METHODS: Mice were sensitized and challenged with ovalbumin (OVA). Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day for 3 days to air (control, C) or SDCCE containing particulate matter (PM) at low (L; 100 µg/m³), medium (M; 300 µg/m³), or high (H; 1000 µg/m³) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after SDCCE exposure, mice received another OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air/SDCCE. Measurement of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed ~24 h after the last exposure. RESULTS: SDCCE significantly increased BAL macrophages and eosinophils in OVA-sensitized mice from the post-OVA protocol. However, there was no effect of SDCCE on BAL macrophages or eosinophils in OVA-sensitized mice from the pre-OVA protocol. BAL neutrophils were elevated following SDCCE in both protocols in nonsensitized mice. These changes were not altered by filtering out the PM. In the post-OVA protocol, SDCCE decreased OVA-specific IgG1 in OVA-sensitized mice but increased levels of total IgE, OVA-specific IgE and OVA-specific IgG1 and IgG(2a) in non-sensitized animals. In the pre-OVA protocol, SDCCE increased OVA-specific IgE in both sensitized and non-sensitized animals. Additionally, BAL IL-4, IL-13, and IFN-γ levels were elevated in sensitized mice. CONCLUSION: These results suggest that acute exposure to either the particulate or gaseous phase of SDCCE can exacerbate various features of allergic airway responses depending on the timing of exposure in relation to allergen challenge.

Health effects of subchronic inhalation exposure to simulated downwind coal combustion emissions.

CONTEXT: There have been no animal studies of the health effects of repeated inhalation of mixtures representing downwind pollution from coal combustion. Environmental exposures typically follow atmospheric processing and mixing with pollutants from other sources. OBJECTIVE: This was the fourth study by the National Environmental Respiratory Center to create a database for responses of animal models to combustion-derived pollutant mixtures, to identify causal pollutants-regardless of source. METHODS: F344 and SHR rats and A/J, C57BL/6, and BALB/c mice were exposed 6 h/day 7 days/week for 1 week to 6 months to three concentrations of a mixture simulating key components of "downwind" coal combustion emissions, to the highest concentration filtered to remove particulate matter (PM), or to clean air. Emissions from low-sulfur subbituminous coal were modified to create a mixture recommended by an expert workshop. Sulfur dioxide, nitrogen oxides, and PM were the dominant components. Nonanimal-derived PM mass concentrations of nominally 0, 100, 300, and 1000 µg/m(3) were mostly partially neutralized sulfate. RESULTS: Only 17 of 270 species-gender-time-outcome comparisons were significantly affected by exposure; some models showed no effects. There was strong evidence that PM participated meaningfully in only three responses. CONCLUSION: On a total mass or PM mass basis, this mixture was less toxic overall than diesel and gasoline exhausts or wood smoke. The largely sulfate PM contributed to few effects and was the sole cause of none. The study did not allow identification of causal pollutants, but the potential role of NOx in some effects is suggested by the literature.

Biomass smoke exposures: toxicology and animal study design.

The International Biomass Smoke Health Effects (IBSHE) conference was convened in Missoula, MT, to define our current knowledge of smoke exposure and the potential health effects. In an effort to ascertain the relative health effects of an exposure to biomass smoke, numerous studies have utilized either animal or in vitro systems. A wide variety of systems that have been employed ranged from more mainstream animal models (i.e., rodents) and transformed cell lines to less common animal (piglets and dogs) and explant models. The Toxicology and Animal Study Design Workgroup at IBSHE was tasked with an analysis of the use of animal models in the assessment of the health effects of biomass smoke exposure. The present article contains a mini-review of models utilized historically, in addition to the adverse health effects assessed, and an overview of the discussion within the breakout session. The most common question that arose in discussions at the IBSHE conference was from local and federal health departments: What level of smoke is unhealthy? The present workgroup determined categories of exposure, common health concerns, and the availability of animal models to answer key health questions.

Is the air pollution health research community prepared to support a multipollutant air quality management framework?

Ambient air pollution is always encountered as a complex mixture, but past regulatory and research strategies largely focused on single pollutants, pollutant classes, and sources one-at-a-time. There is a trend toward managing air quality in a progressively "multipollutant" manner, with the idealized goal of controlling as many air contaminants as possible in an integrated manner to achieve the greatest total reduction of adverse health and environmental impacts. This commentary considers the current ability of the environmental air pollution exposure and health research communities to provide evidence to inform the development of multipollutant air quality management strategies and assess their effectiveness. The commentary is not a literature review, but a summary of key issues and information gaps, strategies for filling the gaps, and realistic expectations for progress that could be made during the next decade. The greatest need is for researchers and sponsors to address air quality health impacts from a truly multipollutant perspective, and the most limiting current information gap is knowledge of personal exposures of different subpopulations, considering activities and microenvironments. Emphasis is needed on clarifying the roles of a broader range of pollutants and their combinations in a more forward-looking manner; that is not driven by current regulatory structures. Although advances in research tools and outcome data will enhance progress, the greater need is to direct existing capabilities toward strategies aimed at placing into proper context the contributions of multiple pollutants and their combinations to the health burdens, and the relative contributions of pollutants and other factors influencing the same outcomes. The authors conclude that the research community has very limited ability to advise multipollutant air quality management and assess its effectiveness at this time, but that considerable progress can be made in a decade, even at current funding levels, if resources and incentives are shifted appropriately.

Is there evidence for synergy among air pollutants in causing health effects?

BACKGROUND: Environmental air pollutants are inhaled as complex mixtures, but the long dominant focus of monitoring and research on individual pollutants has provided modest insight into pollutant interactions that may be important to health. Trends toward managing multiple pollutants to maximize aggregate health gains place increasing value on knowing whether the effects of combinations of pollutants are greater than the sum of the effects of individual pollutants (synergy). OBJECTIVE: We reviewed selected published literature to determine whether synergistic effects of combinations of pollutants on health outcomes have actually been demonstrated. METHODS AND RESULTS: We reviewed 36 laboratory studies of combinations of ozone with other pollutants that were reported in the recent U.S. Environmental Protection Agency Ozone Criteria Document. We examined original reports to determine whether the experimental design tested for synergy and whether synergy was demonstrated. Fourteen studies demonstrated synergism, although synergistic, additive, and antagonistic effects were sometimes observed among different outcomes or at different times after exposure. CONCLUSIONS: Synergisms involving O3 have been demonstrated by laboratory studies of humans and animals. We conclude that the plausibility of synergisms among environmental pollutants has been established, although comparisons are limited, and most involved exposure concentrations much higher than typical of environmental pollutants. Epidemiologic research has limited ability to address the issue explicitly.

Radiotoxicity of inhaled (239)PuO(2) in dogs.

Beagle dogs inhaled graded exposure levels of insoluble plutonium dioxide ((239)PuO(2)) aerosols in one of three monodisperse particle sizes at the Lovelace Respiratory Research Institute (LRRI) to study the life-span health effects of different degrees of alpha-particle dose non-uniformity in the lung. The primary noncarcinogenic effects seen were lymphopenia, atrophy and fibrosis of the thoracic lymph nodes, and radiation pneumonitis and pulmonary fibrosis. Radiation pneumonitis/ pulmonary fibrosis occurred from 105 days to more than 11 years after exposure, with the lowest associated alpha-particle dose being 5.9 Gy. The primary carcinogenic effects also occurred almost exclusively in the lung because of the short range of the alpha-particle emissions. The earliest lung cancer was observed at 1086 days after the inhalation exposure. The most common type seen was papillary adenocarcinoma followed by bronchioloalveolar carcinoma. These lung cancer results indicate that a more uniform distribution of alpha-particle dose within the lung has an equal or possibly greater risk of neoplasia than less uniform distributions of alpha-particle dose. The results are consistent with a linear relationship between dose and response, but these data do not directly address the response expected at low dose levels. No primary tumors were found in the tracheobronchial and mediastinal lymph nodes despite the high alpha-particle radiation doses to these lymph nodes, and no cases of leukemia were observed.

Oxidative stress, inflammation, and pulmonary function assessment in rats exposed to laboratory-generated pollutant mixtures.

Oxidative stress may mediate adverse health effects of many inhaled pollutants. Cardiopulmonary responses of Sprague-Dawley rats to inhalation of whole or filtered gasoline engine exhaust (GEE, FGEE); simulated downwind coal emission atmospheres (SDCAs) from two types of coal, each tested at two concentrations; and two concentrations of re-aerosolized paved road dust (RD) were evaluated. In situ chemiluminescence and thiobarbituric acid-reactive substances (TBARS) were used to evaluate oxidative reactions in the lungs, heart, and liver immediately following exposures. Pulmonary inflammatory responses were measured by bronchoalveolar lavage (BAL) cell counts. Respiratory function parameters during exposure were measured by plethysmography. Only GEE significantly enhanced in situ chemiluminescence (all three organs), but only exposure to the high RD concentration increased TBARS (hearts only). There was a weak trend toward increased macrophages recovered in lavage fluid from both SDCAs, and macrophages were significantly elevated by both FGEE and the lower concentration of RD. Respiratory function effects were small, though the effects of the Central Appalachian low-sulfur SDCA on enhanced pause and the effects of the Powder River Basin SCDA on tidal volume were significant. The discordance between the oxidative stress indicators may relate to the use of a single time point in the context of dynamic changes in compensatory mechanisms. These results further suggest that inflammatory responses measured by BAL cellularity may not always correlate with oxidative stress. Overall, the toxicological effects from exposure to these pollutant mixtures were subtle, but the results show differences in the effects of atmospheres having different physical/chemical characteristics.

Health effects of organic aerosols.

Carbonaceous aerosol, a major component of particulate matter (PM), gases, and vapors in the atmosphere, has been associated with natural and anthropogenic air pollution, reduced visibility, climate modulation, material and ecosystem damage, and adverse health effects. More recently, epidemiological studies have indicated associations between organic fractions of ambient PM and adverse respiratory and cardiovascular health outcomes. The effects of the non-PM components of the organic aerosol have received less attention because their measurement in the general environment is not mandated. This article summarizes current knowledge of the nature, prevalence, and health effects of organic aerosols encountered in the outdoor environment, identifies key information gaps, and presents a conceptual framework for research priorities for resolving those gaps. The broad, diverse class of air contaminants comprising organic aerosols may be more important to public health than the modest attention given to them. This review focuses on hazard identification and exposure assessment for evaluating risks to public health from ambient organic aerosols. Current knowledge is insufficient to support a quantitative characterization of the aggregate risk from organic air contaminants. Assessments should be done for individual species or mixtures. Efforts should be taken to assemble and evaluate a common set of standard reference materials for both organic speciation and health response assays. A greater standardization of approaches across studies and laboratories would be useful to achieve uniformity in assessing health effects. Multidisciplinary research efforts are needed to improve the current regulatory-driven air quality monitoring networks for epidemiological studies. The limited array of biomarkers linking organic aerosols to health effects needs to be expanded and specific organic compounds or classes that are associated with biological effects in human cells or animal studies need to be tested for better understanding of the exposure-response relationship.

Health effects of inhaled gasoline engine emissions.

Despite their prevalence in the environment, and the myriad studies that have shown associations between morbidity or mortality with proximity to roadways (proxy for motor vehicle exposures), relatively little is known about the toxicity of gasoline engine emissions (GEE). We review the studies conducted on GEE to date, and summarize the findings from each of these studies. While there have been several studies, most of the studies were conducted prior to 1980 and thus were not conducted with contemporary engines, fuels, and driving cycles. In addition, many of the biological assays conducted during those studies did not include many of the assays that are conducted on contemporary inhalation exposures to air pollutants, including cardiovascular responses and others. None of the exposures from these earlier studies were characterized at the level of detail that would be considered adequate today. A recent GEE study was conducted as part of the National Environmental Respiratory Center (www.nercenter.org). In this study several in-use mid-mileage General Motors (Chevrolet S-10) vehicles were purchased and utilized for inhalation exposures. An exposure protocol was developed where engines were operated with a repeating California Unified Driving Cycle with one cold start per day. Two separate engines were used to provide two cold starts over a 6-h inhalation period. The exposure atmospheres were characterized in detail, including detailed chemical and physical analysis of the gas, vapor, and particle phase. Multiple rodent biological models were studied, including general toxicity and inflammation (e.g., serum chemistry, lung lavage cell counts/differentials, cytokine/chemokine analysis, histopathology), asthma (adult and in utero exposures with pulmonary function and biochemical analysis), cardiovascular effects (biochemical and electrocardiograph changes in susceptible rodent models), and susceptibility to infection (Pseudomonas bacteria challenge). GEE resulted in significant biological effects for upregulation of MIP-2, clearance of Pseudomonas bacteria, development of allergic response after in utero exposure, and cardiovascular indicators of vasoconstriction, oxidant stress, and damage.

Lung toxicity of ambient particulate matter from southeastern U.S. sites with different contributing sources: relationships between composition and effects.

BACKGROUND: Exposure to air pollution and, more specifically, particulate matter (PM) is associated with adverse health effects. However, the specific PM characteristics responsible for biological effects have not been defined. OBJECTIVES: In this project we examined the composition, sources, and relative toxicity of samples of PM with aerodynamic diameter

Modulators of cigarette smoke-induced pulmonary emphysema in A/J mice.

Mice develop pulmonary emphysema after chronic exposure to cigarette smoke (CS). In this study, the influence of gender, exposure duration, and concentration of CS on emphysema, pulmonary function, inflammation, markers of toxicity, and matrix metalloproteinase (MMP) activity was examined in A/J mice. Mice were exposed to CS at either 100 or 250 mg total particulate material/m(3) (CS-100 or CS-250, respectively) for 10, 16, or 22 weeks. Evidence of emphysema was first seen in female mice after 10 weeks of exposure to CS-250, while male mice did not develop emphysema until 16 weeks. Female mice exposed to CS-100 did not have emphysema until 16 weeks, suggesting that disease development depends on the concentration and duration of exposure. Airflow obstruction and increased pulmonary compliance were observed in mice exposed to CS-250 for 22 weeks. Decreased elasticity was likely the major contributor to airflow obstruction because substantial remodeling of the conducting airways, beyond mild mucous cell hyperplasia, was lacking. Exposure to CS increased the number of macrophages, neutrophils, lymphocytes (B cells and activated CD4- and CD8-positive T cells), and activity of MMP-2 and -9 in the bronchoalveolar lavage fluid (BALF). Treatment with antioxidants N-acetylcysteine or epigallocatechin gallate (EGCG) did not decrease emphysema severity, but EGCG slightly decreased BALF inflammatory cell numbers and lactate dehydrogenase activity. Inflammation and emphysema persisted after a 17-week recovery period following exposure to CS-250 for 22 weeks. The similarities of this model to the human disease make it promising for studying disease pathogenesis and assessing new therapeutic interventions.

Composition, toxicity, and mutagenicity of particulate and semivolatile emissions from heavy-duty compressed natural gas-powered vehicles.

Particulate matter (PM) and vapor-phase semivolatile organic compounds (SVOC) were collected from three buses fueled by compressed natural gas. The bus engines included a well-functioning, conventional engine; a "high emitter" engine; and a new technology engine with an oxidation catalyst. Chemical analysis of the emissions showed differences among these samples, with the high emitter sample containing markers of engine oil constituents. PM + SVOC samples were also collected for mutagenicity and toxicity testing. Extraction efficiencies from the collection media were lower than for similarly collected samples from gasoline or diesel vehicles. Responses to the recovered samples were compared on the basis of exhaust volume, to incorporate the emission rates into the potency factors. Mutagenicity was assessed by Salmonella reverse mutation assay. Mutagenicity was greatest for the high emitter sample and lowest for the new technology sample. Metabolic activation reduced mutagenicity in strain TA100, but not TA98. Toxicity, including inflammation, cytotoxicity, and parenchymal changes, was assessed 24 h after intratracheal instillation into rat lungs. Lung responses were generally mild, with little difference between the responses to equivalent volumes of emissions from the normal emitter and the new technology, but greater responses for the high emitter. These emission sample potencies are further compared on the basis of recovered mass with previously reported samples from normal and high-emitter gasoline and diesel vehicles. While mutagenic potencies for the CNG emission samples were similar to the range observed in the gasoline and diesel emission samples, lung toxicity potency factors were generally lower than those for the gasoline and diesel samples.

Low-level subchronic exposure to wood smoke exacerbates inflammatory responses in allergic rats.

Epidemiological studies have implicated wood smoke as a risk factor for exacerbating asthma. However, comparisons of findings in animal models with those in humans are currently not possible, because detailed clinically relevant measurements of pulmonary function are not available in animal studies. Brown Norway rats were immunized with ovalbumin and exposed to either filtered air or wood smoke at 1 mg particulate matter/m(3) for 70 days and challenged with allergen during the last 4 days of exposure. Baseline values for dynamic lung compliance were lower while functional residual capacity was increased in rats exposed to wood smoke compared to rats exposed to filtered air. IFN-gamma levels were reduced and IL-4 levels increased in the bronchoalveolar lavage fluid and blood plasma, inflammatory lesions in the lungs were 21% greater, and airway mucous cells/mm basal lamina were non-significantly increased in rats exposed to wood smoke compared to controls. Collectively, these studies suggest that the pulmonary function was affected in rats by exposure to wood smoke and this decline was associated with only minor increases in inflammation of the lung. Therefore, this animal model may be useful to elucidate the mechanisms of the decline in pulmonary function caused by environmental pollutants when asthmatics are exposed to allergen.

In vitro versus in vivo exposure to combustion emissions.

Defining the key components of a potentially hazardous aerosol mixture, whether at the level of source contributions or specific physical or chemical constituents, is a challenging but important problem. From the economic, efficiency, and ethical points of view, in vitro techniques have considerable appeal for these types of studies. However, no in vitro technique will ever have the complexity of the whole animal or person. Even for lung-specific endpoints, inhalation of aerosol mixtures by a living animal has the potential to induce systemic effects such as recruitment of inflammatory cells and neurogenic effects that can secondarily affect the lung, as well as local respiratory system responses such as cytokine production, oxidant stress, and cell death. Recent studies also demonstrate the potential for the inhaled materials to cause diverse systemic effects, which are not easily modeled in vitro. To evaluate the effects of aerosol toxicants on lung cells, various in vitro testing techniques have been developed. These methods differ in the cell types used (human or other, primary cells or transformed cell lines, epithelial or other), the exposure method (suspension of collected materials, exposure of conventional cultures to aerosols, or air-liquid interface exposures), and the toxicological endpoints assessed (cell viability, cytokine production, oxidant stress, cell type-specific function). Because these studies may have implications for regulatory decisions, it is critical that any in vitro screening process of comparative toxicology be validated by comparisons with the effects in animal models, and where possible, by controlled human exposures. This article discusses relevant parallels and discrepancies between exposures of cells and living animals.

Effects of low sulfur fuel and a catalyzed particle trap on the composition and toxicity of diesel emissions.

In this study we compared a "baseline" condition of uncontrolled diesel engine exhaust (DEE) emissions generated with current (circa 2003) certification fuel to an emissions-reduction (ER) case with low sulfur fuel and a catalyzed particle trap. Lung toxicity assessments (resistance to respiratory viral infection, lung inflammation, and oxidative stress) were performed on mice (C57Bl/6) exposed by inhalation (6 hr/day for 7 days). The engine was operated identically (same engine load) in both cases, and the inhalation exposures were conducted at the same exhaust dilution rate. For baseline DEE, this dilution resulted in a particle mass (PM) concentration of approximately 200 microg/m3 PM, whereas the ER reduced the PM and almost every other measured constituent [except nitrogen oxides (NOx)] to near background levels in the exposure atmospheres. These measurements included PM, PM size distribution, PM composition (carbon, ions, elements), NOx, carbon monoxide, speciated/total volatile hydrocarbons, and several classes of semivolatile organic compounds. After exposure concluded, one group of mice was immediately sacrificed and assessed for inflammation and oxidative stress in lung homogenate. Another group of mice were intratracheally instilled with respiratory syncytial virus (RSV), and RSV lung clearance and inflammation was assessed 4 days later. Baseline DEE produced statistically significant biological effects for all measured parameters. The use of low sulfur fuel and a catalyzed trap either completely or nearly eliminated the effects.