JeanClare Seagrave

The Role of a Mitochondrial Pathway in the Induction of Apoptosis by Chemicals Extracted from Diesel Exhaust Particles

We are interested in the cytotoxic and proinflammatory effects of particulate pollutants in the respiratory tract. We demonstrate that methanol extracts made from diesel exhaust particles (DEP) induce apoptosis and reactive oxygen species (ROS) in pulmonary alveolar macrophages and RAW 264.7 cells. The toxicity of these organic extracts mimics the cytotoxicity of the intact particles and could be suppressed by the synthetic sulfhydryl compounds, N-acetylcysteine and bucillamine. Because DEP-induced apoptosis follows cytochrome c release, we studied the effect of DEP chemicals on mitochondrially regulated death mechanisms. Crude DEP extracts induced ROS production and perturbed mitochondrial function before and at the onset of apoptosis. This mitochondrial perturbation follows an orderly sequence of events, which commence with a change in mitochondrial membrane potential, followed by cytochrome c release, development of membrane asymmetry (annexin V staining), and propidium iodide uptake. Structural damage to the mitochondrial inner membrane, evidenced by a decrease in cardiolipin mass, leads to O⨪2 generation and uncoupling of oxidative phosphorylation (decreased intracellular ATP levels). N-Acetylcysteine reversed these mitochondrial effects and ROS production. Overexpression of the mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress apoptosis. Taken together, these results suggest that DEP chemicals induce apoptosis in macrophages via a toxic effect on mitochondria.

Histone Deacetylase 2 Is Phosphorylated, Ubiquitinated, and Degraded by Cigarette Smoke

Cigarette smoke (CS)-induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome-dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke.

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 ≥2.5 μm (PM2.5) collected from sites within the Southeastern Aerosol Research and Characterization (SEARCH) air monitoring network during two seasons. These sites represent four areas with differing sources of PM2.5, including local urban versus regional sources, urban areas with different contributions of transportation and industrial sources, and a site influenced by Gulf of Mexico weather patterns. Methods We collected samples from each site during the winter and summer of 2004 for toxicity testing and for chemical analysis and chemical mass balance–based source apportionment. We also collected PM2.5 downwind of a series of prescribed forest burns. We assessed the toxicity of the samples by instillation into rat lungs and assessed general toxicity, acute cytotoxicity, and inflammation. Statistical dose–response modeling techniques were used to rank the relative toxicity and compare the seasonal differences at each site. Projection-to-latent-surfaces (PLS) techniques examined the relationships among sources, chemical composition, and toxicologic end points. Results and conclusions Urban sites with high contributions from vehicles and industry were most toxic.

Relationship between composition and toxicity of motor vehicle emission samples.

In this study we investigated the statistical relationship between particle and semivolatile organic chemical constituents in gasoline and diesel vehicle exhaust samples, and toxicity as measured by inflammation and tissue damage in rat lungs and mutagenicity in bacteria. Exhaust samples were collected from “normal” and “high-emitting” gasoline and diesel light-duty vehicles. We employed a combination of principal component analysis (PCA) and partial least-squares regression (PLS; also known as projection to latent structures) to evaluate the relationships between chemical composition of vehicle exhaust and toxicity. The PLS analysis revealed the chemical constituents covarying most strongly with toxicity and produced models predicting the relative toxicity of the samples with good accuracy. The specific nitro-polycyclic aromatic hydrocarbons important for mutagenicity were the same chemicals that have been implicated by decades of bioassay-directed fractionation. These chemicals were not related to lung toxicity, which was associated with organic carbon and select organic compounds that are present in lubricating oil. The results demonstrate the utility of the PCA/PLS approach for evaluating composition–response relationships in complex mixture exposures and also provide a starting point for confirming causality and determining the mechanisms of the lung effects.

Vehicular Emissions Induce Vascular MMP-9 Expression and Activity Associated With Endothelin-1–Mediated Pathways

Objective—Mechanisms of air pollution–induced exacerbation of cardiovascular disease are currently unknown, thus we examined the roles of vascular endothelin-1 (ET-1) and reactive oxygen species (ROS) in regulating mediators of vascular remodeling, namely matrix metalloproteinases (MMPs), after exposure to vehicle engine emissions. Methods and Results—ApoE−/− mice were exposed by inhalation to filtered air or gasoline engine exhaust (GEE, 1:12 dilution) 6 hours per day for 1 or 7 days. Concurrently, mice were treated with either ETA receptor antagonist BQ-123 (100 ng/kg/d) via osmotic minipumps, Tempol (≈41 mg/kg/d, orally), or vehicle. GEE-exposure increased vascular MMP-2 and -9, endothelin-1 (ET-1), tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and ROS levels. Aortic MMP protein and plasma MMP-9 were similarly upregulated. GEE-mediated increases in vascular ROS were attenuated by Tempol-treatment, as were MMP-2 and TIMP-2; whereas BQ-123 ameliorated GEE-induced vascular expression of MMP-9, MMP-2, ROS, and ET-1. In a parallel study, diesel exhaust exposure in volunteer human subjects induced significant increases in plasma ET-1 and MMP-9 expression and activity. Conclusions—These findings demonstrate that acute exposure to vehicular source air pollutants results in upregulation of circulating and vascular factors associated with progression of atherosclerosis, mediated in part through activation of ET-1–ETA receptor pathways.

Inhaled Diesel Emissions Alter Atherosclerotic Plaque Composition in ApoE−/− Mice

Recent epidemiological studies suggest that traffic-related air pollution may have detrimental effects on cardiovascular health. Previous studies reveal that gasoline emissions can induce several enzyme pathways involved in the formation and development of atherosclerotic plaques. As a direct comparison, the present study examined the impact of diesel engine emissions on these pathways, and further examined the effects on vascular lesion pathology. Apolipoprotein E-null mice were simultaneously placed on a high-fat chow diet and exposed to four concentrations, plus a high concentration exposure with particulates (PM) removed by filtration, of diesel emissions for 6 h/day for 50 days. Aortas were subsequently assayed for alterations in matrix metalloproteinase-9, endothelin-1, and several other biomarkers. Diesel induced dose-related alterations in gene markers of vascular remodeling and aortic lipid peroxidation; filtration of PM did not significantly alter these vascular responses, indicating that the gaseous portion of the exhaust was a principal driver. Immunohistochemical analysis of aortic leaflet sections revealed no net increase in lesion area, but a significant decrease in lipid-rich regions and increasing trends in macrophage accumulation and collagen content, suggesting that plaques were advanced to a more fragile, potentially more vulnerable state by diesel exhaust exposure. Combined with previous studies, these results indicate that whole emissions from mobile sources may have a significant role in promoting chronic vascular disease.

ANIMAL MODELS OF ASTHMA: Potential Usefulness for Studying Health Effects of Inhaled Particles

Asthma is now recognized to be a chronic inflammatory disease that affects the whole lung. Incidence appears to be increasing despite improved treatment regimens. There is substantial epidemiological evidence suggesting a relationship between the incidence and severity of asthma (e.g., hospitalizations) and exposure to increased levels of air pollution, especially fine and ultrafine particulate material, in susceptible individuals. There have been a few studies in animal models that support this concept, but additional animal studies to test this hypothesis are needed. However, such studies must be performed with awareness of the strengths and weaknesses of the currently available animal models. For studies in mice, the most commonly used animal, a broad spectrum of molecular and immunological tools is available, particularly to study the balance between Th1 and Th2 responses, and inbred strains may be useful for genetic dissection of susceptibility to the disease. However, the mouse is a poor model for bronchoconstriction or localized immune responses that characterize the human disease. In contrast, allergic lung diseases in dogs and cats may more accurately model the human condition, but fewer tools are available for characterization of the mechanisms. Finally, economic issues as well as reagent availability limit the utility of horses, sheep, and primates.Asthma is now recognized to be a chronic inflammatory disease that affects the whole lung. Incidence appears to be increasing despite improved treatment regimens. There is substantial epidemiological evidence suggesting a relationship between the incidence and severity of asthma (e.g., hospitalizations) and exposure to increased levels of air pollution, especially fine and ultrafine particulate material, in susceptible individuals. There have been a few studies in animal models that support this concept, but additional animal studies to test this hypothesis are needed. However, such studies must be performed with awareness of the strengths and weaknesses of the currently available animal models. For studies in mice, the most commonly used animal, a broad spectrum of molecular and immunological tools is available, particularly to study the balance between Th1 and Th2 responses, and inbred strains may be useful for genetic dissection of susceptibility to the disease. However, the mouse is a poor model for bronchoconstriction or localized immune responses that characterize the human disease. In contrast, allergic lung diseases in dogs and cats may more accurately model the human condition, but fewer tools are available for characterization of the mechanisms. Finally, economic issues as well as reagent availability limit the utility of horses, sheep, and primates.

EFFECTS OF CIGARETTE SMOKE EXPOSURE AND CESSATION ON INFLAMMATORY CELLS AND MATRIX METALLOPROTEINASE ACTIVITY IN MICE

B6C3F1 female mice were exposed to cigarette smoke (CS) (250 mg/m3 total particulate material) or filtered air (FA), 6 hours/day, 5 days/week, for 6, 7, or 10 weeks, or to CS for 6 weeks, then FA for 1 or 4 additional weeks. Exposure to CS increased macrophages, neutrophils, lymphocytes, and matrix metalloproteinase (MMP)-2 and MMP-9 content in bronchoalveolar lavage fluid. Partial recovery of most lavage parameters (except lymphocytes) was observed 1 week after cessation of CS exposure with further reductions after 4 weeks, but interstitial inflammation persisted longer. These results support a role for MMPs in CS-induced emphysema and indicate that smoking cessation allows restoration toward normal homeostasis.

Health Effects of Subchronic Exposure to Environmental Levels of Diesel Exhaust

Diesel exhaust is a public health concern and contributor to both ambient and occupational air pollution. As part of a general health assessment of multiple anthropogenic source emissions conducted by the National Environmental Respiratory Center (NERC), a series of health assays was conducted on rats and mice exposed to environmentally relevant levels of diesel exhaust. This article summarizes the study design and exposures, and reports findings on several general indicators of toxicity and carcinogenic potential. Diesel exhaust was generated from a commonly used 2000 model 5.9-L, 6-cylinder turbo diesel engine operated on a variable-load heavy-duty test cycle burning national average certification fuel. Animals were exposed to clean air (control) or four dilutions of whole emissions based on particulate matter concentration (30, 100, 300, and 1000 μg/m3). Male and female F344 rats and A/J mice were exposed by whole-body inhalation 6 h/day, 7 days/wk, for either 1 wk or 6 mo. Exposures were characterized in detail. Effects of exposure on clinical observations, body and organ weights, serum chemistry, hematology, histopathology, bronchoalveolar lavage, and serum clotting factors were mild. Significant exposure-related effects occurring in both male and female rats included decreases in serum cholesterol and clotting Factor VII and slight increases in serum gamma-glutamyl transferase. Several other responses met screening criteria for significant exposure effects but were not consistent between genders or exposure times and were not corroborated by related parameters. Carcinogenic potential as determined by micronucleated reticulocyte counts and proliferation of adenomas in A/J mice were unaffected by 6 mo of exposure. Parallel studies demonstrated effects on cardiac function and resistance to viral infection; however, the results reported here show few and only modest health hazards from subchronic or shorter exposures to realistic concentrations of contemporary diesel emissions.

Diesel Particulate Material Binds and Concentrates a Proinflammatory Cytokine That Causes Neutrophil Migration

Exposure to combustion emissions is associated with adverse health effects, but the properties of the emissions that induce these effects are not fully understood. To examine the direct effects of diesel particulate material (DPM) on alveolar epithelial cells, A549 cells were exposed to DPM. Low concentrations of DPM increased the interleukin-8 (IL-8) detected in the conditioned medium. Higher doses appeared to suppress the response, although this suppression was not related to acute DPM toxicity. In a cell-free system, incubation of IL-8 with DPM resulted in loss of immunoreactive IL-8 from the supernatant of the reaction. In contrast, carbon black did not reduce the concentration of IL-8 in the mixture. The DPM-induced loss was only weakly blocked by a large excess of bovine serum albumin (BSA). High concentrations of salts partially prevented the loss, but extraction of the soot with organic solvents had no effect. To determine biological implications, human blood neutrophils were exposed to DPM that had been preincubated with IL-8, then washed to remove free IL-8. The neutrophils changed shape in a manner suggesting directed movement toward the particles. No morphological change was observed either with carbon black that had been incubated with IL-8 or with DPM alone. These results suggest that DPM not only induces the production of IL-8 by epithelial cells, but also binds biologically active chemokine in a particle- and protein-selective manner. DPM-induced inflammatory responses may therefore be more focused or sustained as a result of this binding of inflammatory mediators by DPM.