Richard H. Jaskot

Soluble transition metals mediate residual oil fly ash induced acute lung injury

Identification of constituents responsible for the pulmonary toxicity of fugitive combustion emission source particles may provide insight into the adverse health effects associated with exposure to these particles as well as ambient air particulate pollution. Herein, we describe results of studies conducted to identify constituents responsible for the acute lung injury induced by residual oil fly ash (ROFA) and to assess physical-chemical factors that influence the pulmonary toxicity of these constituents. Biochemical and cellular analyses performed on bronchoalveolar lavage fluid obtained from rats following intratracheal instillation of ROFA suspension demonstrated the presence of severe inflammation, an indicator of pulmonary injury, which included recruitment of neutrophils, eosinophils, and monocytes into the airway. A leachate prepared from ROFA, containing predominantly Fe, Ni, V, Ca, Mg, and sulfate, produced similar lung injury to that induced by ROFA suspension. Depletion of Fe, Ni, and V from the ROFA leachate abrogated its pulmonary toxicity. Correspondingly, minimal lung injury was observed in animals exposed to saline-washed ROFA particles. A surrogate transition metal sulfate solution containing Fe, V, and Ni largely reproduced the lung injury induced by ROFA. Metal interactions and pH were found to influence the severity and kinetics of lung injury induced by ROFA and soluble transition metals. These findings provide direct evidence for the role of soluble transition metals in the pulmonary injury induced by the combustion emission source particulate, ROFA.

PULMONARY PROINFLAMMATORY GENE INDUCTION FOLLOWING ACUTE EXPOSURE TO RESIDUAL OIL FLY ASH: ROLES OF PARTICLE-ASSOCIATED METALS

Residual oil fly ash (ROFA), an emission source particulate, has been shown to induce acute lung injury and fibrosis in the rat. However, the mechanism(s) and the identities of various inflammatory mediators induced by ROFA are not known. Also the extent to which ROFA-associated metals contribute to proinflammatory gene induction is yet to be determined. To examine the mechanism of ROFA-induced lung injury, male SpragueDawley rats (60 days old) were intratracheally instilled with 0.3 ml of either acidified saline (brought to pH 2.5 using H2 SO4, similar to the pH of the ROFA suspension in saline), ROFA (2.5 mg/rat in saline, yields pH of 2.5), or predominant ROFA-associated metals such as Fe2 (SO4)3 (Fe, 0.54 mol/rat), VSO4 (V, 1.7 mol/rat), and NiSO4 (Ni, 1.0 mol/rat), individually or as a mixture (Fe + V + Ni). The quantity of metals instilled reflected the amount present in the leachable material of ROFA. Histopathological findings indicated marked and progressive acute focal lung injury characterized ...

Vascular and Cardiac Impairments in Rats Inhaling Ozone and Diesel Exhaust Particles

Background Mechanisms of cardiovascular injuries from exposure to gas and particulate air pollutants are unknown. Objective We sought to determine whether episodic exposure of rats to ozone or diesel exhaust particles (DEP) causes differential cardiovascular impairments that are exacerbated by ozone plus DEP. Methods and results Male Wistar Kyoto rats (10–12 weeks of age) were exposed to air, ozone (0.4 ppm), DEP (2.1 mg/m3), or ozone (0.38 ppm) + DEP (2.2 mg/m3) for 5 hr/day, 1 day/week for 16 weeks, or to air, ozone (0.51 or 1.0 ppm), or DEP (1.9 mg/m3) for 5 hr/day for 2 days. At the end of each exposure period, we examined pulmonary and cardiovascular biomarkers of injury. In the 16-week study, we observed mild pulmonary pathology in the ozone, DEP, and ozone + DEP exposure groups, a slight decrease in circulating lymphocytes in the ozone and DEP groups, and decreased platelets in the DEP group. After 16 weeks of exposure, mRNA biomarkers of oxidative stress (hemeoxygenase-1), thrombosis (tissue factor, plasminogen activator inhibitor-1, tissue plasminogen activator, and von Willebrand factor), vasoconstriction (endothelin-1, endothelin receptors A and B, endothelial NO synthase) and proteolysis [matrix metalloprotease (MMP)-2, MMP-3, and tissue inhibitor of matrix metalloprotease-2] were increased by DEP and/or ozone in the aorta, but not in the heart. Aortic LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) mRNA and protein increased after ozone exposure, and LOX-1 protein increased after exposure to ozone + DEP. RAGE (receptor for advanced glycation end products) mRNA increased in the ozone + DEP group. Exposure to ozone or DEP depleted cardiac mitochondrial phospholipid fatty acids (DEP > ozone). The combined effect of ozone and DEP exposure was less pronounced than exposure to either pollutant alone. Exposure to ozone or DEP for 2 days (acute) caused mild changes in the aorta. Conclusions In animals exposed to ozone or DEP alone for 16 weeks, we observed elevated biomarkers of vascular impairments in the aorta, with the loss of phospholipid fatty acids in myocardial mitochondria. We conclude that there is a possible role of oxidized lipids and protein through LOX-1 and/or RAGE signaling.

The Role of Particulate Matter-Associated Zinc in Cardiac Injury in Rats

Background Exposure to particulate matter (PM) has been associated with increased cardiovascular morbidity; however, causative components are unknown. Zinc is a major element detected at high levels in urban air. Objective We investigated the role of PM-associated zinc in cardiac injury. Methods We repeatedly exposed 12- to 14-week-old male Wistar Kyoto rats intratracheally (1×/week for 8 or16 weeks) to a) saline (control); b) PM having no soluble zinc (Mount St. Helens ash, MSH); or c) whole-combustion PM suspension containing 14.5 μg/mg of water-soluble zinc at high dose (PM-HD) and d ) low dose (PM-LD), e) the aqueous fraction of this suspension (14.5 μg/mg of soluble zinc) (PM-L), or f ) zinc sulfate (rats exposed for 8 weeks received double the concentration of all PM components of rats exposed for 16 weeks). Results Pulmonary inflammation was apparent in all exposure groups when compared with saline (8 weeks > 16 weeks). PM with or without zinc, or with zinc alone caused small increases in focal subepicardial inflammation, degeneration, and fibrosis. Lesions were not detected in controls at 8 weeks but were noted at 16 weeks. We analyzed mitochondrial DNA damage using quantitative polymerase chain reaction and found that all groups except MSH caused varying degrees of damage relative to control. Total cardiac aconitase activity was inhibited in rats receiving soluble zinc. Expression array analysis of heart tissue revealed modest changes in mRNA for genes involved in signaling, ion channels function, oxidative stress, mitochondrial fatty acid metabolism, and cell cycle regulation in zinc but not in MSH-exposed rats. Conclusion These results suggest that water-soluble PM-associated zinc may be one of the causal components involved in PM cardiac effects.

OXIDATIVE STRESS MEDIATES AIR POLLUTION PARTICLE-INDUCED ACUTE LUNG INJURY AND MOLECULAR PATHOLOGY

Insight into the mechanism(s) by which ambient air particulate matter (PM) mediates adverse health effects is needed to provide biological plausibility to epidemiological studies demonstrating associations between PM exposure and increased morbidity and mortality. Although in vitro PM studies provide an understanding of mechanisms by which PM affects pulmonary cells, it is difficult to extrapolate from in vitro to in vivo mechanisms of PM-induced lung injury. We examined in vivo mechanisms of lung injury generated by oil combustion particles. Rats were pretreated with dimethylthiourea (DMTU) before intratracheal instillation of residual oil fly ash (ROFA). Animals were examined by bronchoalveolar lavage for biomarkers of lung injury, and lung tissues were examined by immunohistochemical, biochemical, and molecular approaches to identify ROFA-induced alterations in intracellular signaling pathways and proinflammatory gene expression. Significant increases in pulmonary inflammation, cytotoxicity, activation of ERK mitogen-activated protein kinase (MAPK), and increases in mRNA levels encoding macrophage inflammatory protein (MIP)-2, interleukin (IL)-6, tumor necrosis factor (TNF)-α, MCP-1 and matrilysin were observed. DMTU pretreatment inhibited ROFA-induced pulmonary inflammation, cytotoxicity, ERK MAPK activation, and cytokine gene expression. Our findings provide coherence with in vitro PM mechanistic information, allow direct in vitro to in vivo extrapolation, and demonstrate a critical role for oxidative stress in ROFA-induced lung injury and associated molecular pathology.

A model of the regional uptake of gaseous pollutants in the lung

An ozone (O3) dosimetry model is presented that takes into account convection and diffusion of O3 in the lumen and airspaces of the lower respiratory tract and transport and chemical reactions in the mucous and surfactant layers and in the underlying tissue and capillaries. The model was applied to human airway morphometric data. Values for the chemical and physical parameters that define the liquid tissue and blood compartments were based on reported experimental data. Simulation results illustrate the variability of results due to an uncertainty in the knowledge of transport parameters, liquid, tissue, and blood compartment thicknesses, and chemical reaction rates. Results were most sensitive to mucous compartment thickness and reaction rate constant and least sensitive to transport and blood parameters. Exercise was simulated, showing little effect on tracheobronchial uptake but a pronounced effect on pulmonary uptake.

One-month diesel exhaust inhalation produces hypertensive gene expression pattern in healthy rats.

Background Exposure to diesel exhaust (DE) is linked to vasoconstriction, endothelial dysfunction, and myocardial ischemia in compromised individuals. Objective We hypothesized that DE inhalation would cause greater inflammation, hematologic alterations, and cardiac molecular impairment in spontaneously hypertensive (SH) rats than in healthy Wistar Kyoto (WKY) rats. Methods and results Male rats (12–14 weeks of age) were exposed to air or DE from a 30-kW Deutz engine at 500 or 2,000 μg/m3, 4 hr/day, 5 days/week for 4 weeks. Neutrophilic influx was noted in the lung lavage fluid of both strains, but injury markers were minimally changed. Particle-laden macrophages were apparent histologically in DE-exposed rats. Lower baseline cardiac anti-oxidant enzyme activities were present in SH than in WKY rats; however, no DE effects were noted. Cardiac mitochondrial aconitase activity decreased after DE exposure in both strains. Electron microscopy indicated abnormalities in cardiac mitochondria of control SH but no DE effects. Gene expression profiling demonstrated alterations in 377 genes by DE in WKY but none in SH rats. The direction of DE-induced changes in WKY mimicked expression pattern of control SH rats without DE. Most genes affected by DE were down-regulated in WKY. The same genes were down-regulated in SH without DE producing a hypertensive-like expression pattern. The down-regulated genes included those that regulate compensatory response, matrix metabolism, mitochondrial function, and oxidative stress response. No up-regulation of inflammatory genes was noted. Conclusions We provide the evidence that DE inhalation produces a hypertensive-like cardiac gene expression pattern associated with mitochondrial oxidative stress in healthy rats.

IN VIVO AND IN VITRO CORRELATION OF PULMONARY MAP KINASE ACTIVATION FOLLOWING METALLIC EXPOSURE

Residual oil fly ash (ROFA) is a particulate pollutant produced in the combustion of fuel oil. Exposure to ROFA is associated with adverse respiratory effects in humans, induces lung inflammation in animals, and induces inflammatory mediator expression in cultured human airway epithelial cells (HAEC). ROFA has a high content of transition metals, including vanadium, a potent tyrosine phosphatase inhibitor that we have previously shown to disregulate phosphotyrosine metabolism and activate mitogen-activated protein kinase (MAPK) signaling cascades in HAEC. In order to study MAPK activation in response to in vivo metal exposure, we used immunohistochemical methods to detect levels of phosphorylated protein tyrosines (P-Tyr) and the MAPKs ERK1/ 2, JNK, and P38 in lung sections from rats intratracheally exposed to ROFA. After a 1-h exposure to 500 µg ROFA, rat lungs showed no histological changes and no significant increases in immunostaining for either P-Tyr or phospho-(P-) MAPKs compared to saline-instilled controls. At 4 h of exposure, there was mild and variable inflammation in the lung, which was accompanied by an increase in specific immunostaining for P-Tyr and P-MAPKs in airway and alveolar epithelial cells and resident macrophages. By 24 h of exposure, there was a pronounced inflammatory response to ROFA instillation and a marked increase in levels of P-Tyr and P-MAPKs present within the alveolar epithelium and in the inflamma tory cells, while the airway epithelium showed a continued increase in the expression of P-ERK1/2. By comparison, HAEC cultures exposed to 100 µg/ml ROFA for 20 min resulted in marked increases in P-Tyr and P-MAPKs, which persisted after 24 h of exposure. PTyr levels continued to accumulate for up to 24 h in HAEC exposed to ROFA. These results demonstrate in vivo activation in cell signaling pathways in response to pulmonary exposure to particulate matter, and support the relevance of in vitro studies in the identification of mechanisms of lung injury induced by pollutant inhalation.Residual oil fly ash (ROFA) is a particulate pollutant produced in the combustion of fuel oil. Exposure to ROFA is associated with adverse respiratory effects in humans, induces lung inflammation in animals, and induces inflammatory mediator expression in cultured human airway epithelial cells (HAEC). ROFA has a high content of transition metals, including vanadium, a potent tyrosine phosphatase inhibitor that we have previously shown to disregulate phosphotyrosine metabolism and activate mitogen-activated protein kinase (MAPK) signaling cascades in HAEC. In order to study MAPK activation in response to in vivo metal exposure, we used immunohistochemical methods to detect levels of phosphorylated protein tyrosines (P-Tyr) and the MAPKs ERK1/2, JNK, and P38 in lung sections from rats intratracheally exposed to ROFA. After a 1-h exposure to 500 microg ROFA, rat lungs showed no histological changes and no significant increases in immunostaining for either P-Tyr or phospho-(P-) MAPKs compared to saline-instilled controls. At 4 h of exposure, there was mild and variable inflammation in the lung, which was accompanied by an increase in specific immunostaining for P-Tyr and P-MAPKs in airway and alveolar epithelial cells and resident macrophages. By 24 h of exposure, there was a pronounced inflammatory response to ROFA instillation and a marked increase in levels of P-Tyr and P-MAPKs present within the alveolar epithelium and in the inflammatory cells, while the airway epithelium showed a continued increase in the expression of P-ERK1/2. By comparison, HAEC cultures exposed to 100 microg/ml ROFA for 20 min resulted in marked increases in P-Tyr and P-MAPKs, which persisted after 24 h of exposure. P-Tyr levels continued to accumulate for up to 24 h in HAEC exposed to ROFA. These results demonstrate in vivo activation in cell signaling pathways in response to pulmonary exposure to particulate matter, and support the relevance of in vitro studies in the identification of mechanisms of lung injury induced by pollutant inhalation.

A comparative study of the effects of inhaled cadmium chloride and cadmium oxide: Pulmonary response

The effects of aerosols of cadmium chloride (CdCl2) and cadmium oxide (CdO) on pulmonary biochemical function were compared. Rats and rabbits were exposed to 0.25, 0.45, or 4.5 mg Cd/m3 for 2 h. Pulmonary toxicity was determined histologically and biochemically. Cadmium chloride and CdO showed a deposition response that was linearly related to the chamber concentration. Both compounds caused multifocal, interstitial pneumonitis 72 h after exposure, but the CdO lesion was more severe with proliferation of fibrocytic-like cells as well as pneumocytes. Comparing the two Cd compounds at the highest concentration (4.5 mg Cd/m3), the biochemical responses in the rat were similar. The majority of the effects occurred 72 h after exposure, with significant increases in lung weight, lung-to-body weight ratio, GSH reductase, GSH transferase, and G-6-PDH. However, GSH peroxidase was inhibited immediately after the CdO exposure. Cadmium oxide-related alterations in the parameters studied could easily be distinguished from those of CdCl2 at the exposure concentration of 0.45 mg Cd/m3. The response pattern in the rabbit resembled that of the rat. In both species Cd had a consistent inhibitory effect on pulmonary GSH peroxidase, even at the lowest concentration of 0.25 mg Cd/m3. Based on these findings, inhaled CdO appeared to be more toxic to the lung than inhaled CdCl2.

Systemic Imbalance of Essential Metals and Cardiac Gene Expression in Rats Following Acute Pulmonary Zinc Exposure

It was recently demonstrated that particulate matter (PM) containing water-soluble zinc produces cardiac injury following pulmonary exposure. To investigate whether pulmonary zinc exposure produces systemic metal imbalance and direct cardiac effects, male Wistar Kyoto (WKY) rats (12–14 wk age) were intratracheally (IT) instilled with saline or 2 μmol/kg zinc sulfate. Temporal analysis was performed for systemic levels of essential metals (zinc, copper, and selenium), and induction of zinc transporter-2 (ZT-2) and metallothionein-1 (MT-1) mRNA in the lung, heart, and liver. Additionally, cardiac gene expression profile was evaluated using Affymetrix GeneChips (rat 230A) arrays to identify zinc-specific effects. Pulmonary zinc instillation produced an increase in plasma zinc to ∼20% at 1 and 4 h postexposure with concomitant decline in the lung levels. At 24 and 48 h postexposure, zinc levels rose significantly (∼35%) in the liver. At these time points, plasma and liver levels of copper and selenium also increased significantly, suggesting systemic disturbance in essential metals. Zinc exposure was associated with marked induction of MT-1 and ZT-2 mRNA in lung, heart, and liver, suggesting systemic metal sequestration response. Given the functional role of zinc in hundreds of proteins, the gene expression profiles demonstrated changes that are expected based on its physiological role. Zinc exposure produced an increase in expression of kinases and inhibition of expression of phosphatases; up- or downregulation of genes involved in mitochondrial function; changes in calcium regulatory proteins suggestive of elevated intracellular free calcium and increases in sulfotransferases; upregulation of potassium channel genes; and changes in free radical-sensitive proteins. Some of these expression changes are reflective of a direct effect of zinc on myocardium following pulmonary exposure, which may result in impaired mitochondrial respiration, stimulated cell signaling, altered Ca2+ homeostasis, and increased transcription of sulfotransferases. Cardiotoxicity may be an outcome of acute zinc toxicosis and occupational exposures to metal fumes containing soluble zinc. Imbalance of systemic metal homeostasis as a result of pulmonary zinc exposure may underlie the cause of extrapulmonary effects.