Longitudinal Impact of WTC Dust Inhalation on Rat Cardiac Tissue Transcriptomic Profiles.

First responders (FR) exposed to the World Trade Center (WTC) Ground Zero air over the first week after the 9/11 disaster have an increased heart disease incidence compared to unexposed FR and the general population. To test if WTC dusts were causative agents, rats were exposed to WTC dusts (under isoflurane [ISO] anesthesia) 2 h/day on 2 consecutive days; controls received air/ISO or air only. Hearts were collected 1, 30, 240, and 360 d post-exposure, left ventricle total RNA was extracted, and transcription profiles were obtained. The data showed that differentially expressed genes (DEG) for WTC vs. ISO rats did not reach any significance with a false discovery rate (FDR) < 0.05 at days 1, 30, and 240, indicating that the dusts did not impart effects beyond any from ISO. However, at day 360, 14 DEG with a low FDR were identified, reflecting potential long-term effects from WTC dust alone, and the majority of these DEG have been implicated as having an impact on heart functions. Furthermore, the functional gene set enrichment analysis (GSEA) data at day 360 showed that WTC dust could potentially impact the myocardial energy metabolism via PPAR signaling and heart valve development. This is the first study showing that WTC dust could significantly affect some genes that are associated with the heart/CV system, in the long term. Even > 20 years after the 9/11 disaster, this has potentially important implications for those FR exposed repeatedly at Ground Zero over the first week after the buildings collapsed.

Longitudinal impact on rat cardiac tissue transcriptomic profiles due to acute intratracheal inhalation exposures to isoflurane.

Isoflurane (ISO) is a widely used inhalation anesthetic in experiments with rodents and humans during surgery. Though ISO has not been reported to impart long-lasting side effects, it is unknown if ISO can influence gene regulation in certain tissues, including the heart. Such changes could have important implications for use of this anesthetic in patients susceptible to heart failure/other cardiac abnormalities. To test if ISO could alter gene regulation/expression in heart tissues, and if such changes were reversible, prolonged, or late onset with time, SHR (spontaneously hypertensive) rats were exposed by intratracheal inhalation to a 97.5% air/2.5% ISO mixture on two consecutive days (2 hr/d). Control rats breathed filtered air only. On Days 1, 30, 240, and 360 post-exposure, rat hearts were collected and total RNA was extracted from the left ventricle for global gene expression analysis. The data revealed differentially-expressed genes (DEG) in response to ISO (compared to naïve control) at all post-exposure timepoints. The data showed acute ISO exposures led to DEG associated with wounding, local immune function, inflammation, and circadian rhythm regulation at Days 1 and 30; these effects dissipated by Day 240. There were other significantly-increased DEG induced by ISO at Day 360; these included changes in expression of genes associated with cell signaling, differentiation, and migration, extracellular matrix organization, cell-substrate adhesion, heart development, and blood pressure regulation. Examination of consistent DEG at Days 240 and 360 indicated late onset DEG reflecting potential long-lasting effects from ISO; these included DEG associated with oxidative phosphorylation, ribosome, angiogenesis, mitochondrial translation elongation, and focal adhesion. Together, the data show acute repeated ISO exposures could impart variable effects on gene expression/regulation in the heart. While some alterations self-resolved, others appeared to be long-lasting or late onset. Whether such changes occur in all rat models or in humans remains to be investigated.

Impact on rats from acute intratracheal inhalation exposures to WTC dusts.

Background: Studies have revealed the increased incidence of health disorders in First Responders (FR) who were at Ground Zero over the initial 72 hr after the World Trade Center (WTC) collapses. Previous studies in rats exposed to WTC dusts using exposure scenarios that mimicked FR mouthbreathing showed exposure led to altered expression of genes whose products could be involved in lung ailments. Nevertheless, it was uncertain if repeated exposures (as occurred in earliest days post-disaster) might have given rise to long-term changes in the lungs/other organs, in white blood cell (WBC) profiles, and/or systemic expression of select (mostly immune-related) proteins.Methods: To examine this, rats were exposed on 2 consecutive days (2 hr/d, intratracheal inhalation) to WTC dusts and then examined over a 1-yr period thereafter. At select times post-exposure, organ (lung, heart, liver, kidney, spleen) weights, WBC profiles, and blood levels of a variety of proteins were evaluated.Results: The study showed that over the 1-yr period, there were nominal effects on organ weights (absolute, index) as a result of the dust exposures. There were significant changes (relative to in naïve rats) in WBC profiles, with exposed rats having increased monocyte-macrophage and decreased lymphocyte percentages. The study also found that dust exposure led to significant systemic increases in many proteins, including MCP-1, RANTES, MMP-9, RAGE, and Galectin-3.Conclusions: These results provide further support for our longstanding hypothesis that the WTC dusts could potentially have acted as direct inducers of many of the health effects that have been seen in the exposed FR.

Complementary biobank of rodent tissue samples to study the effect of World Trade Center exposure on cancer development.

World Trade Center (WTC) responders were exposed to mixture of dust, smoke, chemicals and carcinogens. New York University (NYU) and Mount Sinai have recreated WTC exposure in rodents to observe the resulting systemic and local biological responses. These experiments aid in the interpretation of epidemiological observations and are useful for understanding the carcinogenesis process in the exposed human WTC cohort. Here we describe the implementation of a tissue bank system for the rodents experimentally exposed to WTC dust. NYU samples were experimentally exposed to WTC dust via intratracheal inhalation that mimicked conditions in the immediate aftermath of the disaster. Tissue from Mount Sinai was derived from genetically modified mice exposed to WTC dust via nasal instillation. All processed tissues include annotations of the experimental design, WTC dust concentration/dose, exposure route and duration, genetic background of the rodent, and method of tissue isolation/storage. A biobank of tissue from rodents exposed to WTC dust has been compiled representing an important resource for the scientific community. The biobank remains available as a scientific resource for future research through established mechanisms for samples request and utilization. Studies using the WTC tissue bank would benefit from confirming their findings in corresponding tissues from organs of animals experimentally exposed to WTC dust. Studies on rodent tissues will advance the understanding of the biology of the tumors developed by WTC responders and ultimately impact the modalities of treatment, and the probability of success and survival of WTC cancer patients.

Artery-Associated Sympathetic Innervation Drives Rhythmic Vascular Inflammation of Arteries and Veins.

BACKGROUND: The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. METHODS: We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. RESULTS: In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of ß2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. CONCLUSIONS: Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.

Prostate Cancer in World Trade Center Responders Demonstrates Evidence of an Inflammatory Cascade.

An excess incidence of prostate cancer has been identified among World Trade Center (WTC) responders. In this study, we hypothesized that WTC dust, which contained carcinogens and tumor-promoting agents, could facilitate prostate cancer development by inducing DNA damage, promoting cell proliferation, and causing chronic inflammation. We compared expression of immunologic and inflammatory genes using a NanoString assay on archived prostate tumors from WTC Health Program (WTCHP) patients and non-WTC patients with prostate cancer. Furthermore, to assess immediate and delayed responses of prostate tissue to acute WTC dust exposure via intratracheal inhalation, we performed RNA-seq on the prostate of normal rats that were exposed to moderate to high doses of WTC dust. WTC prostate cancer cases showed significant upregulation of genes involved in DNA damage and G2-M arrest. Cell-type enrichment analysis showed that Th17 cells, a subset of proinflammatory Th cells, were specifically upregulated in WTC patients. In rats exposed to WTC dust, we observed upregulation of gene transcripts of cell types involved in both adaptive immune response (dendritic cells and B cells) and inflammatory response (Th17 cells) in the prostate. Unexpectedly, genes in the cholesterol biosynthesis pathway were also significantly upregulated 30 days after acute dust exposure. Our results suggest that respiratory exposure to WTC dust can induce inflammatory and immune responses in prostate tissue. IMPLICATIONS: WTC-related prostate cancer displayed a distinct gene expression pattern that could be the result of exposure to specific carcinogens. Our data warrant further epidemiologic and cellular mechanistic studies to better understand the consequences of WTC dust exposure.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/8/1605/F1.large.jpg.

Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats.

Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12-13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6-11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled at Ground Zero in the critical initial 72-h period.

Acute high-level exposure to WTC particles alters expression of genes associated with oxidative stress and immune function in the lung.

First responders (FR) present at Ground Zero in the first 72 h after the World Trade Center (WTC) collapsed have progressively exhibited significant respiratory injuries. The few toxicology studies performed to date evaluated effects from just fine (< 2.5 µm) WTC dusts; none examined health effects/toxicities from atmospheres bearing larger particle sizes, despite the fact the majority (> 96%) of dusts were > 10 µm and most FR likely entrained dusts by mouth breathing. Using a system that generated/delivered supercoarse (10-53 µm) WTC dusts to F344 rats (in a manner that mimicked FR exposures), this study sought to examine potential toxicities in the lungs. In this exploratory study, rats were exposed for 2 h to 100 mg WTC dust/m(3) (while under isoflurane [ISO] anesthesia) or an air/ISO mixture; this dose conservatively modeled likely exposures by mouth-breathing FR facing ≈750-1000 mg WTC dust/m(3). Lungs were harvested 2 h post-exposure and total RNA extracted for subsequent global gene expression analysis. Among the > 1000 genes affected by WTC dust (under ISO) or ISO alone, 166 were unique to the dust exposure. In many instances, genes maximally-induced by the WTC dust exposure (relative to in naïve rats) were unchanged/inhibited by ISO only; similarly, several genes maximally inhibited in WTC dust rats were largely induced/unchanged in rats that received ISO only. These outcomes reflect likely contrasting effects of ISO and the WTC dust on lung gene expression. Overall, the data show that lungs of rats exposed to WTC dust (under ISO) - after accounting for any impact from ISO alone - displayed increased expression of genes related to lung inflammation, oxidative stress, and cell cycle control, while several involved in anti-oxidant function were inhibited. These changes suggested acute inflammogenic effects and oxidative stress in the lungs of WTC dust-exposed rats. This study, thus, concludes that a single very high exposure to WTC dusts could potentially have adversely affected the respiratory system - in terms of early inflammatory and oxidative stress processes. As these changes were not compared with other types of dusts, the uniqueness of these WTC-mediated effects remains to be confirmed. It also still remains to be determined if these effects might have any relevance to chronic lung pathologies that became evident among FR who encountered the highest dust levels on September 11, 2001 and the 2 days thereafter. Ongoing studies using longer-range post-exposure analyses (up to 1-year or more) will help to determine if effects seen here on genes were acute, reversible, or persistent, and associated with corresponding histopathologic/biochemical changes in situ.

A novel system to generate WTC dust particles for inhalation exposures.

First responders (FRs) present at Ground Zero within the critical first 72 h after the World Trade Center (WTC) collapse have progressively exhibited significant respiratory injury. The majority (>96%) of WTC dusts were >10 µm and no studies have examined potential health effects of this size fraction. This study sought to develop a system to generate and deliver supercoarse (10-53 µm) WTC particles to a rat model in a manner that mimicked FR exposure scenarios. A modified Fishing Line generator was integrated onto an intratracheal inhalation (ITIH) system that allowed for a bypassing of the nasal passages so as to mimic FR exposures. Dust concentrations were measured gravimetrically; particle size distribution was measured via elutriation. Results indicate that the system could produce dusts with 23 µm mass median aerodynamic diameter (MMAD) at levels up to ≥1200 mg/m(3). To validate system utility, F344 rats were exposed for 2 h to ≈100 mg WTC dust/m(3). Exposed rats had significantly increased lung weight and levels of select tracer metals 1 h after exposure. Using this system, it is now possible to conduct relevant inhalation exposures to determine adverse WTC dusts impacts on the respiratory system. Furthermore, this novel integrated Fishing Line-ITIH system could potentially be used in the analyses of a wide spectrum of other dusts/pollutants of sizes previously untested or delivered to the lungs in ways that did not reflect realistic exposure scenarios.

Hydroxyurea and a cGMP-amplifying agent have immediate benefits on acute vaso-occlusive events in sickle cell disease mice.

Inhibition of leukocyte adhesion to the vascular endothelium represents a novel and important approach for decreasing sickle cell disease (SCD) vaso-occlusion. Using a humanized SCD-mouse-model of tumor necrosis factor-α-induced acute vaso-occlusion, we herein present data demonstrating that short-term administration of either hydroxyurea or the phosphodiesterase 9 (PDE9) inhibitor, BAY73-6691, significantly altered leukocyte recruitment to the microvasculature. Notably, the administration of both agents led to marked improvements in leukocyte rolling and adhesion and decreased heterotypic red blood cell-leukocyte interactions, coupled with prolonged animal survival. Mechanistically, these rheologic benefits were associated with decreased endothelial adhesion molecule expression, as well as diminished leukocyte Mac-1-integrin activation and cyclic guanosine monophosphate (cGMP)-signaling, leading to reduced leukocyte recruitment. Our findings indicate that hydroxyurea has immediate beneficial effects on the microvasculature in acute sickle-cell crises that are independent of the drug's fetal hemoglobin-elevating properties and probably involve the formation of intravascular nitric oxide. In addition, inhibition of PDE9, an enzyme highly expressed in hematopoietic cells, amplified the cGMP-elevating effects of hydroxyurea and may represent a promising and more tissue-specific adjuvant therapy for this disease.

Comparison of WTC dust size on macrophage inflammatory cytokine release in vivo and in vitro.

BACKGROUND: The WTC collapse exposed over 300,000 people to high concentrations of WTC-PM; particulates up to ∼50 mm were recovered from rescue workers' lungs. Elevated MDC and GM-CSF independently predicted subsequent lung injury in WTC-PM-exposed workers. Our hypotheses are that components of WTC dust strongly induce GM-CSF and MDC in AM; and that these two risk factors are in separate inflammatory pathways. METHODOLOGY/PRINCIPAL FINDINGS: Normal adherent AM from 15 subjects without WTC-exposure were incubated in media alone, LPS 40 ng/mL, or suspensions of WTC-PM(10-53) or WTC-PM(2.5) at concentrations of 10, 50 or 100 µg/mL for 24 hours; supernatants assayed for 39 chemokines/cytokines. In addition, sera from WTC-exposed subjects who developed lung injury were assayed for the same cytokines. In the in vitro studies, cytokines formed two clusters with GM-CSF and MDC as a result of PM(10-53) and PM(2.5). GM-CSF clustered with IL-6 and IL-12(p70) at baseline, after exposure to WTC-PM(10-53) and in sera of WTC dust-exposed subjects (n = 70) with WTC lung injury. Similarly, MDC clustered with GRO and MCP-1. WTC-PM(10-53) consistently induced more cytokine release than WTC-PM(2.5) at 100 µg/mL. Individual baseline expression correlated with WTC-PM-induced GM-CSF and MDC. CONCLUSIONS: WTC-PM(10-53) induced a stronger inflammatory response by human AM than WTC-PM(2.5). This large particle exposure may have contributed to the high incidence of lung injury in those exposed to particles at the WTC site. GM-CSF and MDC consistently cluster separately, suggesting a role for differential cytokine release in WTC-PM injury. Subject-specific response to WTC-PM may underlie individual susceptibility to lung injury after irritant dust exposure.

Interactive effect of cigarette smoke extract and world trade center dust particles on airway cell cytotoxicity.

Rescue workers and residents exposed to the environment surrounding the collapse of the World Trade Center (WTC) on September 11, 2001, have suffered a disproportionate incidence of chronic lung disease attributed to the inhalation of airborne dust. To date, the pathophysiology of this lung disease is poorly understood. The aim of this study was to examine whether airborne dust contaminants recovered from the surrounding area 24-48 h after the collapse of the WTC demonstrate direct cytotoxicity to two airway cell types that were most directly exposed to inhaled dust, airway epithelial and smooth muscle cells. It was also of interest to determine whether the presence of these dusts could modulate the effects of cigarette smoke on these cell types in that some of the individuals who responded to the collapse site were also smokers. Human cultured airway epithelial (BEAS-2B) cells were exposed to 10% cigarette smoke extract (CSE), WTC dust particles (10-53 µm; 0.01-0.5 µg/µl), or a combination of the two for 2-24 h. Cell viability was measured by determining mitochondrial integrity (MTT assays) and apoptosis (poly-ADP-ribose polymerase [PARP] immunoblotting). Conditioned cell culture media recovered from the CSE- and/or WTC dust-exposed BEAS-2B cells were then applied to cultured human airway smooth muscle cells that were subsequently assayed for mitochondrial integrity and their ability to synthesize cyclic AMP (a regulator of airway smooth muscle constriction). BEAS-2B cells underwent necrotic cell death following exposure to WTC dust or CSE for 2-24 h without evidence of apoptosis. Smooth muscle cells demonstrated cellular toxicity and enhanced cyclic AMP synthesis following exposure to conditioned media from WTC- or CSE-exposed epithelial cells. These acute toxicity assays of WTC dust and CSE offer insights into lung cell toxicity that may contribute to the pathophysiology of chronic lung disease in workers and residents exposed to WTC dust. These studies clearly showed that WTC dust (at least the supercoarse particle fraction) or CSE alone exerted direct adverse effects on airway epithelial and smooth muscle cells, and altered the signaling properties of airway smooth muscle cells. In addition the combination of CSE and WTC exerted an interactive effect on cell toxicity. It remains to be determined whether these initial cell death events might account, in part, for the chronic lung effects associated with WTC dust exposure among First Responders and others.

Physiological contribution of CD44 as a ligand for E-Selectin during inflammatory T-cell recruitment.

Endothelial selectins guide the migration of inflammatory T cells to extralymphoid tissues. Whereas P-selectin glycoprotein ligand-1 (PSGL-1) functions as the exclusive ligand for P-selectin, it acts in coordination with additional glycoproteins to mediate E-selectin binding. CD44 can act as one such ligand in neutrophils, but its contribution in inflammatory T lymphocytes remains unexplored. We have used real-time in vivo imaging of the cremasteric and dermal microcirculations to explore the dynamics of leukocyte recruitment, as well as the physiological contribution of CD44 in a model of Th1-driven inflammation. CD4(+) T-cell rolling frequency and kinetics, as well as arrest, were dependent on endothelial selectins and were markedly altered under inflammatory conditions. CD44 extracted from Th1 cells bound to soluble E-selectin in vitro and cooperated with PSGL-1 by controlling rolling velocities and promoting firm arrest. Using several competitive recruitment assays in a delayed-type hypersensitivity model, we show that the combined absence of CD44 and PSGL-1 impairs inflammatory T-cell recruitment beyond that of PSGL-1 alone. Differential expression of leukocyte fucosyltransferases in these cells may account for the differential use of E-selectin ligands relative to neutrophils. Our results identify additional mechanisms by which CD44 modulates the inflammatory response.

Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche.

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1(hi) monocytes (MOs), Gr-1(lo) MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin(+) niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169(+) MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin(+) niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.

Roles of MAPK pathway activation during cytokine induction in BEAS-2B cells exposed to fine World Trade Center (WTC) dust.

The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter (PM) into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM2.5 fraction or WTC2.5). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the mitogen-activated protein kinase (MAPK) signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC2.5. Our results showed that exposure to WTC2.5 for 5 hr increased interleukin-6 (IL-6) mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, cytokine multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal protein kinase, signaling pathways were found to be activated in cells exposed to WTC2.5. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC2.5; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC2.5-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other inflammation-associated respiratory dysfunctions in the individuals exposed to the dusts released from the WTC collapse.

Effects of metal compounds with distinct physicochemical properties on iron homeostasis and antibacterial activity in the lungs: chromium and vanadium.

In situ reactions of metal ions or their compounds are important mechanisms by which particles alter lung immune responses. The authors hypothesized that major determinants of the immunomodulatory effect of any metal include its redox behavior/properties, oxidation state, and/or solubility, and that the toxicities arising from differences in physicochemical parameters are manifest, in part, via differential shifts in lung iron (Fe) homeostasis. To test the hypotheses, immunomodulatory potentials for both pentavalent vanadium (VV; as soluble metavanadate or insoluble vanadium pentoxide) and hexavalent chromium (CrVI; as soluble sodium chromate or insoluble calcium chromate) were quantified in rats after inhalation (5h/day for 5 days) of each at 100 microg metal/m3. Differences in effects on local bacterial resistance between the two VV, and between each CrVI, agents suggested that solubility might be a determinant of in situ immunotoxicity. For the soluble forms, VV had a greater impact on resistance than CrVI, indicating that redox behavior/properties was likely also a determinant. The soluble VV agent was the strongest immunomodulant. Regarding Fe homeostasis, both VV agents had dramatic effects on airway Fe levels. Both also impacted local immune/airway epithelial cell Fe levels in that there were significant increases in production of select cytokines/chemokines whose genes are subject to regulation by HIF-1 (whose intracellular longevity is related to cell Fe status). Our findings contribute to a better understanding of the role that metal compound properties play in respiratory disease pathogenesis and provide a rationale for differing pulmonary immunotoxicities of commonly encountered ambient metal pollutants.

Pulmonary immunotoxic potentials of metals are governed by select physicochemical properties: vanadium agents.

The in situ reactions of metal ions/complexes are important in understanding the mechanisms by which environmental and occupational metal particles alter lung immune responses. A better understanding of these reactions in situ will also allow for the improved specificity and controlled toxicity of novel metallocompounds to be used as inhaled diagnostics or therapeutics. Our previous work showed that inhalation of metals (e.g., chromium, vanadium, nickel) caused altered lung immune cell function and host resistance. The data also suggested that the degree of immunomodulation induced depended not only on the amount of metal deposited, but also the compound used. If specificity governs pulmonary immunomodulatory potential, it follows that physicochemical properties inherent to the metal have a role in the elicited effects. We hypothe-size that major determinants of any metal compound's potential are its redox behavior, valency (generally referred to as oxidation state and considered speciation in chemical literature), and/or solubility. In accord with the extensive work carried out with vanadium (chemical symbol V) compounds showing the importance of form used, differences in potential for a range of V agents (pentavalent [V(V)] insoluble vanadium pentoxide and soluble sodium metavanadate, tetravalent [V(IV)] vanadyl dipicolinate, and trivalent [V(III)] bis(dipicolinato)vanadium) were quantified based on induced changes in local bacterial resistance after host inhalation of each agent at 100 mu g V/m(3) (5 hr/d for 5 d). Differences in effect between V(V) forms indicated that solubility was a critical property in in situ pulmonary immunotoxicity. Among the soluble forms, oxidizing vanadate had the greatest impact on resistance; reducing V(III) altered resistance to a lesser extent. Both the V(IV) and insoluble V(V) had no effect. When data was analyzed in the context of pre-infection lung V burdens, soluble V agents with different oxidation states induced varying responses, supporting the hypothesis that differences in immunomodulatory potential might be attributed to redox behavior or valency. Our findings both provide a basis for understanding why some metals could be a greater health risk than others (when encountered in equal amounts) and will assist in the design of inhalable metallopharmaceuticals by allowing researchers to preempt selection of certain metal ions or complexes for use in such products.

Effects of select PM-associated metals on alveolar macrophage phosphorylated ERK1 and -2 and iNOS expression during ongoing alteration in iron homeostasis.

It was hypothesized that relative mass relationships among select constituent metals and iron (Fe3+) govern the pulmonary immunotoxic potential of any PM(2.5) sample, as these determine the extent to which Fe3+ binding by transferrin is affected (resulting in altered alveolar macrophage [AM] Fe status and subsequent antibacterial function). Iron response protein (IRP) binding activity is a useful indirect measurement of changes in Fe status, as reductions in cell Fe levels lead to increases in IRP binding. However, AM IRP activity can be affected by an increased presence of nitric oxide generated by inducible nitric oxide synthase (iNOS). This study sought to determine if any changes in AM IRP activity induced by PM(2.5) constituents V, Mn, or Al were independent from effects of the metals on cell NO formation. NR8383 rat AM were exposed to Fe3+ alone or combined with V, Mn, or Al at metal:Fe ratios representative of those in PM(2.5) collected in New York City, Los Angeles, and Seattle during fall 2001. Cells were then assessed for changes in IRP activity and iNOS expression. Phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2 levels were also measured since activated ERKs are involved in signaling pathways that lead to increased iNOS expression. The results indicate that V and Al, and to a lesser extent Mn, altered IRP activity, though the effects were not consistently concentration dependent. Furthermore, while V and Mn treatments did not induce iNOS expression, Al did. These results confirmed our hypothesis that certain metals associated with PM(2.5) might alter the pulmonary immunocompetence of exposed hosts by affecting the Fe status of AM, a major class of deep lung defense cells.

Pulmonary immunotoxic potentials of metals are governed by select physicochemical properties: chromium agents.

Increasing the understanding of how metal ions/complexes react in situ will allow for the improved specificity and controlled toxicity of novel synthetic metallocompounds that will be used as inhaled diagnostics or therapeutics. Our previous work showed that inhalation of select metals (e.g., chromium, vanadium, nickel, iron) caused alterations in lung immune cell function and in local bacterial resistance. The data also suggested that variations in the degree of immuno-modulation induced were not solely dependent on the amount of metal deposited in the lung, but also on the specific compound. If specificity governs immunomodulatory potential, it follows that physicochemical properties inherent to the metal may have a role in the elicited effects. We hypothesize that major determinants of any metal compound's immunomodulatory potential in situ are its redox behavior, valency, and/or solubility. Using changes in local bacterial resistance as an endpoint, differences in immunotoxic potential in the lungs were quantified for a range of chromium agents (insoluble calcium chromate(VI), and soluble sodium chromate(VI), potassium bis(dipicolinato)chromate(III) and sodium bis(dipicolinato)chromate(II)). Results indicated that among the latter three forms of Cr, strongly oxidizing hexavalent Cr (Cr[VI]) had the greatest impact on resistance, while reducing divalent and fairly unreactive trivalent forms of Cr had no effect at an equal exposure level (i.e., 100 microg Cr/m(3), 5 hr/d, for 5 d). Insoluble Cr(VI) had a greater effect than its soluble form. When data was analyzed in the context of pre-infection lung Cr burdens, it was seen that immunomodulatory potentials for both Cr(VI) agents did not differ significantly; however, complexes with different oxidation states did induce varying responses, suggesting that differences in potential might be attributed to redox behavior. From this it was concluded that for Cr, certain physicochemical properties are likely more important to any in situ pulmonary immunotoxicity than others (i.e., redox behavior is more critical than solubility). Our findings, in part, will help provide a basis for understanding why certain metals could be a greater health risk than others, even when encountered in equal amounts. This, in turn, will help researchers in the design of inhalable diagnostic/therapeutic metallopharmaceuticals by pre-empting the selection of certain metal ions/complexes for potential use in these products.

Chemical analysis of World Trade Center fine particulate matter for use in toxicologic assessment.

The catastrophic destruction of the World Trade Center (WTC) on 11 September 2001 caused the release of high levels of airborne pollutants into the local environment. To assess the toxicity of fine particulate matter [particulate matter with a mass median aerodynamic diameter < 2.5 microm (PM2.5)], which may adversely affect the health of workers and residents in the area, we collected fallen dust samples on 12 and 13 September 2001 from sites within a half-mile of Ground Zero. Samples of WTC dust were sieved, aerosolized, and size-separated, and the PM2.5 fraction was isolated on filters. Here we report the chemical and physical properties of PM2.5 derived from these samples and compare them with PM2.5 fractions of three reference materials that range in toxicity from relatively inert to acutely toxic (Mt. St. Helens PM; Washington, DC, ambient air PM; and residual oil fly ash). X-ray diffraction of very coarse sieved WTC PM (< 53 microm) identified calcium sulfate (gypsum) and calcium carbonate (calcite) as major components. Scanning electron microscopy confirmed that calcium-sulfur and calcium-carbon particles were also present in the WTC PM2.5 fraction. Analysis of WTC PM2.5 using X-ray fluorescence, neutron activation analysis, and inductively coupled plasma spectrometry showed high levels of calcium (range, 22-33%) and sulfur (37-43% as sulfate) and much lower levels of transition metals and other elements. Aqueous extracts of WTC PM2.5 were basic (pH range, 8.9-10.0) and had no evidence of significant bacterial contamination. Levels of carbon were relatively low, suggesting that combustion-derived particles did not form a significant fraction of these samples recovered in the immediate aftermath of the destruction of the towers. Because gypsum and calcite are known to cause irritation of the mucus membranes of the eyes and respiratory tract, inhalation of high doses of WTC PM2.5 could potentially cause toxic respiratory effects.