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Exposure to dust from active and abandoned mining operations may be a very significant health hazard, especially to sensitive populations. We have previously reported that inhalation of real-world mine tailing dusts during lung development can alter lung function and structure in adult male mice. These real-world dusts contain a mixture of metal(loid)s, including arsenic. To determine whether arsenic in inhaled dust plays a role in altering lung development, we exposed C57Bl/6 mice to a background dust (0 arsenic) or to the background dust containing either 3% or 10% by mass, calcium arsenate. Total level of exposure was kept at 100 μg/m 3 . Calcium arsenate was selected since arsenate is the predominant species found in mine tailings. We found that inhalation exposure during in utero and postnatal lung development led to significant increases in pulmonary baseline resistance, airway hyper-reactivity, and airway collagen and smooth muscle expression in male C57Bl/6 mice. Responses were dependent on the level of calcium arsenate in the simulated dust. These changes were not associated with increased expression of TGF-β1, a marker of epithelial to mesenchymal transition. However, responses were correlated with decreases in the expression of club cell protein 16 (CC16). Dose-dependent decreases in CC16 expression and increases in collagen around airways was seen for animals exposed in utero only (GD), animals exposed postnatally only (PN) and animals continuously exposed throughout development (GDPN). These data suggest that arsenic inhalation during lung development can decrease CC16 expression leading to functional and structural alterations in the adult lung., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tungsten (W) occurrence and speciation was investigated in sediments collected from Fallon, Nevada where previous studies have linked elevated W levels in human body fluids to an unusual cluster of childhood leukemia cases. The speciation of sedimentary W was determined by μ-XRF mapping and μ-XANES. The W content of the analyzed surface sediments ranged between 81 and 25,908 mg/kg, which is significantly higher than the W content in deeper sediments which ranged from 37 to 373 mg/kg at 30 cm depth. The μ-XANES findings reveal that approximately 20-50% of the total W in the shallow sediment occurs in the metallic form (W 0 ); the rest occurs in the oxide form (W VI O 3 ). Because W 0 does not occur naturally, its elevated concentrations in surface sediments point toward a possible local anthropogenic origin. The oxidation of metallic W 0 with meteoric waters likely leads to the formation of W VI O 3 . The chief water-soluble W species was identified as WO 4 2- by chromatographic separation and speciation modeling. These results led us to postulate that W 0 particles from a currently unknown but local source(s) is (are) deposited onto the soils and/or surface sediments. The W 0 in interaction with meteoric water is oxidized to W VI O 3 , and as these sediment-water interactions progress, WO 4 2- is formed in the water at pH ∼7. Under pH < 7, and sufficient W concentrations, tungstate tends to polymerize, and polymerized species are less likely to adsorb onto sediments. Polymerized species have lower affinity than monomers, which leads to enhanced mobility of W., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Exposure to mine tailings dust from active and abandoned mining operations may be a very significant health hazard, especially to sensitive populations living in arid and semi-arid climates like the desert southwest of the US. It is anticipated that early life exposures during sensitive times of development can lead to adult disease. However, very few studies have investigated the effects of inhalation exposure to real world dusts during lung development. Using a mouse model, we have examined the effect(s) of inhalation of real world mine tailing dusts under three separate conditions: (1) Exposure only during in utero development (exposure of the pregnant moms) (2) exposure only after birth and (3) exposures that occurred continuously during in utero development, through gestation and birth until the mice reached adulthood (28 days old). We found that the most significant changes in lung structure and function were observed in male mice when exposure occurred continuously throughout development. These changes included increased airway hyper-reactivity, increased expression of epithelial to mesenchymal (EMT) transition protein markers and increased expression of cytokines related to eosinophils. The data also indicate that in utero exposures through maternal inhalation can prime the lung of male mice for more severe responses to subsequent postnatal exposures. This may be due to epigenetic alterations in gene regulation, immune response, molecular signaling, and growth factors involved in lung development that may make the neonatal lung more susceptible to continued dust exposure., (Copyright © 2019. Published by Elsevier Inc.)

The etiology of childhood leukemia is not known. Strong evidence indicates that precursor B-cell Acute Lymphoblastic Leukemia (Pre-B ALL) is a genetic disease originating in utero. Environmental exposures in two concurrent, childhood leukemia clusters have been profiled and compared with geographically similar control communities. The unique exposures, shared in common by the leukemia clusters, have been modeled in C57BL/6 mice utilizing prenatal exposures. This previous investigation has suggested in utero exposure to sodium tungstate (Na2WO4) may result in hematological/immunological disease through genes associated with viral defense. The working hypothesis is (1) in addition to spontaneously and/or chemically generated genetic lesions forming pre-leukemic clones, in utero exposure to Na2WO4 increases genetic susceptibility to viral influence(s); (2) postnatal exposure to a virus possessing the 1FXXKXFXXA/V9 peptide motif will cause an unnatural immune response encouraging proliferation in the B-cell precursor compartment. This study reports the results of exposing C57BL/6J mice to Na2WO4 in utero via water (15 ppm, ad libetum) and inhalation (mean concentration PM5 3.33 mg/m3) and to Respiratory Syncytial Virus (RSV) within 2 weeks of weaning. Inoculation of C57BL/6J mice with RSV was associated with a neutrophil shift in 56% of 5-month old mice. When the RSV inoculation was combined with Na2WO4-exposure, significant splenomegaly resulted (p=0.0406, 0.0184, 0.0108 for control, Na2WO4-only and RSV-only, respectively) in addition to other hematological pathologies which were not significant. Exposure to Na2WO4 and RSV resulted in hematological/immunological disease, the nature of which is currently inconclusive. Further research is needed to characterize this potential leukemia mouse model., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Previously reported dendrochemical data showed temporal variability in concentration of tungsten (W) and cobalt (Co) in tree rings of Fallon, Nevada, US. Criticism of this work questioned the use of the Mann-Whitney test for determining change in element concentrations. Here, we demonstrate that Mann-Whitney is appropriate for comparing background element concentrations to possibly elevated concentrations in environmental media. Given that Mann-Whitney tests for differences in shapes of distributions, inter-tree variability (e.g., "coefficient of median variation") was calculated for each measured element across trees within subsites and time periods. For W and Co, the metals of highest interest in Fallon, inter-tree variability was always higher within versus outside of Fallon. For calibration purposes, this entire analysis was repeated at a different town, Sweet Home, Oregon, which has a known tungsten-powder facility, and inter-tree variability of W in tree rings confirmed the establishment date of that facility. Mann-Whitney testing of simulated data also confirmed its appropriateness for analysis of data affected by point-source contamination. This research adds important new dimensions to dendrochemistry of point-source contamination by adding analysis of inter-tree variability to analysis of central tendency. Fallon remains distinctive by a temporal increase in W beginning by the mid 1990s and by elevated Co since at least the early 1990s, as well as by high inter-tree variability for W and Co relative to comparison towns., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Environmental links to disease are difficult to uncover because environmental exposures are variable in time and space, contaminants occur in complex mixtures, and many diseases have a long time delay between exposure and onset. Furthermore, individuals in a population have different activity patterns (e.g., hobbies, jobs, and interests), and different genetic susceptibilities to disease. As such, there are many potential confounding factors to obscure the reasons that one individual gets sick and another remains healthy. An important method for deducing environmental associations with disease outbreak is the retrospective case-control study wherein the affected and control subject cohorts are studied to see what is different about their previous exposure history. Despite success with infectious diseases (e.g., food poisoning, and flu), case-control studies of cancer clusters rarely have an unambiguous outcome. This is attributed to the complexity of disease progression and the long-term latency between exposure and disease onset. In this article, we consider strategies for investigating cancer clusters and make some observations for improving statistical power through broader non-parametric approaches wherein sub-populations (i.e., whole towns), rather than individuals, are treated as the cases and controls, and the associated cancer rates are treated as the dependent variable. We subsequently present some ecological data for tungsten and cobalt from studies by University of Arizona researchers who document elevated levels of tungsten and cobalt in Fallon, NV. These results serve as candidates for future hybrid ecologic case-control investigations of childhood leukemia clusters., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

There is emerging evidence that tungsten has toxic health effects. We summarize the recent tungsten toxicity research in this short review. Tungsten is widely used in many commercial and military applications because it has the second highest melting temperature of any element. Consequently, it is important to elucidate the potential health effects of tungsten., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

To improve understanding of possible connections between airborne tungsten and public health, size and geography of airborne tungsten particles collected in Fallon, Nevada, and Sweet Home, Oregon, were compared. Both towns have industrial tungsten facilities, but only Fallon has experienced a cluster of childhood leukemia. Fallon and Sweet Home are similar to one another by their particles of airborne tungsten being generally small in size. Meteorologically, much, if not most, of residential Fallon is downwind of its hard metal facility for at least some fraction of time at the annual scale, whereas little of residential Sweet Home is downwind of its tungsten facility. Geographically, most Fallon residents potentially spend time daily within an environment containing elevated levels of airborne tungsten. In contrast, few Sweet Home residents potentially spend time daily within an airborne environment with elevated levels of airborne tungsten. Although it cannot be concluded from environmental data alone that elevated airborne tungsten causes childhood leukemia, the lack of excessive cancer in Sweet Home cannot logically be used to dismiss the possibility of airborne tungsten as a factor in the cluster of childhood leukemia in Fallon. Detailed modeling of all variables affecting airborne loadings of heavy metals would be needed to legitimately compare human exposures to airborne tungsten in Fallon and Sweet Home.

In this study, we examined the role of neprilysin (NEP), a key membrane-bound endopeptidase, in the inflammatory response induced by diesel exhaust emissions (DEE) in the airways through a number of approaches: in vitro, animal, and controlled human exposure. Our specific aims were (1) to examine the role of NEP in inflammatory injury induced by diesel exhaust particles (DEP) using Nep-intact (wild-type) and Nep-null mice; (2) to examine which components of DEP are associated with NEP downregulation in vitro; (3) to determine the molecular impact of DEP exposure and decreased NEP expression on airway epithelial cells' gene expression in vitro, using a combination of RNA interference (RNAi) and microarray approaches; and (4) to evaluate the effects on NEP activity of human exposure to DEE. We report four main results: First, we found that exposure of normal mice to DEP consisting of standard reference material (SRM) 2975 via intratracheal installation can downregulate NEP expression in a concentration-dependent manner. The changes were accompanied by increases in the number of macrophages and epithelial cells, as well as proinflammatory cytokines, examined in bronchoalveolar lavage (BAL) fluid and cells. Nep-null mice displayed increased and/or additional inflammatory responses when compared with wild-type mice, especially in response to exposure to the higher dose of DEP that we used. These in vivo findings suggest that loss of NEP in mice could cause increased susceptibility to injury or exacerbate inflammatory responses after DEP exposure via release of specific cytokines from the lungs. Second, we found evidence, using in vitro studies, that downregulation of NEP by DEP in cultured human epithelial BEAS-2B cells was mostly attributable to DEP-adsorbed organic compounds, whereas the carbonaceous core and transition metal components of DEP had little or no effect on NEP messenger RNA (mRNA) expression. This NEP downregulation was not a specific response to DEP or its contents because the change also occurred after exposure to urban dust (SRM 1649a), which differs in physical and chemical composition from DEP. Third, we also collected the transcriptome profiles of the concentration-effects of SRM 2975 in cultured BEAS-2B cells through a 2 X 3 factorial design. DEP exposure upregulated 151 genes and downregulated 59 genes. Cells with decreased NEP expression (accomplished by transfecting an NEP-specific small interfering RNA [siRNA]) substantially altered the expression of genes (upregulating 17 and downregulating 14) associated with DNA/protein binding, calcium channel activities, and the cascade of intracellular signaling by cytokines. Data generated from the combined RNAi and microarray approaches revealed that there is a complex molecular cascade mediated by NEP in different subcellular compartments, possibly influencing the inflammatory response. Fourth, in a controlled human exposure study, we observed significant increases in soluble NEP in sputum after acute exposure to DEE, with an average net increase of 31%. We speculate that the change in NEP activity in sputum, if confirmed in larger epidemiologic investigations at ambient exposure levels to DEE, may provide a useful endpoint and promote insight into the mechanism of DEE-induced airway alterations.

This study was designed to characterize and compare the effects of jet propellant-8 (JP-8) fuel and synthetic-8 (S-8) on cell viability and nitric oxide synthesis in cultured alveolar type II epithelial cells of rats. Exposure times varied from 0.25, 0.5, 1, and 6 hours at the following concentrations of jet fuel: 0.0, 0.1, 0.4, and 2.0 microg/mL. Data indicate that JP-8 presents a gradual decline in cell viability and steady elevation in nitric oxide release as exposure concentrations increase. At a 2.0 microg/mL concentration of JP-8, nearly all of the cells are not viable. Moreover, S-8 exposure to rat type II lung cells demonstrated an abrupt fall in percentage cell viability and increases in nitric oxide measurement, particularly after the 2.0 microg/mL was reached at 1 and 6 hours. At 0.0, 0.2, and 0.4 microg/mL concentrations of S-8, percentage viability was sustained at steady concentrations. The results suggest different epithelial toxicity and mechanistic effects of S-8 and JP-8, providing further insight concerning the impairment imposed at specific levels of lung function and pathology induced by the different fuels.

In vitro bioaccessibility (IVBA) studies were carried out on samples of mercury (Hg) mine-waste calcine (roasted Hg ore) by leaching with simulated human body fluids. The objective was to estimate potential human exposure to Hg due to inhalation of airborne calcine particulates and hand-to-mouth ingestion of Hg-bearing calcines. Mine waste calcines collected from Hg mines at Almaden, Spain, and Terlingua, Texas, contain Hg sulfide, elemental Hg, and soluble Hg compounds, which constitute primary ore or compounds formed during Hg retorting. Elevated leachate Hg concentrations were found during calcine leaching using a simulated gastric fluid (as much as 6200 microg of Hg leached/g sample). Elevated Hg concentrations were also found in calcine leachates using a simulated lung fluid (as much as 9200 microg of Hg leached/g), serum-based fluid (as much as 1600 microg of Hg leached/g), and water of pH 5 (as much as 880 microg of Hg leached/g). The leaching capacity of Hg is controlled by calcine mineralogy; thus, calcines containing soluble Hg compounds contain higher leachate Hg concentrations. Results indicate that ingestion or inhalation of Hg mine-waste calcine may lead to increased Hg concentrations in the human body, especially through the ingestion pathway.

Neprilysin (NEP) is a key cell surface peptidase in the maintenance of airway homeostasis and the development of pulmonary disorders. However, little information is available about the effect of particulate matter (PM) on airway NEP. In this controlled human exposure study, changes in induced sputum were measured in 11 subjects at baseline, overshot (OS) mucking, and diesel exhaust (DE) exposure days. Neither OS condition nor DE exposure was found to induce significant changes in total protein, but DE induced significant increases in cell numbers of macrophages and epithelium. Moreover, significant increases in soluble NEP were observed following OS mining dust particulates (0.43 +/- 0.06 nmol/microg protein/min; p = .023) and DE exposure (0.40 +/- 0.03 nmol/microg protein/min; p = .035) when compared with the baseline control (0.30 +/- 0.04 nmol/microg protein/min), with 42% and 31% average net increase, respectively. Pearson's correlation analyses indicated that sputum NEP activity was significantly associated with personal exposure product (elemental carbon concentration [mg/m(3)] x time [min]; C x T). The data suggest that changes in NEP activity may be an early, accurate endpoint for airway epithelial injury and provide a new insight into the mechanism of airway effects following particulate exposure.

No current studies have systematically examined pulmonary health effects associated with Syntroleum S-8 synthetic jet fuel (S-8). In order to gain an understanding about the threshold concentration in which lung injury is observed, C57BL/6 male mice were nose-only exposed to S-8 for 1 h/day for 7 days at average concentrations of 0 (control), 93, 352, and 616 mg/m(3). Evaluation of pulmonary function, airway epithelial barrier integrity, and pathohistology was performed 24 h after the final exposures. Significant decreases were detected in expiratory lung resistance and total lung compliance of the 352 mg/m(3) group, for which no clear concentration-dependent alterations could be determined. No significant changes in respiratory permeability were exhibited, indicating that there was no loss of epithelial barrier integrity following S-8 exposure. However, morphological examination and morphometric analysis of distal lung tissue, by using transmission electron microscopy, revealed cellular damage in alveolar type II epithelial cells, with significant increases in volume density of lamellar bodies/vacuoles at 352 and 616 S-8 mg/m(3). Moreover, terminal bronchiolar Clara injury, as evidenced by apical membrane blebs, was observed at relatively low concentrations, suggesting if this synthetic jet fuel is utilized, the current permissible exposure limit of 350 mg/m(3) for hydrocarbon fuels should cautiously be applied.

In addition to cancer endpoints, arsenic exposures can also lead to non-cancerous chronic lung disease. Exposures during sensitive developmental time points can contribute to the adult disease. Using a mouse model, in utero and early postnatal exposures to arsenic (100 ppb or less in drinking water) were found to alter airway reactivity to methacholine challenge in 28 day old pups. Removal of mice from arsenic exposure 28 days after birth did not reverse the alterations in sensitivity to methacholine. In addition, adult mice exposed to similar levels of arsenic in drinking water did not show alterations. Therefore, alterations in airway reactivity were irreversible and specific to exposures during lung development. These functional changes correlated with protein and gene expression changes as well as morphological structural changes around the airways. Arsenic increased the whole lung levels of smooth muscle actin in a dose dependent manner. The level of smooth muscle mass around airways was increased with arsenic exposure, especially around airways smaller than 100 microm in diameter. This increase in smooth muscle was associated with alterations in extracellular matrix (collagen, elastin) expression. This model system demonstrates that in utero and postnatal exposure to environmentally relevant levels of arsenic can irreversibly alter pulmonary structure and function in the adults.

This study was designed to characterize and compare the pulmonary effects in distal lung from a low-level exposure to jet propellant-8 fuel (JP-8) and a new synthetic-8 fuel (S-8). It is hypothesized that both fuels have different airway epithelial deposition and responses. Consequently, male C57BL/6 mice were nose-only exposed to S-8 and JP-8 at average concentrations of 53mg/m(3) for 1h/day for 7 days. A pulmonary function test performed 24h after the final exposure indicated that there was a significant increase in expiratory lung resistance in the S-8 mice, whereas JP-8 mice had significant increases in both inspiratory and expiratory lung resistance compared to control values. Neither significant S-8 nor JP-8 respiratory permeability changes were observed compared to controls, suggesting no loss of epithelial barrier integrity. Morphological examination and morphometric analysis of airway tissue demonstrated that both fuels showed different patterns of targeted epithelial cells: bronchioles in S-8 and alveoli/terminal bronchioles in JP-8. Collectively, our data suggest that both fuels may have partially different deposition patterns, which may possibly contribute to specific different adverse effects in lung ventilatory function.

Spatial patterns of tungsten and cobalt are described for surface dust of Fallon, Nevada, where a cluster of childhood leukemia has been ongoing since 1997. In earlier research, airborne tungsten and cobalt was shown to be elevated in total suspended particulates in Fallon. To fine-tune the spatial patterns of tungsten and cobalt deposition in Fallon, surface dust was collected in a grid pattern within as well as outside of Fallon to establish background concentrations of metals. In surface dust, tungsten and cobalt show sharp peaks (934 ppm and 98 ppm, respectively) within Fallon just north of highway 50 and west of highway 95. These two peaks overlap spatially, and given the grid pattern used for collecting surface dust, the source area of these two airborne metals can be pinpointed to the vicinity of hard-metal industry located north of highway 50 and west of highway 95. Fallon is distinctive in west central Nevada because of high airborne tungsten and cobalt particulates, and given its cluster of childhood leukemia, it stands to reason that additional biomedical research is in order to test directly the leukogenicity of combined airborne tungsten and cobalt particulates.

Morphological and chemical characteristics were determined for airborne tungsten particles in Fallon, Nevada, a town that is distinguishable environmentally by elevated airborne tungsten and cobalt. From samples of airborne dust collected previously at six different places in Fallon, tungsten-rich dust particles were isolated and analyzed with automated electron microprobe and wavelength-dispersive spectrometry. Representative W particles were further analyzed using transmission electron microscopy. Morphologically, Fallon W particles are angular and small, with minimum and maximum sizes of < or = 1 microm and 5.9 microm in diameter, respectively. The number and size of tungsten-rich particles decrease in Fallon with distance from a hard-metal facility located near the center of town. Chemically, Fallon airborne W particles include mixtures of tungsten with cobalt plus other metals such as chromium, iron, and copper. No W-rich particles were identifiable as CaWO4 (scheelite) or MnWO4 (huebnerite). From d-spacings, Fallon particles are most consistent with identification as tungsten carbide. Based on these multiple lines of evidence, airborne W particles in Fallon are anthropogenic in origin, not natural. The hard-metal facility in Fallon processes finely powdered W and W-Co, and further investigation using tracer particles is recommended to definitively identify the source of Fallon's airborne tungsten.

Four groups of Fischer Brown Norway hybrid rats were exposed for 5, 10, 15, or 20 d to aerosolized-vapor jet propulsion fuel 8 (JP-8) compared to freely moving (5 and 10-d exposures) or sham-confined controls (15 and 20-d exposures). Behavioral testing utilized the U.S. Environmental Protection Agency Functional Observational Battery. Exploratory ethological factor analysis identified three salient factors (central nervous system [CNS] excitability, autonomic 1, and autonomic 2) for use in profiling JP-8 exposure in future studies. The factors were used as dependent variables in general linear modeling. Exposed animals were found to engage in more rearing and hyperaroused behavior compared to controls, replicating prior JP-8 exposure findings. Exposed animals also showed increasing but rapidly decelerating stool output (autonomic 1), and a significant increasing linear trend for urine output (autonomic 2). No significant trends were noted for either of the control groups for the autonomic factors. Rats from each of the groups for each of the time frames were randomly selected for tissue assay from seven brain regions for neurotransmitter levels. Hippocampal DOPAC was significantly elevated after 4-wk JP-8 exposure compared to both control groups, suggesting increased dopamine release and metabolism. Findings indicate that behavioral changes do not appear to manifest until wk 3 and 4 of exposure, suggesting the need for longitudinal studies to determine if these behaviors occur due to cumulative exposure, or due to behavioral sensitization related to repeated exposure to aerosolized-vapor JP-8.

This paper describes the use of lichen chemistry to assess airborne tungsten and cobalt in Fallon, Nevada, where a cluster of childhood leukemia has been on going since 1997. Lichens and their rock substrates were collected from Rattlesnake Hill within Fallon as well as from four different rock outcrops located north, east, south, and west of Fallon and at least 20 km away from the town center. In the lichens themselves, W and Co are significantly higher within Fallon than in the combined control site outside of Fallon. In the rock substrates of the lichens, no differences exist in W and Co. The W and Co differences in lichens cannot be attributed to substrate geochemistry. Fallon is distinctive in west central Nevada for high airborne W and Co, and given its cluster of childhood leukemia, it stands to reason that additional biomedical research is in order to test directly the leukogenicity of combined airborne W and Co.

Background: Since 1997, Fallon, Nevada, has experienced a cluster of childhood leukemia that has been declared "one of the most unique clusters of childhood cancer ever reported." Multiple environmental studies have shown airborne tungsten and cobalt to be elevated within Fallon, but the question remains: Have these metals changed through time in correspondence with the onset of the leukemia cluster?, Methods: We used dendrochemistry, the study of element concentrations through time in tree rings, in Fallon to assess temporal variability of airborne tungsten and cobalt since the late 1980s. The techniques used in Fallon were also tested in a different town (Sweet Home, OR) that has airborne tungsten from a known source., Results: The Sweet Home test case confirms the accuracy of dendrochemistry for showing temporal variability of environmental tungsten. Given that dendrochemistry works for tungsten, tree-ring chemistry shows that tungsten increased in Fallon relative to nearby comparison towns beginning by the mid-1990s, slightly before the onset of the cluster, and cobalt has been high throughout the last approximately 15 years. Other metals do not show trends through time in Fallon., Discussion: Results in Fallon suggest a temporal correspondence between the onset of excessive childhood leukemia and elevated levels of tungsten and cobalt. Although environmental data alone cannot directly link childhood leukemia with exposure to metals, research by others has shown that combined exposure to tungsten and cobalt can be carcinogenic to humans., Conclusion: Continued biomedical research is warranted to directly test for linkage between childhood leukemia and tungsten and cobalt.

This paper describes the application of the chemistry of total suspended particulates, lichens/mosses, and surface dust for assessing spatial patterns of airborne tungsten and other metals. These techniques were used recently in Fallon, NV, where distinctive spatial patterns of airborne tungsten were demonstrated. However, doubt has been raised about the extent of airborne tungsten in Fallon. Therefore, these techniques were tested specifically for W in another town that has a small industry known to emit tungsten particles. Airborne particulates were collected in Sweet Home, OR, as well as in nearby comparison towns to provide baseline data. Lichens/mosses were collected in Sweet Home near the known source of W as well as outside of Sweet Home. Surface dust was collected throughout Sweet Home to map concentrations of metals. All three of these environmental monitoring techniques confirm that W is elevated right near the known source of airborne W in Sweet Home but no where else in Sweet Home. This test should allay doubts about the multiple findings of elevated airborne W in Fallon, NV, and this should also instill confidence in these techniques generally for assessing W and other metals in urban environments.

The effects of JP-8 on pro-inflammatory cytokine interleukin (IL)-1alpha,beta and nitric oxide (NO) secretion as well as the role of substance P (SP) in these processes were examined in cultured alveolar macrophages (AM), type II epithelial cells (AIIE), and AM/AIIE co-cultures. Exposure of AM to JP-8 for 24 hr exhibited release of IL-1alpha,beta, whereas exposure to AIIE showed a concentration-dependent NO overproduction. Data indicate that there are cell-dependent inflammatory mechanisms responsible for the actual level of JP-8 exposure in alveoli. However, treatment with substance P significantly attenuated JP-8 induced the IL-1alpha,beta secretion. This finding was confirmed by using [Sar(9) Met (O(2))(11)] SP (10(- 10) M), an agonist of substance P, suggesting that substance P may have signal pathway(s) to AM in the inflammatory response mediated by IL-1. Moreover, AM/AIIE co-culture obviously reduced NO overproduction observed in AIIE alone, suggesting that there may be cell interactions or communications between AM and AIIE in response to the JP-8 exposure.

Lung epithelial cells are critical in the regulation of airway inflammation in response to environmental pollutants. Altered activation of NF-kappaB is associated with expression of several proinflammatory factors in respiratory epithelial cells in response to an insult. Here we show that a low threshold dose (8 microg/ml) of the jet fuel JP-8 induces in a rat alveolar epithelial cell line (RLE-6TN) a prolonged activation of NF-kappaB as well as the increased expression of the proinflammatory cytokines TNF-alpha and IL-8, which are regulated by NF-kappaB. The up-regulation of IL-6 mRNA in cells exposed to JP-8 appears to be a reaction of RLE-6TN cells to reduce the enhancement of proinflammatory mediators in response to the fuel. Moreover, lung tissues from rats exposed to occupational levels of JP-8 by nasal aerosol also showed dysregulated expression of TNF-alpha, IL-8, and IL-6, confirming the in vitro data. The poly(ADP-ribosyl)ation of PARP-1, a coactivator of NF-kappaB, was coincident with the prolonged activation of NF-kappaB during JP-8 treatment. These results evidenced that a persistent exposure of the airway epithelium to aromatic hydrocarbons may have deleterious effects on pulmonary function.

C57BL/6 mice were nose-only exposed to JP-8 jet fuel at average concentrations of 45, 267, and 406 mg JP-8/m(3) for 1 hr/d for 7 days to further test the hypothesis that exposure to JP-8 concentrations below the current permissible exposure level (PEL) of 350 mg/m(3) will induce lung injury, and to validate a new "in-line, real-time" total hydrocarbon analysis system capable of measuring both JP-8 vapor and aerosol concentrations. Pulmonary function and respiratory permeability tests were performed 24 to 30 hr after the final exposures. No significant effects were observed at 45 or 267 mg/m(3). The only significant effect observed at 406 mg/m(3) was a decrease in inspiratory dynamic lung compliance. Morphological examination and morphometric analysis of distal lung tissue demonstrated that alveolar type II epithelial cells showed limited cellular damage with the notable exception of a significant increase in the volume density of lamellar bodies (vacuoles), which is indicative of increased surfactant production, at 45 and 406 mg/m(3). The terminal bronchial epithelium showed initial signs of cellular damage, but the morphometric analysis did not quantify these changes as significant. The morphometric analysis techniques appear to provide an increased sensitivity for detecting the deleterious effects of JP-8 as compared to the physiological evidence offered by pulmonary function or respiratory permeability tests. These observations suggest that the current 350 mg/m(3) PEL for both JP-8 jet fuel and for other more volatile petroleum distillates should be reevaluated and a lower, more accurate PEL should be established with regard human occupational exposure limits.

Epidemiological evidence has indicated that arsenic and cigarette smoking exposure act synergistically to increase the incidence of lung cancer. Since oxidative damage of DNA has been linked to cancer, our hypothesis is that aerosolized arsenic and cigarette smoke work synergistically to increase oxidative stress and increase DNA oxidation in the lung. To test this hypothesis male Syrian golden hamsters were exposed to room air (control), aerosolized arsenic compounds (3.2 mg/m3 for 30 minutes), cigarette smoke (5 mg/m3 for 30 minutes), or both smoke and arsenic. Exposures were for 5 days/week for 5 or 28-days. Animals were sacrificed one day after the last exposure. In the 28-day group, glutathione levels and DNA oxidation (8-oxo-2'-deoxyguanosine (8-oxo-dG)) were determined. Our results show that in the 28-day arsenic/smoke group there was a significant decrease in both the reduced and total glutathione levels compared with arsenic or smoke alone. This correlated with a 5-fold increase in DNA oxidation as shown by HPLC. Immunohistochemical localization of 8-oxo-dG showed increase staining in nuclei of airway epithelium and subadjacent interstitial cells. These results show that dual exposure of arsenic and cigarette smoke at environmentally relevant levels can act synergistically to cause DNA damage.

Introduction: Previous research indicates that a large cohort of veterans from the 1991 Gulf War report polysymptomatic conditions. These syndromes often involve neurocognitive complaints, fatigue, and musculoskeletal symptoms, thus overlapping with civilian illnesses from low levels of environmental chemicals, chronic fatigue syndrome, and fibromyalgia., Methods: To test for time-dependent changes over repeated intermittent exposures, we evaluated objective performance on a computerized visual divided attention test in chronically unhealthy Gulf War veterans (n = 22 ill with low-level chemical intolerance (CI); n = 24 ill without CI), healthy Gulf War veterans (n = 23), and healthy Gulf War era veterans (n = 20). Testing was done before and after each of three weekly, double blind, low-level JP-8 jet fuel or clean air sham exposure laboratory sessions, including acoustic startle stimuli., Results: Unhealthy veterans receiving jet fuel had faster mean peripheral reaction times over sessions compared with unhealthy veterans receiving sham clean air exposures. Unhealthy Gulf veterans with CI exhibited faster post- vs. pre-session mean central reaction times compared with unhealthy Gulf veterans without CI. Findings were controlled for psychological distress variables., Discussion: These data on unhealthy Gulf veterans show an acceleration of divided attention task performance over the course of repeated low-level JP-8 exposures. The present faster reaction times are consistent with rat neurobehavioral studies on environmental toxicant cross-sensitization and nonlinear dose-response patterns with stimulant drugs, as well as some previous civilian studies using other exposure agents. Together with previous research findings, the data suggest involvement of central nervous system dopaminergic pathways in affected Gulf veterans.

Jet Propellant 8 (JP-8) jet fuel is a kerosene-based fuel containing hundreds of hydrocarbons used by the military in NATO countries. Previous rodent inhalation studies carried out with aerosolized JP-8 never evaluated the exposure chamber atmosphere. For this reason, our laboratory developed an analytical method, with an accuracy of better than 80% and precision of better than 20%, for JP-8 aerosol and vapor samples using gas chromatography/mass spectrometry (GC/MS). A method was developed for quantification of selected individual components of JP-8 and for the total amount of JP-8 in aerosolized fuel. A 34 component surrogate hydrocarbon mixture (SHM) was developed and used for simultaneous analysis of the individual components. Three separate runs containing a standard curve and five replicates each at the selected concentrations were analyzed for both the SHM and neat JP-8. The resulting interday accuracy (100-percent relative error) and precision (relative standard deviation) values for the SHM were 86.5% or better and 8.0% or better, respectively. The intraday accuracy and precision values ranged from 99.29% to 84.50% and 0.97% to 12.4%, respectively. For the total amount of JP-8 in aerosol and vapor, the interday accuracy was 83.7% or better and interday precision was 7.0% or better. The intraday accuracy and precision values ranged from 94.8% to 80.4% and 2.4% to 10.5%, respectively. We then used this method to analyze samples collected from an inhalation chamber. From the data obtained, we are able to account for approximately 40-44% of the mass of the aerosol portion and 68-70% of the mass of the vapor portion. The aerosol represented 6-10% of the total mass of the aerosolized JP-8 fuel with the remaining portion being the vapor. From these experiments individual components were identified for further in vivo and in vitro toxicological testing.

Many military personnel are at risk of lung damage or systemic toxicity as a result of exposure to the jet fuel JP-8. We have now used microarray analysis to characterize changes in the gene expression profile of lung tissue induced by exposure of rats to JP-8 at a concentration of 171 or 352 mg/m(3) for 1 h/d for 7 d, with the higher dose estimated to mimic the level of occupational exposure in humans. The expression of 56 genes was significantly affected by a factor of /= 1.5 by JP-8 at the low dose. Eighty-six percent of these genes were downregulated by JP-8. The expression of 66 genes was similarly affected by JP-8 at the higher dose, with the expression of 42% of these genes being upregulated. Prominent among the latter genes was that for the centrosome-associated protein gamma-synuclein, whose expression was consistently increased. The expression of various genes related to antioxidant responses and detoxification, including those for glutathione S-transferases and cytochrome P450 proteins, were also upregulated. The microarray data were confirmed by quantitative RT-PCR analysis. Our extensive data set may thus provide important insight into the pulmonary response to occupational exposure to JP-8 in humans.

Using an in-line, real-time, in vivo exposure system, we investigated whether acute adverse effects of diesel exhaust (DE*) exposure involve neurogenic inflammation in the lungs via sensory nerve C fibers. A total of 168 female F344 rats (175 g, 8 weeks old) were randomly assigned to pretreatment with capsaicin or saline to deplete C-fiber neurotransmitters. In a 2 x 3 factorial design, groups of animals were then exposed nose-only to a low level of DE (LDE, 35.3 microg/m3), a high level of DE (HDE, 632.9 microg/m3), or side-stream cigarette smoke (CS, 0.4 mg/m3). Two control groups were exposed whole body to filtered air in the animal room (fRA) or unfiltered air in the diesel engine room (eRA), respectively. DE was taken directly from a heavy-duty Cummins N14 research engine operated at 75% throttle (California Air Resources Board [CARB] 8, mode 6). Exposure to DE or air was 4 hours/day, 5 days/week, for 3 weeks. Exposure to CS was for 4 hours/day for 7 days. Involvement of neurogenic inflammation in the response to DE or CS was assessed via comparison of plasma extravasation, a sensitive endpoint of neurogenic inflammation, between rats with and without capsaicin pretreatment. Lung injury was assessed via analysis of proinflammatory cytokines, respiratory permeability, and histopathology. Moreover, whether DE exposure affected the molecular mechanisms of neurogenic inflammation was analyzed through quantification of substance P (SP) and its primary neurokinin-1 (NK1) receptor at the gene and protein levels and through neutral endopeptidase (NEP) activity. DE and CS exposure induced dose-dependent plasma extravasation, which may play an important role in initiating the associated lung inflammation and injury. Exposure of rats to DE affected the SP signaling pathway as indicated by overexpression of the NK1 receptor or reduction of SP in the lung tissue. DE exposure consistently inactivated tissue NEP, a key factor that switches neurogenic inflammation from its physiological and protective functions to a role that increases and perpetuates lung injury. The roles of these overlapping neurokininergic mechanisms in the initiation of DE-associated lung injury are plausible, and these changes may contribute to DE-associated respiratory disorders. Capsaicin rats followed the same trends as those of saline animals when exposed to DE or CS: capsaicin rats did not have significantly different plasma extravasation in the airways or lung parenchyma compared to their corresponding controls. Histopathology evaluation likewise demonstrated the same degree of tissue changes, such as edema and alveolar macrophage collection, in capsaicin and saline rats after the same level of DE exposure. In summary, our data suggest that neurokininergic mechanisms may have been involved in DE-induced inflammatory conditions in rat lung but that C fibers did not appear to be involved under these exposure conditions. We believe that time-course or protein knockdown/knockout animal studies are required to characterize further the role of neurokininergic mechanisms in DE-induced lung injury.

Introduction: The purpose of this study was to examine the effects of substance P (SP) on the immune system in a condition similar to microgravity. We analyzed immune disturbances caused by subjecting Fischer 344 rats to a 45 degrees antiorthostatic suspension technique, otherwise known as the hindlimb unloading (HU) model., Methods: Four groups of rats were assigned to either the prone control non-substance P group (P-NSP), prone control substance P group (P-SP), hindlimb unloaded non-substance P (HU-NSP) or the hindlimb unloaded substance P group (HU-SP). SP was administered at 10 ml of a 1 micromol x L(-1) concentration for 15 min x d(-1). HU and SP exposure for all groups lasted 16 d. After 16 d, 500 microl of blood was obtained to assay for both T-cell phenotype and corticosterone (CS) levels. Thymus lobes were excised in order to examine T-cell phenotype. Thymocytes were counted and stained for lymphocyte markers (CD4, CD8, and CD3). An analysis of variance (ANOVA) test was used to determine significance between groups (p < or = 0.05)., Results: HU-NSP rats showed a decrease in thymic CD4+CD8 +/- cells from 85.51 +/- 1.9% to 62.06 +/- 1.9% when compared with P-NSP rats. SP reversed these effects and returned CD4+CD8+ cells to control levels (76.60 +/- 1.9%)., Discussion: Daily SP treatment was found to reverse the deleterious effects caused by HU and corticosterone in rat thymic immune cells. SP could prove to be an effective means for keeping the immune system functioning at normal levels in microgravity, allowing astronauts to stay in space longer and maintain a more productive immune system.

To characterize the tachykininergic effects in fire smoke (FS)-induced acute respiratory distress syndrome (ARDS), we designed a series of studies in rats. Initially, 20 min of FS inhalation induced a significant increase of substance P (SP) in bronchoalveolar lavage fluid (BALF) at 1 h and persisted for 24 h after insult. Conversely, FS disrupted 51.4, 55.6, 46.3, and 43.0% enzymatic activity of neutral endopeptidase (NEP, a primary hydrolyzing enzyme for SP) 1, 6, 12, and 24 h after insult, respectively. Immunolabeling density of NEP in the airway epithelium largely disappeared 1 h after insult due to acute cell damage and shedding. These changes were also accompanied by extensive influx of albumin and granulocytes/lymphocytes in BALF. Furthermore, levels of BALF SP and tissue NEP activity dose dependently increased and decreased, respectively, following 0, low (10 min), and high (20 min) levels of FS inhalation. However, neither the time-course nor the dose-response study observed a significant change in the highest affinity neurokinin-1 receptor (NK-1R) for SP. Finally, treatment (10 mg/kg im) with SR-140333B, an NK-1R antagonist, significantly prevented 20-min FS-induced hypoxemia and pulmonary edema 24 h after insult. Further examination indicated that SR-140333B (1.0 or 10.0 mg/kg im) fully abolished early (1 h) plasma extravasation following FS. Collectively, these findings suggest that a combination of sustained SP and NEP inactivity induces an exaggerated neurogenic inflammation mediated by NK-1R, which may lead to an uncontrolled influx of protein-rich edema fluid and cells into the alveoli as a consequence of increased vascular permeability.

To evaluate the role of substance P (SP)-containing C-fiber nerves in the development of the inflammatory responses to sidestream cigarette smoke (SSCS), female Fischer 344 rats were randomly assigned into vehicle and capsaicin groups, respectively. Then, half the number in each group (N = 24) was nose-only exposed to air or 0.4 mg/m3 total particulate matter of SSCS for 4 h/day for 7 days. Exposure of the vehicle rats to SSCS induced obvious pulmonary neurogenic inflammation as indicated by elevations in plasma extravasation and proinflammatory cytokine secretions [interieukin (IL)-1beta and IL-12]. In addition, except for SP release, SSCS exposure significantly induced the tachykininergic toxicities at the gene level: upregulation of beta-preprotachykinin-I (beta-PPT-I) mRNA. However, neither SSCS exposure nor capsaicin pretreatment affects the immunolabeling density of neurokinin-1 receptor (NK-1R) in airway epithelium. SSCS also significantly inactivated pulmonary neutral endopeptidase (NEP) in lung tissue. Moreover, pretreatment with capsaicin significantly exacerbated the SSCS-induced inflammatory responses mentioned above as well as the release of plasma protein. Considering that capsaicin did not affect the normal control baselines of these parameters except for a decrease in NK-1R mRNA, we conclude that the degree of SSCS-induced inflammatory response was exacerbated because of the depletion of stored SP and/or inactivation of capsaicin-sensitive C-fiber nerves. Our data suggest the loss of afferent tachykinin SP signaling may lead to dysfunction of the sensory C-fiber nerve reflexes during exposure to SSCS, suggesting that SP serves a protective role.

To examine the hypothesis that Jet Propulsion Fuel (JP-8) inhalation potentiates influenza virus-induced inflammatory responses, we randomly divided female C57BL/6 mice (4-weeks old, weighing approximately 24.6g) into the following groups: air control, JP-8 alone (1023 mg/m(3) of JP-8 for 1h/day for 7 days), A/Hong Kong/8/68 influenza virus (HKV) alone (a 10 microl aliquot of 2000 viral titer in the nasal passages), and a combination of JP-8 with HKV (JP-8 + HKV). The HKV alone group exhibited significantly increased total cell number/granulocyte differential in bronchoalveolar lavage fluid (BALF) compared to controls whereas the JP-8 alone group did not. The JP-8 + HKV group further exacerbated the HKV alone-induced response. However, increases in pulmonary microvascular permeability and pathological alterations in JP-8 + HKV just matched the sum of JP-8 alone- and HKV alone-induced response. Increases in BALF substance P in the JP-8 alone group and BALF leukotriene B4 or total lung compliance in the HKV alone group, respectively were similar to the changes in the JP-8 + HKV group. These findings suggest that changes in the JP-8 + HKV group may be attributed to either JP-8 inhalation or HKV treatment and indicate the different physiological responses to either JP-8 or HKV exposure. Taken together, most of the data did not provide supporting evidence that JP-8 inhalation synergizes influenza virus-induced inflammatory responses.

In an established rat model of smoke inhalation injury, we conducted a dose-response study to examine the protective effects of Xigris [drotrecogin alfa (activated) (DrotAA)], a recombinant form of human activated protein C (APC). DrotAA is a serine protease (approximately 55 kD molecular weight) with the same amino acid sequence and the glycosylation site as human plasma-derived APC. A total of 120 F344/NH rats (half each gender, approximately 175 g body weight) were randomly divided into five groups and exposed nose-only to air or diesel fuel smoke for 20 min. These rats were then i.v. administered with DrotAA in 0, 5, 10, and 20 mg/kg body weight, respectively, immediately following smoke exposure. Treatment with DrotAA significantly attenuated smoke inhalation injury in a dose-dependent manner at 2 hours after insult, as indicated by preserving microvascular permeability and proinflammatory cytokine IL-1beta (but not TNF-alpha and neuropeptide substance P) in bronchoalveolar lavage fluid (BALF). Moreover, the rats treated with 20 mg/ kg of DrotAA had an improvement of the expiration phase of pulmonary dynamic compliance. At all dosages, however, DrotAA also significantly increased all phases of pulmonary resistance compared with either the controls or to smoke inhalation alone. Generally, these data suggest that DrotAA may exert an anti-inflammatory effect by inhibiting cytokine-mediated inflammatory amplification. However, additional studies following a clinical course are needed to confirm the maximum efficiency and possible side effects of this recombined human activated protein C.