Immunotoxicological and neurotoxicological profile of health effects following subacute exposure to geogenic dust from sand dunes at the Nellis Dunes Recreation Area, Las Vegas, NV Authors: Deborah Keila, Brenda Buckb, Dirk Goossens, Yuanxin Teng, Mallory Leetham, Lacey Murphy, James Pollard, Margaret Eggers, Brett McLaurin, Russell Gerads, & Jamie DeWitt NOTICE: this is the author’s version of a work that was accepted for publication in Toxicology and Applied Pharmacology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Toxicology and Applied Pharmacology, [Volume 291, January 2016] DOI#10.1016/j.taap.2015.11.020 Keil D, Buck B, Goossens D, Teng Y, Leetham M, Murphy L, Pollard J, Eggers M, McLaurin B, Gerads R, DeWitt J, "Immunotoxicological and neurotoxicological profile of health effects following subacute exposure to geogenic dust from sand dunes at the Nellis Dunes Recreation Area, Las Vegas, NV," Toxicol Appl Pharmacol. 2016 Jan 15 291:1-12. Made available through Montana State University’s ScholarWorks scholarworks.montana.edu Immunotoxicological and neurotoxicological profile of health effects following subacute exposure to geogenic dust from sand dunes at the Nellis Dunes Recreation Area, Las Vegas, NV Deborah Keil a,⁎, Brenda Buck b, Dirk Goossens b,c, M Ja in a te b , N c onm d oze e l S Bl f B g Department of Pharmacology and Toxicology, East Carolina University, Greenville, NC 27834, USA A Exposure to geogenic particulate matter ( human health effects. However, very littl du There ch t colle a p ated n ex of mi du media bu concen we . ICP- alu (70 μ (2 (6 as ex pe thi su 1. so as m he w hu PM di ni al al RA) is a popular managed by the cated just 6 km years, the NDRA Clark County for ithin national borders. In China, Brazil, Spain and India, man health effects have also been re-ported with exposure to legal off-road driving, with annual visitation estimated at over 300,000 (Goossens and Buck, 2009). Recently, dust at NDRA The U.S. National Research Council has identified emission urce characterization and hazardous component assessment two of the top ten research priorities for airborne particulate atter (PM; National Research Council, 2004). PM is a global alth concern, as these airborne particulates are not contained The Nellis Dunes Recreation Area (ND off-road vehi-cle (ORV) driving destination Bureau of Land Manage-ment (BLM), lo northeast of Las Vegas, NV. For the past 40 has provided the public an accessible area in As(V). Mice received four exposures, once/week over 28-days to mimic a month of weekend posures. Descriptive and functional assays to assess immunotoxicity and neurotoxicity were rformed 24 h after the final exposure. The primary observation was that 0.1 to 100 mg/kg of s sand dune derived dust dose-responsively reduced antigen-specific IgM antibody responses, ggesting that dust from this area of NDRA may present a potential health risk. Introduction Moreover, metals on PM are bioavailable for uptake via human inhala-tion and ingestion (Morman et al., 2009).2.5 or PM10 (particulate matter b2.5 ameter, respectively) containing metals ckel, iron, manganese, chromium, and co ., 2009; Aldabe et al., 2011; de Miranda e ., 2013).(69 μg/g), zinc (79 μg/g), arsenic (62 μg/g), strontium /g) and uranium (4.7 μg/g). Arsenic was present only 1,600 μg/g), cobalt (9.4 μg/g), copper 20 μg/g), cesium (13 μg/g), lead 25 μgnes in NDRA. Dust samples ( ffered sa-line and delivered at ight by oropharyngeal aspiration minum (55,090 μg/g), vanadiumPM) comprised of mineral particles has been linked to e data exist on health effects associated with geogenic fore, we characterized particulate matter size, metal cted from the Nellis Dunes Recreation Area (NDRA), ear Las Vegas, NV. Adult female B6C3F1 mice were neral dust collected from active and veg-etated sand n diameter: 4.4 μm) were suspended in phosphate- trations ranging from 0.01 to 100 mg dust/kg body MS analyses of total dissolution of the dust resulted in g/g), chromium (33 μg/g), manganese (511 μg/g), iron Keywords: Geogenic dust Heavy metals Minerals Lung exposure Immunotoxicity Neurotoxicityst exposure in natural set-tings. emistry, and health effects of dus opular off-road vehicle area loc posed to several concentrations B S T R A C Turphy a, mes Pollard b, Margaret Eggers d, Brett McLaur Department of Microbiology and Immunology, Montana Sta Department of Geoscience, University of Nevada, Las Vegas Geography Research Group, Department of Earth and Envir Center for Biofilm Engineering, Montana State University, B Department of Environmental, Geographical, and Geologica oomsburg, PA 17815, USA rooks Rand Labs, LLC, Bothell, WA 98011, USA μm or b10 μm in such as lead, zinc, pper (Kulshrestha et t al., 2012; Wang et w (S (U he th to su Yuanxin Teng b, Mallory Leetham a, Lacey e, Russell Gerads f, Jamie DeWitt g University, Bozeman, MT 59717, USA V 89154, USA ental Sciences, KU Leuven, Belgium man, MT 59717, USA ciences, Bloomsburg University of Pennsylvania, as found to have abnormally high concentrations of arsenic oukup et al., 2012), a metal classified as a human carcinogen SEPA, 1988). Due to the high visitor rate and potential alth risks associated with geogenic dust inhalation exposure, e current study examined the tox-icological effects specific geogenic dust collected from easily erodible sand dune rfaces at NDRA, designated here as “CBN 1”. CBN 1 surfaces at NDRA are characterized by sand dunes with and without vegetation. Dunes are one of the most popular areas in NDRA, utilized by both individual recreationists as well as by companies that provide ORV services. They cover an area of approximately 317 ha, among which 57 ha dunes without vegetation and 260 ha partly vege- tated dunes. Both dune types are prone to severe wind erosion year- round. Soils at the CBN 1 surfaces contain the typical assortment of arid-soilminerals including quartz, calcite, gypsum, amphibole, feldspar and many clay minerals such as palygorskite, illite, smectite, kaolinite and chlorite (Soukup et al., 2011). These minerals have within their scleroderma, and glomerulonephritis (Lim et al., 2012). Those exposed to crystalline silica in occupational environments present with a disrup- tion in immunological homeostasis that includes increased lymphopro- liferative responses, increased levels of the pro-inflammatory cytokines (IFN-γ, IL-1α, TNF-α, IL-6), and increased anti-inflammatory cytokines (IL-10 and TGF-β; Rocha-Parise et al., 2014). Crystalline silica exposure is also associatedwith excessmortality from acute renal disease and can also be associated with an increased risk of end-stage renal disease QL.crystal structure or adsorbed on their surfaces, many different heavy metals and metalloids (see Table 1). Dust in this area is generated by both natural wind erosion and ORV activity (Goossens et al., 2012). The geologic source of the dune sand is the yellow sandstone facies of theMuddy Creek Formation, which contains significant arsenic concen- trations, especially in the finer grain-size fractions (Goossens et al., 2015). These finer grain-size fractions become airborne as a result of wind or ORV activities, leading to inhalation exposures. It is only recently that health effects of dust generated from soil sources has emerged as an increasingly important health concern (Sharratt and Lauer, 2006; Whicker et al., 2006; Buck et al., 2013; Derbyshire, 2007). Morman and Plumlee (2013) recognized that parti- cle size alone is insufficient for understanding the risks of particulate matter and that the relative contributions of toxic trace metals and other components of dust to respiratory morbidity and mortality de- serve further research. In a large study across 25 U.S. communities, Franklin et al. (2008) used data that combined air quality information from the U.S. Environmental Protection Agency (EPA), mortality data for more than 1.3million deaths, and U.S. National Climatic Data Center meteorological data, to examine how the association of mortality with PM2.5 differed among communities as a result of variation in the chem- ical composition of the particulate matter. Mortality increased when nickel, aluminum, sulfate, silicon, and arsenic were in a higher propor- tion while the combination of sulfate, nickel, and aluminum modified the association between PM2.5 mass and mortality. Furthermore, Ruckerl et al. (2011) cite multiple studies documenting that the metal components of natural dust, such as vanadium, chromium, nickel, cop- per, and iron, cause oxidative stress thereby inducing proinflammatory effects in the lung. Many studies have found that exposure to airborne PM increases risks of respiratory illness, cardiovascular disease, and mortality (Dockery et al., 1993; Pope et al., 1995; Samet et al., 2000; Lee et al., 2006; Beelen et al., 2008; Samoli et al., 2008; Ruckerl et al., 2011). Exten- sive reports identify that a number of air pollutants and particulates cause local pulmonary immune deficits. This is further supported with numerous epidemiologic studies describing increased PM air pollution with increased frequency of respiratory complaints such as pneumonia, asthma, croup, bronchitis and viral pulmonary infections (Brauer et al., 2002; Dockery & Pope, 1994; Gilmour & Koren, 2000; Lambert et al., 2003). While it is well-established that airborne PM affects local im- mune responses in the lung, less is known about systemic immunolog- ical effects due to PM exposure (Albright and Goldstein, 1996; Leonardi et al., 2000; Hassani et al., 2004). One study demonstrates that systemic IgG antibody production is suppressed in an autoimmune mouse fol- lowing exposure to PM (Hassani et al., 2004). Geogenic dust also almost always has significant amounts of crystal- line silica, primarily in the formof themineral quartz and any number of other silicateminerals. Silica exposure is associatedwith a variety of au- toimmune problems, including rheumatoid arthritis, lupus, Table 1 Total elemental concentration in dry geogenic dust sample (μg/g) from CBN 1. Mediana Al V Cr Mn Fe Co Cu Zn 4.39 55,100 70 33 511 21,600 9.4 69 79 b indicates value is below method quantitation limit (MQL) and that value presented is M Data are reported with a maximum of three significant figures. a Median diameter (μm).(reviewed by Ghahramani, 2010). Even fewer studies have characterized neurotoxicity following PM exposure. Calderón-Garcidueñas et al. (2008) report that exposure to elevated concentrations of air pollutants including ultrafine PM and PM2.5 causes neuroinflammation and alters innate immune responses in the brain of children and young adults. Ultrafine PM appears to en- hance formation of protein fibrils in the brain affecting beta-amyloid (β42) and α-synuclein (Linse et al. 2007). Extrapulmonary transloca- tion of inhaled PM and metals to the brain have also been reported (Oberdorster et al., 2004; Tjalve and Henriksson, 1999). Calderón- Garcidueñas et al. (2008) argue that neuroinflammation as a result of exposure to air pollution could have a causative role in both Alzheimer's and Parkinson's diseases. As we learn more about geogenic dust and its complexity, it is in- creasingly important to characterize health effects due to exposures, es- pecially as recreational and developmental pressures increase the number of humans exposed to these dusts. Therefore, this studywasde- signed to establish a toxicological profile following lung exposure via oropharyngeal aspiration to geogenic dust, characterized by size as well as silica andmetal content. A suite of immunotoxicological, clinical chemistry, and neurotoxicological evaluations were used to determine the dose–response profile of exposure to NDRA geogenic dust. 2. Methods 2.1. Collection of geogenic dust Composite samples were collected from the topsoil (upper 0–4 cm) using a plastic scoop and placed into a clean plastic bag, whichwas her- metically closed after collection. GPS position of the center of the collec- tion areawas recorded for each sampling point. Samples were collected from active sand dunes and sand sheets with no vegetation and dune sands with sparse and isolated shrubs; these correspond to an internal designation for our study described as surface units 1.1 and 1.2 in McLaurin et al., 2011. The sampleswere treated in a Soil Fine Particle Ex- tractor (seeGoossens, 2012) to extract particleswith amediandiameter of approximately 4 μm. The extracts were combined to create combina- tion surface unit designated as “CBN 1” based on the areal extent of that surface unit within the dune field: surface unit 1.1: 13.7%, and surface unit 1.2: 86.3%. 2.2. ICP-MS analyses of geogenic dust All samples were digested in accordance with the USGS Four-Acids Method (Briggs and Meier, 1999) and subsequently analyzed using an Agilent 7700 inductively coupled plasma/mass spectrometry (ICP-MS) device (Agilent Technologies, Santa Clara, USA). To ensure quality con- trol for the ICP-MS analyses, all quality control procedures set forth by US EPA Method 6020A (USEPA, 2007) were followed. In addition, NIST As Sr Cd Sb Cs Tl Pb U Si 62 620 b0.47 b3.0 13 b8.3 25 4.7 197,000 SRM 8704 (Buffalo River Sediment) and NIST SRM 2711a (Montana II Soil) were used as standard reference materials (SRMs). 2.3. Arsenic speciation Arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid quantification speciation was performed using IC-ICP-CRC-MS at Brooks Rand Labs, LLC (formerly Applied Speciation and Consulting, LLC) according to an in-house developed method based on Kubachka et al. (2012). 2.4. Quantitation of silicon in geogenic dust Analysis for the element silicon utilized a Thermo Scientific Niton XL3t GOLDD+ portable XRF (X-ray fluorescence) instrument. For cali- bration the NIST Standard Reference Material 2711A was run and the XRF results are in agreement with the certified values for silicon and within the margins of uncertainty for the soil standard. A total of six samples were analyzed from CBN 1 and each sample was run twice for a total of 120 s for each analysis. 2.5. Preparation, stability and verification of geogenic dust for animal exposures Dust samples were carefully labeled, stored in sealed and dry con- tainers, protected from light, and secured in a lock box in the laboratory. CBN 1 dust was prepared in sterile, endotoxin-free, phosphate buffered saline (ETF-PBS) at concentrations of 0.01, 0.1, 1, 10, or 100 mg of dust/ kg of body weight and was administered to mice within 1–2 h of prep- aration. Some elements have various degrees of solubility in water and addition of dust samples to saline for delivery into themousemay have changed the distribution of insoluble elements versus concentration of those elements in solution. To verify that adding the dust samples to PBS did not substantially alter the solubility of elements, a stability study was performed with the lowest concentration (0.01 mg/kg) and a higher concentration (10 mg/kg). CBN 1 dust was added to ETF-PBS to ascertain stable time frames in which the solution could be used for mouse exposures. Solutionswere prepared and samples of the solutions were collected immediately after preparation, and then at 1, 2, 4, and 6 h. Samples were immediately centrifuged, supernatants removed, and examined using an ICP-MS to quantitate total soluble element con- centrations. The analysis indicated that leaving the dust samples in an ETF-PBS solution for up to six hours did not substantially change the dis- tribution of elements in solution. At 6 h, soluble element concentrations in supernatant began to increase, indicating that insoluble:soluble por- tions remained constant for 6 h in solution. We did not test for changes in speciation, but only total values of elemental metals. This additional quality controlmeasure verified our dosing solution concentrations, po- tential for flux, and accounted for potential contamination from PBS or other steps in our preparation process. To control for contamination in this preparation process, no metal spatulas or any other metal items were used for weighing, storage, manipulation, or transport of dust samples. 2.6. Animals Micewere obtained from Charles River Laboratories (headquartered inWilmington, MA) andwere acclimated for 7 days to the conditions of the treatment room (12-h light/dark cycle, 22 ± 2 °C, 60–65% relative humidity) at the University of Nevada Las Vegas (UNLV) animal facili- ties, which are accredited by the Association for Assessment and Ac- creditation of Laboratory Animal Care International. The UNLV Institutional Animal Care and Use Committee approved all experiments. Mice were housed in ventilated polycarbonate shoebox cages with corncob bedding and were given unlimited access to food and water.2.7. Animal exposures To simulate the potential health impacts of a month of weekend ex- posures to geogenic dust from the NDRA, adult female B6C3F1 mice were exposed to CBN 1 dust samples with a median diameter of 4.39 μm (Table 1) at 0, 0.01, 0.1, 1.0, 10, or 100 mg/kg of body weight once weekly for four weeks. Each dose administered was adjusted to body weight. Therefore, based on a 20 g mouse, 20μg was administered to the lung via oropharyngeal aspiration. Mice in the 0 mg/kg group re- ceived PBS only and served as a vehicle control group. To ensure avail- ability of tissue for toxicology assays, each dose group was comprised of 12 mice, housed six per cage. In addition, three separate groups of micewere used for a total of three replicates for each exposure. Samples for toxicity studies were collected from each group three days in a row. Fig. 1 depicts the basic arrangement for replicates and sample collection. Mice were exposed by oropharyngeal aspiration using isoflurane as an anesthetic agent. The dose was based on current body weight of the mouse. An average volume of 10 μL was administered to each mouse; however, based on individual bodyweight changes, this volume was adjusted to between 9 and 13 μL per mouse. One day after the final dosewas delivered, samples for toxicity studies were collected from an- imals euthanized by carbon dioxide asphyxiation. The following assays were performed to determine a profile of toxicological effects specific to NDRA geogenic dust from CBN 1. 2.8. Body weight, organ weights, and immune organ cellularity Body weight was monitored weekly during the study and terminal body weights were collected for all animals the day of euthanasia. The brain, kidney, liver, lung, spleen, and thymus were removed and weighed. Weights were adjusted for terminal body weights to deter- mine absolute and relative organ weights. Spleens and thymuses were suspended in complete medium (RPMI, 10% fetal calf serum, 50 IU pen- icillin and 50 μg streptomycin) and were aseptically processed into single-cell suspensions by gentle grinding between two sterile, frosted microscope slides. An aliquot of each spleen or thymus suspension was manually counted on a hemocytometer to determine the number of live cells (viability of cells for each organ was generally greater than 95%). The total number of cells per spleen and thymus (cellularity), ad- justed by the weight of each organ, was determined for each animal from Set B (see Fig. 1). 2.9. Hematology, clinical chemistry, and blood metals For hematology and clinical chemistry endpoints, blood from anes- thetized animals was collected into a microtainer tube containing EDTA, which kept the blood from coagulating or in a microtainer with no anticoagulant for serum collection. Once collected, samples were sent overnight to the Montana Veterinary Diagnostic Laboratory (MVDL) in Bozeman,MT, for hematology (whole EDTA blood) and clin- ical chemistry analysis (serum). Hematology parameters included: white blood cells (WBC; 109/L), red blood cells (RBC; 109/L), hemoglo- bin (HGB; g/dl), hematocrit (HCT; %), mean corpuscular volume (MCV; fl), mean corpuscular hemoglobin (MCH; pg), mean corpuscular hemoglobin concentration (MCHC; %), red cell distribution (RDW; %), platelet count (109/L), neutrophils (%), neutrophils (109/L), lympho- cytes (%), lymphocytes (109/L), monocytes (%), monocytes (109/L), eo- sinophils (%), eosinophils (109/L). Clinical chemistry parameters included: creatine phosphokinase (CPK; IU/I), aspartate aminotransfer- ase/serum glutamic oxaloacetic transaminase (AST/SGOT; IU/I), alanine aminotransferase/serum glutamic pyruvate transaminase (ALT/SGPT; IU/I), alkaline phosphatase (ALKP; IU/I), glucose (mg/dl), cholesterol (mg/dl), total protein (g/dl), albumin (g/dl), globulin (g/dl), phosphate (mg/dl), blood urea nitrogen (BUN; mg/dl), creatinine (mg/dl), and total bilirubin (mg/dl). Hematologies were run on all samples; however clinical chemistries performed were dependent on the volume of le csample provided and not all samples were of sufficient volume. For this reason, hematology and clinical chemistry analyseswere not performed in duplicate or triplicate as were other assays. For determination of blood metal and metalloid concentrations, blood from anesthetized mice was collected into a microtainer tube containing heparin. Using an analytical balance, each blood collection tube was weighed before and after collection of blood to determine the weight of each blood sample. Once collected, samples were frozen at−80 °C and then shipped to the Laboratory Services Bureau of the Montana Department of Public Health and Human Services for analysis of total levels of metal and metalloid concentrations. Metals/metalloids included: arsenic, cadmium, chromium, lead, magnesium, manganese, molybdenum, nickel, strontium, vanadium, and zinc. Blood metal/met- alloid concentrations were not performed in duplicate or triplicate as were other assays. Due to limited volumes,whole blood valueswere de- termined only. 2.10. Immunophenotyping of B lymphocytes and CD4/CD8 lymphocytes The number of splenic B cells (B220) and splenic and thymic T cells (CD4+, CD8+, CD4+/CD8+, and CD4−/CD8-) was counted in single- cell suspensions diluted to a concentration of 1 × 107 cells/mL. Optimal concentrations of flow antibodies and reagentswere determined in pre- vious experiments. All experimental replicates included isotype con- trols (to estimate non-specific binding), unstained cells as negative controls, and single color controls as positive controls to determine color compensation. Flow cytometric analysis was performed using a BD FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) and 10,000 events were collected from each sample. The total number of each cell type was determined from the spleen or thymus cellularity. 2.11. Immunophenotyping of regulatory T lymphocytes (Tregs) Fig. 1. Toxicology sampSplenic lymphocytes were adjusted to a concentration of 1 × 106 cells per well and depleted of red blood cells via a 5-minute incubation in NH4Cl lysis buffer at 37 °C. Monoclonal antibodies coupled to fluoro- chromes specific for the followingmarkerswere used at a concentration of 1 μg/106 cells: anti-mouse CD25-FITC, rat IgG1-PE isotype control, rat IgG2b-AF647 isotype control, and rat IgG2b-FITC isotype control (BD Pharmingen, San Diego, CA, USA). FoxP3, CD4, and IL-17 cells were stained using a commercial kit (BD Pharmingen, San Diego, CA, USA or eBiosciences, San Diego, CA, USA) according to manufacturer's instruc- tions. Appropriate positive, negative, and isotype controls were added to wells containing cells only. Treg subsets were quantified using a BD FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). 10,000 events were acquired for each sample. CD4+ lymphocytes in the lymphocyte fraction were gated, and the percentages of CD25+- foxP3+ cells, CD25+foxP3− cells, IL-17+, and IL-17− cells were calculated.2.12. Plaque forming cell (PFC) assay The primary IgM response to sheep red blood cells (SRBC; Rockland, Gilbertsville, PA) was determined using a modified hemolytic plaque assay (Jerne and Nordin, 1963), which allows for an estimate of the number of B cells (“plaque forming cells” or “PFCs”) producing antibody against SRBC. Five days before euthanasia, micewere given an intraper- itoneal injection of 100 μL of 25% SRBC in PBS. Single-cell suspensions (as previously described) were prepared from spleens of mice injected with SRBC and diluted to a concentration of 1.0 × 106 cells/mL. A 10 μL aliquot of the single-cell suspension was added to a tube contain- ing 100 μL of 25% SRBC in PBS, 40 μL of RPMI medium (without addi- tives), and 50 μL of guinea pig complement. Aliquots of the solution were placed into Cunningham chamber slides. The slides were sealed with paraffin and were incubated at 37 °C and 5% CO2 for 1–2 h. PFCs were counted microscopically and were reported as PFCs/million splenocytes. 2.13. Natural killer cell activity Natural killer (NK) cell activity was assessed via an in vitro cytotox- icity assay using 51Cr-labeled Yac-1 cells as described previously (Duke et al., 1985; Holsapple et al., 1988). To minimize radioactive waste, the procedure was adapted to 96-well plates that were read on a Packard Top Count scintillation counter. Spleen single-cell suspensionswere ad- justed to 2 × 107 cells/mL in completemediumand then the spleen cells and Yac-1 cells were added, in triplicate wells, in ratios of 100:1, 50:1, 25:1, and 12.5:1 spleen cells:labeled Yac-1 cells, and in a final volume of 0.2 mL per well. Maximum release was determined by lysing 51Cr- labeled Yac-1 cells with 0.1% Triton X-100 in completemedium. Sponta- neous release was determined by incubating Yac-1 cells only in com- plete medium. After a four hour incubation at 37 °C and 5% CO2, the plates were centrifuged (1200 rpm, 3 min), and 25 μL of supernatant ollection arrangement.was then transferred to a 96-well plate containing solid scintillant (LumaPlate). Plates were air dried overnight and within 24 h, were counted for 5 min, after a 10-min dark delay, using a Packard Top Count-NXT. The results are expressed in lytic units per 107 splenocytes using 10% lysis as the reference point as described by Bryant et al., 1992; equation 10. Essentially, thismeasure considers the target tumor cell ac- tivity in the context of both maximum and spontaneous release. This method has been validated in interlaboratory studies by the National Toxicology Program (Luster, et al., 1988). 2.14. Neuronal autoantibody formation Blood was collected, held at room temperature for at least 30 min, and then centrifuged at 4 °C to separate serum. Sera were then frozen at −80 °C until analysis of IgM and IgG antibody concentrations. Flatbottom 96-well high binding microtiter plates were coated with 2.17. Statistical analysis Data were tested for normality and homogeneity and, if needed, ap- propriate transformations were made. A one-way analysis of variance (ANOVA)was used to determine differences among doses for each end- point using JMP 9 (SAS Institute Inc., Cary, NC) in which the standard error used a pooled estimate of error variance. When significant differ- ences were detected by the F-test (p b 0.05), Dunnett's t-test was used to compare treatment groups to the 0 mg/kg group. A Dunnett's t-test100 μL of 1 mg/mL of purified neuronal protein for either glial fibrillary acidic protein (GFAP; American Research Products, Waltham,MA), my- elin basic protein (MBP; Sigma-Aldrich, St. Louis,MO), or neurofilament 68 (NF-68; American Research Products, Waltham, MA) and then incu- bated at 4 °C overnight (at least 16 h). After washing, blocking of nonspecific binding, and addition of serum samples (serially diluted 1:100), secondary antibody (alkaline phosphatase goat anti-mouse IgM or IgG; ABCAM, Cambridge, MA) was added. Each set of two plates also included mouse monoclonal primary antibodies directed against each of the three neuronal proteins instead of serum samples; these pri- mary antibodies were added in known concentrations and served as standard curves. All plates also included blank wells. Following washes and addition of substrate (p-nitrophenyl-phosphate; Sigma-Aldrich, St. Louis, MO), plates were incubated for 30 min at room temperature and then stop solution (0.4 N sodium hydroxide) was added. Plates were read at 405 nm on a BioTek Synergy HT plate reader. Optical density values were converted to ng/mL concentrations using values obtained from the standard curve. All sera were assayed twice to verify results. Values that fell below the limits of detection were assigned a value of zero. Values that fell above the limits of detection were diluted upon the second evaluation. Values that still remained above the limits of detection after dilution were eliminated from the overall calculations due to insufficient amounts of remaining sample for further analysis. 2.15. Brain histology Immediately following euthanasia, the brains were removed, weighed, and immersion fixed in 10% neutral buffered formalin. After 24 h, the brains were transferred to 70% ethanol for storage at room temperature and were ultimately shipped to East Carolina University (ECU). Once received, the brains were processed and embedded by the Histology Laboratory Core in the Department of Anatomy and Cell Biology at ECU. Two sections of the cerebellum, each 10 μm thick, were cut and mounted onto glass slides. One set of sections was stained with anti-CD3+ antibody (abcam, Cam- bridge, MA) and the other set was stained with anti-myelin basic protein (MBP) antibody (abcam, Cambridge, MA). In sections stained with anti-CD3+, the number of T cells present throughout both sections was counted at 20× magnification. In sections stained with anti-MBP, the relative intensity of the stain was gauged relative to the intensity of the staining of the sections from the control brains. Control brains were scored as weak (1), mild (2), moderate (3), or strong (4). The intensity of the stain in the brains from exposed animals was assigned a numerical value according to the following scale: 0 = no change in staining intensi- ty relative to controls; 1 = very weak staining intensity relative to controls; 2 = mild intensity in staining relative to controls; 3 = moderate intensity in staining relative to controls; 4 = strong in- tensity in staining relative to controls; 5 = very strong intensity in staining relative to controls. 2.16. Particle positive control To test “elemental metal effects” vs. “particle effects”, separate groups of mice were exposed to titanium dioxide (TiO2). TiO2 is used often as a particle control as it is considered “neutral”with no associated heavymetals. It was approximately 21 nm in size andwas administered to separate groups of mice via the same exposure paradigm as mice exposed to geogenic dust samples from CBN 1. TiO2 was prepared in sterile, endotoxin-free, phosphate buffered saline (ETF-PBS) at concen- trations of 0.01, 0.1, 1, 10, or 100 mg of particle/kg of body weight and was administered to mice within 1–2 h of preparation. Toxicity testing was evaluated in the TiO2 exposed mice in parallel with CBN 1 exposed mice.alsowas used to compare results of the 0mg/kg group to the TiO2 group. 2.18. Quality assurance This studywas conducted as under the conceptual guidance of Good Laboratory Practices (GLP). Within this guidance, periodic audits of all aspects of the project were conducted as well as extensive independent reviewof all documentation anddata. In addition, each of theparticipat- ing university sites conducting experiments (UNLV, MSU and ECU) were audited by an internal but independent Quality Assurance team. All final notebooks were reviewed and initialed by the Quality Assur- ance Team. 3. Results 3.1. CBN 1 geogenic dust characterization Dust from CBN 1 used in this study had a median diameter of 4.39 μm as determined by laser diffraction (Table 1 and Fig. 7). Total di- gestion chemical composition of the dust is also shown in Table 1. All of the arsenic was As (V) (Table 2). 3.2. Body weight, organ weights, and immune organ cellularity Overall, mice in the dosed groups gainedweight over the 28-day ex- posure and did not demonstrate significant, dose-related changes in body weight when compared to mice from the 0 mg/kg group. No sig- nificant changes in immune organweights, thymic and splenic cellular- ities, kidney, lung, or brain weights, were observed. In two of the three replicates, relative liver weights were significantly decreased in mice exposed to 0.1, 1, 10, or 100 mg/kg of geogenic dust. This decrease ranged from 6%–14% relative to the 0 mg/kg group and changes were not dose-responsive. No significant bodyweight or organweight chang- es were observed in mice exposed to TiO2. 3.3. Hematology, clinical chemistry, and blood metals Hematological endpoints measured in mice dosed with dust sam- ples from CBN 1 did not vary statistically significantly relative to re- sponses in the 0 mg/kg group nor were any changes dose-responsive in nature. Similarly, most clinical chemistry endpoints did not vary by dose; however, plasma creatinine was dose-responsively increased in mice exposed to 0.01 mg/kg to 100 mg/kg (Fig. 2). We did not measure these endpoints in TiO2 exposed mice due to limited blood volume available in this smaller subset of mice. Table 2 Arsenic speciation of dry geogenic dust sample from CBN 1. As (III) As(V) MMAs DMAs Units ND (b0.27) 21.4 ND (b0.20) ND (b0.18) μg/g All results reflect the applied dilution. All results reported as received (Wet Weight). ND = Not detected at the applied dilution. MMAs =Monomethylarsonic acid. DMAs = Dimethylarsinic acid. gea sta kg gWhole blood collected from mice 24 h after the final exposure had detectable concentrations of themetals/metalloids (Table 3).Mean con- centrations did not differ by dose. 3.4. Immunophenotyping The total number of splenic B cells and the total number of splenic and thymic T CD4/CD8 lymphocyte subpopulations did not statistically change by exposure to CBN 1 geogenic dust (Table 4). The number of + + − Fig. 2. Serum creatinine levels measured in adult female B6C3F1 mice following oropharyn samples from NDRA each week for 28 days. Data are presented as mean creatinine levels ± sentative of one trial day. The (*) indicates a response statistically different from the 0 mg/CD4 CD25 foxP3 lymphocytes was statistically reduced at all ad- ministered doses (Table 4) by 88.4%, on average, relative to the 0 mg/kg group. No statistically significant lymphocyte subpopulation changes were observed in mice exposed to TiO2. 3.5. Plaque forming cell (PFC) assay Exposure to geogenic dust samples fromCBN 1 significantly reduced the number of plaque forming cells secreting IgM antibody to SRBC at all administered doses (Fig. 3). PFC/million of spleen cells was reduced by 45–74% in dosed groups relative to the 0mg/kg group. As even the low- est dose group was reduced relative to the 0 mg/kg group, a NOAEL Table 3 Total elemental concentration (μg/g in wet sample) inwhole blood of animals dosed with CBN1 geogenic dust fromvegetated and non-vegetated sanddunes fromNDRA eachweek for 28 days. 0 mg/kg 0.01 mg/kg 0.1 mg/kg 1 mg/kg 10 mg/kg 100 mg/kg As 0.0050 0.0037 0.0063 0.0033 0.0040 0.0037 Cd a a a a 0.0077 a Cr a a a a a 0.0358 Pb a 0.0013 0.0018 0.0029 0.0016 a Mg 36.9 35.9 45.5 35.2 35.2 35.7 Mn 0.0231 0.0202 0.0268 0.0206 0.0204 0.0218 Mo 0.0602 0.0929 0.0448 0.0571 0.0225 0.0430 Ni 0.0176 0.0366 a a a a Sr 0.0092 0.0074 0.0124 0.0084 0.0083 0.0084 V 0.0108 0.0084 0.0135 0.0101 0.0106 0.0106 Zn 4.32 4.26 5.55 4.33 4.21 4.30 a Indicates values not on the standard curve were not used.could not be determined. Therefore, the LOAEL was identified as 0.01 mg/kg for this response. No statistically significant changes in the PFC response were observed in mice exposed to TiO2. 3.6. Natural killer cell activity Natural killer cell activity following exposure to geogenic dust sam- ples from CBN1 (Fig. 4) did not change dose-responsively. Lytic activity of the 1.0 and 100mg/kg exposure groupwas statistically increased rel- l aspiration exposure to CBN 1 (vegetated and non-vegetated sand dunes) geogenic dust ndard deviations. Sample size for each group was 5–6 animals. Data presented are repre- roup (p b 0.05).ative to the activity of the 0 mg/kg group. No statistically significant changes were observed in mice exposed to TiO2. 3.7. Neuronal autoantibody formation Exposure to geogenic dust from CBN 1 reduced IgM antibody produc- tion against NF-68 and GFAP and IgG antibody production against GFAP, relative to production in the 0 mg/kg group. NF-68 IgM in the 0.1– 100 mg/kg dosed groups (Fig. 5A) was reduced by 84%, on average, and GFAP IgM in the 100 mg/kg dosed group (Fig. 5B) was reduced by 28.9% relative to the 0 mg/kg group. GFAP IgG in the 10 and 100 mg/kg dosed groups (Fig. 5C) was reduced by 42.2%, on average, relative to the 0 mg/kg group. No other changes were statistically different. 3.8. Brain histology The brains of animals exposed to geogenic dust from CBN 1 did not show CD3+T cell infiltration (data not shown). On average, MBP stain- ing in the groups exposed to 0.1, 10, and 100mg/kgwas less intense rel- ative to staining observed in the 0 mg/kg group (Fig. 6). 4. Discussion Understanding health impacts of geogenic dust exposure is both a local and global concern (Kulshrestha et al., 2009; Aldabe et al., 2011; de Miranda et al., 2012; Wang et al., 2013). Yet, there is insufficient ev- idence to identify health effects of exposure to geogenic dust with dif- ferent chemical compositions (Plumlee et al., 2006; Stanek et al., 2011). Further complicating this understanding is that dust in natural Table 4 Spleen and thymus B and T cell lymphocytes in adult female B6C3F1 mice following oropharyngeal aspiration exposure to CBN 1 geogenic dust from vegetated and non-vegetated sand dunes from NDRA each week for 28 days. Geogenic dust (mg/kg) Spleen CD4+ (cells × 107) CD8+ (cells × 107) CD4+/CD8+ (cells × 106) CD4−/CD8- (cells × 108) B220 (cells × 107) 0 4.14 ± 0.954 1.48 ± 0.158 7.77 ± 2.93 1.08 ± 0.282 6.76 ± 1.58 0.01 4.83 ± 1.38 1.91 ± 0.543 8.46 ± 3.07 1.28 ± 0.267 8.14 ± 1.83 0.1 4.00 ± 0.0554 1.54 ± 0.168 5.53 ± 3.03 1.03 ± 0.124 6.45 ± 0.867 1 3.64 ± 0.0576 1.38 ± 0.166 5.79 ± 1.54 0.917 ± 0.144 6.07 ± 1.14 10 4.33 ± 2.26 1.53 ± 0.757 7.40 ± 3.89 1.17 ± 0.583 7.19 ± 3.62 100 4.17 ± 1.06 1.60 ± 0.415 5.37 ± 4.66 1.14 ± 0.156 7.00 ± 0.783 Geogenic dust (mg/kg) Thymus CD4+ (cells × 107) CD8+ (cells × 106) CD4+/CD8+ (cells × 107) CD4−/CD8- (cells × 106) 0 1.19 ± 0.255 5.52 ± 0.148 8.17 ± 2.29 5.33 ± 1.21 0.01 1.23 ± 0.256 6.72 ± 0.165 9.09 ± 1.57 5.86 ± 1.23 0.1 0.949 ± 0.275 5.33 ± 0.102 7.73 ± 3.03 4.90 ± 1.61 1 1.21 ± 0.335 7.30 ± 0.402 7.88 ± 2.70 5.89 ± 2.21 10 1.09 ± 0.280 6.50 ± 0.124 8.42 ± 1.53 6.10 ± 0.956 100 1.15 ± 0.383 6.36 ± 0.190 9.06 ± 2.95 5.85 ± 1.74 Geogenic dust (mg/kg) Spleen 4+/25+/fp3+ (cells × 106) 4+/25-fp3+ (cells × 107) 4+/25+/fp3- (cells × 106) 4+/IL17A+ (cells × 106) 4−/IL17A+ (cells × 106) 0 7.03 ± 2.67 1.09 ± 0.458 8.38 ± 5.07 0.993 ± 0.388 0.682 ± 0.166 0.01 5.82 ± 2.60 1.33 ± 0.417 1.15 ± 0.715* 0.979 ± 0.522 0.657 ± 0.214 0.1 6.05 ± 2.59 1.18 ± 0.573 0.501 ± 0.321* 0.851 ± 0.375 0.509 ± 0.134 p wa term1 6.94 ± 1.49 1.35 ± 0.366 10 5.63 ± 1.63 1.09 ± 0.403 100 6.34 ± 1.90 1.21 ± 0.360 Data are presented as mean cell number ± standard deviations. Sample size for each grou * indicates a response statistically different from the 0 mg/kg group (p b 0.05) and was desettings is often a complex mixture of mineral and components with highly variable chemical compositions and particle sizes ranging from nanometers to several tens of micrometers. This study focused on sand dunes, themost popular sections for rec- reationists and the most emissive soil type of the NDRA. Human Fig. 3. Sheep red blood cell-specific-IgM antibody production in adult female B6C3F1 mice fo dunes) geogenic dust samples from NDRA each week for 28 days. Data are presented as mea Data presented are representative of three trial days. The (*) indicates a response statistically di1.05 ± 0.495* 1.00 ± 0.277 1.80 ± 0.239 0.932 ± 0.317* 0.905 ± 0.236 0.578 ± 0.139 1.22 ± 0.497* 1.05 ± 0.324 0.526± s 5–6 animals. Data presented are representative of three trial days. ined from log transformed data.exposure to geogenic dust in the sand dune area is remarkable because these surfaces produce the greatest amount of respirable dust during wind erosion (Goossens et al., 2012). These surfaces also are highly emissive when driving an ORV up to 40 km/h. Mineral dusts are also commonly high in crystalline silica and aluminum, because they are llowing oropharyngeal aspiration exposure to CBN 1 (vegetated and non-vegetated sand n PFC/million cells ± standard deviations. Sample size for each group was 5–6 animals. fferent from the 0mg/kg group (p b 0.05) and was determined from log transformed data. ing nit pectprimarily composed of common crustal aluminum silicateminerals. The concentration of metals andmetalloids will vary based on geologic pro- cesses that can concentrate specific elements in rocks, and the chemical reactivity of the minerals and surface area of the particles themselves, which affects adsorption. In addition, airborne arsenic concentrations were of particular concern in this study due to its carcinogen classifica- tion (Anders et al., 2004; USEPA, 1988). Even though arsenic concentra- tions in the sand dune sediment are relatively low (b10 μg/g), onwindy days, airborne arsenic concentrations can be quite high (0.01 to 3 Fig. 4. Splenic natural killer cell activity was assessed in adult female B6C3F1 mice follow geogenic dust samples fromNDRA eachweek for 28 days. Data are presented asmean lytic u are representative of three trial days. The (*) indicates data significantly different from resN0.03 μg/m ). This poses concerns as a substantial amount of dust is emitted from the dunes, and because arsenic concentrations are greater in the finer-sized fractions (Goossens et al., 2015). During calm condi- tions, the airborne arsenic concentrations in the dunes are similar to background levels in rural areas elsewhere in the world, approximately 0.0004 μg/m3 (Suta, 1978; Ball et al., 1983). The primary focus of this studywas to define a range of health effects following geogenic dust exposure using a mouse model. A strength of this study is that we expanded the dose–response range to provide an opportunity to define a NOAEL and LOAEL. The selection of immunotoxicology parameters, PFC assay, NK cell assay, and flow cyto- metric evaluation of lymphocytic subpopulations, were included in this assessment due to their strength in predicting alterations in immune function (Luster et al., 1992; Luster et al., 1993; Keil et al., 1999; Keil et al., 2001). While a NOAEL could not be determined, the LOAEL established in this study was 0.01 mg/kg was based on both an immu- nological and kidney measure. A dose-responsive suppression of IgM antibody production and increasing levels of serum creatinine were the most sensitive parameters altered by exposure to geogenic dust from CBN 1. It is important to communicate that immunotoxicity oc- curred at geogenic dust concentrations corresponding with no overt toxicity defined as a 10% change in body weight from animals exposed to 0mg/kg. Greater than a 10% change in mouse body weight is an indi- cator of overt or systemic toxicity, often blurring direct effects on im- mune function. The LOAEL of 0.01 mg/kg was associated with a 45% reduction in IgM antibody production relative to the responses mea- sured in the 0 mg/kg group. The large reduction in IgM antibody pro- duction at low concentrations of geogenic dust from CBN 1 indicates that this mixture is capable of interfering with the ability of B cells toproduce immune protective antibodies. A limitation of this study is that we did not have the budget to also measure organic compounds on the dust. Even though quantitated metals were notably high on the geogenic dust, we acknowledge the possibility of unmeasured organic compounds as an underlying part of the geogenic dust mixture that may also contribute to the effects reported in our model. These observations are consistent with reports demonstrating that single exposures to lead, arsenic or crystalline silica suppress antibody responses. Specifically, arsenic and crystalline silica suppress the IgM re- oropharyngeal aspiration exposure to CBN 1 (vegetated and non-vegetated sand dunes) ± standard error of themean. Sample size for each groupwas 5–6 animals. Data presented ive 0 mg/kg (p b 0.05) was determined from log transformed data.sponse to T cell-dependent antigens, including SRBC and keyhole limpet hemocyanin (KLH) (Burchiel et al., 2009). Lead exposure suppressed serum levels of IgA, IgM and IgG and caused oxidative damage in Fischer 344 rats (Ercal et al., 2000). With regard to human studies, little is known about howmetals-containing dusts affect serum antibody levels. Bencko and colleagues reported that workers exposed to arsenic in a coal-burning plant had reduced serum levels of IgM, IgA or IgG that were less than 1/10 the levels of matched controls (Bencko et al., 1988). Like the geogenic dusts used in these experiments, welding fumes vary with respect to the type and quantity of metals that they contain (Anderson et al., 2007). Anderson et al. (2007) evaluated the ef- fects of stainless steel welding fumes that predominantly contain chro- mium andmanganese. By oropharyngeal aspiration inmice, the highest administered concentration (20 mg/kg) suppressed the primary IgM antibody response to SRBCswithout a concomitant reduction in theper- centage of B or T cells (Anderson et al., 2007). An in vitro study in the same publication demonstrated that when splenic lymphocytes were exposed to either a soluble portion, an insoluble portion, or a mixture of the soluble and insoluble portion of the welding fume particles, only the soluble portion of the welding fume particles were capable of reducing primary antibody responses. As chromium and manganese were higher in the soluble fraction of welding fume particles than in the insoluble portion, Anderson et al. concluded that these metals were associated with the observed immune suppression (Anderson et al., 2007). Although our environmental mixture was more complex, results of experiments with TiO2, a particle with no associated heavy metals, sug- gest that the metals and/or the minerals in the mixture, or the mixture of the metals and minerals with the particles, rather than the particles alone, were likely contributing to the observed changes in immune function. Additionally, although each individual metal had a relatively low concentration in the blood (Table 3), we cannot discount that the cumulative concentration of the metals in the blood in combination with the particles may have produced the observed effects. Very little Fig. 5. Autoantibody production against neuronal proteins in adult female B6C3F1 mice following oropharyngeal aspiration exposure to CBN 1 (vegetated and non-vegetated sand dunes) geogenic dust samples fromNDRA eachweek for 28 days. Data are presented as mean autoantibody levels ± standard deviations. Sample size for each group was 5–6 animals. The (*) indicates a response statistically different from the 0 mg/kg group (p b 0.05). A) NF-68 IgM; B) GFAP IgM; C) GFAP IgG.is known about environmental mixtures; this study is one of the first to evaluate such a complex mixture. In summary, at least five compo- nents in our geogenic dust, arsenic, lead, crystalline silica, chromium and manganese, are known to suppress IgM antibody production and this is consistent with our observations. The average decrease in activated T helper cells (CD4+CD25+- foxP3−) by approximately 88.4% in the exposure groups can account for reduced IgM antibody production. These lymphocytes are key to an- tigen presentation and subsequent antibody production (Vazquez et al., 2015). Of the elements in this complex geogenic dust mixture (Table 1), arsenic has been reported to reduce the CD4+ lymphocyte count (Soto- Pena et al., 2006), suppress proliferative lymphocytic responses, and de- crease IL-2 production (Biswas et al., 2008). Crystalline silica exposures in an occupational environment led to workers with increased lympho- proliferative responses and decreased production of IL-2 (Rocha-Parise et al., 2014). IL-2 is key to activating T cells. Both silica and arsenic are reported to suppress this cytokine. Although this change to activated T helper cells was not reflected in the LOAEL established in this study, it is an important observation that may explain in part, the decreases in T cell-dependent IgM antibody production. In some cases elevated levels of creatinine have been reported with exposure to inorganic arsenic (Moore et al., 1994). Furthermore, animal studies have reported that the urinary system is a more sensitive target for DMA than for MMA (Cohen et al., 2001). Evidently, our study differs in that we examined an exposure to silica and metals, arsenic included. In our mixture, dose-responsive increases in serum creatinine were ob- served following exposure to geogenic dust from CBN 1, comprising of 62 μg/g arsenic, 25 μg/g lead, 511 μg/gmanganese, and 4.7 μg/g uranium (Table 1). Serum creatinine is a marker of kidney function and increas- ing levels suggest nephrotoxicity. Increased creatinine levels in the blood may also suggest diseases or conditions that affect kidney func- tion such as acute tubular necrosis or glomerulonephritis from infection or autoimmune disease. The kidneys are particularly susceptible not only to metal toxicity, but also to silica. Silica exposure is associated with excess mortality from acute renal disease and an increased risk of end-stage renal disease (reviewed by Ghahramani, 2010). Therefore, it is plausible that the combined metals and silica exposure in our geogenic dust would alter a kidney function. It is important to remark that the increase in creatinine developed during ‘weekend’ exposures over onemonth. This is a swift change in creatinine and suggests dimin- ished kidney function. Therefore, examination of chronic exposure to geogenic dust may be needed to understand the full extent of effects on the kidney. Although our results indicate that Tregswere not depleted following an exposure to geogenic dust from CBN 1, previous research reported that arsenic or silica has been shown to affect immune regulation by specifically targeting Tregs. Tregs are recognized as a specialized subset of T lymphocytes responsible for suppression of immune responses and a key cell in preventing autoimmune disease. Silica exposure is associat- ed with a variety of autoimmune problems, including rheumatoid ar- thritis, lupus, scleroderma, and glomerulonephritis (Lim et al., 2012). Tregs are characterized by high surface expression of CD25 (the IL-2 re- ceptor alpha chain) and intracellular expression of the master switch transcription factor forkhead box protein P3 (FoxP3) (Fontenot et al., 2003). Studies have reported both increases and decreases in Treg pop- ulations following arsenic exposure (Hernandez-Castro et al. 2009; Thomas-Schoemann et al., 2012). This contrasts our studies where no change in numbers was observed. At this time, the reasons for this dif- ference are unclear. Upon injury to cells within the nervous system, liberated proteins may induce an autoimmune response measurable as serum autoanti- bodies (El-Fawal et al., 1999). El-Fawal and O'Callaghan reported in- creases in IgM and IgG antibodies against neural and glial proteins in rats exposed to only one dose of trimethyltin that were detectable up to three weeks post-exposure (El-Fawal and O'Callaghan, 2008), so this endpoint was chosen to evaluate the potential neurotoxicity of gea icathe complex geogenic dust in this study. While statistically significant changes in autoantibodies against neural proteins were observed, they were reduced in animals from dosed groups, rather than increased rel- ative to levels in the 0 mg/kg group. This is likely indicative of the over- whelming immune suppression observed in the PFC assay. Additionally, no infiltration of T cells was observed in the brains of dosedmice, which suggests that the geogenic dust from CBN 1 was not given at a concen- tration or for a duration sufficient to induce a strong enough inflamma- tory response to attract T cells from the periphery. The relative intensity ofMBPwas evaluated in brain sections to determine if autoantibodies or inflammatory processes were directed against this common brain pro- Fig. 6. Histological markers in the brains of adult female B6C3F1 mice following oropharyn samples from NDRA each week for 28 days. Data are presented as mean immunohistochem (*) indicates a response statistically different from the 0 mg/kg group (p b 0.05).tein that often is a target in neurodegeneration. The observed decrease in MBP in dosed groups (0.1, 10, and 100 mg/kg) relative to the control group can indicate inflammation-induced demyelination, which was detectable with immunohistochemistry, but not sufficient to attract im- mune cells from outside of the central nervous system. Additionally, the inconsistency and variability in this particular response indicate that the exposure paradigm or concentrations were not sufficient to induce strong reductions in MBP. While neurotoxicity cannot be ruled out as a concern, under the conditions of this study, overt neurotoxicity was not evident. Fig. 7. Particle size distribution of the CBN1 geogenic dust used in this study.The authors acknowledge that exposure and bioavailability of the adsorbed components in this geogenic dust are fundamental to bridging these data to human health risk. The complex interactions in the human body upon exposure to these substances are further complicated by the different bioaccessibility, biodurability, and bioreactivity of these min- erals and their adsorbed components (Plumlee et al., 2006; Morman and Plumlee, 2013). In addressing this topic more completely, forth- coming publications from this research team will report on the bioac- cessibility in fluids and bioavailability of the geogenic dust components in both mice and humans. In conclusion, exposure to geogenic dust from sand dunes of the l aspiration exposure to CBN 1 (vegetated and non-vegetated sand dunes) geogenic dust l intensity score ± standard deviations. Sample size for each group was 5–6 animals. TheNDRA dose-responsively and consistently reduced the ability of the im- mune system to produce IgM antibodies against a T cell-dependent an- tigen with corresponding decreases in activated T helper cell populations. Based on the IgM antibody response data, the LOAEL established in this study was 0.01 mg/kg, while a NOAEL could not be determined. The LOAELwas based on a dose-responsive immunological effect and a kidney measure. The immunological parameters affected in this study are known to be predictive of increased disease susceptibility (Luster et al., 1992; Luster et al., 1993) and, therefore, are regarded as reliable markers for immunotoxicity. Based on the lack of statistical findings with a “neutral particle” (TiO2), our results suggest that the metal/mineral components associated with the geogenic dust in CBN 1 are likely to contribute to the reduction in antigen-specific antibody production. Suppression of this type of immune function is a sensitive indicator of immunotoxicity in experimental animal systems and is sug- gestive of the risk of immunotoxicity in other exposed organisms, in- cluding humans. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgments This project was funded by the U.S. Bureau of Land Management (URL: http://www.blm.gov/nv/st/en/fo/lvfo.html), Grant number: L11AC20058. The funders had no role in study design, data collection Ercal, N., Neal, R., Treeratphan, P., Lutz, P.M., Hammond, T.C., Dennery, P.A., Spitz, D.R., 2000. A role for oxidative stress in suppressing serum immunoglobulin levels in lead-exposed Fisher 344 rats. Arch. Environ. Contam. Toxicol. 39, 251–256. Fontenot, J.D., Gavin, M.A., Rudensky, A.Y., 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336. Franklin, M., Koutrakis, P., Schwartz, P., 2008. The role of particle composition on the as- sociation between PM2.5 and mortality. Epidemiology 19, 680–689. Ghahramani, N., 2010. Silica nephropathy. The International Journal of Occupational and Environmental Medicine 1 (3 July).and analysis, decision to publish, or preparation of the manuscript. We are grateful for the assistance of Margie Peden-Adams, Ph.D., Sharon Young, Ph.D., Qing Hu, Corey Boles, Hakan Gürleyük, Ph.D., Deborah Ellis, Ph.D., Henry LeTang, MLT(ASCP) and our grant legal consultant, Curtis Coulter. References Albright, J.F., Goldstein, R.A., 1996. Airborne pollutants and the immune system. Otolaryngol. Head Neck Surg. 114 (2), 232–238. Aldabe, J., Elustondo, D., SantamarÃa, C., Lasheras, E., Pandolfi, M., Alastuey, A., Querol, X., SantamarÃa, J.M., 2011. Chemical characterisation and source apportionment of PM2.5 and PM10 at rural, urban and traffic sites in Navarra (North of Spain). Atmo- spheric Research 102, 191–205. Anders, W.M., Bull, R.J., Cantor, K.P., Chakraborti, D., Chen, C., DeAngelo, A.B., DeMarini, D.M., Ferreccio, C., Fukushima, S., Gebel, T.W., 2004. Some drinking-water disinfec- tants and contaminants, including arsenic. World Health Organization. Anderson, S.E., Meade, B.J., Butterworth, L.F., Munson, A.E., 2007. The humoral immune response of mice exposed to manual metal arc stainless steel-welding fumes. J. Immunotoxicol. 4, 15–23. Ball, A.L., Rom, W.N., Glenne, B., 1983. Arsenic distribution in soils surrounding the Utah copper smelter. Am. Ind. Hyg. Assoc. J. 44, 341–348. Beelen, R., Hoek, G., van den Brandt, P.A., Goldbohm, R.A., Fischer, P., Schouten, L.J., Armstrong, B., Brunekreef, B., 2008. Long-term exposure to traffic-related air pollu- tion and lung cancer risk. Epidemiology 19, 702–710. Bencko, V., Wagner, V., Wagnerova, M., Batora, J., 1988. Immunological profiles inworkers of a power plant burning coal rich in arsenic content. J Hyg Epidemiol Microbiol Immunol 32, 137–146. Biswas, R., Ghosh, P., Banerjee, N., Das, J.K., Sau, T., Banerjee, A., Roy, S., Ganguly, S., Chatterjee, M., Mukherjee, A., Giri, A.K., 2008. Analysis of T-cell proliferation and cytokine secretion in the individuals exposed to arsenic. Hum Exp Toxicol 27, 381–386. Brauer, M., Hoek, G., Van Vliet, P., Meliefste, K., Fischer, P.H., Wijga, A., Koopman, L.P., Neijens, H.J., Gerritsen, J., Kerkhof, M., Heinrich, J., Bellander, T., Brunekreef, B., 2002. Air pollution from traffic and the development of respiratory infections and asthmatic and allergic symptoms in children. Am. J. Respir. Crit. Care Med. 166, 1092–1098. Briggs, P.H., Meier, A.L., 1999. The Determination of Forty Two Elements in Geological Ma- terials by Inductively Coupled Plasma-Mass Spectrometry. US Department of the In- terior, US Geological Survey. Bryant, J., Day, R., Whiteside, T.L., Herberman, R.B., 1992. Calculation of lytic units for the expression of cell-mediated cytotoxicity. J. Immunol. Methods 146, 91–103. Buck, B.J., Goossens, D., Metcalf, R.V., McLaurin, B., Ren, M., Freudenberger, F., 2013. Natu- rally occurring asbestos: potential for human exposure, Southern Nevada, USA. Soil Sci. Soc. Am. J. 77, 2192–2204. Burchiel, S.W., Mitchell, L.A., Lauer, F.T., Sun, X., McDonald, J.D., Hudson, L.G., Liu, K.J., 2009. Immunotoxicity and biodistribution analysis of arsenic trioxide in C57Bl/6 mice following a 2-week inhalation exposure. Toxicol. Appl. Pharmacol. 241, 253–259. Calderón-Garcidueñas, L., Solt, A.C., Henríquez-Roldán, C., Torres-Jardón, R., Nuse, B., Herritt, L., Villarreal-Calderón, R., et al., 2008. Long-term air pollution exposure is as- sociated with neuroinflammation, an altered innate immune response, disruption of the blood–brain barrier, ultrafine particulate deposition, and accumulation of amy- loid β-42 and α-synuclein in children and young adults. Toxicologic Pathology 36 (2), 289–310. Cohen, S.M., Yamamoto, S., Cano, M., Arnold, L.L., 2001. Urothelial cytotoxicity and regen- eration induced by dimethylarsinic acid in rats. Toxicol. Sci. 59, 68–74. deMiranda, R.M., de Fatima Andrade, M., Fornaro, A., Astolfo, R., de Andre, P.A., Saldiva, P., 2012. Urban air pollution: a representative survey of PM(2.5) mass concentrations in six Brazilian cities. Air Qual Atmos Health 5, 63–77. Derbyshire, E., 2007. Natural minerogenic dust and human health. Ambio 36, 73–77. Dockery, D.W., Pope 3rd, C.A., Xu, X., Spengler, J.D., Ware, J.H., Fay, M.E., Ferris Jr., B.G., Speizer, F.E., 1993. An association between air pollution and mortality in six U.S. cit- ies. N Engl J Med 329, 1753–1759. Dockery, D.W., Pope, C., 1994. Acute respiratory effects of particulate air pollution. Annu. Rev. Public Health 15, 107–132. Duke, S.S., Schook, L.B., Holsapple, M.P., 1985. Effects of N-nitrosodimethylamine on tumor susceptibility. J. Leukoc. Biol. 37, 383–394. El-Fawal, H.A., O'Callaghan, J.P., 2008. Autoantibodies to neurotypic and gliotypic proteins as biomarkers of neurotoxicity: assessment of trimethyltin (TMT). Neurotoxicology 29, 109–115. El-Fawal, H.A., Waterman, S.J., De Feo, A., Shamy, M.Y., 1999. Neuroimmunotoxicology: humoral assessment of neurotoxicity and autoimmune mechanisms. Environ Health Perspect 107 (Suppl. 5), 767–775.Gilmour, M.I., Koren, H., 2000. Interaction of inhaled particles with the immune system. In: Gehr, P.H. (Ed.), Particle–lung interactions. Marcel Dekker, New York, pp. 629–652. Goossens, D., 2012. A method for dry extracting large volumes of fine particulate matter from bulk soil samples. Air Qual. Atmos. Health 5, 425–431. Goossens, D., Buck, B., 2009. Dust emission by off-road driving: experiments on 17 arid soil types, Nevada, USA. Geomorphology 107, 118–138. Goossens, D., Buck, B.J., McLaurin, B., 2012. Contributions to atmospheric dust production of natural and anthropogenic emissions in a recreational area designated for off-road vehicular activity (Nellis Dunes, Nevada, USA). J. Arid Environ. 78, 80–99. Goossens, D., Buck, B., Teng, Y., McLaurin, B., 2015. Surface and airborne arsenic concen- trations in a recreational site near Las Vegas, Nevada, USA. PLoS One 10 (4), e0124271. http://dx.doi.org/10.1371/journal.pone.0124271. Hassani, M., Brown, J.M., Morandi, M.T., Holian, A., 2004. Particulate matter immunomod- ulatory effects on autoantibody development in New Zealand mixed mice. J. Immunotoxicol. 1 (2), 95–102. Hernandez-Castro, B., Doniz-Padilla, L.M., Salgado-Bustamante, M., Rocha, D., Ortiz-Perez, M.D., Jimenez-Capdeville, M.E., Portales-Perez, D.P., Quintanar-Stephano, A., Gonzalez-Amaro, R., 2009. Effect of arsenic on regulatory T cells. J. Clin. Immunol. 29, 461–469. Holsapple, M.P., White Jr., K.L., McCay, J.A., Bradley, S.G., Munson, A.E., 1988. An immunotoxicological evaluation of 4,4′-thiobis-(6-t-butyl-m-cresol) in female B6C3F1 mice. 2. Humoral and cell-mediated immunity, macrophage function, and host resistance. Fundam Appl Toxicol 10, 701–716. Jerne, N.K., Nordin, A.A., 1963. Plaque formation in agar by single antibody-producing cells. Science 140, 405. Keil, D.E., Luebke, R.W., Ensley, M., Gerard, P., Pruett, S.B., 1999. Evaluation of multivariate statistical methods for analysis and modeling of immunotoxicology data. Toxicol. Sci. 51, 245–258. Keil, D.E., Leubke, R.W., Pruett, S.B., 2001. Quantifying the relationship between immuno- logical parameters and host resistance. J. Immunol. 167, 4543–4552. Kubachka, K.M., Shockey, N.V., Hanley, T.A., Conklin, S.D., Heitkemper, D.T., 2012. FDA El- emental Analysis Manual: Section 4.11: Arsenic Speciation in Rice and Rice Products Using High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometric Determination" Version 1.1. (http://www.fda.gov/Food/ FoodScienceResearch/LaboratoryMethods/ucm328363.htm). Kulshrestha, A., Satsangi, P.G., Masih, J., Taneja, A., 2009. Metal concentration of PM2.5 and PM10 particles and seasonal variations in urban and rural environment of Agra, India. Sci Total Environ 407, 6196–6204. Lambert, A.L., Trasti, F.S., Mangum, J.B., Everitt, J.I., 2003. Effect of preexposure to ultrafine carbon black on respiratory syncytial virus infection in mice. Toxicol. Sci. 72 (2), 331–338. Lee, S.C., Cheng, Y., Ho, K.F., Cao, J.J., Louie, P.K.K., Chow, J.C., Watson, J.G., 2006. PM1.0 and PM2.5 Characteristics in the Roadside Environment of Hong Kong. Aerosol Science and Technology 40, 157–165. Leonardi, G.S., Houthuijs, D., Steerenberg, P.A., Fletcher, T., Armstrong, B., Antova, T., Lochman, I., Lochmanova, A., Rudnai, P., Erdei, E., Musial, J., Jazwiec-Kanyion, B., Niciu, E.M., Durbaca, S., Fabianova, E., Koppova, K., Lebret, E., Brunekreef, B., van Loveren, H., 2000. Immune biomarkers in relation to exposure to particulate matter: a cross- sectional survey in 17 cities of Central Europe. Inhal. Toxicol. 12 (Suppl. 4), 1–14. Lim, S.S., Agan, M., Drenkard, C.M., 2012. Impact of systemic lupus erythematosus organ damage on unemployment or disability from a population-based cohort. Arthritis Re- search & Therapy 14 (3), 1–28. Linse, S., Cabaleiro-Lago, C., Xue,W.F., Lynch, I., Lindman, S., Thulin, E., et al., 2007. Nucleation of protein fibrillation by nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 104, 8691–8696. Luster, M.I., Munson, A.E., Thomas, P.T., Holsapple, M.P., Fenters, J.D., White, K.L., ... Dean, J.H., 1988. Development of a testing battery to assess chemical-induced immunotoxicity: National Toxicology Program's guidelines for immunotoxicity eval- uation in mice. Toxicol. Sci. 10 (1), 2–19. Luster, M.I., Portier, C., Pait, D.G., Rosenthal, G.J., Germolec, D.R., Corsini, E., Blaylock, B.L., Pollock, P., Kouchi, Y., Craig, W., et al., 1993. Risk assessment in immunotoxicology. II. Relationships between immune and host resistance tests. Fundam Appl Toxicol 21, 71–82. Luster, M.I., Portier, C., Pait, D.G., White Jr., K.L., Gennings, C., Munson, A.E., Rosenthal, G.J., 1992. Risk assessment in immunotoxicology. I. Sensitivity and predictability of im- mune tests. Fundam Appl Toxicol 18, 200–210. McLaurin, B.T., Goossens, D., Buck, B.J., 2011. Combining surface mapping and process data to assess, predict, and manage dust emissions from natural and disturbed land surfaces. Geosphere 7, 260–275. Moore, M.M., Harrington-Brock, K., Doerr, C.L., 1994. Genotoxicity of arsenic and its meth- ylated metabolites. Environ. Geochem. Health 16, 191–198. Morman, S.A., Garrison, V.H., Plumlee, G.S., Lowers, H.A., Bunnell, J.E., 2009. Assessing con- centration and bioaccessibly of potentially toxic elements in African dust. Geological Society of America Annual Meeting, p. 544 Portland, Oregon. Morman, S.A., Plumlee, G.S., 2013. The role of airborne mineral dusts in human disease. Aeolian Res. 9, 203–212. National Research Council. 2004. accessed 06-01-2015. http://epa.gov/ncer/science/pm/ 2008sab/exec_summary_2004_nrc_report.pdf. Oberdorster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Kreyling, W., Cox, C., 2004. Translocation of inhaled ultrafine particles to the brain. Inhal. Toxicol. 16, 437–445. Plumlee, G.S., Morman, S.A., Ziegler, T.L., 2006. The toxicological geochemistry of earth materials: an overview of processes and the interdisciplinarymethods used to under- stand them. Rev. Mineral. Geochem. 64, 5–57. Pope 3rd, C.A., Thun, M.J., Namboodiri, M.M., Dockery, D.W., Evans, J.S., Speizer, F.E., Heath Jr., C.W., 1995. Particulate air pollution as a predictor of mortality in a prospective study of U.S. adults. Am J Respir Crit Care Med 151, 669–674. Rocha-Parise, M., Santos, L.M., Damoiseaux, J.G., Bagatin, E., Lido, A.V., Torello, C.O., ... Queiroz, M.L., 2014. Lymphocyte activation in silica-exposed workers. International Journal of Hygiene and Environmental Health 217 (4), 586–591. Ruckerl, R., Schneider, A., Breitner, S., Cyrys, J., Peters, A., 2011. Health effects of particulate air pollution: a review of epidemiological evidence. Inhal. Toxicol. 23, 555–592. Samet, J.M., Dominici, F., Curriero, F.C., Coursac, I., Zeger, S.L., 2000. Fine particulate air pol- lution and mortality in 20 U.S. cities, 1987–1994. N Engl J Med 343, 1742–1749. Samoli, E., Peng, R., Ramsay, T., Pipikou, M., Touloumi, G., Dominici, F., Burnett, R., Cohen, A., Krewski, D., Samet, J., Katsouyanni, K., 2008. Acute effects of ambient particulate matter on mortality in Europe and North America: results from the APHENA study. Environ. Health Perspect. 116, 1480–1486. Sharratt, B.S., Lauer, D., 2006. Particulate matter concentration and air quality affected by windblown dust in the Columbia plateau. J. Environ. Qual. 35, 2011–2016. Soto-Pena, G.A., Luna, A.L., Acosta-Saavedra, L., Conde, P., Lopez-Carrillo, L., Cebrian, M.E., Bastida, M., Calderon-Aranda, E.S., Vega, L., 2006. Assessment of lymphocyte subpop- ulations and cytokine secretion in children exposed to arsenic. FASEB J. 20, 779–781. Soukup, D., Buck, B., Goossens, D., Teng, Y., Baron, D., 2011. Mineralogical composition of soil samples in the Nellis Dunes Recreational Area. In: Goossens, D., Buck, B. (Eds.), Assessment of Dust Emissions, Chemistry, and Mineralogy for Management of Natu- ral and Disturbed Surfaces at Nellis Dunes Recreational Area, Nevada, Final Report to Bureau of Land Management for Task Agreement Number FAA010017. Soukup, D., Buck, B., Goossens, D., Ulery, A., McLaurin, B.T., Baron, D., Teng, Y., 2012. Arse- nic concentrations in dust emissions from wind erosion and off-road vehicles in the Nellis Dunes Recreational Area, Nevada, USA. Aeolian Res. 5, 77–89. Stanek, L.W., Brown, J.S., Stanek, J., Gift, J., Costa, D.L., 2011. Air pollution toxicology—a brief review of the role of the science in shaping the current understanding of air pol- lution health risks. Toxicol. Sci. 120 (Suppl. 1), S8–27. Suta, B.E., 1978. Human exposures to atmospheric arsenic. Center for Resource and Envi- ronmental Systems Studies, CRESS Report No. 50. Tjalve, H., Henriksson, J., 1999. Uptake of metals in the brain via olfactory pathways. Neurotoxicol. 20, 181–195. Thomas-Schoemann, A., Batteux, F., Mongaret, C., Nicco, C., Chereau, C., Annereau, M., Dauphin, A., Goldwasser, F., Weill, B., Lemare, F., Alexandre, J., 2012. Arsenic trioxide exerts antitumor activity through regulatory T cell depletion mediated by oxidative stress in a murine model of colon cancer. J. Immunol. 189, 5171–5177. USEPA. 1988 Arsenic, inorganic (CASRN 7440-38-2) http://www.epa.gov/iris/subst/0278. htm. USEPA. 2007. Method 6020A. Inductively coupled plasma-mass spectrometry. 6000 series methods. www.epa.gov/osw/hazard/testmethods/sw846/pdfs/6020a.pdf. Vazquez, M.I., Catalan-Dibene, J., Zlotnik, A., 2015. B cells responses and cytokine produc- tion are regulated by their immune microenvironment. Cytokine 74 (2), 318–326. Wang, J., Hu, Z., Chen, Y., Chen, Z., Xu, S., 2013. Contamination characteristics and possible sources of PM10 and PM2. 5 in different functional areas of Shanghai, China. Atmo- spheric Environment 68, 221–229. Whicker, J.J., Pinder III, J.E., Breshears, D.D., Eberhart, C.F., 2006. From dust to dose: effects of forest disturbance on increase inhalation exposure. Sci. Total Environ. 368, 519–530.