Comparison of circulating and excreted metals and of autoimmunity between two Great Plains Tribal communities.

Metals contaminants of the environment from mine waste have been implicated as contributing agents in autoimmune disease. The current study compares metals and autoimmunity in two Tribal communities residing in the Black Hills and the Bighorn Mountains geographical regions that are scattered with extant hard rock mines. With documented drinking water contamination in both communities, in vivo levels of more than half of the measured serum and urine metals differed between the two communities and were substantially different from their national median values. Serum autoantibodies associated with systemic autoimmune disease were rare or at low-level, but antibodies to denatured (single-stranded) DNA and thyroid-specific autoantibodies were commonly elevated, especially in women. A three-tier statistical modeling process was carried out to examine individual metals exposure as predictors of autoantibody levels. For the most part only weak positive associations between individual metals and systemic autoantibodies were found, although univariate quantile regression analysis showed positive statistical associations of serum lead and antimony with anti-chromatin and anti-histone autoantibodies. Using age and gender-adjusted multivariable statistical models, metals did not predict anti-thyroglobulin or -thyroid peroxidase significantly and metals were generally negative predictors of the other autoantibodies. Overall these results suggest that elevated levels of environmental metals and metalloids in these communities may result in suppression of autoantibodies associated with systemic autoimmune disease.

Effects of an environmentally relevant PCB-mixture on immune function, clinical chemistry, and thyroid hormone levels in adult female B6C3F1 mice.

Polychlorinated biphenyls (PCBs) have been assessed for immunotoxicity; however, humans and wildlife are exposed to multiple PCBs environmentally. Therefore, the aim of this study was to examine the effects of a complex 37 PCB congener mixture identified in blubber specific to dolphins residing in the estuarine waters of Charleston, South Carolina. Immunotoxicity was determined in adult female B6C3F1 mice by assessing lymphocyte proliferation, splenic and thymic immunophenotypes, and IgM production. Mice were exposed via oral gavage to the PCB-mixture (0, 1.8, 3.6, 7.1, or 14.3 mg/kg/day) for 28 days to yield a targeted total administered dose (TAD) 0, 50, 100, 200, or 400 mg/kg. Significant increased liver weight occurred at the highest treatment. IgM production was suppressed compared to control for all treatments. Numbers of thymic CD4+/CD8+, CD4-/CD8-, and CD4+/CD8- cells were not altered, but numbers of thymic CD4-/CD8+ cells were significantly increased in the highest treatment. Lymphocyte proliferation was not markedly affected by any treatment. The numbers of splenic CD4/CD8 T-cells or MHCII+ cells were not significantly changed. Humoral immunity using the plaque-forming cell assay for determining the specific IgM antibody-forming cell response appeared to be the most sensitive endpoint affected. As the lowest concentration tested resulted in decreased IgM production and total and free thyroxine (T4) serum levels a NOAEL was not identified. The calculated ED50 for suppression of IgM production was 2.4 mg/kg/day.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits Libby amphibole fiber-induced acute inflammation in mice.

BACKGROUND: Exposure to the Libby amphibole (LA) asbestos-like fibers found in Libby, Montana, is associated with inflammatory responses in mice and humans, and an increased risk of developing mesothelioma, asbestosis, pleural disease, and systemic autoimmune disease. Flaxseed-derived secoisolariciresinol diglucoside (SDG) has anti-inflammatory, anti-fibrotic, and antioxidant properties. We have previously identified potent protective properties of SDG against crocidolite asbestos exposure modeled in mice. The current studies aimed to extend those findings by evaluating the immunomodulatory effects of synthetic SDG (LGM2605) on LA-exposed mice. METHODS: Male and female C57BL/6 mice were given LGM2605 via gavage initiated 3 days prior to and continued for 3 days after a single intraperitoneal dose of LA fibers (200 µg) and evaluated on day 3 for inflammatory cell influx in the peritoneal cavity using flow cytometry. RESULTS: LA exposure induced a significant increase (p < 0.0001) in spleen weight and peritoneal influx of white blood cells, all of which were reduced with LGM2605 with similar trends among males and females. Levels of peritoneal PMN cells were significantly (p < 0.0001) elevated post LA exposure, and were significantly (p < 0.0001) blunted by LGM2605. Importantly, LGM2605 significantly ameliorated the LA-induced mobilization of peritoneal B1a B cells. CONCLUSIONS: LGM2605 reduced LA-induced acute inflammation and WBC trafficking supporting its possible use in mitigating downstream LA fiber-associated diseases. SUMMARY: Following acute exposure to Libby amphibole (LA) asbestos-like fibers, synthetic SDG (LGM2605), a small synthetic molecule, significantly reduced the LA-induced increase in spleen weight and peritoneal inflammation in C57BL/6 male and female mice. Our findings highlight that LGM2605 has immunomodulatory properties and may, thus, likely be a chemopreventive agent for LA-induced diseases.

Comparative health effects in mice of Libby amphibole asbestos and a fibrous amphibole from Arizona.

This project developed from studies demonstrating that Libby Amphibole Asbestos (LAA) causes a non-typical set of health outcomes not generally reported for asbestos, including systemic autoimmunity and an unusual and devastating lamellar pleural thickening that progresses to severe pulmonary dysfunction and death. Further, mineral fiber mixtures with some similarities to LAA have recently been discovered in southern Nevada and northwestern Arizona, where the material exists in extensive recreational areas and is present in yards, roads, parking lots and school yards. The objective was to compare the health outcomes in mice exposed to either LAA or the fibrous amphiboles collected in Arizona at the Lake Mead National Recreational Area at very low doses to represent environmental exposures. In this study, the fibrous amphibole asbestos sample from Arizona (AzA) is composed of winchite (69%), actinolite (22%), and non-amphibole minerals (9%) and has a mean aspect ratio of 16.7±0.9. Fibrous amphibole asbestos from Libby (LAA) is composed of winchite (70%), richterite (9%), tremolite (5%), and non-amphibole minerals (16%) with a mean aspect ratio of 8.4±0.7. C57BL/6 mice were exposed by oropharyngeal aspiration to fiber suspensions at a very low dose of 3µg/mouse. After seven months, both LAA- and AzA-exposed mice had indices of chronic immune dysfunction related to a TH17 cytokine profile, with B cell activation, autoantibody production and proteinuria, suggesting kidney involvement. In addition, both exposures led to significant lung and pleural fibrosis. These data suggest that there is risk of pulmonary disease and autoimmune outcomes with environmental exposure to amphibole asbestos, and that this is not limited to Libby, Montana.

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.

Exposure to geogenic particulate matter (PM) comprised of mineral particles has been linked to human health effects. However, very little data exist on health effects associated with geogenic dust exposure in natural settings. Therefore, we characterized particulate matter size, metal chemistry, and health effects of dust collected from the Nellis Dunes Recreation Area (NDRA), a popular off-road vehicle area located near Las Vegas, NV. Adult female B6C3F1 mice were exposed to several concentrations of mineral dust collected from active and vegetated sand dunes in NDRA. Dust samples (median diameter: 4.4 µm) were suspended in phosphate-buffered saline and delivered at concentrations ranging from 0.01 to 100 mg dust/kg body weight by oropharyngeal aspiration. ICP-MS analyses of total dissolution of the dust resulted in aluminum (55,090 µg/g), vanadium (70 µg/g), chromium (33 µg/g), manganese (511 µg/g), iron (21,600 µg/g), cobalt (9.4 µg/g), copper (69 µg/g), zinc (79 µg/g), arsenic (62 µg/g), strontium (620 µg/g), cesium (13 µg/g), lead 25 µg/g) and uranium (4.7 µg/g). Arsenic was present only as As(V). Mice received four exposures, once/week over 28-days to mimic a month of weekend exposures. Descriptive and functional assays to assess immunotoxicity and neurotoxicity were performed 24 h after the final exposure. The primary observation was that 0.1 to 100 mg/kg of this sand dune derived dust dose-responsively reduced antigen-specific IgM antibody responses, suggesting that dust from this area of NDRA may present a potential health risk.

Health effects following subacute exposure to geogenic dusts from arsenic-rich sediment at the Nellis Dunes Recreation Area, Las Vegas, NV.

Geogenic dust from arid environments is a possible inhalation hazard for humans, especially when using off-road vehicles that generate significant dust. This study focused on immunotoxicological and neurotoxicological effects following subacute exposure to geogenic dust generated from sediments in the Nellis Dunes Recreation Area near Las Vegas, Nevada that are particularly high in arsenic; the naturally-occurring arsenic concentrations in these surficial sediments ranged from 4.8 to 346µg/g. Dust samples from sediments used in this study had a median diameter of 4.5µm and also were a complex mixture of naturally-occurring metals, including aluminum, vanadium, chromium, manganese, iron, cobalt, copper, zinc, strontium, cesium, lead, uranium, and arsenic. Adult female B6C3F1 mice exposed via oropharyngeal aspiration to 0.01 to 100mg dust/kg body weight, four times, a week apart, for 28days, were evaluated 24h after the last exposure. Peripheral eosinophils were increased at all concentrations, serum creatinine was dose responsively increased beginning at 1.0mg/kg/day, and blood urea nitrogen was decreased at 10 and 100mg/kg/day. Antigen-specific IgM responses and natural killer cell activity were dose-responsively suppressed at 0.1mg/kg/day and above. Splenic CD4+CD25+ T cells were decreased at 0.01, 0.1, 10, and 100mg/kg/day. Antibodies against MBP, NF-68, and GFAP were selectively reduced. A no observed adverse effect level of 0.01mg/kg/day and a lowest observed adverse effect level of 0.1mg/kg/day were determined from IgM responses and natural killer cell activity, indicating that exposure to this dust, under conditions similar to our design, could affect these responses.

Current status of developmental immunotoxicity: early-life patterns and testing.

Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and therefore may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. When evaluating DIT in an animal model, specific endpoints are assessed, which can reveal the potential for a risk factor to alter immune system development. However, linking DIT evaluation in an animal model with clinical realities observed in human populations requires that DIT testing regimens evaluate critical windows in immune system development. In addition, pathways leading to DIT may not be apparent without the stressors that induce aberrant and detectable responses. This review contains brief descriptions of recently published work that addresses disease patterns associated with DIT and solutions for altering such patterns of disease. We also comment on gaps between DIT testing in animal models and the clinical manifestation of immune-based diseases in children that can be filled by a better understanding of critical windows in immune system development and DIT testing that includes multiple functional assays.

Discouraging academic dishonesty in online courses.

With the development of distance education and blended course delivery formats, our faculty faced new issues related to academic integrity in online testing. Current students often differ in their understanding of what is appropriate academic behavior and what is considered cheating. Enhancing quiz formats and educating faculty and students about academic integrity policies has minimized the situation in our program.

Wastewater-based epidemiology pilot study to examine drug use in the Western United States.

The extent of prescription and illicit drug abuse in geographically isolated rural and micropolitan communities in the intermountain western United States (US) has not been well tracked. The goal of this pilot study was to accurately measure drug dose consumption rates (DCR) between two select populations, normalize the data and compare the DCRs to similar communities. To learn about patterns of drug abuse between the two disparate communities, we used the emergent field of wastewater-based epidemiology (WBE). A rapid, quantitative and systematic process for the determination of multiple classes of prescribed and illicit drugs was applied to influent wastewater samples. Influent samples were collected over the course of three months (April to June 2019) at two wastewater treatment plants representing a small urban and a rural community. Collection of sewage influent included 24-h composite samples and the use of polar organic chemical integrative samplers (POCIS), time-weighted samplers. Using the results from the composite sampling data, DCRs per 1000 population could be calculated from the concentration data and the use of excretion correction factors. The following 18 compounds: amphetamine, methamphetamine, MDA, MDMA, morphine, 6-acetylmorphine, methadone, EDDP, codeine, benzoylecgonine, hydrocodone, hydromorphone, oxycodone, noroxycodone, ketamine, fluoxetine, tramadol, and ritalinic acid; represent a subset of the targeted analytes that were consistently measured at detectable concentration levels, and present at both sites. Following normalization of the drug measurements to influent flow rates and per capita, the small urban community demonstrated greater collective excretion rates (CER) than the rural community, with the exceptions of amphetamine and methamphetamine.

Uptake and effect of mercury on amino acid losses from the gills of the bivalve mollusks Mytilus californianus and Anodonta californiensis.

Inorganic mercury (Hg(2+)) and herbicides are important contaminants of world water systems with effects on aquatic organisms and humans. The uptake of Hg(2+) and glycine by the gills of the bivalve mollusks Mytilus californianus and Anodonta californiensis was determined. Additionally, the effects of glycine, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4-dinitrophenol (DNP) on the uptake of Hg(2+) were also determined. The loss of primary amines from the excised gills of both species was measured in the presence and absence of Hg(2+) or MeHg(+). The results indicate that (1) the uptake of Hg(2+) is approximately equivalent in both species; (2) comparison of the uptakes with that of inulin, which occupies only extracellular space, shows that Hg(2+) is taken up; (3) the uptake of Hg(2+) is slightly altered by the presence of glycine and herbicides such as 2,4-D and DNP; (4) the rate of loss of primary amines was highly increased relative to the control by the presence of Hg(2+) and to a lesser extent MeHg(+) for both species. These results showed that both inorganic and MeHg(+) are effective in disrupting the permeability of cell membranes, causing leakage of essential amino acids from the cell. This could result in discharge of potential gradients, reduced efficiency of energy coupling, and consequently cell death.

Nevada desert dust with heavy metals suppresses IgM antibody production.

Systemic health effects from exposure to a complex natural dust containing heavy metals from the Nellis Dunes Recreation Area (NDRA) near Las Vegas, NV, were evaluated. Several toxicological parameters were examined following lung exposure to emissive dust from three geologic sediment types heavily used for recreational off-road activities: yellow sand very rich in arsenic (termed CBN 5); a shallow cover of loose dune sand overlying a gravelly subsoil bordering dune fields (termed CBN 6); and brown claystone and siltstone (termed CBN 7). Adult female B6C3F1 mice were exposed by oropharyngeal administration to these three types of geogenic dusts at 0.01-100 mg of dust/kg of body weight, once per week for four weeks. The median grain sizes were 4.6, 3.1, and 4.4 µm, for CBN 5, 6, and 7, respectively. Each type of dust contained quantifiable amounts of aluminum, vanadium, chromium, manganese, iron, cobalt, copper, zinc, arsenic, strontium, cesium, lead, uranium, and others. Descriptive markers of immunotoxicity, neurotoxicity, hematology, and clinical chemistry parameters were assessed. Notable among all three CBN units was a systemic, dose-responsive decrease in antigen-specific IgM antibody responses. Geogenic dust from CBN 5 produced more than a 70% suppression in IgM responses, establishing a lowest adverse effect level (LOAEL) of 0.01 mg/kg. A suppression in IgM responses and a corresponding increase in serum creatinine determined a LOAEL of 0.01 mg/kg for CBN 6. The LOAEL for CBN 7 was 0.1 mg/kg and also was identified from suppression in IgM responses. These results are of concern given the frequent off-road vehicle traffic and high visitor rates at the NDRA, estimated at 300,000 each year.

Suppression of humoral immunity following exposure to the perfluorinated insecticide sulfluramid.

Perfluorinated hydrocarbons have been manufactured for over 40 yr and have numerous applications in industry. This group of compounds has recently generated much interest, as some of these compounds such as perfluorooctane sulfonate (PFOS) and perfluoroctanic acid (PFOA) are persistent in the environment and detectable in blood samples of both wildlife and humans. Studies show that these perfluorinated compounds induce peroxisomal proliferation, induce hepatomegaly, alter steroidogenesis, and decrease body weight, accompanied by a wasting syndrome; however, effects on immune function have not been addressed at length. This study examined sulfluramid, a perfluorinated pesticide that is currently available in the marketplace and is a representative member of this class of chemicals. Adult female B6C3F1 mice were exposed via gavage to either an oil carrier control or sulfluramid for 14 d (1, 3, 10, or 30 mg/kg/d) or 28 d (0.3, 1, 3, or 10 mg/kg/d). Although responses were normal in natural killer cell activity and lymphocyte proliferation, dose-responsive suppression was noted in the plaque forming cell (PFC) response at exposure levels as low as 3 mg/kg/d in the 14-d exposure and 0.3 mg/kg/d for 28 d. Dose-responsive increases in liver mass were observed following treatment with 1, 3, 10, or 30 mg/kg/d for 14 d and 0.3, 1, 3, or 10 mg/kg/d for 28 d. A significant reduction in body weight was observed at the highest dose level in each study. Novel findings in this study indicate that sulfluramid suppresses immunoglobulin (Ig) M production. Additional immunotoxicity studies are required to understand potential mechanisms of suppression and determine potential health risks associated with exposure to perfluorinated hydrocarbons.

Health effects following subacute exposure to geogenic dust collected from active drainage surfaces (Nellis Dunes Recreation Area, Las Vegas, NV).

The specific health effects of direct inhalation of fine minerogenic dusts generated by natural soil surfaces remain poorly known and relatively little researched. To learn more about this exposure and its contribution to human health effects, we surveyed surface sediment and characterized dust from the Nellis Dunes Recreation Area (NDRA) in Clark County, Nevada, a popular off-road vehicle (ORV) recreational site. Dry drainage systems at NDRA are commonly used as natural trail systems for ORV recreation; these surfaces also are characterized by high concentrations of heavy metals. Geogenic dust with a median diameter of 4.05 µm, collected from drainage surfaces at NDRA contained a total elemental concentration of aluminum (79,651 µg/g), vanadium (100 µg/g), chromium (54 µg/g), manganese (753 µg/g), iron (33,266 µg/g), cobalt (14 µg/g), copper (37 µg/g) zinc (135 µg/g), arsenic (71 µg/g), strontium (666 µg/g), cesium (15 µg/g), lead (34 µg/g), and uranium (54.9 µg/g). Adult female B6C3F1 mice exposed via oropharyngeal aspiration to 0.01-100 mg dust/kg body weight, four times, a week apart, for 28-days, were evaluated for immuno- and neurotoxicological outcomes 24 h after the last exposure. Antigen-specific IgM responses were dose-responsively suppressed at 0.1, 1.0, 10 and 100 mg/kg. Splenic lymphocytic subpopulations, hematological and clinical chemistry parameters were affected. In brain tissue, antibodies against NF-68, and GFAP were not affected, whereas IgM antibodies against MBP were reduced by 26.6% only in the highest dose group. A lowest observed adverse effect level (LOAEL) of 0.1 mg/kg/day and a no observed adverse effect level (NOAEL) of 0.01 mg/kg/day were derived based on the antigen primary IgM responses after subacute exposure to this geogenic dust.

Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments.

Several laboratory and field studies indicate that organochlorine contaminants (OCs), such as polychlorinated biphenyls (PCBs) and pesticides, modulate immune responses in rodents, wildlife, and humans. In the present study we examined the effects of OCs on immunity in free-ranging loggerhead sea turtles (Caretta caretta). Mitogen-induced lymphocyte proliferation responses, lysozyme activity, and OC concentrations were measured from blood samples. Mitogens chosen in the lymphocyte proliferation assay were phytohemagglutinin (PHA) and concanavalin A (ConA) for T-lymphocyte stimulation, and lipopolysaccharide (LPS) and phorbol 12,13-dibutyrate (PDB) for B-lymphocyte stimulation. Lysozyme activity was significantly and negatively correlated with whole-blood concentrations of 4,4 -dichlorodiphenyldichloroethylene (4,4 -DDE) and the sum of chlordanes. Lymphocyte proliferation responses stimulated by PHA, LPS, and PDB were significantly and positively correlated with concentrations of the sum of PCBs measured in whole blood. LPS- and PDB-induced proliferation were also significantly and positively correlated with 4,4 -DDE blood concentrations. These correlative observations in free-ranging turtles suggest that current, chronic exposure to OCs may suppress innate immunity and enhance certain lymphocyte functions of loggerhead sea turtles. To further test this hypothesis, lymphocyte proliferation was measured after in vitro exposure of peripheral blood leukocytes from 16 turtles to Aroclor 1254 (0-13.5 microg/mL) or 4,4 -DDE (0-13.4 microg/mL). Both contaminants increased PHA- and PDB-induced proliferation at concentrations below those that affected cell viability. Moreover, the concentrations that enhanced PDB-induced proliferation in vitro were similar to concentrations measured in turtles with the highest proliferative responses. The similarities between the in vitro experiments and the correlative field study suggest that OC exposure modulates immunity in loggerhead turtles.

Essiac tea: scavenging of reactive oxygen species and effects on DNA damage.

Essiac, a tea reportedly developed by the Ojibwa tribe of Canada and widely publicized as a homeopathic cancer treatment, is prepared from a mixture of four herbs Arctium lappa, Rumex acetosella, Ulmus rubra and Rheum officinale. Each of these herbs has been reported to possess antioxidant and anti-cancer activity. Essiac itself has also been reported to demonstrate anti-cancer activity in vitro, although its effects in vivo are still a matter of debate. We prepared an extract of Essiac tea from a concentration of 25mg/mL and boiled it for 10 min. From this preparation we used concentrations of 5, 10, 25 and 50% to measure Essiac effects. In this study, we examined the effects of Essiac on free radical scavenging and DNA damage in a non-cellular system, as well as the effects Essiac on lipid peroxidation using the RAW 264.7 cell line. We observed, using electron spin resonance, that Essiac effectively scavenged hydroxyl, up to 84% reduction in radical signal at the 50% tea preparation concentration, and superoxide radicals, up to 82% reduction in radical signal also at the 50% tea preparation concentration, as well as prevented hydroxyl radical-induced DNA damage. In addition, Essiac inhibited hydroxyl radical-induced lipid peroxidation by up to 50% at the 50% tea preparation concentration. These data indicate that Essiac tea possesses potent antioxidant and DNA-protective activity, properties that are common to natural anti-cancer agents. This study may help to explain the mechanisms behind the reported anti-cancer effects of Essiac.

Health effects from exposure to atmospheric mineral dust near Las Vegas, NV, USA.

Desert areas are usually characterized by a continuous deposition of fine airborne particles. Over time, this process results in the accumulation of silt and clay on desert surfaces. We evaluated health effects associated with regional atmospheric dust, or geogenic dust, deposited on surfaces in the Nellis Dunes Recreation Area (NDRA) in Clark County, Nevada, a popular off-road vehicle (ORV) recreational site frequented daily by riders, families, and day campers. Because of atmospheric mixing and the mostly regional origin of the accumulated particles, the re-suspended airborne dust is composed of a complex mixture of minerals and metals including aluminum, vanadium, chromium, manganese, iron, cobalt, copper, zinc, arsenic, strontium, cesium, lead, uranium, and others. Geogenic dust with a median diameter of 4.1 µm was administered via oropharyngeal aspiration to female B6C3F1 mice at doses of 0.01 to 100 mg dust/kg body weight, four times, a week apart, for 28-days. Immuno- and neurotoxicological outcomes 24 h following the last exposure were evaluated. Antigen-specific IgM responses were dose-responsively suppressed at 0.1, 1.0, 10 and 100 mg/kg/day. Splenic and thymic lymphocytic subpopulations and natural killer cell activity also were significantly reduced. Antibodies against MBP, NF-68, and GFAP were not affected, while brain CD3+ T cells were decreased in number. A lowest observed adverse effect level (LOAEL) of 0.1 mg/kg/day and a no observed adverse effect level (NOAEL) of 0.01 mg/kg/day were derived based on the antigen-specific IgM responses.

Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity.

A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.

A Test Procedure for the Determination of (2-Methoxyethoxy)acetic Acid in Urine from Jet Fuel-Exposed Mice.

A test procedure for the determination of (2-methoxyethoxy)acetic acid (MEAA) was adapted and applied to urine samples from jet fuel (JP-8)-exposed mice using capillary gas chromatography with a mass selective detector (MSD). MEAA is a metabolite and proposed biomarker for exposure to 2-(2-methoxyethoxy)ethanol, a glycol ether component in the formulation of JP-8. The collected urine samples were spiked with deuterated butoxyacetic acid internal standard, and extracted with ethyl acetate, and esterified with ethanol and sulfuric acid, and the esters of the glycol ethers were extracted with methylene chloride. The chromatographic conditions used easily separate the MEAA ethyl ester from interferences within mouse urine. The application of this procedure to urine samples collected from mice demonstrated that MEAA was detectable after oral (2000 mg/kg) or dermal (50 mu L) exposure for 7 days to JP-8 at levels as high as 8.5 or 6.5 mu g/mL, respectively. This pilot demonstration indicated that total urinary MEAA was a viable biomarker for the two routes of JP-8 exposure in laboratory mice.

Immunological function in mice exposed to JP-8 jet fuel in utero.

Immunological parameters, host resistance, and thyroid hormones were evaluated in F1 mice exposed in utero to jet propulsion fuel-8 (JP-8). C57BL/6 pregnant dams (mated with C3H/HeJ males) were gavaged daily on gestation days 6-15 with JP-8 in a vehicle of olive oil at 0, 1000, or 2000 mg/kg. At weaning (3 weeks of age), no significant differences were observed in body, liver, spleen, or thymus weight, splenic and thymic cellularity, splenic CD4/CD8 lymphocyte subpopulations, or T-cell proliferation. Yet, lymphocytic proliferative responses to B-cell mitogens were suppressed in the 2000 mg/kg treatment group. In addition, thymic CD4-/CD8+ cells were significantly increased. By adulthood (8 weeks of age), lymphocyte proliferative responses and the alteration in thymic CD4-/CD8+ cells had returned to normal. However, splenic weight and thymic cellularity were altered, and the IgM plaque forming cell response was suppressed by 46% and 81% in the 1000 and 2000 mg/kg treatment groups, respectively. Furthermore, a 38% decrease was detected in the total T4 serum hormone level at 2000 mg/kg. In F1 adults, no significant alterations were observed in natural killer cell activity, T-cell lymphocyte proliferation, bone marrow cellularity and proliferative responses, complete blood counts, peritoneal and splenic cellularity, liver, kidney, or thymus weight, macrophage phagocytosis or nitric oxide production, splenic CD4/CD8 lymphocyte subpopulations, or total T3 serum hormone levels. Host resistance models in treated F1 adults demonstrated that immunological responses were normal after challenge with Listeria monocytogenes, but heightened susceptibility to B16F10 tumor challenge was seen at both treatment levels. This study demonstrates that prenatal exposure to JP-8 can target the developing murine fetus and result in impaired immune function and altered T4 levels in adulthood.

Developmental immunotoxicity (DIT): assays for evaluating effects of exogenous agents on development of the immune system.

Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. Immunotoxicity testing guidelines established by the Environmental Protection Agency for adult animals (OPPTS 8703.7800) require functional tests and immunophenotyping that are suitable for detecting immunomodulation, especially immunosuppression. However, evaluating immune function in offspring that are not fully immunocompetent yields results that are challenging to interpret. Therefore, this unit will describe an optimum exposure scenario, reference two assays (immunophenotyping and histopathology) appropriate for detecting immunomodulation in weaning-age offspring, and reference four assays (immunophenotyping, histopathology, T cell-dependent antibody responses, and delayed-type hypersensitivity) appropriate for detecting immunomodulation in immunocompetent offspring. The protocol also will reference other assays (natural killer cell and cytotoxic T lymphocyte) with potential utility for assessing DIT.