Immunotoxicity of N-butylbenzenesulfonamide: impacts on immune function in adult mice and developmentally exposed rats.

N-butylbenzenesulfonamide (NBBS) is a high-production volume plasticizer that is an emerging contaminant of concern for environmental and human health. To understand the risks and health effects of exposure to NBBS, studies were conducted in adult-exposed mice and developmentally exposed rats to evaluate the potential for NBBS to modulate the immune system. Beginning between 8 and 9 weeks of age, dosed feed containing NBBS at concentrations of 0, 313, 625, 1250, 2500, and 5000 ppm was continuously provided to B6C3F1/N female mice for 28 days. Dosed feed was also continuously provided to time-mated Harlan Sprague Dawley (Sprague Dawley SD) rats at concentrations of 0-, 250-, 500-, and 1000-ppm NBBS from gestation day 6 to postnatal day 28 and in F1 rats until 11-14 weeks of age. Functional assessments of innate, humoral, and cell-mediated immunity were conducted in adult female mice and F1 rats following exposure to NBBS. In female mice, NBBS treatment suppressed the antibody-forming cell (AFC) response to SRBC with small increases in T-cell responses and natural killer (NK)-cell activity. In developmentally exposed rats, NBBS treatment-related immune effects were sex dependent. A positive trend in NK-cell activity occurred in male F1 rats while a negative trend occurred in female F1 rats. The AFC response to SRBC was decreased in female F1 rats but not in male F1 rats. These data provide evidence that oral exposure to NBBS has the potential to produce immunomodulatory effects on both innate and adaptive immune responses, and these effects appear to have some dependence on species, sex, and period of exposure (developmental vs adult).

Consensus on the Key Characteristics of Immunotoxic Agents as a Basis for Hazard Identification.

BACKGROUND: Key characteristics (KCs), properties of agents or exposures that confer potential hazard, have been developed for carcinogens and other toxicant classes. KCs have been used in the systematic assessment of hazards and to identify assay and data gaps that limit screening and risk assessment. Many of the mechanisms through which pharmaceuticals and occupational or environmental agents modulate immune function are well recognized. Thus KCs could be identified for immunoactive substances and applied to improve hazard assessment of immunodulatory agents. OBJECTIVES: The goal was to generate a consensus-based synthesis of scientific evidence describing the KCs of agents known to cause immunotoxicity and potential applications, such as assays to measure the KCs. METHODS: A committee of 18 experts with diverse specialties identified 10 KCs of immunotoxic agents, namely, 1) covalently binds to proteins to form novel antigens, 2) affects antigen processing and presentation, 3) alters immune cell signaling, 4) alters immune cell proliferation, 5) modifies cellular differentiation, 6) alters immune cell-cell communication, 7) alters effector function of specific cell types, 8) alters immune cell trafficking, 9) alters cell death processes, and 10) breaks down immune tolerance. The group considered how these KCs could influence immune processes and contribute to hypersensitivity, inappropriate enhancement, immunosuppression, or autoimmunity. DISCUSSION: KCs can be used to improve efforts to identify agents that cause immunotoxicity via one or more mechanisms, to develop better testing and biomarker approaches to evaluate immunotoxicity, and to enable a more comprehensive and mechanistic understanding of adverse effects of exposures on the immune system. https://doi.org/10.1289/EHP10800.

Identifying environmental factors that influence immune response to SARS-CoV-2: Systematic evidence map protocol.

BACKGROUND: Widespread environmental contamination can directly interact with human immune system functions. Environmental effects on the immune system may influence human susceptibility to respiratory infections as well as the severity of infectious diseases, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the efficacy of vaccines to respiratory diseases may be impacted by environmental exposures through immune perturbations. Given the quick pace of research about COVID-19 and associated risk factors, it is critical to identify and curate the streams of evidence quickly and effectively. OBJECTIVE: We developed this systematic evidence map protocol to identify and organize existing human and animal literature on high-priority environmental chemical classes (Per- and polyfluoroalkyl substances, pesticides, phthalates, quaternary ammonium compounds, and air pollutants) and their potential to influence three key outcomes: (1) susceptibility to respiratory infection, including SARS-CoV-2 (2) severity of the resultant disease progression, and (3) impact on vaccine efficacy. The result of this project will be an online, interactive database which will show what evidence is currently available between involuntary exposures to select environmental chemicals and immune health effects, data gaps that require further research, and data rich areas that may support further analysis. SEARCH AND STUDY ELIGIBILITY: We will search PubMed for epidemiological or toxicological literature on select toxicants from each of the chemical classes and each of the three outcomes listed above. STUDY APPRAISAL AND SYNTHESIS OF METHODS: For each study, two independent reviewers will conduct title and abstract screening as well as full text review for data extraction of study characteristics. Study quality will not be evaluated in this evidence mapping. The main findings from the systematic evidence map will be visualized using a publicly available and interactive database hosted on Tableau Public.

Evaluation of skin sensitization induced by four ionic liquids.

Ionic liquids (ILs) are synthetic solvents used as replacements for volatile organic solvents. Human exposure occurs through dermal or oral routes. In rodents, several ILs were reported to induce dermal toxicity, irritation, and sensitization. Due to the potential for occupational exposure, and industrial use as nonvolatile solvents, 1-ethyl-3-methylimidazolium chloride (EMIM, 6.25% to 50% v/v), 1-butyl-3-methylimidazolium chloride (BMIM, 3.12% to 12.5% v/v), 1-butyl-1-methylpyrrolidinium chloride (BMPY, 0.825% to 6.25% v/v), and N-butylpyridinium chloride (NBuPY, 0.825% to 12.5% v/v) were nominated to the National Toxicology Program and evaluated for skin sensitization. The test compound was applied to the ears of female BALB/c mice daily for 3 days in a primary irritancy (IRR)/local lymph node assay (LLNA). Sensitization was assessed in vitro in the direct peptide reactivity assay (DPRA), KeratinoSens™ assay, and human cell line activation test (h-CLAT). In the LLNA, the butylated ILs, BMIM, and BMPY were more potent than NBuPY (butylated) or EMIM (ethylated), which was neither an irritant nor a sensitizer. NBuPY induced skin irritation in vivo at ≥3.12% (p ≤ 0.01), and sensitization in vitro in the KeratinoSens™ assay and h-CLAT, but was negative for sensitization in vivo and in the DPRA. Although SI3 was not achieved, dermal treatment with 12.5% BMIM or 6.25% BMPY increased (p ≤ 0.01) lymph node cell proliferation in the LLNA. In vitro, BMIM was positive for sensitization in the h-CLAT, and BMPY was positive in the h-CLAT and KeratinoSens™ assay; both were negative in the DPRA. Integrated data analyses, weighted toward in vivo data, suggested that BMIM and BMPY may induce weak to mild sensitization.

Immunotoxicity studies of sulfolane following developmental exposure in Hsd:Sprague Dawley SD rats and adult exposure in B6C3F1/N mice.

Sulfolane is a solvent used in the petrochemical industry and a groundwater contaminant in areas near refineries. The current studies were conducted to assess the impact of oral exposure to sulfolane on the immune system using two models: (1) a perinatal drinking water exposure to 0, 30, 100, 300, or 1000 mg/L from gestation day (GD) 6 until ∼13 weeks-of-age in Harlan Sprague Dawley rats; and, (2) a 90-day gavage exposure of adult female B6C3F1/N mice to 0, 1, 10, 30, 100, or 300 mg/kg/day. Immune parameters evaluated included measurement of antibody production against sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), ex vivo measurements of natural killer (NK) cell activity, cytotoxic T-cell (CTL) activity, and T-cell proliferation, as well as measures of splenic immune cell populations, hematological parameters, and histopathology of immune tissues. A decrease in ex vivo NK cell activity was observed in cells from female - but not male - F1 rats following developmental exposure. In adult female mice, splenic NK cell number was lower than the vehicle controls at doses ≥ 100 mg/kg; however, ex vivo NK cell activity was not affected by sulfolane treatment. In female mice, a decrease in the number of large unstained cells at doses ≥ 30 mg/kg was observed. In F1 rats, effects on white blood cells (WBC) were limited to a decreasing trend in leukocytes in females; no effects were observed in males. Under the conditions of this study, a no-observed-effect level (NOEL) of 3 mg/kg/day was identified based on reduced NK cell activity in female F1 rats. Overall, these findings suggest that oral exposure to sulfolane in rodents had minimal effects on the immune system.

Harnessing In Silico, In Vitro, and In Vivo Data to Understand the Toxicity Landscape of Polycyclic Aromatic Compounds (PACs).

Polycyclic aromatic compounds (PACs) are compounds with a minimum of two six-atom aromatic fused rings. PACs arise from incomplete combustion or thermal decomposition of organic matter and are ubiquitous in the environment. Within PACs, carcinogenicity is generally regarded to be the most important public health concern. However, toxicity in other systems (reproductive and developmental toxicity, immunotoxicity) has also been reported. Despite the large number of PACs identified in the environment, research attention to understand exposure and health effects of PACs has focused on a relatively limited subset, namely polycyclic aromatic hydrocarbons (PAHs), the PACs with only carbon and hydrogen atoms. To triage the rest of the vast number of PACs for more resource-intensive testing, we developed a data-driven approach to contextualize hazard characterization of PACs, by leveraging the available data from various data streams (in silico toxicity, in vitro activity, structural fingerprints, and in vivo data availability). The PACs were clustered on the basis of their in silico toxicity profiles containing predictions from 8 different categories (carcinogenicity, cardiotoxicity, developmental toxicity, genotoxicity, hepatotoxicity, neurotoxicity, reproductive toxicity, and urinary toxicity). We found that PACs with the same parent structure (e.g., fluorene) could have diverse in silico toxicity profiles. In contrast, PACs with similar substituted groups (e.g., alkylated-PAHs) or heterocyclics (e.g., N-PACs) with varying ring sizes could have similar in silico toxicity profiles, suggesting that these groups are better candidates for toxicity read-across analysis. The clusters/regions associated with certain in silico toxicity, in vitro activity, and structural fingerprints were identified. We found that genotoxicity/carcinogenicity (in silico toxicity) and xenobiotic homeostasis and stress response (in vitro activity), respectively, dominate the toxicity/activity variation seen in the PACs. The "hot spots" with enriched toxicity/activity in conjunction with availability of in vivo carcinogenicity data revealed regions of either data-poor (hydroxylated-PAHs) or data-rich (unsubstituted, parent PAHs) PACs. These regions offer potential targets for prioritization of further in vivo assessment and for chemical read-across efforts. The analysis results are searchable through an interactive web application (https://ntp.niehs.nih.gov/go/pacs_tableau), allowing for alternative hypothesis generation.

In vivo assessment of respiratory burst inhibition by xenobiotic exposure using larval zebrafish.

Currently, assessment of the potential immunotoxicity of a given agent involves a tiered approach for hazard identification and mechanistic studies, including observational studies, evaluation of immune function, and measurement of susceptibility to infectious and neoplastic diseases. These studies generally use costly low-throughput mammalian models. Zebrafish, however, offer an excellent alternative due to their rapid development, ease of maintenance, and homology to mammalian immune system function and development. Larval zebrafish also are a convenient model to study the innate immune system with no interference from the adaptive immune system. In this study, a respiratory burst assay (RBA) was utilized to measure reactive oxygen species (ROS) production after developmental xenobiotic exposure. Embryos were exposed to non-teratogenic doses of chemicals and at 96 h post-fertilization, the ability to produce ROS was measured. Using the RBA, 12 compounds with varying immune-suppressive properties were screened. Seven compounds neither suppressed nor enhanced the respiratory burst; five reproducibly suppressed global ROS production, but with varying potencies: benzo[a]pyrene, 17ß-estradiol, lead acetate, methoxychlor, and phenanthrene. These five compounds have all previously been reported as immunosuppressive in mammalian innate immunity assays. To evaluate whether the suppression of ROS by these compounds was a result of decreased immune cell numbers, flow cytometry with transgenic zebrafish larvae was used to count the numbers of neutrophils and macrophages after chemical exposure. With this assay, benzo[a]pyrene was found to be the only chemical that induced a change in the number of immune cells by increasing macrophage but not neutrophil numbers. Taken together, this work demonstrates the utility of zebrafish larvae as a vertebrate model for identifying compounds that impact innate immune function at non-teratogenic levels and validates measuring ROS production and phagocyte numbers as metrics for monitoring how xenobiotic exposure alters the innate immune system.

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies.

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10-12 %), compared to control, occurred at 5,000-10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9-13 %) were increased at 2,500-5,000 ppm and 1250-10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000-10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0-10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5-50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10-50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10-50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250-1000 µg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Markers of Inflammation.

Inflammation is a complex and necessary component of the response to biological, chemical, or physical stimuli, and the cellular and molecular events that initiate and regulate the interactions between the various players in the inflammatory process remain a source of ongoing investigation. In the acute phase of the inflammatory response, cells of the immune system migrate to the site of injury in a carefully orchestrated sequence of events that is facilitated by soluble mediators such as cytokines, chemokines, and acute-phase proteins. Depending on the degree of injury, this acute phase may be sufficient to resolve the damage and initiate healing processes. Persistent inflammation, either as a result of prolonged exposure to stimulation or an inappropriate reaction against self-molecules, can lead to the chronic phase, in which tissue damage and fibrosis can occur. Chronic inflammation has been reported to contribute to numerous diseases, including arthritis, asthma, atherosclerosis, autoimmune diseases, diabetes, and cancer, and to conditions of aging. Hematology and clinical chemistry data from standard toxicology studies can provide an initial indication of the presence and sometimes the location of inflammation. These data may suggest more specific immune function assays that are necessary to determine the presence and/or mechanism(s) of immunomodulation. Although changes in hematology dynamics, acute-phase proteins, complement factors, and cytokines are common to virtually all inflammatory conditions, and can be measured by a variety of techniques, individual biomarkers have yet to be strongly associated with specific pathologic events. Thus, although sensitive indicators of inflammation, these factors generally lack the specificity to identify the offending cause. The profile seen in a given inflammatory condition is dependent on the severity, chronicity, and mechanisms involved in the inflammatory process, as well as the species and the capacity of the individual's immune system to respond and adapt.

Aspergillus fumigatus viability drives allergic responses to inhaled conidia.

BACKGROUND: Aspergillus fumigatus-induced allergic airway disease has been shown to involve conidial germination in vivo, but the immunological mechanisms remain uncharacterized. OBJECTIVE: A subchronic murine exposure model was used to examine the immunological mediators that are regulated in response to either culturable or nonculturable A fumigatus conidia. METHODS: Female B6C3F1/N mice were repeatedly dosed via inhalation with 1 × 105 viable or heat-inactivated conidia (HIC), twice per week for 13 weeks (26 exposures). Control mice inhaled high-efficiency particulate arrestor-filtered air. The influence of A fumigatus conidial germination on the pulmonary immunopathological outcomes was evaluated by flow cytometry analysis of cellular infiltration in the airways, assessment of lung messenger RNA expression, quantitative proteomics, and histopathology of whole lung tissue. RESULTS: Repeated inhalation of viable conidia, but not HIC, resulted in allergic inflammation marked by vascular remodeling, extensive eosinophilia, and accumulation of alternatively activated macrophages (AAMs) in the murine airways. More specifically, mice that inhaled viable conidia resulted in a mixed TH1 and TH2 (IL-13) cytokine response. Recruitment of eosinophils corresponded with increased Ccl11 transcripts. Furthermore, genes associated with M2 or alternatively activated macrophage polarization (eg, Arg1, Chil3, and Retnla) were significantly up-regulated in viable A fumigatus-exposed mice. In mice inhaling HIC, CD4+ T cells expressing IFN-γ (TH1) dominated the lymphocytic infiltration. Quantitative proteomics of the lung revealed metabolic reprogramming accompanied by mitochondrial dysfunction and endoplasmic reticulum stress stimulated by oxidative stress from repetitive microbial insult. CONCLUSION: Our studies demonstrate that A fumigatus conidial viability in vivo is critical to the immunopathological presentation of chronic fungal allergic disease.

Assessment of immunotoxicity in female Fischer 344/N and Sprague Dawley rats and female B6C3F1 mice exposed to hexavalent chromium via the drinking water.

Sodium dichromate dihydrate (SDD), an inorganic compound containing hexavalent chromium (Cr(VI)), is a common environmental contaminant of groundwater sources due to widespread industrial use. There are indications in the literature that Cr(VI) may induce immunotoxic effects following dermal exposure, including acting as both an irritant and a sensitizer; however, the potential immunomodulatory effects of Cr(VI) following oral exposure are relatively unknown. Following the detection of Cr(VI) in drinking water sources, the National Toxicology Program (NTP) conducted extensive evaluations of the toxicity and carcinogenicity of SDD following drinking water exposure, including studies to assess the potential for Cr(VI) to modulate immune function. For the immunotoxicity assessments, female Fischer 344/N (F344/N) and Sprague Dawley (SD) rats and female B6C3F1 mice were exposed to SDD in drinking water for 28 consecutive days and evaluated for alterations in cellular and humoral immune function as well as innate immunity. Rats were exposed to concentrations of 0, 14.3, 57.3, 172, or 516 ppm SDD while mice were exposed to concentrations of 0, 15.6, 31.3, 62.5, 125, or 250 ppm SDD. Final mean body weight and body weight gain were decreased relative to controls in 250 ppm B6C3F1 mice and 516 ppm SD rats. Water consumption was significantly decreased in F344/N and SD rats exposed to 172 and 516 ppm SDD; this was attributed to poor palatability of the SDD drinking water solutions. Several red blood cell-specific parameters were significantly (5-7%) decreased in 250 ppm mice; however, these parameters were unaffected in rats. Sporadic increases in the spleen IgM antibody response to sheep red blood cells (SRBC) were observed, however, these increases were not dose-dependent and were not reproducible. No significant effects were observed in the other immunological parameters evaluated. Overall, exposure to Cr(VI) in drinking water had limited effects on the immune system in both rats and mice.

Evaluation of 4-methylcyclohexanemethanol (MCHM) in a combined irritancy and Local Lymph Node Assay (LLNA) in mice.

4-Methylcyclohexanemethanol (MCHM) is a flotation reagent used in fine coal beneficiation. On January 9, 2014, crude MCHM, a mixture containing predominantly MCHM, was inadvertently released into the Elk River, a municipal water source that serves about 300,000 people in the Charleston, WV area, resulting in temporary contamination of 15 percent of the state's tap water and causing significant dermal exposure. The current studies were undertaken to determine whether crude MCHM or MCHM has the potential to produce dermal irritancy and/or sensitization. BALB/c female mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 µL of various concentrations of crude MCHM or MCHM to the dorsum of each ear. A mouse ear-swelling test was used to determine irritancy potential and was undertaken in combination with the standardized Local Lymph Node Assay (LLNA) to determine skin sensitizing potential. MCHM was found to produce skin irritation at concentrations above 20% and did not produce sensitization. Crude MCHM also produced irritation, although weaker, and in addition was found to be a weak to moderate skin sensitizer. The results are discussed in terms of potential human health hazard.

Respiratory toxicity and immunotoxicity evaluations of microparticle and nanoparticle C60 fullerene aggregates in mice and rats following nose-only inhalation for 13 weeks.

C60 fullerene (C60), or buckminsterfullerene, is a spherical arrangement of 60 carbon atoms, having a diameter of approximately 1 nm, and is produced naturally as a by-product of combustion. Due to its small size, C60 has attracted much attention for use in a variety of applications; however, insufficient information is available regarding its toxicological effects. The effects on respiratory toxicity and immunotoxicity of C60 aggregates (50 nm [nano-C60] and 1 µm [micro-C60] diameter) were examined in B6C3F1/N mice and Wistar Han rats after nose-only inhalation for 13 weeks. Exposure concentrations were selected to allow for data evaluations using both mass-based and particle surface area-based exposure metrics. Nano-C60 exposure levels selected were 0.5 and 2 mg/m3 (0.033 and 0.112 m2/m3), while micro-C60 exposures were 2, 15 and 30 mg/m3 (0.011, 0.084 and 0.167 m2/m3). There were no systemic effects on innate, cell-mediated, or humoral immune function. Pulmonary inflammatory responses (histiocytic infiltration, macrophage pigmentation, chronic inflammation) were concentration-dependent and corresponded to increases in monocyte chemoattractant protein (MCP)-1 (rats) and macrophage inflammatory protein (MIP)-1α (mice) in bronchoalveolar lavage (BAL) fluid. Lung overload may have contributed to the pulmonary inflammatory responses observed following nano-C60 exposure at 2 mg/m3 and micro-C60 exposure at 30 mg/m3. Phenotype shifts in cells recovered from the BAL were also observed in all C60-exposed rats, regardless of the level of exposure. Overall, more severe pulmonary effects were observed for nano-C60 than for micro-C60 for mass-based exposure comparisons. However, for surface-area-based exposures, more severe pulmonary effects were observed for micro-C60 than for nano-C60, highlighting the importance of dosimetry when evaluating toxicity between nano- and microparticles.

Immunotoxic effects of sodium tungstate dihydrate on female B6C3F1/N mice when administered in drinking water.

Tungsten is a naturally occurring, high-tensile strength element that has been used in a number of consumer products. Tungsten has been detected in soil, waterways, groundwater, and human tissue and body fluids. Elevated levels of tungsten in urine were reported for populations exposed to tungstate in drinking water in areas where natural tungsten formations were prevalent. Published reports indicated that sodium tungstate may modulate hematopoiesis, immune cell populations, and immune responses in rodent models. The objective of this study was to assess potential immunotoxicity of sodium tungstate dihydrate (STD), a drinking water contaminant. Female B6C3F1/N mice received 0-2000 mg STD/L in their drinking water for 28 d, and were evaluated for effects on immune cell populations in spleen and bone marrow, and humoral-mediated, cell-mediated, and innate immunity. Three different parameters of cell-mediated immunity were similarly affected at 1000 mg STD/L. T-cell proliferative responses against allogeneic leukocytes and anti-CD3 were decreased 32%, and 21%, respectively. Cytotoxic T-lymphocyte activity was decreased at all effector:target cell ratios examined. At 2000 mg STD/L, the absolute numbers of CD3(+) T-cell progenitor cells in bone marrow were increased 86%, but the alterations in B-lymphocyte and other progenitor cells were not significant. There were no effects on bone marrow DNA synthesis or colony forming capabilities. STD-induced effects on humoral-mediated immunity, innate immunity, and splenocyte sub-populations were limited. Enhanced histopathology did not detect treatment-related lesions in any of the immune tissues. These data suggest exposure to STD in drinking water may adversely affect cell-mediated immunity.

Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment.

This overview is an update of the unit originally published in 2004. While the basic tenets of immunotoxicity have not changed in the past 10 years, several publications have explored the application of immunotoxicological data to the risk assessment process. Therefore, the goal of this unit is still to highlight relationships between xenobiotic-induced immunosuppression and risk of clinical diseases progression. In immunotoxicology, this may require development of models to equate moderate changes in markers of immune functions to potential changes in incidence or severity of infectious diseases. For most xenobiotics, exposure levels and disease incidence data are rarely available, and safe exposure levels must be estimated based on observations from experimental models or human biomarker studies. Thus, it is important to establish a scientifically sound framework that allows accurate and quantitative interpretation of experimental or biomarker data in the risk assessment process.

Lung deposition and clearance of microparticle and nanoparticle C60 fullerene aggregates in B6C3F1 mice and Wistar Han rats following nose-only inhalation for 13 weeks.

C60 fullerenes (C60) are spherical structures consisting of 60 carbon atoms that are generated via combustion from both natural and anthropogenic sources. C60 are also synthesized intentionally for industrial applications. Individual C60 structures have an approximate diameter of 1nm; however, C60 readily forms aggregates and typically exist as larger particles that range from nanometers to micrometers in diameter. In this report, lung and extrapulmonary tissue deposition and lung clearance of C60 nanoparticles (nano-C60, 50nm) and microparticles (micro-C60, 1µm) were examined in Wistar Han rats and B6C3F1/N mice after nose-only inhalation for 90 days. Exposure concentrations were 0.5 and 2mg/m(3) (nano-C60) and 2, 15, and 30mg/m(3) (micro-C60). For both C60 particle sizes, the C60 lung burden increased proportionally to exposure concentration. The C60 lung burden was greater in both species at all time points following exposure to nano-C60 particle exposure compared to micro-C60 exposure at the common exposure concentration 2mg/m(3). The calculated C60 particle lung retention half-times were similar for both nano-C60 and micro-C60 exposure at 2mg/m(3) in male mice (15-16 days). In contrast, in male rats, the half-time of C60 particles following nano-C60 exposure (61 days) was roughly twice as long as the half-time following micro-C60 exposure (27 days) at the same exposure concentration (2mg/m(3)) and was similar to the clearance following micro-C60 exposure at higher exposure concentrations (15 and 30mg/m(3)). C60 was detected in bronchial lymph nodes but the burden was not quantified due to the high variability in the data. C60 concentrations were below the experimental limit of quantitation (ELOQ) in liver, spleen, blood, brain and kidney tissues. These tissue burden data provide information for comparison between nanometer and micrometer sized C60 particle exposure and will aid in the interpretation of toxicity data.

Genistein protects female nonobese diabetic mice from developing type 1 diabetes when fed a soy- and alfalfa-free diet.

The objective of this study was to determine the effects of the phytoestrogen genistein (GEN) on the time of onset and/or the incidence of type 1 diabetes (T1D) in female nonobese diabetic (NOD) mice, when administered GEN by gavage once every day for up to 180 days. Five groups of mice (approximately 24 animals/group; 6-7 weeks of age) were included: naive control, vehicle control (25 mM Na2CO3 in water), and 3 GEN treatment groups (2 mg/kg, 6 mg/kg, and 20 mg/kg). Mice were maintained on a soy- and alfalfa-free diet (5K96) during the study and were monitored for blood glucose changes every week. When compared to the vehicle control, exposure to 2-mg/kg GEN produced significant decreases ranging from 55 to 79% in the total incidences of diabetes (blood glucose ≥ 250 mg/dl) and severe diabetes (blood glucose ≥ 400 mg/dl) starting at week 14 of the study. However, during the later stages of the study (i.e., after week 23), the 2-mg/kg dose had no effect on disease incidence. In animals treated with 6-mg/kg and 20-mg/kg GEN, significant decreases in the total incidence of diabetes were observed starting at week 16, while the incidence of severe diabetes was significantly decreased with the changes being observed initially at weeks 18 and 17 for the 6-mg/kg and 20-mg/kg GEN treatment groups, respectively. Several lines of evidence, including histopathological analysis, suggested that GEN protected the pancreas from autoimmune destruction. However, this protective effect of GEN was absent when female NOD mice were maintained on NTP-2000 rodent diet, which contained 5% soybean meal and 7.5% alfalfa meal (the total concentrations of phytoestrogens ranged between 95 and 134 mg/kg). In summary, oral dosing of GEN reduced the incidence and increased the time to onset of T1D in female NOD mice but only when fed a soy- and alfalfa-free diet.

A murine inhalation model to characterize pulmonary exposure to dry Aspergillus fumigatus conidia.

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.

Genetic variants in the major histocompatibility complex class I and class II genes are associated with diisocyanate-induced Asthma.

OBJECTIVE: To investigate the association between single nucleotide polymorphisms (SNPs) located across the major histocompatibility complex and susceptibility to diisocyanate-induced asthma (DA). METHODS: The study population consisted of 140 diisocyanate-exposed workers. Genotyping was performed using the Illumina GoldenGate major histocompatibility complex panels. RESULTS: The HLA-E rs1573294 and HLA-DPB1 rs928976 SNPs were associated with an increased risk of DA under dominant (odds ratio [OR], 6.27; 95% confidence interval [CI], 2.37 to 16.6; OR, 2.79, 95% CI, 0.99 to 7.81, respectively) and recessive genetic models (OR, 6.27, 95% CI, 1.63 to 24.13; OR, 10.10, 95% CI, 3.16 to 32.33, respectively). The HLA-B rs1811197, HLA-DOA rs3128935, and HLA-DQA2 rs7773955 SNPs conferred an increased risk of DA in a dominant model (OR, 7.64, 95% CI, 2.25 to 26.00; OR, 19.69, 95% CI, 2.89 to 135.25; OR, 8.43, 95% CI, 3.03 to 23.48, respectively). CONCLUSION: These results suggest that genetic variations within HLA genes play a role in DA risk.