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.

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.

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.

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.

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.

Metabolism and disposition of [(14)C]dimethylamine borane in male Harlan Sprague Dawley rats following gavage administration, intravenous administration and dermal application.

1. Dimethylamine borane (DMAB) is used as a reducing agent in the manufacturing of a variety of products and in chemical synthesis. National Toxicology Program is evaluating the toxicity of DMAB in rodents following dermal application. The objective of this study was to evaluate the metabolism and disposition of DMAB in male Harlan Sprague Dawley (HSD) rats. 2. Disposition of radioactivity was similar between gavage and intravenous administration of 1.5 mg/kg [(14)C] DMAB, with nearly 84%-89% of the administered radioactivity recovered in urine 24 h post dosing. At 72 h, only 1% or less was recovered in feces, 0.3% as CO2, and 0.5%-1.4% as volatiles and 0.3%-0.4 % in tissues. 3. The absorption of [(14)C]DMAB following dermal application was moderate; percent dose absorbed increased with the dose, with 23%, 32% and 46% of dose absorbed at 0.15, 1.5 and 15 mg/kg, respectively. Urinary and fecal excretion ranged from 18%-37% and 2%-4% of dose, respectively, and 0.1%-0.2% as CO2, and 1%-3% as volatiles. Tissue retention of the radiolabel was low ∼1%, but was higher than following the gavage or intravenous administration. 4. Following co-adminsitration of DMAB and sodium nitrite by gavage, N-nitrosodimethylamine was not detected in blood or urine above the limit of quantitation of the analytical method of 10 ng/mL. 5. Absorption of DMAB in fresh human skin in vitro was ∼41% of the applied dose: the analysis of the receptor fluid shows that the intact DMAB complex can be absorbed through the skin.

Prevalence and sociodemographic correlates of antinuclear antibodies in the United States.

OBJECTIVE: To estimate the prevalence, types, and sociodemographic and biobehavioral correlates of antinuclear antibodies (ANAs) in the US. METHODS: We conducted a cross-sectional analysis of 4,754 individuals from the National Health and Nutrition Examination Survey 1999-2004. ANAs were assessed by indirect immunofluorescence. In ANA-positive individuals, cellular staining patterns were determined, and specific autoantibody reactivities were assessed by immunoprecipitation. RESULTS: The ANA prevalence in the US population of individuals ages 12 years and older was 13.8% (95% confidence interval [95% CI] 12.2-15.5%). ANA prevalence increased with age (P=0.01), and ANAs were more prevalent among females than males (17.8% versus 9.6%; P<0.001), with the female-to-male ratio peaking at 40-49 years of age. ANA prevalence was modestly higher in African Americans compared with whites (age-adjusted prevalence odds ratio [POR] 1.30, 95% CI 1.00-1.70). Remarkably, ANAs were less common in overweight and obese individuals (age-adjusted POR 0.74) than in persons of normal weight. No significant associations of ANA with education, family income, alcohol use, smoking history, serum levels of cotinine, or C-reactive protein were observed. In ANA-positive individuals, nuclear patterns were seen in 84.6%, cytoplasmic patterns were seen in 21.8%, and nucleolar patterns were seen in 6.1%; the most common specific autoantibodies were anti-Ro (3.9%) and anti-Su (2.4%). CONCLUSION: These findings suggest that more than 32 million persons in the US have ANAs, and that the prevalence is higher among females, older individuals, African Americans, and those with a normal body weight. These data will serve as a useful baseline for future investigations of predictors and changes in ANA prevalence over time.

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).

Severely blunted allergen-induced pulmonary Th2 cell response and lung hyperresponsiveness in type 1 transient receptor potential channel-deficient mice.

Transient receptor potential channels (TRPCs) are widely expressed and regulate Ca²âº entry in the cells that participate in the pathophysiology of airway hyperreactivity, inflammation, and remodeling. In vitro studies point to a role for TRPC1-mediated Ca²âº signaling in several of these cell types; however, physiological evidence is lacking. Here we identify TRPC1 signaling as proinflammatory and a regulator of lung hyperresponsiveness during allergen-induced pulmonary response. TRPC1-deficient (Trpc1(-/-)) mice are hyposensitive to methacholine challenge and have significantly reduced allergen-induced pulmonary leukocyte infiltration coupled with an attenuated T helper type 2 (Th2) cell response. Upon in vitro allergen exposure, Trpc1(-/-) splenocytes show impaired proliferation and T cell receptor-induced IL-2 production. A high number of germinal centers in spleens of Trpc1(-/-) mice and elevated levels of immunoglobulins in their serum are indicative of dysregulated B cell function and homeostasis. Thus we propose that TRPC1 signaling is necessary in lymphocyte biology and in regulation of allergen-induced lung hyperresponsiveness, making TRPC1 a potential target for treatment of immune diseases and asthma.

Criteria for environmentally associated autoimmune diseases.

Increasing evidence supports a role for the environment in the development of autoimmune diseases, as reviewed in the accompanying three papers from the National Institute of Environmental Health Sciences Expert Panel Workshop. An important unresolved issue, however, is the development of criteria for identifying autoimmune disease phenotypes for which the environment plays a causative role, herein referred to as environmentally associated autoimmune diseases. There are several different areas in which such criteria need to be developed, including: 1) identifying the necessary and sufficient data to define environmental risk factors for autoimmune diseases meeting current classification criteria; 2) establishing the existence of and criteria for new environmentally associated autoimmune disorders that do not meet current disease classification criteria; and 3) identifying in clinical practice specific environmental agents that induce autoimmune disease in individual patients. Here we discuss approaches that could be useful for developing criteria in these three areas, as well as factors that should be considered in evaluating the evidence for criteria that can distinguish individuals with such disorders from individuals without such disorders with high sensitivity and specificity. Current studies suggest that multiple lines of complementary evidence will be important and that in many cases there will be clinical, serologic, genetic, epigenetic, and/or other laboratory features that could be incorporated as criteria for environmentally associated autoimmune diseases to improve diagnosis and treatment and possibly allow for preventative strategies in the future.

In vitro characterization of the immunotoxic potential of several perfluorinated compounds (PFCs).

We have previously shown that PFOA and PFOS directly suppress cytokine secretion in immune cells, with different mechanisms of action. In particular, we have demonstrated a role for PPAR-α in PFOA-induced immunotoxicity, and that PFOS has an inhibitory effect on LPS-induced I-κB degradation. These studies investigate the immunomodulatory effects of four other PFCs, namely PFBS, PFOSA, PFDA, and fluorotelomer using in vitro assays. The release of the pro-inflammatory cytokines IL-6 and TNF-α was evaluated in lipolysaccharide (LPS)-stimulated human peripheral blood leukocytes (hPBL) and in the human promyelocytic cell line THP-1, while the release of IL-10 and IFN-γ was evaluated in phytohemagglutinin (PHA)-stimulated hPBL. All PFCs suppressed LPS-induced TNF-α production in hPBL and THP-1 cells, while IL-6 production was suppressed by PFOSA, PFOS, PFDA and fluorotelomer. PFBS, PFOSA, PFOS, PFDA and fluorotelomer inhibited PHA-induced IL-10 release, while IFN-γ secretion was affected by PFOSA, PFOS, PFDA and fluorotelomer. Leukocytes obtained from female donors appear to be more sensitive to the in vitro immunotoxic effects of PFCs when their responses are compared to the results obtained using leukocytes from male donors. Mechanistic investigations demonstrated that inhibition of TNF-α release in THP-1 cells occurred at the transcriptional level. All PFCs, including PFOA and PFOS, decreased LPS-induced NF-κB activation. With the exception of PFOA, none of the PFCs tested was able to activate PPARα driven transcription in transiently transfected THP-1 cells, excluding a role for PPARα in the immunomodulation observed. PFBS and PFDA prevented LPS-induced I-κB degradation. Overall, these studies suggest that PFCs affect NF-κB activation, which directly suppresses cytokine secretion by immune cells. Our results indicate that PFOA is the least active of the PFCs examined followed by PFBS, PFDA, PFOS, PFOSA and fluorotelomer.

Immunotoxicity of perfluorinated compounds: recent developments.

Perfluorinated compounds (PFCs) are environmentally widespread, persistent, and bioaccumulative chemicals with multiple toxicities reported in experimental models and wildlife, including immunomodulation. The two most commonly detected compounds, which also generally occur in the highest concentrations in environmentally exposed organisms, are perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). PFOA and PFOS have been reported to alter inflammatory responses, production of cytokines, and adaptive and innate immune responses in rodent models, avian models, reptilian models, and mammalian and nonmammalian wildlife. Mounting evidence suggests that immune effects in laboratory animal models occur at serum concentrations below, within the reported range, or just above those reported for highly exposed humans and wildlife. Thus, the risk of immune effects for humans and wildlife exposed to PFCs cannot be discounted, especially when bioaccumulation and exposure to multiple PFCs are considered. This review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA, PFOS, and other PFCs in rodent models, alternative laboratory models, and wildlife.

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.

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.

In vitro evaluation of the immunotoxic potential of perfluorinated compounds (PFCs).

There is evidence from both epidemiology and laboratory studies that perfluorinated compounds may be immunotoxic, affecting both cell-mediated and humoral immunity. The overall goal of this study was to investigate the mechanisms underlying the immunotoxic effects of perfluorooctane sulfonate (PFOS) and perfluorooctane acid (PFOA), using in vitro assays. The release of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was evaluated in lipolysaccharide (LPS)-stimulated human peripheral blood leukocytes and in the human promyelocytic cell line THP-1, while the release of IL-4, IL-10 and IFN-γ was evaluated in phytohaemagglutinin (PHA)-stimulated peripheral blood leukocytes. PFOA and PFOS suppressed LPS-induced TNF-α production in primary human cultures and THP-1 cells, while IL-8 was suppressed only in THP-1 cells. IL-6 release was decreased only by PFOS. Both PFOA and PFOS decreased T-cell derived, PHA-induced IL-4 and IL-10 release, while IFN-γ release was affected only by PFOS. In all instances, PFOS was more potent than PFOA. Mechanistic investigations carried out in THP-1 cells demonstrated that the effect on cytokine release was pre-transcriptional, as assessed by a reduction in LPS-induced TNF-α mRNA expression. Using siRNA, a role for PPAR-α could be demonstrated for PFOA-induced immunotoxicity, while an inhibitory effect on LPS-induced I-κB degradation could explain the immunomodulatory effect of PFOS. The dissimilar role of PPAR-α in PFOA and PFOS-induced immunotoxicity was consistent with the differing effects observed on LPS-induced MMP-9 release: PFOA, as the PPAR-α agonist fenofibrate, modulated the release, while PFOS had no effect. Overall, these studies suggest that PFCs directly suppress cytokine secretion by immune cells, and that PFOA and PFOS have different mechanisms of action.

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.

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.

Immunotoxicological profile of chloroform in female B6C3F1 mice when administered in drinking water.

Chloroform can be formed as a disinfection by-product during water chlorination, one of the primary modalities for purifying municipal water supplies for human consumption. The aim of this study was to characterize the immunotoxic effects of chloroform in female B6C3F1 mice when exposure occurred via the drinking water. Consistent with human exposure, female B6C3F1 mice were exposed to chloroform-containing drinking water at 2.5, 10, 25, 100, and 250 ppm for 28 days. The examined endpoints included the effects of chloroform on body and organ weights, water consumption, hematology, innate immunity, humoral immunity, and cell-mediated immunity. The functions of natural killer, B-, and T-cells were not altered by chloroform in drinking water at the concentrations tested, except that an increase in splenocyte basal proliferation was observed at chloroform levels of 100 and 250 ppm. Following chloroform administration, there was a decreased number of circulating neutrophils in the blood in all treatment groups, but neutrophil function in lung homogenates, as evaluated using an assay for myeloperoxidase activity following lipopolysaccharide and N-Formyl-Met-Leu-Phe stimulation, was not compromised. Further, the results of host resistance to Listeria monocytogenes infection also suggested that neutrophil function was normal. At the highest treatment level of chloroform (250 ppm), erythrocyte number and hemoglobin levels were significantly decreased. Some significant changes were also observed for body weights, water consumption, and organ weights; however, most of these effects were only observed at the highest treatment level of chloroform (250 ppm). Taken together, the results demonstrate that while chloroform administered via the drinking water affects body weight and selected hematological parameters at high dose levels, overall immune responses, as measured in several tests for immune function, are not compromised.

Meeting report: moving upstream-evaluating adverse upstream end points for improved risk assessment and decision-making.

BACKGROUND: Assessing adverse effects from environmental chemical exposure is integral to public health policies. Toxicology assays identifying early biological changes from chemical exposure are increasing our ability to evaluate links between early biological disturbances and subsequent overt downstream effects. A workshop was held to consider how the resulting data inform consideration of an "adverse effect" in the context of hazard identification and risk assessment. OBJECTIVES: Our objective here is to review what is known about the relationships between chemical exposure, early biological effects (upstream events), and later overt effects (downstream events) through three case studies (thyroid hormone disruption, antiandrogen effects, immune system disruption) and to consider how to evaluate hazard and risk when early biological effect data are available. DISCUSSION: Each case study presents data on the toxicity pathways linking early biological perturbations with downstream overt effects. Case studies also emphasize several factors that can influence risk of overt disease as a result from early biological perturbations, including background chemical exposures, underlying individual biological processes, and disease susceptibility. Certain effects resulting from exposure during periods of sensitivity may be irreversible. A chemical can act through multiple modes of action, resulting in similar or different overt effects. CONCLUSIONS: For certain classes of early perturbations, sufficient information on the disease process is known, so hazard and quantitative risk assessment can proceed using information on upstream biological perturbations. Upstream data will support improved approaches for considering developmental stage, background exposures, disease status, and other factors important to assessing hazard and risk for the whole population.