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).

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.

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.

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.

Immunotoxic and hepatotoxic effects of perfluoro-n-decanoic acid (PFDA) on female Harlan Sprague-Dawley rats and B6C3F1/N mice when administered by oral gavage for 28 days.

Poly- and perfluoroalkyl substances (PFAS) are chemically and thermally stable, hydrophobic, lipophobic compounds used in stain repellants and water and oil surfactants, and associated with immunosuppression and peroxisome proliferator activity. Perfluoro-n-decanoic acid (PFDA, (CF3(CF2)8COOH), a fluorinated straight chain fatty acid compound, is reported to induce thymic atrophy and reversible bone marrow hypocellularity in rodent models. The objective of this study was to assess potential immunotoxicity of PFDA, due to its structural similarity to other immunosuppressive PFASs. Female Harlan Sprague-Dawley rats were exposed to 0-2.0 mg PFDA/kg by oral gavage daily for 28 d. Female B6C3F1/N mice were exposed once/week to 0-5.0 mg PFDA/kg by gavage for 4 weeks. Animals were evaluated for effects on immune cell populations in spleen and bone marrow, and innate, humoral-, and cell-mediated immunity. Mice were also evaluated for resistance to Influenza virus. Treatment-related hepatocyte necrosis and hepatomegaly were observed in rats treated with 0.5 mg PFDA/kg/d. In mice, hepatomegaly (26-89%) was observed following exposure to ≥0.625 mg PFDA/kg/week, while splenic atrophy (20%) was observed at 5.0 mg PFDA/kg/week. At 5.0 mg PFDA/kg/week, total spleen cells, and Ig + and NK + cells were decreased (17.6-27%). At ≥ 1.25 mg PFDA/kg/week the numbers of splenic CD3+, CD4+, CD8+, and Mac3+ cells were decreased (10.5-39%). No changes were observed in leukocyte subpopulations in PFDA-exposed rats. Phagocytosis by fixed-tissue macrophages was decreased in liver (specific activity, 24-39%) at ≥0.25 mg PFDA/kg/d in rats. PFDA-induced effects on humoral- and cell-mediated immunity, host resistance, and bone marrow progenitor cells were limited. These data suggest that exposure to PFDA may induce adverse effects in rat liver in a manner consistent with the PFAS class, and may also alter the balance of immune cell populations in lymphoid tissues in mice.

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.

Immunomodulatory effects of black cohosh (Actaea racemosa) extract in female B6C3F1/N mice.

Black cohosh extracts (BCE; Actaea racemosa) are being used worldwide as an alternative to hormone replacement therapy for the management of menstrual and menopausal symptoms, yet the effects of BCE on the immune system are largely unknown. Female B6C3F1/N mice were treated daily with BCE (0, 62.5, 125, 250, 500, or 1000mg/kg) for 28 days by oral gavage. Liver weights were significantly increased (26-32%) at the 1000mg/kg dose. Dose-related increases in mean corpuscular volume and mean corpuscular hemoglobin were observed. Decreasing trends were observed in all thymic T cell populations, with the most notable dose-responsive effects on immature thymocytes. In the spleen, dose-related decreases were observed in all cell phenotypes evaluated, reaching the level of statistical significance at the 1000mg/kg BCE dose. Splenic natural killer (NK) cell numbers were significantly decreased at all BCE doses, with the exception of absolute NK numbers at the 125mg/kg dose. No effects were observed on T-dependent antibody responses of the humoral immune system, including the antibody-forming cell response to sheep erythrocytes (sRBC) and IgM antibody levels to both sRBC and keyhole limpet hemocyanin. Cytotoxic T cell (TCTL) activity was increased, as was the mixed leukocyte response in one of two studies. Anti-CD3 mediated proliferation and the delayed-type hypersensitivity response were unaffected. No effects were observed on innate immunity or on bone marrow cellularity and colony-forming units. Overall, BCE exposure in B6C3F1/N mice for 28 days at doses up to 1000mg/kg had minimal immune effects, with the exception of an increased TCTL response.

Markers of inflammation.

Inflammation is a complex and necessary component of an organism's response to biological, chemical or physical stimuli. In the acute phase, cells of the immune system migrate to the site of injury in a carefully orchestrated sequence of events that is mediated by cytokines and acute phase proteins. Depending upon the degree of injury, this acute phase may be sufficient to resolve the damage and initiate healing. Persistent inflammation as a result of prolonged exposure to stimulus or an inappropriate reaction to self molecules can lead to the chronic phase, in which tissue damage and fibrosis can occur. Chronic inflammation is reported to contribute to numerous diseases including allergy, 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 location of inflammation in the absence of specific data on the immune tissues. These data may suggest more specific immune function assays are necessary to determine the existence 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. The specific profile in a given inflammatory condition is dependent upon species, mechanisms, severity, chronicity, and capacity of the immune system to respond and adapt.