Contact sensitizing potential of annatto extract and its two primary color components, cis-bixin and norbixin, in female BALB/c mice

The present studies were performed to examine the contact allergenic effects of an annatto extract (ANT) in female BALB/c mice. ANT at 5-10% induced a greater than threefold increase in lymph node cell proliferation when compared to the control in the LLNA. Moreover, a significant increase in the percent ear swelling at 24 h after ANT challenge was observed in the MEST. A significant increase in the percentage of B cells was also observed. To determine which of the two predominant coloring components (norbixin and bixin) in ANT was responsible for the sensitizing effects of ANT, norbixin was subsequently examined, with negative results being observed in both the LLNA and MEST following treatment with norbixin (1-20%). These findings suggested that perhaps bixin was responsible for the positive responses in both the LLNA and MEST following exposure to ANT. Therefore, further studies using a partially purified cis-bixin extract were conducted. Positive responses in both the LLNA and MEST were observed in mice treated with cis-bixin at the concentrations as low as 0.1-0.5%. These results have demonstrated that cis-bixin, but not norbixin, is likely a contact sensitizer and contributes to the contact hypersensitivity effects observed following dermal exposure to ANT in mice.     

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.     

Immunotoxicological profile of chloramine in female B6C3F1 mice when administered in the drinking water for 28 days

Monochloramine has been used to provide a disinfecting residual in water distribution systems where it is difficult to maintain an adequate free-chlorine residual or where disinfection by-product formation is of concern. The goal of this study was to characterize the immunotoxic effects of chloramine in female B(6)C(3)F(1) mice when administered via the drinking water. Mice were exposed to chloramine-containing deionized tap water at 2, 10, 20, 100, or 200 ppm for 28 days. No statistically significant differences in drinking water consumption, body weight, body weight gain, organ weights, or hematological parameters between the exposed and control animals were noted during the experimental period. There were no changes in the percentages and numbers of total B-lymphocytes, T-lymphocytes, CD4(+) and CD8(+) T-lymphocytes, natural killer (NK) cells, and macrophages in the spleen. Exposure to chloramine did not affect the IgM antibody-forming cell response to sheep red blood cells (SRBC) or anti-SRBC IgM antibody production. Minimal effects, judged to be biologically insignificant, were observed in the mixed-leukocyte response and NK activity. In conclusion, chloramine produced no toxicological and immunotoxic effects in female B(6)C(3)F(1) mice when administered for 28 days in the drinking water at concentrations ranging from 2-200 ppm.     

Differential surface expression of CD18 and CD44 by neutrophils in bone marrow and spleen contributed to the neutrophilia in thalidomide-treated female B6C3F1 mice

Previously, we have reported that thalidomide (Thd) can enhance neutrophil function in female B6C3F1 mice. The present study was intended to evaluate the mechanisms underlying the enhanced neutrophil responses following Thd treatment intraperitoneally (100 mg/kg) for 14 or 28 days. Treatment with Thd increased the numbers of neutrophils in the spleen, peripheral blood, bone marrow, peritoneal cavity and lungs of female B6C3F1 mice when compared to the vehicle control mice. Thd treatment for 14 days increased the percentage and the number of neutrophils in the spleen in the first 8 h (peaking at 2 h) after the last Thd treatment, and it returned to the baseline after 24 h. However, Thd treatment for 28 days increased the percentage and number of neutrophils in the spleen even at the 24-h time point after the last Thd treatment. These neutrophils were demonstrated to be functional by the myeloperoxidase activity assay. Further studies have ruled out the possibility of an increased bone marrow granulopoiesis following Thd treatment. Flow cytometric analysis of the surface expression of adhesion molecules suggested that Thd treatment for either 14 or 28 days decreased the surface expression of either CD18 or CD44 by bone marrow neutrophils. On the other hand, the surface expression of both CD18 and CD44 by splenic neutrophils was increased following Thd treatment for 28 days but not for 14 days. No effect was produced for other cell surface molecules such as CD62L and CD11a. It was possible that decreased surface expressions of CD18 and CD44 facilitated neutrophils' release from the bone marrow; increased surface expressions of CD44 and CD18 by splenic neutrophils after 28 days of Thd treatment increased their ability to remain in the periphery. Taken together, Thd treatment increased neutrophils in female B6C3F1 mice, at least partially, through differentially modulating the surface expression of CD18 and CD44 by the neutrophils in the bone marrow 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.     

Immunomodulation by Dok Din Daeng (Aeginetia indica Roxb.) extracts in female B6C3F1 mice: (I): stimulation of T cells

Aeginetia indica Roxbert (Dok Din Daeng, DDD), a parasitic plant that grows on bamboo, is extensively used in Thai traditional medicine to treat various diseases. There have been no published studies on the pharmacological, toxicological or immunological effects of DDD, indigenous to Thailand. The study reported here was focused on the immunological effects (T cells) of the whole plant extract using water (WDDD) or ethanol (EDDD) as a solvent. The extracts were administered to female B6C3F1 mice by gavage for WDDD (10-100%) and intraperitoneally (i.p.) for EDDD (0.25-250 mg/kg) for 28 days. Only mice administrated the highest dose of EDDD exhibited an increase in absolute spleen and liver weights. Three T cell functional assays, including anti-CD3 antibody-mediated T cell proliferation, the mixed leukocyte response (MLR) and the cytotoxic T lymphocyte (CTL) response, were employed to determine the effects of DDD extracts on splenic T cell activities. Exposure to WDDD enhanced the responses in all three assays with significant changes observed in the anti-CD3 and MLR assays. Exposure to EDDD also enhanced the responses in all three assays with significant changes observed in the MLR and CTL assays. Additionally, significant increases in the MLR and anti-CD3 responses were also observed when EDDD was used to treat cells in vitro. Finally, exposure to WDDD decreased both the percentage and absolute number of regulatory CD4(+)CD25(+) T cells in the spleen, which was consistent with a significant increase in interferon-gamma (IFN-gamma) production from Con A-stimulated splenocytes. Overall, this study demonstrated that the extracts from A. indica Roxbert had a T cell stimulatory activity.     

Immunotoxicity of dibromoacetic acid administered via drinking water to female B₆C₃F₁ mice

Dibromoacetic acid (DBA) is a disinfection by-product commonly found in drinking water as a result of chlorination/?ozonation processes. The Environmental Protection Agency estimates that more than 200 million people consume disinfected water in the United States. This study was conducted to evaluate the potential immunotoxicological effects of DBA exposure when administered for 28 days via drinking water to B?C?F? mice, at concentrations of 125, 500, and 1000?mg/L. Multiple endpoints were evaluated to assess innate, humoral, and cell-mediated immune components, as well as host resistance. Standard toxicological parameters were unaffected, with the exception of a dose-responsive increase in liver weight and a decrease in thymus weight at the two highest exposure levels. Splenocyte differentials were affected, although the effects were not dose-responsive. Exposure to DBA did not significantly affect humoral immunity (immunoglobulin M [IgM] plaque assay and serum IgM anti-sheep erythrocyte titers) or cell-mediated immunity (mixed-leukocyte response). No effects were observed on innate immune function in either interferon-γ-induced in vitro macrophage cytotoxic activity or basal natural killer (NK)-cell activity. Augmented NK-cell activity (following exposure to polyinosinic-polycytidylic acid) was decreased at the low dose, however the effect was not dose-responsive. Finally, DBA exposure had no effect on resistance to infection with either Streptococcus pneumoniae or Plasmodium yoelii, or challenge with B16F10 melanoma cells. With the exception of changes in thymus weight, these results indicate that DBA exposure resulted in no immunotoxic effects at concentrations much larger than those considered acceptable in human drinking water.     

Immunomodulation by Dok Din Daeng (Aeginetia indica Roxb.) extracts in female B6C3F1 mice: II. Humoral immunity, innate immunity and hematology

In the previous report, we have provided evidence that Aeginetia indica Roxbert (DDD) extracts enhance T cell-mediated immune responses. The study reported here was focused on the hematological and immunological effects, including B cells, natural killer (NK) cells, macrophages and neutrophils, of the whole plant extract using water (WDDD) or ethanol (EDDD) as the solvent. The extracts were administered to female B6C3F1 mice by gavage for WDDD (10-100%) and intraperitoneally for EDDD (0.25-250 mg/kg) for 28 days. In addition to hematological evaluation, several quantitative measures and functional assays (e.g., the splenic phenotypic analysis, IgM antibody-forming cell responses, natural killer cell activity, mononuclear phagocyte system [MPS] and neutrophil activity) were employed to examine the effects of DDD extracts on the innate and humoral immunities. The results from this study demonstrated that exposure to WDDD and EDDD produced minimal changes in the activities of B cells and natural killer cells, macrophages and neutrophils. Overall, hematological parameters were not affected by exposure to WDDD or EDDD. Taken together, the enhancing effect of DDD extracts on T cells may be primarily responsible for the successful and long-time use of this traditional herbal medicine in Thailand.     

Validation of the Candida albicans delayed-type hypersensitivity (DTH) model in the female B₆C₃F₁ mouse for use in immunotoxicological investigations

Although numerous models are used to evaluate the immunotoxic effects of xenobiotics on cell-mediated immunity (CMI), no holistic model for evaluating such effects on the delayed-type hypersensitivity (DTH) response has gained widespread acceptance. Due to a lack of interference from antigen-specific antibody production, the Candida albicans DTH model has recently been demonstrated to be a more appropriate model for assessing effects on CMI than other DTH models that utilize different sensitizing antigens, such as sheep erythrocytes (SRBC) or keyhole limpet hemocyanin (KLH). The present studies were conducted to validate the C. albicans DTH model for its ability to detect suppression (or the lack thereof) of CMI following exposure for 28 days to well-characterized immunosuppressive drugs, each having a different mechanism of action. The compounds evaluated included azathioprine (AZA), cyclophosphamide (CPS), cyclosporin A (CSA), dexamethasone (DEX), and the non-immunotoxic compound, benzo[e]pyrene (B[e]P). Exposure to each of the four known immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans. Footpad swelling was decreased following exposure to AZA at ≥ 20?mg/kg but not at 10?mg/kg, CPS at ≥ 10?mg/kg but not at 5?mg/kg, CSA at ≥ 3?mg/kg but not at 1?mg/kg, or DEX at ≥ 0.3?mg/kg (intermittently at 0.1?mg/kg) but not at 0.03?mg/kg. As expected, exposure to B[e]P for 28 days at doses up to 40?mg/kg had no effect on the DTH response. These results demonstrated that the C. albicans DTH assay in the B?C?F? mouse was capable of appropriately classifying each test article as to its immunotoxic effects on CMI. Furthermore, comparisons of these results with previous reports of effects on ex vivo CMI end points suggest that this DTH assay may be more sensitive than standard ex vivo assays at detecting immunosuppressive effects.