Evaluation of immunotoxicity induced by pirimiphos-methyl in male Balb/c mice following exposure to for 28 days.

Pirimiphos-methyl (O-2-diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate: POM) is widely used organophosphorous (OP) insecticide as a grain protectant to control insects during storage. This study was carried out to assess the immunologic effects of POM in Balb/c mice after 28-day oral exposure. Three dose levels of POM (10, 60, or 120 mg/kg/day) were administered orally to mice for 4 weeks. At autopsy after 28-day exposure, there were significant decreases in relative spleen weight and splenic cellularity found at 120 mg POM, but body weight, relative thymic weight, thymic cellularity, and splenic and thymic subsets were not affected. T cell proliferation response induced by Con A was significantly decreased at all dosages though no statistical differences were observed in splenic B cell proliferation. Significant increases in the production of cytokines (IL-2, IL-4, IL-6, IFN-gamma, and IL-10) were evident on the whole, but the increase in production of inflammatory cytokines overwhelmed that of the T(H)1 cell suppressive cytokine (IL-10). The relative levels of three types of autoantibodies, anti-dsDNA, anti-histone, and antinuclear antibody (ANA) were dose-dependently decreased in serum. Oral exposure to POM induced a significant decrease in Immunoglobulin M production capability in Balb/c mice. This decrease in antibody production capability may result from disturbances in cytokine balance produced by splenic immune cells. These results show that POM may induce allergic responses by relatively enhancing T(H)2 development and additionally contribute to chronic inflammation by attracting macrophage by IFN-gamma.

Modulation of intracellular cytokines in draining lymph node cells following allergen and irritant.

The murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. However, there is a need to develop a non-radioisotopic endpoint for the LLNA because of the radioisotopic method's requiring the use of special facilities. In this study, we investigated to evaluate the populations of intracellular cytokine producing cells and to analyze the expression of mRNA levels in the lymph node (LN) cells following allergen and irritant. Female Balb/c mice were treated by the topical application on the dorsum of both ears with strong sensitizers, 2,4-dinitrochlorobenzene (DNCB) and toluene diisocyanate (TDI) and a strong irritant, sodium lauryl sulfate (SLS), once daily for 3 consecutive days. The lymph node cells were harvested 72h after the final treatment. The analysis of intracellular cytokine cell in LN cells was performed with a flow cytometry. Mice were treated with DNCB and TDI showed a preferential increase in the percentage of CD4+IL-2+ cells compared with vehicle and irritant-treated mice. There was an increase in CD4+IFN-g+ cells of mice treated with DNCB and TDI, but no significant increases were observed in mice treated with SLS. Mice were treated with DNCB and TDI showed an increase in the percentage of CD4+IL-4+ cells compared with vehicle and irritant-treated mice. There was an increase in the mRNA level for interleukin 4 (IL-4) in mice treated with DNCB and TDI, but no significant increases were observed in mice treated with SLS. These results suggest that the population of interferon-gamma (IFN-g+) and IL-4+ cells on CD4+ cells and the mRNA expression for IL-4 in lymphocytes could be selectively modulated in allergen-treated mice.

Evaluation of the potential immunotoxicity of 3-monochloro-1,2-propanediol in Balb/c mice. I. Effect on antibody forming cell, mitogen-stimulated lymphocyte proliferation, splenic subset, and natural killer cell activity.

3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. MCPD has been reported genotoxic in vitro, and reproductive toxicity and carcinogenicity in rats. However, no previous studies have investigated MCPD-induced alterations in the immune system. In the present study, MCPD was administered by gavage for 14 days at 0, 25, 50, and 100 mg/kg per day to female Balb/c mice. The antibody-mediated immune response to sheep red blood cells (SRBC) was assessed using the antibody-forming cell (AFC) assay, and splenic cell phenotypes were quantified by flow cytometry. Hematological and histopathological changes were assessed. Mitogen-stimulated spleen lymphocyte proliferation and natural killer (NK) cell activity were evaluated. The T-lymphocyte blastogenesis by concanavalin A (Con A) or anti-CD3 and B-lymphocyte blastogenesis by lipopolysaccharide (LPS) were not significantly changed. There were no significant changes in the hematological and histopathological findings of MCPD-treated mice. However, the significant decrease in thymus weight was observed in 100 mg dose group, even though that did not change body weight gain. The cellularities of spleen and thymus were significantly reduced in high-dose group. Exposure to high dose of MCPD decreased the AFC response to SRBC in mice. There was a significant decrease in NK cell activity of mice treated with high dose of MCPD. These results indicate that MCPD could modulate the immune function in Balb/c mice.

Immunomodulatory effect of caffeic acid phenethyl ester in Balb/c mice.

Caffeic acid phenethyl ester (CAPE), an the active component of propolis, is known to have anticarcinogenic, antiviral and various biological activities; however, the effect of CAPE on the immunomodulatory activity in vivo remains unknown. We have investigated the effect of CAPE on the immune system in female Balb/c mice. CAPE (0, 5, 10, 20 mg/kg) was given to mice orally for 14 days. Immunomodulatory activity was evaluated by assessment of body and organ weight, lymphocyte blastogenesis, plaque-forming cell (PFC) assay, lymphocyte subpopulation by flow cytometry and cytokine production. Even though the change of body weight was not observed in CAPE-administered group, thymus weight and/or cellularity of thymus and spleen are decreased at the all dose groups of CAPE (5, 10, 20 mg/kg). On the other hand, CAPE had no effect on B lymphocyte proliferation induced by lipopolysaccharide (LPS) but increased T lymphocyte blastogenesis induced by concanavalin A (Con A) at the dose of 20 mg/kg. In the case of lymphocyte subpopulation, the population of T and B cells was not changed but CD4(+) T cell subsets are significantly increased in exposure to CAPE. The antibody responses to T lymphocyte dependent antigen, sheep red blood cell and keyhole limpet hemocyanin (KLH) were increased more than 10 mg/kg in CAPE-treated group. Likewise, the cytokine, IL-2, IL-4 and IFN-gamma were significantly increased at the dose of 20 mg/kg CAPE group. These results suggest that CAPE could have immunomodulatory effects in vivo.

Evaluation of cell proliferation in ear and lymph node using BrdU immunohistochemistry for mouse ear swelling test.

The mouse ear swelling test (MEST) was developed as an alternative to guinea pig models for measuring the contact sensitization potential. However, the MEST relies on the quantitative measurement of ear swelling by micrometer as the means of determining the endpoint. The purpose of this study was to investigate the possibility of using cell proliferation in the ear and lymph node by bromodeoxyuridine (BrdU) immunohistochemistry as a reliable marker for MEST. Female Balb/c mice were treated by the topical application of various sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI) and α-hexylcinnamaldehyde (HCA) and an irritant, sodium lauryl sulfate (SLS) following the protocol of MEST. The proliferation of cells in the ear and auricular lymph node was analyzed by BrdU incorporations into cells. There were significant increases in the cell proliferations of the ear and auricular lymph node in mice treated with DNCB and TDI compared to the vehicle control. All allergens and the irritant were correctly identified by the MEST using BrdU immunohistochemistry of lymph node responses. The standard MEST assay showed positive results in the case of the strong sensitizers, DNCB and TDI. However, HCA and SLS were not correctly identified in the ear swelling assay. These results suggest that the measurement of cell proliferation in the auricular lymph node using BrdU immunohistochemistry could provide a reliable marker for MEST.