Immunotoxicity of the organochlorine pesticide methoxychlor in female ICR, BALB/c, and C3H/He mice.

Several types of pesticides, including organochlorines, are known to suppress or modulate immune responses. The present study evaluated the immunotoxicity of the organochlorine pesticide methoxychlor (MXC) in female BALB/c, C3H/He, and ICR mice. Mice were given oral MXC doses of 0, 30, 100, and 300 mg/kg each day for 7 consecutive days. On day 4, the mice also received an intravenous injection of sheep red blood cells (SRBC). The splenic plaque-forming cell (PFC) IgM response and the serum anti-SRBC IgM antibody titer were evaluated while splenic lymphocytes were counted by flow cytometry and the spleen underwent histopathological analysis. Significant decreases in IgM PFC responses were seen in BALB/c, C3H/He, and ICR mice that received MXC doses of 100 and 300 mg/kg. Similar changes in serum anti-SRBC IgM antibody titers occurred in three strain mice. Flow cytometric analysis revealed significantly decreased splenic T-cell (CD3+) populations in a dose dependent manner in BALB/c mice, and in the 300 mg/kg of MXC-treated group of C3H/He mice. Germinal center (GC) B-cell (CD19+PNA+) populations were significantly decreased in the 300 mg/kg of MXC-treated groups of all three mouse strains and in the 30 and 100 mg/kg of MXC-treated groups of BALB/c and C3H/He strain mice. Histopathological analysis revealed decreased cellularity of the periarteriolar lymphoid sheath (PALS; T-cell area) and decreased GC development in all three strains of mice treated with 300 mg/kg MXC. These results suggest that MXC has an immune-suppressive effect in mice, and that our protocol may be useful for rapidly detecting immunosuppression induced by environmental chemicals.

Immunotoxicity in mice induced by short-term exposure to methoxychlor, parathion, or piperonyl butoxide.

Exposure to environmental agents can compromise numerous immunological functions. Immunotoxicology focuses on the evaluation of the potential adverse effects of xenobiotics on immune mechanisms that can lead to harmful changes in host responses such as: increased susceptibility to infectious diseases and tumorigenesis; the induction of hypersensitivity reactions; or an increased incidence of autoimmune disease. In order to assess the immunosuppressive response to short-term exposure to some commonly used pesticides, the studies here focused on the response of mice after exposures to the organochlorine pesticide methoxychlor, the organophosphorus pesticide parathion, or the agricultural insecticide synergist piperonyl butoxide. In these studies, 7-week-old mice were orally administered (by gavage) methoxychlor, parathion, or piperonyl butoxide daily for five consecutive days. On Day 2, all mice in each group were immunized with sheep red blood cells (SRBC), and their SRBC-specific IgM responses were subsequently assessed. In addition, levels of B-cells in the spleen of each mouse were also analyzed via surface antigen expression. The results of these studies indicated that treatments with these various pesticides induced marked decreases in the production of SRBC-specific IgM antibodies as well as in the expression of surface antigens in IgM- and germinal center-positive B-cells. Based on these outcomes, it is concluded that the short-term exposure protocol was able to detect potential immunosuppressive responses to methoxychlor, parathion, and piperonyl butoxide in situ, and, as a result, may be useful for detecting other environmental chemical-related immunotoxicities.

Prior exposure to immunosuppressive organophosphorus or organochlorine compounds aggravates the T(H)1- and T(H)2-type allergy caused by topical sensitization to 2,4-dinitrochlorobenzene and trimellitic anhydride.

Immunosuppressive environmental chemicals may increase the potency of allergens and thereby play a role in the development of allergic diseases. This study's primary objective was to examine the mechanisms behind the relationship between allergic diseases and the immunosuppression induced by some environmental chemicals. We focused on the modulation of allergic potential in vitro and in mice by the organophosphorus pesticide O,O-diethyl-O-4-nitrophenyl-thiophosphate (parathion) and the organochlorine pesticide 1,1,1-trichloro-2,2-bis(4-methoxy-phenyl)ethane (methoxychlor), with respect to the T(H)1-type allergen 2,4-dinitrochlorobenzene (DNCB) and the T(H)2-type allergen trimellitic anhydride (TMA). Mice (4-week-old) were orally administered parathion or methoxychlor. Four weeks after the final dosing, the mice were sensitized to DNCB or TMA, and T-lymphocyte proliferation measured in their (using a local lymph node assay [LLNA]). In addition, we analyzed T-lymphocytes via surface antigen expression and local cytokine production in auricular lymph nodes after treatment with 0.1% DNCB or 0.3% TMA. The estimated concentration of DNCB and TMA to yield a stimulation index (SI) of cell proliferation of three decreased markedly in parathion- and methoxychlor-pre-treated mice. Pesticide pre-treatment induced marked increases in the number of helper and cytotoxic T-cells, levels of T(H)1 and T(H)2 cytokines, and gene expression in lymph node cells. According to our results, T(H)1- and T(H)2-type allergies are aggravated by prior exposure to immunosuppressive environmental chemicals.

Prior exposure to organophosphorus and organochlorine pesticides increases the allergic potential of environmental chemical allergens in a local lymph node assay.

The dysregulation of immune functions by some pesticides leads to various immune disorders, including immunodeficiency, tumorigenesis, allergies, and autoimmunity. This study's primary objective was to examine the relationship between immune disorders and the immunosuppression induced by immunosuppressive pesticides. We focused on the modulation of allergic potential by the organophosphorus pesticide parathion, organochlorine pesticide methoxychlor, phenoxyacetic acid herbicide 2,4-d-butyl, and benzoic acid fungicide eugenol, as detected by a local lymph node assay (LLNA), which was developed initially for hazard identification of skin sensitization. Parathion and methoxychlor are immunosuppressive chemicals, and 2,4-d-butyl and eugenol are contact allergens. After the immunosuppressive characteristics of parathion and methoxychlor were confirmed in a pilot study, 4-week-old mice were orally administered parathion (0, 0.4, 1.2mg/kg) or methoxychlor (0, 100, 300 mg/kg). Four weeks after the last administration, an LLNA was conducted using 2,4-d-butyl (0%, 2.5%, 5%, and 10%) and eugenol (0%, 5%, 10%, and 25%). In addition, detailed analysis of their auricular lymph nodes for number of surface antigen expression of T cells and local cytokine production were performed using 5% 2,4-d-butyl and 5% eugenol treatment groups. EC3 values (estimated concentration to yield a stimulation index of 3) of 2,4-d-butyl and eugenol decreased markedly in parathion- and methoxychlor-pretreated groups. Parathion- and methoxychlor-pretreated groups induced marked increase in number of surface antigen expression of T cells and levels of Th1 cytokines (IFN-γ, TNF-α, and IL-17) produced by ex vivo restimulated lymph node cells. According to our results, the allergic potentials of 2,4-d-butyl and eugenol are increased by prior exposure to parathion and methoxychlor.

A method for measuring mouse respiratory allergic reaction to low-dose chemical exposure to allergens: an environmental chemical of uncertain allergenicity, a typical contact allergen and a non-sensitizing irritant.

Our aim was to improve a method for detecting respiratory hypersensitivity by testing three confirmed respiratory allergens (trimellitic anhydride [TMA], phthalic anhydride [PA] and toluene diisocyanate [TDI]), an environmental chemical of uncertain allergenicity (2,4-d-butyl [DB]), a confirmed contact allergen (2,4-dinitrochlorobenzene [DNCB]) and a standard irritant (sodium dodecyl sulfate [SDS]). BALB/c mice were topically sensitized (nine times in 3 weeks) with these chemicals, then challenged via the trachea. One day post-challenge, samples were taken from the mice to assay for total immunoglobulin (IgE and IgG(1)) levels in serum and bronchoalveolar lavage fluid (BALF); differential cell counts and cytokine/chemokine levels in BALF; lymphocyte counts and surface antigen expression on B-cells within lung-associated lymph nodes (LNs); ex situ cytokine production by cells from these LNs; and gene expression in BALF (CCR3) and LNs (CCR4, STAT6 and GATA-3). The three confirmed respiratory allergens and DB induced immune response characteristic of immediate-type respiratory reactions, as evidenced by increased total IgE and IgG(1) levels; influx of eosinophils, neutrophils, chemokines and cytokines in BALF; increased surface antigen expression on B-cells in LNs; increased Th2 cytokine production in LNs; and increased respiratory allergy-related gene expression in both BALF and LNs. In contrast, DNCB and SDS treatments yielded, at most, insignificant increases in all respiratory allergic parameters. Thus, the protocol was equally suitable for use with an environmental chemical of unknown allergenicity and for typical respiratory allergens. Since the protocol differentiated respiratory allergens from contact allergens and irritants, it may be useful for detecting respiratory allergy related to environmental chemicals.

Apoptosis in immunocytes induced by several types of pesticides.

Several types of pesticides, such as organophosphates and organochlorines, can induce thymocyte apoptosis, resulting in thymic atrophy and predisposing the highly sensitive fetal immune system to loss of tolerance to self-antigens and subsequent increased risk for autoimmune disease and allergies. In the studies here, mouse primary thymocytes and a human acute T-cell leukemia cell line (J45.01) were employed to examine potential thymocyte apoptosis induced by several types of chemicals, including several commonly-used pesticides. Thymocytes and J45.01 cells were treated for 4 or 8 hr with varying doses of metamidophos, parathion, PNMC, or methoxychlor; dexamethasone was used as a positive control. Apoptosis, cell viability, the proportion of Annexin-V+ cells, the activities of caspases 3/7, 8, and 9, and the levels of DNA fragmentation in both the J45.01 cells and thymocytes were then examined. The results here show that with both cell types, there was an increase in the proportion of annexin-V+ cells and levels of DNA fragmentation following exposure to parathion, PNMC, methoxychlor, or dexamethasone (positive control); however, the levels of sensitivity appeared to differ between the cell types. Furthermore, caspase-7 and -8 activities also differed between the J45.01 cells and thymocytes when treated with PNMC, methoxychlor, or dexamethasone. A more precise characterization of these inter-cellular differences is the logical next step in our studies of the effects of these (and other) pesticides on immune cell integrity. These specific types of follow-on mechanistic experiments are currently underway in our laboratories.

Investigation of the chemical-induced selective type II (T(H)2) allergic response in mice: effect of the length of the sensitizing phase.

Allergies are immune system disorders characterized by abnormal, acquired sensitivity to various environmental chemicals. We investigated the mechanism of chemical-induced selective type II (T(H)2) allergy by using three different sensitization protocols and the well-known respiratory sensitizer trimellitic anhydride (TMA). Mice were sensitized for either 1, 2, or 3 weeks. For each sensitization schedule, the mice were allocated into 3 or 4 groups: -/- group, both sensitized and challenged with vehicle; -/+ group, sensitized with vehicle and challenged with 0.1% TMA; +/- group, sensitized with 1% TMA and challenged with vehicle; and +/+ group, both sensitized and challenged with 0.1% TMA. After challenge, we assayed the auricular lymph nodes of all mice for number of lymphocytes, surface antigen expression of B-cells, and local cytokine production, and we measured TMA-specific serum IgE levels. Some parameters in mice sensitized for 1 or 2 wk showed, at most, mild changes. In contrast, all parameters in animals receiving 3-wk sensitization showed marked increases, as well as marked increases in the IgE/major histocompatibility complex (MHC) Class II-positive B-cell population and T(H)2 cell production of IL-10 and IL-13. These results indicate that 3 wk of sensitization according to our protocol led to overt respiratory allergic reactions. While these studies showed that using the approach here, positive reactions were elicited using a typical allergen; whether the same events occur after sensitization by other chemicals that are found in the environment remains uncertain. These findings here should be regarded moreover as preliminary in scope and that additional studies with irritants, dermal sensitizers and other respiratory sensitizers are needed to further evaluate the overall sensitivity and selectivity of this novel protocol.

Allergic reaction induced by dermal and/or respiratory exposure to low-dose phenoxyacetic acid, organophosphorus, and carbamate pesticides.

Several types of pesticides, such as organophosphates, phenoxyacetic acid, and carbamate have a high risk of affecting human health, causing allergic rhinitis and bronchial asthma-like diseases. We used our long-term sensitization method and a local lymph node assay to examine the allergic reactions caused by several types of pesticides. BALB/c mice were topically sensitized (9 times in 3 weeks), then challenged dermally or intratracheally with 2,4-D, BRP, or furathiocarb. One day post-challenge, the mice were processed to obtain biologic materials for use in assays of total IgE levels in serum and bronchoalveolar lavage fluid (BALF); differential cell counts and chemokine levels in BALF; lymphocyte counts and surface antigen expression on B-cells within regional lymph nodes (LNs); and, ex situ cytokine production by cells from these LNs. 2,4-D-induced immune responses characteristic of immediate-type respiratory reactions, as evidenced by increased total IgE levels in both serum and BALF; an influx of eosinophils, neutrophils, and chemokines (MCP-1, eotaxin, and MIP-1beta) in BALF; increased surface antigen expression on B-cells IgE and MHC class II production) in both auricular and the lung-associated LNs; and increased Th2 cytokine production (IL-4, IL-5, IL-10, and IL-13) in both auricular and the lung-associated LN cells. In contrast, BRP and furathiocarb treatment yielded, at most, non-significant increases in all respiratory allergic parameters. BRP and furathiocarb induced marked proliferation of MHC Class II-positive B-cells and Th1 cytokines (IL-2, TNF-alpha, and IFN-gamma) in only auricular LN cells. These results suggest that 2,4-D is a respiratory allergen and BRP and furathiocarb are contact allergens. As our protocol detected classified allergic responses to low-molecular-weight chemicals, it thus may be useful for detecting environmental chemical-related allergy.

Use of long term dermal sensitization followed by intratracheal challenge method to identify low-dose chemical-induced respiratory allergic responses in mice.

The inhalation of many types of chemicals, including pesticides, perfumes, and other low-molecular weight chemicals, is a leading cause of allergic respiratory diseases. We attempted to develop a new test protocol to detect environmental chemical-related respiratory hypersensitivity at low and weakly immunogenic doses. We used long-term dermal sensitization followed by a low-dose intratracheal challenge to evaluate sensitization by the well-known respiratory sensitizers trimellitic anhydride (TMA) and toluene diisocyanate (TDI) and the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them intratracheally with TMA, TDI, or DNCB, we assayed differential cell counts and chemokine levels in bronchoalveolar lavage fluid (BALF); lymphocyte counts, surface antigen expression of B cells, and local cytokine production in lung-associated lymph nodes (LNs); and antigen-specific IgE levels in serum and BALF. TMA induced marked increases in antigen-specific IgE levels in both serum and BALF, proliferation of eosinophils and chemokines (MCP-1, eotaxin, and MIP-1beta) in BALF, and proliferation of Th2 cytokines (interleukin (IL)-4, IL-10, and IL-13) in restimulated LN cells. TDI induced marked increases in levels of cytokines (IL-4, IL-10, IL-13, and IFN-gamma) produced by restimulated LN cells. In contrast, DNCB treatment yielded, at most, small, nonsignificant increases in all parameters. Our protocol thus detected respiratory allergic responses to low-molecular weight chemicals and may be useful for detecting environmental chemical-related respiratory allergy.

Detection of low-level environmental chemical allergy by a long-term sensitization method.

Multiple chemical sensitivity (MCS) is characterized by various signs, including neurological disorders and allergy. Exposure may occur through a major event, such as a chemical spill, or from long-term contact with chemicals at low levels. We are interested in the allergenicity of MCS and the detection of low-level chemical-related hypersensitivity. We used long-term sensitization followed by low-dose challenge to evaluate sensitization by well-known Th2 type sensitizers (trimellitic anhydride (TMA) and toluene diisocyanate (TDI)) and a Th1 type sensitizer (2,4-dinitrochlorobenzene (DNCB)). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them with TMA, TDI or DNCB, we assayed their auricular lymph nodes (LNs) for number of lymphocytes, surface antigen expression of B cells, and local cytokine production, and measured antigen-specific serum IgE levels. TMA and TDI induced marked increases in levels of antigen-specific serum IgE and of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) produced by ex vivo restimulated lymph node cells. DNCB induced a marked increase in Th1 cytokine (IL-2, IFN-gamma, and TNF-alpha) levels, but antigen-specific serum IgE levels were not elevated. All chemicals induced significant increases in number of lymphocytes and surface antigen expression of B cells. Our mouse model enabled the identification and characterization of chemical-related allergic reactions at low levels. This long-term sensitization method would be useful for detecting environmental chemical-related hypersensitivity.

Sensitizing potential of chromated copper arsenate in local lymph node assays differs with the solvent used.

We used the local lymph node assay to evaluate the abilities of chromated copper arsenate (CCA), a commonly used wood preservative, and its components to cause sensitizing reactions after their dilution in acetone-olive oil (AOO) and dimethyl sulfoxide (DMSO). After CBA/J mice were treated topically with 0.3 to 10% CCA, 0.3-3% chromium oxide, 0.3-3% arsenic oxide, or 0.3-3% copper oxide, their auricular lymph nodes (LN) were weighed and used in lymphocyte proliferation assays. In addition, total levels of chromium and arsenic in blood samples were measured. In all groups treated with CCA in AOO or DMSO, all parameters, including LN weight and lymphocyte proliferation, increased in a dose-dependent manner. The stimulation index (SI; the mean [3H]-TdR incorporation of the treatment group divided by that of the control group) showed a positive response (SI > 3) in all treatment groups; the EC3 values (estimated concentration to yield SI of 3) of CCA in AOO and DMSO were 1.86% and < 0.3%, respectively. In addition, we confirmed that the three components of CCA--chromium oxide, arsenic oxide and copper oxide--each individually exerted sensitizing ability. Mice treated with arsenic oxide in AOO or DMSO yielded nearly equal positive responses; however, the LLNA responses in mice treated with chromium oxide and copper oxide was much higher in the DMSO groups than in the AOO groups. The total chromium level in blood was higher in DMSO groups than AOO groups, whereas arsenic levels were comparable between the DMSO and AOO groups. Our findings suggest that CCA has sensitizing activity and that the type of solvent used can influence the results of sensitization assays evaluating metals.