Di-(n-butyl)-phthalate-induced oxidative stress and depression-like behavior in mice with or without ovalbumin immunization.

OBJECTIVE: To investigate the relationship between atopic allergy and depression and the role of DBP in the development of depression. METHODS: BALB/c mice were randomly divided into eight groups: saline; ovalbumin (OVA)-immunized; saline+DBP (0.45 mg/kg•d); saline+DBP (45 mg/kg•d); DBP (0.45 mg/kg•d) OVA-immunized; DBP (45 mg/kg•d) OVA-immunized; saline+hydrocortisone (30 mg/kg•d); and hydrocortisone (30 mg/kg•d)-exposed OVA-immunized. Behavior (e.g. open-field, tail suspension, and forced swimming tests), viscera coefficients (brain and spleen), oxidative damage [e.g. reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH)], as well as levels of IgE and IL-4, were then analyzed. RESULTS: In the saline and OVA groups, the degree of depression symptoms in mice increased with increasing DBP concentration. Additionally, the OVA-immunity groups were associated with more serious depressive behavior compared with the same exposure concentration in the saline group. Oxidative damage was associated with a dose-dependent increase in DBP in the different groups. IL-4 and IgE levels were associated with low-dose DBP stimulation, which changed to high-dose inhibition with increasing DBP exposure, possibly due to spleen injury seen at high DBP concentrations. CONCLUSION: Development of an atopic allergy has the potential to increase the risk of depression in mice, and it seems that DBP helps OVA to exert its effect in our present model. Moreover, the results of our study implicate a certain connection between brain oxidative stress and depression, which deserves a further exploration.

The immunotoxicity of dibutyl phthalate on the macrophages in mice.

OBJECTIVE: Dibutyl phthalate (DBP), a widely used phthalate chemical, is commonly used as plasticizer. It is well known that DBP causes reproductive and developmental diseases, but the effect of DBP on the immune system remains to be determined. We assessed the effect of DBP on immune functions of murine macrophages, which constitute a key component in the immune response. MATERIALS AND METHODS: Murine peritoneal exudate macrophages (PEMs) were treated with 0, 1, 5, 10, 50 or 100 µM DBP in vitro for 24 h and then the viability of PEMs were measured by flow cytometry (FCM) and trypan blue count. To investigate the effect of DBP on the functions of PEMs, we treated the PEMs with moderate dose of DBP (0, 1, 5 or 10 µM) in vitro for 24 h. The phenotypes, phagocytosis and cytokine production of PEMs were measured by FCM or real-time PCR. The immunogenicity and antigen presenting capacity of PEMs treated with DBP in vitro were assessed both by the mixed lymphocytereaction (MLR) in vitro assay and through the injection of exposed cells in mice by the delayed-type hypersensitivity (DTH) assay. RESULTS: High dose of DBP (50-100 µM) showed cytotoxicity on PEMs, whereas after the treatment with moderate dose of DBP (1-10 µM) in vitro, PEMs expressed low level of CD36, CD80 and MHC-II molecules, and showed significantly decreased phagocytosis on apoptotic cells and Escherichia coli. In addition, DBP treatment exhibited a decrease in the cytokine production, immunogenicity and antigen-presenting capacity of PEMs. CONCLUSIONS: The present study shows the effects of DBP on macrophages, demonstrating immunogenicity and decreased antigen presentation in vitro.

An immunoassay for dibutyl phthalate based on direct hapten linkage to the polystyrene surface of microtiter plates.

BACKGROUND: Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed. METHODOLOGY/PRINCIPAL FINDINGS: A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC(50)=106 ng/mL), the direct hapten coated format (IC(50)=14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples. CONCLUSIONS/SIGNIFICANCE: The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples.

Dibutyl phthalate-induced thymic stromal lymphopoietin is required for Th2 contact hypersensitivity responses.

Thymic stromal lymphopoietin (TSLP) is an IL-7-related cytokine, produced by epithelial cells, that has been linked to atopic dermatitis and asthma; however, it remains unclear how TSLP shapes the adaptive immune response that causes these allergic disorders. In this study, we demonstrate a role for TSLP in a Th2 model of contact hypersensitivity in mice. TSLP is required for the development of Th2-type contact hypersensitivity induced by the hapten FITC in combination with the sensitizing agent dibutyl phthalate. TSLPR-deficient mice exhibited a dramatically reduced response, including markedly reduced local infiltration by eosinophils, Th2 cytokine production, and serum IgE levels, following FITC sensitization and challenge. The reduced response by TSLPR-deficient mice is likely due to decreased frequency and reduced T cell stimulatory function of skin-derived Ag-bearing FITC(+)CD11c(+) dendritic cells in draining lymph nodes following FITC sensitization. These data suggest that skin-derived dendritic cells are direct or indirect targets of TSLP in the development of type 2 immune responses in the skin, where TSLP drives their maturation, accumulation in skin draining lymph nodes, and ability to induce proliferation of naive allergen-specific T cells.

Effects of phthalate esters on the sensitization phase of contact hypersensitivity induced by fluorescein isothiocyanate.

BACKGROUND: Many different types of phthalate ester are used as plasticizers and are thus found in the air. There have been several studies that suggest an association between allergies and phthalate esters. We previously found that di-butyl phthalate (DBP) has an adjuvant effect in a mouse contact hypersensitivity model, in which fluorescein isothiocyanate (FITC) is involved as an immunogenic hapten. OBJECTIVE: We examined whether other phthalate esters enhance the process of sensitization to FITC by facilitating the trafficking of FITC-presenting dendritic cells or macrophages from skin sites to draining lymph nodes. METHODS: Mice were epicutaneously sensitized with FITC dissolved in acetone containing a phthalate ester. Sensitization was evaluated as ear swelling after a challenge with FITC. Draining lymph node cells obtained 24 h after skin sensitization were examined for FITC fluorescence by means of flow cytometry. FITC-positive cells were characterized with anti-CD11c and anti-CD11b by three-colour flow cytometry. RESULTS: When mice were sensitized with FITC in acetone containing DBP or di-n-propyl phthalate (DPP), strong enhancement of the ear-swelling response was observed. Di-methyl phthalate (DMP) and di-ethyl phthalate (DEP) were less effective but produced some enhancement. Consistent enhancement was not observed with di-(2-ethylhexyl) phthalate or di-isononyl phthalate. Upon sensitization in the presence of DBP or DPP, the number of FITC-positive dendritic cells (total CD11c+ as well as CD11c+/CD11b+) was increased in draining lymph nodes. As to the other four phthalate esters, there was no significant increase in the FITC-positive cell number in the draining lymph nodes. CONCLUSION: During the process of sensitization to FITC, DBP, and DPP exert strong adjuvant effects that are associated with enhancement of trafficking of antigen-presenting dendritic cells from the skin to draining lymph nodes.