Huihui You

Pulmonary Toxicity and Adjuvant Effect of Di-(2-exylhexyl) Phthalate in Ovalbumin-Immunized BALB/c Mice

Background Asthma is a complex pulmonary inflammatory disease, which is characterized by airway hyperresponsiveness, variable airflow obstruction and inflammation in the airways. The majority of asthma is allergic asthma, which is a disease caused by type I hypersensitivity mediated by IgE. Exposures to a number of environmental chemicals are suspected to lead to asthma, one such pollutant is di-(2-ethylheyl) phthalate (DEHP). DEHP is a manufactured chemical that is commonly added in plastic products to make them flexible. Epidemiological studies have revealed a positive association between DEHP exposure and asthma prevalence. Methodology/Principal Findings The present study was aimed to determine the underlying role of DEHP exposure in airway reactivity, especially when combined with allergen exposure. The biomarkers include pulmonary histopathology, airway hyperresponsiveness (lung function), IgE, IL-4, IFN-γ and eosinophils. Healthy balb/c mice were randomly divided into eight exposure groups (n = 8 each): (1) saline control, (2) 30 µg/(kg•d) DEHP, (3) 300 µg/(kg•d) DEHP, (4) 3000 µg/(kg•d) DEHP, and (5) ovalbumin (OVA)-sensitized group, (6) OVA-combined with 30 µg/(kg•d) DEHP, (7) OVA-combined with 300 µg/(kg•d) DEHP, and (8) OVA-combined with 3000 µg/(kg•d) DEHP. Experimental tests were conducted after 52-day DEHP exposure and subsequently one week of challenge with aerosolized OVA. The principal findings include: (1) Strong postive associations exist between OVA-combined DEHP exposure and serum total IgE (T-IgE), as well as histological findings. These positive associations show a dose-dependent low dose sensitive effect of DEHP. (2) IL-4, eosinophil recruitment and lung function are also indicators for adjuvant effect of DEHP. Conclusions/Significance Our results suggest that except the significant changes of immunological and inflammatory biomarkers (T-IgE, IL-4, IFN-γ and eosinophils), the pulmonary histological (histopathological examination) and physiological (lung function) data also support that DEHP may promote and aggravate allergic asthma by adjuvant effect.

Role of Transient Receptor Potential Ion Channels and Evoked Levels of Neuropeptides in a Formaldehyde-Induced Model of Asthma in Balb/c Mice

Objective Asthma is a complex pulmonary inflammatory disease characterized by the hyper-responsiveness, remodeling and inflammation of airways. Formaldehyde is a common indoor air pollutant that can cause asthma in people experiencing long-term exposure. The irritant effect and adjuvant effect are the two possible pathways of formaldehyde promoted asthma. Methodology/Principal Findings To explore the neural mechanisms and adjuvant effect of formaldehyde, 48 Balb/c mice in six experimental groups were exposed to (a) vehicle control; (b) ovalbumin; (c) formaldehyde (3.0 mg/m3); (d) ovalbumin+formaldehyde (3.0 mg/m3); (e) ovalbumin+formaldehyde (3.0 mg/m3)+HC-030031 (transient receptor potential ankyrin 1 antagonist); (f) ovalbumin+formaldehyde (3.0 mg/m3)+ capsazepine (transient receptor potential vanilloid 1 antagonist). Experiments were conducted after 4 weeks of combined exposure and 1-week challenge with aerosolized ovalbumin. Airway hyper-responsiveness, pulmonary tissue damage, eosinophil infiltration, and increased levels of interleukin-4, interleukin-6, interleukin-1β, immunoglobulin E, substance P and calcitonin gene-related peptide in lung tissues were found in the ovalbumin+formaldehyde (3.0 mg/m3) group compared with the values seen in ovalbumin -only immunized mice. Except for interleukin-1β levels, other changes in the levels of biomarker could be inhibited by HC-030031 and capsazepine. Conclusions/Significance Formaldehyde might be a key risk factor for the rise in asthma cases. Transient receptor potential ion channels and neuropeptides have important roles in formaldehyde promoted-asthma.

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 (IC50 = 106 ng/mL), the direct hapten coated format (IC50 = 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.

The adjuvant effect induced by di-(2-ethylhexyl) phthalate (DEHP) is mediated through oxidative stress in a mouse model of asthma

Di-(2-ethylhexyl) phthalate, as the most commonly used plasticizer, is considered to be related to the asthma prevalence. There are studies affirming that the DEHP has an adjuvant effect in the pathogenesis of allergy asthma. Oxidative stress is one possible pathway for DEHP-adjuvant effect. Thus, this study explored whether DEHP could induce adjuvant effect in mouse asthma model via oxidative stress pathway. Male BALB/c mice were randomly divided into six groups: (1) saline group, (2) DEHP group, (3) ovalbumin (OVA) group, (4) DEHP+OVA group, (5) OVA+vitamin E (Vit E) group, (6) DEHP+OVA+Vit E group. The exposure dose of DEHP was 30 mg/kg body weight (bw)/day. After 18 days of the exposure protocol. Reactive oxygen species (ROS), glutathione (GSH) and malonaldehyde (MDA) levels and biomarkers related to asthma model were measured. Collectively, these data indicated higher ROS and MDA levels and lower GSH contents in DEHP+OVA group than that in OVA group, while Vit E, an antioxidant, could restore ROS, MDA and GSH levels to control levels and attenuate the DEHP and/or OVA effects. Our observations suggested that there was a relationship between oxidative stress and the adjuvant effect induced by DEHP in this mouse asthma model.

Thymic Stromal Lymphopoietin Neutralization Inhibits the Immune Adjuvant Effect of Di-(2-Ethylhexyl) Phthalate in Balb/c Mouse Asthma Model

Di-(2-ethylhexyl) phthalate (DEHP), a commonly used plasticizer, has an adjuvant effect in combination with ovalbumin (OVA). The adjuvant effect of DEHP has already been verified in our previous studies. In this study, to further investigate whether thymic stromal lymphopoietin (TSLP) was involved in the DEHP-adjuvant effect, DEHP was administered through a daily gavage exposure route. Mice were sensitized with ovalbumin (OVA) to trigger allergic responses, and an anti-TSLP monoclonal antibody was used to neutralize the effect of TSLP. Biomarkers including cytokines in bronchoalveolar lavage fluid (BALF), serum total IgE and TSLP content in the lung were detected. In addition, airway hyperreactivity and lung sections were examined. Collectively, these data indicated a salient Th2 response which was characterized by the upregulation of Th2-type cytokines, such as interleukin 4 (IL-4), IL-5 and IL-13. Moreover, the eosinophil number in BALF and the eosinophil cationic protein (ECP) in the lung were seen to have increased significantly. However, neutralization of TSLP with an anti-TSLP mAb reversed the adjuvant effect of DEHP on airway inflammation, structural alterations in the airway wall and increased airway hyperresponsiveness (AHR) to methacholine induced by the OVA allergen, suggesting that TSLP was an effective target site for suppressing the adjuvant effect of DEHP co-exposure.

Dibutyl phthalate induced oxidative stress does not lead to a significant adjuvant effect on a mouse asthma model

The prevalence of allergic diseases around the world has been increasing dramatically in recent years. Epidemiological and experimental studies have suggested the involvement of phthalate esters (PAEs) in this increase in allergic diseases. It has been previously reported that PAEs may act as adjuvants, thus contributing to an increase of these diseases. In this study we focus on examining whether dibutyl phthalate (DBP) exhibits an adjuvant effect mediated via an oxidative stress mechanism in a murine asthma model. The DBP was applied through a daily gavage exposure route. Mice were immunized with ovalbumin (OVA) to initiate immune responses, and melatonin (MT) was used as an antioxidant. However, we did not see a significant difference in the level of oxidative stress, which suggested that the possible adjuvant effect of DBP is not via an oxidative stress mechanism. Additionally, the level of immunoglobulin E (IgE) in serum, cytokines and inflammatory cells in bronchoalveolar lavage fluid (BALF) showed no significant difference between the OVA positive control group and the DBP and OVA combined exposure group. The significant difference that was observed in the expiratory resistance of airway hyperresponsiveness (AHR) may be attributed to changes in the three dimensional structure of the airway wall and the slight shrinkage of the airway. The administration of MT significantly reversed all these effects. Taken together with our data, these results suggest that DBP has little or no adjuvant effect in a murine mouse model and is not mediated through an oxidative stress mechanism.

A smart multi-functional coating based on anti-pathogen micelles tethered with copper nanoparticles via a biosynthesis method using L-vitamin C

A multi-functional anti-pathogen coating with “release-killing”, “contact-killing” and “anti-adhesion” properties was prepared from biocompatible polymer encapsulated chlorine dioxide (ClO2) which protected the active ingredient from the outside environment. A slow sustained-release of ClO2 from micelles over fifteen days was detected for long-term release-killing. Micelles only release ClO2 on demand in minimum inhibitory concentrations. We prepared nanoparticles which were covalently clustered on micelle surfaces to improve contact-killing as well as to improve the stability of the micelle. Copper nanoparticles were generated using the biosynthesis method including L-vitamin C, which avoids the toxicity and allows for the preparation of copper nanoparticles in a green environment. Synergistic anti-pathogen activity could be generated by a combination of micelle released ClO2 and ascorbic acid. In addition to release-killing and contact-killing, a pluronic polymer coated surface also provides an additional “anti-adhesion” property through its protein-repelling ability. In this research, the designed coating demonstrated a broad-spectrum of activity to kill drug-resistant bacteria, viruses and spores in short period of time. Based on scanning electron microscopy (SEM), transmission electron microscopy (TEM) and anti-oxidase assays, we found that the designed coatings killed the pathogens via bio-oxidation. We also carried out acute respiratory toxicity tests in this research. Analysis of blood samples, lung function and histopathological slices indicated that the synthesized micelles allowed a controlled and sustained release of ClO2 to kill pathogens while maintaining an overall ClO2 concentration in the air within a safe range.

Development of two enzyme-linked immunosorbent assay formats for thifluzamide residues’ analysis based on distinct polyclonal antibodies

ABSTRACT Haptens 2-Methyl-4-(trifluoromethyl)thiazole-5-carboxylic acid and 2,6-Dibromo-4-(trifluoromethoxy)aniline, the two moieties of thifluzamide, were conjugated with carrier proteins for the synthesis of artificial antigens. Two distinct anti-thifluzamide polyclonal antibodies (PAb-1 and PAb-2) were produced from the immunized female Balb/c mice. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) in two formats based on the PAbs was developed for thifluzamide analysis. The concentration of 50% inhibition (IC50) of ELISA-1 was 1.39 mg L−1 and its limit of detection (LOD) was 0.082 mg L−1. Meanwhile, ELISA-2 had a similar IC50 of 1.96 mg L−1 and a LOD of 0.074 mg L−1 as ELISA-1. Both the raised PAbs exhibited high specificity to thifluzamide. The recoveries for spiked samples including water and wheat ranged from 72.0% to 128.4%, and the accuracy of ELISA was confirmed by high-performance liquid chromatography. In summary, the ic-ELISA might be a promising tool for simple, sensitive and rapid detection of thifluzamide residues in real samples.

Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

ABSTRACT Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post‐exposure stage is crucial for a deep understanding of FA‐induced hematotoxicity. BALB/c mice were exposed to 3 mg/m3 FA for 2 weeks, mimicking occupational exposure, and were monitored for another 7 days post‐exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co‐exposure occurs frequently. After 7‐day recovery, colonies of progenitors for CFU‐GM and BFU‐E, and nucleated bone marrow cells in FA‐exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) in CFU‐GM and BFU‐E from FA‐exposed mice were higher than controls, although the increase in 8‐OHdG was not significant. Granulocyte‐macrophage colony stimulating factor (GM‐CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA‐exposed mice respond to a small amount of GM‐CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co‐exposure with BZ was more potent for promoting CFU‐GM formation and inducing ROS in BFU‐E and 8‐OHdG in CFU‐GM during the post‐exposure period. The compensation of myeloid progenitors with elevated ROS and 8‐OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co‐exposure may pose a greater leukemia risk. HIGHLIGHTSNucleated bone marrow cell count recovered after 7 days post‐FA and/or BZ exposure.CFU‐GM showed an increase in colonies and 8‐OHdG after 7 days post‐FA + BZ exposure.Levels of ROS in CFU‐GM and BFU‐E were increased by FA or FA + BZ during recovery.Levels of GM‐CSF and EPOR were suppressed after 7 days post‐FA or FA + BZ exposure.Co‐exposure was more potent for some endpoints and may pose a greater leukemia risk.

Mono-butyl phthalate-induced mouse testis injury is associated with oxidative stress and down-regulated expression of Sox9 and Dazl

Mono-butyl phthalate (MBP) has reproductive toxicity but the related mechanisms have not been fully elucidated in vivo. We exposed male Balb/c mice to MBP by gavage at doses of 0, 25, 50, 100, 200 mg/kg for 14 days, and then evaluated the testicular alterations at the histological and molecular levels. MBP reduced mouse sperm count along with sperm malformation and seminiferous tubule degeneration in a dose-dependent manner. MBP dosed at 200 mg/kg significantly increased reactive oxygen species and malondialdehyde content in mouse testes. High doses of MBP (200 mg/kg) also significantly reduced mRNA expressions of testis growth and function related genes (Sox9 and Dazl). Our findings suggest that oxidative stress and down-regulated expression of Sox9 and Dazl may play important roles in MBP-induced testis injury.