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

Airway hyperresponsiveness (AHR) measurements in mice.

<p>Covariance analyses (SPSS) were used in these tests. (a) Ri values of saline control and DEHP groups; (b) Re values of saline control and DEHP groups; (c) Cldyn values of saline control and DEHP groups; (d) Ri values of OVA only and OVA+DEHP groups; (e) Re values of OVA only and OVA+DEHP groups; (f) Cldyn values of OVA only and OVA+DEHP groups. Group data were expressed as means±standard deviation (SD).</p

Serum IgE.

<p>(a) Total IgE levels in the eight different exposure groups. ** p<0.01, compared with the saline control; ## p<0.01, compared with the OVA only group; (b) Dose-response relationship between DEHP (+OVA) and total serum IgE levels; (c) OVA-IgE levels in the eight different exposure groups. Group data were expressed as means±standard deviation (SD). * p<0.05, ** p<0.01, compared with the saline control.</p

The relative number of eosinophil to total cell numbers in BALF.

<p>Group data were expressed as means±standard deviation (SD). * p<0.05 and ** p<0.01, compared with the saline control; ^# p<0.05, compared with the OVA only group.</p

IFN-γ and IL-4 levels in lung tissue.

<p>(a) pulmonary tissue IFN-γ levels in each group; (b) pulmonary tissue IL-4 levels in each group; (c) IL-4/IFN-γ ratios in each group. Group data were expressed as means±standard deviation (SD). * p<0.05, ** p<0.01, compared with the saline control; ^# p<0.05, ^## p<0.01, compared with OVA only group.</p

Summary of the experimental results.

<p> <b>–: As statistical control group; Compared with saline control: NS p>0.05,</b></p>*<p> <b>p<0.05,</b></p>**<p> <b>p<0.01; Compared with OVA only group: ns p>0.05,</b></p>#<p> <b>p<0.05,</b></p>##<p> <b>p<0.01.</b></p

Multiple covariance analyses for AHR measurements.

<p>Covariance analyses were used for AHR testing. Dependent variable: biomarkers; Fixed factor: DEHP doses; Covariate: MCh doses.</p

Exposure and immunization schedule.

<p>Exposure and immunization schedule.</p