Impairment of Di ( 2-Ethylhexyl ) Phthalate on Cellular Immunity in Kunming Mice

Immunity is crucial to the health of animals and it can determine their survival and fitness. Di(2ethylhexyl) phthalate (DEHP) is widely used as a plasticizer and hence is the most abundant phthalate in the environment. Exposure to DEHP is of great concern for human health. In the present study, we tested the hypothesis that exposure to DEHP would suppress T cell-mediated immunity in mice. Twenty adult male Kunming mice were randomly assigned into the control (n = 10) and the DEHP treatment (n = 10) groups. Both groups have free access to food and water, while the mice in the latter group drank DEHP solution (2000 mg/L) for 42 days. T cell-mediated immunity assessed by phytohaemagglutinin (PHA) response was depressed in the DEHP treated mice compared with the controls, however, wet thymus and spleen mass, white blood cells were not influenced by DEHP treatment. Taken together, different immunological parameters responded differently to DEHP treatment in Kunming mice.


Introduction
The immune system protects animals against environmental pathogens, which is crucial for their health and is important to determine their survival and fitness [1] [2].However, immune function is influenced by many factors including endocrine disruptors such as di(2-ethylhexyl) phthalate (DEHP) [3]- [6].
DEHP, produced at annual quantities of 2 million tons and widely used in medical devices and plastics, is one of the principal phthalates causing human health concerns [7]- [9].DEHP has endocrine-disrupting property [10]- [12].Many researchers have focused on the reproductive toxicology of DEHP in rodents (25; 26) and human [13] [14].The ability of DEHP to impact on immune and allergic responses has been examined [5] [6] [15].Moreover, some investigators have found that DEHP treatment could inhibit B cell proliferation and reduces the abundance of IgM-secreting cells in cultured immune tissues [16].However, whether DEHP treatment could suppress cellular immunity still remains unclear.Phytohaemagglutinin (PHA) response can be used to evaluate mammalian cellular immunity, which is one arm of adaptive immune system and generally responsible for intracellular pathogen control [17] [18].Immune organs including thymus and spleen are also indicative of immune function [19]- [21].Thymus is essential for primary T cell development [19], and a larger spleen is representative of stronger immunity [21].Total white blood cells (or leukocytes, WBC), which are crucial for immune responses against pathogens, are useful to assess the overall health [20].
In the present study, we tested the hypothesis that DEHP would have great influences on immune function in Kunming mice.We expected that immunological parameters including cellular immunity, thymus and spleen mass and white blood cells would be suppressed in DEHP treated mice compared with the controls.

Animals and Experimental Design
All animal procedures were licensed under the Institutional Animal Care and Use Committee of Qufu Normal University.Adult male Kunming mice (age: 6 months) used in this study were obtained from the Experiment Animal Center in Jining Medical College of Shangdong province.The experiment was carried out from September 6 to October 25 in 2012.The animals were housed individually in plastic cages (30 cm × 15 cm × 20 cm) with sawdust as bedding.The raising conditions are semi-natural in which maximum and minimum ambient temperature and humidity were described in   and the DEHP treated group (n = 10) in which each mouse drank DEHP solution (2000 mg/L).The period of the experiment was 42 days.Day 0 and day n represented initial day and n days of treatment, respectively.4 mice in the DEHP group died on day 7, 20, 34, 40 respectively, and 2 mice in the control group died on day 15, 35 respectively.The data of the six mice were not included in the subsequent statistical analysis.

Cellular Immunity Assays
PHA response was measured as described previously [18] [22].Specifically, mice in the control and DEHP groups on day 39 were caught, then we measured their footpad thickness of the left hind foot with a micrometer (Digimatic Indicator ID-C Mitutoyo Absolute cod.547-301, Japan) to ±0.01 mm.Immediately thereafter, mice in both groups were injected subcutaneously 0.1 mg of PHA (PHA-P, Sigma L-8754) dissolved in 0.03 mL of sterile saline (pH 7.4) in the middle of the footpad.After 6 h, 24 h, 48 h and 72 h injection, we measured footpad thickness.The PHA response (i.e., cellular immunity) was calculated as the difference between pre-and postinjection measurements divided by initial footpad thickness (PHA response = (post PHA − pre PHA)/pre PHA).Six measures of footpad thickness were taken to obtain the value of each mouse [22].

Organs
Organs were measured as described previously [22].In brief, the visceral organs, including heart, thymus, lungs, liver, spleen, kidneys, adrenal glands, testes, epididymis, seminal vesicals and the digestive organs with contents (i.e., stomach, small intestine, caecum and colon) were dissected and weighed (±1 mg).The stomach, small intestine, caecum and colon were rinsed with saline to eliminate all the gut contents, before being weighed.

White Blood Cells Assays
At the end of the experiment, after collecting trunk blood, 20 µL whole blood was diluted immediately in 0.38 mL solution containing 1.5% glacial acetic acid, 1% crystal violet (Sigma) and the leukocytes were counted in an improved Neubauer chamber using microscope.The total number of WBC was determined by counting all leucocytes in the four corner large-squares of the Neubauer chamber, and multiplying the raw data by 5 × 10 7 to obtain the final values (10 9 cells/L) [23].

Statistical Analysis
Data were analyzed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA).Prior to all statistical analyses, data were examined for normality and homogeneity of variance, using Kolmogorov-Smirnov and Levene tests, respectively.The ratio values such as PHA response were subjected to arcsine transformation.The differences of body mass between the control and DEHP treated groups were analyzed by independent-samples t-test.Group differences in wet organ mass with body mass as the covariate were analyzed by General Linear Model multivariate analysis followed by Bonferroni post hoc tests.Group differences in other parameters (PHA response, WBC) were analyzed by independent-samples t-test.Results were expressed as mean ± SE, and P < 0.05 was considered to be statistically significant.

Organs
DEHP treatment decreased the masses of stomach with contents and seminal vesical while increased the mass of colon (Table 1).The masses of thymus, spleen and other organs were all not influenced by DEHP treatment (Table 1).

Discussion
As expected, cellular immunity was depressed in the DEHP treated mice compared with the control mice.However other immunological parameters such as thymus and spleen mass, white blood cells were not affected by DEHP treatment.
Influences of DEHP on immune responses were carried out in rodents both in vitro and in vivo.For example, Koike et al. (2009) investigated the effects of DEHP on immune cells from atopic prone NC/Nga mice in vitro, and found that DEHP enhances bone marrow-derived dendritic cells differentiation and also enhances Th2 response in splenocytes [5].However, DEHP treatment could inhibit B cell proliferation and reduces the abundance of IgM-secreting cells in cultured immune tissues [16].Tonk et al. (2012) have found the immune system in juvenile and adult wistar rats had different sensitivity to DEHP exposure, in which more immune parameters were affected in juvenile rats compared to adult rats [6].In our study, cellular immunity was depressed in Kunming mice after DEHP exposure.The disparate results with other researches might be due to the differences in species used, the treatment method and the immune parameters measured [5]

DEHP White blood cells (×10
The major response to stress is the increase of stress hormones [24].We found that adrenal gland increased nearly significantly after 42 days of DEHP treatment, which might imply the activation of the hypothalamic-pituitary-adrenal (HPA) axis.DEHP treatment might be a stressor to mice, although we did not detect the concentration of stress hormones such as corticosterone.Generally, corticosterone had suppressive effect on immune function.Therefore, the impairment of cellular immunity might be due to the increase of adrenal gland.Future work is required to examine the levels of stress hormones and their relationships with immune function.
In the present study, DEHP treatment decreased seminal vesical mass, which indicated its suppressive effect on reproductive function.This result was consistent with other researches [25] [26].However, DEHP treatment had no effect on body mass and most organ masses in mice.

Conclusion
In conclusion, different immunological indices have different sensitivity to DEHP.DEHP had significant suppressive effect cellular immune response, whereas it did not affect thymus and spleen and white blood cells.

Figure A .
Figure A. The changes of maximum and minimum temperature during the course of the experiment.

Figure B .
Figure B. The changes of humidity during the course of the experiment.

Figure 1 .
Figure 1.Changes of body mass in mice during DEHP treatment.Values are means ± SE.Body mass on day 0 between the control and DEHP treatment groups did not differ significantly.

Figure 2 .
Figure2.Effect of DEHP treatment on PHA response in mice.Values are means ± SE.PHA response was significantly higher in the control group than in the DEHP treated group.The white column represents the control group and solid column stands for the DEHP treated group.An asterisk ( * ) indicates statistical differences at P < 0.05.A pound (#) indicates nearly approaching the significant level.

Figure 3 .
Figure 3.Effect of DEHP treatment on white blood cells in mice.Values are means ± SE.WBC did not differ between the control and the DEHP treated groups.

Table 1 .
Effect of DEHP treatment on wet organ mass in Kunming mice.Values are means ± SE.Values for a specific parameter that share different superscripts are significantly different at P < 0.05, determined by General Linear Model multivariate analysis followed by Bonferroni post hoc tests with body mass as the covariate.