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 Figure A and Figure B, and the photoperiod was natural light. Standard rat pellets chow (Animal Breeding Center in Jining Medical College, Jining, China) and water were provided ad libitum. After body mass stabilized, 20 mice were randomly divided into the control group (n = 10)

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.

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 post- injection 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 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.

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 × 10⁷ to obtain the final values (10⁹ cells/L) [23] .

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.

On day 0, body mass between the control and DEHP groups was not different (t = −0.666, df = 12, P = 0.518). There was no difference of body mass between these two groups from day 1 (t = −0.437, df = 12, P = 0.670) to day 42 (t = −0.064, df = 12, P = 0.950) (Figure 1).

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).

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.

WBC did not differ between the control and DEHP treated mice (t = 0.342, df = 12, P = 0.738) (Figure 2).

Compared with the control mice, PHA response in the DEHP treated mice was suppressed significantly after 24 h (t = −2.822, df = 12, P = 0.015), 48 h (t = −2.859, df = 12, P = 0.014) and 72 h (t = −4.126, df = 12, P = 0.001) of PHA injection (Figure 3). Impact of DEHP treatment on PHA response after 6h of postimmunization in DEHP treated mice was near to the significant level in contrast with the control mice (t = −2.152, df = 12, P = 0.052) (Figure 3).