Developmental Immunotoxicology of Di-(2-Ethylhexyl)phthalate (DEHP): Age-Based Assessment in the Female Rat

Figures & data

TABLE 1 Pregnancy outcome following fetal exposure to di-(2-ethylhexyl)phthalate for the last 16 days of gestation¹

	Di-(2-ethylhexyl)phthalate (mg/kg/d)
	0	37.5	75	150	300
Litter size	11.3 ± 0.8	10.5 ± 0.6	12.0 ± 0.3	10.5 ± 0.6	11.7 ± 0.6
Stillbirths/litter^2	0.00	0.00	0.08 ± 0.08	0.08 ± 0.08	0.00
Early deaths^2, ^3	0.17 ± 0.17	0.23 ± 0.17	0.15 ± 0.10	0.08 ± 0.08	0.00
Sex ratio	1.99 ± 0.36	1.38 ± 0.22	0.96 ± 0.16	1.56 ± 0.39	1.92 ± 0.48
Females/litter	7.33 ± 0.88	5.54 ± 0.50	5.31 ± 0.60	5.58 ± 0.56	6.69 ± 0.50

¹All pups were born between the morning of day 22 and the morning of day 23 of gestation therefore no differences in gestation length could be detected.

²Because stillbirths and early deaths were low only the mean ± SEM is presented. No further statistics were performed on these values.

³This number includes the pups that died within 3 days of birth plus all stillbirths.

TABLE 2 Body weights and ano-genital distances (AGD) measured in offspring of Sprague-Dawley CD rats exposed to di- (2-ethylhexyl)phthalate for the last 16 days of gestation

Di-(2-ethylhexyl)phthalate (mg/kg/day)^1
	0	37.5	75	150	300
At 7 days old
Body weight (g)	15.6 ± 0.2	15.9 ± 0.2	15.6 ± 0.2	16.2 ± 0.2	15.5 ± 0.3
AGD (mm)	2.94 ± 0.04^ab	2.87 ± 0.04^b	3.06 ± 0.03^ac	3.20 ± 0.06^cd	3.27 ± 0.04^d
AGD/BW (mm/g)	0.19 ± 0.003^ab	0.18 ± 0.002^b	0.20 ± 0.003^ac	0.20 ± 0.003^ac	0.21 ± 0.003^d
AGD/BW^1/3(mm/g^1/3)	1.18 ± 0.02^ab	1.14 ± 0.01^b	1.23 ± 0.01^ac	1.27 ± 0.02^cd	1.31 ± 0.01^d
At 21 days old
Body weight (g)	52.8 ± 0.7^a	56.8 ± 0.7^b	56.6 ± 0.4^b	57.9 ± 0.8^b	55.9 ± 0.7^b
AGD (mm)	8.59 ± 0.09^a	9.34 ± 0.11^b	9.83 ± 0.08^c	10.07 ± 0.13^c	10.67 ± 0.07^d
AGD/BW (mm/g)	0.16 ± 0.002^a	0.16 ± 0.002^ab	0.17 ± 0.001^bc	0.18 ± 0.002^c	0.19 ± 0.002^d
AGD/BW^1/3(mm/g^1/3)	2.29 ± 0.02^a	2.43 ± 0.02^b	2.54 ± 0.02^c	2.60 ± 0.03^c	2.79 ± 0.02^d

Values represent mean ± SEM. Means within a row with different letters are significantly different at p < 0.05. Measurements were made on all available female pups in each litter (2 to 13 pups/litter).

FIG. 1 Nitrite (top) levels and tumor necrosis factor alpha (TNFα, bottom) produced by adherent splenocytes isolated from 5-week-old female offspring exposed to di-(2-ethylhexyl) phthalate for the last 16 days of gestation. Cells were stimulated with 10 ng/ml LPS and supernatants were collected after 24 hours of incubation at 37°C. The minimum detection limit for TNFα was 0.7 pg/ml. There were no statistical differences (p > 0.05).

FIG. 2 Cytokines produced by unseparated splenocytes isolated from 5-week-old offspring exposed to di-(2-ethylhexyl)phthalate in utero for the last 16 days of gestation. Cells were incubated with 5 μg/ml Con A for 72 hours. The minimum detection limit for each of the cytokines was as follows: IFN-γ < 13 pg/ml, IL-2 < 5 pg/ml IL-4 < 1.3 pg/ml, IL-10 < 5 pg/ml, IL-12 < 3 pg/ml. There were no significant differences between treatments (p > 0.05).

FIG. 3 Cytokines produced by unseparated splenocytes isolated from 13-week-old offspring exposed to di-(2-ethylhexyl)phthalate in utero for the last 16 days of gestation. Cells were incubated with 5 μg/ml Con A for 72 hours. The minimum detection limit for each of the cytokines was IFN-γ < 13 pg/ml, IL-2 < 5 pg/ml, IL-4 < 1.3 pg/ml, IL-10 < 5 pg/ml, IL-12 < 3 pg/ml. There were no significant differences between treatments (p > 0.05).

FIG. 4 Cytokines produced by unseparated splenocytes isolated from non-pregnant adults exposed to di-(2-ethylhexyl)phthalate for 16 days concurrently with pregnant dams. Cells were incubated with 5 μg/ml Con A for 72 hours. The minimum detection limit for each of the cytokines was as follows: Interferon gamma < 13 pg/ml, IL-2 < 5 pg/ml IL-4 < 1.3 pg/ml, IL-10 < 5 pg/ml, IL-12 < 3 pg/ml. There were no significant differences between treatments (p > 0.05).

TABLE 3 Flow cytometry profile of cell populations in 5-week-old offspring exposed to di-(2-ethylhexyl)phthalate in utero for the last 16 days of gestation

	Di-(2-etheylhexyl)phthalate (mg/kg/d)
	0	37.5	75	150	300
5-week-old^1			% Gated cells
CD4^+CD8^+	1.91 ± 0.20	1.85 ± 0.17	1.64 ± 0.10	1.75 ± 0.21	1.94 ± 0.18
CD4^−CD8^+	16.74 ± 1.32	18.80 ± 0.99	16.38 ± 1.00	16.75 ± 1.55	17.18 ± 1.00
CD4^+CD8^−	19.08 ± 2.68	18.70 ± 1.97	17.07 ± 1.71	19.71 ± 2.69	20.73 ± 2.31
Natural killer	2.21 ± 0.39	2.52 ± 0.30	2.39 ± 0.39	2.69 ± 0.47	2.64 ± 0.29
Macrophage	5.05 ± 0.82	5.37 ± 0.67	4.34 ± 0.50	4.70 ± 0.33	4.97 ± 0.27
B-cells	53.72 ± 3.48	55.73 ± 2.67	55.46 ± 3.15	54.16 ± 2.77	54.10 ± 2.74

¹The “n” for each treatment was 11, 13, 13, 12, and 13 for 0, 37.5, 75, 150, 300 mg/kg/d, respectively.