Effect of maternal exposure to ozone on reproductive outcome and immune, inflammatory, and allergic responses in the offspring

Figures & data

Figure 1.  Experimental regime. Timed-pregnant BALB/c dams were exposed to either O₃ or air from gestational day (GD) 9 to GD18 for 4 h/day. The offspring were assessed at postnatal day (PND) 42 before sensitization, or following sensitization with ovalbumin (OVA) at (A) PND2 and PND3, or at (B) PND42 and PND43 with both groups challenged at PND54, PND55, and PND56. Bovine serum albumin (BSA) or sheep red blood cell (SRBC) were injected into the separate groups of the offspring at PND42 to evaluate systemic responses.

Table 1.  Effect of maternal exposure to ozone on reproductive outcomes.

Treatment	Percentage of delivered pregnancy (dams giving birth/total dams per group)				Number and sex of offspring (♂/♀)				Litter size
	I	II	III	Average	I	II	III	Average ♂/♀ ratio
	(exposures)				(exposures)
Air	75% (15/20)	45% (9/20)	55% (11/20)	58%	25/53	23/33	25/21	1:1.5	5.3 ± 0.4
0.4 ppm O3	70% (14/20)	20% (4/20)	45% (9/20)	45%	34/45	11/14	14/29	1:1.6	5.4 ± 0.5
0.8 ppm O3	50% (10/20)	30% (6/20)	55% (11/20)	45%	21/37	11/16	21/33	1:1.7	5.3 ± 0.3
1.2 ppm O3	45% (9/20)	30% (6/20)	25% (5/20)	33%**	25/28	11/17	11/14	1:1.3	5.3 ± 0.2

**P < 0.005 significant compared with control group.

Table 2.  Effect of maternal exposure to ozone on rate of weight gain in the offspring.

Treatment	Body weight (g)
	PND1	PND3	PND7	PND14	PND21	PND42
Air	1.50 ± 0.06	2.30 ± 0.10	4.6 ± 0.1	8.9 ± 0.3	10.0 ± 0.2	♂ 23.0 ± 0.6
						♀ 18.2 ± 0.7
0.4 ppm O3	1.40 ± 0.10	2.10 ± 0.08	4.5 ± 0.1	8.8 ± 0.3	9.8 ± 0.4	♂ 22.7 ± 0.6
						♀ 17.7 ± 0.4
0.8 ppm O3	1.40 ± 0.04	2.10 ± 0.05	4.6 ± 0.1	9.1 ± 0.2	10.0 ± 0.2	♂ 22.7 ± 0.4
						♀ 18.0 ± 0.5
1.2 ppm O3	1.30 ± 0.04*	1.80 ± 0.06*	3.9 ± 0.1**	8.1 ± 0.3	9.5 ± 0.2	♂ 20.9 ± 0.6*
						♀ 17.6 ± 0.2

*P < 0.05 and **P < 0.005, significant compared with control group.

Data are presented as the mean (±SEM) in three experiments until postnatal day (PND) 21, excluding ovalbumin (OVA)-sensitized offspring and 3–7 offspring at PND42 in two Experiments → Experiment I (n = 3–6) and Experiment III (n = 6–7).

Figure 2.  Splenic T-cell populations and bronchoalveolar lavage fluid (BALF) cytokines in non-allergic offspring born from dams exposed to O₃. A representative data of a total 10,000 events of the spleen cells screened by FACS with appropriate antibodies for (A) CD4⁺ T and (B) iNKT cells, and (C) IFN-γ and (D) IL-17 measured by enzyme-linked immunosorbent assay (ELISA) in BALF at postnatal day (PND) 42 has been shown. Data are the mean (±SEM) of 3–6 offspring for each sex per group in Experiments I (n = 3) and III (n = 6).

Figure 3.  Inflammatory responses in non-allergic offspring born from dams exposed to O₃. Bronchoalveolar lavage fluid (BALF) was assessed for (A) lactate dehydrogenase (LDH) and (B) protein at postnatal day (PND) 42. Data are the mean (±SEM) of 3–7 offspring for each sex per group in Experiments I (n = 3–6) and III (n = 6–7). *P < 0.05; significantly different from air group.

Figure 4.  Systemic immunotoxicity in the offspring born from dams exposed to O₃. (A) DTH was determined by difference in thickness between saline- and bovine serum albumin (BSA)-injected footpads. Data are the mean (±SEM) of 5–6 offspring for each sex in a group, in Experiment I only. *P < 0.05, significantly different from air group. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of sheep red blood cell (SRBC)-specific (B) IgM and (C) IgG in the sera. Data are the mean (±SEM) of 6–8 offspring per sex in Experiment I only.

Table 3.  Cell differentials in bronchoalveolar lavage fluid (BALF) of allergic offspring.

	Treatment group	× 10^4 cells/mL
		Total cells		Macrophages		Lymphocytes		Neutrophils		Eosinophils
		♂	♀	♂	♀	♂	♀	♂	♀	♂	♀
Early (<PND3) sensitization	Air + ovalbumin (OVA)	13 ± 3	17 ± 4	11 ± 2	12 ± 2	0.80 ± 0.40	1.70 ± 0.70	1.0 ± 0.1	1.6 ± 0.3^##	0.50 ± 0.30	2.00 ± 1.00^^#
	0.8 ppm + OVA	13 ± 1	13 ± 1	11 ± 1	10 ± 1	0.40 ± 0.10	0.80 ± 0.20	1.3 ± 0.2	1.9 ± 0.3	0.20 ± 0.10	1.10 ± 0.50
	1.2 ppm + OVA	10 ± 2	9 ± 1*	9 ± 2	7 ± 1*	0.20 ± 0.05	0.30 ± 0.07**	0.8 ± 0.2	0.9 ± 0.2	0.10 ± 0.03	0.10 ± 0.02**
Late (>PND42) sensitization	Air + OVA	16 ± 1	18 ± 1	15 ± 1	14 ± 1	0.50 ± 0.07	0.80 ± 0.07	1.2 ± 0.2	3.1 ± 0.3	0.20 ± 0.05	0.70 ± 0.07
	0.8 ppm + OVA	15 ± 1	14 ± 1	14 ± 1	12 ± 1	0.30 ± 0.06	0.50 ± 0.08	0.9 ± 0.2	1.1 ± 0.2**	0.50 ± 0.30	0.20 ± 0.03
	1.2 ppm + OVA	15 ± 2	17 ± 2	14 ± 2	15 ± 2	0.40 ± 0.08	0.70 ± 0.10	0.9 ± 0.2	1.1 ± 0.2**	0.10 ± 0.03	0.40 ± 0.08

Data are presented as mean (± SEM) of 3–7 offspring in three experiments (offspring numbers per sex in a group were n = 3–5 in Experiment I, n = 5–6 in Experiment II, and n = 5–7 in Experiment III).

*P < 0.05 and **P < 0.005 significant compared with the same sex in control group.

^#P < 0.05 and ^##P < 0.005 significant compared with the same sex in control group of late sensitization.

^{^}P < 0.05 significant compared with males within the group.

Figure 5.  Ovalbumin (OVA)-specific IgE and inflammatory responses in allergic offspring of dams exposed to O₃ 1 day after the last OVA challenge, (A) OVA-specific IgE in the serum was detected by enzyme-linked immunosorbent assay (ELISA). (B) Levels of lactate dehydrogenase (LDH) and (C) protein in bronchoalveolar lavage fluid (BALF) were assessed. All data are the mean (±SEM) of 3–7 offspring for each sex per group in three experiments (offspring numbers per sex in a group were n = 3–5 in Experiment I, n = 5–6 in Experiment II and n = 5–7 in Experiment III). *P < 0.05; significantly different from other groups.

Figure 6.  Effect of maternal O₃ exposure on airway responsiveness to methacholine (MCh) in the offspring. One day after the last ovalbumin (OVA) challenge, Penh to MCh was recorded in a 12-chamber Buxco system. Data are the mean (±SEM) of 3–6 offspring for (A) males and (B) females per group in three experiments (offspring numbers per sex in a group were n = 3–5 in Experiment I, n = 5–6 in Experiment II, and n = 5–7 in Experiment III). *P < 0.05 and **P < 0.005; significantly different from other groups.