Impact of maternal cigarette smoke exposure on brain inflammation and oxidative stress in male mice offspring

Abstract

Maternal cigarette smoke exposure (SE) during gestation can cause lifelong adverse effects in the offspring's brain. Several factors may contribute including inflammation, oxidative stress and hypoxia, whose changes in the developing brain are unknown. Female Balb/c mice were exposed to cigarette smoke prior to mating, during gestation and lactation. Male offspring were studied at postnatal day (P) 1, P20 and 13 weeks (W13). SE dams had reduced inflammatory mediators (IL-1β, IL-6 and toll like receptor (TLR)4 mRNA), antioxidant (manganese superoxide dismutase (MnSOD)), and increased mitochondrial activities (OXPHOS-I, III and V) and protein damage marker nitrotyrosine. Brain hypoxia-inducible factor (HIF)1α and its upstream signalling molecule early growth response factor (EGR)1 were not changed in the SE dams. In the SE offspring, brain IL-1R, IL-6 and TLR4 mRNA were increased at W13. The translocase of outer mitochondrial membrane, and MnSOD were reduced at W13 with higher nitrotyrosine staining. HIF-1α was also increased at W13, although EGR1 was only reduced at P1. In conclusion, maternal SE increased markers of hypoxia and oxidative stress with mitochondrial dysfunction and cell damage in both dams and offspring, and upregulated inflammatory markers in offspring, which may render SE dams and their offspring vulnerable to additional brain insults.

Figure 1. Brain mRNA expression of inflammatory markers in the SHAM and SE dams (n = 8).

Results are expressed as mean ± S.E.M. Data were analysed by student’s unpaired t-test. *P < 0.05; **P < 0.01. SE: smoke exposed.

Figure 2. Brain protein levels of MnSOD (a), TOM20 (b) and OXPHOS complexes (CI, CII, CIII, CIV and CV) (c) in the SHAM and SE dams.

Whole gel images of (a–c) in Supplementary Fig. 1. Immunohistochemistry for cortex nitrotyrosine in the dams (d) Scale bar = 50 μm (n = 4). Results are expressed as mean ± S.E.M. Data were analysed by student’s unpaired t-test. *P < 0.05; **P < 0.01. MnSOD: manganese superoxide dismutase; OXPHOS: oxidative phosphorylation; SE: smoke exposed; TOM20: translocase of the mitochondrial outer membrane.

Figure 3. Brain protein levels of HIF-1α (a) and EGR1 (b) in the SHAM and SE dams (n = 3).

Whole gel images of (a,b) in Supplementary Fig. 2. Results are expressed as mean ± S.E.M. Data were analysed by student’s unpaired t-test. EGR1: early growth response factor: HIF-1α: hypoxia-inducible factor; SE: smoke exposed.

Figure 4. Brain mRNA expression of inflammatory markers in the offspring of SHAM and SE mothers at different ages (n = 8).

Results are expressed as mean ± S.E.M. Data were analysed by student’s unpaired t-test. *P < 0.05; **P < 0.01. SE: smoke exposed.

Figure 5. Protein expression of MnSOD (a), TOM20 (b) and OXPHOS complexes (CI, CII, CIII, CIV and CV) (c) in the brain mitochondria in the offspring of SHAM and SE mothers at different ages (n = 3).

Whole gel images of (a–c) in Supplementary Fig. 3. Immunohistochemistry of cortex nitrotyrosine in week 13 offspring (d) Scale bar = 50 μm (n = 3). Results are expressed as mean ± S.E.M. Data were analysed by student’s unpaired t-test. *P < 0.05; **P < 0.01. MnSOD: manganese superoxide dismutase; OXPHOS: oxidative phosphorylation; SE: smoke exposed; TOM20: translocase of the mitochondrial outer membrane.

Figure 6. Brain protein levels of hypoxia markers in the offspring of SHAM and SE mothers at different ages (a–f) (n = 3).

Whole gel images of (a–c) in Supplementary Fig. 4. Results are expressed as mean ± S.E.M. Data were analysed by student’s unpaired t-test. **P < 0.01. EGR1: Early growth response factor; HIF-1α: hypoxia-inducible factor; SE: smoke exposed.