Early-life exposure to cigarette smoke primes lung function and DNA methylation changes at Cyp1a1 upon exposure later in life

Abstract

Prenatal and early-life exposure to cigarette smoke (CS) has repeatedly been shown to induce stable, long-term changes in DNA methylation (DNAm) in offspring. It has been hypothesized that these changes might be functionally related to the known outcomes of prenatal and early-life CS exposure, which include impaired lung development, altered lung function, and increased risk of asthma and wheeze. However, to date, few studies have examined DNAm changes induced by prenatal CS in tissues of the lung, and even fewer have attempted to examine the specific influences of prenatal versus early postnatal exposures. Here, we have established a mouse model of CS exposure which isolates the effects of prenatal and early postnatal CS exposures in early life. We have used this model to measure the effects of prenatal and/or postnatal CS exposures on lung function and immune cell infiltration as well as DNAm and expression of Cyp1a1, a candidate gene previously observed to demonstrate DNAm differences on CS exposure in humans. Our study revealed that exposure to CS prenatally and in the early postnatal period causes long-lasting differences in offspring lung function, gene expression, and lung Cyp1a1 DNAm, which wane over time but are reestablished on reexposure to CS in adulthood. This study creates a testable mouse model that can be used to investigate the effects of prenatal and early postnatal CS exposures and will contribute to the design of intervention strategies to mediate these detrimental effects.NEW & NOTEWORTHY Here, we isolated effects of prenatal from early postnatal cigarette smoke and showed that exposure to cigarette smoke early in life causes changes in offspring DNA methylation at Cyp1a1 that last through early adulthood but not into late adulthood. We also showed that smoking in adulthood reestablished these DNA methylation patterns at Cyp1a1, suggesting that a mechanism other than DNA methylation results in long-term memory associated with early-life cigarette smoke exposures at this gene.

Keywords: DNA methylation; cigarette smoke; early life; lung function; priming.

Graphical abstract

Figure 1.

Development of a mouse model to study the effects of early-life CS exposure. A: experimental design: Adult female mice were exposed to CS for 9 wk, starting 3 wk before mating and ending 3 wk after birth. Half of control offspring were cross-fostered at birth with half of the CS-exposed offspring to generate four groups of offspring: control (N = 43), prenatal CS-exposed (N = 42), postnatal CS-exposed (N = 42), and combined prenatal and postnatal CS-exposed (N = 36) groups. Lungs, blood, and other tissues were collected from dams (N = 16 control, N = 16 CS) and offspring at birth (N = 19 control, N = 13 CS-exposed) and at 16 wk of age (N = 136), after lung function measurement. At age 60 wk, half of the remaining female offspring were reexposed (N = 13) to CS for 3 wk, followed by lung function and tissue collection. The remaining half (N = 14) were not reexposed to CS and served as adult controls. B: litter size of control and smoke-exposed dams. Student’s t test was used to measure differences in litter size between control and smoke-exposed dams. C: weight of control and CS-exposed male (N = 23 control, N = 16 CS) and female (N = 44 control, N = 30 CS) offspring at birth. Differences in birth weight were analyzed using Student’s t tests within sexes. CS, cigarette smoke.

Figure 2.

Early-life exposure to CS alters sensitivity to methacholine up to 13 wk after cessation of smoke exposure. Lung function parameters: total lung resistance at baseline (A) and airway resistance at baseline (B). Female offspring exposed to prenatal CS had significantly higher Rn (mean = 0.34, SD = 0.03) compared with female controls (mean = 0.26, SD = 0.06). C: tissue resistance at baseline. D: alveolar elastance at baseline. Male offspring exposed to prenatal CS had significantly higher H (mean = 17.50, SD = 0.63) compared with male controls (mean = 16.40, SD = 0.91). E: total lung resistance with methacholine. F: airway resistance with methacholine. G: tissue resistance with methacholine. H: alveolar elastance with methacholine. Lung function with methacholine (E–H): *P < 0.05, control versus full CS; †P < 0.05 control versus postnatal CS; #P < 0.05 control versus prenatal CS groups. Group numbers: control females = 6, postnatal females = 7, prenatal females = 5, full females = 9, control males = 7, postnatal males = 8, prenatal males = 9, full males = 6. Lung function was assessed by taking 90th percentile values in response to injection of saline into the lungs (baseline) and in response to increasing doses of methacholine. Lung function data were analyzed in a sex-disaggregated manner using a two-way ANOVA, followed by multiple comparisons between groups at each methacholine dose where significant. CS, cigarette smoke; H, elastance; Rn, Newtonian resistance.

Figure 3.

Early-life exposure to CS increases immune cell infiltration into offspring lungs, up to 13 wk after smoke exposure. A: total immune cells per mL lavage. B: eosinophils per mL lavage. C: macrophages per mL lavage. D: lymphocytes per mL lavage. Group numbers: control females = 17, postnatal females = 14, prenatal females = 13, full females = 8, control males = 10, postnatal males = 10, prenatal males = 10, full males = 7. Differential cell counts were normalized to lavage volume. Pairwise comparisons were conducted using t tests. CS, cigarette smoke.

Figure 4.

Early-life exposure to CS significantly alters lung Cyp1a1 expression at birth and Cyp1a1 DNAm 13 wk after smoke cessation. A: Cyp1a1 DNAm in offspring lungs at birth (N = 10 control, N = 10 CS). B: Cyp1a1 expression in offspring lungs at birth was significantly increased in CS-exposed males (N = 5, mean = 19.00, SD = 10.30) and females (N = 5, mean = 27.50, SD = 4.54) offspring, compared with control males (N = 5, mean = 1.36, SD = 0.93) and control females (N = 5, mean = 1.47, SD = 0.43). C: Ahrr DNAm in offspring lungs at birth (N = 8 control, N = 10 CS). D: Ahrr expression in offspring lungs at birth (N = 10 control, N = 10 CS). E: Cyp1a1 DNAm in offspring lungs at 16 wk of age. Prenatal and postnatal CS exposure caused an increase in female lung Cyp1a1 DNAm, whereas fully exposed offspring showed a decrease. Only prenatally exposed female offspring had significantly elevated Cyp1a1 DNAm (mean = 18.80, SD = 1.42) compared with control females (mean = 14.10, SD = 0.26). N = 3 per group. F: Cyp1a1 expression in offspring lungs at 16 wk of age. Prenatally exposed female offspring had significantly elevated lung Cyp1a1 expression levels (mean = 3.28, SD = 1.44) compared with control females (mean = 1.48, SD = 0.47). N = 3–6 per group. G: Ahrr DNAm in offspring lungs at 16 wk of age. N = 3 per group. H: Ahrr expression in offspring lungs at 16 wk of age. N = 3–6 per group. One-way ANOVA was used to compare DNAm values between groups (P < 0.05 was significant), followed by two-group comparisons where significant. CS, cigarette smoke.

Figure 5.

Acute reexposure to CS at 60 wk induced significant alterations in baseline lung function compared with early-life control offspring which were exposed to CS in adulthood. Lung function parameters at baseline. A: total lung resistance. Offspring with full early-life CS exposure and reexposed to CS at 63 wk (N = 3) had higher Rrs (mean = 0.88, SD = 0.62) compared with early-life control offspring exposed to CS at 63 wk only (N = 4, mean = 0.35, SD = 0.11). B: airway resistance. C: tissue resistance. Offspring with full early-life CS exposure and reexposed to CS at 63 wk (N = 3) had higher G (mean = 6.04, SD = 3.60) compared with early-life control offspring exposed to CS at 63 wk only (N = 4, mean = 2.74, SD = 0.20). Offspring exposed to prenatal CS early in life and reexposed to CS at 63 wk (N = 3) had higher G (mean = 3.40, SD = 0.51) compared with early-life control offspring exposed to CS at 63 wk only. D: alveolar elastance. E: total immune cells per mL lavage. F: eosinophils per mL lavage. G: macrophages per mL lavage. H: lymphocytes per mL lavage. For lung function, 90th percentile values were calculated in response to saline administered into the lungs. Differential cell counts were normalized to lavage volume. CS, cigarette smoke; G, tissue resistance; Rrs, total airway resistance.

Figure 6.

Reexposure of offspring to CS at 60 wk induced changes in lung DNAm which were similar to those observed at 16 wk. A: Cyp1a1 DNAm in offspring lungs at 63 wk. Offspring with full early-life CS exposure and reexposed to CS at 63 wk (N = 3) had higher lung Cyp1a1 DNAm (mean = 17.00, SD = 0.65) compared with early-life control offspring exposed to CS at 63 wk only (N = 5, mean = 19.20, SD = 1.37). B: Cyp1a1 expression in offspring lungs at 63 wk (N = 27). Differences in DNAm and expression were calculated using a one-way ANOVA, followed by two-group comparisons. CS, cigarette smoke.

Figure 7.

Chromatin state and genomic locations of nine Cyp1a1 and four Ahrr positions on the Illumina mouse methylation microarray. I: Cyp1a1 sites from the Illumina mouse array are highlighted in gray and labeled A–I, whereas the chosen candidate Cyp1a1 position (not contained in the mouse array) is highlighted in red. The genomic locations of the nine Cyp1a1 array sites are as follows: A at position 57687270, B at position 57694012, C at position 57696335, D at position 57696629, E at position 57697560, F at position 57697641, G at position 57698266, H at position 57700489, and I at position 57702263. The genomic location of the candidate CpG is at position 57696231. II: Ahrr sites from the Illumina mouse array are highlighted in gray and labeled A–D, whereas the chosen candidate Ahrr position (not contained in the mouse array) is highlighted in red. The genomic locations of the four Ahrr array sites are as follows: A at position 74245665, B at position 74248917, C at position 74292502, and D at position 74292541. The genomic location of the candidate CpG is at position 74260517. Information on genomic locations and chromatin state was obtained from UCSC genome browser. Chromatin state displayed here is of mouse lungs at postnatal day 0, as this is the latest and most relevant period currently available.