Background
Since the first Surgeon General’s Report on Smoking and Health in 1964 [
1], tobacco use has been recognized as a leading public health concern. Currently, over 5 million deaths worldwide occur annually due to direct tobacco use, with second-hand smoke (SHS) exposures being responsible for approximately 600,000 of those deaths [
1,
2]. In 1994, the US EPA classified SHS as a class A carcinogen. SHS aerosols are a complex mixture of highly toxic particles and gases that contain more than 4000 different chemicals, including polynuclear aromatic hydrocarbons (PAHs), 250 cytotoxic compounds and at least 50 substances that are classified as known or possibly carcinogenic to humans, such as formaldehyde, cadmium, nickel and benzo(a)pyrene [
2‐
4]. Annually, in the United States, over 126 million people, including pregnant women and women of childbearing age, are exposed to SHS [
5]. This is in addition to the 10% of women who smoke during their pregnancy [
6]. Thus, unborn children are a vulnerable subpopulation involuntarily exposed to cigarette smoke and SHS. It is now well documented that active smoking during pregnancy causes altered lung function, including decreased compliance and expiratory flow rates, as well as increased resistance, and increased lower respiratory diseases in the offspring [
7‐
12]. However, few studies have investigated the contribution of
in utero SHS exposures to these same outcomes. Currently, there is a growing body of epidemiological evidence showing that
in utero exposures to SHS can affect fetal development and result in adverse effects ranging from low birth weight to increased disease susceptibility in adulthood [
13‐
15].
As early as 1967, the health consequences of SHS to children, particularly on the respiratory tract, were recognized and documented [
16]. More recent studies have shown that
in utero exposure to environmental pollutants, including SHS, results in alterations of physical and physiological features of the fetus, as well as increased disease susceptibility in adulthood [
17‐
20]. Studies from our lab and others have shown that
in utero SHS exposure exacerbates adult responses to environmental stressors, including house dust mites,
Aspergillus fumigatus and SHS [
18,
21,
22]. We have previously established that
in utero exposure to SHS aggravates airway hyperresponsiveness, and increases expression of chemokines, cytokines, and acute phase response genes, which results in establishment of a pro-asthmatic milieu in ovalbumin-challenged adult mice [
20,
23]. We also showed that upon re-exposure to SHS as adult, mice exposed
in utero to SHS exhibited altered lung structure [
21]. Overall, we have demonstrated that
in utero exposure to SHS 1) elicits persistent well-documented pathophysiological and molecular changes in various adult lung disease models, upon re-exposure to an irritant as an adult, and 2) modulates responses related to lung function and structure, as well as transcriptomic alterations. Meanwhile, epidemiological studies associated
in utero exposure to cigarette smoke with sustained lung deficiencies, which could lead to impaired lung function following additional postnatal exposures [
8,
24‐
26]. Moreover, epidemiological studies have demonstrated that
in utero SHS exposures are a risk factor for pulmonary diseases, including asthma, as well as for altered lung function [
27,
28]. Despite those clear associations, little is known about whether
in utero SHS exposure alone permanently alters lung function and/or structure in the offspring. This is due mostly to the fact the human lung is exposed to a multitude of irritants throughout life, e.g. allergens and bacteria, making the contribution of
in utero SHS exposure alone on lung health challenging to assess [
29‐
31]. Thus, there is still insufficient evidence to define the long-term contribution of
in utero SHS exposure alone, as a sole or baseline factor, to modification of lung function and structure, thereby predisposing to the development of lung diseases, such as asthma, emphysema, chronic bronchitis, fibrosis or cancer [
29‐
31]. To address this knowledge gap, in the present study we asked whether
in utero SHS exposure alone, without any postnatal re-exposure to an irritant, is sufficient to alter lung structure and function in adult 15-week old mice.
Discussion
SHS is a major indoor air pollutant and increasing numbers of epidemiological and experimental studies have associated
in utero exposure to SHS with adverse outcomes in newborns [
2,
23,
25,
37‐
39]. Here we investigated whether
in utero SHS exposure alone is sufficient to alter lung structure and function in adult mice. To the best of our knowledge, this is the first report of lung structural changes in adult mice exposed only to SHS
in utero. We showed that
in utero SHS exposure significantly increased Lm and decreased the SApUV of the lungs in both males and females (Fig.
2), indicating perturbation in alveolar developmental processes. Consequent to the lung structural alterations, the tidal volume, minute volume and inspiratory capacity were significantly decreased in male mice exposed
in utero to SHS compared with the controls (Fig.
3), suggesting that males are more sensitive than females to an SHS insult during lung development. Additionally, the
in utero SHS exposure dysregulated the expression of 33 genes (Fig.
4), including
Serpina1a, the mouse ortholog of the human gene
A1AT, with deficiency, or lower levels, of
A1AT being known genetic risk factors for emphysema. Here
A1AT was significantly down-regulated in SHS-exposed male mice. This suggests that
in utero SHS exposure may predispose to emphysema development in adulthood. Furthermore,
Dnmt3a, which is involved in DNA methylation, was significantly down-regulated at both gene (10 fold) and protein (2.7 fold) expression levels in SHS-exposed male mice (Figs.
4 and
5), suggesting a hypomethylated state of the lung tissue, which is known to be associated with activation of oncogenes [
40‐
43]. In female mice,
in utero exposures to SHS up-regulated the protein expression of DNMT3A 5.5 fold compared with air controls (Fig.
5), suggesting hypermethylation, which is related to silencing of tumor suppressors [
40‐
43]. Overall, these data strongly indicate that
in utero SHS exposure alone has significant persistent repercussions on the respiratory system, suggesting that
in utero SHS exposure can predispose to at least some incurable adult lung diseases.
Morphometric measurements determined that, following
in utero SHS exposure, both male and female mice at 15 weeks of age had altered lung structure, as evidenced by elevated L
m values, indicating 1) increased airspace size, and 2) diminished alveolar surface area (Fig.
2). Whether the observed SHS-induced alterations in lung structure in this mouse model are due to compromised alveolar development or to damaged septa walls is unknown [
44]; however, the RNA-sequencing data suggest that
in utero SHS affects extracellular matrix-associated gene expression, with the inclusion of 3 differentially expressed genes in the fibronectin type III cluster (Fig.
4). This cluster includes proteins involved in extracellular matrix remodeling (
Fsd1l,
Obsl1 and
Trim46; Fig.
4). A previous report noted that imbalances in the production of extracellular matrix proteins, including connective tissue growth factor, collagen and fibronectin, resulted in airway remodeling [
45]. Moreover,
Serpina1a, the mouse ortholog of the human gene
A1AT, protects the lungs against elastase activity, which hydrolyzes proteins of the extracellular matrix, and thus contributes to lung structural breakdown [
46].
Serpina1a was down-regulated at the gene expression level in the
in utero SHS-exposed males and at the protein level in both male and female SHS-exposed mice (Figs.
4 and
5). At 15 weeks post-
in utero SHS exposure, the expression of genes involved in lung development was not significantly dysregulated; however, at this time-point, expression of genes included in the fibronectin type III cluster was significantly changed (Fig.
4). This suggests that effects of
in utero SHS on genes involved in the fibronectin type III cluster are important, regarding persistence of structural and functional alterations in adulthood. Overall, our data suggest that the airspace enlargement observed in the
in utero SHS-exposed mice may be consequent to lung tissue remodeling, rather than solely subsequent to impaired lung growth and development. These results are supported by epidemiological studies and animal models, where it was previously shown that
in utero cigarette smoke, as well as SHS, exposures affect the lungs of the offspring in terms of reduced growth, as well as enlarged alveoli that are present in fewer numbers (hypoalveolarization) [
10,
25,
47‐
49]. Hence, in line with previous published reports, our study shows that
in utero SHS exposure alone is a direct cause of alveolar structural defects, and therefore may increase the risk of developing respiratory diseases, including obstructive pulmonary diseases, e.g. emphysema, later in life.
Our study also showed that 15-week old male mice exposed
in utero to SHS had significantly decreased lung capacities, including tidal and minute volumes, as well as inspiratory capacity (Fig.
3). These decrements in lung function seem to be secondary to changes in the lung architecture, as suggested by previous studies where
in utero cigarette smoke-induced lung structural alterations impaired lung function by restricting airflow in small conducting airways [
10,
25,
47‐
49]. In our model, it is unlikely that restricted airflow is responsible for the decreases in lung volumes, since
in utero SHS had no significant effect on respiratory system resistance or Newtonian resistance (resistance of the conducting airways) at 15 weeks of age (data not shown). Since
in utero SHS exposure alone significantly affected the lungs’ geometrical architecture, measured by alveolar airspace enlargement (Fig.
2), and gene and protein expression changes were largely associated with matrix airway-remodeling (Figs
4 and
5) in both male and female mice, with significant decline in lung function observed only in male mice (Fig.
3), the data suggest that changes in lung function may only be indirectly associated with lung structural damage. In fact, decline in lung function is multifactorial [
50,
51] and our data suggest that lung structural changes may be necessary, but not sufficient, to impair lung function. Since all SHS fetuses were exposed simultaneously in the same chamber, these results also suggest that male mice are more susceptible than female mice to
in utero SHS-induced damage, and that sex differences during lung development may play a pivotal role in healthy pulmonary function.
It is well recognized that for many mammalian species, including mice and humans, critical factors in lung development, including surfactant production and fetal breathing, are events beginning earlier in the lung maturation process of female fetuses compared with males [
52‐
56]. Lung surfactant synthesis provides the pulmonary phenotype with adequate air flows, resistance and compliance, and thus plays a key role in optimal pulmonary function and lung homeostasis [
56‐
60]. This suggests that the synthesis of surfactant occurring at an earlier developmental time point in females may protect the lungs against environmental insults, such as SHS, with regard to lung function. Epidemiological studies support this sex bias, with females being preferentially protected from early lung environmental irritants. The prevalence for wheeze and asthma is higher in boys than in girls [
61‐
65]. These findings correlate with results of a previous study from our laboratory showing that male mice exposed
in utero to SHS exhibited enhanced asthmatic responses following ovalbumin treatment compared with their female counterparts at 23 weeks of age [
23]. Whether there is potential for lung recovery following
in utero SHS exposure cannot be determined from our study since we only analyzed one time-point. In accordance, however, with the results of the present study, epidemiological evidence showed declines in lung function in offspring exposed
in utero to cigarette smoke or SHS in childhood [
8,
66‐
73]. These declines were sustained until early adulthood (21 years) in males [
74]. This suggests that even though there may be some lung recovery,
in utero cigarette smoke or SHS pulmonary effects persist into childhood and early adulthood. This study provides additional data demonstrating that lung function alterations that are sustained through early adulthood seem to be sex-specific, with males being more susceptible than females to
in utero SHS effects; thus, potentially increasing the risk of males to develop respiratory diseases, including restrictive lung diseases.
Overall, we showed that even at 15 weeks of age, the effects of
in utero SHS exposure can be seen in terms of altered lung structure and function, as well as altered gene and protein expression in male mice (Figs
2,
3,
4 and
5). The exact mechanisms linking the
in utero SHS exposure to those adverse lung effects in adulthood are unknown; however, it is widely recognized that several types of environmental exposures, including air pollution [
75], maternal diet [
76], and cigarette smoke [
77], induce epigenetic alterations that could impact lung development, as well as lung repair following acute injury [
78]. Epigenetic mechanisms can modify the epigenome in a transitory manner or permanently into adulthood [
79]. DNA methylation regulates gene expression through an epigenetic silencing mechanism and is involved in tumorigenesis by influencing, among others, the expression of oncogenes or tumor suppressor genes [
41]. DNA (Cytosine-5-)-Methyltransferase 3 Alpha (Dnmt3a), an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, is responsible for
de novo DNA methylation, and thus that plays a central role in this mechanism [
74]. The methylation status of DNA can either be hyper- or hypomethylated, although global DNA hypomethylation and specific locus hypermethylation frequently co-occur in human cancers [
40,
42]. Numerous studies [
80‐
83] showed that DNA methylation has roles in both early and late stages of tumorigenesis. In early stages, increased levels of DNA methylation lead to hypermethylation of tumor suppressor genes and thus, facilitate tumor initiation. In late stages, decreased levels of DNA methylation lead to hypomethylation of oncogenes, which promotes cancer progression [
43]. Our RNA-sequencing results, confirmed by protein expression (Figs.
4 and
5), indicate that
in utero SHS exposure alone significantly down-regulated (10.4 fold change at the gene level and 2.7 fold change at the protein level; Figs.
4 and
5) the expression of Dnmt3a in males, which suggests the possibility of DNA hypomethylation of other lung genes. This hypomethylation finding is supported by an epidemiological study conducted by Breton et al. [
77] that used buccal and aerodigestive tissue cells (as surrogates for lung cells) of children exposed
in utero to cigarette smoke and showed that this exposure resulted in significantly decreased global methylation, i.e. hypomethylation, as well as gene-specific DNA methylation alterations [
77]. Global DNA hypomethylation is associated with development of cancer [
84], including lung cancer, which has been associated with Dnmt3a deficiency in mice [
85,
86]. Furthermore, in our study, while
in utero SHS-exposed male mice exhibited down-regulation of the DNMT3A protein (2.7 fold), female mice that underwent the same treatment showed up-regulation of this protein by 5.5 fold (Fig.
5). In a previous study, it was observed that methylation levels can be influenced by sex, although no mechanisms have been proposed [
40]. As mentioned previously, it is well known that both types of alteration are associated with tumor formation, with hypomethylation status being associated with activation of oncogenes and hypermethylation with silencing of tumor suppressors [
40]. Thus, in addition to our results, there is increasing evidence for epigenetic effects of
in utero SHS exposure alone on lung development with persistence into adult life, and this may predispose to respiratory morbidity, including lung cancer [
39].