Prenatal exposure to environmental tobacco smoke alters gene expression in the developing murine hippocampus

Abstract

Background: Little is known about the effects of passive smoke exposures on the developing brain.

Objective: The purpose of the current study was to identify changes in gene expression in the murine hippocampus as a consequence of in utero exposure to sidestream cigarette smoke (an experimental equivalent of environmental tobacco smoke (ETS)) at exposure levels that do not result in fetal growth inhibition.

Methods: A whole body smoke inhalation exposure system was utilized to deliver ETS to pregnant C57BL/6J mice for 6 h/day from gestational days 6-17 (gd 6-17) [for microarray] or gd 6-18.5 [for fetal phenotyping].

Results: There were no significant effects of ETS exposure on fetal phenotype. However, 61 "expressed" genes in the gd 18.5 fetal hippocampus were differentially regulated (up- or down-regulated by 1.5-fold or greater) by maternal exposure to ETS. Of these 61 genes, 25 genes were upregulated while 36 genes were down-regulated. A systems biology approach, including computational methodologies, identified cellular response pathways, and biological themes, underlying altered fetal programming of the embryonic hippocampus by in utero cigarette smoke exposure.

Conclusions: Results from the present study suggest that even in the absence of effects on fetal growth, prenatal smoke exposure can alter gene expression during the "early" period of hippocampal growth and may result in abnormal hippocampal morphology, connectivity, and function.

Figure 1

Photographs of the dorsal view of the gestational day 17 murine brain. (A) The intact fetal brain on gestational day 17. (B) A unilateral view of the interior portion of the fetal cerebral cortex on gestational day 17. The cerebral cortex was opened along the midline and the hippocampal region was identified, removed and frozen on dry ice. The region demarcated by the white line represents the hippocampal tissue that was bilaterally excised from the cerebral cortex of the gestational day 17 murine fetus for RNA isolation. (CB) Cerebellum; (CTX) Cerebral cortex/Cerebrum; (OL) Olfactory lobes; (HI) Hippocampus.

Figure 2

Heat map illustrating the differentially regulated genes in the murine hippocampus following exposure to sham and sidestream smoke exposure. Each row of the heat map represents a gene while each column represents the experimental treatment (labeled at the bottom). The color saturation scale, shown to the right, represents the level of gene expression, with red indicating an increase in gene expression and blue indicating a decrease in gene expression. Only genes whose expression demonstrated a 1.5 fold or greater increase or decrease are depicted. The list of genes comprising the heat map is found in Table 3.

Figure 3

Ontogeny of up-regulated genes in the hippocampus following exposure to ETS. The pie chart depicts the predicted function of genes that demonstrated an increase in expression in the hippocampus following ETS exposure. Numbers beside each pie segment indicate the percentage of the total number of upregulated genes assigned to a given functional category. Functional gene category assignments were determined through an extensive literature search in PubMed. Note that individual genes may be present in more than one category.

Figure 4

Ontogeny of down-regulated genes in the hippocampus following exposure to ETS. The pie chart depicts the predicted function of genes that demonstrated a decrease in expression in the hippocampus following ETS exposure. Numbers on each pie segment indicate the percentage of the total number of down-regulated genes assigned to a given functional category. Functional gene category assignments were determined through an extensive literature search in PubMed. Note that individual genes may be present in more than one category.

Figure 5

Computational gene interaction predictions: selected gene networks (A, B, C) in the fetal hippocampus following ETS exposure. Gene networks were constructed with Ingenuity Systems Pathway Analysis (IPA) software. Several differentially regulated genes from the study were used to construct gene association maps for predicting effects of prenatal exposure to ETS on various cellular and molecular events in the developing mouse hippocampus. The first statistically significant network (Fig. 5A) that was generated, includes p53 tumor suppressor (TP53), Hras oncogene, β-catenin (CTNNB1) and Ubiquitin-B (UBB), and also consists of several genes such as those encoding HDAC2, Semaphorin-5A, Aldehyde dehydrogenase 1A3 (ALDH1A3), Rho guanine nucleotide exchange factor 15 (ARHGEF15), Apolipoprotein D, Polymerase kappa, and Neurexin-3 (NRXN3) among others, which demonstrated significant differential expression within the hippocampus as a consequence of prenatal ETS exposure (Table 3). The second statistically significant network (Fig. 5B) consists of – Rous sarcoma oncogene (SRC), Angiotensinogen (AGT), Zinc finger protein 36 (ZFP36) and Hoxa9, in addition to a number of genes encoding proteins such as Somatostatin receptor 5 (SSTR5), Potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11), Musashi-2 (MSI2), and Phospholipase A2, group VI (PLA2G6) among others, which are either up- or down-regulated in the developing hippocampus exposed to in utero sidestream tobacco smoke (Table 3). The third statistically significant network (Fig. 5C) is composed of – p53, RhoA, β-catenin, Hras, as well as several differentially expressed genes – detected in the present study – such as those encoding – Insulin-like growth factor 2 mRNA binding protein 1 (Igf2bp1), HDAC2, Apolipoprotein D (APOD), ATPase Class V, type 10D (ATP10D), Ring finger protein (RNF168), ATPase family, AAA domain containing 2 (ATAD2), etc. (Table 3). Solid lines specify direct relationships whereas dotted lines indicate indirect interactions.

Figure 5

Computational gene interaction predictions: selected gene networks (A, B, C) in the fetal hippocampus following ETS exposure. Gene networks were constructed with Ingenuity Systems Pathway Analysis (IPA) software. Several differentially regulated genes from the study were used to construct gene association maps for predicting effects of prenatal exposure to ETS on various cellular and molecular events in the developing mouse hippocampus. The first statistically significant network (Fig. 5A) that was generated, includes p53 tumor suppressor (TP53), Hras oncogene, β-catenin (CTNNB1) and Ubiquitin-B (UBB), and also consists of several genes such as those encoding HDAC2, Semaphorin-5A, Aldehyde dehydrogenase 1A3 (ALDH1A3), Rho guanine nucleotide exchange factor 15 (ARHGEF15), Apolipoprotein D, Polymerase kappa, and Neurexin-3 (NRXN3) among others, which demonstrated significant differential expression within the hippocampus as a consequence of prenatal ETS exposure (Table 3). The second statistically significant network (Fig. 5B) consists of – Rous sarcoma oncogene (SRC), Angiotensinogen (AGT), Zinc finger protein 36 (ZFP36) and Hoxa9, in addition to a number of genes encoding proteins such as Somatostatin receptor 5 (SSTR5), Potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11), Musashi-2 (MSI2), and Phospholipase A2, group VI (PLA2G6) among others, which are either up- or down-regulated in the developing hippocampus exposed to in utero sidestream tobacco smoke (Table 3). The third statistically significant network (Fig. 5C) is composed of – p53, RhoA, β-catenin, Hras, as well as several differentially expressed genes – detected in the present study – such as those encoding – Insulin-like growth factor 2 mRNA binding protein 1 (Igf2bp1), HDAC2, Apolipoprotein D (APOD), ATPase Class V, type 10D (ATP10D), Ring finger protein (RNF168), ATPase family, AAA domain containing 2 (ATAD2), etc. (Table 3). Solid lines specify direct relationships whereas dotted lines indicate indirect interactions.

Figure 5

Computational gene interaction predictions: selected gene networks (A, B, C) in the fetal hippocampus following ETS exposure. Gene networks were constructed with Ingenuity Systems Pathway Analysis (IPA) software. Several differentially regulated genes from the study were used to construct gene association maps for predicting effects of prenatal exposure to ETS on various cellular and molecular events in the developing mouse hippocampus. The first statistically significant network (Fig. 5A) that was generated, includes p53 tumor suppressor (TP53), Hras oncogene, β-catenin (CTNNB1) and Ubiquitin-B (UBB), and also consists of several genes such as those encoding HDAC2, Semaphorin-5A, Aldehyde dehydrogenase 1A3 (ALDH1A3), Rho guanine nucleotide exchange factor 15 (ARHGEF15), Apolipoprotein D, Polymerase kappa, and Neurexin-3 (NRXN3) among others, which demonstrated significant differential expression within the hippocampus as a consequence of prenatal ETS exposure (Table 3). The second statistically significant network (Fig. 5B) consists of – Rous sarcoma oncogene (SRC), Angiotensinogen (AGT), Zinc finger protein 36 (ZFP36) and Hoxa9, in addition to a number of genes encoding proteins such as Somatostatin receptor 5 (SSTR5), Potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11), Musashi-2 (MSI2), and Phospholipase A2, group VI (PLA2G6) among others, which are either up- or down-regulated in the developing hippocampus exposed to in utero sidestream tobacco smoke (Table 3). The third statistically significant network (Fig. 5C) is composed of – p53, RhoA, β-catenin, Hras, as well as several differentially expressed genes – detected in the present study – such as those encoding – Insulin-like growth factor 2 mRNA binding protein 1 (Igf2bp1), HDAC2, Apolipoprotein D (APOD), ATPase Class V, type 10D (ATP10D), Ring finger protein (RNF168), ATPase family, AAA domain containing 2 (ATAD2), etc. (Table 3). Solid lines specify direct relationships whereas dotted lines indicate indirect interactions.