The effect of (prenatal) smoke exposure on lung progenitor cell behavior and Cyp2a5 gene regulation

Lung progenitor cell behavior and Cyp2a5 gene regulationPrenatal smoke exposure (PSE) is associated with an increased risk for chronic obstructive pulmonary disease (COPD) and cigarette smoke addiction in later in life. One of the proposed underlying mechanisms for PSE-induced risk for COPD and aberrant smoking behavior could be epigenetic modifications of particular genes. UMCG researcher Khosbayar Lkhagvadorj investigated the effect of (prenatal) smoke exposure on lung epithelial cell development and regeneration linked with epidermal growth factor receptor (EGFR) signaling. This was addressed in PSE neonatal mouse offspring and patients with COPD. In addition, he investigated the effect of pre- and/or postnatal smoke exposure on Cyp2a5 DNA methylation in relation to the in vitro nicotine conversion in liver and lung. He has provided evidence in mouse and human lung tissue and organoid models that (prenatal) smoke exposure triggers abnormal lung development, tissue regeneration, and nicotine dependence. Firstly, (prenatal) smoke exposure impaired lung epithelial cell differentiation in mouse offspring, which was associated with EGFR signaling. Moreover, lower expression of the EGFR on alveolar type 2 cells could be related to impaired alveolar progenitor cell function in COPD patients, which could explain impaired lung regeneration in these patients. Secondly, PSE increased the nicotine metabolism, which could be linked to a persistent change of PSE-induced Cyp2a5 methylation across the three developmental stages. If our results in the mouse were translated to the human situation, smoking during pregnancy would pose a threat to the unborn child, as it not only affects the development of the fetal lung but may also increase the risk of nicotine addiction when the offspring starts smoking

Prenatal smoke effect on mouse offspring Igf1 promoter methylation from fetal stage to adulthood is organ- and sex-specific

Prenatal smoke exposure (PSE) is associated with reduced birth weight, impaired fetal development, and increased risk for diseases later in life. Changes in DNA methylation may be involved, as multiple large-scale epigenome-wide association studies showed that PSE is robustly associated with DNA methylation changes in blood among offspring in early life. Insulin-like growth factor-1 (IGF1) is important in growth, differentiation, and repair processes after injury. However, no studies investigated the organ-specific persistence of PSE-induced methylation change of Igf1 into adulthood. Based on our previous studies on the PSE effect on Igf1 promoter methylation in fetal and neonatal mouse offspring, we now have extended our studies to adulthood. Our data show that basal Igf1 promoter methylation generally increased in the lung but decreased in the liver (except for 2 persistent CpG sites in both organs) across three different developmental stages. PSE changed Igf1 promoter methylation in all three developmental stages, which was organ and sex specific. The PSE effect was less pronounced in adult offspring compared with the fetal and neonatal stages. In addition, the PSE effect in the adult stage was more pronounced in the lung compared with the liver. For most CpG sites, an inverse correlation was found for promoter methylation and mRNA expression when the data of all three stages were combined. This was more prominent in the liver. Our findings provide additional evidence for sex- and organ-dependent prenatal programming, which supports the developmental origins of health and disease (DOHaD) hypothesis

Prenatal smoke exposure induces persistent Cyp2a5 methylation and increases nicotine metabolism in the liver of neonatal and adult male offspring

Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5. We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life

Postnatal Smoke Exposure Further Increases the Hepatic Nicotine Metabolism in Prenatally Smoke Exposed Male Offspring and Is Linked with Aberrant Cyp2a5 Methylation

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.</p

Postnatal Smoke Exposure Further Increases the Hepatic Nicotine Metabolism in Prenatally Smoke Exposed Male Offspring and Is Linked with Aberrant Cyp2a5 Methylation

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life

Cytochrome P450 2A5 methylation and gene expression profile is organ-specific and affected by maternal smoking during pregnancy

Background: Prenatal smoke exposure (PSE) is a risk factor for COPD. One susceptibility gene for COPD is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher CYP2A6 activity is associated nicotine dependence. However, no research has addressed the PSE effects on CYP2A6, or CYP2A5 in the mouse. Aim: To investigate the PSE effect on Cyp2a5 gene expression and promoter methylation in fetal and neonatal offspring. Methods: C57BL/6 mice were exposed to fresh air or smoke from 3 weeks before conception until delivery. From liver and lung tissue, DNA and RNA were isolated for gene expression and promoter methylation analysis of Cyp2a5 in fetuses (E17.5) and neonates (D3). Results: Cyp2a5 mRNA expression was increased in male neonatal liver from PSE offspring. In contrast, in the lung, Cyp2a5 expression was reduced in both fetal and neonatal male PSE offspring. Promoter methylation was sex-dependently induced in both fetal and neonatal liver from PSE offspring when compared to their corresponding control groups. In the fetal lung, methylation was downregulated by PSE. Promoter methylation of several CpG sites (inversely) correlated with mRNA expression in liver and lung in both fetal and neonatal offspring. Conclusions: This study presents a PSE effect on Cyp2a5 expression, which is opposite in liver and lung and associated with promoter methylation. Induced Cyp2a5 gene expression in the neonatal liver may indicate a higher nicotine metabolism which, when translated to the human situation, could lead to an increased risk for COPD and higher nicotine dependence later in life

Prenatal smoke exposure dysregulates lung epithelial cell differentiation in mouse offspring - Role for AREG-induced EGFR signaling

Prenatal smoke exposure is a risk factor for impaired lung development in children. Recent studies have indicated that amphiregulin (AREG), which is a ligand of the epidermal growth factor receptor (EGFR), has a regulatory role in airway epithelial cell differentiation. In this study, we investigated the effect of prenatal smoke exposure on lung epithelial cell differentiation and linked this with AREG-EGFR signaling in 1-day-old mouse offspring. Bronchial and alveolar epithelial cell differentiations were assessed by immunohistochemistry. Areg, epidermal growth factor (Egf), and mRNA expressions of specific markers for bronchial and alveolar epithelial cells were assessed by RT-qPCR. The results in neonatal lungs were validated in an AREG-treated three-dimensional mouse lung organoid model. We found that prenatal smoke exposure reduced the number of ciliated cells and the expression of the cilia-related transcription factor Foxj1, whereas it resulted in higher expression of mucus-related transcription factors Spdef and Foxm1 in the lung. Moreover, prenatally smoke-exposed offspring had higher numbers of alveolar epithelial type II cells (AECII) and lower expression of the AECI-related Pdpn and Gramd2 markers. This was accompanied by higher expression of Areg and lower expression of Egf in prenatally smoke-exposed offspring. In bronchial organoids, AREG treatment resulted in fewer ciliated cells and more basal cells when compared with non-treated bronchiolar organoids. In alveolar organoids, AREG treatment led to more AECII cells than non-treated AECII cells. Taken together, the observed impaired bronchial and alveolar cell development in prenatally smoke-exposed neonatal offspring may be induced by increased AREG-EGFR signaling