A 3D Epithelial-Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by ongoing inflammation, impaired tissue repair, and aberrant interplay between airway epithelium and fibroblasts, resulting in an altered extracellular matrix (ECM) composition. The ECM is the three-dimensional (3D) scaffold that provides mechanical support and biochemical signals to cells, now recognized not only as a consequence but as a potential driver of disease progression. To elucidate how the ECM influences pathophysiological changes occurring in COPD, in vitro models are needed that incorporate the ECM. ECM hydrogels are a novel experimental tool for incorporating the ECM in experimental setups. We developed an airway wall model by combining lung-derived ECM hydrogels with a co-culture of primary human fibroblasts and epithelial cells at an air-liquid interface. Collagen IV and a mixture of collagen I, fibronectin, and bovine serum albumin were used as basement membrane-mimicking coatings. The model was initially assembled using porcine lung-derived ECM hydrogels and subsequently with COPD and non-COPD human lung-derived ECM hydrogels. The resulting 3D construct exhibited considerable contraction and supported co-culture, resulting in a differentiated epithelial layer. This multi-component 3D model allows the investigation of remodelling mechanisms, exploring ECM involvement in cellular crosstalk, and holds promise as a model for drug discovery studies exploring ECM involvement in cellular interactions.

Alterations of gut microbiota and its metabolomics in children with 6PPDQ, PBDE, PCB, and metal(loid) exposure.

The composition and metabolites of the gut microbiota can be altered by environmental pollutants. However, the effect of co-exposure to multiple pollutants on the human gut microbiota has not been sufficiently studied. In this study, gut microorganisms and their metabolites were compared between 33 children from Guiyu, an e-waste dismantling and recycling area, and 34 children from Haojiang, a healthy environment. The exposure level was assessed by estimating the daily intake (EDI) of polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), 6PPD-quinone (6PPDQ), and metal(loid)s in kindergarten dust. Significant correlations were found between the EDIs of 6PPDQ, BDE28, PCB52, Ni, Cu, and the composition of gut microbiota and specific metabolites. The Bayesian kernel machine regression model showed negative correlations between the EDIs of five pollutants (6PPDQ, BDE28, PCB52, Ni, and Cu) and the composition of gut microbiota. The EDIs of these five pollutants were positively correlated with the levels of the metabolite 2,4-diaminobutyric acid, while negatively correlated with the levels of d-erythro-sphingosine and d-threitol. Our study suggests that exposure to 6PPDQ, BDE28, PCB52, Ni, and Cu in kindergarten dust is associated with alterations in the composition and metabolites of the gut microbiota. These alterations may be associated with children's health.

Altered Extracellular Vesicle-Derived Protein and microRNA Signatures in Bronchoalveolar Lavage Fluid from Patients with Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease for which there is no cure. Accumulating research results suggest a role for extracellular vesicles (EVs) in the pathogenesis of COPD. This study aimed to uncover the involvement of EVs and their molecular cargo in the progression of COPD by identification of EV-associated protein and microRNA (miRNA) profiles. We isolated EVs from the bronchial alveolar lavage fluid (BALF) of 18 patients with COPD and 11 healthy controls using size-exclusion chromatography. EV isolates were characterized using nanoparticle tracking analysis and protein content. Proteomic analysis revealed a higher abundance of 284 proteins (log2FC > 1) and a lower abundance of 3 proteins (log2FC < -1) in EVs derived from patients with COPD. Ingenuity pathway analysis showed that proteins enriched in COPD-associated EVs trigger inflammatory responses, including neutrophil degranulation. Variances in surface receptors and ligands associated with COPD EVs suggest a preferential interaction with alveolar cells. Small RNAseq analysis identified a higher abundance of ten miRNAs and a lower abundance of one miRNA in EVs from COPD versus controls (Basemean > 100, FDR < 0.05). Our data indicate that the molecular composition of EVs in the BALF of patients with COPD is altered compared to healthy control EVs. Several components in COPD EVs were identified that may perpetuate inflammation and alveolar tissue destruction.

Associations of co-exposure to polycyclic aromatic hydrocarbons and lead (Pb) with IGF1 methylation in peripheral blood of preschool children from an e-waste recycling area.

BACKGROUND: Childhood exposure to polycyclic aromatic hydrocarbons (PAHs) or lead (Pb) is associated with epigenetic modifications. However, the effects of their co-exposures on IGF1 (Insulin-like growth factor 1) methylation and the potential role in child physical growth are unclear. METHODS: From our previous children study (N = 238, ages of 3-6), 75 children with higher total concentrations of urinary ten hydroxyl PAH metabolites (∑10OH-PAHs) from an e-waste recycling area, Guiyu, and 75 with lower ∑10OH-PAHs from Haojiang (reference area) were included. Pb and IGF1 P2 promoter methylation in peripheral blood were also measured. Multivariable linear regression analyses were performed to estimate individual associations, overall effects and interactions of co-exposure to OH-PAHs and Pb on IGF1 methylation were further explored using Bayesian kernel machine regression. RESULTS: Methylation of IGF1 (CG-232) was lower (38.00 vs. 39.74 %, P < 0.001), but of CG-207 and CG-137 were higher (59.94 vs. 58.41 %; 57.60 vs. 56.28 %, both P < 0.05) in exposed children than the reference. The elevated urinary 2-OHPhe was associated with reduced methylation of CG-232 (B = -0.051, 95 % CI: -0.096, -0.005, P < 0.05), whereas blood Pb was positively associated with methylation of CG-108 (B = 0.106, 95 %CI: 0.013, 0.199, P < 0.05), even after full adjustment. Methylations of CG-224 and 218 significantly decreased when all OH-PAHs and Pb mixtures were set at 35th - 40th and 45th - 55th percentile compared to when all fixed at 50th percentile. There were bivariate interactions of co-exposure to the mixtures on methylations of CG-232, 224, 218, and 108. Methylations correlated with height, weight, were observed in the exposed children. CONCLUSIONS: Childhood co-exposure to high PAHs and Pb from the e-waste may be associated with IGF1 promoter methylation alterations in peripheral blood. This, in turn, may interrupt the physical growth of preschool children.

Association between 6PPD-quinone exposure and BMI, influenza, and diarrhea in children.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) has received extensive attention due to its ubiquitous distribution and potential toxicity. However, the distribution characteristics of 6PPD-quinone in dust from e-waste recycling areas and the consequential health risks to children are unclear. A total of 183 dust samples were collected from roads (n = 40), homes (n = 91), and kindergartens (n = 52) in Guiyu (the e-waste-exposed group) and Haojiang (the reference group) from 2019 to 2021. The results show that the concentrations of 6PPD-quinone in kindergarten and house dust from the exposed group were significantly higher than those from the reference group (P < 0.001). These findings show that e-waste may be another potential source of 6PPD-quinone, in addition to rubber tires. The exposure risk of 6PPD-quinone in children was assessed using their daily intake. The daily intake of 925 kindergarten children was calculated using the concentration of 6PPD-quinone in kindergarten dust. The daily intake of 6PPD-quinone via ingestion was approximately five orders of magnitude higher than via inhalation. Children in the exposed group had a higher exposure risk to 6PPD-quinone than the reference group. A higher daily intake of 6PPD-quinone from kindergarten dust was associated with a lower BMI and a higher frequency of influenza and diarrhea in children. This study reports the distribution of 6PPD-quinone in an e-waste recycling town and explores the associated health risks to children.

3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro. The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-ß1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels.

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

MicroRNAs Associated with Chronic Mucus Hypersecretion in COPD Are Involved in Fibroblast-Epithelium Crosstalk.

We recently identified microRNAs (miRNAs) associated with chronic mucus hypersecretion (CMH) in chronic obstructive pulmonary disease (COPD), which were expressed in both airway epithelial cells and fibroblasts. We hypothesized that these miRNAs are involved in communication between fibroblasts and epithelium, contributing to airway remodeling and CMH in COPD. Primary bronchial epithelial cells (PBECs) differentiated at the air-liquid interface, and airway fibroblasts (PAFs) from severe COPD patients with CMH were cultured alone or together. RNA was isolated and miRNA expression assessed. miRNAs differentially expressed after co-culturing were studied functionally using overexpression with mimics in mucus-expressing human lung A549 epithelial cells or normal human lung fibroblasts. In PBECs, we observed higher miR-708-5pexpression upon co-culture with fibroblasts, and miR-708-5p expression decreased upon mucociliary differentiation. In PAFs, let-7a-5p, miR-31-5p and miR-146a-5p expression was significantly increased upon co-culture. miR-708-5p overexpression suppressed mucin 5AC (MUC5AC) secretion in A549, while let-7a-5poverexpression suppressed its target gene COL4A1 in lung fibroblasts. Our findings suggest that let-7a-5p, miR-31-5p and miR-146a-5p may be involved in CMH via fibroblasts-epithelium crosstalk, including extracellular matrix gene regulation, while airway epithelial expression of miR-708-5p may be involved directly, regulating mucin production. These findings shed light on miRNA-mediated mechanisms underlying CMH, an important symptom in COPD.

Ubiquitin carboxyl-terminal hydrolase isozyme L1/UCHL1 suppresses epithelial-mesenchymal transition and is under-expressed in cadmium-transformed human bronchial epithelial cells.

Cadmium (Cd), a highly toxic heavy metal, is widespreadly distributed in the environment. Chronic exposure to Cd is associated with the development of several diseases including cancers. Over the decade, many researches have been carried on various models to examine the acute effects of Cd; yet, limited knowledge is known about the long-term Cd exposure, especially in the human lung cells. Previously, we showed that chronic Cd-exposed human bronchial epithelial BEAS-2B cells exhibited transformed cell properties, such as anchorage-independent growth, augmented cell migration, and epithelial-mesenchymal transition (EMT). To study these Cd-transformed cells more comprehensively, here, we further characterized their subproteomes. Overall, a total of 63 differentially expressed proteins between Cd-transformed and passage-matched control cells among the five subcellular fractions (cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal) were identified by mass spectrometric analysis and database searching. Interestingly, we found that the thiol protease ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) is one of the severely downregulated proteins in the Cd-transformed cells. Notably, the EMT phenotype of Cd-transformed cells can be suppressed by forced ectopic expression of UCHL1, suggesting UCHL1 as a crucial modulator in the maintenance of the proper differentiation status in lung epithelial cells. Since EMT is considered as a critical step during malignant cell transformation, finding novel cellular targets that can antagonize this transition may lead to more efficient strategies to inhibit cancer development. Our data report for the first time that UCHL1 may play a function in the suppression of EMT in Cd-transformed human lung epithelial cells, indicating that UCHL1 might be a new therapeutic target for chronic Cd-induced carcinogenesis. Graphical abstract.

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.

PAH exposure is associated with enhanced risk for pediatric dyslipidemia through serum SOD reduction.

BACKGROUND: Exposure to polycyclic aromatic hydrocarbons (PAHs) is linked to abnormal lipid metabolism, but evidence regarding PAHs as risk factors for dyslipidemia is lacking. OBJECTIVE: To investigate the respective role and interaction of PAH exposure and antioxidant consumption in the risk for pediatric dyslipidemia. METHODS: We measured the concentrations of serum lipids, superoxide dismutase (SOD) and urinary hydroxylated PAHs (OH-PAHs) in 403 children, of which 203 were from an e-waste-exposed area (Guiyu) and 200 were from a reference area (Haojiang). Biological interactions were calculated by additive models. RESULTS: Guiyu children had higher serum triglyceride concentration and dyslipidemia incidence, and lower serum concentration of high-density lipoprotein (HDL) than Haojiang children. Elevated OH-PAH concentration, and concomitant SOD reduction, were both associated with lower HDL concentration and higher hypo-HDL risk (∑3OH-Phes: B for lgHDL = -0.048, P < 0.01; OR for hypo-HDL = 3.708, 95% CI: 1.200, 11.453; SOD: BT3 for lgHDL = 0.061, P < 0.01; ORT3 for hypo-HDL = 0.168, 95% CI: 0.030, 0.941; all were adjusted for confounders). Biological interaction between phenanthrol exposure and SOD reduction was linked to dyslipidemia risk (RERI = 2.783, AP = 0.498, S = 2.537). Children with both risk factors (higher ∑3OH-Phes and lower SOD) had 5.594-times (95% CI: 1.119, 27.958) the dyslipidemia risk than children with neither risk factors (lower ∑3OH-Phes and higher SOD). CONCLUSION: High PAH exposure combined with SOD reduction is recommended for predicting elevated risk for pediatric dyslipidemia. Risk assessment of PAH-related dyslipidemia should take antioxidant concentration into consideration.

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.

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.

Elevated expression of AhR and NLRP3 link polycyclic aromatic hydrocarbon exposure to cytokine storm in preschool children.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs), as a group of persistent organic pollutants, are linked to impaired immune function and low-grade inflammation in adults and children. However, the potential of PAHs to lead to a cytokine storm associated with AhR (aryl hydrocarbon receptor) and NLRP3 (NLR family pyrin domain containing 3) in humans has been poorly studied. OBJECTIVES: We aimed to investigate the associations between PAH exposure, AhR and NLRP3 expression, and cytokines associated with a cytokine storm in healthy preschoolers. METHODS: Basic demographic surveys and physical examinations were conducted on 248 preschoolers from an electronic waste (e-waste) recycling area (Guiyu, n = 121) and a reference area (Haojiang, n = 127). Ten urinary PAH metabolite (OH-PAH) concentrations were measured. We also measured the expression levels of AhR and NLRP3 and seventeen serum cytokine levels. RESULTS: The concentrations of multiple OH-PAHs were significantly higher in the exposed group than those in the reference group, especially 1-hydroxynaphthalene (1-OH-Nap) and 2-hydroxynaphthalene (2-OH-Nap). PAH exposure was closely related to a child's living environment and hygiene habits. Expression levels of AhR and NLRP3 were significantly higher in the exposed group than in the reference group. Similarly, serum IL-1ß, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-22, IL-23, and IFN-γ levels were notably higher in the e-waste-exposed children than in the reference children. After adjusting for age, gender, BMI, family income, parental education level, and second-hand smoke exposure, we found that increased PAH exposure was associated with higher AhR and NLRP3 expression and elevated IL-4, IL-10, IL-12p70, IL-18, IL-22, IL-23, TNF-α, and IFN-γ levels. The associations between PAH exposure and IL-1ß, IL-18, IFN-γ, and TNF-ß were mediated by NLRP3 expression, and the relationships between PAH exposure and IL-4, IL-10, IL-12p70, IL-22, IL-23, and TNF-α were mediated by AhR expression. CONCLUSIONS: Our findings suggest that the association between PAH exposure and a cytokine storm may be mediated by AhR and NLRP3 expression among preschoolers.

PM2.5-bound PAHs exposure linked with low plasma insulin-like growth factor 1 levels and reduced child height.

BACKGROUND: Exposure to atmospheric fine particle matter (PM2.5) pollution and the absorbed pollutants is known to contribute to numerous adverse health effects in children including to growth. OBJECTIVE: The aim of this study was to evaluate exposure levels of atmospheric PM2.5-bound polycyclic aromatic hydrocarbons (PAHs) in an electronic waste (e-waste) polluted town, Guiyu, and to investigate the associations between PM2.5-PAH exposure, insulin-like growth factor 1 (IGF-1) levels and child growth. METHODS: This study recruited 238 preschool children (3-6 years of age), from November to December 2017, of which 125 were from Guiyu (an e-waste area) and 113 were from Haojiang (a reference area). Levels of daily PM2.5 and PM2.5-bound ∑16 PAHs were assessed to calculate individual chronic daily intakes (CDIs). IGF-1 and IGF-binding protein 3 (IGFBP-3) concentrations in child plasma were also measured. The associations and further mediation effects between exposure to PM2.5 and PM2.5-bound PAHs, child plasma IGF-1 concentration, and child height were explored by multiple linear regression models and mediation effect analysis. RESULTS: Elevated atmospheric PM2.5-bound ∑16 PAHs and PM2.5 levels were observed in Guiyu, and this led to more individual CDIs of the exposed children than the reference (all P < 0.001). The median level of plasma IGF-1 in the exposed group was lower than in the reference group (91.42 ng/mL vs. 103.59 ng/mL, P < 0.01). IGF-1 levels were negatively correlated with CDIs of PM2.5, but not with CDIs of PM2.5-bound ∑16 PAHs after adjustment. An increase of 1 µg/kg of PM2.5 intake per day was associated with a 0.012 cm reduction of child height (95% CI: -0.014, -0.009), and similarly, an elevation of 1 ng/kg of PM2.5-bound ∑16 PAHs intake per day was associated with a 0.022 cm decrease of child height (95% CI: -0.029, -0.015), both after adjustment of several potential confounders (age, gender, family cooking oil, picky eater, eating sweet food, eating fruits or vegetables, parental education level and monthly household income). The decreased plasma IGF-1 concentration mediated 15.8% of the whole effect associated with PM2.5 exposure and 23.9% of the whole effect associated with PM2.5-bound ∑16 PAHs exposure on child height. CONCLUSION: Exposure to atmospheric PM2.5-bound ∑16 PAHs and PM2.5 is negatively associated with child height, and is linked to reduced IGF-1 levels in plasma. This may suggest a causative negative role of atmospheric PM2.5-bound exposures in child growth.

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.

Differential DNA methylation in newborns with maternal exposure to heavy metals from an e-waste recycling area.

This study explored the effects of maternal exposure to e-waste environmental heavy metals on neonatal DNA methylation patterns. Neonatal umbilical cord blood (UCB) samples were collected from participants that resided in an e-waste recycling area, Guiyu and a nearby non-e-waste area, Haojiang in China. The concentrations of UCB lead (Pb), cadmium (Cd), manganese (Mn) and chromium (Cr) were measured by graphite furnace atomic absorption spectrometry. Epigenome-wide DNA methylation at 473, 844 CpG sites (CpGs) were assessed by Illumina 450 K BeadChip. The differential methylation of CpG sites from the microarray were further validated by bisulfite pyrosequencing. Bioinformatics analysis showed that 125 CpGs mapped to 79 genes were differential methylation in the e-waste exposed group with higher concentrations of heavy metals in neonatal UCB. These genes mainly involve in multiple biological processes including calcium ion binding, cell adhesion, embryonic morphogenesis, as well as in signaling pathways related to NFkB activation, adherens junction, TGF beta and apoptosis. Among them, BAI1 and CTNNA2 (involving in neuron differentiation and development) were further verified to be hyper- and hypo-methylated, respectively, which were associated with maternal Pb exposure. These results suggest that maternal exposure to e-waste environmental heavy metals (particularly lead) during pregnancy are associated with peripheral blood differential DNA methylation in newborns, specifically the genes involving in brain neuron development.

In utero exposure to cigarette smoke and effects across generations: A conference of animals on asthma.

BACKGROUND: The prevalence of asthma and chronic obstructive pulmonary disease (COPD) has risen markedly over the last decades and is reaching epidemic proportions. However, underlying molecular mechanisms are not fully understood, hampering the urgently needed development of approaches to prevent these diseases. It is well established from epidemiological studies that prenatal exposure to cigarette smoke is one of the main risk factors for aberrant lung function development or reduced fetal growth, but also for the development of asthma and possibly COPD later in life. Of note, recent evidence suggests that the disease risk can be transferred across generations, that is, from grandparents to their grandchildren. While initial studies in mouse models on in utero smoke exposure have provided important mechanistic insights, there are still knowledge gaps that need to be filled. OBJECTIVE: Thus, in this review, we summarize current knowledge on this topic derived from mouse models, while also introducing two other relevant animal models: the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. METHODS: This review is based on an intensive review of PubMed-listed transgenerational animal studies from 1902 to 2018 and focuses in detail on selected literature due to space limitations. RESULTS: This review gives a comprehensive overview of mechanistic insights obtained in studies with the three species, while highlighting the remaining knowledge gaps. We will further discuss potential (dis)advantages of all three animal models. CONCLUSION/CLINICAL RELEVANCE: Many studies have already addressed transgenerational inheritance of disease risk in mouse, zebrafish or fly models. We here propose a novel strategy for how these three model organisms can be synergistically combined to achieve a more detailed understanding of in utero cigarette smoke-induced transgenerational inheritance of disease risk.